Cytocidal amino acid starvation of Saccharomyces cerevisiae and Candida albicans acetolactate synthase (ilv2{Delta}) mutants is influenced by the carbon source and rapamycin.

The isoleucine and valine biosynthetic enzyme acetolactate synthase (Ilv2p) is an attractive antifungal drug target, since the isoleucine and valine biosynthetic pathway is not present in mammals, Saccharomyces cerevisiae ilv2Delta mutants do not survive in vivo, Cryptococcus neoformans ilv2 mutants are avirulent, and both S. cerevisiae and Cr. neoformans ilv2 mutants die upon isoleucine and valine starvation. To further explore the potential of Ilv2p as an antifungal drug target, we disrupted Candida albicans ILV2, and demonstrated that Ca. albicans ilv2Delta mutants were significantly attenuated in virulence, and were also profoundly starvation-cidal, with a greater than 100-fold reduction in viability after only 4 h of isoleucine and valine starvation. As fungicidal starvation would be advantageous for drug design, we explored the basis of the starvation-cidal phenotype in both S. cerevisiae and Ca. albicans ilv2Delta mutants. Since the mutation of ILV1, required for the first step of isoleucine biosynthesis, did not suppress the ilv2Delta starvation-cidal defects in either species, the cidal phenotype was not due to alpha-ketobutyrate accumulation. We found that starvation for isoleucine alone was more deleterious in Ca. albicans than in S. cerevisiae, and starvation for valine was more deleterious than for isoleucine in both species. Interestingly, while the target of rapamycin (TOR) pathway inhibitor rapamycin further reduced S. cerevisiae ilv2Delta starvation viability, it increased Ca. albicans ilv1Delta and ilv2Delta viability. Furthermore, the recovery from starvation was dependent on the carbon source present during recovery for S. cerevisiae ilv2Delta mutants, reminiscent of isoleucine and valine starvation inducing a viable but non-culturable-like state in this species, while Ca. albicans ilv1Delta and ilv2 Delta viability was influenced by the carbon source present during starvation, supporting a role for glucose wasting in the Ca. albicans cidal phenotype.

Multiple phenotypic changes in mice after knockout of the B3gnt5 gene, encoding Lc3 synthase--a key enzyme in lacto-neolacto ganglioside synthesis.

BACKGROUND: Ganglioside biosynthesis occurs through a multi-enzymatic pathway which at the lactosylceramide step is branched into several biosynthetic series. Lc3 synthase utilizes a variety of galactose-terminated glycolipids as acceptors by establishing a glycosidic bond in the beta-1,3-linkage to GlcNaAc to extend the lacto- and neolacto-series gangliosides. In order to examine the lacto-series ganglioside functions in mice, we used gene knockout technology to generate Lc3 synthase gene B3gnt5-deficient mice by two different strategies and compared the phenotypes of the two null mouse groups with each other and with their wild-type counterparts. RESULTS: B3gnt5 gene knockout mutant mice appeared normal in the embryonic stage and, if they survived delivery, remained normal during early life. However, about 9% developed early-stage growth retardation, 11% died postnatally in less than 2 months, and adults tended to die in 5-15 months, demonstrating splenomegaly and notably enlarged lymph nodes. Without lacto-neolacto series gangliosides, both homozygous and heterozygous mice gradually displayed fur loss or obesity, and breeding mice demonstrated reproductive defects. Furthermore, B3gnt5 gene knockout disrupted the functional integrity of B cells, as manifested by a decrease in B-cell numbers in the spleen, germinal center disappearance, and less efficiency to proliferate in hybridoma fusion. CONCLUSIONS: These novel results demonstrate unequivocally that lacto-neolacto series gangliosides are essential to multiple physiological functions, especially the control of reproductive output, and spleen B-cell abnormality. We also report the generation of anti-IgG response against the lacto-series gangliosides 3'-isoLM1 and 3',6'-isoLD1.

High glucose decreases intracellular glutathione concentrations and upregulates inducible nitric oxide synthase gene expression in intestinal epithelial cells

Diabetes is associated with oxidative stress and increased levels of inflammatory cytokines. The aim of the study was to assess the effects of inflammatory cytokines and oxidative stress associated with raised glucose levels on inducible nitric oxide synthase (iNOS) promoter activity in intestinal epithelial cells. High glucose (25 mmol/l) conditions reduced glutathione (GSH) levels in the human intestinal epithelial cell line, DLD-1. Addition of the antioxidant alpha-lipoic acid resulted in the restoration of GSH levels to normal. Upregulation of basal iNOS promoter activity was observed when cells were incubated in high glucose alone. This effect was significantly reduced by the addition of the antioxidant, alpha-lipoic acid and completely blocked with inhibition of NFkappa B activity. Cytokine stimulation [interleukin-1 beta, tumor necrosis factor-alpha, interferon-gamma] induced iNOS promoter activity in all conditions and this was accompanied by an increase in nitric oxide (NO) production. Inhibition of NFkappa-B activity decreased but did not completely inhibit cytokine-induced iNOS promoter activity and subsequent NO production. In conclusion, high glucose-induced iNOS promoter activity is mediated in part through intracellular GSH and NFkappa-B.

Characterization of a thymidylate synthase (TS)-inducible cell line: a model system for studying sensitivity to TS- and non-TS-targeted chemotherapies

Thymidylate synthase (TS) is responsible for the de novo synthesis of thymidylate, which is required for DNA synthesis and repair and which is an important target for fluoropyrimidines such as 5-fluorouracil (5-FU), and antifolates such as Tomudex (TDX), ZD9331, and multitargeted antifolate (MTA). To study the importance of TS expression in determining resistance to these agents, we have developed an MDA435 breast cancer-derived cell line with tetracycline-regulated expression of TS termed MTS-5. We have demonstrated that inducible expression of TS increased the IC(50) dose of the TS-targeted therapeutic agents 5-FU, TDX, and ZD9331 by 2-, 9- and 24-fold respectively. An IC(50) dose for MTA was unobtainable when TS was overexpressed in these cells, which indicated that MTA toxicity is highly sensitive to increased TS expression levels. The growth inhibitory effects of the chemotherapeutic agents CPT-11, cisplatin, oxaliplatin, and Taxol were unaffected by TS up-regulation. Cell cycle analyses revealed that IC(50) doses of 5-FU, TDX and MTA caused an S-phase arrest in cells that did not overexpress TS, and this arrest was overcome when TS was up-regulated. Furthermore, the S-phase arrest was accompanied by 2- to 4-fold increased expression of the cell cycle regulatory genes cyclin E, cyclin A, and cyclin dependent kinase 2 (cdk2). These results indicate that acute increases in TS expression levels play a key role in determining cellular sensitivity to TS-directed chemotherapeutic drugs by modulating the degree of S-phase arrest caused by these agents. Moreover, CPT-11, cisplatin, oxaliplatin, and Taxol remain highly cytotoxic in cells that overexpress TS.

The Role of Thymidylate Synthase Induction in Modulating p53-regulated Gene Expression in Response to 5-Fluorouracil and Antifolates

Thymidylate synthase (TS) is a critical target for chemotherapeutic agents such as 5-fluorouracil (5-FU) and antifolates such as tomudex (TDX),multitargeted antifolate, and ZD9331. Using the MCF-7 breast cancer line, we have developed p53 wild-type (M7TS90) and null (M7TS90-E6) isogenic lines with inducible TS expression (approximately 6-fold induction compared with control after 48 h). In the M7TS90 line, inducible TS expression resulted in a moderate approximately 3-fold increase in 5-FU IC-50(72 h) dose and a dramatic >20-fold increase in the IC-50(72 h) doses of TDX, multitargeted antifolate, and ZD9331. S-phase cell cycle arrest and apoptosis induced by the antifolates were abrogated by TS induction. In contrast, cell cycle arrest and apoptosis induced by 5-FU was unaffected by TS expression levels. Inactivation of p53 significantly increased resistance to 5-FU and the antifolates with IC-50(72 h) doses for 5-FU and TDX of >100 and >10 microM, respectively, in the M7TS90-E6 cell line. Furthermore, p53 inactivation completely abrogated the cell cycle arrest and apoptosis induced by 5-FU. The antifolates induced S-phase arrest in the p53 null cell line; however, the induction of apoptosis by these agents was significantly reduced compared with p53 wild-type cells. Both inducible TS expression and the addition of exogenous thymidine (10 microM) blocked p53 and p21 induction by the antifolates but not by 5-FU in the M7TS90 cell line. Similarly, inducible TS expression and exogenous thymidine abrogated antifolate but not 5-FU-mediated up-regulation of Fas/CD95 in M7TS90 cells. Our results indicate that in M7TS90 cells, inducible TS expression modulates p53 and p53 target gene expression in response to TS-targeted antifolate therapies but not to 5-FU.

Functional comparison of the endothelial nitric oxide synthase Glu298Asp polymorphic variants in human endothelial cells

The G894T endothelial nitric oxide synthase (eNOS) polymorphism results in a Glu to Asp substitution at position 298. This position is located externally on the protein and as the regulation of eNOS is dependent on its subcellular localization and interaction with modulatory proteins, we aimed to address whether the substitution of Asp at 298 had any effect on these mechanisms. Initially, we developed a novel method to accurately determine molar quantities of each variant by expressing them as green fluorescent protein (GFP) fusion proteins and using recombinant adenoviruses to facilitate transient infection of human microvascular endothelial cells. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting of eNOS298Asp revealed a 135-kDa proteolytic fragment which was not present with eNOS298Glu. This proteolysis was prevented by using LDS buffer confirming that this differential cleavage is an artefact of sample preparation and unlikely to occur intracellularly. Nitric oxide was measured following stimulation with calcium ionophore or oestrogen in the presence of varying sepiapterin concentrations. GFP fluorescence was used to quantify the amount of fusion protein and calculate intracellular specific activity. There was no significant difference in intracellular specific activity between Glu298 and Asp298 eNOS in response to calcium ionophore or oestrogen. Tetrahydrobiopterin supplementation increased eNOS activity of both variants in an identical manner. The presence of the GFP also facilitated the visualization of the variants by confocal microscopy and demonstrated that both localized to the plasma membrane and the Golgi. These findings demonstrate that the Asp substitution at 298 does not have a major effect in modulating eNOS activity in vivo.

Truncated estrogen receptor alpha 46-kDa isoform in human endothelial cells: relationship to acute activation of nitric oxide synthase.

Background Estrogen acutely activates endothelial nitric oxide synthase (eNOS). However, the identity of the receptors involved in this rapid response remains unclear. Methods and Results We detected an estrogen receptor (ER) transcript in human endothelial cells that encodes a truncated 46-kDa ER (1a-hER-46). A corresponding 46-kDa ER protein was identified in endothelial cell lysates. Transfection of cDNAs encoding the full-length ER (ER-66) and 1a-hER-46 resulted in appropriately sized recombinant proteins identified by anti-ER antibodies. Confocal microscopy revealed that a proportion of both ER-66 and hER-46 was localized outside the nucleus and mediated specific cell-surface binding of estrogen as assessed by FITC-conjugated, BSA-estrogen binding studies. Both ER isoforms colocalized with eNOS and mediated acute activation of eNOS in response to estrogen stimulation. However, estrogen-stimulated transcriptional activation mediated by 1a-hER-46 was much less than with ER-66. Furthermore, 1a-hER-46 inhibited classical hER-66 mediated transcriptional activation in a dominant-negative fashion. Conclusions These findings suggest that expression of an alternatively spliced, truncated ER isoform in human endothelial cells confers a unique ability to mediate acute but not transcriptional responses to estrogen.

GTP Cyclohydrolase I Gene Transfer Augments Intracellular Tetrahydrobiopterin in Human Endothelial Cells: Effects on Nitric Oxide Synthase Activity and Dimerisation.

Objectives: Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS) activity. BH4 levels are regulated by de novo biosynthesis; the rate-limiting enzyme is GTP cyclohydrolase I (GTPCH). BH4 activates and promotes homodimerisation of purified eNOS protein, but the intracellular mechanisms underlying BH4-mediated eNOS regulation in endothelial cells remain less clear. We aimed to investigate the role of BH4 levels in intracellular eNOS regulation, by targeting the BH4 synthetic pathway as a novel strategy to modulate intracellular BH4 levels. Methods: We constructed a recombinant adenovirus, AdGCH, encoding human GTPCH. We infected human endothelial cells with AdGCH, investigated the changes in intracellular biopterin levels, and determined the effects on eNOS enzymatic activity, protein levels and dimerisation. Results: GTPCH gene transfer in EAhy926 endothelial cells increased BH4 >10-fold compared with controls (cells alone or control adenovirus infection), and greatly enhanced NO production in a dose-dependent, eNOS-specific manner. We found that eNOS was principally monomeric in control cells, whereas GTPCH gene transfer resulted in a striking increase in eNOS homodimerisation. Furthermore, the total amounts of both native eNOS protein and a recombinant eNOS–GFP fusion protein were significantly increased following GTPCH gene transfer. Conclusions: These findings suggest that GTPCH gene transfer is a valid approach to increase BH4 levels in human endothelial cells, and provide new evidence for the relative importance of different mechanisms underlying BH4-mediated eNOS regulation in intact human endothelial cells. Additionally, these observations suggest that GTPCH may be a rational target to augment endothelial BH4 and normalise eNOS activity in endothelial dysfunction states.