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Cinatl, J., J. Cinatl, V. Gerein, B. Kornhuber, and H. W. Doerr. "The establishment and characterization of mouse L-929 cells in protein-free Eagle's Minimal Essential Medium." Journal of Biological Standardization 16, no. 4 (January 1988): 249–57. http://dx.doi.org/10.1016/0092-1157(88)90012-1.
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Hnat, Michael, and Roger E. Bawdon. "Transfer of Meropenem in the ex Vivo Human Placenta perfusion Model." Infectious Diseases in Obstetrics and Gynecology 13, no. 4 (2005): 223–27. http://dx.doi.org/10.1155/2005/961356.
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Objectives.To determine maternal-fetal transplacental passage of meropenem in the ex vivo human perfusion model.Study design.Term placentae (n= 6) were collected immediately after delivery. A single cotyledon was localized, perfused and stabilized with physiologic Eagles minimal essential medium containing 3% bovine albumin and heparin as described by Chalier (Chalier JC. Criteria for evaluating perfusion experiments and presentation results. Contrib Gynecol Obstet 1985; 13:32–39). Meropenem was added to the maternal medium in concentrations similar to maternal serum peak and trough levels, then perfused through the maternal circulation of the cotyledon. To assess transfer and accumulation, fluid aliquots from both the maternal and fetal compartments were collected over an hour at defined intervals in an open and closed system. AntipyrineC14was added to the medium in order to calculate the transport fraction and clearance indexes. Meropenem and antipyrineC14concentrations were determined by High-pressure Liquid Chromatography and liquid scintillation, respectively.Results.Mean antipyrine transport fraction was 2.33 + 0.25. Maternal and fetal mean meropenem peak concentrations were 54.3 + 3.3μg/ml and 2.2 + 0.18μg/ml, respectively. Whereas, maternal and fetal mean trough concentrations were 12.7 + 1.3μg/ml and 0.41 + 0.10μg/ml, respectively. Mean peak clearance index was 0.077 + 0.007 and the mean trough was 0.052 + 0.015. Mean accumulation for the peak and trough concentrations of meropenem were 0.9 and 2.95μg/ml, respectively.Conclusions.Transplacental passage of meropenem was incomplete in the ex vivo human placental perfusion model. Accumulation was also noted in the fetal compartment
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Seino, Satoshi, Yasuo Imoto, Tomoya Kosaka, Tomoki Nishida, Takashi Nakagawa, and Takao A. Yamamoto. "Antiviral Activity of Silver Nanoparticles Immobilized onto Textile Fabrics Synthesized by Radiochemical Process." MRS Advances 1, no. 11 (2016): 705–10. http://dx.doi.org/10.1557/adv.2016.43.
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ABSTRACTAntiviral activity of metallic Ag nanoparticles immobilized on textile fabrics were investigated. The Ag nanoparticles synthesized by radiochemical process are firmly immobilized on the surface of support textile fabrics of cotton. Small Ag particles of about 2–4 nm were observed together with relatively large particles of more than 10 nm. The Ag nanoparticles showed antiviral activity against Influenza A and Feline Calicivirus. The antiviral activity significantly depended on the concentration of the Eagle’s minimal essential medium. It was implied that the surface passivation by inhibitory agent lead to the deactivation of metallic Ag nanoparticles.
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Tung, L. C. "Immunocytochemistrical Studies on Cytoskeletal Change in Cultured Cardiomyocyte of Tilapia." Microscopy and Microanalysis 6, S2 (August 2000): 888–89. http://dx.doi.org/10.1017/s143192760003693x.
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In the present study, the myofibril regeneration in the long-term cultured fish cardiomyocytes was studied with immunocytochemistry.Adult Tilapia heart was dissociated into a single-cell suspension with collagenase and protease-minced tissue method. The culture medium was Eagle's minimal essential medium (MEM) with Earle's salts, supplemented with 10% fetal calf serum, 1 x nonessential amino acid mixture, 100 IU/ml penicillin G, and 100 μg/ml streptomycin. The cultured cells were grown in a humidified CO2 incubator at 28°Cand in a medium without glutamine for eliminating fibroblast contamination. In the initial 24 h culture, the elongated-shape cells gradually shortened from their both ends and rounded up. Over 5 to 6 days postcultivation, the cells attached to the bottom of the culture flask and began to protrude pseudopodia. The cells could not be subcultured and also proliferated indefinitely. The life span of cells in culture was 30 to 60 days.
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Heredia, Nelly Solfania, Ann Sabrina Ávila, and Luz Elena Velásquez. "In vitro culture of L3 larvae of nematodes obtained from the African giant snail Lissachatina fulica (Mollusca: Gastropoda) in Santa Fe de Antioquia." Biomédica 38 (August 1, 2018): 24–29. http://dx.doi.org/10.7705/biomedica.v38i3.3408.
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Introducción. Más de 170 municipios colombianos están invadidos por Lissachatina fulica, caracol africano que puede portar larvas de nematodos de interés en salud humana y veterinaria. Los parásitos entran al caracol huésped intermediario en el estadio de larva L1, y allí cambian a L2 y L3, formas estas capaces de infectar a vertebrados.Objetivo. Estandarizar el cultivo in vitro de las L3 portadas por especímenes de L. fulica recolectados en Santa Fe de Antioquia.Materiales y métodos. Entre julio y noviembre de 2014 se recolectaron 10 caracoles, se sacrificaron y se digirieron con ácido clorhídrico al 0,7 %. Las larvas se recuperaron mediante la técnica de Baermann; se cultivaron 36 días en los medios Schneider, mínimo esencial de Eagle modificado por Dulbecco (Dulbecco’s Modified Eagles Minimal Essential Medium, DMEM), y Roswell Park Memorial Institute (RPMI), con suero fetal bovino (SFB) al 20 % y sin este, y agua destilada con SFB al 20 %. Los medios de cultivo se cambiaron cada 36 horas. Las larvas se midieron con el microscopio utilizando reglilla ocular, y se evaluaron la supervivencia, la longitud y el ancho. Se calcularon datos estadísticos de resumen y se hicieron gráficos de cajas y bigotes, así como la prueba t de Student. El nivel de significación (p) se estableció como menor de 0,05.Resultados. El 50 % de las larvas sobrevivió, 85 % en DMEM con SFB al 20 %, el 70 % con RPMI más SFB al 20 %, el 60 % en RPMI, el 50 % en Schneider más SFB al 20 %, el 45 % en Schneider y el 40 % en DMEM. El control sobrevivió diez días. Hubo diferencias significativas entre la longitud inicial promedio de las larvas y la longitud final promedio en los medios con suplementos: inicial, 645,83 μm; final en DMEM más SFB al 20 %, 732,65 μm (p<0,001); en RPMI más SFB al 20 %, 718,79 μm (p<0,001), y en Schneider más SFB al 20 %, 696,12 μm (p<0,01). No hubo diferencias significativas entre la anchura inicial promedio, de 24,99 μm, y la final.Conclusiones. El mejor medio para cultivar las L3 de L. fulica fue el DMEM más SFB al 20 %. En la evaluación del crecimiento larval, la longitud fue más informativa que la anchura. Las larvas estudiadas no correspondieron a Angiostrongylus cantonensis, A. costaricensis ni Aelurostrongylus abstrusus.
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Mouat, M. F., A. C. Cantrell, and K. L. Manchester. "Membrane potential of rat hepatoma cells in culture: Influence of factors affecting amino acid transport." Bioscience Reports 15, no. 4 (August 1, 1995): 173–84. http://dx.doi.org/10.1007/bf01540451.
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The effect has been studied of various media, hormones and of amino acids on the membrane potential of rat hepatoma cells in culture measured by microelectrode impalement. Cells in Eagle's minimal essential medium plus 5% serum had a value which varied daily from about 5–8 mV, inside negative. The membrane potential of rat hepatocytes was measured to be 8.7 ± 0.2mV, inside negative. The membrane potential of the hepatoma cells was decreased by insulin and increased by glucagon. Membrane potential was unaffected by change of medium to Hanks' or Earle's balanced salt solutions or deprivation of serum. It was, however, reduced in cells in phosphate-buffered saline and by reduction of pH. The former effect was shown to be due to the higher [Na+] of phosphat-buffered saline as opposed to the other media. Addition of alanine, glycine, serine, proline and methylaminoisobutyrate all reduced membrane potential by 2–3 mV. Smaller decreases were seen with methionine, leucine and phenylalanine, but none with glutamine, threonine, BCH (2-aminonorborane-2-carboxylic acid) and D-alanine. The results are compared with the effects of similar conditions on aminoisobutyrate uptake. Whilst there was a correlation under some conditions there was not under others. It is concluded that for the hepatoma cells factors additional to the membrane potential must exert some influence on the capacity for amino acid transport.
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Al-Nazhan, Saad, and Afaf Al-Nasser. "Viability of Human Periodontal Ligament Fibroblasts in Tissue Culture After Exposure to Different Contact Lens Solutions." Journal of Contemporary Dental Practice 7, no. 4 (2006): 37–44. http://dx.doi.org/10.5005/jcdp-7-4-37.
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Abstract Aim The viability of the periodontal ligament (PDL) cells is critical for successful healing of replanted avulsed teeth. Viability is primarily dependent on the duration of the extra-alveolar time and storage medium used to preserve teeth. Several storage media have been suggested but milk ranks highest. It would be desirable to evaluate other media as a suitable alternative for milk. The purpose of this study was to determine the viability of human PDL fibroblasts and their morphology after storage in different types of contact lens solutions. Methods and Materials PDL fibroblasts were cultured from a healthy extracted impacted human tooth and exposed to Bausch and Lomb (Renu), Ciba Vision (Titmus), and Alcon (Opti-free) contact lens solutions. Eagle's minimal essential medium served as control. The experiment was performed in plastic tissue culture clusters containing 24 wells. The PDL fibroblasts were grown in each well for three days. On the day of the experiment the culture medium was decanted, the cells were washed with phosphate buffered saline solution (PBS), and 1 ml of the tested solution was placed in each culture well. All tissue culture clusters were incubated at 37°C in 5% CO2 and 95% air for one, four, and 24 hrs. At the end of the incubation period, the cells were fixed and prepared for scanning electron microscope (SEM) examination. Results The results indicated Renu and Opti-free solutions were superior to Titmus solution in terms of their capacity to maintain the viability and normal morphology of PDL fibroblasts. Conclusion Contact lens solution is a good storage medium to maintain the viability of PDL fibroblasts for a short-term period. Citation Al-Nazhan S, Al-Nasser A. Viability of Human Periodontal Ligament Fibroblasts in Tissue Culture After Exposure to Different Contact Lens Solutions. J Contemp Dent Pract 2006 September;(7)4:037-044.
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Kellermayer, Miklós S. Z., Tamás Henics, György Szücs, and Gerald H. Pollack. "Electron microscopic analysis of the microtubular structure of HEp-2 cells." Proceedings, annual meeting, Electron Microscopy Society of America 48, no. 3 (August 12, 1990): 470–71. http://dx.doi.org/10.1017/s0424820100159898.
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The HEp-2 cell line was first established by Moore, et al. and today it serves as a widely used subject for a large variety of microbiological and cell-biological experiments. A tubular structure of unknown origin and function has been described first in virally infected, and later in uninfected HEp-2 cells. However, this tubular structure has not been further analyzed. We performed transmission (TEM) and whole cell mount electron microscopic studies of monolayer HEp-2 cells to morphologically describe the structure and relate it to other organelles of the cell.For embedding, HEp-2 cells were grown on glass coverslips in Eagle's minimal essential medium. 24 hours after passage, three types of procedures were carried out: (1) The cells were fixed with 2.5% glutaraldehyde in 0.14 M Na-cacodylate buffer. (2) The cells were treated for 10 min. with Hank's solution containing 0.2% Brij-58 and 2.5% glutaraldehyde. Fixation was completed with 2.5% glutaraldehyde in 0.14 M Na-cacodylate. (3) Cells were treated with 0.2% Brij-58 in Hank’s solution for 10 min. Subsequently, they were fixed with 2.5% glutaraldehyde in 0.14 M Na-cacodylate.
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Tifrea, Delia F., Pooja Ralli-Jain, Sukumar Pal, and Luis M. de la Maza. "Vaccination with the Recombinant Major Outer Membrane Protein Elicits Antibodies to the Constant Domains and Induces Cross-Serovar Protection against Intranasal Challenge with Chlamydia trachomatis." Infection and Immunity 81, no. 5 (March 11, 2013): 1741–50. http://dx.doi.org/10.1128/iai.00734-12.
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ABSTRACTTo determine the ability of the major outer membrane protein (MOMP) to elicit cross-serovar protection, groups of mice were immunized by the intramuscular (i.m.) and subcutaneous (s.c.) routes with recombinant MOMP (rMOMP) fromChlamydia trachomatisserovars D (UW-3/Cx), E (Bour), or F (IC-Cal-3) orChlamydia muridarumstrain Nigg II using CpG-1826 and Montanide ISA 720 VG as adjuvants. Negative-control groups were immunized i.m. and s.c. withNeisseria gonorrhoeaerecombinant porin B (Ng-rPorB) or i.n. with Eagle's minimal essential medium (MEM-0). Following vaccination, the mice developed antibodies not only against the homologous serovar but also against heterologous serovars. The rMOMP-vaccinated animals also mounted cell-mediated immune responses as assessed by a lymphoproliferative assay. Four weeks after the last immunization, mice were challenged i.n. with 104inclusion-forming units (IFU) ofC. muridarum. The mice were weighed for 10 days and euthanized, and the number of IFU in their lungs was determined. At 10 days postinfection (p.i.), mice immunized with the rMOMP ofC. muridarumorC. trachomatisD, E, or F had lost 4%, 6%, 8%, and 8% of their initial body weight, respectively, significantly different from the negative-control groups (Ng-rPorB, 13%; MEM-0, 19%;P< 0.05). The median number of IFU recovered from the lungs of mice immunized withC. muridarumrMOMP was 0.13 × 106. The median number of IFU recovered from mice immunized with rMOMP from serovars D, E, and F were 0.38 × 106, 7.56 × 106, and 11.94 × 106IFU, respectively. All the rMOMP-immunized animals had significantly less IFU than theNg-rPorB (40 × 106)- or MEM-0 (70 × 106)-immunized mice (P< 0.05). In conclusion, vaccination with rMOMP can elicit protection against homologous and heterologousChlamydiaserovars.
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Reader, S. C. J., B. Davison, J. G. Ratcliffe, and W. R. Robertson. "Measurement of low concentrations of bovine thyrotrophin by iodide uptake and organification in porcine thyrocytes." Journal of Endocrinology 106, no. 1 (July 1985): 13—NP. http://dx.doi.org/10.1677/joe.0.1060013.
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ABSTRACT Thyrocytes isolated from porcine thyroids by mechanical and enzymatic dispersion and cultured in Eagle's minimal essential medium, supplemented with 5% (v/v) fetal calf serum, glutamine and cortisol, formed a continuous monolayer within 48 h. This monolayer was without cytochemical peroxidase and diaphorase (NADPH reoxidation) activity. In the presence of bovine thyrotrophin (bTSH; 50 mu./l) the cells developed a follicular-like architecture which was maximal at 4 days before reverting back to a uniform monolayer at 6 days. There were no detectable changes in the total DNA content over this period. The follicular structures had marked diaphorase and peroxidase activity, the latter being apically distributed. Concomitant with follicle formation bTSH induced uptake and organification of iodide presented to the cells during the last 6 h of culture. The extent of this process depended on the dose of bTSH and the duration of stimulation. The most sensitive effects for both iodide uptake and organification occurred with 1 mu. bTSH/1 and were maximal with 100 mu./l. Uptake and organification were increased 20±8-fold and 9·6±2- fold (n = 10) respectively over the control with 100 mu./l and the doses of bTSH at which a half maximal response was seen (ED50) were 15±2 and 7±1 (s.d) mu./l (n = 10) respectively. On changing the culture medium to a serum-free system using HB101 culture medium the stimulation time for the most sensitive bTSH effect was reduced to 2·5 days. Moreover the sensitivity of iodide uptake to bTSH was dramatically increased being significantly stimulated by 0·1 mu./l, saturated with 10 mu./l, and having an ED50 of 7 ± 0·2 mu. bTSH/1 (n = 7). Over the dose range 0·1–10 mu. bTSH/l intra- and interassay coefficients of variation were 7%±1·5 (n = 15) and 11% ± 2·5 (n = 15) respectively. Cyclic AMP production in cultures incubated under similar conditions (i.e. after chronic TSH stimulation) was also stimulated by bTSH doses in the range 15·6–125 mu./l. Cells stimulated for 2·5 days with dibutyryl cyclic AMP (range 1 μmol/1–2 mmol/l) also actively concentrated iodide in a dose-dependent fashion. The presence of normal human serum in the medium yielded a progressive and dose-related decrease in TSH-stimulated iodide uptake. This inhibitory effect occurred with serum concentrations as low as 0·1%. In conclusion, a porcine cell culture system is described in which iodide uptake is significantly stimulated by 0·1 mu. bTSH/l, a sensitivity inferior only to that reported with the cytochemical bioassay. J. Endocr. (1985) 106, 13–20
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Knight, Susan K., and Richard L. Knight. "Vigilance Patterns of Bald Eagles Feeding in Groups." Auk 103, no. 2 (April 1, 1986): 263–72. http://dx.doi.org/10.1093/auk/103.2.263.
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Abstract Patterns of vigilant behavior of wintering Bald Eagles (Haliaeetus leucocephalus) feeding on spawned salmon were examined in 1983-1984 on the Nooksack River in northwestern Washington. Vigilance in feeding birds has, in general, been attributed to predator detection; however, we proposed an additional function of vigilance in socially feeding birds that are vulnerable to food robbery and possible injury by conspecifics. We tested predictions of two nonexclusive hypotheses: (1) eagles look up while feeding to detect danger from humans, and (2) eagles look up while feeding to detect pirating attempts or avoid injury by conspecifics. Results suggest that the function of vigilance varies, depending on the size of the feeding group. Vigilance patterns of eagles feeding in small groups (1-4 eagles) and medium groups (5-7 eagles0 are consistent with hypothesis 1, whereas those of eagles feeding in large groups (8-14 eagles) are consistent with hypothesis 2. Eagles in small groups were more vigilant (measured as scanning time and rate of head raising) when feeding near potential danger (riverbank cover) than when far from danger. Adult eagles feeding in areas of intense human activity were more vigilant than immatures feeding at the same site and were more vigilant than both adults and immatures feeding at secluded sites. Vigilance declined as group size increased from 1 to 4 eagles, and increased as group size ranged from 8 to 14 eagles. Feeding eagles that were looking up at the time of a pirating attempt were more successful in keeping their food than eagles with their heads down. In feeding areas where human activity was minimal, eagles formed larger groups than at more disturbed sites.
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Ellsworth, J. L., C. Chandrasekaran, and A. D. Cooper. "Evidence for sterol-independent regulation of low-density lipoprotein receptor activity in Hep-G2 cells." Biochemical Journal 279, no. 1 (October 1, 1991): 175–87. http://dx.doi.org/10.1042/bj2790175.
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The relationship between the serum factor(s)-mediated induction of low-density lipoprotein (LDL) receptor activity and changes in cellular cholesterol metabolism was examined in the human hepatoma cell line Hep-G2. Relative to incubation with serum-free media [Eagle's minimal essential medium (MEM) control], short-term (less than 8 h) incubation with medium containing 15% of either calf serum (MEM + serum) or the d greater than 1.25 fraction of calf serum (MEM + d greater than 1.25) produced a time- and concentration-dependent increase in the uptake of 125I-LDL. Immunoblotting with anti-(LDL receptor) antibodies demonstrated that this was correlated with a 2-fold increase in the amount of the mature 136,000 Da LDL receptor protein in detergent-solubilized Hep-G2 cell membranes. Incubation with MEM + serum, but not MEM + d greater than 1.25, increased the efflux of radiolabelled cholesterol from Hep-G2 cells. However, the induction of 125I-LDL uptake by MEM + d greater than 1.25 (2.3-fold) and MEM + serum (2.2-fold) was virtually identical. Addition of the d less than 1.063 lipoproteins of calf serum to MEM + d greater than 1.25 at their original or three times their serum concentration decreased the induction of 125I-LDL uptake by MEM + d greater than 1.25 by only 20-30%. Together, these results suggest that the stimulation of 125I-LDL uptake was not due to the presence of high-density lipoprotein, the absence of LDL or the stimulation of cholesterol efflux. MEM + serum stimulated 125I-LDL uptake in cells cholesterol-loaded by incubation with rat very-low-density lipoprotein with beta electrophoretic mobility (beta-VLDL). Compared to incubation with the MEM control, either MEM + serum or MEM + d greater than 1.25 produced time-dependent increases in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase which also occurred in cholesterol-loaded cells. However, cholesterol biosynthesis, whether measured from 3H2O, [14C]acetate or [3H]mevalonic acid, was not increased. Incubation with MEM + serum or MEM + d greater than 1.25 did not affect [3H]oleate incorporation into cellular cholesteryl esters, hydrolysis of intracellular [3H]cholesteryl esters or the cellular mass of unesterified or esterified cholesterol. Incubation with MEM + serum or MEM + d greater than 1.25 produced a transient increase in the level of LDL receptor mRNA, reaching a maximum of 5-10-fold by 2 h and decreasing to near baseline levels by 4 h. Actinomycin D blocked the serum-factor-mediated induction of LDL receptor mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)
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Stewart, R. R., D. J. Zou, J. M. Treherne, K. Mollgard, N. R. Saunders, and J. G. Nicholls. "The intact central nervous system of the newborn opossum in long-term culture: fine structure and GABA-mediated inhibition of electrical activity." Journal of Experimental Biology 161, no. 1 (November 1, 1991): 25–41. http://dx.doi.org/10.1242/jeb.161.1.25.
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1. The entire central nervous system (CNS) of the newly born, South American opossum (Monodelphis domestica) was isolated and maintained in basal medium, Eagle's (BME) with 0.2% foetal calf serum and antibiotics. Isolated CNS preparations remained electrically excitable for up to 10 days. The fine structure of the spinal cord was normal after 5 days in culture: axons, synapses, dendrites and glia were virtually unchanged. Signs of degeneration were evident only in dorsal areas of the spinal cord, which had been denervated by removal of the dorsal root ganglia during dissection. 2. Amino acid transmitters such as glycine, glutamate, N-methyl-D-aspartate (NMDA) and gamma-aminobutyric acid (GABA), applied to the bathing fluid, rapidly and reversibly inhibited synaptic transmission in cervical segments of the spinal cord. GABA (10–100 mumol l-1) produced a dose-dependent reduction in the magnitude of ventral root responses evoked by dorsal root stimulation. GABA also inhibited synaptically activated compound action potentials produced by spinal cord stimulation. Dose-response curves for GABA obtained in different preparations were highly reproducible. 3. Both GABAA and GABAB receptors were reversibly activated by selective agonists and inhibited by specific antagonists. The actions of GABA were potentiated by benzodiazepines, competitively antagonised by bicuculline (a selective GABAA antagonist) and mimicked by muscimol (a GABAA agonist). Baclofen (a specific GABAB agonist) also inhibited electrical activity and was competitively antagonised by the GABAB antagonist, CGP 35348. 4. After 5 days of culture in BME or minimal essential medium (MEM), GABA dose-response curves were unchanged from those observed immediately after removal of the CNS. The inhibitory potency of baclofen was also unaffected by culture in BME. By contrast, after 5 days of culture in MEM, baclofen no longer inhibited electrical activity. This difference between BME and MEM could be attributed to the higher content of L-histidine in MEM. Thus, addition of 150 mumol l-1 L-histidine to BME produced similar results to culture in MEM: the inhibitory action of baclofen was virtually abolished after 3–5 days. L-Histidine had no effect on freshly dissected preparations. Chronic application of L-histidine did not affect glycine or glutamate responses after 5 days. Addition of D-histidine or other amino acids, such as arginine, to BME did not abolish the responses to baclofen. 5. These results show that the isolated CNS of the newborn opossum survives well in long-term culture and that it provides a useful preparation to study receptor development and plasticity of an intact mammalian CNS in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)
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Rubessa, M., R. Rocha, L. Lima, R. Winters, J. R. Figueiredo, and M. B. Wheeler. "136 COMPARISON OF NCSU-23 AND ALPHA-MINIMAL ESSENTIAL MEDIA IN THE DEVELOPMENT OF ISOLATED PORCINE PREANTRAL FOLLICLES IN VITRO." Reproduction, Fertility and Development 28, no. 2 (2016): 198. http://dx.doi.org/10.1071/rdv28n2ab136.
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To develop a preantral follicular culture system that will support follicular growth and result in fertilizable oocytes, we conducted an experiment designed to determine the best medium for culture. In our preliminary experiment, we compared 2 common base media used for porcine oocytes: α-minimal essential medium and NCSU-23. Ovaries were collected from prepubertal gilts at a local abattoir and transported to the laboratory in saline solution (0.9% NaCl) maintained at 30–35°C. The ovaries were cut into small pieces (1–3 mm), and preantral follicles were isolated mechanically. Preantral follicles from 280 to 300 μm in diameter were collected into a small dish containing medium TCM199 (Lonza 12–117F) supplemented with 5% fetal bovine serum. The follicles were transferred from the collecting medium to the culture medium that consisted of base medium (NCSU23 or α-minimal essential medium) supplemented with 3.5 μg mL–1 of insulin, 10 μg mL–1 of transferrin, 100 μg mL–1 of l-ascorbic acid, 7.5% porcine serum, and 1.5 ng mL–1 of FSH. The follicles were randomly distributed to the different experimental treatments and cultured for 6 days in 24-well cell culture plates, with 3 follicles per well in 280 μL of culture medium. The culture was carried out at 38.5°C in 5% CO2 in air. Culture medium was changed every 2 days with freshly prepared medium. The diameters of follicles were measured every 2 days, and each follicle was photographed and evaluated at 20× magnification. Forty-two follicles per group were analysed and collected in 4 replicates. Data were statistically analysed with ANOVA using the Generalized Linear Model (GLM) procedure (SPSS, version 18, SPSS Inc., Chicago, IL, USA), where the independent variable was the sample (group and day of culture). Tukey's post hoc test was used to perform multiple comparisons; the α level was set at 0.05. All data were expressed as quadratic means with standard error of the means. Only the antrum formation was evaluated by chi-square test. The results, reported in Table 1, show that there was no statistical differences between follicle size between NCSU23 or α-minimal essential media, but at Day 6 there was a positive trend (P = 0.08). Otherwise, when we compared the size inside the groups, we observed that the preantral follicles grew more in α-minimal essential media than in NCSU23. The percentages of antrum formation were 65 v. 76% (NCSU23 and α-minimal essential media, respectively). These results support the use of α-minimal essential media because it had a positive effect on the antrum formation, and that after Day 4 some follicles could undergo a regression phase. Future studies will be necessary to evaluate the molecular status and the hormone production. Table 1.Follicle size (μm) from Day 0 to Day 6
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Luo, Jun, Yasuhiro Matsuo, Galina Gulis, Haylee Hinz, Jana Patton-Vogt, and Stevan Marcus. "Phosphatidylethanolamine Is Required for Normal Cell Morphology and Cytokinesis in the Fission Yeast Schizosaccharomyces pombe." Eukaryotic Cell 8, no. 5 (March 13, 2009): 790–99. http://dx.doi.org/10.1128/ec.00029-09.
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ABSTRACT To investigate the contributions of phosphatidylethanolamine to the growth and morphogenesis of the fission yeast Schizosaccharomyces pombe, we have characterized three predicted genes in this organism, designated psd1, psd2, and psd3, encoding phosphatidylserine decarboxylases, which catalyze the conversion of phosphatidylserine to phosphatidylethanolamine in both eukaryotic and prokaryotic organisms. S. pombe mutants carrying deletions in any one or two psd genes are viable in complex rich medium and synthetic defined minimal medium. However, mutants carrying deletions in all three psd genes (psd1-3Δ mutants) grow slowly in rich medium and are inviable in minimal medium, indicating that the psd1 to psd3 gene products share overlapping essential cellular functions. Supplementation of growth media with ethanolamine, which can be converted to phosphatidylethanolamine by the Kennedy pathway, restores growth to psd1-3Δ cells in minimal medium, indicating that phosphatidylethanolamine is essential for S. pombe cell growth. psd1-3Δ cells produce lower levels of phosphatidylethanolamine than wild-type cells, even in medium supplemented with ethanolamine, indicating that the Kennedy pathway can only partially compensate for the loss of phosphatidylserine decarboxylase activity in S. pombe. psd1-3Δ cells appear morphologically indistinguishable from wild-type S. pombe cells in medium supplemented with ethanolamine, but when cultured in nonsupplemented medium, they produce high frequencies of abnormally shaped cells as well as cells exhibiting severe septation defects, including multiple, mispositioned, deformed, and misoriented septa. Our results demonstrate that phosphatidylethanolamine is essential for cell growth and for normal cytokinesis and cellular morphogenesis in S. pombe, and they illustrate the usefulness of this model eukaryote for investigating potentially conserved biological and molecular functions of phosphatidylethanolamine.
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Meuleman, N., T. Tondreau, D. Bron, and L. Lagneaux. "Human marrow mesenchymal stem cell culture: serum-free medium allows better expansion than classical alpha-minimal essential medium (MEM)." European Journal of Haematology 78, no. 2 (January 5, 2007): 168. http://dx.doi.org/10.1111/j.1600-0609.2006.00795.x.
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Hasona, Adnan, Youngnyun Kim, F. G. Healy, L. O. Ingram, and K. T. Shanmugam. "Pyruvate Formate Lyase and Acetate Kinase Are Essential for Anaerobic Growth of Escherichia coli on Xylose." Journal of Bacteriology 186, no. 22 (November 15, 2004): 7593–600. http://dx.doi.org/10.1128/jb.186.22.7593-7600.2004.
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ABSTRACT During anaerobic growth of bacteria, organic intermediates of metabolism, such as pyruvate or its derivatives, serve as electron acceptors to maintain the overall redox balance. Under these conditions, the ATP needed for cell growth is derived from substrate-level phosphorylation. In Escherichia coli, conversion of glucose to pyruvate yields 2 net ATPs, while metabolism of a pentose, such as xylose, to pyruvate only yields 0.67 net ATP per xylose due to the need for one (each) ATP for xylose transport and xylulose phosphorylation. During fermentative growth, E. coli produces equimolar amounts of acetate and ethanol from two pyruvates, and these reactions generate one additional ATP from two pyruvates (one hexose equivalent) while still maintaining the overall redox balance. Conversion of xylose to acetate and ethanol increases the net ATP yield from 0.67 to 1.5 per xylose. An E. coli pfl mutant lacking pyruvate formate lyase cannot convert pyruvate to acetyl coenzyme A, the required precursor for acetate and ethanol production, and could not produce this additional ATP. E. coli pfl mutants failed to grow under anaerobic conditions in xylose minimal medium without any negative effect on their survival or aerobic growth. An ackA mutant, lacking the ability to generate ATP from acetyl phosphate, also failed to grow in xylose minimal medium under anaerobic conditions, confirming the need for the ATP produced by acetate kinase for anaerobic growth on xylose. Since arabinose transport by AraE, the low-affinity, high-capacity, arabinose/H+ symport, conserves the ATP expended in pentose transport by the ABC transporter, both pfl and ackA mutants grew anaerobically with arabinose. AraE-based xylose transport, achieved after constitutively expressing araE, also supported the growth of the pfl mutant in xylose minimal medium. These results suggest that a net ATP yield of 0.67 per pentose is only enough to provide for maintenance energy but not enough to support growth of E. coli in minimal medium. Thus, pyruvate formate lyase and acetate kinase are essential for anaerobic growth of E. coli on xylose due to energetic constraints.
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Scollan, N. D., B. W. McBride, N. S. Jessop, G. F. Allan, and J. D. Oldham. "The influence of sex, feeding and genotype on sodium-potassium ATPase activity in erythrocytes from young dairy calves." Canadian Journal of Animal Science 73, no. 4 (December 1, 1993): 981–85. http://dx.doi.org/10.4141/cjas93-100.
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The activity of sodium-potassium ATPase was assessed in erythrocytes from calves of both sexes with either low or high pedigree indices for milk yield. Animals were sampled in both fasted and fed states, and enzyme activity was determined in minimal essential medium ± fetal calf serum. Genotype did not influence enzyme activity, but effects of sex, feeding level and incubation medium were observed. Key words: Calves (dairy), genotype, sodium-potassium ATPase, erythrocyte, sex, incubation medium
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Knels, Lilla, Doris Goetze, Katrin Engelmann, and Monika Valtink. "Serum-free medium and hydroxyethyl starch supports cell survival better than Minimal Essential Medium and dextran in organ-cultured mouse corneas." British Journal of Ophthalmology 98, no. 12 (October 6, 2014): 1744–50. http://dx.doi.org/10.1136/bjophthalmol-2014-305450.
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Joyce, Andrew R., Jennifer L. Reed, Aprilfawn White, Robert Edwards, Andrei Osterman, Tomoya Baba, Hirotada Mori, Scott A. Lesely, Bernhard Ø. Palsson, and Sanjay Agarwalla. "Experimental and Computational Assessment of Conditionally Essential Genes in Escherichia coli." Journal of Bacteriology 188, no. 23 (September 29, 2006): 8259–71. http://dx.doi.org/10.1128/jb.00740-06.
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ABSTRACT Genome-wide gene essentiality data sets are becoming available for Escherichia coli, but these data sets have yet to be analyzed in the context of a genome scale model. Here, we present an integrative model-driven analysis of the Keio E. coli mutant collection screened in this study on glycerol-supplemented minimal medium. Out of 3,888 single-deletion mutants tested, 119 mutants were unable to grow on glycerol minimal medium. These conditionally essential genes were then evaluated using a genome scale metabolic and transcriptional-regulatory model of E. coli, and it was found that the model made the correct prediction in ∼91% of the cases. The discrepancies between model predictions and experimental results were analyzed in detail to indicate where model improvements could be made or where the current literature lacks an explanation for the observed phenotypes. The identified set of essential genes and their model-based analysis indicates that our current understanding of the roles these essential genes play is relatively clear and complete. Furthermore, by analyzing the data set in terms of metabolic subsystems across multiple genomes, we can project which metabolic pathways are likely to play equally important roles in other organisms. Overall, this work establishes a paradigm that will drive model enhancement while simultaneously generating hypotheses that will ultimately lead to a better understanding of the organism.
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Sohlenkamp, Christian, Karel E. E. de Rudder, and Otto Geiger. "Phosphatidylethanolamine Is Not Essential for Growth of Sinorhizobium meliloti on Complex Culture Media." Journal of Bacteriology 186, no. 6 (March 15, 2004): 1667–77. http://dx.doi.org/10.1128/jb.186.6.1667-1677.2004.
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ABSTRACT In addition to phosphatidylglycerol (PG), cardiolipin (CL), and phosphatidylethanolamine (PE), Sinorhizobium meliloti also possesses phosphatidylcholine (PC) as a major membrane lipid. The biosynthesis of PC in S. meliloti can occur via two different routes, either via the phospholipid N-methylation pathway, in which PE is methylated three times in order to obtain PC, or via the phosphatidylcholine synthase (Pcs) pathway, in which choline is condensed with CDP-diacylglycerol to obtain PC directly. Therefore, for S. meliloti, PC biosynthesis can occur via PE as an intermediate or via a pathway that is independent of PE, offering the opportunity to uncouple PC biosynthesis from PE biosynthesis. In this study, we investigated the first step of PE biosynthesis in S. meliloti catalyzed by phosphatidylserine synthase (PssA). A sinorhizobial mutant lacking PE was complemented with an S. meliloti gene bank, and the complementing DNA was sequenced. The gene coding for the sinorhizobial phosphatidylserine synthase was identified, and it belongs to the type II phosphatidylserine synthases. Inactivation of the sinorhizobial pssA gene leads to the inability to form PE, and such a mutant shows a greater requirement for bivalent cations than the wild type. A sinorhizobial PssA-deficient mutant possesses only PG, CL, and PC as major membrane lipids after growth on complex medium, but it grows nearly as well as the wild type under such conditions. On minimal medium, however, the PE-deficient mutant shows a drastic growth phenotype that can only partly be rescued by choline supplementation. Therefore, although choline permits Pcs-dependent PC formation in the mutant, it does not restore wild-type-like growth in minimal medium, suggesting that it is not only the lack of PC that leads to this drastic growth phenotype.
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Shi, Wanliang. "Activity of Pyrazinamide against Mycobacterium tuberculosis at Neutral pH in PZA-S1 Minimal Medium." Antibiotics 10, no. 8 (July 26, 2021): 909. http://dx.doi.org/10.3390/antibiotics10080909.
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Susceptibility testing of tuberculosis (TB) drugs on Mycobacterium tuberculosis is essential for the rapid detection of strains resistant to the drugs, providing the patient with effective treatment, and preventing the spread of drug-resistant TB strains. Pyrazinamide (PZA) is one of the first-line agents used for the treatment of TB. However, current phenotypic PZA susceptibility testing is unreliable due to its performance in acidic pH conditions. The aims of this study were to develop minimal media to determine the activity of PZA at a neutral pH at 37 °C to avoid problems caused by an acidic pH, which is currently used in PZA susceptibility tests, and to identify PZA-resistant M. tuberculosis in media with reproducibility and accuracy. Different minimal media were used to determine the activity of PZA using the broth microdilution method with M. tuberculosis H37Ra as the reference strain. The PZA-S1 minimal medium was proposed as the most suitable medium. PZA inhibited the growth of M. tuberculosis in PZA-S1 at a neutral pH of 6.8, which is the optimal pH for M. tuberculosis growth. Moreover, PZA showed activity at a neutral pH on a PZA-S1 agar plate when using the disk diffusion method. PZA-resistant M. tuberculosis could be identified at a neutral pH in PZA-S1 minimal medium. This study establishes valuable information regarding the testing of PZA’s susceptibility in relation to M. tuberculosis at a neutral pH of 6.8 with reliability and accuracy in clinical settings.
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Miethke, Marcus, Helga Westers, Evert-Jan Blom, Oscar P. Kuipers, and Mohamed A. Marahiel. "Iron Starvation Triggers the Stringent Response and Induces Amino Acid Biosynthesis for Bacillibactin Production in Bacillus subtilis." Journal of Bacteriology 188, no. 24 (September 29, 2006): 8655–57. http://dx.doi.org/10.1128/jb.01049-06.
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ABSTRACT Iron deprivation in bacteria causes the derepression of genes controlled by the ferric uptake regulator (Fur). The present microarray analysis of iron-starved Bacillus subtilis cells grown in minimal medium unveils additional physiological effects on a large number of genes linked to stringent-response regulation and to genes involved in amino acid biosynthesis associated with pathways essential for bacillibactin production.
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King, W. A., P. Guay, and L. Picard. "A cytogenetical study of 7-day-old bovine embryos of poor morphological quality." Genome 29, no. 1 (February 1, 1987): 160–64. http://dx.doi.org/10.1139/g87-027.
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Seven-day-old embryos were collected from Canadian Holstein and Ayrshire heifers after superovulation with pregnant mare's serum gonadotropin (PMSG) or follicle-stimulating hormone (FSH). A total of 103 morphologically abnormal (type C) and 23 morphologically normal (type A) embryos were cytogenetically analyzed after 4, 20–24, or 44–48 h of culture in enriched phosphate-buffered saline or Eagles minimum essential medium. Twenty-one of 23 (91.3%) type A and 75 of 103 (72.8%) type C embryos had cells in metaphase. Among the 21 type C embryos produced by PMSG stimulation, 17 (80.9%) could be analyzed: 6 were mixoploid (two 2n/3n, three 2n/4n, one 2n/6n), 2 were aneuploid (61 XXY), and 9 were diploid. Among the 82 type C embryos produced by FSH stimulation, 58 (70.7%) could be analyzed: 6 were mixoploid (one n/2n, one 2n/3n, three 2n/4n, one 2n/4n/8n), 2 were polyploid (4n), and 50 were diploid. No abnormalities were observed in the type A embryos. Key words: bovine embryo, chromosome, mixoploid, aneuploid.
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Zhu, Jiajun, Simon Schwörer, Mirela Berisa, Yeon Ju Kyung, Keun Woo Ryu, Junmei Yi, Xuejun Jiang, Justin R. Cross, and Craig B. Thompson. "Mitochondrial NADP(H) generation is essential for proline biosynthesis." Science 372, no. 6545 (April 22, 2021): 968–72. http://dx.doi.org/10.1126/science.abd5491.
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The coenzyme nicotinamide adenine dinucleotide phosphate (NADP+) and its reduced form (NADPH) regulate reductive metabolism in a subcellularly compartmentalized manner. Mitochondrial NADP(H) production depends on the phosphorylation of NAD(H) by NAD kinase 2 (NADK2). Deletion of NADK2 in human cell lines did not alter mitochondrial folate pathway activity, tricarboxylic acid cycle activity, or mitochondrial oxidative stress, but rather led to impaired cell proliferation in minimal medium. This growth defect was rescued by proline supplementation. NADK2-mediated mitochondrial NADP(H) generation was required for the reduction of glutamate and hence proline biosynthesis. Furthermore, mitochondrial NADP(H) availability determined the production of collagen proteins by cells of mesenchymal lineage. Thus, a primary function of the mitochondrial NADP(H) pool is to support proline biosynthesis for use in cytosolic protein synthesis.
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Scarcia, P., L. Palmieri, G. Agrimi, F. Palmieri, and H. Rottensteiner. "Three mitochondrial transporters of Saccharomyces cerevisiae are essential for ammonium fixation and lysine biosynthesis in synthetic minimal medium." Molecular Genetics and Metabolism 122, no. 3 (November 2017): 54–60. http://dx.doi.org/10.1016/j.ymgme.2017.07.004.
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Oedekoven, Caroline Anna, Miriam Belmonte, Evangelia Diamanti, Hugo Bastos, Daniel Bode, Maike Paramor, and David Kent. "Retention of Single Long Term Hematopoietic Stem Cells in Minimal Culture Medium Reveals Essential Components of HSC Function." Experimental Hematology 64 (August 2018): S90. http://dx.doi.org/10.1016/j.exphem.2018.06.109.
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Hartman, Hassan B., David A. Fell, Sergio Rossell, Peter Ruhdal Jensen, Martin J. Woodward, Lotte Thorndahl, Lotte Jelsbak, et al. "Identification of potential drug targets in Salmonella enterica sv. Typhimurium using metabolic modelling and experimental validation." Microbiology 160, no. 6 (June 1, 2014): 1252–66. http://dx.doi.org/10.1099/mic.0.076091-0.
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Salmonella enterica sv. Typhimurium is an established model organism for Gram-negative, intracellular pathogens. Owing to the rapid spread of resistance to antibiotics among this group of pathogens, new approaches to identify suitable target proteins are required. Based on the genome sequence of S. Typhimurium and associated databases, a genome-scale metabolic model was constructed. Output was based on an experimental determination of the biomass of Salmonella when growing in glucose minimal medium. Linear programming was used to simulate variations in the energy demand while growing in glucose minimal medium. By grouping reactions with similar flux responses, a subnetwork of 34 reactions responding to this variation was identified (the catabolic core). This network was used to identify sets of one and two reactions that when removed from the genome-scale model interfered with energy and biomass generation. Eleven such sets were found to be essential for the production of biomass precursors. Experimental investigation of seven of these showed that knockouts of the associated genes resulted in attenuated growth for four pairs of reactions, whilst three single reactions were shown to be essential for growth.
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Cavalcante, A. Y. P., B. B. Gouveia, R. S. Barberino, T. L. B. G. Lins, L. P. Santos, R. J. S. Gonçalves, J. J. H. Celestino, and M. H. T. Matos. "Kit ligand promotes the transition from primordial to primary follicles afterin vitroculture of ovine ovarian tissue." Zygote 24, no. 4 (October 27, 2015): 578–82. http://dx.doi.org/10.1017/s0967199415000556.
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SummaryThis study evaluated the effects of kit ligand (KL) on the morphology and development of ovine preantral follicles (fresh control) and after 7 days ofin vitroculture in α-Minimal Essential Medium (α-MEM; control medium) or the presence of KL (1, 10, 50, 100 or 200 ng/ml). There was an increase in the percentage of primary follicles at the concentration of 100 ng/ml KL, compared with the fresh control, control medium (α-MEM) and the other KL concentrations. Follicle diameter was significantly higher than the control medium only at concentrations of 50 and 100 ng/ml KL. In conclusion, 100 ng/ml KL promoted the transition from primordial to primary follicles (follicular activation) afterin vitroculture of ovine ovarian tissue.
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Thomaides, Helena B., Ella J. Davison, Lisa Burston, Hazel Johnson, David R. Brown, Alison C. Hunt, Jeffery Errington, and Lloyd Czaplewski. "Essential Bacterial Functions Encoded by Gene Pairs." Journal of Bacteriology 189, no. 2 (November 17, 2006): 591–602. http://dx.doi.org/10.1128/jb.01381-06.
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ABSTRACT To address the need for new antibacterials, a number of bacterial genomes have been systematically disrupted to identify essential genes. Such programs have focused on the disruption of single genes and may have missed functions encoded by gene pairs or multiple genes. In this work, we hypothesized that we could predict the identity of pairs of proteins within one organism that have the same function. We identified 135 putative protein pairs in Bacillus subtilis and attempted to disrupt the genes forming these, singly and then in pairs. The single gene disruptions revealed new genes that could not be disrupted individually and other genes required for growth in minimal medium or for sporulation. The pairwise disruptions revealed seven pairs of proteins that are likely to have the same function, as the presence of one protein can compensate for the absence of the other. Six of these pairs are essential for bacterial viability and in four cases show a pattern of species conservation appropriate for potential antibacterial development. This work highlights the importance of combinatorial studies in understanding gene duplication and identifying functional redundancy.
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Inaoka, Takashi, Yoshinobu Matsumura, and Tetsuaki Tsuchido. "SodA and Manganese Are Essential for Resistance to Oxidative Stress in Growing and Sporulating Cells ofBacillus subtilis." Journal of Bacteriology 181, no. 6 (March 15, 1999): 1939–43. http://dx.doi.org/10.1128/jb.181.6.1939-1943.1999.
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ABSTRACT We constructed a sodA-disrupted mutant ofBacillus subtilis 168, BK1, by homologous recombination. The mutant was not able to grow in minimal medium without Mn(II). The spore-forming ability of strain BK1 was significantly lower in Mn(II)-depleted medium than that of the wild-type strain. These deleterious effects caused by the sodA mutation were reversed when an excess of Mn(II) was used to supplement the medium. Moreover, the growth inhibition by superoxide generators in strain BK1 and its parent strain was also reversed by the supplementation with excess Mn(II). We therefore estimated the Mn-dependent superoxide-scavenging activity in BK1 cells. Whereas BK1 cells have no detectable superoxide dismutase (Sod) on native gel, the superoxide-scavenging activity in crude extracts of BK1 cells grown in Mn(II)-supplemented LB medium (10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl per liter) was significantly detected by the modified Sod assay method without using EDTA. The results obtained suggest that Mn, as a free ion or a complex with some cellular component, can catalyze the elimination of superoxide and that both SodA and Mn(II) are involved not only in the superoxide resistance of vegetative cells but also in sporulation.
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Reuß, Daniel R., Fabian M. Commichau, Jan Gundlach, Bingyao Zhu, and Jörg Stülke. "The Blueprint of a Minimal Cell: MiniBacillus." Microbiology and Molecular Biology Reviews 80, no. 4 (September 28, 2016): 955–87. http://dx.doi.org/10.1128/mmbr.00029-16.
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SUMMARYBacillus subtilisis one of the best-studied organisms. Due to the broad knowledge and annotation and the well-developed genetic system, this bacterium is an excellent starting point for genome minimization with the aim of constructing a minimal cell. We have analyzed the genome ofB. subtilisand selected all genes that are required to allow life in complex medium at 37°C. This selection is based on the known information on essential genes and functions as well as on gene and protein expression data and gene conservation. The list presented here includes 523 and 119 genes coding for proteins and RNAs, respectively. These proteins and RNAs are required for the basic functions of life in information processing (replication and chromosome maintenance, transcription, translation, protein folding, and secretion), metabolism, cell division, and the integrity of the minimal cell. The completeness of the selected metabolic pathways, reactions, and enzymes was verified by the development of a model of metabolism of the minimal cell. A comparison of theMiniBacillusgenome to the recently reported designed minimal genome ofMycoplasma mycoidesJCVI-syn3.0 indicates excellent agreement in the information-processing pathways, whereas each species has a metabolism that reflects specific evolution and adaptation. The blueprint ofMiniBacilluspresented here serves as the starting point for a successive reduction of theB. subtilisgenome.
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Kurokawa, Masaomi, and Bei-Wen Ying. "Experimental Challenges for Reduced Genomes: The Cell Model Escherichia coli." Microorganisms 8, no. 1 (December 18, 2019): 3. http://dx.doi.org/10.3390/microorganisms8010003.
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Genome reduction, as a top-down approach to obtain the minimal genetic information essential for a living organism, has been conducted with bacterial cells for decades. The most popular and well-studied cell models for genome reduction are Escherichia coli strains. As the previous literature intensively introduced the genetic construction and application of the genome-reduced Escherichia coli strains, the present review focuses the design principles and compares the reduced genome collections from the specific viewpoint of growth, which represents a fundamental property of living cells and is an important feature for their biotechnological application. For the extended simplification of the genomic sequences, the approach of experimental evolution and concern for medium optimization are newly proposed. The combination of the current techniques of genomic construction and the newly proposed methodologies could allow us to acquire growing Escherichia coli cells carrying the extensively reduced genome and to address the question of what the minimal genome essential for life is.
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Kimura, Keitarou, Lam-Son Phan Tran, and Yoshifumi Itoh. "Roles and regulation of the glutamate racemase isogenes, racE and yrpC, in Bacillus subtilis." Microbiology 150, no. 9 (September 1, 2004): 2911–20. http://dx.doi.org/10.1099/mic.0.27045-0.
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Many bacteria, including Escherichia coli, have a unique gene that encodes glutamate racemase. This enzyme catalyses the formation of d-glutamate, which is necessary for cell wall peptidoglycan synthesis. However, Bacillus subtilis has two glutamate racemase genes, named racE and yrpC. Since racE appears to be indispensable for growth in rich medium, the role of yrpC in d-amino acid synthesis is vague. Experiments with racE- and yrpC-knockout mutants confirmed that racE is essential for growth in rich medium but showed that this gene was dispensable for growth in minimal medium, where yrpC executes the anaplerotic role of racE. LacZ fusion assays demonstrated that racE was expressed in both types of media but yrpC was expressed only in minimal medium, which accounted for the absence of yrpC function in rich medium. Neither racE nor yrpC was required for B. subtilis cells to synthesize poly-γ-dl-glutamate (γ-PGA), a capsule polypeptide of d- and l-glutamate linked through a γ-carboxylamide bond. Wild-type cells degraded the capsule during the late stationary phase without accumulating the degradation products, d-glutamate and l-glutamate, in the medium. In contrast, racE or yrpC mutant cells accumulated significant amounts of d- but not l-glutamate. Exogenous d-glutamate utilization was somewhat defective in the mutants and the double mutation of race and yrpc severely impaired d-amino acid utilization. Thus, both racemase genes appear necessary to complete the catabolism of exogenous d-glutamate generated from γ-PGA.
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Seipke, Ryan F., and Rosemary Loria. "Hopanoids Are Not Essential for Growth of Streptomyces scabies 87-22." Journal of Bacteriology 191, no. 16 (June 5, 2009): 5216–23. http://dx.doi.org/10.1128/jb.00390-09.
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ABSTRACT Hopanoids are triterpenoic, pentacyclic compounds that are structurally similar to sterols, which are required for normal cell function in eukaryotes. Hopanoids are thought to be an important component of bacterial cell membranes because they control membrane fluidity and diminish passive diffusion of ions, and a few taxons modulate their hopanoid content in response to environmental stimuli. However, to our knowledge, mutational studies to assess the importance of hopanoids in bacterial physiology have never been performed. Genome sequencing of the potato scab pathogen, Streptomyces scabies 87-22, revealed a hopanoid biosynthetic gene cluster (HBGC) that is predicted to synthesize hopene and aminotrihydroxybacteriohopane products. Hopene was produced by fully sporulated cultures of S. scabies on solid ISP4 (International Streptomyces Project 4) medium as well as by submerged mycelia grown in liquid minimal medium. The elongated hopanoid aminotrihydroxybacteriohopane was not detected under either growth condition. Transcription of the S. scabies HBGC was upregulated during aerial growth, which suggests a link between hopanoid production and morphological development. Functional analysis of the S. scabies Δhop615-1 and Δhop615-7 mutant strains, the first hopanoid mutants created in any bacterial taxon, revealed that hopanoids are not required for normal growth or for tolerance of ethanol, osmotic and oxidative stress, high temperature, or low pH. This suggests that hopanoids are not essential for normal streptomycete physiology.
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Lupo, Domenico, and Robin Ghosh. "The Reaction Center H Subunit Is Not Required for High Levels of Light-Harvesting Complex 1 in Rhodospirillum rubrum Mutants." Journal of Bacteriology 186, no. 17 (September 1, 2004): 5585–95. http://dx.doi.org/10.1128/jb.186.17.5585-5595.2004.
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ABSTRACT The gene (puhA) encoding the H subunit of the reaction center (RC) was deleted by site-directed interposon mutagenesis by using a kanamycin resistance cassette lacking transcriptional terminators to eliminate polar effects in both the wild-type strain Rhodospirillum rubrum S1 and the carotenoid-less strain R. rubrum G9. The puhA interposon mutants were incapable of photoheterotrophic growth but grew normally under aerobic chemoheterotrophic conditions. Absorption spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the RCs were absent. In minimal medium and also in modified medium containing succinate and fructose, the light-harvesting 1 complex (LH1) levels of the S1-derived mutants were about 70 to 100% of the wild-type levels in the same media. The correct assembly of LH1 in the membrane and the pigment-pigment interaction were confirmed by near-infrared circular dichroism spectroscopy. LH1 formation was almost absent when the carotenoid-less G9-derived puhA mutants were grown in standard minimal medium, suggesting that carotenoids may stabilize LH1. In the fructose-containing medium, however, the LH1 levels of the G9 mutants were 70 to 100% of the parental strain levels. Electron micrographs of thin sections of R. rubrum revealed photosynthetic membranes in all mutants grown in succinate-fructose medium. These studies indicate that the H subunit of the RC is necessary neither for maximal formation of LH1 nor for photosynthetic membrane formation but is essential for functional RC assembly.
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Furman, Eugeny L., Arcady B. Finkelstein, and Maxim L. Cherny. "The Anisotropy of Replicated Aluminum Foams." Advances in Materials Science and Engineering 2014 (2014): 1–6. http://dx.doi.org/10.1155/2014/230767.
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The replication casting process gives the open-cell aluminum foams that can be used in many industrial applications as well as in filtering technology. The essential requirement for filters is the uniformity of filtering degree which is defined by the minimal pore size. However the structure of replication castings is often inhomogeneous and the minimal pore radius is decreasing in the direction of melt infiltration. The objective of this investigation is to study the dynamics of melt impregnation of the porous medium by vacuum suction to identify the possibility of reducing the anisotropy. Theoretical data illustrate the processes at the boundary between melt and gas medium. The experiments were carried out using the replication aluminum samples produced according to commercial technology. It was found that the permeability coefficient varies throughout the height of castings. A method for estimation of pressure on the line of melt movement was proposed. The resistance of NaCl layer and circular vents of the mold causes the inhomogeneity of castings. Finally the ways of minimizing the anisotropy were offered.
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Sambamoorthy, Gayathri, and Karthik Raman. "MinReact: a systematic approach for identifying minimal metabolic networks." Bioinformatics 36, no. 15 (May 14, 2020): 4309–15. http://dx.doi.org/10.1093/bioinformatics/btaa497.
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Abstract Motivation Genome-scale metabolic models are widely constructed and studied for understanding various design principles underlying metabolism, predominantly redundancy. Metabolic networks are highly redundant and it is possible to minimize the metabolic networks into smaller networks that retain the functionality of the original network. Results Here, we establish a new method, MinReact that systematically removes reactions from a given network to identify minimal reactome(s). We show that our method identifies smaller minimal reactomes than existing methods and also scales well to larger metabolic networks. Notably, our method exploits known aspects of network structure and redundancy to identify multiple minimal metabolic networks. We illustrate the utility of MinReact by identifying multiple minimal networks for 77 organisms from the BiGG database. We show that these multiple minimal reactomes arise due to the presence of compensatory reactions/pathways. We further employed MinReact for a case study to identify the minimal reactomes of different organisms in both glucose and xylose minimal environments. Identification of minimal reactomes of these different organisms elucidate that they exhibit varying levels of redundancy. A comparison of the minimal reactomes on glucose and xylose illustrates that the differences in the reactions required to sustain growth on either medium. Overall, our algorithm provides a rapid and reliable way to identify minimal subsets of reactions that are essential for survival, in a systematic manner. Availability and implementation Algorithm is available from https://github.com/RamanLab/MinReact. Supplementary information Supplementary data are available at Bioinformatics online.
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Deng, Yinyue, Calvin Boon, Leo Eberl, and Lian-Hui Zhang. "Differential Modulation of Burkholderia cenocepacia Virulence and Energy Metabolism by the Quorum-Sensing Signal BDSF and Its Synthase." Journal of Bacteriology 191, no. 23 (October 2, 2009): 7270–78. http://dx.doi.org/10.1128/jb.00681-09.
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ABSTRACT Burkholderia cenocepacia produces the molecule cis-2-dodecenoic acid (BDSF), which was previously shown to play a role in antagonism against the fungal pathogen Candida albicans by interfering with its morphological transition. In this study, we show that production of BDSF is under stringent transcriptional control and the molecule accumulates in a cell density-dependent manner, typically found with quorum-sensing (QS) signals. B. cenocepacia mutant strain J2315 with a deleted Bcam0581 gene, which encodes an enzyme essential for BDSF production, exhibited a growth defect in minimal medium but not in rich medium, decreased virulence gene expression, and attenuated virulence in a zebrafish infection model. Exogenous addition of BDSF to the mutant rescues virulence gene expression but fails to restore its growth defect in minimal medium. We show that Bcam0581, but not BDSF, is associated with B. cenocepacia ATP biogenesis. We also provide evidence that some of the BDSF-regulated genes are also controlled by the acyl-homoserine-lactone-dependent QS system and are thus coregulated by two cell-to-cell signaling systems. These data demonstrate that in addition to the role in cross-kingdom signal interference, BDSF and its synthase are also important for the virulence and physiology of B. cenocepacia.
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García-Estepa, Raúl, Montserrat Argandoña, Mercedes Reina-Bueno, Nieves Capote, Fernando Iglesias-Guerra, Joaquín J. Nieto, and Carmen Vargas. "The ectD Gene, Which Is Involved in the Synthesis of the Compatible Solute Hydroxyectoine, Is Essential for Thermoprotection of the Halophilic Bacterium Chromohalobacter salexigens." Journal of Bacteriology 188, no. 11 (June 1, 2006): 3774–84. http://dx.doi.org/10.1128/jb.00136-06.
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ABSTRACT The halophilic bacterium Chromohalobacter salexigens synthesizes and accumulates compatible solutes in response to salt and temperature stress. 13C-nuclear magnetic resonance analysis of cells grown in minimal medium at the limiting temperature of 45°C revealed the presence of hydroxyectoine, ectoine, glutamate, trehalose (not present in cells grown at 37°C), and the ectoine precursor, Nγ-acetyldiaminobutyric acid. High-performance liquid chromatography analyses showed that the levels of ectoine and hydroxyectoine were maximal during the stationary phase of growth. Accumulation of hydroxyectoine was up-regulated by salinity and temperature, whereas accumulation of ectoine was up-regulated by salinity and down-regulated by temperature. The ectD gene, which is involved in the conversion of ectoine to hydroxyectoine, was isolated as part of a DNA region that also contains a gene whose product belongs to the AraC-XylS family of transcriptional activators. Orthologs of ectD were found within the sequenced genomes of members of the proteobacteria, firmicutes, and actinobacteria, and their products were grouped into the ectoine hydroxylase subfamily, which was shown to belong to the superfamily of Fe(II)- and 2-oxoglutarate-dependent oxygenases. Analysis of the ectoine and hydroxyectoine contents of an ectABC ectD mutant strain fed with 1 mM ectoine or hydroxyectoine demonstrated that ectD is required for the main ectoine hydroxylase activity in C. salexigens. Although in minimal medium at 37°C the wild-type strain grew with 0.5 to 3.0 M NaCl, with optimal growth at 1.5 M NaCl, at 45°C it could not cope with the lowest (0.75 M NaCl) or the highest (3.0 M NaCl) salinity, and it grew optimally at 2.5 M NaCl. The ectD mutation caused a growth defect at 45°C in minimal medium with 1.5 to 2.5 M NaCl, but it did not affect growth at 37°C at any salinity tested. With 2.5 M NaCl, the ectD mutant synthesized 38% (at 37°C) and 15% (at 45°C) of the hydroxyectoine produced by the wild-type strain. All of these data reveal that hydroxyectoine synthesis mediated by the ectD gene is thermoregulated and essential for thermoprotection of C. salexigens.
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Carvalho, André Lacerda Ulysses de, Fábio Henrique Portella Corrêa de Oliveira, Rosa de Lima Ramos Mariano, Ester Ribeiro Gouveia, and Ana Maria Souto-Maior. "Growth, sporulation and production of bioactive compounds by Bacillus subtilis R14." Brazilian Archives of Biology and Technology 53, no. 3 (June 2010): 643–52. http://dx.doi.org/10.1590/s1516-89132010000300020.
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The physiology of B. subtilis R14 was investigated in minimal medium under excess-oxygen and oxygen-limited conditions. Growth and efficient sporulation could be achieved in excess-oxygen culture on medium with readily metabolizable carbon and nitrogen sources, which allowed high growth rate and high biomass yield. A short transition phase between the exponential growth and sporulation could be attained by formulating a medium with a well-balanced C/N ratio. Under oxygen-limitation, but in the presence of essential nutrients (i.e. excess-nutrient cultivation), B. subtilis R14 produced bioactive compounds, which showed activity against several phytopathogenic bacteria. Under anaerobic condition, the organism did not grow neither through fermentation nor anaerobic respiration. However, addition of pyruvate to the medium allowed its growth through fermentation and anaerobic respiration. The knowledge acquired in this work could be relevant both for the design of a production process as well as for the formulation of an effective commercial biocontrol product.
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Jackel, T., L. Knels, M. Valtink, R. H. W. Funk, and K. Engelmann. "Serum-free corneal organ culture medium (SFM) but not conventional minimal essential organ culture medium (MEM) protects human corneal endothelial cells from apoptotic and necrotic cell death." British Journal of Ophthalmology 95, no. 1 (October 24, 2010): 123–30. http://dx.doi.org/10.1136/bjo.2010.183418.
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Hottes, Alison K., Maliwan Meewan, Desiree Yang, Naomi Arana, Pedro Romero, Harley H. McAdams, and Craig Stephens. "Transcriptional Profiling of Caulobacter crescentus during Growth on Complex and Minimal Media." Journal of Bacteriology 186, no. 5 (March 1, 2004): 1448–61. http://dx.doi.org/10.1128/jb.186.5.1448-1461.2004.
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ABSTRACT Microarray analysis was used to examine gene expression in the freshwater oligotrophic bacterium Caulobacter crescentus during growth on three standard laboratory media, including peptone-yeast extract medium (PYE) and minimal salts medium with glucose or xylose as the carbon source. Nearly 400 genes (approximately 10% of the genome) varied significantly in expression between at least two of these media. The differentially expressed genes included many encoding transport systems, most notably diverse TonB-dependent outer membrane channels of unknown substrate specificity. Amino acid degradation pathways constituted the largest class of genes induced in PYE. In contrast, many of the genes upregulated in minimal media encoded enzymes for synthesis of amino acids, including incorporation of ammonia and sulfate into glutamate and cysteine. Glucose availability induced expression of genes encoding enzymes of the Entner-Doudoroff pathway, which was demonstrated here through mutational analysis to be essential in C. crescentus for growth on glucose. Xylose induced expression of genes encoding several hydrolytic exoenzymes as well as an operon that may encode a novel pathway for xylose catabolism. A conserved DNA motif upstream of many xylose-induced genes was identified and shown to confer xylose-specific expression. Xylose is an abundant component of xylan in plant cell walls, and the microarray data suggest that in addition to serving as a carbon source for growth of C. crescentus, this pentose may be interpreted as a signal to produce enzymes associated with plant polymer degradation.
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Gonçalves, Rodrigo José Sousa, Agnes Yasmin Pitombeira Cavalcante, Bruna Bortoloni Gouveia, Thae Lanne Barbosa Lins, Ricássio Sousa Barberino, Vanúzia Gonçalves Menezes, Vanessa Raquel Pinto Barros, et al. "Follicular survival, activation of primordial follicles and DNA fragmentation after storage of goat ovaries at 35ºC in supplemented Minimal Essential Medium." Acta Veterinaria Brasilica 11, no. 1 (March 31, 2017): 50–56. http://dx.doi.org/10.21708/avb.2017.11.1.6660.
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Alam, Md Kausar, and Susan G. W. Kaminskyj. "Aspergillus galactose metabolism is more complex than that of Saccharomyces: the story of GalDGAL7 and GalEGAL1." Botany 91, no. 7 (July 2013): 467–77. http://dx.doi.org/10.1139/cjb-2012-0270.
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Saccharomyces cerevisiae Hansen GAL1 (galactokinase) generates galactose-1-phosphate; GAL7 (galactose-1-phosphate uridylyltransferase) transfers UDP between galactose or glucose and their respective sugar-1-phosphate conjugates, and both are essential on galactose. Aspergillus nidulans ANID_04957 has 41% amino acid sequence identity with GAL1; ANID_06182 has 50% sequence identity with GAL7. The names Aspergillus nidulans GalE (galactokinase) and GalD (galactose-1-phosphate uridylyltransferase) are consistent with prior studies. Complemented galDΔ:ScGAL7 and galEΔ:ScGAL1 strains had wild-type phenotype, demonstrating functional homology. The galD5 and galE9 alleles were truncated. Strains galDΔ and galD5 were impaired on minimal medium containing 1% galactose (MM-Gal) at pH 7.5 and did not grow on MM-Gal pH 4.5. Strains galEΔ and galE9 grew on MM-Gal at both pH levels. Strains galDΔ and galEΔ produced wild-type conidiophores on minimal medium containing 1% glucose (MM-Glu) but few spores; for both, sporulation was lower on MM-Gal pH 7.5. GalD-GFP (green fluorescent protein) and GalE-GFP were cytosolic and upregulated on MM-Gal, consistent with quantitative real-time polymerase chain reaction. Galactofuranose immunolocalization in galDΔ resembled wild type on MM-Glu but was reduced on MM-Gal. The galEΔ strains had immunolocalizable Galf on all these media. Strains galDΔ and galEΔ were more sensitive to calcofluor, caspofungin, and itraconazole on MM-Gal. Neither galD nor galE is essential on galactose at high pH, implying additional routes for galactose metabolism in Aspergillus. Aspergillus galactose metabolism is more complex than that of S. cerevisiae.
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Stülke, Jörg, and Larissa Krüger. "Cyclic di-AMP Signaling in Bacteria." Annual Review of Microbiology 74, no. 1 (September 8, 2020): 159–79. http://dx.doi.org/10.1146/annurev-micro-020518-115943.
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The second messenger molecule cyclic di-AMP (c-di-AMP) is formed by many bacteria and archaea. In many species that produce c-di-AMP, this second messenger is essential for viability on rich medium. Recent research has demonstrated that c-di-AMP binds to a large number of proteins and riboswitches, which are often involved in potassium and osmotic homeostasis. c-di-AMP becomes dispensable if the bacteria are cultivated on minimal media with low concentrations of osmotically active compounds. Thus, the essentiality of c-di-AMP does not result from an interaction with a single essential target but rather from the multilevel control of complex homeostatic processes. This review summarizes current knowledge on the homeostasis of c-di-AMP and its function(s) in the control of cellular processes.
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Kim, Beum Jun, Joon Ho Park, Tai Hyun Park, Philip A. Bronstein, David J. Schneider, Samuel W. Cartinhour, and Michael L. Shuler. "Effect of Iron Concentration on the Growth Rate of Pseudomonas syringae and the Expression of Virulence Factors in hrp-Inducing Minimal Medium." Applied and Environmental Microbiology 75, no. 9 (March 6, 2009): 2720–26. http://dx.doi.org/10.1128/aem.02738-08.
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ABSTRACT Although chemically defined media have been developed and widely used to study the expression of virulence factors in the model plant pathogen Pseudomonas syringae, it has been difficult to link specific medium components to the induction response. Using a chemostat system, we found that iron is the limiting nutrient for growth in the standard hrp-inducing minimal medium and plays an important role in inducing several virulence-related genes in Pseudomonas syringae pv. tomato DC3000. With various concentrations of iron oxalate, growth was found to follow Monod-type kinetics for low to moderate iron concentrations. Observable toxicity due to iron began at 400 μM Fe3+. The kinetics of virulence factor gene induction can be expressed mathematically in terms of supplemented-iron concentration. We conclude that studies of induction of virulence-related genes in P. syringae should control iron levels carefully to reduce variations in the availability of this essential nutrient.
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Gravbrot, Nicholas, Daniel F. Kelly, John Milligan, Chester F. Griffiths, Garni Barkhoudarian, Heidi Jahnke, William L. White, and Andrew S. Little. "The Minimal Clinically Important Difference of the Anterior Skull Base Nasal Inventory-12." Neurosurgery 83, no. 2 (August 4, 2017): 277–80. http://dx.doi.org/10.1093/neuros/nyx401.
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Abstract BACKGROUND The minimal clinically important difference (MCID) is defined as the smallest change in health-related quality of life (QOL) that patients consider meaningful. The MCID is essential for determining clinically significant changes, rather than simply statistically significant changes, in QOL scores. The Anterior Skull Base Nasal Inventory-12 (ASK Nasal-12), a site-specific sinonasal QOL instrument, has emerged as a standard instrument for assessing QOL in patients who have undergone endonasal transsphenoidal surgery. OBJECTIVE To determine the MCID for the ASK Nasal-12. METHODS Distribution- and anchor-based methods were used to determine the MCID for the ASK Nasal-12 based on raw data from a multicenter prospective QOL study of 218 patients. RESULTS Two distribution-based statistical methods, the one-half standard deviation method and the effect-size method, both yielded MCIDs of 0.37 (medium effect). The first anchor-based method, using the 2-wk postoperative overall nasal functioning item as the anchor, yielded an MCID of 0.31. The second anchor-based method, using the 2-wk postoperative Short Form Health Survey 8 bodily pain item as the anchor, yielded an MCID of 0.29. CONCLUSION The largest MCID obtained for the ASK Nasal-12 using 4 statistical methods 2 wk postoperatively was 0.37. This information provides clinicians with an essential context for determining the clinical significance of changes in QOL scores after interventions. Our results will help clinicians better interpret QOL scores and design future studies that are powered to detect meaningful QOL changes.
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Pinna, Andrea, and Luca Massidda. "A Procedure for Complete Census Estimation of Rooftop Photovoltaic Potential in Urban Areas." Smart Cities 3, no. 3 (August 12, 2020): 873–93. http://dx.doi.org/10.3390/smartcities3030045.
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Rooftop photovoltaic solar systems can be an essential tool to support the energy transition of Europe. The assessment of solar power generation potential in urban areas, necessary for smart grid planning, requires the processing of data of different types, such as building cadastral information, a detailed description of available roof areas, and solar irradiation data. We introduce an algorithm for the fast calculation of the building’s shadows and a procedure for the integration of solar irradiation in time. We therefore develop a methodology that allows a fast evaluation with minimal computational resources, and we apply it to an urban scenario of a medium-sized European city obtaining an estimate of the complete census PV power generation potential, with a spatial resolution of 1 m. We validate the results by comparison with a reference procedure, obtaining minimal deviation with a much lower demand for computational resources.
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Safi, Mazen, and Ayman Al-Mariri. "In vitro antibacterial activity of several plant extracts and essential oils against Brucella melitensis." Herba Polonica 60, no. 1 (March 1, 2014): 29–38. http://dx.doi.org/10.2478/hepo-2014-0003.
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Summary Medicinal plants are considered to be new resources for the production of agents that could act as alternatives to antibiotics in the treatment of antibiotic-resistant bacteria. The aim of this study was to evaluate the efficacy of some plants native to Syria in the treatment of brucellosis. In vitro activities of some essential oils and plant extracts of some medicinal plants against 89 Brucella melitensis isolates was determined by disc diffusion method at a concentration of 5%. The microdilution assay in the fluid medium was used to determine the MICs of essential oils and plant extracts. Among the evaluated herbs, only Thymus syriacus and Cinnamomum zeylanicum essential oils and Laurus nobilis plant extract showed a high activity against B. melitensis strains. Thus, minimal inhibitory concentration (MIC50) values for T. syriacus, C. zeylanicum, and L. nobilis against B. melitensis were 6.25, 3.125 and 6.25 μl/ml, respectively. Among studied essential oils and plant extracts, T. syriacus and C. zeylanicum essential oils, and L. nobilis plant extract were the most effective ones. Moreover, T. syriacus - C. zeylanicum combination was more effective than use of each of them alone. Then, T. syriacus and C. zeylanicum essential oils and L. nobilis plant extract could act as bactericidal agents against B. melitensis.