Diminished reproductive potential of male mice in response to selenium‐induced oxidative stress: Involvement of HSP70, HSP70‐2, and MSJ‐1

The oxidative stress imposed by nutritional variations in selenium (Se) has plausible role in reproductive toxicology and affects the reproductive potential. Also, the expression of heat shock proteins (HSPs) is a highly regulated event throughout the process of spermatogenesis and is modulated by stressful stimuli. This prompted us to investigate the possibility that Se‐induced oxidative stress may affect the fertility status by altering the expressions of the constitutive and inducible HSP70 proteins, having crucial role in spermatogenesis. Different Se status‐deficient, adequate, and excess, male Balb/c mice were created by feeding yeast‐based Se‐deficient diet (group I) and deficient diet supplemented with Se as sodium selenite at 0.2 and 1 ppm Se (group II and III) for a period of 8 weeks. After completion of the diet‐feeding schedule, a significant decrease in the Se and glutathione peroxidase (GSH‐Px) levels was observed in the Se‐deficient group (I), whereas Se‐excess group (III) demonstrated an increase. Increased levels of reactive oxygen species, malondialdehyde, and alterations in the redox status in both groups I and III indicated oxidative‐stressed conditions. There was an overall reduced fertility status in mice supplemented with Se‐deficient and Se‐excess diet. The mRNA and protein expression of HSP70 was found to be elevated in these two groups, whereas the expression patterns of HSP70‐2 and MSJ‐1 demonstrated a reverse trend. In vitro CDC2 kinase assay showed reduced kinase activity in group I and group III. These findings suggest that Se‐induced oxidative stress by differentially regulating various HSP70s can affect its downstream factors having crucially important role in differentiation of germ cells and completion of spermatogenesis. Therefore, it can provide an insight into the mechanism(s) by which the oxidative stress–induced reproductive toxicity can lead to increased apoptosis/growth arrest and infertility. This will thus add new dimensions to the molecular mechanism underlying the human male infertility and open new vistas in the development of various chemo‐preventive methods. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:125–136, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20276     

Morphogenesis in Isolated Microspore Cultures of <Emphasis Type="Italic">Brassica juncea</Emphasis>

Androgenesis is a phenomenon in which microspores are made to bypass the sexual pathway and follow the sporophytic mode of development to generate new plants without the intervention of fertilization under specialized in vitro conditions. Microspore culture provides an ideal system, with a large, relatively uniform population of haploid cells, for use in mutant selection, genetic transformation and in studies on the molecular mechanism of induction of androgenesis and embryogenesis. This paper involves a study on establishing a reproducible and efficient protocol for microspore embryogenesis in various varieties of Brassica juncea. The genotype had a pronounced effect on androgenic response in microspore cultures. The cultivar Rajat exhibited the most response, producing around 3500 embryos/100 buds. The microspores of B. juncea cv. PR-45 from ed plants maintained at a day/night temperature of 10 °C/5 °C form embryos with suspensors with varied morphology. The microspore embryos germinated to produce plants with frequencies. These plants exhibited 52% survival and 74% fertility.     

Sequence preference for BI/BII conformations in DNA: MD and crystal structure data analysis

Deciphering sequence information from sugar-phosphate backbone is finely tuned through the conformational substates of DNA. BII conformation, one of the conformational substates of B-DNA, is known to play a key role in DNA-protein recognition. BI and BII are identified by the epsilon-zeta difference, which is negative in BI and positive in BII. Our analysis of MD and crystal structures shows that BII conformation is sequence specific and dinucleotides GC, CG, CA, TG, TA show high preference to take up BII conformation, while TT, TC, CT, CC dinucleotides rarely take up this conformation. Significant changes were observed in the dinucleotide parameters viz. twist, roll, and slide for the steps having BII conformation. Interestingly, the magnitude of variation in the dinucleotide parameters is seen to depend mainly on two factors, the magnitude of epsilon-zeta difference and the presence or absence of BII conformation in the second strand, across the WC base-paired dinucleotide step. Based on these two factors, the conformational substate of a dinucleotide step can be further classified as BI.BI (BI conformation in both strands), BI.BII (BI conformation in one strand and BII conformation in the other), and BII.BII (BII conformation in both strands). The occurrence of BII in both strands was found to be quite rare and thus, it can be concluded that BI.BI and BI.BII hybrid steps are more favorable than a BII.BII step. In conformity with the sequence preference seen for dinucleotides in each strand, BII.BII combination of backbone conformation was observed only for GC, CG, CA, and TG containing dinucleotide steps. We further classified BII.BII step as strong BII and weak BII depending on the magnitude of the average epsilon-zeta difference. The dinucleotide steps which belong to the category of strong BII, have large twist, high positive slide and negative roll values, while those in the weak BII group have roll, twist, and slide values similar to that of hybrid BI.BII steps. This conformational property could be contributing to the groove opening/closing and thus can modulate protein-DNA interaction.     

NMR studies of exchange between intra- and extracellular glutathione in human erythrocytes

Glutathione is the main source of intracellular antioxidant protection in the human erythrocyte and its redox status has frequently been used as a measure of oxidative stress. Extracellular glutathione has been shown to enhance intracellular reduced glutathione levels in some cell types. However, there are conflicting reports in the literature and it remains unclear as to whether erythrocytes can utilise extracellular glutathione to enhance the intracellular free glutathione pool. We have resolved this issue using a 13C-NMR approach. The novel use of L-gamma-glutamyl-L-cysteinyl-[2-13C]glycine allowed the intra- and extracellular glutathione pools to be distinguished unequivocally, enabling the direct and non-invasive observation over time of the glutathione redox status in both compartments. The intracellular glutathione redox status was measured using 1H spin-echo NMR, while 13C[1H-decoupled] NMR experiments were used to measure the extracellular status. Extracellular glutathione was not oxidised in the incubations, and did not affect the intracellular glutathione redox status. Extracellular glutathione also did not affect erythrocyte glucose metabolism, as measured from the lactate-to-pyruvate ratio. The results reported here refute the previously attractive hypothesis that, in glucose-starved erythrocytes, extracellular GSH can increase intracellular GSH concentrations by releasing bound glutathione from mixed disulfides with membrane proteins.     

Effect of cholesterol and 7-beta hydroxycholesterol on glutathione status and nitric oxide production in murine peritoneal macrophages

Present study was conducted to observe the effect of cholesterol and oxidized cholesterol (7beta-hydroxycholesterol,7beta-OH) on the nitric oxide (NO) production and the redox ratio by lipopolysaccharide-stimulated macrophages. Dose-dependent decrease in NO levels was seen with both cholesterol and 7beta-OH at different incubation intervals (6,12,18,24 hr) and concentrations (2.5,5,7.5microg/ml). On comparison, a significant decrease in the NO was observed at 24 hr interval in 7beta-OH exposed cells with all respective concentrations of cholesterol. Incubation with 7beta-OH also resulted in significant increase in levels of oxidized glutathione (GSSG) and decrease in reduced glutathione (GSH), while cholesterol showed no effect on GSSG levels. Moreover, GSH levels were lowered only at highest concentration (7.5microg/ml), and at longer incubation intervals (18,24 hr) with cholesterol exposure. This altered the redox status in both cholesterol/7beta-OH treated macrophages. Increased redox ratio and decreased NO levels indicated increased oxidative stress and decreased vasodilation by 7beta-OH compared to cholesterol.     

Phospholipid metabolism and protein kinase C mediated protein phosphorylation in dietary protein deficiency in rat lung

Nutritional deprivation of proteins decreases the protein kinase C (PKC) activity in rat lung. The activity of (PKC) is influenced by lipid metabolism. Changes in PKC activity may influence phosphorylation of its substrate proteins in the tissues. Therefore, alterations in phospholipid metabolism and PKC mediated protein phosphorylation in dietary protein deficiency in rat lung were envisaged. The study was conducted on rats fed on three different types of diet viz., casein (20% protein), deficient (4% protein, rice flour as source of protein) and supplemented (deficient diet supplemented with L-lysine and DL-threoning). Feeding of protein deficient diet caused reduction in incorporation of [3H] myo-inositol in the total phosphoinositides in lungs and an increase in total inositol phosphate pool. There was a significant reduction in the contents and turnover rate of phosphatidyl inositol and phosphatidyl inositol monophosphate. Supplementation of diet with L-lysine and DL-threonine had a reversing effect on total pool of phosphoinositides and, the metabolism of phosphatidyl inositol bisphosphate and phosphatidyl inositol. In phosphatidyl choline metabolism, the dietary protein deficiency led to a decrease in incorporation of [14C-methyl] choline-chloride in total phospholipids. In contrast, its incorporation increased in phosphatidyl choline pool. The contents of phosphatidyl choline and residue, incorporation of [14C-methyl] choline-chloride in them and their turnover rate also increased. Supplementation of diet had a reversal effect on most of these parameters. Phosphorylation of proteins of 84, 47, 35 and 16 kDa was identified to be mediated by PKC. In dietary protein deficiency, phosphorylation of all these proteins, except that of 47 kDa, increased. Supplementation of diet reversed the pattern except that of 84 kDa. The findings suggest that changes in phospholipid metabolism in dietary protein deficiency may effect the activity of PKC thereby influencing the phosphorylation of its substrate proteins and hence associated functions that may lead to pathophysiology of lung.     

Role of p-glycoprotein expression in predicting response to neoadjuvant chemotherapy in breast cancer-a prospective clinical study

Background

Lung cancer still remains one of the most commonly occurring solid tumors and even in stage Ia, surgery fails in 30% of patients who develop distant metastases. It is hypothesized that these must have developed from occult circulating tumor cells present at the time of surgery, or before. The aim of the present study was to detect such cells in the peripheral blood and to monitor these cells following surgery.

Methods

30 patients treated for lung cancer with surgery were monitored for circulating epithelial cells (CEC) by taking peripheral blood samples before, 2 weeks and 5 months after surgery and/or radiotherapy (RT) chemotherapy (CT) or combined RT/CT using magnetic bead enrichment and laser scanning cytometry (MAINTRAC^®) for quantification of these cells.

Results

In 86% of the patients CEC were detected before surgery and in 100% at 2 weeks and 5 months after surgery. In the control group, which consisted of 100 normal donors without cancer, 97 % were negative for CEC. A significantly higher number of CEC was found preoperatively in patients with squamous cell carcinoma than in those with adenocarcinoma. In correlation to the extent of parenchymal manipulation 2 weeks after surgery, an increase in numbers of CEC was observed with limited resections (18/21) whereas pneumonectomy led to a decrease (5/8) of CEC, 2 weeks after surgery. The third analysis done 5 months after surgery identified 3 groups of patients. In the group of 5 patients who received neo- or adjuvant chemo/radiotherapy there was evidence that monitoring of CEC can evaluate the effects of therapy. Another group of 7 patients who underwent surgery only showed a decrease of CEC and no signs of relapse. A third group of 11 patients who had surgery only, showed an increase of CEC (4 with an initial decrease after surgery and 7 with continuous increase). In the group with a continuous increase during the following 24 months, 2 early relapses in patients with stage Ia adenocarcinoma were observed. The increase of CEC preceded clinical detection by six months.

Conclusion

We consider, therefore, that patients with adenocarcinoma and a continuous increase of CEC after complete resection for lung cancer are at an increased risk of early relapse.     

Cleavage of disulfide-linked fetuin-bisphosphonate conjugates with three physiological thiols

An effective therapeutic agent for treatment of bone diseases is expected to exhibit a high affinity to bone. Conjugating proteins to bisphosphonates (BPs), a class of molecules with an exceptional affinity to bone mineral hydroxyapatite (HA), is a feasible means to impart such a bone affinity. Protein-BP conjugates with cleavable linkages, which allow protein release from the mineral, are preferable over conjugates with stable linkages. To this end, 2-(3-mercaptopropylsulfanyl)-ethyl-1,1-bisphosphonic acid (thiolBP) was conjugated onto fetuin, a model protein, using N-succinimidyl-3-(2-pyridyldithio)propionate to create disulfide-linked conjugates. Although the fetuin-thiolBP conjugates were stable under aqueous conditions, the disulfide linkage was readily cleaved in the presence of the physiological thiols l-cysteine, dl-homocysteine, and l-glutathione. dl-Homocysteine exhibited the highest cleavage of the disulfide linkage among these thiols. The imparted bone affinity as a result of thiolBP conjugation, as assessed by HA binding in vitro, was eliminated upon cleavage of the disulfide linkage. The cleavage of the conjugates bound to HA was as effective as the conjugate cleavage in solution, and even more so at high concentrations of l-glutathione. In conclusion, disulfide-linked fetuin-thiolBP conjugates exhibited a high affinity to HA, which was readily lost upon cleavage with thiols found in physiological milieu.