Synthesis and peptide incorporation of an unnatural amino acid containing activity-based probe for protein tyrosine phosphatases

An unnatural amino acid was synthesized to incorporate a quinone methide-generating activity-based probe for protein tyrosine phosphatases (PTPs) and then integrated into a PTP1B-specific substrate. The resulting probe led to preferential labeling of PTP1B in cell lysates in the presence of PTP4A3.     

Crystal structures of human pyridoxal kinase in complex with the neurotoxins, ginkgotoxin and theophylline: insights into pyridoxal kinase inhibition

Several drugs and natural compounds are known to be highly neurotoxic, triggering epileptic convulsions or seizures, and causing headaches, agitations, as well as other neuronal symptoms. The neurotoxic effects of some of these compounds, including theophylline and ginkgotoxin, have been traced to their inhibitory activity against human pyridoxal kinase (hPL kinase), resulting in deficiency of the active cofactor form of vitamin B(6), pyridoxal 5'-phosphate (PLP). Pyridoxal (PL), an inactive form of vitamin B(6) is converted to PLP by PL kinase. PLP is the B(6) vitamer required as a cofactor for over 160 enzymatic activities essential in primary and secondary metabolism. We have performed structural and kinetic studies on hPL kinase with several potential inhibitors, including ginkgotoxin and theophylline. The structural studies show ginkgotoxin and theophylline bound at the substrate site, and are involved in similar protein interactions as the natural substrate, PL. Interestingly, the phosphorylated product of ginkgotoxin is also observed bound at the active site. This work provides insights into the molecular basis of hPL kinase inhibition and may provide a working hypothesis to quickly screen or identify neurotoxic drugs as potential hPL kinase inhibitors. Such adverse effects may be prevented by administration of an appropriate form of vitamin B(6), or provide clues of how to modify these drugs to help reduce their hPL kinase inhibitory effects.     

Evaluation of complementary effects of 9,10-anthraquinone and fumaric acid on methanogenesis and ruminal fermentation in vitro

The objective of the present study was to investigate the hypothesis that 9,10-anthraquinone (AQ) in combination with fumaric acid (FMA) may provide complementary effects to inhibit methanogens and enhance rumen's capacity for better utilisation of FMA towards propionate production. Three levels of AQ and four levels of FMA were tested in a 3 x 4 factorial design using in vitro gas production technique. AQ reduced the total gas and methane production significantly. The combination of 4 ppm AQ with FMA had additive effect on concentration of propionate. Supplementation of AQ alone resulted in hydrogen accumulation (p < 0.001), whereas presence of FMA (up to 6.5 mM) along with AQ declined hydrogen concentration (p < 0.001). The level of 4 ppm AQ did not affect in vitro digestibility, however, a reduction of organic matter digestibility was caused by 8 ppm AQ (p < 0.001), which was partially compensated by the addition of FMA (p = 0.06). The optimum FMA level depended on the AQ concentration. At 4 ppm AQ, a FMA level of 3.5 mM had best possible effect on partitioning factor and microbial biomass production (p < 0.001), though, at 8 ppm AQ the higher level of FMA (6.5 mM) responded better. Overall, FMA in combination with AQ provided an alternative hydrogen sink and might be introduced as a novel strategy for mitigation of enteric methane emission. Nevertheless, the result should be proved by in vivo experiments.     

Avian scavengers and the threat from veterinary pharmaceuticals

Veterinary use of the non-steroidal anti-inflammatory drug diclofenac on domesticated ungulates caused populations of resident Gyps vultures in the Indian sub-continent to collapse. The birds died when they fed on carrion from treated animals. Veterinary diclofenac was banned in 2006 and meloxicam was advocated as a ‘vulture-safe’ alternative. We examine the effectiveness of the 2006 ban, whether meloxicam has replaced diclofenac, and the impact of these changes on vultures. Drug residue data from liver samples collected from ungulate carcasses in India since 2004 demonstrate that the prevalence of diclofenac in carcasses in 2009 was half of that before the ban and meloxicam prevalence increased by 44%. The expected vulture death rate from diclofenac per meal in 2009 was one-third of that before the ban. Surveys at veterinary clinics show that diclofenac use in India began in 1994, coinciding with the onset of rapid Gyps declines ascertained from measured rates of declines. Our study shows that one pharmaceutical product has had a devastating impact on Asia''s vultures. Large-scale research and survey were needed to detect, diagnose and quantify the problem and measure the response to remedial actions. Given these difficulties, other effects of pharmaceuticals in the environment may remain undetected.     

Temporal synergism of neurotransmitters (serotonin and dopamine) affects testicular development in mice

The temporal phase relation of circadian oscillations is reported to regulate reproduction in many seasonally breeding avian and mammalian species, but its role in the reproductive regulation of continuous breeders is not yet known. Hence in the present study, six experimental groups of 3-week-old male Parkes strain mice, Mus musculus, were injected with 5-hydroxytryptophan (5-HTP, serotonin precursor) and l-dihydroxyphenylalanine (l-DOPA, dopamine precursor) at intervals of 0, 4, 8, 12, 16 or 20 hr (5 mg/100 g body weight per day for 13 days). Control mice received two daily injections of normal saline. When observed 24 days post-treatment, 8-hr mice exhibited low body weight and suppression of gonadal activity (spermatogenesis, sperm count/motility/viability and plasma testosterone concentration), while body weight and degree of gonadal development were higher in the 12-hr mice as compared to the controls. It is concluded that normal somatic and gonadal growth of pre-puberal mice may be suppressed with an 8-hr phase relation of circadian serotonergic and dopaminergic oscillations. On the other hand, a 12-hr phase relation accelerated the rate of gonadal maturation, while other relations led to more or less similar gonadal development as in the control mice. This study suggests the importance of circadian organization as a function of specific temporal phase relations of neural oscillations in the maturation of gonads. Although the exact mechanism still needs to be investigated, this seems to be mediated via effects on the neuroendocrine axis.     

Functional Surfaces on the Actin-binding Protein Coronin Revealed by Systematic Mutagenesis

Coronin is a conserved actin-binding protein that co-functions with ADF/cofilin and Arp2/3 complex to govern cellular actin dynamics. Despite emerging roles for coronin in a range of physiological processes and disease states, a detailed understanding of the molecular interactions of coronin with actin and other binding partners has been lacking. Here, we performed a systematic mutational analysis of surfaces on the yeast coronin β-propeller domain, which binds to F-actin and is conserved in all coronin family members. We generated 21 mutant alleles and analyzed their biochemical effects on actin binding and ADF/cofilin activity. Conserved actin-binding residues mapped to a discrete ridge stretching across one side of the β-propeller. Mutants defective in actin binding showed loss of synergy with ADF/cofilin in severing filaments, diminished localization to actin structures in vivo, and loss of coronin overexpression growth defects. In addition, one allele showed normal actin binding but impaired functional interactions with ADF/cofilin. Another allele showed normal actin binding but failed to cause coronin overexpression defects. Together, these results indicate that actin binding is critical for many of the biochemical and cellular functions of coronin and that the β-propeller domain mediates additional functional interactions with ADF/cofilin and possibly other ligands. Conservation of the actin-binding surfaces across distant species and in all three major classes of coronin isoforms suggests that the nature of the coronin-actin association may be similar in other family members.     

Chemi-refiner mechanical pulping of flax shives: refining energy and fiber properties.

Oil-seed flax shive has been promoted as a raw material for low-end paper making because of its overall cost benefit over hardwood and groundwood pulp and increasing demand for low-cost pulp in rigid packaging applications. We have made refiner mechanical pulp from oil-seed shives by using a 300 mm Sprout-Bauer pilot refiner. The factors controlled during refining were: consistency, multi-pass refining, temperature, spacing between the plates and polyol-anhydride reactant concentration. Based on the results in this study it is apparent that unmodified shives develop low mechanical strength, shorter fibers and large amount of fines. Chemical modification of shives with a pre-reacted mixture of polyol-anhydride reduces refining energy consumption, improves strength performance but decreases scattering coefficient. From the results of investigation it is suggested that chemically modified refiner pulp from shives cannot be used for high-grade paper manufacture but it can be used as a low-cost filler for low-end applications in packaging or low-performance decorative papers.     

Inhibition of squalene synthase upregulates PCSK9 expression in rat liver

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a critical role in cholesterol metabolism by enhancing the degradation of the LDL receptor protein in the liver. It has previously been shown that administration of zaragozic acid A (ZA), a potent inhibitor of squalene synthase, also significantly increases the rate of degradation of hepatic LDL receptor protein. Thus, we decided to determine whether ZA administration might act to up regulate hepatic expression of the rat PCSK9 gene. Administration of ZA resulted in increased PCSK9 mRNA and protein levels in rat liver surprisingly in concert with an increase in hepatic LDL receptor mRNA levels, LDL receptor protein turnover, and decreased serum cholesterol levels. These observations suggest an involvement of PCSK9 in hepatic LDL receptor protein degradation and perhaps, in increasing the rate of LDL receptor cycling resulting in lower serum cholesterol levels in response to cholesterol biosynthesis inhibitors.     

Gene expression patterns of Helicoverpa armigera gut proteases

Relative quantification of reported gut proteinase cDNAs from Helicoverpa armigera larvae fed on various host plants (cotton, chickpea, pigeonpea, tomato and okra), non-host plant PIs (winged bean, bitter gourd, ground nut, and capsicum) and during larval development has been carried out using semi-quantitative RT-PCR. Five trypsin-like and three chymotrypsin-like proteinases were categorized as insensitive or sensitive to most of the proteinase inhibitors (PIs) and insensitive/sensitive to specific PIs based on their expression analysis. These results were supported by amino acid sequence analysis, where diverged amino acids were observed in the regions, which are reported to be involved in typical trypsin-trypsin inhibitor interactions and critical for proteinase inhibitor resistance. Among exopeptidases (five aminopeptidase and three carboxypeptidase), HaAmi4 and HaAmi5 of aminopeptidase and HaCar1 of carboxypeptidase exhibited considerable differential expression. Elastase and cathepsin B-like proteinases were expressed at negligible levels. The proteases identified in the study would be ideal candidates for further interactions studies with protease inhibitors to understand the structural reasons of protease inhibitor insensitivity.