Effect of retinoic acid on adenosine diphosphate and collagen-induced alterations in enzymes of GSH-linked antioxidant defence system of human blood platelets in vitro

Collagen stimulation of blood platelets resulted in significant increases in malondialdehyde (MDA) formation and activity of glucose-6-phosphate dehydrogenase (G6PDH) and a decrease in catalase and glutathione peroxidase (GPx). Retinoic acid (RA) pretreatment did not show any appreciable changes except for a decrease in G6PDH activity as compared with collagen alone. RA pretreatment of human blood platelets resulted in an increase in the activities of catalase and GPx, two important radical scavenging enzymes, with significant decrease in MDA formation when compared with ADP alone. It is suggested that RA has a significant effect on the antioxidant defence system in ADP stimulated platelets but not in the collagen stimulated platelets.     

Intracellular fate of fibrinogen B beta chain expressed in COS cells

Full-length fibrinogen B beta cDNA was subcloned into an expression vector, pBC12BI, and transfected into COS cells. B beta chain expression was measured by pulse-labelling cells with L-[35S]methionine, immunoprecipitating the B beta chain with antibody to fibrinogen and separating the nascent radioactive protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). B beta chain was expressed in transfected COS cells but was not secreted into the medium. Treatment with endoglycosidase H showed that non-secreted B beta chain contains mannose-rich carbohydrates rather than the complex form of carbohydrate which occurs in plasma fibrinogen and indicates that B beta chain is not transported to the Golgi apparatus. In transfected COS cells, antibody to fibrinogen co-immunoprecipitated B beta chain and 78 kDa immunoglobulin-binding protein (BiP) and antibody to BiP immunoprecipitated BiP and nascent B beta chains. Non-secreted B beta chain was degraded intracellularly with a half-life of 5 h by enzymes which were not affected by incubating transfected cells with NH4Cl, which indicates a non-lysosomal pathway of degradation. These studies indicate that B beta chain by itself does not contain the signal for fibrinogen secretion and that non-secreted B beta chain is associated with BiP and degraded in the rough endoplasmic reticulum.     

Characterization of an adenosine triphosphatase from myeloblasts infected with the avian myeloblastosis virus.

An adenosine triphosphatase (ATPase EC 3.6.1.3) was partially purified from myeloblasts of chicken infected with the avian myeloblastosis virus and some of its molecular, catalytic and immunological properties were compared with that of the ATPase purified from the virus. Both the enzymes possessed almost same electrophoretic mobility, molecular weight, S20,w value, substrate specificity, metal-ion requirement, apparent Km value and sensitivity to inhibitors and activator. Evidence also indicated immunological identity of the two enzymes. The insensitivity of this enzyme to rutamycin or ouabain and extreme sensitivity to most of the detergents, trypsin and mercurials are the remarkable properties of this enzyme.     

Size regulation of multiple organelles competing for a limiting subunit pool

How cells regulate the size of intracellular structures and organelles is a longstanding question. Recent experiments suggest that size control of intracellular structures is achieved through the depletion of a limiting subunit pool in the cytoplasm. While the limiting pool model ensures organelle-to-cell size scaling, it does not provide a mechanism for robust size control of multiple co-existing structures. Here we develop a generalized theory for size-dependent growth of intracellular structures to demonstrate that robust size control of multiple intracellular structures, competing for a limiting subunit pool, is achieved via a negative feedback between the growth rate and the size of the individual structure. This design principle captures size maintenance of a wide variety of subcellular structures, from cytoskeletal filaments to three-dimensional organelles. We identify the feedback motifs for structure size regulation based on known molecular processes, and compare our theory to existing models of size regulation in biological assemblies. Furthermore, we show that positive feedback between structure size and growth rate can lead to bistable size distribution and spontaneous size selection.     

Association of RAGE gene polymorphism with circulating AGEs level and paraoxonase activity in relation to macro-vascular complications in Indian type 2 diabetes mellitus patients

Background and aims

Sustained interaction of advanced glycation end products (AGEs) with their receptor RAGE and subsequent signaling plays an important role in the development of diabetic complications. Genetic variation of RAGE gene may be associated with the development of vascular complications in type 2 diabetes mellitus (T2DM).

Objectives

The present study aimed to explore the possible association of RAGE gene polymorphisms namely − 374T/A, − 429T/C and G82S with serum level of AGEs, paraoxonase (PON1) activity and macro-vascular complications (MVC) in Indian type 2 diabetes mellitus patients (T2DM).

Methods

A total of 265 diabetic patients, including DM without any complications (n = 135), DM-MVC (n = 130) and 171 healthy individuals were enrolled. Genotyping of RAGE variants were assessed by polymerase chain reaction-restriction fragment length polymorphism. Serum AGEs were estimated by ELISA and fluorometrically. and PON1 activity was assessed spectrophotometrically.

Results

Of the three examined SNPs, association of − 429T/C polymorphism with MVC in T2DM was observed (OR = 3.001, p = 0.001) in the dominant model. Allele ‘A’ of − 374T/A polymorphism seems to confer better cardiac outcome in T2DM. Patients carrying C allele (− 429T/C) and S allele (G82S) had significantly higher AGEs levels. − 429T/C polymorphism was also found to be associated with low PON1 activity. Interaction analysis revealed that the risk of development of MVC was higher in T2DM patients carrying both a CC genotype of − 429T/C polymorphism and a higher level of AGEs (OR = 1.343, p = 0.040).

Conclusion

RAGE gene polymorphism has a significant effect on AGEs level and PON1 activity in diabetic subjects compared to healthy individuals. Diabetic patients with a CC genotype of − 429T/C are prone to develop MVC, more so if AGEs levels are high and PON1 activity is low.     

N-(1,3-Diaryl-3-oxopropyl)amides as a new template for xanthine oxidase inhibitors

A series of forty two N-(1,3-diaryl-3-oxopropyl)amides were synthesized via an efficient, modified Dakin-West reaction and were evaluated for in vitro xanthine oxidase inhibitory activity for the first time. Structure-activity relationship analyses have been presented. Selected active xanthine oxidase inhibitors (3r, 3s, and 3zh) were assessed in vivo to study their anti-hyperuricemic effect in potassium oxonate induced hyperuricemic mice model. Compound 3s emerged as the most potent xanthine oxidase inhibitor (IC(50)=2.45 μM) as well as the most potent anti-hyperuricemic agent. The basis of significant inhibition of xanthine oxidase by 3s was rationalized by its molecular docking into catalytic site of xanthine oxidase.     

The frequency and accuracy of replication past a thymine-thymine cyclobutane dimer are very different in Saccharomyces cerevisiae and Escherichia coli.

We have compared the mutagenic properties of a T-T cyclobutane dimer in baker's yeast, Saccharomyces cerevisiae, with those in Escherichia coli by transforming each of these species with the same single-stranded shuttle vector carrying either the cis-syn or the trans-syn isomer of this UV photoproduct at a unique site. The mutagenic properties investigated were the frequency of replicational bypass of the photoproduct, the error rate of bypass, and the mutation spectrum. In SOS-induced E. coli, the cis-syn dimer was bypassed in approximately 16% of the vector molecules, and 7.6% of the bypass products had targeted mutations. In S. cerevisiae, however, bypass occurred in about 80% of these molecules, and the bypass was at least 19-fold more accurate (approximately 0.4% targeted mutations). Each of these yeast mutations was a single unique event, and none were like those in E. coli, suggesting that in fact the difference in error rate is much greater. Bypass of the trans-syn dimer occurred in about 17% of the vector molecules in both species, but with this isomer the error rate was higher in S. cerevisiae (21 to 36% targeted mutations) than in E. coli (13%). However, the spectra of mutations induced by the latter photoproduct were virtually identical in the two organisms. We conclude that bypass and error frequencies are determined both by the structure of the photoproduct-containing template and by the particular replication proteins concerned but that the types of mutations induced depend predominantly on the structure of the template. Unlike E. coli, bypass in S. cerevisiae did not require UV-induced functions.