Academic literature on the topic 'Subunit vector'

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

This thesis focuses on qualitative and quantitative aspects of some nonlinear PDEs arising in optimal control and differential games, ranging from regularity issues to maximum principles. More precisely, it is concerned with the analysis of some fully nonlinear second order degenerate PDEs over Hörmander vector fields that can be written in Hamilton-Jacobi-Bellman and Isaacs form and those arising in the recent theory of Mean Field Games, where the prototype model is described by a coupled system of PDEs involving a backward Hamilton-Jacobi and a forward Fokker-Planck equation. The thesis is divided in three parts. The first part is devoted to analyze strong maximum principles for fully nonlinear second order degenerate PDEs structured on Hörmander vector fields, having as a particular example fully nonlinear subelliptic PDEs on Carnot groups. These results are achieved by introducing a notion of subunit vector field for these nonlinear degenerate operators in the spirit of the seminal works on linear equations. As a byproduct, we then prove some new strong comparison principles for equations that can be written in Hamilton-Jacobi-Bellman form and Liouville theorems for some second order fully nonlinear degenerate PDEs. The second part of the thesis deals with time-dependent fractional Mean Field Game systems. These equations arise when the dynamics of the average player is described by a stable Lévy process to which corresponds a fractional Laplacian as diffusion operator. More precisely, we establish existence and uniqueness of solutions to such systems of PDEs with regularizing coupling among the equations for every order of the fractional Laplacian $sin(0,1)$. The existence of solutions is addressed via the vanishing viscosity method and we prove that in the subcritical regime the equations are satisfied in classical sense, while if $sleq1/2$ we find weak energy solutions. To this aim, we develop an appropriate functional setting based on parabolic Bessel potential spaces. We finally show uniqueness of solutions both under the Lasry-Lions monotonicity condition and for short time horizons. The last part focuses on the regularizing effect of evolutive Hamilton-Jacobi equations with Hamiltonian having superlinear growth in the gradient and unbounded right-hand side. In particular, the analysis is performed both for viscous Hamilton-Jacobi equations and its fractional counterpart in the subcritical regime via a duality method. The results are accomplished exploiting the regularity of solutions to Fokker-Planck-type PDEs with rough velocity fields in parabolic Sobolev and Bessel potential spaces respectively.

Gliomas are the most frequent brain tumors worldwide. Glioblastoma is classified by the World Health Organization as a high-grade glioma with predominantly astrocytic differentiation and is characterized by large intratumoral heterogeneity, microvascular proliferation and high infiltrative capacity. This brain tumor represents the most common form of glioma and is currently associated with a median survival of only 14 months from diagnosis. The standard therapy for glioblastoma patients include surgical resection of the tumor mass, radiotherapy and chemotherapy with the alkylating agent temozolomide. Despite this aggressive treatment, glioblastoma is still considered an incurable cancer and less than 5% of patients survive after five years. Tumor relapse is frequently observed and is probably related to the presence of a subpopulation of cancer stem cells, which represent one of the major obstacle to glioblastoma therapy. Indeed, cancer stem cells are known to be resistant to radiotherapy and chemotherapy thanks to their recently described plasticity, a strong DNA repair capacity, overexpression of DNA checkpoints and drug efflux proteins. Based on these characteristics, cell cultures of cancer stem cells as gliomaspheres are a recognized in vitro model for the study of glioblastoma and for identification of new therapeutic targets. Recent advancement in glioma etiopathogenesis allowed introduction of molecular markers in addition to histological criteria for glioma classification, pointing to a future personalized molecular therapy. In the last decades, many innovative therapeutic strategies and potential targets among molecules of signaling pathways specifically altered in glioblastoma have been investigated. Most of these therapeutic options are currently under preclinical and clinical evaluation showing promising results, but the high complexity and heterogeneity of glioma tumors underlie the continuous need for novel therapeutic targets. In this direction, protein kinase A (PKA) has recently emerged due to its involvement in many distinctive features of cancer cells and to its peculiarities in glioblastoma cells compared to brain parenchyma. PKA is a multifunctional enzyme, whose activation depends on binding of cAMP molecules. In its inactive form PKA is a tetramer consisting of two catalytic subunits and a regulatory subunit dimer. Up to now three catalytic isoforms and four regulatory isoforms have been identified and all of them present a specific expression pattern among different cell types and tissues. The cAMP/PKA pathway regulates a wide range of cellular processes, such as metabolism, gene transcription, cell cycle progression, cell proliferation, cell differentiation and apoptosis. The isoform-specific, spatio-temporal regulated PKA distribution allows a fine control of PKA signaling and is of primary importance for maintenance of physiological state. Indeed, several alterations of protein kinase A, including unbalanced expression of its subunits, dysregulation of the kinase activity and PKA delocalization, have been associated with many human diseases, including cancer. In particular, in human glioblastoma PKA is ten-times more abundant than normal brain. What is more, in rodent and human glioblastoma cells the regulatory subunit R2A presents a peculiar intracellular localization. In these cells perinuclear clusters of R2A co-localized with the Golgi apparatus have been described, whereas they have not been reported in the brain parenchyma, where R1A and R2B aggregates are instead detected. The functional significance of Golgi-associated PKA is still unknown, but this observation points to a potential role of R2A subunit as a tumor marker or a novel therapeutic target. Based on these considerations, the present PhD project aimed at providing additional insights into the role of protein kinase A, particularly R2A subunit, in human glioblastoma. For this purpose, the study of PKA R2A intracellular localization was extended to human glioblastoma stem like cells, as up to now no data are reported as regard to R2A localization in cancer stem cells. Immunofluorescence experiments were performed on human gliomaspheres, confirming in glioblastoma stem like cells the same peculiar R2A intracellular distribution that had been previously described in non-stem glioblastoma cell lines and primary cell cultures. This observation further supports the peculiarity of R2A subunit in human glioblastoma compared to brain parenchyma, as well as the need to further characterize the specific functional role of this peculiarity. In order to develop new molecular tools for study of PKA, a set of recombinant plasmids for expression of PKA subunits fused to EGFP fluorescent protein were generated and preliminarily validated in human glioblastoma cells. Despite a low transfection efficiency, these plasmids proved to be useful tools for functional studies of PKA in different cellular models. Moreover, in the first part of the research project, effects of transient interference with PKA pathway were analyzed in human glioblastoma cells through multiple approaches. Alterations of PKA activity, expression and intracellular distribution were induced by drug treatments, small interfering RNA (siRNA) transfection, overexpression of wild type and mutant PKA isoforms and expression of peptide inhibitors for PKA dislocation. Consistently with other scientific studies, these experiments demonstrated that transient interference with PKA differentially affects human glioblastoma cells, particularly cell viability and cell motility. In addition, as previously reported in other human cell lines, siRNA-mediated downregulation of PKA R2A expression proved to induce Golgi fragmentation and dispersion of R2A in the cell cytoplasm of glioblastoma cells, an effect that deserves further investigation. As a complementary approach, to investigate the relationship between PKA R2A and Golgi apparatus, effects of disrupted Golgi function on human glioblastoma cells and R2A intracellular localization were also analyzed. Increased cell death, reduced cell motility and redistribution of R2A subunit in the cell cytosol were observed in glioblastoma cells following treatment with the Golgi disruptor brefeldin A. These results support the relevant relationship between R2A subunit and Golgi complex in glioblastoma cells, but future studies are needed to better understand its functional meaning. The second part of the PhD project was focused on the development of a third-generation lentiviral system for downregulation of PKA R2A expression through short hairpin RNA (shRNA) delivery. Lentiviral vectors for expression of shRNA targeting different exons of R2A gene, their co-expression with EGFP reporter protein, as well as expression of a scrambled shRNA were thus generated and validated in non-stem and stem like glioblastoma cells. In both cell cultures PKA R2A gene silencing lentiviral vectors proved able to induce an efficient and stable downregulation of R2A expression. Preliminary experiments also evaluated the effects of PKA R2A knockdown on glioblastoma cell proliferation and cell death. As regard to non-stem glioblastoma cells, data from cell growth assays are still controversial due to a possible cytotoxicity of the scrambled shRNA sequence, not detected by previous bioinformatic validation of the shRNA and that is currently under investigation. Conversely, in glioblastoma stem like cells the scrambled vector did not affect cell viability compared to untransduced cells. Moreover, a significantly different distribution between viable and dead cells, particularly an increase in dead cells, was reported for glioblastoma stem like cells with downregulated PKA R2A expression compared to scrambled transduced cells. These results thus point to a potential relationship between PKA R2A silencing and cell survival of glioblastoma stem like cells, an interesting aspect that should be further investigated. In conclusion, the present PhD project investigated the multiple functions of PKA in human glioblastoma by means of many complementary approaches and different in vitro models. PKA transient interference exhibited some anti-tumor effects and preliminary data indicated a potential relationship between PKA R2A downregulation and glioblastoma stem like cell growth. What is more, a lentiviral system for efficient R2A silencing was developed and successfully validated, proving to be a promising tool for future study of PKA in several cellular models.
I gliomi sono i più frequenti tumori cerebrali a livello mondiale. Il glioblastoma è classificato dall'Organizzazione Mondiale della Sanità come glioma ad alto grado con prevalente differenziazione astrocitica ed è caratterizzato da elevata eterogeneità intratumorale, proliferazione microvascolare e grande capacità infiltrativa. Il glioblastoma è la forma più comune di glioma ed è attualmente associato ad una sopravvivenza media di soli 14 mesi dalla diagnosi. La terapia standard per questo tipo di tumore prevede la resezione chirurgica della massa tumorale, radioterapia e chemoterapia con l'agente alchilante temozolomide. Nonostante questo approccio aggressivo, il gliobastoma è ad oggi ancora considerato un tumore incurabile e meno del 5% dei pazienti sopravvive a cinque anni dalla diagnosi. Nei pazienti affetti da glioblastoma, le ricadute si verificano frequentemente e sono probabilmente dovute alla presenza di una sottopopolazione di cellule staminali tumorali, che rappresentano uno dei maggiori ostacoli alla terapia. È infatti noto che queste cellule sono resistenti a radioterapia e chemioterapia grazie alla loro plasticità, un'elevata capacità di riparare i danni al DNA, un'elevata espressione di proteine checkpoint che controllano la progressione del ciclo cellulare in risposta a danni genetici e di proteine che sono in grado di espellere all'esterno della cellula diverse molecole tra cui i farmaci. Proprio per queste caratteristiche di resistenza alle terapie convenzionali, le colture cellulari di cellule staminali tumorali sono oggi un modello in vitro riconosciuto per lo studio del glioblastoma e per l'identificazione di nuovi target terapeutici. Le cellule staminali tumorali vengono mantenute in particolari condizioni di coltura, che ne permettono la crescita selettiva sottoforma di aggregati sferici (sfere) in sospensione. Grazie alla ricerca scientifica, la conoscenza dell'eziopatogenesi dei gliomi è recentemente cresciuta e l'introduzione di marcatori molecolari, oltre ai criteri istologici per la classificazione dei gliomi, rappresenta un avanzamento verso una terapia molecolare personalizzata. Negli ultimi vent'anni, sono state sviluppate molte strategie terapeutiche innovative e sono stati identificati potenziali nuovi target tra i componenti delle vie di segnalazione intracellulari specificamente alterate nel glioblastoma. La maggior parte di questi nuovi approcci terapeutici è attualmente in fase di valutazione preclinica e clinica, i risultati sembrano promettenti, ma la complessità e l'eterogeneità dei gliomi sottolineano la necessità di cercare continuamente nuovi bersagli terapeutici. Proprio in questa direzione, la proteina chinasi A (PKA) è recentemente emersa per il suo coinvolgimento in molte caratteristiche distintive delle cellule tumorali e per le sue peculiarità nelle cellule di glioblastoma rispetto al parenchima cerebrale. PKA è un enzima che svolge molte funzioni all'interno della cellula e la cui attivazione dipende dal secondo messaggero cAMP. Nella sua forma inattiva PKA è un tetramero costituito da due subunità catalitiche e un dimero di subunità regolatorie. Fino ad oggi sono state identificate tre isoforme catalitiche e quattro isoforme regolatorie. Ciascuna subunità, sia catalitica che regolatoria, presenta un pattern di espressione specifico tra i diversi tipi cellulari e i diversi tessuti dell'organismo. La via mediata da cAMP/PKA regola una vasta gamma di processi cellulari, tra i quali il metabolismo, la progressione del ciclo cellulare, la proliferazione cellulare, la differenziazione cellulare, la trascrizione genica e l'apoptosi. La distribuzione isoforma-specifica di PKA e la sua regolazione spazio-temporale permettono un fine controllo dei segnali mediati dalla via di cAMP/PKA, che risulta essere di primaria importanza per il mantenimento di uno stato cellulare fisiologico. Infatti, molte alterazioni di PKA, come una sbilanciata espressione delle sue isoforme, una disregolazione della sua attività chinasica e la delocalizzazione dell'enzima stesso, sono state associate a molte malattie umane, compresi diversi tumori. In particolare, nel glioblastoma umano PKA è dieci volte più abbondante rispetto al cervello sano. Inoltre, nelle cellule di glioblastoma di roditore e umane la subunità regolatoria R2A di PKA presenta una peculiare localizzazione intracellulare sottoforma di cluster perinucleari co-localizzati con l'apparato di Golgi. Questa particolare distribuzione della subunità R2A non è stata riscontrata nel parenchima cerebrale, dove invece sono stati individuati aggregati delle subunità R1A e R2B. Il significato funzionale della proteina chinasi A associata all'apparato di Golgi non è ancora chiara, ma questa osservazione indica un potenziale ruolo della subunità R2A come marcatore tumorale o come nuovo target terapeutico. Sulla base di quanto esposto finora, questo progetto di dottorato ha lo scopo di approfondire il ruolo della proteina chinasi A, ed in particolare della subunità R2A, nel glioblastoma umano. Per questo motivo, lo studio della localizzazione intracellulare di R2A è stato esteso, per la prima volta, alle cellule staminali tumorali di glioblastoma. Esperimenti di immunofluorescenza hanno dimostrato che anche in queste cellule la subunità R2A presenta la stessa distribuzione intracellulare che era stata precedentemente descritta in linee cellulari e colture primarie non staminali di glioblastoma umano. Questa osservazione rafforza ulteriormente la peculiarità di R2A rispetto al parenchima sano e sottolinea la necessità di caratterizzare lo specifico ruolo funzionale di questa subunità. Al fine di sviluppare nuovi strumenti molecolari per lo studio di PKA, è stato generato un set di plasmidi ricombinanti per l'espressione delle subunità di PKA in fusione alla proteina verde fluorescente (EGFP). Questi plasmidi sono stati validati in cellule di glioblastoma umano e, nonostante la bassa efficienza di trasfezione, si sono rivelati degli utili strumenti per studi funzionali di PKA in vari modelli cellulari. Nella prima parte del progetto di ricerca sono inoltre stati analizzati in cellule di glioblastoma umano gli effetti di una interferenza transiente con la via di PKA. Alterazioni dell'attività, dell'espressione e della distribuzione intracellulare della proteina chinasi A sono state indotte mediante diversi approcci, come il trattamento con agenti chimici, la trasfezione con small interfering RNA (siRNA), l'overespressione di forme wild type e mutate delle subunità di PKA e l'espressione di peptidi inibitori che causano la delocalizzazione di PKA. Coerentemente con quanto riportato in letteratura, questi esperimenti hanno dimostrato che l'interferenza transiente con PKA può avere effetti diversi sulle cellule di glioblastoma umano, in particolare sulla loro vitalità e capacità migratoria. Come già riportato in altre linee cellulari umane, si è osservato che la ridotta espressione di PKA R2A indotta dalla trasfezione con siRNA induce la frammentazione dell'apparato di Golgi e la dispersione di R2A nel citoplasma delle cellule di glioblastoma, un effetto che deve essere ulteriormente approfondito. Per studiare più a fondo la relazione tra PKA R2A e apparato di Golgi, sono stati anche analizzati gli effetti indotti dall'interferenza con l'apparato di Golgi sulle cellule di glioblastoma e in particolare sulla localizzazione intracellulare di R2A. Il trattamento con brefeldina A, che distrugge la funzione dell'apparato di Golgi, causa un aumento della mortalità cellulare, riduce la motilità e induce la ridistribuzione della subunità R2A nel citosol delle cellule di glioblastoma. Questi risultati sono a sostegno dell'importante relazione tra PKA R2A e l'apparato di Golgi, ma ulteriori studi sono necessari per comprenderne meglio il significato funzionale. La seconda parte del progetto di ricerca riguarda lo sviluppo di un sistema basato su vettori lentivirali di terza generazione per la riduzione dell'espressione della subunità R2A di PKA mediante short hairpin RNA (shRNA). Sono quindi stati generati dei vettori lentivirali per l'espressione di shRNA aventi come bersaglio diversi esoni del gene di R2A, per la co-espressione degli stessi shRNA e del gene reporter EGFP e per l'espressione di uno shRNA scrambled per la valutazione di eventuali effetti off-target. Tutti i vettori sono stati validati sia in cellule non staminali che in cellule staminali di glioblastoma umano, dimostrandosi in grado di indurre un silenziamento efficiente e stabile dell'espressione di R2A in entrambi i tipi di colture cellulari. Esperimenti preliminari hanno inoltre valutato gli effetti del silenziamento della subunità R2A sulla proliferazione e sulla morte cellulare. Per quanto riguarda le cellule non staminali di glioblastoma umano, i dati ottenuti sono controversi a causa di una possibile citotossicità della sequenza scrambled, che non era stata rilevata dalla precedente validazione bioinformatica e che è tuttora in fase di studio. Al contrario, nelle cellule staminali di glioblastoma la stessa sequenza scrambled non risulta essere tossica in quanto non induce una riduzione della vitalità cellulare rispetto alle cellule non trasdotte. Ancora più interessante è l'osservazione che le cellule staminali di glioblastoma con una ridotta espressione di R2A dovuta alla trasduzione con il vettore recante la sequenza shRNA contro il gene di R2A, presentano una distribuzione di cellule vive/morte significativamente diversa, nello specifico un aumento della morte cellulare, rispetto alle cellule trasdotte con il vettore scrambled. Questi risultati suggeriscono quindi una potenziale relazione tra il silenziamento della subunità R2A e la sopravvivenza delle cellule staminali di glioblastoma, un aspetto che merita di essere ulteriormente approfondito. In conclusione, questo progetto di dottorato ha indagato le molteplici funzioni di PKA nel glioblastoma umano, attraverso approcci tra loro complementari e diversi modelli in vitro. È stato dimostrato che l'interferenza transiente con la via di PKA può avere effetti anti-tumorali sulle cellule di glioblastoma. Dati preliminari indicano anche una possibile relazione tra la riduzione dell'espressione della subunità R2A e la crescita delle cellule staminali di glioblastoma. Inoltre, è stato messo a punto un sistema lentivirale per il silenziamento efficiente e stabile di PKA R2A. Questo strumento è stato validato con successo nelle cellule di glioblastoma umano e può essere considerato uno strumento promettente per futuri studi della proteina chinasi A in diversi modelli cellulari.

碩士
國立中興大學
分子生物學研究所
103
The baculovirus display system has been applied with vaccines development in the past decade years. The advantages of this system is the high expression of efficiency exogenous proteins in the insect host cells, post-translational modifications, and able to carry large fragment of DNA. We have successfully developed baculovirus display expression vector with multiple promotors, which applies to construct the genetics recombinant baculovirus, including canine distemper virus of H gene, canine parvovirus virus of VP2 gene, canine adenovirus of Fiber head gene, canine parainfluenza virus of HN gene, canine coronavirus of S1 gene, and canine leptospires of LigB gene from the canine pathogen genes. In this study, the viral genes descried were amplified by polymerase chain reaction (PCR), and then cloned into the pBacDual 4Display-EGFP vector. After the verification by retraction enzyme digestion and DNA sequencing, these recombinant plasmids harboring the target genes were transformed respectively into DH10Bac E.coli for selection of the recombinant Bacmid DNA. The resultant recombinant Bacmid DNA was transfected into insect host cell (Sf9) for the production of baculovirus which has the expressed target protein that was anchored on the baculovirus envelop. Finally, four genetics recombinant baculovirus named BacD4D-4H(d58), BacD4D-2VP2-2H(d58), BacD4D-VP2-2H(d58)-LigB and BacD4D-S1-HN(d55)-Fiber head have constructed. The results of western blot assay showed that all of target proteins (CDV-H(d58), CPV-VP2, CAV-fiber head, CPiV-HN(d55), CCoV S1,and CL-LigB) were successfully expressed in each recombinant baculovirus infected-cells. The present study has demonstrated that expressed protein level is positive related to the copy number of the target gene, except the H(d58) protein from the BacD4D-4H(d58). A possible explanation for the decrease expression of H(d58) protein is the occurrence of spontaneous self-gene recombination during the replication of BacD4D-4H(d58), leading to knockout of H(d58) gene. These results showed that the important canine pathogen proteins could be anchored on cell membrane. In order to estimate the potential of recombinant baculovirus as a vaccine for the prevention of virus infection, the insect host cell infected by genetics recombinant baculovirus are going to be used to immunize mice, and the mice serum will be analyzed for the determination of the specific antibody by ELISA.

碩士
國立中興大學
分子生物學研究所
100
Porcine circovirus type 2 (PCV2) was the mainly infectious agent of postweaning multisystemic wasting syndrome (PMWS), one of the most important swine disease with a negative impact concerning on economic losses worldwide. PMWS was clinically characterized by wasting and growth retardation, although other signs such as fever, pallor of the skin, respiratory distress and diarrhoea could be observed as well. The PCV2 capsid proteins (Cap) were the major structural viral proteins encoded by ORF2 could induce host-specific neutralizing antibody. Recombinant baculovirus expression systems have been exploited for the production of vaccines. However, the higher costs in eukaryotic expression systems will be the obstacles when the products are undertaked to the commercially application in the future. Thus, the aim of this study was to construct high efficient recombinant baculoviruses, improving the target protein expression level and immunogenicity in a cost-effective manner. The baculovirus surface display system was used as the platform. After infection, the proteins were expressed and anchored on the plasma membrane of Sf-9 cells. In this study, recombinant baculovirus, BacSC-Cap(d41) with deletion of 41 amino acids in N-terminal side of the Cap protein, hvae been confirmed to express more stably in insect cells than BacSC-Cap(d41)-mcherry with Cap(d41) fused with mCherry by Western blot analysis. Furthermore, four different recombinant baculovirus shuttle vectors, pBacSC-Cap(d41), pBacDD-2Cap(d41), pBacDD-3Cap(d41) and pBacDD-4Cap(d41) for construction of recombinant baculoviruses, namely BacSC-Cap(d41), BacDD-2Cap(d41), BacDD-3Cap(d41) and BacDD-4Cap(d41) respectively, hve been constructed. The results indicated that BacDD-4Cap (d41) was able to express the highest level of Cap(d41) paoteins by Western blot analysis. For the highest efficiency of protein expression, we have tested different conditions, such as infection days, mutiplicity of infection (MOI) and cell numbers. Our results reveal that three days post infection under MOI 5 or 10 showed the highest protein expression level. In vivo experiments, the cell numbers of 10^7, 10^6 and 10^5 infected with recombinant baculoviruses were used as the antigens to immunize the mice, and the results showed all the BacSC-Cap(d41), BacDD-4Cap(d41) and commercial vaccine could elicit the immune response by ELISA analysis. The Cap(d41) protein produced from recombinant baculovirus BacDD-4Cap(d41) could elicit anti-PCV2 neutralizing antibodies, as confirmed by virus neutralization test. Importantly, it also induces IFN-γ further confirming cellular immunity mediated at PCV2-Cap(d41) protein. Our results suggest that the PCV2-Cap(d41) protein can elicite both cellular and humoral immune responses. Taken together, the high efficiency of recombinant baculovirus expression vectors and recombinant baculoviruses have been constructed to develope the PCV2 Cap subunit vaccine.

Anhui Zhifei Longcom ‘s Zifivax, also known as ZF2001 (ZF-UZ-VAC-2001) is a protein subunit vaccine using a dimeric form of the receptor-binding domain (RBD) as the antigen, a harmless piece of the SARS-Cov-2 virus. As of June, 2022, over 300 million doses of Zifivax have been vaccinated with localized production in China base and Tashkent, Uzbekistan. At present, the severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) is constantly mutating and evolving, and the coronavirus disease 2019 (COVID-19) epidemic is seriously threatening human health. Vaccination is the most effective and economical method to prevent and control the COVID-19 pandemic. Research institutions and companies around the world are employing various techniques to develop COVID-19 vaccines. According to the preparation technology, COVID-19 vaccines can be classified as inactivated virus vaccines, live attenuated vaccines, mRNA vaccines, DNA vaccines, viral vector vaccines, virus-like particle vaccines and protein subunit vaccines. Among these, viral protein subunit vaccines based on in vitro production of key viral proteins or peptides from bacterial, yeast, insect or mammalian cells have been drawing attention owing to their advantages of high safety and effectiveness, low cost of production, storage and transportation. Givrn this, this study reviewed the research and development status of ZF2001, as a reference for the development of protein subunit vaccines against SARS-Cov-2.

Vaccines are undoubtedly one of the great triumphs of medical science. The global eradication of smallpox, once one of the most devastating infectious diseases of humankind, and the extensive control of various other infectious diseases bear testimony to the efficacy of vaccines. Much of this success employed classical vaccine designs, namely live attenuated vaccines as used for smallpox, measles, mumps and rubella (MMR), the BCG tuberculosis vaccine and the Sabin polio vaccine, and inactivated vaccines such as the Salk polio vaccine. Since then, subunit vaccines based on isolated macromolecules, including toxoid vaccines against tetanus, diphtheria and pertussis and conjugate vaccines against several forms of bacterial meningitis, have been developed. The advent of recombinant DNA technology and the first recombinant protein vaccine, the hepatitis B vaccine introduced in the 1980s, heralded a paradigm shift in vaccine design – no longer was it necessary to culture the pathogen. This millennium saw the introduction of recombinant protein vaccines against human papillomavirus (HPV) and meningitis B (MenB). Despite these successes, the persistence of malaria, HIV/AIDS and hepatitis C along with the emergence of novel zoonotic infections such as the devastating outbreaks of Ebola virus disease and the coronavirus outbreaks, including severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS) and Covid-19, serve as a reminder of the need for new vaccine technologies. At the forefront of this are recombinant vector vaccines and nucleic acid vaccines supported by dedicated bioinformatics tools. This chapter provides an overview of the immunology of vaccines and the range of vaccine design strategies currently being employed.

Vaccines can be classified according to their nature into the following types: ● Inactivated vaccines: ■ Whole organism; ■ Acellular extracts. ● Live attenuated vaccines. ● Toxoid vaccines. ● Subunit vaccines. ● Conjugate vaccines. ● DNA vaccines. ● Recombinant vector vaccines. Inactivation of the whole organism is the most basic form of vaccine produced by killing the micro-organism causing the disease using heat, chemical or radiation and presents all the antigens in the inactivated organism as a vaccine to induce immunity in the recipient. Other methods to produce an inactivated vaccine is by extracting acellular components of the organism through filtration. Examples of inactivated bacterial vaccines currently in use include: ● Anthrax—sterile filtrate from cultures of the Sterne strain of B. anthracis. ● Cholera—oral inactivated vaccine with 1mg of recombinant cholera toxin B (rCTB) in a liquid suspension of four strains of killed V. cholerae O1, representing subtypes Inaba and Ogawa and biotypes El Tor and classical. ● Pertussis—acellular vaccine has replaced previously used whole cell vaccine. ● Typhoid—purified Vi capsular polysaccharide from S. typhi; NB: the injectable, killed, whole-cell typhoid vaccine which contains heat-inactivated, phenol-preserved S. typhi organisms is no longer in use in the UK. Examples of inactivated viral vaccines currently in use in the UK include: ● Hepatitis A virus. ● Hepatitis E virus. ● Influenza A and B viruses. ● Japanese encephalitis virus. ● Polio viruses 1, 2, and 3 (IPV). ● Rabies virus. ● Tick-borne encephalitis virus. ● Bacterial vaccines: Bacillus Calmette-Guerin (BCG) vaccine is a live attenuated vaccine against tuberculosis derived from a Mycobacterium bovis strain. The oral typhoid vaccine contains a live attenuated strain of S. typhi (Ty21a) in an enteric-coated capsule. ● Viral vaccines: The measles, mumps, and rubella (MMR) vaccine contain live attenuated strains of measles, mumps, and rubella viruses, which are cultured separately and mixed before being lyophilized. Oral polio vaccine (OPV) against polio viruses 1, 2, and 3—OPV contains live attenuated strains of poliomyelitis virus types 1, 2, and 3 grown in cell cultures.

The platelet membrane GPIIb/GPIIIa complex on activated platelets contains receptors for fibrinogen, von Willebrand factor, and fibronectin. GPIIb and GPIlia also appear to be members of a family of membrane receptors involved in cell-cell and cell-matrix interactions. To study the structure of GPIIb and GPIIIa, we have constructed an expression library in the vector lambda gtll using mRNA from the HEL cell line and screened it with polyclonal antibody against each platelet protein. HEL cells constitutively express proteins similar to platelet GPIIb and GPIIIa. A 3.2kb GPIIb cDNA clone was identified that encodes for all 1008 amino acids of GPIIb including the known N-terminal amino acids of the α Cand βsubunits. This confirms that GPIIb is synthesized as a single chain polypeptide that is cleaved into two disulfide-linked subunits posttranslation. Analysis of the amino acid sequence revealed a major C-terminal transmembrane domain in the βsubunit, two potential transmembrane domains near the N-terminus of the αsubunit, and four possible N-linked glycosylation sites. Approximately 30% amino acid identity was found between GPIIb and the available amino acid sequences for the larger chains of the fibronectin and vitronectin receptors. Initial sequence analysis of a 3.8kb cDNA for GPIIIa included the known N-terminal amino acids of the platelet protein. Northern blot analysis was performed using HEL cell total RNA. The GPIIb cDNA hybridized to a 4.1kb mRNA while the GPIIIa cDNA hybridized to a 5.8kb mRNA. This indicates that the two cDNAs do not cross-hybridize and suggests that GPIIb and GPIIIa are encoded by separate genes. The availability of these cDNA for GPIIb and GPIIIa will facilitate study of the structure and function of the proteins and will aid in clarifying their relationship to other adhesive protein receptors.

The black carp (bc)GtH IIb cDNA was amplified and isolated, cloned and sequenced. Comparison of the bcGtH IIb deduced a.a. sequence with that of GtH IIb from other teleosts revealed high homology to cyprinid species and a lower homology to salmonid or perciform fish. The gene coding for the GtH IIb was isolated and sequenced. Three bc recombinant phages which hybridized to the goldfish GtH Ib cDNA probe were isolated and are currently being characterized. The region coding for the mature GtH IIb was expressed in a bacterial expression vector resulting in the production of a recombinant protein. In vitro folding resulted in a protein only 1.3% of which displaced the native common carp GtH II in a RIA. Therefore, the common carp GtH RIA was utilized for the physiological studies at the current phase of the project. Two non-functional sites were identified along the brain-pituitary gonadal axis in the immature black carp. The pituitary is refractory to GnRH stimulation due to a block proximal to the activation of PKA and PKC probably at the level of GnRH receptors. The gonads, although capable of producing steroids, are refractory to gonadotropic stimulation but do respond to cAMP antagonists, indicating a block at the GtH receptor level. Attempts to advance puberty in 2 and 3 y old black carp showed that testosterone (T) stimulates GtH synthesis in the pituitary and increases its sensitivity to GnRh. A 2 month treatment combining T+GnRH increased the circulating GFtH level in 3 y old fish. Addition of domperidone to such a treatment facilitated both the accumulation of GtH in the pituitary and its response to GnRH. The cDNA of striped bass GtH a, Ib and IIb subunits were amplified, isolated, cloned and sequenced, and their deduced a.a. sequences were compared with those of other teleosts. A ribonuclease protection assay was developed for a sensitive and simultaneous determination of all GtH subunits, and of b-actin mRNAs of the striped bass. GnRH stimulated dramatically the expression of the a and GtH IIb subunits but the level of GtH Ib mRNA increased only moderately. These findings suggest that GtH-II, considered in salmonids to be involved only in final stages of gametogenesis, can be induced by GnRH to a higher extent than GtH-I in juvenile striped bass. The native GtH II of the striped bass was isolated and purified, and an ELISA for its determination was developed. The production of all recombinant striped bass GtH subunits is in progress using the insect cell (Sf9) culture and the BAC-TO-BAC baculovirus expression system. A recombinant GtH IIb subunit has been produced already, and its similarity to the native subunit was confirmed. The yield of the recombinant glycoprotein can reach 3.5 mg/ml after 3 days culture. All male striped bass reach puberty after 3 y. However, precocious puberty was discovered in 1 and 2 y old males. Females become vitellogenic during their 4th year. In immature 2 y old females, T treatment elevates the pituitary GtH II content while GnRH only potentiates the effect. However, in males GnRH and not T affects GtH accumulation in the pituitary. Neither GnRH, nor T treatment resulted in gonadal growth in 2 y old striped bass, indicating that either the accumulated GtH II was not released, or if released, the gonads were refractory to GtH stimulation, similar to the situation in the immature black carp. In 3 y old female striped bass, 150 day GnRHa treatment resulted in an increase in GSI, while T treatment, with or without GnRHa, resulted in a decrease in oocyte diameter, similar to the effect seen in the black carp. Further attempts to advance puberty in both fish species should take into account the positive effect of T on pituitary GtH and its negative effect of ovarian growth.

The aim of this research project was to obtain information on the role of the cytochrome b559 in the function of Photosystem-II (PSII) with special emphasis on the light induced photo inactivation of PSII and turnover of the photochemical reaction center II protein subunit RCII-D1. The major goals of this project were: 1) Isolation and sequencing of the Chlamydomonas chloroplast psbE and psbF genes encoding the cytochrome b559 a and b subunits respectively; 2) Generation of site directed mutants and testing the effect of such mutation on the function of PSII under various light conditions; 3) To obtain further information on the mechanism of the light induced degradation and replacement of the PSII core proteins. This information shall serve as a basis for the understanding of the role of the cytochrome b559 in the process of photoinhibition and recovery of photosynthetic activity as well as during low light induced turnover of the D1 protein. Unlike in other organisms in which the psbE and psbF genes encoding the a and b subunits of cytochrome b559, are part of an operon which also includes the psbL and psbJ genes, in Chlamydomonas these genes are transcribed from different regions of the chloroplast chromosome. The charge distribution of the derived amino-acid sequences of psbE and psbF gene products differs from that of the corresponding genes in other organisms as far as the rule of "positive charge in" is concerned relative to the process of the polypeptide insertion in the thylakoid membrane. However, the sum of the charges of both subunits corresponds to the above rule possibly indicating co-insertion of both subunits in the process of cytochrome b559 assembly. A plasmid designed for the introduction of site-specific mutations into the psbF gene of C. reinhardtii. was constructed. The vector consists of a DNA fragment from the chromosome of C. reinhardtii which spans the region of the psbF gene, upstream of which the spectinomycin-resistance-conferring aadA cassette was inserted. This vector was successfully used to transform wild type C. reinhardtii cells. The spectinomycin resistant strain thus obtained can grow autotrophically and does not show significant changes as compared to the wild-type strain in PSII activity. The following mutations have been introduced in the psbF gene: H23M; H23Y; W19L and W19. The replacement of H23 involved in the heme binding to M and Y was meant to permit heme binding but eventually alter some or all of the electron transport properties of the mutated cytochrome. Tryptophane W19, a strictly conserved residue, is proximal to the heme and may interact with the tetrapyrole ring. Therefore its replacement may effect the heme properties. A change to tyrosine may have a lesser affect on the potential or electron transfer rate while a replacement of W19 by leucine is meant to introduce a more prominent disturbance in these parameters. Two of the mutants, FW19L and FH23M have segregated already and are homoplasmic. The rest are still grown under selection conditions until complete segregation will be obtained. All mutants contain assembled and functional PSII exhibiting an increased sensitivity of PSII to the light. Work is still in progress for the detailed characterization of the mutants PSII properties. A tobacco mutant, S6, obtained by Maliga and coworkers harboring the F26S mutation in the b subunit was made available to us and was characterized. Measurements of PSII charge separation and recombination, polypeptide content and electron flow indicates that this mutation indeed results in light sensitivity. Presently further work is in progress in the detailed characterization of the properties of all the above mutants. Information was obtained demonstrating that photoinactivation of PSII in vivo initiates a series of progressive changes in the properties of RCII which result in an irreversible modification of the RCII-D1 protein leading to its degradation and replacement. The cleavage process of the modified RCII-D1 protein is regulated by the occupancy of the QB site of RCII by plastoquinone. Newly synthesized D1 protein is not accumulated in a stable form unless integrated in reassembled RCII. Thus the degradation of the irreversibly modified RCII-D1 protein is essential for the recovery process. The light induced degradation of the RCII-D1 protein is rapid in mutants lacking the pD1 processing protease such as in the LF-1 mutant of the unicellular alga Scenedesmus obliquus. In this case the Mn binding site of PSII is abolished, the water oxidation process is inhibited and harmful cation radicals are formed following light induced electron flow in PSII. In such mutants photo-inactivation of PSII is rapid, it is not protected by ligands binding at the QB site and the degradation of the inactivated RCII-D1 occurs rapidly also in the dark. Furthermore the degraded D1 protein can be replaced in the dark in absence of light driven redox controlled reactions. The replacement of the RCII-D1 protein involves the de novo synthesis of the precursor protein, pD1, and its processing at the C-terminus end by an unknown processing protease. In the frame of this work, a gene previously isolated and sequenced by Dr. Pakrasi's group has been identified as encoding the RCII-pD1 C-terminus processing protease in the cyanobacterium Synechocystis sp. PCC 6803. The deduced sequence of the ctpA protein shows significant similarity to the bovine, human and insect interphotoreceptor retinoid-binding proteins. Results obtained using C. reinhardtii cells exposes to low light or series of single turnover light flashes have been also obtained indicating that the process of RCII-D1 protein turnover under non-photoinactivating conditions (low light) may be related to charge recombination in RCII due to back electron flow from the semiquinone QB- to the oxidised S2,3 states of the Mn cluster involved in the water oxidation process.

The broad objective of the project was to develop a feasible approach to combat diarrheal disease caused by ETEC through the development of a low-cost oral immunogen in tomato fruit, expressed in the context of a prototype tomato that would answer the shortcomings of plant oral vaccines, especially in terms of produce handling and control of gene escape. Specifically, the goals for Boyce Thompson Institute (BTI) on this project were to develop transgenic tomato lines that express the enterotoxigenic E. coli (ETEC) heat-labile enterotoxin (LT) subunits A and/or B for use in oral edible vaccines, and to optimize expression and assembly of these antigens in tomato fruits.LT-B is a useful vaccine antigen against ETEC disease, since antibodies against LT-B can prevent binding and delivery of the holotoxinLT. Mutant forms of the toxic LT-A subunit that have reduced toxicity can be co-expressed and assembled with LT-Bpentamers to form mutant LT (mLT) complexes that could be used as mucosaladjuvants for other oral vaccines. Work on the project is continuing at Arizona State University, after Dr. Mason moved there in August 2002. A number of approaches were taken to ensure the expression of both subunits and bring about their assembly inside the transgenic fruits. Initially, expression was driven by the fruit-specific E-8 promoter for LT-B and the constitutive CaMV 35S promoter for LT-A(K63). While LT-B accumulated up to 7 µg per gram ripe fruit, assembled LT-K63 was only 1 µg per gram. Since promoter activities for the two genes likely differed in cell type and developmental stage specificity, the ratios of A and B subunits was not optimal for efficient assembly in all cells. In order to maximize the chance of assembly of mLT in fruit, we focused on constructs in which both genes are driven by the same promoter. These included co-expression plasmids using the 35S promoter for both, while switching to attenuated mLTs (LT-R72 and LT-G192) that have shown greater potential for oral adjuvanticity than the initial LT-K63, and thus are better candidates for a plant-derived adjuvant. Other, more novel approaches were then attempted, including several new vectors using the tomato fruit-specific E8 promoter driving expression of both LT-B and mutant LT-A, as well as a dicistronic construct for co-expression of both LT-B and mutant LT-A genes from a single promoter, and a geminivirusreplicon construct. We describe in the Appendix the results obtained in transgenic tomato lines transformed with these constructs. Overall, each contributed to enhanced expression levels, but the assembly itself of the holotoxin to high levels was not observed in the fruit tissues. The Israeli lab’s specific objective was to develop transgenic tomato lines expressing the LTholotoxin antigen bearing attributes to prevent gene escape (male sterility and orange fruit color) and to improve the dissemination of the oral vaccine (long shelf-life tomato cherry fruit or tomato processing background). Breeding lines bearing a number of attributes to prevent gene escape were developed by combining material and backcrossing either to a tomato cherry background, or two different processing backgrounds. Concomitantly, (these lines can be utilized for the creation of any future oral vaccine or other therapeutic-expressing tomato, either by crosses or transformation), the lines were crossed to the holotoxin-expressing tomatoes received from the United States, and this transgenic material was also incorporated into the backcrossing programs. To date, we have finalized the preparation of the cherry tomato material, both non-transgenic (bearing all the desired attributes), and transgenic, expressing the holotoxin. The level of expression of LT-B in the cherry fruits was comparable to the original transgenic tomatoes. Since it was not higher, this would necessitate the consumption of more fruits to reach a desired dose. A final backcross has been made for both the non-transgenic and the transgenic material in the processing lines. Auxin sprays resulted in high percentages of fruit set, but the processing genotypes gave many puffed fruits.