Biochemical characterization of some pyrazolopyrimidine-based inhibitors of xanthine oxidase

Inhibition of xanthine oxidase-catalyzed conversion of xanthine to uric acid by various pyrazolopyrimidine-based inhibitors (allopurinol derivatives) was evaluated and compared with the standard inhibitor allopurinol. Three compounds out of the seven compounds used in the study were found to be reasonably good inhibitors of xanthine oxidase (XO). 4-Amino-6-mercaptopyrazolo-3,4-d-pyrimidine was found to be the most potent inhibitor of XO (IC50 = 0.600 +/- 0.009 microM). 4-Mercapto-1H-pyrazolo-3,4-d-pyrimidine (IC50 = 1.326 +/- 0.013 microM) and 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine (IC50 = 1.564 +/- 0.065 microM) also showed comparable inhibitory activity to that of allopurinol (IC50 = 0.776 +/- 0.012 microM). All three compounds showed competitive type of inhibition with comparable Ki values. Induction of the electron transfer reaction catalyzed by XO in the presence of these compounds monitored as reduction of 2,6-dichlorophenolindophenol (DCPIP) revealed that electron transfer by 4-amino-6-mercaptopyrazolo-3,4-d-pyrimidine is comparable to that obtained by allopurinol or xanthine. However, 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine and 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine did not show DCPIP reduction. On the other hand, enzymatic reduction of cytochrome c in the presence of the three compounds was found to be insignificant and much less in comparison to allopurinol and xanthine. Therefore, both 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine and 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine displayed the inhibitory property and also did not produce XO-mediated reactive oxygen species (ROS). Since 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine was found to have some toxicity, the effect of 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine on the enzymatic formation of uric acid and ROS was investigated and it was found that this compound was inhibiting enzymatic generation of both uric acid and ROS. It can be noted that the standard inhibitor, allopurinol, inhibits uric acid formation but produces ROS.     

Brd4 is required for recovery from antimicrotubule drug-induced mitotic arrest: preservation of acetylated chromatin

The mammalian bromodomain protein Brd4 interacts with mitotic chromosomes by binding to acetylated histone H3 and H4 and is thought to play a role in epigenetic memory. Mitotic cells are susceptible to antimicrotubule drugs. These drugs activate multiple response pathways and arrest cells at mitosis. We found that Brd4 was rapidly released from chromosomes upon treatment with antimicrotubule drugs, including the reversible agent nocodazole. Yet, when nocodazole was withdrawn, Brd4 was reloaded onto chromosomes, and cells proceeded to complete cell division. However, cells in which a Brd4 allele was disrupted (Brd4+/-), and expressing only half of the normal Brd4 levels, were defective in reloading Brd4 onto chromosomes. Consequently, Brd4+/- cells were impaired in their ability to recover from nocodazole-induced mitotic arrest: a large fraction of +/- cells failed to reach anaphase after drug withdrawal, and those that entered anaphase showed an increased frequency of abnormal chromosomal segregation. The reloading defect observed in Brd4+/- cells coincided with selective hypoacetylation of lysine residues on H3 and H4. The histone deacetylase inhibitor trichostatin A increased global histone acetylation and perturbed nocodazole-induced Brd4 unloading. Brd4 plays an integral part in a cellular response to drug-induced mitotic stress by preserving a properly acetylated chromatin status.     

Chronic hypobaric hypoxia mediated skeletal muscle atrophy: role of ubiquitin–proteasome pathway and calpains

The most frequently reported symptom of exposure to high altitude is loss of body mass and decreased performance which has been attributed to altered protein metabolism affecting skeletal muscles mass. The present study explores the mechanism of chronic hypobaric hypoxia mediated skeletal muscle wasting by evaluating changes in protein turnover and various proteolytic pathways. Male Sprague-Dawley rats weighing about 200 g were exposed to hypobaric hypoxia (7,620 m) for different durations of exposure. Physical performance of rats was measured by treadmill running experiments. Protein synthesis, protein degradation rates were determined by (14)C-Leucine incorporation and tyrosine release, respectively. Chymotrypsin-like enzyme activity of the ubiquitin-proteasome pathway and calpains were studied fluorimetrically as well as using western blots. Declined physical performance by more than 20%, in terms of time taken in exhaustion on treadmill, following chronic hypobaric hypoxia was observed. Compared to 1.5-fold increase in protein synthesis, the increase in protein degradation was much higher (five-folds), which consequently resulted in skeletal muscle mass loss. Myofibrillar protein level declined from 46.79 ± 1.49 mg/g tissue at sea level to 37.36 ± 1.153 (P < 0.05) at high altitude. However, the reduction in sarcoplasmic proteins was less as compared to myofibrillar protein. Upregulation of Ub-proteasome pathway (five-fold over control) and calpains (three-fold) has been found to be important factors for the enhanced protein degradation rate. The study provided strong evidences suggesting that elevated protein turnover rate lead to skeletal muscle atrophy under chronic hypobaric hypoxia via ubiquitin-proteasome pathway and calpains.     

Reduction of Salmonella enterica serovar enteritidis colonization in 20-day-old broiler chickens by the plant-derived compounds trans-cinnamaldehyde and eugenol

The efficacies of trans-cinnamaldehyde (TC) and eugenol (EG) for reducing Salmonella enterica serovar Enteritidis colonization in broiler chickens were investigated. In three experiments for each compound, 1-day-old chicks (n = 75/experiment) were randomly assigned to five treatment groups (n = 15/treatment group): negative control (-ve S. Enteritidis, -ve TC, or EG), compound control (-ve S. Enteritidis, +ve 0.75% [vol/wt] TC or 1% [vol/wt] EG), positive control (+ve S. Enteritidis, -ve TC, or EG), low-dose treatment (+ve S. Enteritidis, +ve 0.5% TC, or 0.75% EG), and high-dose treatment (+ve S. Enteritidis, +ve 0.75% TC, or 1% EG). On day 0, birds were tested for the presence of any inherent Salmonella (n = 5/experiment). On day 8, birds were inoculated with ～8.0 log(10) CFU S. Enteritidis, and cecal colonization by S. Enteritidis was ascertained (n = 10 chicks/experiment) after 24 h (day 9). Six birds from each treatment group were euthanized on days 7 and 10 after inoculation, and cecal S. Enteritidis numbers were determined. TC at 0.5 or 0.75% and EG at 0.75 or 1% consistently reduced (P < 0.05) S. Enteritidis in the cecum (≥3 log(10) CFU/g) after 10 days of infection in all experiments. Feed intake and body weight were not different for TC treatments (P > 0.05); however, EG supplementation led to significantly lower (P < 0.05) body weights. Follow-up in vitro experiments revealed that the subinhibitory concentrations (SICs, the concentrations that did not inhibit Salmonella growth) of TC and EG reduced the motility and invasive abilities of S. Enteritidis and downregulated expression of the motility genes flhC and motA and invasion genes hilA, hilD, and invF. The results suggest that supplementation with TC and EG through feed can reduce S. Enteritidis colonization in chickens.     

Genetic Diversity and Population Structure of Indian Golden Silkmoth (Antheraea assama)

Background

The Indian golden saturniid silkmoth (Antheraea assama), popularly known as muga silkmoth, is a semi-domesticated silk producing insect confined to a narrow habitat range of the northeastern region of India. Owing to the prevailing socio-political problems, the muga silkworm habitats in the northeastern region have not been accessible hampering the phylogeography studies of this rare silkmoth. Recently, we have been successful in our attempt to collect muga cocoon samples, although to a limited extent, from their natural habitats. Out of 87 microsatellite markers developed previously for A. assama, 13 informative markers were employed to genotype 97 individuals from six populations and analyzed their population structure and genetic variation.

Methodology/Principal Findings

We observed highly significant genetic diversity in one of the populations (WWS-1, a population derived from West Garo Hills region of Meghalaya state). Further analysis with and without WWS-1 population revealed that dramatic genetic differentiation (global F_ST = 0.301) was due to high genetic diversity contributed by WWS-1 population. Analysis of the remaining five populations (excluding WWS-1) showed a marked reduction in the number of alleles at all the employed loci. Structure analysis showed the presence of only two clusters: one formed by WWS-1 population and the other included the remaining five populations, inferring that there is no significant genetic diversity within and between these five populations, and suggesting that these five populations are probably derived from a single population. Patterns of recent population bottlenecks were not evident in any of the six populations studied.

Conclusions/Significance

A. assama inhabiting the WWS-1 region revealed very high genetic diversity, and was genetically divergent from the five populations studied. The efforts should be continued to identify and study such populations from this region as well as other muga silkworm habitats. The information generated will be very useful in conservation of dwindling muga culture in Northeast India.     

Concise synthesis of the pentasaccharide O-antigen of Escherichia coli O83:K24:H31 present in the Colinfant vaccine

A block synthetic approach is presented for the synthesis of the pentasaccharide repeating unit of the O-antigen of E. coli O83:K24:H31 strain, present in the “Colifant” vaccine. The target pentasaccharide has been synthesized by coupling a disaccharide with a trisaccharide in excellent yield. Yields are quite satisfactory in all intermediate steps. A concise synthesis of the pentasaccharide repeating unit of the O-antigen of E. coli O83:K24:H31 strain, present in the COLINFANT vaccine is presented. The target pentasaccharide has been synthesized following a block synthetic strategy by coupling a disaccharide with a trisaccharide in excellent yield.