Biomolecular Characterization of Bacillus tequilensis A1C1 Isolated from Soil and Chromatographic Analysis of D-serine in its Cellular Fraction

The current work was carried out to investigate serine enantiomers in bacterial cells. The bacteria isolated from the pomace dumping soil site (bacteria id A1C1) showed maximum growth (O.D600 = 1.97±0.4 X 109cells/ml) within 48h in the minimal salt media supplemented with L-serine. The isolated strain was identified as ‘Bacillus tequilensis’ through 16sRNA sequencing. The study’s peculiarity reflects the fact that the isolated strain was explored for the first time to detect the presence of serine enantiomers. The strain was quantified for D-serine content by using RP-HPLC. The D-serine concentration was calculated as 0.919±0.02 nM in the bacterial cellular fraction by using a standard curve plot and linear curve equation. Further, recovery % was also calculated for the spiked samples which vary from 85-90%. The optimum growth parameters were recorded as 37℃±0.5, 150±0.5 RPM, and 7±0.5pH. The strain was Gram-positive, rod shape, large, irregular, off-white-coloured, and synthesized endospores. A1C1 showed positive results (within 14±2h of incubation) for indole production, lactose fermentation, and protease (0.9 mm, clear zone). The antibacterial assay showed 5% and 2% efficacy of the extracellular fraction against MTCC 40 and MTCC 11949 respectively within 12h of incubation. These results demonstrate that Bacillus tequilensis A1C1 has antibacterial activity, the potential to secrete extracellular enzymes, and D-serine content in the intracellular fraction of the cultivated cells. Given results demonstrate the industrial significance and implication of the isolated strain for the synthesis of commercially valuable products.

Biomolecular Characterization of Bacillus tequilensis A1C1 Isolated from Soil and Chromatographic Analysis of D-serine in its Cellular Fraction

The bacteria isolated from the pomace dumping soil site (bacteria id A1C1) showed maximum growth (O.D600 = 1.97±0.4 X 109 cells/ml) within 48h in the minimal salt media supplemented with L-serine. The isolated strain was identified as ‘Bacillus tequilensis’ through 16sRNA sequencing. The strain was quantified for D-serine content by using RP-HPLC. The D-serine concentration was calculated as 0.919±0.02 nM in the bacterial cellular fraction by using a standard curve plot and linear curve equation. Further, recovery % was also calculated for the spiked samples which vary from 85-90%. The study’s peculiarity reflects the fact that the isolated strain was explored for the first time to detect the presence of serine enantiomers. The biochemical features also showed 70% similarity to the standard strain Bacillus tequilensis 10bT. The optimum growth parameters were recorded as 37℃±0.5, 150±0.5 RPM, and 7±0.5pH. The strain was Gram-positive and synthesized endospores. Morphological results showed its rod shape, large, irregular, and off-white-coloured colonies. A1C1 was also tested for the production of secondary metabolites and enzymes. A1C1 showed positive results for indole production, lactose fermentation, protease, and gelatinase whereas, negative results for catalase, MR-VP, citrate utilization, cellulase, amylase, and pectinase. Further, the strain was assayed for PGPR attributes and showed a negative phosphate solubilization index and IAA production. The antibacterial assay showed 5% and 2% efficacy of the extracellular fraction against MTCC 40 and MTCC 11949 respectively. These results demonstrate that Bacillus tequilensis A1C1 has antibacterial activity, the potential to secrete extracellular enzymes, and D-serine content in the intracellular fraction of the cultivated cells.

Biomolecular Characterization of Bacillus tequilensis A1C1 Isolated from Soil and Chromatographic Analysis of D-serine in its Cellular Fraction

The bacteria isolated from the pomace dumping soil site (bacteria id A1C1) showed maximum growth (O.D600 = 1.97±0.4 X 109 cells/ml) within 48h in the minimal salt media supplemented with L-serine. The isolated strain was identified as ‘Bacillus tequilensis’ through 16sRNA sequencing. The strain was quantified for D-serine content by using RP-HPLC. The D-serine concentration was calculated as 0.919±0.02 nM in the bacterial cellular fraction by using a standard curve plot and linear curve equation. Further, recovery % was also calculated for the spiked samples which vary from 85-90%. The study’s peculiarity reflects the fact that the isolated strain was explored for the first time to detect the presence of serine enantiomers. The biochemical features also showed 70% similarity to the standard strain Bacillus tequilensis 10bT. The optimum growth parameters were recorded as 37℃±0.5, 150±0.5 RPM, and 7±0.5pH. The strain was Gram-positive and synthesized endospores. Morphological results showed its rod shape, large, irregular, and off-white-coloured colonies. A1C1 was also tested for the production of secondary metabolites and enzymes. A1C1 showed positive results for indole production, lactose fermentation, protease, and gelatinase whereas, negative results for catalase, MR-VP, citrate utilization, cellulase, amylase, and pectinase. Further, the strain was assayed for PGPR attributes and showed a negative phosphate solubilization index and IAA production. The antibacterial assay showed 5% and 2% efficacy of the extracellular fraction against MTCC 40 and MTCC 11949 respectively. These results demonstrate that Bacillus tequilensis A1C1 has antibacterial activity, the potential to secrete extracellular enzymes, and D-serine content in the intracellular fraction of the cultivated cells.

Biomolecular Characterization of Bacillus tequilensis A1C1 Isolated from Soil and Chromatographic Analysis of D-serine in its Cellular Fraction

The bacteria isolated from the pomace dumping soil site (bacteria id A1C1) showed maximum growth (O.D600 = 1.97 ± 0.4 X 109 cells/ml) within 48h in the minimal salt media supplemented with L-serine. The isolated strain was identified as ‘Bacillus tequilensis’ through 16sRNA sequencing. The strain was quantified for D-serine content by using RP-HPLC. The D-serine concentration was calculated as 0.919 ± 0.02 nM in the bacterial cellular fraction by using a standard curve plot and linear curve equation. Further, recovery % was also calculated for the spiked samples which vary from 85–90%. The study’s peculiarity reflects the fact that the isolated strain was explored for the first time to detect the presence of serine enantiomers. The biochemical features also showed 70% similarity to the standard strain Bacillus tequilensis 10bT. The optimum growth parameters were recorded as 37℃±0.5, 150 ± 0.5 RPM, and 7 ± 0.5pH. The strain was Gram-positive and synthesized endospores. Morphological results showed its rod shape, large, irregular, and off-white-coloured colonies. A1C1 was also tested for the production of secondary metabolites and enzymes. A1C1 showed positive results for indole production, lactose fermentation, protease, and gelatinase whereas, negative results for catalase, MR-VP, citrate utilization, cellulase, amylase, and pectinase. Further, the strain was assayed for PGPR attributes and showed a negative phosphate solubilization index and IAA production. The antibacterial assay showed 5% and 2% efficacy of the extracellular fraction against MTCC 40 and MTCC 11949 respectively. These results demonstrate that Bacillus tequilensis A1C1 has antibacterial activity, the potential to secrete extracellular enzymes, and D-serine content in the intracellular fraction of the cultivated cells.

Arabidopsis PAP17 is a dual-localized purple acid phosphatase up-regulated during phosphate deprivation, senescence, and oxidative stress

A 35 kDa monomeric purple acid phosphatase (APase) was purified from cell wall extracts of Pi starved (–Pi) Arabidopsis thaliana suspension cells and identified as AtPAP17 (At3g17790) by mass spectrometry and N-terminal microsequencing. AtPAP17 was de novo synthesized and dual-localized to the secretome and/or intracellular fraction of –Pi or salt-stressed plants, or senescing leaves. Transiently expressed AtPAP17–green fluorescent protein localized to lytic vacuoles of the Arabidopsis suspension cells. No significant biochemical or phenotypical changes associated with AtPAP17 loss of function were observed in an atpap17 mutant during Pi deprivation, leaf senescence, or salinity stress. Nevertheless, AtPAP17 is hypothesized to contribute to Pi metabolism owing to its marked up-regulation during Pi starvation and leaf senescence, broad APase substrate selectivity and pH activity profile, and rapid repression and turnover following Pi resupply to –Pi plants. While AtPAP17 also catalyzed the peroxidation of luminol, which was optimal at pH 9.2, it exhibited a low Vmax and affinity for hydrogen peroxide relative to horseradish peroxidase. These results, coupled with absence of a phenotype in the salt-stressed or –Pi atpap17 mutant, do not support proposals that the peroxidase activity of AtPAP17 contributes to the detoxification of reactive oxygen species during stresses that trigger AtPAP17 up-regulation.

Effect of TNFα stimulation on expression of kidney risk inflammatory proteins in human umbilical vein endothelial cells cultured in hyperglycemia

We recently identified a kidney risk inflammatory signature (KRIS), comprising 6 TNF receptors (including TNFR1 and TNFR2) and 11 inflammatory proteins. Elevated levels of these proteins in circulation were strongly associated with risk of the development of end-stage kidney disease (ESKD) during 10-year follow-up. It has been hypothesized that elevated levels of these proteins in circulation might reflect (be markers of) systemic exposure to TNFα. In this in vitro study, we examined intracellular and extracellular levels of these proteins in human umbilical vein endothelial cells (HUVECs) exposed to TNFα in the presence of hyperglycemia. KRIS proteins as well as 1300 other proteins were measured using the SOMAscan proteomics platform. Four KRIS proteins (including TNFR1) were down-regulated and only 1 protein (IL18R1) was up-regulated in the extracellular fraction of TNFα-stimulated HUVECs. In the intracellular fraction, one KRIS protein was down-regulated (CCL14) and 1 protein was up-regulated (IL18R1). The levels of other KRIS proteins were not affected by exposure to TNFα. HUVECs exposed to a hyperglycemic and inflammatory environment also showed significant up-regulation of a distinct set of 53 proteins (mainly in extracellular fraction). In our previous study, circulating levels of these proteins were not associated with progression to ESKD in diabetes.

A Cell-Free Approach Based on Phospholipid Characterization for Determination of the Cell Specific Unbound Drug Fraction (fu,cell)

Purpose The intracellular fraction of unbound compound (fu,cell) is an important parameter for accurate prediction of drug binding to intracellular targets. fu,cell is the result of a passive distribution process of drug molecules partitioning into cellular structures. Initial observations in our laboratory showed an up to 10-fold difference in the fu,cell of a given drug for different cell types. We hypothesized that these differences could be explained by the phospholipid (PL) composition of the cells, since the PL cell membrane is the major sink of unspecific drug binding. Therefore, we determined the fu,cell of 19 drugs in cell types of different origin. Method The cells were characterized for their total PL content and we used mass spectrometric PL profiling to delineate the impact of each of the four major cellular PL subspecies: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI). The cell-based experiments were compared to cell-free experiments that used beads covered by PL bilayers consisting of the most abundant PL subspecies. Results PC was found to give the largest contribution to the drug binding. Improved correlations between the cell-based and cell-free assays were obtained when affinities to all four major PL subspecies were considered. Together, our data indicate that fu,cell is influenced by PL composition of cells. Conclusion We conclude that cellular PL composition varies between cell types and that cell-specific mixtures of PLs can replace cellular assays for determination of fu,cell as a rapid, small-scale assay covering a broad dynamic range.

Cytoplasmic Overexpression of HER2: A Key Factor in Colorectal Cancer

Trastuzumab, a humanized mouse monoclonal antibody directed against HER2 (Human Epidermal Growth Factor Receptor 2), is currently a keystone in the treatment of breast cancer. Meanwhile, trastuzumab has been validated for use in other types of cancer too. But the data on HER2 expression in colorectal cancer are ambiguous, with reported overexpression of HER2 varying between zero and 84%. In this review these studies are evaluated and compared. It shows that many factors influence the determination of HER2-expression, especially of the intracellular fraction of HER2. It is concluded that although membranous overexpression of HER2 is low in colorectal cancer with only 5% of all patients being positive, a significant proportion of the patients (30%) shows cytoplasmic HER2 overexpression. The clinical impact of enhanced intracellular HER2 is not known, because the nature and origin have not completely been revealed yet. Enlightening this process could be a stepping stone towards targeting of intracellular HER2 as a treatment option.

Acquired Tolerance to Oxidative Stress in Bifidobacterium longum 105-A via Expression of a Catalase Gene

ABSTRACTFor improvement of tolerance to oxidative stress inBifidobacterium longum105-A, we introduced theBacillus subtiliscatalase gene (katE) into it. The transformant showed catalase activity (39 U/mg crude protein) in the intracellular fraction, which increased survival by ∼100-fold after a 1-h exposure to 4.4 mM H2O2, decreasedde novoH2O2accumulation, and increased survival in aerated cultures by 105-fold at 24 h. The protection level was better than that conferred by exogenously added catalase.