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1
Kano, Itsu, Kanako Satoh, Fumiko Nagai, Keiko Ushiyama, Toshiko Nakao, Yukichi Hara та Kazutaka Kano. "Antigenic determinant of a monoclonal antibody: extracellular domain at the M3–M4 junction of the α-subunit of Na,K-ATPase". Biochemistry and Cell Biology 68, № 11 (1 листопада 1990): 1262–67. http://dx.doi.org/10.1139/o90-187.
Повний текст джерелаАнотація:
The binding site of a monoclonal antibody, M45-80, against the α-subunit of horse Na,K-ATPase was determined. Various sizes of DNA fragments derived from rat Na,K-ATPase α1-subunit cDNA were cloned into pUC19 expression vector and some fragments of horse genomic DNA were cloned into pUC18. Escherichia coli JM83 cells harboring the plasmids were grown and the cell lysates were used as antigens. An enzyme-linked immunosorbent assay revealed that M45-80 recognizes the hexapeptide Glu-Tyr-Thr-Trp-Leu-Glu (which is identical to the rat and horse α1-subunits) at the M3–M4 junction located on the extracellular side. The ouabain-binding site is discussed in relation to the recognition site of M45-80.Key words: Na,K-ATPase α-subunit, monoclonal antibody, pUC19 expression vector, antibody-binding site, ouabain-binding site.
2
HILL, M. Craig, Siew Siew PANG, and G. Ronald DUGGLEBY. "Purification of Escherichia coli acetohydroxyacid synthase isoenzyme II and reconstitution of active enzyme from its individual pure subunits." Biochemical Journal 327, no. 3 (November 1, 1997): 891–98. http://dx.doi.org/10.1042/bj3270891.
Повний текст джерелаАнотація:
The first step in the biosynthesis of branched-chain amino acids is catalysed by acetohydroxyacid synthase (EC 4.1.3.18). The reaction involves the decarboxylation of pyruvate followed by condensation with either a second molecule of pyruvate or with 2-oxobutyrate. The enzyme requires as cofactors thiamine diphosphate, a divalent metal ion and, usually, FAD. In most bacteria the enzyme is a heterotetramer of two large and two small subunits. Escherichia coli contains three active isoenzymes and the present study concerns isoenzyme II, whose large and small subunits are encoded by the ilvG and ilvM genes respectively. Cloning these genes into a plasmid vector and overexpression in E. coli allowed a two-step purification procedure for the native enzyme to be developed. The level of expression is considerably higher from a vector that introduces a 50 residue N-terminal fusion containing an oligohistidine sequence on the large subunit. Purification to homogeneity was achieved in a single step by immobilized-metal-affinity chromatography. The kinetic properties of the native and fusion enzyme are indistinguishable with respect to the substrate pyruvate and the inhibitor chlorsulfuron. The individual subunits were expressed as oligohistidine-tagged fusion proteins and each was purified in a single step. Neither subunit alone has significant enzymic activity but, on mixing, the enzyme is reconstituted. The kinetic properties of the reconstituted enzyme are very similar to those of the fusion enzyme. It is proposed that the reconstitution pathway involves successive, and highly co-operative, binding of two small subunit monomers to a large subunit dimer. None of the cofactors is needed for subunit association although they are necessary for the restoration of enzymic activity.
3
Martinez, Nathan P., Matthew Pinch, Yashoda Kandel, and Immo A. Hansen. "Knockdown of the Sodium/Potassium ATPase Subunit Beta 2 Reduces Egg Production in the Dengue Vector, Aedes aegypti." Insects 14, no. 1 (January 5, 2023): 50. http://dx.doi.org/10.3390/insects14010050.
Повний текст джерелаАнотація:
The Na+/K+ ATPase (NKA) is present in the cellular membrane of most eukaryotic cells. It utilizes energy released by ATP hydrolysis to pump sodium ions out of the cell and potassium ions into the cell, which establishes and controls ion gradients. Functional NKA pumps consist of three subunits, alpha, beta, and FXYD. The alpha subunit serves as the catalytic subunit while the beta and FXYD subunits regulate the proper folding and localization, and ion affinity of the alpha subunit, respectively. Here we demonstrate that knockdown of NKA beta subunit 2 mRNA (nkaβ2) reduces fecundity in female Ae. aegypti. We determined the expression pattern of nkaβ2 in several adult mosquito organs using qRT-PCR. We performed RNAi-mediated knockdown of nkaβ2 and assayed for lethality, and effects on female fecundity. Tissue expression levels of nkaβ2 mRNA were highest in the ovaries with the fat body, midgut and thorax having similar expression levels, while Malpighian tubules had significantly lower expression. Survival curves recorded post dsRNA injection showed a non-significant decrease in survival of nkaβ2 dsRNA-injected mosquitoes compared to GFP dsRNA-injected mosquitoes. We observed a significant reduction in the number of eggs laid by nkaβ2 dsRNA-injected mosquitoes compared to control mosquitoes. These results, coupled with the tissue expression profile of nkaβ2, indicate that this subunit plays a role in normal female Ae. aegypti fecundity. Additional research needs to be conducted to determine the exact role played by NKAβ2 in mosquito post-blood meal nutrient sensing, transport, yolk precursor protein (YPP) synthesis and yolk deposition.
4
ORRISS, George L., Michael J. RUNSWICK, Ian R. COLLINSON, Bruno MIROUX, Ian M. FEARNLEY, J. Mark SKEHEL та John E. WALKER. "The δ- and ε-subunits of bovine F1-ATPase interact to form a heterodimeric subcomplex". Biochemical Journal 314, № 2 (1 березня 1996): 695–700. http://dx.doi.org/10.1042/bj3140695.
Повний текст джерелаАнотація:
The δ-subunit of bovine F1-ATPase was expressed from a bacterial vector at fairly high level in Escherichia coli, but the yield of bovine ε-subunit was rather low under similar conditions. However, co-expression of the proteins from a dicistronic operon δ-ε in the same expression vector, produced both of them in good yield in a soluble form in the bacterial cytoplasm, and by chromatography it was found that the δ- and ε-subunits were associated in a stable complex. The amino groups in the complex were labelled exhaustively by chemical reaction under denaturing conditions with ethyl-[1-14C]acetimidate. The α-amino groups of the proteins were unmodified, but complete reaction of all ε-amino groups in both proteins was demonstrated by determination of the molecular masses of the modified proteins by electrospray MS. The modified subunits were separated by denaturing gel electrophoresis, and from measurements of the ratio of incorporated radioactivities and the lysine contents of the proteins, it was calculated that the subcomplex contains equimolar amounts of the two proteins. As the apparent molecular mass of the complex determined by gel filtration was 29 kDa, it appears that the complex contains one copy of each protein. It is likely that the δ- and ε-subunits are associated in a similar manner in the bovine F1-ATPase complex, and that, like a bacterial homologue of the δ-subunit, they interact with the γ-and β-subunits.
5
Bloch, D. B., J. V. Bonventre, E. J. Neer, and J. G. Seidman. "The G protein alpha o subunit alters morphology, growth kinetics, and phospholipid metabolism of somatic cells." Molecular and Cellular Biology 9, no. 12 (December 1989): 5434–39. http://dx.doi.org/10.1128/mcb.9.12.5434-5439.1989.
Повний текст джерелаАнотація:
The physiological role of the alpha o subunit of guanine nucleotide-binding (G) protein was investigated with a murine adrenal cell line (Y1) transfected with a rat alpha o cDNA cloned in a retroviral expression vector. The parental cell line lacked detectable alpha o subunit. Expression of the alpha o cDNA in transfected cell lines was confirmed by Western blot (immunoblot) analysis. The rat alpha o subunit interacted with murine beta and gamma subunits and associated with cell membranes. Y1 cells containing large amounts of alpha o subunit had altered cellular morphology and reduced rate of cell division. In addition, GTP-gamma S-stimulated release of arachidonic acid from these cells was significantly increased compared with that in control cells. The alpha o subunit appears directly or indirectly to regulate cellular proliferation, morphology, and phospholipid metabolism.
6
Bloch, D. B., J. V. Bonventre, E. J. Neer, and J. G. Seidman. "The G protein alpha o subunit alters morphology, growth kinetics, and phospholipid metabolism of somatic cells." Molecular and Cellular Biology 9, no. 12 (December 1989): 5434–39. http://dx.doi.org/10.1128/mcb.9.12.5434.
Повний текст джерелаАнотація:
The physiological role of the alpha o subunit of guanine nucleotide-binding (G) protein was investigated with a murine adrenal cell line (Y1) transfected with a rat alpha o cDNA cloned in a retroviral expression vector. The parental cell line lacked detectable alpha o subunit. Expression of the alpha o cDNA in transfected cell lines was confirmed by Western blot (immunoblot) analysis. The rat alpha o subunit interacted with murine beta and gamma subunits and associated with cell membranes. Y1 cells containing large amounts of alpha o subunit had altered cellular morphology and reduced rate of cell division. In addition, GTP-gamma S-stimulated release of arachidonic acid from these cells was significantly increased compared with that in control cells. The alpha o subunit appears directly or indirectly to regulate cellular proliferation, morphology, and phospholipid metabolism.
7
Tiburzy, Hans-Jürgen, Martin Zimmermann, Regina Oworah-Nkruma, and Richard J. Berzborn. "Heterologous Overexpression of Membrane-Anchored Subunit II of Spinach Chloroplast ATP Synthase and Its Detergent-Free Purification as a Soluble Protein." Zeitschrift für Naturforschung C 54, no. 3-4 (April 1, 1999): 230–38. http://dx.doi.org/10.1515/znc-1999-3-413.
Повний текст джерелаАнотація:
Subunit II is one of the four nonidentical subunits of the membrane integral, protontransporting moiety (CFₒ) hloroplast ATP synthase. In chloroplasts of spinach leaves, it is the only nuclear-encoded CFₒ subunit. It has been deduced that CFₒII is not an additional subunit typical for photosynthetic organisms with no counterpart in E.coli, but equivalent to E. coli subunit b (Tiburzy, H.-J. and Berzborn, R. J. (1997), Z. Naturforsch. 52c, 789-798). Heterologous expression of subunit II was achieved by using the bacterial expression vector pT7-7. Recombinant subunit II (IIrec) does not integrate into the bacterial membrane nor does it precipitate into inclusion bodies. Gel filtration chromatography indicates that IIrec forms higher order aggregates. In three chromatographic steps approx. 10 mg of soluble IIrec of electrophoretic homogeneity are obtained from one liter of bacterial culture without using detergents. Thus, a eukaryotic membrane-anchored protein has been overexpressed in E. coli and has been purified in a soluble form.
8
Kaufman, David R., Jaap Goudsmit, Lennart Holterman, Bonnie A. Ewald, Matthew Denholtz, Colleen Devoy, Ayush Giri, et al. "Differential Antigen Requirements for Protection against Systemic and Intranasal Vaccinia Virus Challenges in Mice." Journal of Virology 82, no. 14 (April 30, 2008): 6829–37. http://dx.doi.org/10.1128/jvi.00353-08.
Повний текст джерелаАнотація:
ABSTRACT The development of a subunit vaccine for smallpox represents a potential strategy to avoid the safety concerns associated with replication-competent vaccinia virus. Preclinical studies to date with subunit smallpox vaccine candidates, however, have been limited by incomplete information regarding protective antigens and the requirement for multiple boost immunizations to afford protective immunity. Here we explore the protective efficacy of replication-incompetent, recombinant adenovirus serotype 35 (rAd35) vectors expressing the vaccinia virus intracellular mature virion (IMV) antigens A27L and L1R and extracellular enveloped virion (EEV) antigens A33R and B5R in a murine vaccinia virus challenge model. A single immunization with the rAd35-L1R vector effectively protected mice against a lethal systemic vaccinia virus challenge. The rAd35-L1R vector also proved more efficacious than the combination of four rAd35 vectors expressing A27L, L1R, A33R, and B5R. Moreover, serum containing L1R-specific neutralizing antibodies afforded postexposure prophylaxis after systemic vaccinia virus infection. In contrast, the combination of rAd35-L1R and rAd35-B5R vectors was required to protect mice against a lethal intranasal vaccinia virus challenge, suggesting that both IMV- and EEV-specific immune responses are important following intranasal infection. Taken together, these data demonstrate that different protective antigens are required based on the route of vaccinia virus challenge. These studies also suggest that rAd vectors warrant further assessment as candidate subunit smallpox vaccines.
9
Roberson, M. S., A. Misra-Press, M. E. Laurance, P. J. Stork, and R. A. Maurer. "A role for mitogen-activated protein kinase in mediating activation of the glycoprotein hormone alpha-subunit promoter by gonadotropin-releasing hormone." Molecular and Cellular Biology 15, no. 7 (July 1995): 3531–39. http://dx.doi.org/10.1128/mcb.15.7.3531.
Повний текст джерелаАнотація:
Gonadotropin-releasing hormone (GnRH) interacts with a G protein-coupled receptor and increases the transcription of the glycoprotein hormone alpha-subunit gene. We have explored the possibility that mitogen-activated protein kinase (MAPK) plays a role in mediating GnRH effects on transcription. Activation of the MAPK cascade by an expression vector for a constitutively active form of the Raf-1 kinase led to stimulation of the alpha-subunit promoter in a concentration-dependent manner. GnRH treatment was found to increase the phosphorylation of tyrosine residues of MAPK and to increase MAPK activity, as determined by an immune complex kinase assay. A reporter gene assay using the MAPK-responsive, carboxy-terminal domain of the Elk1 transcription factor was also consistent with GnRH-induced activation of MAPK. Interference with the MAPK pathway by expression vectors for kinase-defective MAPKs or vectors encoding MAPK phosphatases reduced the transcription-stimulating effects of GnRH. The DNA sequences which are required for responses to GnRH include an Ets factor-binding site. An expression vector for a dominant negative form of Ets-2 was able to reduce GnRH effects on expression of the alpha-subunit gene. These findings provide evidence that GnRH treatment leads to activation of the MAPK cascade in gonadotropes and that activation of MAPK contributes to stimulation of the alpha-subunit promoter. It is likely that an Ets factor serves as a downstream transcriptional effector of MAPK in this system.
10
Cao, H., Z. M. Lei та Ch V. Rao. "Consequences of antisense human chorionic gonadotrophin-α subunit cDNA expression in human choriocarcinoma JAR cells". Journal of Molecular Endocrinology 14, № 3 (червень 1995): 337–47. http://dx.doi.org/10.1677/jme.0.0140337.
Повний текст джерелаАнотація:
ABSTRACT The biosynthesis of human chorionic gonadotrophin (hCG) is a hallmark endocrine function of human choriocarcinoma cells. The present study investigated the consequences of greatly diminishing this synthesis in JAR cells by stably transfecting them with pRSV-antisense hCG-α cDNA expression vector. The vector directs the synthesis of antisense hCG-α subunit mRNA which would then bind to sense hCG-α subunit mRNA, thus blocking its translation and consequently dimer hCG protein synthesis. The transfection with pRSV-antisense hCG-α cDNA resulted in a dramatic decrease in hCG secretion as compared with untransfected parental cells or those transfected with an empty vector used for the selection of clones. The decreased secretion was due to a decreased synthesis which in turn was due to a fall in steady-state hCG-α and -β subunit mRNA levels. The decrease of hCG-β subunit transcripts was unexpected and it was not due to contamination of antisense hCG-α cDNA construct with hCG-β sequence. The transcription of hCG-α and -β subunit genes was not altered in transfected cells suggesting that increased degradation was responsible for decreased steadystate hCG subunit mRNA levels. Despite the decreased hCG levels, the transfected cells maintained normal hCG receptor levels, responded to epidermal growth factor stimulation of hCG synthesis and secretion and grew at the same rate as the control parental cells and those transfected with an empty vector.
11
Arrington, Joshua, Ralph P. Braun, Lichun Dong, Deborah H. Fuller, Michael D. Macklin, Scott W. Umlauf, Sarah J. Wagner, Mary S. Wu, Lendon G. Payne, and Joel R. Haynes. "Plasmid Vectors Encoding Cholera Toxin or the Heat-Labile Enterotoxin from Escherichia coli Are Strong Adjuvants for DNA Vaccines." Journal of Virology 76, no. 9 (May 1, 2002): 4536–46. http://dx.doi.org/10.1128/jvi.76.9.4536-4546.2002.
Повний текст джерелаАнотація:
ABSTRACT Two plasmid vectors encoding the A and B subunits of cholera toxin (CT) and two additional vectors encoding the A and B subunits of the Escherichia coli heat-labile enterotoxin (LT) were evaluated for their ability to serve as genetic adjuvants for particle-mediated DNA vaccines administered to the epidermis of laboratory animals. Both the CT and the LT vectors strongly augmented Th1 cytokine responses (gamma interferon [IFN-γ]) to multiple viral antigens when codelivered with DNA vaccines. In addition, Th2 cytokine responses (interleukin 4 [IL-4]) were also augmented by both sets of vectors, with the effects of the LT vectors on IL-4 responses being more antigen dependent. The activities of both sets of vectors on antibody responses were antigen dependent and ranged from no effect to sharp reductions in the immunoglobulin G1 (IgG1)-to-IgG2a ratios. Overall, the LT vectors exhibited stronger adjuvant effects in terms of T-cell responses than did the CT vectors, and this was correlated with the induction of greater levels of cyclic AMP by the LT vectors following vector transfection into cultured cells. The adjuvant effects observed in vivo were due to the biological effects of the encoded proteins and not due to CpG motifs in the bacterial genes. Interestingly, the individual LT A and B subunit vectors exhibited partial adjuvant activity that was strongly influenced by the presence or absence of signal peptide coding sequences directing the encoded subunit to either intracellular or extracellular locations. Particle-mediated delivery of either the CT or LT adjuvant vectors in rodents and domestic pigs was well tolerated, suggesting that bacterial toxin-based genetic adjuvants may be a safe and effective strategy to enhance the potency of both prophylactic and therapeutic DNA vaccines for the induction of strong cellular immunity.
12
Cox, Robert H., та Samantha J. Fromme. "A naturally occurring truncated Cav1.2 α1-subunit inhibits Ca2+current in A7r5 cells". American Journal of Physiology-Cell Physiology 305, № 8 (15 жовтня 2013): C896—C905. http://dx.doi.org/10.1152/ajpcell.00217.2013.
Повний текст джерелаАнотація:
Alternative splicing of the voltage-gated Ca2+(CaV) α1-subunit adds to the functional diversity of Ca2+channels. A variant with a 73-nt deletion in exon 15 of the Cav1.2 α1-subunit (Cav1.2Δ73) produced by alternative splicing that predicts a truncated protein has been described, but its function, if any, is unknown. We sought to determine if, by analogy to other truncated CaVα1-subunits, Cav1.2Δ73 acts as an inhibitor of wild-type Cav1.2 currents. HEK-293 cells were transfected with Cav1.2Δ73 in a pIRES vector with CD8 or in pcDNA3.1 with a V5/ his COOH-terminal tag plus β2and α2δ1accessory subunits and pEGFP. Production of Cav1.2Δ73 protein was confirmed by Western blotting and immunofluorescence. Voltage-clamp studies revealed the absence of functional channels in transfected cells. In contrast, cells transfected with full-length Cav1.2 plus accessory subunits and pEGFP exhibited robust Ca2+currents. A7r5 cells exhibited endogenous Cav1.2-based currents that were greatly reduced (>80%) without a change in voltage-dependent activation when transfected with Cav1.2Δ73-IRES-CD8 compared with empty vector or pIRES-CD8 controls. Transfection of A7r5 cells with an analogous Cav2.3Δ73-IRES-CD8 had no effect on Ca2+currents. Immunofluorescence showed intracellular, but not plasma membrane, localization of Cav1.2Δ73-V5/ his, as well as colocalization with an endoplasmic reticulum marker, ER Organelle Lights. Expression of Cav1.2Δ73 α1-subunits in A7r5 cells inhibits endogenous Cav1.2 currents. The fact that this variant arises naturally by alternative splicing raises the possibility that it may represent a physiological mechanism to modulate Cav1.2 functional activity.
13
Capasso, Juan M., Christopher J. Rivard та Tomas Berl. "Silencing and overexpression of the γ-subunit of Na-K-ATPase directly affect survival of IMCD3 cells in response to hypertonic stress". American Journal of Physiology-Renal Physiology 291, № 6 (грудень 2006): F1142—F1147. http://dx.doi.org/10.1152/ajprenal.00077.2006.
Повний текст джерелаАнотація:
The γ-subunit of Na-K-ATPase is robustly expressed in inner medullary collecting duct (IMCD)3 cells either acutely challenged or adapted to hypertonicity but not under isotonic conditions. Circumstantial evidence suggests that this protein may be important for the survival of renal cells in a hypertonic environment. However, no direct proof for such a contention has been forthcoming. The complete mRNA sequences of either γ-subunit isoforms were spliced into an expression vector and transfected into IMCD3 cells. Multiple clones stably expressed γ-subunit protein under isotonic conditions. Clones expressing the γb isoform showed enhanced survival at lethal acute hypertonicity compared with either γa isoform or empty vector (control) expressing clones. We also evaluated the loss of γ-subunit expression on the survival of IMCD3 cells exposed to hypertonicity employing silencing RNA techniques. Multiple stable γ-subunit-specific siRNA clones were obtained and exposed to sublethal hypertonicity. Under these conditions, both the level of γ mRNA and protein was essentially undetectable. The impact of silencing γ-subunit expression resulted in a 70% reduction at 48 h ( P < 0.01) in cell survival compared with empty vector (control) clones. γ siRNA clones showed a 45% decrease in myo-inositol uptake compared with controls after an 18-h exposure to sublethal hypertonicity. Taken together, these data demonstrate a direct and critical role of the γ-subunit on IMCD3 cell survival and/or adaptation in response to ionic hypertonic stress.
14
Sezgin, Gulay, Abdallah Nihrane, Adrianna Henson, Max Wattenberg, Steven Ellis, and Johnson M. Liu. "Impaired Ribosome Maturation In Human Cells Depleted of Shwachman-Diamond Syndrome Protein SBDS." Blood 116, no. 21 (November 19, 2010): 2242. http://dx.doi.org/10.1182/blood.v116.21.2242.2242.
Повний текст джерелаАнотація:
Abstract Abstract 2242 Background: Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by pancreatic exocrine dysfunction, neurocognitive and skeletal abnormalities, and bone marrow failure. Mutations in SBDS have been shown to cause SDS. From experiments on its yeast ortholog (Haematologica 2010. 95:57-64), SBDS has been implicated in maturation and function of the 60S ribosomal subunit. In particular, subunit maturation in the SDS yeast model was associated with delayed export and accumulation of 60S-like particles in the nucleoplasm. Methods and Results: To clarify its role in human cells, erythroleukemia TF-1 cells were transduced with lentiviral vectors expressing short hairpin RNA (shRNA) against SBDS. Immunoblot assays confirmed approximately 60% knockdown in individual TF-1 cell clones expressing different shRNAs. The growth and hematopoietic colony forming potential of TF-1 knockdown cells were markedly hindered when compared to cells stably transduced with shRNA against a scrambled SBDS sequence. Using Hoechst 33342/Pyronin Y staining and flow cytometry, we also found an increased percentage of knockdown cells retained at the G0/G1 cell cycle phase. To address whether near-complete knockdown of SBDS affected ribosome synthesis as it does in yeast cells, we silenced SBDS in A549 cells. Our data revealed a reduction in polysomes but in contrast to what was observed in yeast, there was no evidence of half-mer polysomes indicative of decreased 60S subunits participating in translation. The absence of half-mers is not unusual in mammalian systems, so to better analyze the effect of SBDS on 60S subunit maturation subunit localization was assessed by co-transfection with a vector expressing a fusion between human RPL29 and enhanced GFP. Preliminary studies indicated a higher percentage of SBDS-depleted cells with nuclear localization of 60S subunits, when compared with normal controls (Fig. 1). Conclusions: Disclosures: No relevant conflicts of interest to declare.
15
Bachnoff, Niv, Moshe Cohen-Kutner, and Daphne Atlas. "The Involvement of Ser1898 of the Human L-Type Calcium Channel in Evoked Secretion." International Journal of Endocrinology 2011 (2011): 1–13. http://dx.doi.org/10.1155/2011/746482.
Повний текст джерелаАнотація:
A PKA consensus phosphorylation site S1928 at theα11.2 subunit of the rabbit cardiac L-type channel, CaV1.2, is involved in the regulation of CaV1.2 kinetics and affects catecholamine secretion. This mutation does not alter basal CaV1.2 current properties or regulation of CaV1.2 current by PKA and the beta-adrenergic receptor, but abolishes CaV1.2 phosphorylation by PKA. Here, we test the contribution of the corresponding PKA phosphorylation site of the humanα11.2 subunit S1898, to the regulation of catecholamine secretion in bovine chromaffin cells. Chromaffin cells were infected with a Semliki-Forest viral vector containing either the human wt or a mutated S1898Aα11.2 subunit. Both subunits harbor a T1036Y mutation conferring nifedipine insensitivity. Secretion evoked by depolarization in the presence of nifedipine was monitored by amperometry. Depolarization-triggered secretion in cells infected with either the wtα11.2 orα11.2/S1898A mutated subunit was elevated to a similar extent by forskolin. Forskolin, known to directly activate adenylyl-cyclase, increased the rate of secretion in a manner that is largely independent of the presence of S1898. Our results are consistent with the involvement of additional PKA regulatory site(s) at the C-tail ofα11.2, the pore forming subunit of CaV1.2.
16
Su, Zhong-Liang, Kai-Xian Qian, Cong-Ping Tan, Chun-Xiao Meng, and Song Qin. "Recombination and Heterologous Expression of Allophycocyanin Gene in the Chloroplast of Chlamydomonas reinhardtii." Acta Biochimica et Biophysica Sinica 37, no. 10 (October 1, 2005): 709–12. http://dx.doi.org/10.1111/j.1745-7270.2005.00092.x.
Повний текст джерелаАнотація:
Abstract Heterogeneous expression of multiple genes in the nucleus of transgenic plants requires the introduction of an individual gene and the subsequent backcross to reconstitute multi-subunit proteins or metabolic pathways. In order to accomplish the expression of multiple genes in a single transformation event, we inserted both large and small subunits of allophycocyanin gene (apcA and apcB) into Chlamydomonas reinhardtii chloroplast expression vector, resulting in papc-S. The constructed vector was then introduced into the chloroplast of C. reinhardtii by micro-particle bombardment. Polymerase chain reaction and Southern blot analysis revealed that the two genes had integrated into the chloroplast genome. Western blot and enzyme-linked immunosorbent assay showed that the two genes from the prokaryotic cyanobacteria could be correctly expressed in the chloroplasts of C. reinhardtii. The expressed foreign protein in transformants accounted for about 2%-3% of total soluble proteins. These findings pave the way to the reconstitution of multi-subunit proteins or metabolic pathways in transgenic C. reinhardtii chloroplasts in a single transformation event.
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Wongsantichon, Jantana, and Albert J. Ketterman. "An intersubunit lock-and-key ‘Clasp’ motif in the dimer interface of Delta class glutathione transferase." Biochemical Journal 394, no. 1 (January 27, 2006): 135–44. http://dx.doi.org/10.1042/bj20050915.
Повний текст джерелаАнотація:
Structural investigations of a GST (glutathione transferase), adGSTD4-4, from the malaria vector Anopheles dirus show a novel lock-and-key ‘Clasp’ motif in the dimer interface of the Delta class enzyme. This motif also appears to be highly conserved across several insect GST classes, but differs from a previously reported mammalian lock-and-key motif. The aromatic ‘key’ residue not only inserts into a hydrophobic pocket, the ‘lock’, of the neighbouring subunit, but also acts as part of the ‘lock’ for the other subunit ‘key’. The ‘key’ residues from both subunits show aromatic ring stacking with each other in a pi–pi interaction, generating a ‘Clasp’ in the middle of the subunit interface. Enzyme catalytic and structural characterizations revealed that single amino acid replacements in this ‘Clasp’ motif impacted on catalytic efficiencies, substrate selectivity and stability. Substitutions to the ‘key’ residue create strong positive co-operativity for glutathione binding, with a Hill coefficient approaching 2. The lock-and-key motif in general and especially the ‘Clasp’ motif with the pi–pi interaction appear to play a pivotal role in subunit communication between active sites, as well as in stabilizing the quaternary structure. Evidence of allosteric effects suggests an important role for this particular intersubunit architecture in regulating catalytic activity through conformational transitions of subunits. The observation of co-operativity in the mutants also implies that glutathione ligand binding and dimerization are linked. Quaternary structural changes of all mutants suggest that subunit assembly or dimerization basically manipulates subunit communication.
18
Santangelo, Thomas J., L'ubomíra Čuboňová, and John N. Reeve. "Shuttle Vector Expression in Thermococcus kodakaraensis: Contributions of cis Elements to Protein Synthesis in a Hyperthermophilic Archaeon." Applied and Environmental Microbiology 74, no. 10 (March 31, 2008): 3099–104. http://dx.doi.org/10.1128/aem.00305-08.
Повний текст джерелаАнотація:
ABSTRACT Shuttle vectors that replicate stably and express selectable phenotypes in both Thermococcus kodakaraensis and Escherichia coli have been constructed. Plasmid pTN1 from Thermococcus nautilis was ligated to the commercial vector pCR2.1-TOPO, and selectable markers were added so that T. kodakaraensis transformants could be selected by ΔtrpE complementation and/or mevinolin resistance. Based on Western blot measurements, shuttle vector expression of RpoL-HA, a hemagglutinin (HA) epitope-tagged subunit of T. kodakaraensis RNA polymerase (RNAP), was ∼8-fold higher than chromosome expression. An idealized ribosome binding sequence (5′-AGGTGG) was incorporated for RpoL-HA expression, and changes to this sequence reduced expression. Changing the translation initiation codon from AUG to GUG did not reduce RpoL-HA expression, but replacing AUG with UUG dramatically reduced RpoL-HA synthesis. When functioning as translation initiation codons, AUG, GUG, and UUG all directed the incorporation of methionine as the N-terminal residue of RpoL-HA synthesized in T. kodakaraensis. Affinity purification confirmed that an HA- plus six-histidine-tagged RpoL subunit (RpoL-HA-his6) synthesized ectopically from a shuttle vector was assembled in vivo into RNAP holoenzymes that were active and could be purified directly from T. kodakaraensis cell lysates by Ni2+ binding and imidazole elution.
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Ascón, Miguel A., Javier Ochoa-Repáraz, Nancy Walters, and David W. Pascual. "Partially Assembled K99 Fimbriae Are Required for Protection." Infection and Immunity 73, no. 11 (November 2005): 7274–80. http://dx.doi.org/10.1128/iai.73.11.7274-7280.2005.
Повний текст джерелаАнотація:
ABSTRACTAntibodies to K99 fimbriae afford protection to F5+bovine enterotoxigenicEscherichia coli(ETEC). Previous studies show that murine dams immunized withSalmonellavaccine vectors stably expressing K99 fimbriae confer protection to ETEC-challenged neonatal pups. To begin to address adaptation of the K99 scaffold to display heterologous B- and T-cell epitopes, studies were conducted to determine how much of the assembled K99 fimbria is required to maintain protective immunity. Sequential deletions in the K99 gene clusters were made, resulting in diminished localization of the K99 fimbrial subunit in the outer membrane. As placement of the K99 fimbrial subunit became progressively contained within the vaccine vector, diminished immunoglobulin A (IgA) and IgG1 antibody titers, as well as diminished Th2-type cytokine responses, were observed in orally immunized mice. Deletion offanGH, which greatly reduced the export of the fimbrial subunit to the outer membrane, showed only partial reduction in protective immunity. By contrast, deletion offanDEFGH, which also reduced the export of the fimbrial subunit to the outer membrane but retained more subunit in the cytoplasm, resulted in protective immunity being dramatically reduced. Thus, these studies showed that retention of K99 fimbrial subunit as native fimbriae or with the deletion offanGHis sufficient to confer protection.
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Xia, H., J. Huang, T.-M. Chen та D. Puett. "Lysine residues 2 and 104 of the human chorionic gonadotrophin β subunit influence receptor binding". Journal of Molecular Endocrinology 10, № 3 (червень 1993): 337–43. http://dx.doi.org/10.1677/jme.0.0100337.
Повний текст джерелаАнотація:
ABSTRACT Human chorionic gonadotrophin (hCG), like other members of the glycoprotein hormone family, contains a common α subunit and a hormone-specific β subunit. The latter is a 145 amino acid residue polypeptide with six sites of glycosylation. Positions 2 and 104 are occupied by basic amino acid residues in the 12 known amino acid sequences of mammalian β subunits from CG and LH, a related gonadotrophin that acts through the same receptor. Lysine residues are found in both these positions in hCG-β. Using site-directed mutagenesis, each of these two lysines in hCG-β was replaced with glutamic acid. The mutant and wild-type cDNAs were subcloned into a eukaryotic expression vector, which was then transiently transfected into Chinese hamster ovary cells containing a stably integrated gene for the bovine α subunit. Holoprotein formation occurred with each of the two heterologous gonadotrophin mutants, i.e. the bovine α subunit bound to hCG-β (Glu2) and to hCG-β (Glu104), as well as with the control, i.e. the bovine α subunit bound to the hCG-β wild-type subunit. In two in-vitro assays, one a competitive binding assay with 125I-labelled hCG as bound ligand and the other based on stimulation of progesterone production in a transformed murine Leydig cell line, MA-10, both the heterodimers containing a mutant β subunit exhibited bioactivity, but their potencies were lower than that of the bovine α subunit bound to the hCG-β wild-type subunit. These results suggest that the basic amino acid residues at positions 2 and 104 in hCG-β participate, either directly or indirectly, in receptor binding.
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Frangioni, J. V., and B. G. Neel. "Use of a general purpose mammalian expression vector for studying intracellular protein targeting: identification of critical residues in the nuclear lamin A/C nuclear localization signal." Journal of Cell Science 105, no. 2 (June 1, 1993): 481–88. http://dx.doi.org/10.1242/jcs.105.2.481.
Повний текст джерелаАнотація:
We have constructed a general purpose mammalian expression vector for the study of intracellular protein targeting. The vector, p3PK, facilitates construction of N- and/or C-terminal fusions of an amino acid sequence of interest to the normally cytosolic protein chicken muscle pyruvate kinase (CMPK). The vector has been engineered such that any fusion construct can be subcloned into the versatile pJx omega family of mammalian expression vectors and into pGEX bacterial expression vectors, for the generation of affinity reagents. In this paper, we demonstrate the general utility of p3PK by redirecting CMPK to mitochondria (using the twelve amino acid pre-sequence of yeast cytochrome c oxidase subunit IV) and to the nucleus (using a putative eight amino acid nuclear localization signal from human nuclear lamins A and C). We also report that, contrary to the predictions of previously published work, substitution of a critical residue in the nuclear lamin A/C nuclear localization signal (the equivalent of lysine 128 in the SV40 large T nuclear localization signal) retains nuclear localization, and discuss how amino acid context might affect targeting to the nucleus.
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Cyert, M. S., and J. Thorner. "Regulatory subunit (CNB1 gene product) of yeast Ca2+/calmodulin-dependent phosphoprotein phosphatases is required for adaptation to pheromone." Molecular and Cellular Biology 12, no. 8 (August 1992): 3460–69. http://dx.doi.org/10.1128/mcb.12.8.3460-3469.1992.
Повний текст джерелаАнотація:
By using an assay specific for detection of calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase, this enzyme was purified approximately 5,000-fold from extracts of the yeast Saccharomyces cerevisiae. Cna1p and Cna2p, the products of two yeast genes encoding the catalytic (A) subunits of calcineurin, were major constituents of the purified fraction. A third prominent component of apparent molecular mass 16 kDa displayed several properties, including ability to bind 45Ca2+, that are characteristic of the regulatory (B) subunit of mammalian calcineurin and was recognized by an antiserum raised against bovine calcineurin. These antibodies were used to isolate the structural gene (CNB1) encoding this protein from a yeast expression library in the vector lambda gt11. The nucleotide sequence of CNB1 predicted a polypeptide similar in length and highly related in amino acid sequence (56% identity) to the mammalian calcineurin B subunit. Like its counterpart in higher cells, yeast Cnb1p was myristoylated at its N terminus. Mutants lacking Cnb1p, or all three calcineurin subunits (Cna1p, Cna2p, and Cnb1p), were viable. Extracts of cnb1 delta mutants contained no detectable calcineurin activity, even though Cna1p and Cna2p were present at normal levels, suggesting that the B subunit is required for full enzymatic activity in vitro. As was observed previously for MATa cna1 cna2 double mutants, MATa cnb1 mutants were defective in their ability to recover from alpha-factor-induced growth arrest. Thus, the B subunit also is required for the function of calcineurin in promoting adaptation of haploid yeast cells to pheromone in vivo.
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Cyert, M. S., and J. Thorner. "Regulatory subunit (CNB1 gene product) of yeast Ca2+/calmodulin-dependent phosphoprotein phosphatases is required for adaptation to pheromone." Molecular and Cellular Biology 12, no. 8 (August 1992): 3460–69. http://dx.doi.org/10.1128/mcb.12.8.3460.
Повний текст джерелаАнотація:
By using an assay specific for detection of calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase, this enzyme was purified approximately 5,000-fold from extracts of the yeast Saccharomyces cerevisiae. Cna1p and Cna2p, the products of two yeast genes encoding the catalytic (A) subunits of calcineurin, were major constituents of the purified fraction. A third prominent component of apparent molecular mass 16 kDa displayed several properties, including ability to bind 45Ca2+, that are characteristic of the regulatory (B) subunit of mammalian calcineurin and was recognized by an antiserum raised against bovine calcineurin. These antibodies were used to isolate the structural gene (CNB1) encoding this protein from a yeast expression library in the vector lambda gt11. The nucleotide sequence of CNB1 predicted a polypeptide similar in length and highly related in amino acid sequence (56% identity) to the mammalian calcineurin B subunit. Like its counterpart in higher cells, yeast Cnb1p was myristoylated at its N terminus. Mutants lacking Cnb1p, or all three calcineurin subunits (Cna1p, Cna2p, and Cnb1p), were viable. Extracts of cnb1 delta mutants contained no detectable calcineurin activity, even though Cna1p and Cna2p were present at normal levels, suggesting that the B subunit is required for full enzymatic activity in vitro. As was observed previously for MATa cna1 cna2 double mutants, MATa cnb1 mutants were defective in their ability to recover from alpha-factor-induced growth arrest. Thus, the B subunit also is required for the function of calcineurin in promoting adaptation of haploid yeast cells to pheromone in vivo.
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Johnson, Nicholas, Mark A. Pickett, Peter J. Watt, Ian N. Clarke, and John E. Heckels. "Construction of an epitope vector utilising the diphtheria toxin B-subunit." FEMS Microbiology Letters 146, no. 1 (January 17, 2006): 91–96. http://dx.doi.org/10.1111/j.1574-6968.1997.tb10176.x.
Повний текст джерела
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Bjorgvinsdottir, H., L. Zhen, and MC Dinauer. "Cloning of murine gp91phox cDNA and functional expression in a human X- linked chronic granulomatous disease cell line." Blood 87, no. 5 (March 1, 1996): 2005–10. http://dx.doi.org/10.1182/blood.v87.5.2005.2005.
Повний текст джерелаАнотація:
Abstract The phagocyte cytochrome b558, a heterodimer comprised of gp91phox and p22phox, is a flavocytochrome that mediates the transfer of electrons from NADPH to molecular oxygen in the respiratory burst oxidase. The human gene encoding the glycosylated gp91phox subunit is the site of mutations in X-linked chronic granulomatous disease (CGD). Reverse transcriptase-polymerase chain reaction was used to obtain a full- length clone for the murine gp91phox cDNA, which was 87% identical to the human gp91phox cDNA. The encoded murine protein had 39 amino acids out of 570 that differed from the human, many of which were conservative substitutions. Nonconservative replacements occurred in hydrophilic regions outside of domains previously implicated in binding to NADPH, flavin, and the cytosolic oxidase subunit p47phox. Some substitutions altered potential N-glycosylation sites, which is likely to explain why the glycosylated murine protein migrates with an apparent molecular mass of 58 kD instead of 91 kD as seen for the human protein. Expression of murine gp91phox in a human myeloid cell line with a null gp91phox allele using a mammalian expression plasmid or a retroviral vector rescued stable expression of the p22phox subunit and fully reconstituted respiratory burst activity. This suggests that the murine gp91phox subunit forms a functional cytochrome b558 heterodimer with human oxidase subunits, consistent with the high degree of identity between the mouse and human proteins in domains implicated in cytochrome function.
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Bjorgvinsdottir, H., L. Zhen, and MC Dinauer. "Cloning of murine gp91phox cDNA and functional expression in a human X- linked chronic granulomatous disease cell line." Blood 87, no. 5 (March 1, 1996): 2005–10. http://dx.doi.org/10.1182/blood.v87.5.2005.bloodjournal8752005.
Повний текст джерелаАнотація:
The phagocyte cytochrome b558, a heterodimer comprised of gp91phox and p22phox, is a flavocytochrome that mediates the transfer of electrons from NADPH to molecular oxygen in the respiratory burst oxidase. The human gene encoding the glycosylated gp91phox subunit is the site of mutations in X-linked chronic granulomatous disease (CGD). Reverse transcriptase-polymerase chain reaction was used to obtain a full- length clone for the murine gp91phox cDNA, which was 87% identical to the human gp91phox cDNA. The encoded murine protein had 39 amino acids out of 570 that differed from the human, many of which were conservative substitutions. Nonconservative replacements occurred in hydrophilic regions outside of domains previously implicated in binding to NADPH, flavin, and the cytosolic oxidase subunit p47phox. Some substitutions altered potential N-glycosylation sites, which is likely to explain why the glycosylated murine protein migrates with an apparent molecular mass of 58 kD instead of 91 kD as seen for the human protein. Expression of murine gp91phox in a human myeloid cell line with a null gp91phox allele using a mammalian expression plasmid or a retroviral vector rescued stable expression of the p22phox subunit and fully reconstituted respiratory burst activity. This suggests that the murine gp91phox subunit forms a functional cytochrome b558 heterodimer with human oxidase subunits, consistent with the high degree of identity between the mouse and human proteins in domains implicated in cytochrome function.
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Chebrou, Hervé, Yves Hurtubise, Diane Barriault, and Michel Sylvestre. "Heterologous Expression and Characterization of the Purified Oxygenase Component of Rhodococcus globerulusP6 Biphenyl Dioxygenase and of Chimeras Derived from It." Journal of Bacteriology 181, no. 16 (August 15, 1999): 4805–11. http://dx.doi.org/10.1128/jb.181.16.4805-4811.1999.
Повний текст джерелаАнотація:
ABSTRACT In this work, we have purified the His-tagged oxygenase (ht-oxygenase) component of Rhodococcus globerulus P6 biphenyl dioxygenase. The α or β subunit of P6 oxygenase was exchanged with the corresponding subunit of Pseudomonas sp. strain LB400 or of Comamonas testosteroni B-356 to create new chimeras that were purified ht-proteins and designated ht-αP6βP6, ht-αP6βLB400, ht-αP6βB-356, ht-αLB400βP6, and ht-αB-356βP6. ht-αP6βP6, ht-αP6βLB400, ht-αP6βB-356 were not expressed active in recombinant Escherichia coli cells carrying P6bphA1 and bphA2, P6 bphA1 and LB400bphE, or P6 bphA1 and B-356 bphEbecause the [2Fe-2S] Rieske cluster of P6 oxygenase α subunit was not assembled correctly in these clones. On the other hand ht-αLB400βP6 and ht-αB-356βP6 were produced active inE. coli. Furthermore, active purified ht-αP6βP6, ht-αP6βLB400, ht-αP6βB-356, showing typical spectra for Rieske-type proteins, were obtained from Pseudomonas putidaKT2440 carrying constructions derived from the new shuttle E. coli-Pseudomonas vector pEP31, designed to produce ht-proteins inPseudomonas. Analysis of the substrate selectivity pattern of these purified chimeras toward selected chlorobiphenyls indicate that the catalytic capacity of hybrid enzymes comprised of an α and a β subunit recruited from distinct biphenyl dioxygenases is not determined specifically by either one of the two subunits.
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Bentancor, Leticia V., Marcos Bilen, Romina J. Fernández Brando, María Victoria Ramos, Luis C. S. Ferreira, Pablo D. Ghiringhelli, and Marina S. Palermo. "A DNA Vaccine Encoding the Enterohemorragic Escherichia coli Shiga-Like Toxin 2 A2 and B Subunits Confers Protective Immunity to Shiga Toxin Challenge in the Murine Model." Clinical and Vaccine Immunology 16, no. 5 (January 28, 2009): 712–18. http://dx.doi.org/10.1128/cvi.00328-08.
Повний текст джерелаАнотація:
ABSTRACT Production of verocytotoxin or Shiga-like toxin (Stx), particularly Stx2, is the basis of hemolytic uremic syndrome, a frequently lethal outcome for subjects infected with Stx2-producing enterohemorrhagic Escherichia coli (EHEC) strains. The toxin is formed by a single A subunit, which promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells. Host enzymes cleave the A subunit into the A1 peptide, endowed with N-glycosidase activity to the 28S rRNA, and the A2 peptide, which confers stability to the B pentamer. We report the construction of a DNA vaccine (pStx2ΔAB) that expresses a nontoxic Stx2 mutated form consisting of the last 32 amino acids of the A2 sequence and the complete B subunit as two nonfused polypeptides. Immunization trials carried out with the DNA vaccine in BALB/c mice, alone or in combination with another DNA vaccine encoding granulocyte-macrophage colony-stimulating factor, resulted in systemic Stx-specific antibody responses targeting both A and B subunits of the native Stx2. Moreover, anti-Stx2 antibodies raised in mice immunized with pStx2ΔAB showed toxin neutralization activity in vitro and, more importantly, conferred partial protection to Stx2 challenge in vivo. The present vector represents the second DNA vaccine so far reported to induce protective immunity to Stx2 and may contribute, either alone or in combination with other procedures, to the development of prophylactic or therapeutic interventions aiming to ameliorate EHEC infection-associated sequelae.
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Roche, Catherine, Alfredo J. Zamora, David Taïeb, Esteban Lavaque, Ramahefarizo Rasolonjanahary, Henri Dufour, Claude Bagnis, Alain Enjalbert, and Anne Barlier. "Lentiviral vectors efficiently transduce human gonadotroph and somatotroph adenomas in vitro. Targeted expression of transgene by pituitary hormone promoters." Journal of Endocrinology 183, no. 1 (October 2004): 217–33. http://dx.doi.org/10.1677/joe.1.05759.
Повний текст джерелаАнотація:
Despite important advances in human therapeutics, no specific treatment for both non-functioning gonadotroph and resistant somatotroph adenomas is available. Gene transfer by viral vectors can be considered as a promising way to achieve a specific and efficient treatment. Here we show the possibility of efficient gene transfer in human pituitary adenoma cells in vitro using a human immunodeficiency virus (HIV)-type 1-derived vector. Using enhanced green fluorescent protein (eGFP) gene as a marker placed under the phosphoglycerate kinase (PGK) promoter, gonadotroph and somatotroph adenomas were transduced even with moderate viral loads. The expression started at day 2, reached a peak at day 5, and it was still present at day 90. For targeting somatotroph and gonadotroph adenomas, human growth hormone (GH) promoter (GH −481, +54 bp) and two fragments of the human glycoprotein hormone α-subunit promoter (α-subunit 1 −520, +33 bp, and α-subunit 2 −907, +33 bp) were tested. In gonadotroph adenomas, the percentage of identified fluorescent cells and the fluorescence intensity analyzed by fluorescence-activated cell sorting indicated that the strength of the α-subunit 1 and α-subunit 2 promoters were comparable to that of the PGK promoter. Primary cultures of rat pituitary cells showed that α-subunit 1 is more selective to thyreotroph and gonadotroph phenotypes than α-subunit 2. GH promoter activity appeared weak in somatotroph adenomas. The human GH enhancer did not increase the GH promoter activity at all but the human prolactin promoter (−250 bp) allowed 4-fold more fluorescent cells to be obtained than the GH promoter. Several cell lines appeared too permissive to test cell-specificity of pituitary promoters. However, on human non-pituitary cell cultures, the tested pituitary promoters seemed clearly selective to target endocrine pituitary phenotypes. This study gives a starting point for a gene-therapy program using lentiviral vectors to transfer therapeutic genes in human pituitary adenomas.
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WANG, Minghan, James OFFORD, Dale L. OXENDER та Ti-Zhi SU. "Structural requirement of the calcium-channel subunit α2δ for gabapentin binding". Biochemical Journal 342, № 2 (24 серпня 1999): 313–20. http://dx.doi.org/10.1042/bj3420313.
Повний текст джерелаАнотація:
Gabapentin [Neurontin, 1-(aminomethyl)cyclohexaneacetic acid] is a novel anticonvulsant drug with a high binding affinity for the Ca2+-channel subunit α2δ. In this study, the gabapentin-binding properties of wild-type and mutated porcine brain α2δ proteins were investigated. Removal of the disulphide bonds between the α2 and the δ subunits did not result in a significant loss of gabapentin binding, suggesting that the disulphide linkage between the two subunits is not required for binding. Singly expressed α2 protein remained membrane associated. However, α2 alone was unable to bind gabapentin, unless the cells were concurrently transfected with the expression vector for δ, suggesting that both α2 and δ are required for gabapentin binding. Using internal deletion mutagenesis, we mapped two regions [amino acid residues 339-365 (δF) and 875-905 (δJ)] within the α2 subunit that are not required for gabapentin binding. Further, deletion of three other individual regions [amino acid residues 206-222 (δD), 516-537 (δH) and 583-603 (δI)] within the α2 subunit disrupted gabapentin binding, suggesting the structural importance of these regions. Using alanine to replace four to six amino acid residues in each of these regions abolished gabapentin binding. These results demonstrate that region D, between the N-terminal end and the first putative transmembrane domain of α2, and regions H and I, between the putative splicing acceptor sites (Gln511 and Ser601), may play important roles in maintaining the structural integrity for gabapentin binding. Further single amino acid replacement mutagenesis within these regions identified Arg217 as critical for gabapentin binding.
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Ramotar, K., B. Boyd, G. Tyrrell, J. Gariepy, C. Lingwood, and J. Brunton. "Characterization of Shiga-like toxin I B subunit purified from overproducing clones of the SLT-I B cistron." Biochemical Journal 272, no. 3 (December 15, 1990): 805–11. http://dx.doi.org/10.1042/bj2720805.
Повний текст джерелаАнотація:
The cistron encoding the B subunit of Escherichia coli Shiga-like toxin I (SLT-I) was cloned under control of the tac promoter in the expression vector pKK223-3 and the SLT-I B subunit was expressed constitutively in a wild-type background and inducibly in a lacIq background. The B subunit was located in the periplasmic space, and less than 10% was found in the culture medium after 24 h incubation. Polymyxin B extracts contained as much as 160 micrograms of B subunit/ml of culture. B subunit was purified to homogeneity by ion-exchange chromatography followed by chromatofocusing. Cross-linking analysis of purified native B subunit showed that it exists as a pentamer. In gels containing 0.1% SDS the native protein dissociated into monomers. B subunit was found to have the same glycolipid-receptor-specificity as SLT-I holotoxin. Competitive binding studies showed that B subunit and holotoxin had the same affinity for the globotriosylceramide receptor. We conclude that this recombinant plasmid is a convenient source of large amounts of purified SLT-I B subunit, which could be used for biophysical and structural studies or as a natural toxoid.
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Cawley, K. C., C. G. Akita, and D. A. Walsh. "Expression of a cDNA for the catalytic subunit of skeletal-muscle phosphorylase kinase in transfected 3T3 cells." Biochemical Journal 263, no. 1 (October 1, 1989): 223–29. http://dx.doi.org/10.1042/bj2630223.
Повний текст джерелаАнотація:
Phosphorylase kinase is a multimeric enzyme of composition (alpha, beta, gamma, delta)4 whose catalytic activity resides in the gamma-subunit. As an approach to understand further its regulation, a cDNA for the gamma-subunit of phosphorylase kinase (gamma PhK) has been cloned into a mammalian expression vector behind the mouse metallothionein-1 promoter. NIH 3T3 cells were co-transfected with this construct (pEV gamma PhK) and pSV2neo, G418-resistant clones were selected, and several were found to have stably incorporated the gamma-subunit cDNA into their genomic DNA. Phosphorylase kinase activity was clearly present in extracts from cultures of pEV gamma PhK-transformed cells and increased several-fold after 24 h of incubation with Zn2+, whereas it was undetectable in the parent 3T3 cells. A significant, but variable, proportion (15-70%) of the activity was Ca2+-dependent. We conclude that the phosphorylase kinase activity expressed by the cells transformed with pEV gamma PhK is due to free gamma-subunit and gamma-subunit associated with cellular calmodulin, which replaces the delta-subunit normally associated with the gamma-subunit in the holoenzyme.
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Nguyen, Tu N., Bao N. Nguyen, Ju Huck Lee, Aswini K. Panigrahi, and Arthur Günzl. "Characterization of a Novel Class I Transcription Factor A (CITFA) Subunit That Is Indispensable for Transcription by the Multifunctional RNA Polymerase I of Trypanosoma brucei." Eukaryotic Cell 11, no. 12 (October 26, 2012): 1573–81. http://dx.doi.org/10.1128/ec.00250-12.
Повний текст джерелаАнотація:
ABSTRACT Trypanosoma brucei is the only organism known to have evolved a multifunctional RNA polymerase I (pol I) system that is used to express the parasite's ribosomal RNAs, as well as its major cell surface antigens, namely, the variant surface glycoprotein (VSG) and procyclin, which are vital for establishing successful infections in the mammalian host and the tsetse vector, respectively. Thus far, biochemical analyses of the T. brucei RNA pol I transcription machinery have elucidated the subunit structure of the enzyme and identified the class I transcription factor A (CITFA). CITFA binds to RNA pol I promoters, and its CITFA-2 subunit was shown to be absolutely essential for RNA pol I transcription in the parasite. Tandem affinity purification (TAP) of CITFA revealed the subunits CITFA-1 to -6, which are conserved only among kinetoplastid organisms, plus the dynein light chain DYNLL1. Here, by tagging CITFA-6 instead of CITFA-2, a complex was purified that contained all known CITFA subunits, as well as a novel proline-rich protein. Functional studies carried out in vivo and in vitro , as well as a colocalization study, unequivocally demonstrated that this protein is a bona fide CITFA subunit, essential for parasite viability and indispensable for RNA pol I transcription of ribosomal gene units and the active VSG expression site in the mammalian-infective life cycle stage of the parasite. Interestingly, CITFA-7 function appears to be species specific, because expression of an RNA interference (RNAi)-resistant CITFA-7 transgene from Trypanosoma cruzi could not rescue the lethal phenotype of silencing endogenous CITFA-7 .
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Chahine, K. G., W. Walke, and D. Goldman. "A 102 base pair sequence of the nicotinic acetylcholine receptor delta-subunit gene confers regulation by muscle electrical activity." Development 115, no. 1 (May 1, 1992): 213–19. http://dx.doi.org/10.1242/dev.115.1.213.
Повний текст джерелаАнотація:
Muscle electrical activity suppresses expression of the embryonic-type (alpha, beta, gamma, and delta) nicotinic acetylcholine receptor (nAChR) genes. The molecular mechanism by which electrical activity regulates these genes is not known. One approach to this problem is to identify regions of the nAChR genes that mediate electrical regulation. Here we report results from such a study of the nAChR delta-subunit gene. We cloned the rat delta-subunit promoter region and created an expression vector in which this DNA controlled the expression of a down-stream luciferase structural gene. The effect that muscle electrical activity had on the expression from this promoter was assayed by introducing this expression vector into electrically stimulated and tetrodotoxin (TTX)-treated rat primary myotubes, and assaying for luciferase activity. These myotubes, when stimulated with extracellular electrodes, suppressed endogenous embryonic-type nAChR gene expression compared to those treated with TTX. Transfection of these cells with delta-promoter-luciferase expression vectors resulted in the delta-promoter conferring electrical regulation on luciferase expression. Additional experiments using deletions from the 5′ end of the delta-promoter region have identified an element between −677 and −550 bp that suppressed delta-promoter activity and a minimal 102 bp sequence that promotes and regulates luciferase expression in response to muscle electrical activity. This latter sequence also contains all the necessary elements to confer tissue and developmental stage-specific expression.
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Bowman, Valorie D., Elaine S. Chase, Alexander W. E. Franz, Paul R. Chipman, Xing Zhang, Keith L. Perry, Timothy S. Baker, and Thomas J. Smith. "An Antibody to the Putative Aphid Recognition Site on Cucumber Mosaic Virus Recognizes Pentons but Not Hexons." Journal of Virology 76, no. 23 (December 1, 2002): 12250–58. http://dx.doi.org/10.1128/jvi.76.23.12250-12258.2002.
Повний текст джерелаАнотація:
ABSTRACT Cucumber mosaic virus (CMV), the type member of the genus Cucumovirus (family Bromoviridae), is transmitted by aphids in a nonpersistent manner. Mutagenesis experiments identified the βH-βI loop of the capsid subunit as a potential key motif responsible for interactions with the insect vector. To further examine the functional characteristics of this motif, we generated monoclonal antibodies that bound to native virions but not to βH-βI mutants. Fab fragments from these antibodies were complexed with wild-type CMV and the virus-Fab structure was determined to 12-Å resolution by using electron cryomicroscopy and image reconstruction techniques. The electron density attributed to the bound antibody has a turret-like appearance and protrudes from each of the 12 fivefold axes of the icosahedral virus. Thus, the antibody binds only to the pentameric clusters (pentons) of A subunits of the T=3 quasisymmetric virus and does not appear to bind to any of the B and C subunits that occur as hexameric clusters (hexons) at the threefold (quasi-sixfold) axes. Modeling and electron density comparisons were used to analyze the paratope-epitope interface and demonstrated that the antibody binds to three βH-βI loops in three adjacent A subunits in each penton. This antibody can discriminate between A and B/C subunits even though the βH-βI loop adopts the same structure in all 180 capsid subunits and is therefore recognizing differences in subunit arrangements. Antibodies with such character have potential use as probes of viral assembly. Our results may provide an additional rationale for designing synthetic vaccines by using symmetrical viral particles.
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Pan, Chien-Hsiung, Hui-mei Hu, Yu-Ju Hsiao, and Sze-Hsien Wu. "Heterologous prime-boost vaccination with DNA vaccine and recombinant subunit containing four serotypes of dengue virus envelope protein domain III elicits neutralizing antibodies and specific T-cell responses (P4327)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 123.27. http://dx.doi.org/10.4049/jimmunol.190.supp.123.27.
Повний текст джерелаАнотація:
Abstract Dengue is still an important public health problem and no dengue vaccine is available now. In this study, we characterized prime-boost vaccine regimens using heterologous and homologous dengue DNA vaccine and recombinant subunit vaccine consisted of envelope (E) protein domain III (ED III) from four serotypes of dengue virus (DENV). A broader and balanced IFN-gamma and IL-4 responses was observed in DNA-prime and subunit-boost immunized mice, compared to a narrower but strong IFN-gamma production after homologous DNA vaccination and an IL-4 only response in homologous subunit vaccine immunized mice. In addition, a significant ED III specific antibody response was detected in all immunized mice except vector control. The highest IgG titer was appeared in homologous subunit vaccine immunized mice, followed by heterologous prime-boost regimen and homologous DNA vaccine. However, that heterologous prime-boost immunized mice generated a highest neutralizing antibody than the others, suggested a better protection provided by this DNA-prime and subunit vaccine-boost regimen.
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Mendez-Lopez, Max A., Andrej Besse, Bogdan Florea, Christian Zuppinger, Herman Overkleeft, Christoph Driessen, and Lenka Besse. "Carfilzomib Induces Cardiotoxicity Via ß5/ß2-Specific Proteasome Subunit Inhibition Pattern." Blood 134, Supplement_1 (November 13, 2019): 3110. http://dx.doi.org/10.1182/blood-2019-127987.
Повний текст джерелаАнотація:
BACKGROUND All proteasome inhibitors (PI) approved for Multiple Myeloma (MM) treatment, Bortezomib (BTZ), Ixazomib (IXA) and Carfilzomib (CFZ), by design target the rate-limiting ß5 proteolytic proteasome subunit, but differ in their co-inhibition of the ß1 or ß2 proteolytic subunits at higher doses. CFZ is unique in inducing ß5/ß2 co-inhibition in a dose-dependent manner, which results in increased proteotoxic stress and higher cytotoxicity compared to the ß5/ß1 co-inhibition pattern mediated by BTZ or IXA. CFZ treatment, in particular at higher doses, is not only associated with increased clinical activity, but also with significantly higher cardiac toxicity compared to BTZ. The molecular basis for this difference is poorly understood, and mechanism-based pharmacological alleviation strategies are lacking. We hypothesized that the acute cardiotoxicity of CFZ is related to its unique ß5/ß2 inhibition pattern. METHODS Isolated primary murine cardiomyocytes showing spontaneous rhythmical contractions were used to assess the functional cardiotoxicity of PI in vitro. Cardiomyocytes were treated with the PI BTZ, CFZ or with highly-selective, subunit (ß1, ß2, ß5)-specific PI for 1h. Proteasome inhibition was directly verified using subunit-selective activity-based chemical probes. Contractility was assessed with motion vector image analysis, and calcium transients were examined by confocal microscopy after transduction of cardiomyocytes with a GCaMP vector. Differentially accumulated proteins after 1h PI treatment were quantitatively compared by liquid chromatography/mass spectrometry (LC/MS). In vivo, electrical changes and alterations in heart rate were monitored by electrocardiography (ECG). Calcium transients, ECG signals and LC/MS data were analyzed with R software version 3.5.1 (2018-07-02). RESULTS Treatment of cardiomyocytes with CFZ or with the combination of ß5/ß2-targeting subunit-specific PI impaired contractility in vitro in contrast to BTZ, or to co-inhibition of ß5/ß1 proteasome subunits. In vivo, the CFZ-type proteasome inhibition triggered acute bradycardia. Treatment of cardiomyocytes with CFZ induced a shift of intracellular calcium pools from the endoplasmic reticulum (ER) to the cytosol, consistent with ER-to-cytosol translocation of calcium described upon ER stress induction. Co-treatment with CFZ and cycloheximide, an inhibitor of protein synthesis, rescued cardiomyocytes from CFZ-induced functional impairment in vitro, suggesting that the accumulation of specific proteins is involved in CFZ-induced cardiomyocyte dysfunction. Quantitative proteomic comparison of primary cardiomyocytes treated by either CFZ-type or BTZ-type proteasome inhibition for 1h identified a selective accumulation of proteins of the retinoic acid pathway in CFZ-treated cardiomyocytes. Interestingly, co-treatment of cardiomyocytes with all-trans-retinoic acid (ATRA) prevented CFZ-induced acute cardiotoxicity in vitro. CONCLUSION Our data suggests that CFZ specifically impairs cardiac contractility through its unique ß5/ß2 proteasome subunit inhibition pattern, which results in more effective functional proteasome inhibition, protein accumulation and proteotoxic stress. The shift of intracellular calcium pools in cardiomyocytes upon CFZ treatment mirrors contractility impairment, and the specific changes identified in the retinoic acid pathway suggest ATRA as a potential drug candidate to alleviate CFZ-induced cardiac toxicity. Disclosures No relevant conflicts of interest to declare.
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Segura, María Mercedes, Alain Garnier, Marcos Rafael Di Falco, Gavin Whissell, Angélica Meneses-Acosta, Normand Arcand, and Amine Kamen. "Identification of Host Proteins Associated with Retroviral Vector Particles by Proteomic Analysis of Highly Purified Vector Preparations." Journal of Virology 82, no. 3 (November 21, 2007): 1107–17. http://dx.doi.org/10.1128/jvi.01909-07.
Повний текст джерелаАнотація:
ABSTRACT The Moloney murine leukemia virus (MMLV) belongs to the Retroviridae family of enveloped viruses, which is known to acquire minute amounts of host cellular proteins both on the surface and inside the virion. Despite the extensive use of retroviral vectors in experimental and clinical applications, the repertoire of host proteins incorporated into MMLV vector particles remains unexplored. We report here the identification of host proteins from highly purified retroviral vector preparations obtained by rate-zonal ultracentrifugation. Viral proteins were fractionated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in-gel tryptic digested, and subjected to liquid chromatography/tandem mass spectrometry analysis. Immunogold electron microscopy studies confirmed the presence of several host membrane proteins exposed at the vector surface. These studies led to the identification of 27 host proteins on MMLV vector particles derived from 293 HEK cells, including 5 proteins previously described as part of wild-type MMLV. Nineteen host proteins identified corresponded to intracellular proteins. A total of eight host membrane proteins were identified, including cell adhesion proteins integrin β1 (fibronectin receptor subunit beta) and HMFG-E8, tetraspanins CD81 and CD9, and late endosomal markers CD63 and Lamp-2. Identification of membrane proteins on the retroviral surface is particularly attractive, since they can serve as anchoring sites for the insertion of tags for targeting or purification purposes. The implications of our findings for retrovirus-mediated gene therapy are discussed.
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Hayashi, Y., B. Haimovich, A. Reszka, D. Boettiger, and A. Horwitz. "Expression and function of chicken integrin beta 1 subunit and its cytoplasmic domain mutants in mouse NIH 3T3 cells." Journal of Cell Biology 110, no. 1 (January 1, 1990): 175–84. http://dx.doi.org/10.1083/jcb.110.1.175.
Повний текст джерелаАнотація:
Chicken integrin beta 1 cDNA and its site-directed mutants were cloned into a mammalian expression vector and introduced into mouse NIH 3T3 cells. Stable transfectants expressing the chicken beta 1 subunit or its site-directed mutants were identified by immunostaining with antibodies specific for the chicken integrin beta 1 subunit. The chicken beta 1 proteins were expressed predominately in the endoplasmic reticulum of transfectants and to a lesser degree in the plasma membrane. Immunoblots and immunoprecipitations, using anti-chicken integrin antibodies, revealed three different sizes of the chicken subunit (90, 95, and 120 kD) and a mouse 140-kD alpha subunit. Immunoprecipitations of the cell surface receptors showed only two peptides, an 120-kD beta 1 and an 140-kD alpha subunit. Antibodies perturbing mouse and chicken integrin-specific cell adhesions were used to demonstrate that the chimeric receptors functioned in adhesion to both laminin and fibronectin. Immunofluorescent staining with antibodies specific for either the chicken or mouse receptors showed that both the wild type and the chimeric receptors localized in focal contacts. Several mutations in the cytoplasmic domain were synthesized and used in the transfection experiments. In one mutant the tyrosine (Tyr 788) in the consensus sequence for phosphorylation was replaced by a phenylalanine. In another the lysine (Lys 757) at the end of the membrane spanning region was replaced by a leucine. Both of these mutants formed dimers with mouse alpha subunits, participated in adhesion, localized in focal contacts, and displayed biological properties indistinguishable from the wild-type transfection. In contrast, mutants containing deletions greater than 5-15 amino acids nearest the carboxyl end in the cytoplasmic domain neither promoted adhesion nor localized in focal contacts. They did, however, form heterodimers that were expressed on the cell surface.
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Baekelandt, Veerle, Anje Claeys, Peter Cherepanov, Erik De Clercq, Bart De Strooper, Bart Nuttin, and Zeger Debyser. "DNA-Dependent Protein Kinase Is Not Required for Efficient Lentivirus Integration." Journal of Virology 74, no. 23 (December 1, 2000): 11278–85. http://dx.doi.org/10.1128/jvi.74.23.11278-11285.2000.
Повний текст джерелаАнотація:
ABSTRACT How DNA is repaired after retrovirus integration is not well understood. DNA-dependent protein kinase (DNA-PK) is known to play a central role in the repair of double-stranded DNA breaks. Recently, a role for DNA-PK in retroviral DNA integration has been proposed (R. Daniel, R. A. Katz, and A. M. Skalka, Science 284:644–647, 1999). Reduced transduction efficiency and increased cell death by apoptosis were observed upon retrovirus infection of culturedscid cells. We have used a human immunodeficiency virus (HIV) type 1 (HIV-1)-derived lentivirus vector system to further investigate the role of DNA-PK during integration. We measured lentivirus transduction of scid mouse embryonic fibroblasts (MEF) and xrs-5 or xrs-6 cells. These cells are deficient in the catalytic subunit of DNA-PK and in Ku, the DNA-binding subunit of DNA-PK, respectively. At low vector titers, efficient and stable lentivirus transduction was obtained, excluding an essential role for DNA-PK in lentivirus integration. Likewise, the efficiency of transduction of HIV-derived vectors in scid mouse brain was as efficient as that in control mice, without evidence of apoptosis. We observed increased cell death in scid MEF and xrs-5 or xrs-6 cells, but only after transduction with high vector titers (multiplicity of infection [MOI], >1 transducing unit [TU]/cell) and subsequent passage of the transduced cells. At an MOI of <1 TU/cell, however, transduction efficiency was even higher in DNA-PK-deficient cells than in control cells. Taken together, the data suggest a protective role of DNA-PK against cellular toxicity induced by high levels of retrovirus integrase or integration. Another candidate cellular enzyme that has been claimed to play an important role during retrovirus integration is poly(ADP-ribose) polymerase (PARP). However, no inhibition of lentivirus vector-mediated transduction or HIV-1 replication by 3-methoxybenzamide, a known PARP inhibitor, was observed. In conclusion, DNA-PK and PARP are not essential for lentivirus integration.
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Makanda, Moni, Gladys Kemunto, Lucy Wamuyu, Joel Bargul, Jackson Muema, and James Mutunga. "Diversity and Molecular Characterization of Mosquitoes (Diptera: Culicidae) in selected ecological regions in Kenya." F1000Research 8 (March 6, 2019): 262. http://dx.doi.org/10.12688/f1000research.18262.1.
Повний текст джерелаАнотація:
Mosquitoes play a predominant role as leading agents in the spread of vector-borne diseases and consequent mortality in humans. Despite reports on increase of new and recurrent mosquito borne-disease outbreaks such as chikungunya, dengue fever and Rift valley fever in Kenya little is known about the genetic characteristics and diversity of the vector species that have been incriminated in transmission of disease pathogens. In this study, we identified mosquito species across Kisumu, Kilifi and Nairobi Counties and determined their genetic diversity and phylogenetic relationships. PCR was used to amplify and sequence the partial cytochrome oxidase subunit 1 (CO1) gene of mosquito samples. Molecular-genetic and phylogenetic analysis of the partial cytochrome oxidase subunit 1 (CO1) gene was employed to identify their relationships with known mosquito species. Fourteen (14) haplotypes belonging to genus Aedes, nine (9) haplotypes belonging to genus Anopheles and twelve (12) haplotypes belonging to genus Culex were identified in this study. Findings from this study revealed a potentially new haplotype belonging to Anopheles genus and reported the first molecular characterization of Aedes cummnisii in Kenya. Sequence results revealed variation in mosquito species from Kilifi, Kisumu and Nairobi. Since vector competence varies greatly across species and species-complexes and is strongly associated with specific behavioural adaptations, proper species identification is important for vector control programs.
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Makanda, Moni, Gladys Kemunto, Lucy Wamuyu, Joel Bargul, Jackson Muema, and James Mutunga. "Diversity and Molecular Characterization of Mosquitoes (Diptera: Culicidae) in Selected Ecological Regions in Kenya." F1000Research 8 (September 24, 2019): 262. http://dx.doi.org/10.12688/f1000research.18262.2.
Повний текст джерелаАнотація:
Mosquitoes play a predominant role as leading agents in the spread of vector-borne diseases and the consequent mortality in humans. Despite reports on increase of new and recurrent mosquito borne-disease outbreaks such as chikungunya, dengue fever and Rift Valley fever in Kenya, little is known about the genetic characteristics and diversity of the vector species that have been incriminated in transmission of disease pathogens. In this study, mosquito species were collected from Kisumu city, Kilifi town and Nairobi city and we determined their genetic diversity and phylogenetic relationships. PCR was used to amplify the partial cytochrome oxidase subunit 1 (CO1) gene of mosquito samples. Molecular-genetic and phylogenetic analysis of the partial cytochrome oxidase subunit 1 (CO1) gene were employed to identify their relationship with known mosquito species. Fourteen (14) haplotypes belonging to genus Aedes, nine (9) haplotypes belonging to genus Anopheles and twelve (12) haplotypes belonging to genus Culex were identified in this study. Findings from this study revealed a potentially new haplotype belonging to Anopheles genus and reported the first molecular characterization of Aedes cumminsii in Kenya. Sequence results revealed variation in mosquito species from Kilifi, Kisumu and Nairobi. Since vector competence varies greatly across species as well as species-complexes and is strongly associated with specific behavioural adaptations, proper species identification is important for vector control programs.
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Gaudreault, Natasha N., and Juergen A. Richt. "Subunit Vaccine Approaches for African Swine Fever Virus." Vaccines 7, no. 2 (June 25, 2019): 56. http://dx.doi.org/10.3390/vaccines7020056.
Повний текст джерелаАнотація:
African swine fever virus (ASFV) is the cause of a highly fatal disease in swine, for which there is no available vaccine. The disease is highly contagious and poses a serious threat to the swine industry worldwide. Since its introduction to the Caucasus region in 2007, a highly virulent, genotype II strain of ASFV has continued to circulate and spread into Eastern Europe and Russia, and most recently into Western Europe, China, and various countries of Southeast Asia. This review summarizes various ASFV vaccine strategies that have been investigated, with focus on antigen-, DNA-, and virus vector-based vaccines. Known ASFV antigens and the determinants of protection against ASFV versus immunopathological enhancement of infection and disease are also discussed.
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Jiménez-Cabello, Luis, Sergio Utrilla-Trigo, Eva Calvo-Pinilla, Sandra Moreno, Aitor Nogales, Javier Ortego, and Alejandro Marín-López. "Viral Vector Vaccines against Bluetongue Virus." Microorganisms 9, no. 1 (December 25, 2020): 42. http://dx.doi.org/10.3390/microorganisms9010042.
Повний текст джерелаАнотація:
Bluetongue virus (BTV), the prototype member of the genus Orbivirus (family Reoviridae), is the causative agent of an important livestock disease, bluetongue (BT), which is transmitted via biting midges of the genus Culicoides. To date, up to 29 serotypes of BTV have been described, which are classified as classical (BTV 1–24) or atypical (serotypes 25–27), and its distribution has been expanding since 1998, with important outbreaks in the Mediterranean Basin and devastating incursions in Northern and Western Europe. Classical vaccine approaches, such as live-attenuated and inactivated vaccines, have been used as prophylactic measures to control BT through the years. However, these vaccine approaches fail to address important matters like vaccine safety profile, effectiveness, induction of a cross-protective immune response among serotypes, and implementation of a DIVA (differentiation of infected from vaccinated animals) strategy. In this context, a wide range of recombinant vaccine prototypes against BTV, ranging from subunit vaccines to recombinant viral vector vaccines, have been investigated. This article offers a comprehensive outline of the live viral vectors used against BTV.
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Wilcox, David A., John C. Olsen, Lori Ishizawa, Paul F. Bray, Deborah L. French, Douglas A. Steeber, William R. Bell, Michael Griffith та Gilbert C. White. "Megakaryocyte-targeted synthesis of the integrin β3-subunit results in the phenotypic correction of Glanzmann thrombasthenia". Blood 95, № 12 (15 червня 2000): 3645–51. http://dx.doi.org/10.1182/blood.v95.12.3645.
Повний текст джерелаАнотація:
Abstract Glanzmann thrombasthenia is an inherited bleeding disorder characterized by qualitative or quantitative defects of the platelet-specific integrin, IIbβ3. As a result, IIbβ3 cannot be activated and cannot bind to fibrinogen, leading to a loss of platelet aggregation. Thrombasthenia is clinically characterized by mucocutaneous hemorrhage with episodes of intracranial and gastrointestinal bleeding. To develop methods for gene therapy of Glanzmann thrombasthenia, a murine leukemia virus (MuLV)-derived vector, −889PlA2β3, was transduced into peripheral blood CD34+ cells from 2 patients with thrombasthenia with defects in the β3 gene. The human IIb promoter was used in this vector to drive megakaryocyte-targeted expression of the wild-type β3 subunit. Proviral DNA and IIbβ3 biosynthesis were detected after in vitro differentiation of transduced thrombasthenic CD34+ cells with megakaryocyte growth and development factor. Flow cytometric analysis of transduced patient samples indicated that 19% of megakaryocyte progeny expressed IIbβ3 on the surface at 34% of normal receptor levels. Treatment of transduced megakaryocytes with a combination of agonists including epinephrine and the thrombin receptor-activating peptide induced the IIbβ3 complex to form an activated conformation capable of binding fibrinogen as measured by PAC-1 antibody binding. Transduced cells retracted a fibrin clot in vitro similar to megakaryocytes derived from a normal nonthrombasthenic individual. These results demonstrate ex vivo phenotypic correction of Glanzmann thrombasthenia and support the potential use of hematopoietic CD34+ cells as targets for IIb promoter-driven MuLV vectors for gene therapy of platelet disorders.
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Wilcox, David A., John C. Olsen, Lori Ishizawa, Paul F. Bray, Deborah L. French, Douglas A. Steeber, William R. Bell, Michael Griffith та Gilbert C. White. "Megakaryocyte-targeted synthesis of the integrin β3-subunit results in the phenotypic correction of Glanzmann thrombasthenia". Blood 95, № 12 (15 червня 2000): 3645–51. http://dx.doi.org/10.1182/blood.v95.12.3645.012k51a_3645_3651.
Повний текст джерелаАнотація:
Glanzmann thrombasthenia is an inherited bleeding disorder characterized by qualitative or quantitative defects of the platelet-specific integrin, IIbβ3. As a result, IIbβ3 cannot be activated and cannot bind to fibrinogen, leading to a loss of platelet aggregation. Thrombasthenia is clinically characterized by mucocutaneous hemorrhage with episodes of intracranial and gastrointestinal bleeding. To develop methods for gene therapy of Glanzmann thrombasthenia, a murine leukemia virus (MuLV)-derived vector, −889PlA2β3, was transduced into peripheral blood CD34+ cells from 2 patients with thrombasthenia with defects in the β3 gene. The human IIb promoter was used in this vector to drive megakaryocyte-targeted expression of the wild-type β3 subunit. Proviral DNA and IIbβ3 biosynthesis were detected after in vitro differentiation of transduced thrombasthenic CD34+ cells with megakaryocyte growth and development factor. Flow cytometric analysis of transduced patient samples indicated that 19% of megakaryocyte progeny expressed IIbβ3 on the surface at 34% of normal receptor levels. Treatment of transduced megakaryocytes with a combination of agonists including epinephrine and the thrombin receptor-activating peptide induced the IIbβ3 complex to form an activated conformation capable of binding fibrinogen as measured by PAC-1 antibody binding. Transduced cells retracted a fibrin clot in vitro similar to megakaryocytes derived from a normal nonthrombasthenic individual. These results demonstrate ex vivo phenotypic correction of Glanzmann thrombasthenia and support the potential use of hematopoietic CD34+ cells as targets for IIb promoter-driven MuLV vectors for gene therapy of platelet disorders.
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Lü, Peng, Keping Chen, Qin Yao, Lu Gao, Ye Pan, Yuanqing He, Guoping Huang, and Lin Wang. "Expression and Localization of Bombyx mori V-ATPase 16 kDa Subunit c." Zeitschrift für Naturforschung C 65, no. 1-2 (February 1, 2010): 119–26. http://dx.doi.org/10.1515/znc-2010-1-219.
Повний текст джерелаАнотація:
V-ATPase plays a central role in lepidopteran midgut ion transport physiology, and lepidopteran midgut turned out to be a model tissue for the study of V-ATPase. In the present study, the 5’-RACE method is used to obtain the 5’-UTR of V-ATPase c subunit gene from Bombyx mori. Sequence analysis of the promoter region and 3’-UTR of V-ATPase c subunit gene revealed that the transcription of the V-ATPase c subunit gene may be regulated by multi-ways. RT-PCR analysis showed that B. mori V-ATPase c subunit mRNA expresses in the whole developmental stages of B. mori. We also constructed a transient vector to determine the subcellular localization of the B. mori V-ATPase c subunit, and the result demonstrated that it is located in the membrane and some specifi c regions of BmN cells. Real-time PCR analysis further indicated that the c subunit mRNA expression was upregulated signifi cantly at 24 and 72 h in the midguts of resistant B. mori larvae after being inoculated with B. mori nucleopolyhedrovirus, suggesting that it may be related to the immune response against virus infection.
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Buck, D., and J. R. Guest. "Overexpression and site-directed mutagenesis of the succinyl-CoA synthetase of Escherichia coli and nucleotide sequence of a gene (g30) that is adjacent to the suc operon." Biochemical Journal 260, no. 3 (June 15, 1989): 737–47. http://dx.doi.org/10.1042/bj2600737.
Повний текст джерелаАнотація:
The succinyl-CoA synthetase of Escherichia coli is encoded by two genes, sucC (beta subunit) and sucD (alpha subunit), which are distal genes in the sucABCD operon. They are expressed from the suc promoter, which also expresses the dehydrogenase and dihydrolipoyl succinyl-transferase subunits of the 2-oxoglutarate dehydrogenase complex. Strategies have now been devised for the site-directed mutagenesis and independent expression of the succinyl-CoA synthetase (alpha 2 beta 2 tetramer) and the individual subunits. These involve (1) subcloning a promoterless sucCD fragment downstream of the lac promoter in M13mp10, and (2) precise splicing of the suc coding regions with the efficient atpE ribosome-binding site and expression from the thermoinducible lambda promoters in the pJLA503 vector. Succinyl-CoA synthetase specific activities were amplified 40-60-fold within 5 h of thermoinduction of the lambda promoters, and the alpha and beta subunits accounted for almost 30% of the protein in supernatant fractions of the cell-free extracts. Site-directed mutagenesis of potential CoA binding-site residues indicated that Trp-43 beta and His-50 beta are essential residues in the beta-subunit, whereas Cys-47 beta could be replaced by serine without inactivating the enzyme. No activity was detected after the histidine residue at the phosphorylation site of the alpha-subunit was replaced by aspartate (His-246 alpha----Asp), but this alteration seemed to have a deleterious effect on the accumulation of the enzyme in cell-free supernatant extracts. The nucleotide sequence of an unidentified gene (g30) that is adjacent to the sucABCD operon was defined by extending the sequence of the citric acid cycle gene cluster by 818 bp to 13379 bp: gltA-sdhCDAB-sucABCD-g30. This gene converges on the suc operon and encodes a product (P30) that contains 230 amino acids (Mr 27,251). Highly significant similarities were detected between the N-terminal region of P30 and those of GENA [the product of another unidentified gene (geneA) located upstream of the aceEF-lpd operon], and GNTR (a putative transcriptional repressor of the gluconate operon of Bacillus subtilis). Possible roles for GENA and P30 as transcriptional regulators of the adjacent operons encoding the pyruvate and 2-oxoglutarate dehydrogenase complexes are discussed.
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Takahashi, Masayo, Hiroyuki Miyoshi, Inder M. Verma, and Fred H. Gage. "Rescue from Photoreceptor Degeneration in therd Mouse by Human Immunodeficiency Virus Vector-Mediated Gene Transfer." Journal of Virology 73, no. 9 (September 1, 1999): 7812–16. http://dx.doi.org/10.1128/jvi.73.9.7812-7816.1999.
Повний текст джерелаАнотація:
ABSTRACT Retinitis pigmentosa (RP) is the most common inherited retinal disease, in which photoreceptor cells degenerate, leading to blindness. Mutations in the rod photoreceptor cGMP phosphodiesterase β subunit (PDEβ) gene are found in patients with autosomal recessive RP as well as in the rd mouse. We have recently shown that lentivirus vectors based on human immunodeficiency virus (HIV) type 1 achieve stable and efficient gene transfer into retinal cells. In this study, we evaluated the potential of HIV vector-mediated gene therapy for RP in the rd mouse. HIV vectors containing a gene encoding a hemagglutinin (HA)-tagged PDEβ were injected into the subretinal spaces of newborn rd mouse eyes. One to three rows of photoreceptor nuclei were observed in the eyes for at least 24 weeks postinjection, whereas no photoreceptor cells remained in the eyes of control animals at 6 weeks postinjection. Expression of HA-tagged PDEβ in the rescued photoreceptor cells was confirmed by two-color confocal immunofluorescence analysis using anti-HA and anti-opsin antibodies. HIV vector-mediated gene therapy appears to be a promising means for the treatment of recessive forms of inherited retinal degeneration.
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Hamilton, B. J., M. A. Mortin, and A. L. Greenleaf. "Reverse genetics of Drosophila RNA polymerase II: identification and characterization of RpII140, the genomic locus for the second-largest subunit." Genetics 134, no. 2 (June 1, 1993): 517–29. http://dx.doi.org/10.1093/genetics/134.2.517.
Повний текст джерелаАнотація:
Abstract We have used a reverse genetics approach to isolate genes encoding two subunits of Drosophila melanogaster RNA polymerase II. RpII18 encodes the 18-kDa subunit and maps cytogenetically to polytene band region 83A. RpII140 encodes the 140-kDa subunit and maps to polytene band region 88A10:B1,2. Focusing on RpII140, we used in situ hybridization to map this gene to a small subinterval defined by the endpoints of a series of deficiencies impinging on the 88A/B region and showed that it does not represent a previously known genetic locus. Two recently defined complementation groups, A5 and Z6, reside in the same subinterval and thus were candidates for the RpII140 locus. Phenotypes of A5 mutants suggested that they affect RNA polymerase II, in that the lethal phase and the interaction with developmental loci such as Ubx resemble those of mutants in the gene for the largest subunit, RpII215. Indeed, we have achieved complete genetic rescue of representative recessive lethal mutations of A5 with a P-element construct containing a 9.1-kb genomic DNA fragment carrying RpII140. Interestingly, the initial construct also rescued lethal alleles in the neighboring complementation group, Z6, revealing that the 9.1-kb insert carries two genes. Deleting coding region sequences of RpII140, however, yielded a transformation vector that failed to rescue A5 alleles but continued to rescue Z6 alleles. These results strongly support the conclusion that the A5 complementation group is equivalent to the genomic RpII140 locus.