Human Cdc14B promotes progression through mitosis by dephosphorylating Cdc25 and regulating Cdk1/cyclin B activity

Entry into and progression through mitosis depends on phosphorylation and dephosphorylation of key substrates. In yeast, the nucleolar phosphatase Cdc14 is pivotal for exit from mitosis counteracting Cdk1-dependent phosphorylations. Whether hCdc14B, the human homolog of yeast Cdc14, plays a similar function in mitosis is not yet known. Here we show that hCdc14B serves a critical role in regulating progression through mitosis, which is distinct from hCdc14A. Unscheduled overexpression of hCdc14B delays activation of two master regulators of mitosis, Cdc25 and Cdk1, and slows down entry into mitosis. Depletion of hCdc14B by RNAi prevents timely inactivation of Cdk1/cyclin B and dephosphorylation of Cdc25, leading to severe mitotic defects, such as delay of metaphase/anaphase transition, lagging chromosomes, multipolar spindles and binucleation. The results demonstrate that hCdc14B-dependent modulation of Cdc25 phosphatase and Cdk1/cyclin B activity is tightly linked to correct chromosome segregation and bipolar spindle formation, processes that are required for proper progression through mitosis and maintenance of genomic stability.     

The effect of angiotensin-converting enzyme inhibitors on experimental colitis in rats

The present study was aimed to investigate the effect of ACE inhibition on trinitrobenzene sulphonic acid (TNBS)-induced colonic inflammation in rats by using captopril and lisinopril. In treatment groups, the rats were treated with ACE inhibitors, captopril or lisinopril (0.1 and 1 mg/kg/day; intraperitoneally). The drugs were given 5 min after induction of colitis and the treatment was continued for 3 days. Three days after the induction of colitis, all rats were decapitated. The distal colon was weighed and the mucosal lesions were scored at both macroscopical at microscopic levels. Malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity and collagen content were assessed in tissue samples. Formation of reactive oxygen species in colonic samples was monitored by using chemiluminescence technique. Serum TNF-alphalevel was assessed in trunk blood. Captopril treatment was found to be beneficial in all parameters, except colonic glutathione content. On the other hand, although stimulation of lipid peroxidation and increase in serum TNF-alpha level were successfully prevented by lisinopril, the morphology of the lesions remained unchanged. In conclusion, sulphydryl and non-sulphydryl ACE inhibitors, captopril and lisinopril do not seem to be similarly effective in TNBS-induced colitis model at least at the doses tested in our study.     

Amelioration of sepsis-induced hepatic and ileal injury in rats by the leukotriene receptor blocker montelukast

Background: Sepsis is a generalized inflammatory response, which involves organ systems remote from the locus of the initial infectious insult, involves the release of cytokines and the subsequent formation of reactive oxygen and nitrogen species.Objective: The aim of this study was to investigate the possible protective effect of montelukast, a leukotriene receptor blocker, against oxidative damage in the liver and ileum of septic rats.Methods: Sepsis was induced by cecal ligation and puncture method in female Wistar albino rats. Sepsis and sham operated (control) groups received either saline or montelukast (10 mg/kg, ip) immediately after the operation and at 12 h. Twenty-four hours after the surgery, rats were decapitated and malondialdehyde (MDA) content—an index of lipid peroxidation, glutathione (GSH) levels—a key antioxidant, myeloperoxidase (MPO) activity—an index of neutrophil infiltration, and collagen contents were determined in the liver and ileum. Formation of reactive oxygen species in liver and ileal tissue samples was monitored by using chemiluminescence (CL) technique with luminol and lucigenin probes. Both tissues were also analyzed histologically. Serum lactate dehydrogenase (LDH) and tumor necrosis factor-α (TNF-α) level were assessed in trunk blood.Results: Sepsis resulted in decreased GSH levels, and increased MDA levels, MPO activity, CL levels and collagen contents in both the liver and the ileum (P<0.05–P<0.001) indicating the presence of the oxidative damage. Similarly, serum TNF-α and LDH were elevated in the sepsis group as compared to control group. On the other hand, montelukast treatment reversed all these biochemical indices, as well as histopathological alterations, which were induced by sepsis.Conclusion: Findings of the present study suggest that montelukast possesses an anti-inflammatory effect on sepsis-induced hepatic and intestinal damage and protects against oxidative injury by a neutrophil-dependent mechanism.     

The Skp1 prolyl hydroxylase from Dictyostelium is related to the hypoxia-inducible factor-alpha class of animal prolyl 4-hydroxylases

Skp1 is a cytoplasmic and nuclear protein of eukaryotes best known as an adaptor in SCF ubiquitin-protein isopeptide ligases. In Dictyostelium, Skp1 is subject to 4-hydroxylation at Pro(143) and subsequent O-glycosylation by alpha-linked GlcNAc and other sugars. Soluble cytosolic extracts have Skp1 prolyl 4-hydroxylase (P4H) activity, which can be measured based on hydroxylation-dependent transfer of [(3)H]GlcNAc to recombinant Skp1 by recombinant (Skp1-protein)-hydroxyproline alpha-N-acetyl-d-glucosaminyltransferase. The Dictyostelium Skp1 P4H gene (phyA) was predicted using a bioinformatics approach, and the expected enzyme activity was confirmed by expression of phyA cDNA in Escherichia coli. The purified recombinant enzyme (P4H1) was dependent on physiological concentrations of O(2), alpha-ketoglutarate, and ascorbate and was inhibited by CoCl(2), 3,4-dihydroxybenzoate, and 3,4-dihydroxyphenyl acetate, as observed for known animal cytoplasmic P4Hs of the hypoxia-inducible factor-alpha (HIFalpha) class. Overexpression of phyA cDNA in Dictyostelium yielded increased enzyme activity in a soluble cytosolic extract. Disruption of the phyA locus by homologous recombination resulted in loss of detectable activity in extracts and blocked hydroxylation-dependent glycosylation of Skp1 based on molecular weight analysis by SDS-PAGE, demonstrating a requirement for P4H1 in vivo. The sequence and functional similarities of P4H1 to animal HIFalpha-type P4Hs suggest that hydroxylation of Skp1 may, like that of animal HIFalpha, be regulated by availability of O(2), alpha-ketoglutarate, and ascorbate, which might exert novel control over Skp1 glycosylation.     

Glutathione S-Transferase M1, T1, P1 Genotypes and Risk for Development of Colorectal Cancer

The glutathione S-transferase (GST) supergene family is an important part of cellular enzyme defense against endogenous and exogenous chemicals, many of which have carcinogenic potential. The present investigation was conducted to detect a possible association between polymorphisms at the GSTM1, GSTT1, and GSTP1 genes and the interaction with cigarette smoking and colorectal cancer incidence. We examined 181 patients with colorectal cancer and 204 controls. DNA was extracted from whole blood, and the GSTM1, GSTT1, and GSTP1 polymorphisms were determined using a real-time polymerase chain reaction and fluorescence resonance energy transfer with a Light-Cycler instrument. Associations between specific genotypes and the development of colorectal cancer were examined by use of logistic regression analysis to calculate odds ratios (OR) and 95% confidence intervals (CI). The GSTM1 polymorphism was associated with an increased risk of developing colorectal cancer (OR = 1.62, 95% CI: 1.06–2.46). Also the risk of colorectal cancer associated with the GSTT1 null genotype was 1.64 (95% CI: 1.10–2.59). Statistically no differences were found between patients with colorectal cancer and control groups for the GSTP1 Ile/Ile, Ile/Val and Val/Val genotypes. In addition, the frequencies of the GSTM1 and GSTT1 deletion genotypes differed significantly between the cases and controls for current smokers; the GSTT1 null genotype especially is associated with a greater risk of colorectal cancer (OR = 2.44, 95% CI: 1.24–4.81). The GSTM1 and GSTT1 deletions were associated with an increased risk of developing a transverse or rectal tumor (OR = 1.86, 95% CI: 1.15–3.00; OR = 1.70, 95% CI: 1.02–2.84; respectively). The glutathione S-transferase polymorphisms were not associated with risk in patients stratified by age. The risk of colorectal cancer increased as putative high-risk genotypes increased for the combined genotypes of GSTM1 null, GSTT1 null, and either GSTP1 valine heterozygosity or GSTP1 valine homozygosity (OR = 2.69, 95% CI: 1.02–7.11). In conclusion, the results obtained in this study clearly suggest that those susceptibility factors related to different GST polymorphic enzymes are predisposing for colorectal cancer.     

The Interplay of Proton,Electron, and Metabolite Supply for Photosynthetic H2 Production in Chlamydomonas reinhardtii

To obtain a detailed picture of sulfur deprivation-induced H₂ production in microalgae, metabolome analyses were performed during key time points of the anaerobic H₂ production process of Chlamydomonas reinhardtii. Analyses were performed using gas chromatography coupled to mass spectrometry (GC/MS), two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GCxGC-TOFMS), lipid and starch analysis, and enzymatic determination of fermentative products. The studies were designed to provide a detailed metabolite profile of the solar Bio-H₂ production process. This work reports on the differential analysis of metabolic profiles of the high H₂-producing strain Stm6Glc4 and the wild-type cc406 (WT) before and during the H₂ production phase. Using GCxGC-TOFMS analysis the number of detected peaks increased from 128 peaks, previously detected by GC/MS techniques, to ∼1168. More detailed analysis of the anaerobic H₂ production phase revealed remarkable differences between wild-type and mutant cells in a number of metabolic pathways. Under these physiological conditions the WT produced up to 2.6 times more fatty acids, 2.2 times more neutral lipids, and up to 4 times more fermentation products compared with Stm6Glc4. Based on these results, specific metabolic pathways involving the synthesis of fatty acids, neutral lipids, and fermentation products during anaerobiosis in C. reinhardtii have been identified as potential targets for metabolic engineering to further enhance substrate supply for the hydrogenase(s) in the chloroplast.     

Effects of ovariectomy and tamoxifen on rat bone tissue: histomorphometric and histopathologic study

OBJECTIVE: To investigate possible detrimental effects on bone tissue induced by ovariectomy and tamoxifen (TMX) using bone densitometry and histomorphologic analysis. STUDY DESIGN: Twenty-four rats were allocated into 4 groups: group 1, intact normal rats (n = 6); group 2, ovariectomized rats (n = 6); group 3, normal female rats that received 1 mg/kg/day TMX dissolved in dimethyl sulfoxide (DMSO) for 2 months (n = 6); group 4, normal female rats that received DMSO for the same duration and with a volume equal to that of TMX (n = 6). Results of histomorphometric analysis for trabecular thickness, number of osteoblasts and osteoclasts, trabecular number, and area and cortical thickness were compared. RESULTS: No significant effects of ovariectomy on femoral or lumbar bone mineral density (BMD) were found. In the TMX group, the value of femoral BMD increased significantly compared to control group cellular and pathologic changes. TMX caused significant decrease in osteoblasts compared to the control group. CONCLUSION: TMX has a positive effect on inorganic bone tissue, but a negative effect on number of osteoblasts and osteoclasts. Future studies investigating estrogenic and antiestrogenic effects of TMX should include cellular parameters related to proliferation using histopathologic and histomorphometric analyses.     

N-acetyltransferase 2 gene polymorphism in patients with colorectal carcinoma

The acetylation polymorphism is a common inherited variation in human drug and carcinogen metabolism. Because N- acetyltransferase (NAT2) is important for the detoxification and/or bioactivation of drugs and carcinogens, polymorphisms of this gene have important implications in therapeutics of and susceptibility to cancer. In this study, NAT2 genotype (NAT2*5A (C(481)T), NAT2*6A (G(590)A), NAT2*7A/B (G(857)A)) and NAT2*14A (G(191)A) and phenotype were determined in 125 patients with colorectal carcinoma and 82 healthy control in Mersin, a city located in the southern region of Turkey. Isolation of the subjects' DNA was performed by using a highly purified PCR template preparation kit/(Roche Diagnostics cat. no: 1 796 828) and the NAT2 polymorphism was detected using real-time PCR (Roche Diagnostics, GmbH, Mannheim, Germany). According to this study high protein intake is associated with the increased risk for the development of colon cancer (OR = 1.73; 95% CI, 1.10-3.07). Although only NAT2*14A fast type was associated with increased risk in patients with colorectal carcinoma (OR = 3.03; 95% CI, 1.56-5.86), when a high protein diet was considered, NAT2*7A/B fast genotype was also found to be associated with an increased risk (OR = 2.06, 95% CI for NAT2*7A/B, 1.10-3.86; OR = 2.65; 95% CI, 1.29-5.46 for NAT2*14A). Smoking status did not differ between the control and patient groups. Our data suggest that exposure to carcinogens through consumption of a high-protein diet may increase the risk of colorectal carcinoma only in genetically-susceptible individuals.