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11.
12.
Vishwanathgouda Maralingannavar Dharmeshkumar Parmar Venkateswarlu Panchagnula Mugdha Gadgil 《Biotechnology progress》2019,35(5):e2856
Passaging and expansion of animal cells in lean maintenance medium could result in periods of limitation of some nutrients. Over time, such stresses could possibly result in selection of cells with metabolic changes and contribute to heterogeneity. Here, we investigate whether selection of Chinese Hamster Ovary (CHO) cells under glutamine limitation results in changes in growth under glutamine-replete conditions. In glutamine-limiting medium, compared to control cells passaged in glutamine-rich medium, the selected cells showed higher glutamine synthetase (GS) activity and attained a higher peak viable cell density (PVCD). Surprisingly, in glutamine-replete conditions, selected cells still showed a higher GS activity but a lower PVCD. We show that in glutamine-replete medium, PVCD of selected cells was restored on (a) inhibition of GS activity with methionine sulfoximine, (b) supplementation of aspartate—without affecting GS activity, and (c) supplementation of serine, which is reported to inhibit GS in vitro. Consistent with the reported effect of serine, inhibition of GS activity was observed upon serine supplementation along with reduced growth of cells under glutamine-limiting conditions. The latter observation is important for the design of glutamine-free culture medium and feed used for GS-CHO and GS-NS0. In summary, we show that CHO cells selected under glutamine limitation have superfluous GS activity in glutamine-replete medium, which negatively affects their PVCD. This may be due to its effect on availability of aspartate which was the limiting nutrient for the growth of selected cells in glutamine-replete conditions. 相似文献
13.
Different quaternary structures of human RECQ1 are associated with its dual enzymatic activity 总被引:1,自引:0,他引:1
Muzzolini L Beuron F Patwardhan A Popuri V Cui S Niccolini B Rappas M Freemont PS Vindigni A 《PLoS biology》2007,5(2):e20
RecQ helicases are essential for the maintenance of chromosome stability. In addition to DNA unwinding, some RecQ enzymes have an intrinsic DNA strand annealing activity. The function of this dual enzymatic activity and the mechanism that regulates it is, however, unknown. Here, we describe two quaternary forms of the human RECQ1 helicase, higher-order oligomers consistent with pentamers or hexamers, and smaller oligomers consistent with monomers or dimers. Size exclusion chromatography and transmission electron microscopy show that the equilibrium between the two assembly states is affected by single-stranded DNA (ssDNA) and ATP binding, where ATP or ATPγS favors the smaller oligomeric form. Our three-dimensional electron microscopy reconstructions of human RECQ1 reveal a complex cage-like structure of approximately 120 Å × 130 Å with a central pore. This oligomeric structure is stabilized under conditions in which RECQ1 is proficient in strand annealing. In contrast, competition experiments with the ATPase-deficient K119R and E220Q mutants indicate that RECQ1 monomers, or tight binding dimers, are required for DNA unwinding. Collectively, our findings suggest that higher-order oligomers are associated with DNA strand annealing, and lower-order oligomers with DNA unwinding. 相似文献
14.
15.
Lamb DC Warrilow AG Venkateswarlu K Kelly DE Kelly SL 《Biochemical and biophysical research communications》2001,282(1):48-54
A highly thermostable xylanase (Xyl I) produced by Thermomonospora sp. was purified to homogeneity and was classified as a family 10 xylanase based on its molecular weight (38,000 Da) and isoelectric point (4.1). K2d analysis showed that the secondary structure of Xyl I was made up of 38% alpha-helix and 10% beta-sheet. The optimal temperature for the activity of Xyl I was 80 degrees C. Xyl I was highly thermostable with half-lives of 86, 30, and 15 min at 80, 90, and 100 degrees C respectively. Xyl I was stable in an expansive pH range of 5 to 10 with more than 75% residual activity. Our present investigation using o-phthalaldehyde (OPTA) as the chemical initiator for fluorescent chemoaffinity labeling and trinitrobenzenesulphonic acid (TNBS) as chemical modifier have revealed the presence of a single lysine residue in the active site of Xyl I. The high pK value for the basic limb of the pH profile reflects the ionization of a lysine residue. The higher K(m) values and similar k(cat) values of the TNBS modified enzyme in comparison to native enzyme and the substrate protection against OPTA and TNBS, suggested the presence of the lysine residue in the substrate-binding site. 相似文献
16.
In this study, we address whether TGFbeta signaling mediates vitamin D3 analog-induced growth inhibition in nonmalignant and malignant breast cells. Normal mammary epithelial cells (184), immortalized nonmalignant mammary epithelial cells (184A1 and MCF10A), and breast cancer cells (early passage MCF7: MCF7E) were sensitive to the inhibitory effects of vitamin D3 analogs (EB1089 and MC1288) while late passage MCF7 breast cancer (MCF7L) cells were relatively resistant. A similar pattern of sensitivity to TGFbeta was observed with these cells. Thus, the sensitivity to the vitamin D3 analogs correlated with the sensitivity to TGFbeta. MCF7L TGFbetaRII-transfected cells, which have autocrine TGFbeta activity, were more sensitive to EB1089 than MCF7L cells. TGFbeta neutralizing antibody was found to block the inhibitory effects of these analogs. These results are consistent with the idea that autocrine TGFbeta signaling mediates the anti-proliferative effects of the vitamin D3 analogs in these cells. The expression of TGFbeta isoforms and/or TGFbeta receptors was induced by the analogs in the vitamin D3 and TGFbeta sensitive cells. Vitamin D3 analogs did not induce TGFbeta or TGFbeta receptor expression in the resistant MCF7L cells. Therefore, EB1089 induces autocrine TGFbeta activity through increasing expression of TGFbeta isoforms and/or TGFbeta receptors. In addition, EB1089 induced nuclear VDR protein levels in the sensitive 184A1 cells but not in the resistant MCF7L cells. 184A1 cells were more sensitive to EB1089-induced VDR-dependent transactivation than MCF7L cells as measured by a luciferase reporter construct containing the VDRE, indicating a defect of VDR signaling in MCF7L cells. Smad3, a TGFbeta signaling mediator, coactivated VDR-dependent transactivation in 184A1 cells but not in MCF7L cells. These results indicate that Smad3 coactivates VDR to further enhance TGFbeta signaling and vitamin D3 signaling in the sensitive 184A1 cells. The results also indicate that Smad3 is not of itself sufficient to coactivate VDR in TGFbeta/vitamin D3 resistant MCF7L cells and other factors are required. We found that the PI 3-kinase pathway inhibitor LY29004 inhibited the synergy of TGFbeta and EB1089 on VDR-dependent transactivation activity. This indicates that the crosstalk between TGFbeta and vitamin D signaling is also PI 3-kinase pathway dependent. 相似文献
17.
Lagaud G Karicheti V Knot HJ Christ GJ Laher I 《American journal of physiology. Heart and circulatory physiology》2002,283(6):H2177-H2186
The effects of two structurally distinct inhibitors of gap junction communication were studied by using three different forms of vasoconstriction in pressurized rat middle cerebral arteries. The sensitivity of myogenic tone (at 60 mmHg), vasopressin-induced tone (10 nM, at 20 mmHg), and depolarizing solution-induced tone (80 mM K(+), at 20 mmHg) to inhibition by heptanol (1.0 microM to 3.0 mM) or 18alpha-glycyrrhetinic acid (18alpha-GA, 1.0 to 50 microM) were determined. Pressure-induced myogenic tone was inhibited by heptanol (IC(50) = 0.75 +/- 0.09 mM) and 18alpha-GA ( approximately 30 microM). Vasopressin-induced vasoconstriction was also inhibited by heptanol (IC(50) = 0.4 +/- 0.3 mM) and 18alpha-GA (>1 microM). Depolarizing solution-induced vasoconstriction was less sensitive to inhibition by heptanol compared to vasopressin (P < 0.01) or pressure-induced constriction (P < 0.05). However, 18alpha-GA did not inhibit depolarization-induced constriction. Sharp microelectrode experiments on isolated arteries revealed stable membrane potentials, with no detectable effect of heptanol (1 mM) or 18alpha-GA (20-30 microM) on the average membrane potential at 20 mmHg. However, approximately 20% of impaled cells (5 of 28) exhibited uncharacteristic oscillations in membrane potential after pharmacological uncoupling. At 60 mmHg a approximately 7- to 9-mV hyperpolarization and corresponding vasodilation (approximately 50%) was observed, and the frequency of membrane potential oscillations doubled (9 of 23 cells). These data indicate that gap junctions play an important role in the maintenance and modulation of membrane potential and tone in cerebral resistance arteries. 相似文献
18.
Sharma VM Prasanna P Seshu KV Renuka B Rao CV Kumar GS Narasimhulu CP Babu PA Puranik RC Subramanyam D Venkateswarlu A Rajagopal S Kumar KB Rao CS Mamidi NV Deevi DS Ajaykumar R Rajagopalan R 《Bioorganic & medicinal chemistry letters》2002,12(17):2303-2307
In our endeavor to design and synthesize novel anticancer agents, a new series of indoloquinazoline compounds were prepared and tested initially for anticancer activity in vitro against a panel of human cancer cell lines. Most of these compounds exhibited cytotoxic activity in in vitro screens. Compounds were selected and further evaluated using a modified Hollow Fiber Assay for their preliminary in vivo activity against 12 cell lines implanted in the subcutaneous and intraperitoneal compartments in mice. The results indicate that these compounds may constitute a new class of anticancer agents. 相似文献
19.
Christ GJ Day NS Day M Santizo C Zhao W Sclafani T Zinman J Hsieh K Venkateswarlu K Valcic M Melman A 《American journal of physiology. Regulatory, integrative and comparative physiology》2001,281(5):R1699-R1709
The goal of these studies was to examine the potential utility of bladder instilled K+ channel gene therapy with hSlo cDNA (i.e., the maxi-K channel) to ameliorate bladder overactivity in a rat model of partial urinary outlet obstruction. Twenty-two female Sprague-Dawley rats were subjected to partial urethral (i.e., outlet) obstruction, with 17 sham-operated control rats run in parallel. After 6 wk of obstruction, suprapubic catheters were surgically placed in the dome of the bladder in all rats. Twelve obstructed rats received bladder instillation of 100 microg of hSlo/pcDNA in 1 ml PBS during catheterization, and another 10 obstructed rats received 1 ml PBS (7 rats) or 1 ml PBS containing pcDNA only (3 rats). Two days after surgery cystometry was performed on all animals to examine the characteristics of the micturition reflex in conscious and unrestrained rats. Obstruction was associated with a three- to fourfold increase in bladder weight and alterations in virtually every micturition parameter estimate. PBS-injected obstructed rats routinely displayed spontaneous bladder contractions between micturitions. In contrast, hSlo injection eliminated the obstruction-associated bladder hyperactivity, without detectably affecting any other cystometric parameter. Presumably, expression of hSlo in rat bladder functionally antagonizes the increased contractility normally observed in obstructed animals and thereby ameliorates bladder overactivity. These initial observations indicate a potential utility of gene therapy for urinary incontinence. 相似文献
20.
Murali Krishna PS Venkateswarlu G Venkateswar Rao L 《Letters in applied microbiology》2000,31(1):73-76
The effect of different organic nitrogen compounds on the production of rifamycin SV by Amycolatopsis mediterranei MV35R and their optimum concentrations have been described. Results obtained indicate that rifamycin SV production increased from 4020 mg l-1 to 4575 mg l-1 when organic nitrogen compound uracil was added at 0.2% (w/v) concentration to the fermentation medium by A. mediterranei MV35R. The rifamycin SV yield was enhanced by 505 mg l-1 using uracil (2 g l-1) when compared with barbital. 相似文献