Advanced glycation end products enhance reactive oxygen and nitrogen species generation in neutrophils in vitro

Increased oxidative stress (OS) in diabetes mellitus is one of the major factors leading to diabetic pathology. However, the mediators and mechanism that provoke OS in diabetes is not fully understood, and it is possible that accumulation of advanced glycation end products (AGEs) formed secondary to hyperglycemic conditions may incite circulating polymorphonuclear neutrophils (PMN) to generate reactive oxygen species (ROS). In this report, we aim to investigate the effect of AGE on reactive oxygen and nitrogen species generation and subsequent OS in PMN. AGE-HSA exert dose- and time-dependent enhancement of ROS and reactive nitrogen intermediates (RNI) generation by PMN. Increased ROS and RNI generation were found to be mediated through the upregulation of NADPH oxidase and inducible nitric oxide synthase (iNOS), respectively, as evident from the fact that AGE-treated neutrophils failed to generate ROS and RNI in presence of diphenyleneiodonium, a flavoprotein inhibitor for both enzymes. Further increased generation of ROS and RNI ceased when the cells were incubated with anti-RAGE antibody suggesting the involvement of AGE-RAGE interaction. Also increased malondialdehyde (MDA) and protein carbonyl formation in AGE-exposed PMN suggest induction of OS by AGE. This study provides evidence that AGEs may play a key role in the induction of oxidative stress through the augmentation of PMN-mediated ROS and RNI generation and this may be in part responsible for development of AGE-induced diabetic pathology.     

Discovery of nor-seco himbacine analogs as thrombin receptor antagonists

Discovery of a novel nor-seco himbacine analog as potent thrombin receptor (PAR-1) antagonist is described. Despite low plasma level, these new analogs showed excellent ex vivo efficacy in the monkey platelet aggregation assay. A potent hydroxy metabolite generated in vivo was identified as the agent responsible for the ex vivo efficacy. Following this discovery, the metabolite series was optimized to obtain analogs that showed very good ex vivo efficacy along with excellent pharmacokinetic profile in c. monkey.     

Design of inhibitors of Helicobacter pylori glutamate racemase as selective antibacterial agents: Incorporation of imidazoles onto a core pyrazolopyrimidinedione scaffold to improve bioavailabilty

Structure-activity relationships are presented around a series of pyrazolopyrimidinediones that inhibit the growth of Helicobacter pylori by targeting glutamate racemase, an enzyme that provides d-glutamate for the construction of N-acetylglucosamine-N-acetylmuramic acid peptidoglycan subunits assimilated into the bacterial cell wall. Substituents on the inhibitor scaffold were varied to optimize target potency, antibacterial activity and in vivo pharmacokinetic stability. By incorporating an imidazole ring at the 7-position of scaffold, high target potency was achieved due to a hydrogen bonding network that occurs between the 3-position nitrogen atom, a bridging water molecule and the side chains Ser152 and Trp244 of the enzyme. The lipophilicity of the scaffold series proved important for expression of antibacterial activity. Clearances in vitro and in vivo were monitored to identify compounds with improved plasma stability. The basicity of the imidazole may contribute to increased aqueous solubility at lower pH allowing for improved oral bioavailability.     

Constraints on the coevolution of contemporary human males and females

Because autosomal genes in sexually reproducing organisms spend on average half their time in each sex, and because the traits that they influence encounter different selection pressures in males and females, the evolutionary responses of one sex are constrained by processes occurring in the other sex. Although intralocus sexual conflict can restrict sexes from reaching their phenotypic optima, no direct evidence currently supports its operation in humans. Here, we show that the pattern of multivariate selection acting on human height, weight, blood pressure and glucose, total cholesterol, and age at first birth differs significantly between males and females, and that the angles between male and female linear (77.8 ± 20.5°) and nonlinear (99.1 ± 25.9°) selection gradients were closer to orthogonal than zero, confirming the presence of sexually antagonistic selection. We also found evidence for intralocus sexual conflict demonstrated by significant changes in the predicted male and female responses to selection of individual traits when cross-sex genetic covariances were included and a significant reduction in the angle between male- and female-predicted responses when cross-sex covariances were included (16.9 ± 15.7°), compared with when they were excluded (87.9 ± 31.6°). We conclude that intralocus sexual conflict constrains the joint evolutionary responses of the two sexes in a contemporary human population.     

Dynamics of microRNAs in bull spermatozoa

Background

Degenerative effects of critical regulators of reproduction, the kisspeptin peptides, on cellular aspects of sexually immature male gonads are known but similar information on accessory sex glands remain elusive.

Methods

Prepubertal laboratory rats were injected kisspeptin-10 at three different dosage concentrations (10 pg, 1 ng and 1 microgram) for a period of continuous 12 days at the rate of two doses per day. Control rats were maintained in parallel. The day following the end of the experimental period, seminal vesicles were removed and processed for light and electron microscopic examination using the standard methods. DNA damage was estimated by DNA ladder assay and DNA fragmentation assay.

Results

The results demonstrated cellular degeneration. Epithelial cell height of seminal vesicles decreased significantly at all doses (P < 0.05). Marked decrease in epithelial folds was readily noticeable, while the lumen was dilated. Ultrastructural changes were characterized by dilatation of endoplasmic reticulum and Golgi complex, heterochromatization of nuclei, invagination of nuclear membranes and a decreased number of secretory granules. Percent DNA damage to the seminal vesicle was 19.54 +/- 1.98, 38.06 +/- 2.09 and 58.18 +/- 2.59 at 10 pg, 1 ng and 1 microgram doses respectively.

Conclusion

The study reveals that continuous administration of kisspeptin does not lead to an early maturation but instead severe degeneration of sexually immature seminal vesicles.     

Association of genetic polymorphisms in glutathione S-transferases and susceptibility to head and neck cancer

Polymorphism in glutathione S-transferase (GST) genes (GSTM1, GSTT1 and GSTP1) and interaction with environmental factors such as tobacco (smoking or chewing) and alcohol on susceptibility to head and neck squamous cell carcinoma (HNSCC) was studied in a case-control study. The study group consisted of 175 patients suffering from HNSCC and 200 age matched healthy controls. Statistical analysis showed an increase in risk to HNSCC in the patients with null genotype of GSTM1 (OR: 2.02; 95% CI: 1.32-3.10; P=0.001) or GSTT1 (OR: 1.66; 95% CI: 1.02-2.69; P=0.04), though the risk was not found to be significant when adjusted for age, sex, smoking, tobacco chewing or alcohol use by multivariate logistic regression model. Our data further showed that combination of deletion genotypes of GST (GSTM1 and GSTT1) confer an even higher risk of HNSCC. Interestingly, GSTP1 wild type genotype in combination with GSTM1 null or GSTT1 null genotype increased susceptibility for HNSCC (OR: 2.49 and 2.75, respectively). Likewise a much greater risk for HNSCC was observed in the patients carrying a genotype combination of GSTM1 null, GSTT1 null and GSTP1 (Ile/Ile) (OR: 4.47; 95% CI: 1.62-12.31; P=0.002). Our data have further provided evidence that tobacco chewing and alcohol consumption are the important risk factors for HNSCC. The interaction between tobacco chewing and null genotype of GSTM1 or GSTT1 resulted in about 3.5- and 2.2-fold increase in the risk respectively in the patients when compared to those not chewing tobacco. Alcohol use resulted in more than 4-fold increase in the risk in the patients with null genotype of GSTM1 as compared to those who are non-drinkers. Alcohol consumption also increased the risk (approx. 3-fold) in the cases with null genotype of GSTT1, though the association was not found to be significant when compared to non-drinkers. Our data have provided evidence that GST polymorphism modifies the susceptibility to HNSCC and have further demonstrated importance of gene-environment interaction in modulating the risk to HNSCC.     

Ability of mice to detect estrous odor in bovine urine: roles of hormones and behavior in odor discrimination

This study was designed to evaluate the ability of mice to discriminate cow urinary odor from different reproductive phases, with a view toward detecting the estrous phase. Experiments were also carried out to establish the relationship between androgen and mouse behaviors during estrous detection. Further, the study was also intended to establish the relationship between androgen and behaviors in estrous detection. Bovine urine was collected during estrus and non-estrous periods, i.e., prepubertal, preovulatory, ovulatory, postovulatory, pregnancy, and lactation. Behavioral analyses were carried out in a Y-maze apparatus, in which the mice were acclimatized in before odor-preference tests. The number and duration of visits, and grooming behavior by male responders towards the urine samples, were recorded. Intact male mice showed a higher response towards estrus urine samples than towards non-estrous urine. By contrast, orchidectomized mice failed to discriminate estrous urine, whereas castrated mice treated with testosterone regained the ability to discriminate estrus odor. A higher level of grooming behavior was found in males exposed to estrous urine than to urine of other phases. These results suggest that normal mice have the ability to detect estrus, and that this discriminating ability is androgen dependent. The grooming behavior shown by males in response to estrous urine may be taken as a key parameters in estrous detection. The results further suggest that bovine estrous urine produces specific odors that probably involve both intraspecific and interspecific communication.     

Role of histidine residues in EcoP15I DNA methyltransferase activity as probed by chemical modification and site-directed mutagenesis

Towards understanding the catalytic mechanism of M.EcoP15I [EcoP15I MTase (DNA methyltransferase); an adenine methyltransferase], we investigated the role of histidine residues in catalysis. M.EcoP15I, when incubated with DEPC (diethyl pyrocarbonate), a histidine-specific reagent, shows a time- and concentration-dependent inactivation of methylation of DNA containing its recognition sequence of 5'-CAGCAG-3'. The loss of enzyme activity was accompanied by an increase in absorbance at 240 nm. A difference spectrum of modified versus native enzyme shows the formation of N-carbethoxyhistidine that is diminished by hydroxylamine. This, along with other experiments, strongly suggests that the inactivation of the enzyme by DEPC was specific for histidine residues. Substrate protection experiments show that pre-incubating the methylase with DNA was able to protect the enzyme from DEPC inactivation. Site-directed mutagenesis experiments in which the 15 histidine residues in the enzyme were replaced individually with alanine corroborated the chemical modification studies and established the importance of His-335 in the methylase activity. No gross structural differences were detected between the native and H335A mutant MTases, as evident from CD spectra, native PAGE pattern or on gel filtration chromatography. Replacement of histidine with alanine residue at position 335 results in a mutant enzyme that is catalytically inactive and binds to DNA more tightly than the wild-type enzyme. Thus we have shown in the present study, through a combination of chemical modification and site-directed mutagenesis experiments, that His-335 plays an essential role in DNA methylation catalysed by M.EcoP15I.     

Induction of early blight resistance in tomato by <Emphasis Type="Italic">Quercus infectoria</Emphasis> gall extract in association with accumulation of phenolics and defense-related enzymes

Treatment of tomato leaves with aqueous extract (0.5%) of the galls of Quercus infectoria significantly reduced infection from subsequent inoculation with Alternaria solani, the tomato early blight pathogen. When the leaves were challenge-inoculated with A. solani 3 d after application of Q. infectoria gall extract (QIGE), the percent defoliation decreased from 33.6 to 7.3. Two to three day pre-treatment with QIGE reduced the percent defoliation by 77 percent. The biochemical responses of tomato plants to QIGE were also studied. In tomato plants treated with QIGE, phenolic content increased rapidly, reached the maximum at 2 d after treatment. Phenylalanine ammonia-lyase (PAL) activity increased significantly from 1 d after treatment and the maximum enzyme activity was recorded 2 d after treatment at which period a 3-fold increase in PAL activity was observed when compared to the control. Peroxidase (PO) activity was also significantly increased 1 d after treatment and the maximum activity was reached 2 d after treatment. Peroxidase isozyme analysis indicated that PO-1 was increased dramatically in tomato leaves 1 d after treatment and maintained at the same level throughout the experimental period of 6 d. When tomato leaves were treated with QIGE, a two-fold increase in chitinase and β-1,3-glucanase activities was recorded 2 and 3 d respectively, after treatment. The enhanced activities of defense-related enzymes and elevated levels of phenolics in QIGE-treated tomato plants between 1 and 3 d after treatment suggest that these induced biochemical defenses may be involved in the suppression of early blight by QIGE.