Expression of the gene for large subunit of m-calpain is elevated in skeletal muscle from Duchenne muscular dystrophy patients

Calpain is an intracellular nonlysosomal protease involved in essential regulatory or processing functions of the cell, mediated by physiological concentrations of Ca²⁺. However, in an environment of abnormal intracellular calcium, such as that seen in Duchenne muscular dystrophy (DMD), calpain is suggested to cause degeneration of muscle owing to enhanced activity. To test whether the reported increase in calpain activity in DMD results fromde novo synthesis of the protease, we have assessed the quantitative changes in mRNA specific for m-calpain. mRNA isolated from DMD and control muscle was analysed by dot blot hybridization using a cDNA probe for the large subunit of m-calpain. Compared to control a four-fold increase in specific mRNA was observed in dystrophic muscle. This enhanced expression of the m-calpain gene in dystrophic condition suggests that the reported increase in m-calpain activity results fromde novo synthesis of protease and underlines the important role of m-calpain in DMD.     

Warfarin administration reduces synthesis of sulfatides and other sphingolipids in mouse brain

The modulation of phosphosphingolipid synthesis by vitamin K depletion has been observed in the vitamin K-dependent microorganism, Bacteriodes levii. When cultured briefly without the vitamin, a reduction occurred in the activity of the first enzyme of the sphingolipid pathway, 3-ketodihydrosphingosine synthase. In this report, 16-day-old mice were treated with the vitamin K antagonist, warfarin. Brain microsomes from these animals showed a 19% reduction in synthase activity. Mice treated with warfarin for 2 weeks showed a major reduction in sulfatide level (42%), with a lesser degree or no reduction in levels of gangliosides and cerebrosides. In further experiments, mice were treated with warfarin for 2 weeks and a group was then injected with vitamin K1 (aquamephyton) for 3 days. Enzyme activity returned to a normal level within 2-3 days. Sulfatide levels had increased 33% in the vitamin K-injected group and ganglioside levels also increased, where levels of cerebrosides and sphingomyelin declined. Sulfatide synthesis determined by [35S] sulfate incorporation, showed a 52% increase in incorporation following administration of vitamin K for 3 days. These results suggest a role for vitamin K in the biosynthesis of sulfatides and other sphingolipids in brain. This putative role could be by post-translational protein modification analogous to the role of vitamin K in other systems.     

Regulatory characteristics of phosphoenolpyruvate carboxylase from the extreme thermophile, Thermus aquaticus.

Phosphoenolpyruvate carboxylase from the extremely thermophilic bacterium, Thermus aquaticus YT-1, exhibits a virtually absolute requirement for acetyl CoA and there is strong positive cooperativity in the interaction of this activator with the enzyme. Several tricarboxylic acid cycle intermediates inhibit the enzyme. These findings suggest an anaplerotic role for the enzyme and an allosteric modulation of its activity by acetyl CoA and tricarboxylic acid cycle intermediates.     

Utility of unipolar recordings for complex Wolff–Parkinson–White ablation

Radiofrequency ablation has been shown to be a safe and effective treatment strategy for the management of symptomatic patients with Wolff–Parkinson–White syndrome. It is supported by a success rate of 95% and a recurrence rate of less than 5%. However, ablation of accessory pathways can be challenging at times. The causes for failure can be grouped into three categories – unusual location of the pathway, technical difficulties in delivering the ablation and localization error [1]. In this case report we are reporting a case of a young male who presented to us with symptomatic Wolff–Parkinson–White syndrome with two failed prior ablations at another institution. This case illustrates the importance of knowing accurate localization and course of the accessory pathway by utilizing the unipolar and bipolar electrograms simultaneously during radiofrequency ablation.     

Basal expression of abscisic acid inducing immunity against Karnal bunt of wheat

In order to understand the intricate mechanism of differential immunity against Karnal bunt (KB), basal levels of carotenoids, abscisic acid (ABA), total protease and protease inhibitor were determined in resistant and susceptible genotypes during transition from vegetative to developing stages of wheat spikes. The lower levels of carotenoid precursor of ABA in resistant genotype than in susceptible genotype could be explained by more inter-conversion of carotenoids into ABA pool which was evident from the results of determining ABA by enzyme-linked sorbent assay. The ABA was significantly higher in resistant genotype at all stages than in susceptible genotype, while a sharp increase was observed at S2 stage. The activity of total protease was higher at initial stages of resistant genotype and gradually declined in later stages after anthesis. In contrast to the protease activity, a reverse trend was observed for the levels of cysteine protease inhibitor, suggesting a negative correlation with each other. Level of cysteine protease inhibitor was observed to be three-folds higher at S2 stage in the resistant genotype than in the susceptible genotype. The results provided the clue for the involvement of ABA-dependent pathways in upregulation of cystatin that leads to induction of differential immunity against the KB pathogen.     

Diversity and analysis of sequences encoded by arcelin genes from Indian wild pulses resistant to bruchids

Wild pulse accessions are considered a vital source of genes for insect resistance for crop improvement programmes. Wild pulses resistant to infestation towards the bruchid insect pest, Callosobruchus maculatus from South India were chosen to screen the existence of potent insecticidal protein, arcelin from APA locus (Arcelin/Phytohemagglutinin/α-Amylase inhibitor) to ascertain their nature and functional diversity without any specific indication for insect resistant factors. The DNA sequence coding for arcelin from various species of wild pulses were amplified, sequenced and deduced to their protein sequences. These protein sequences were examined physico-chemically using several bioinformatics tools and docked with various sugars to resolve the nature of arcelin molecules. Results indicated the presence of significant differences in the properties of arcelin molecules from various species of Indian wild pulses with their amino acid sequences, several physico-chemical properties and binding ability with sugars. The differences observed on these arcelin molecules from diverse wild pulses are predicted to provide a prospective insect pest control factors.     

Enhancement of HLA class II-restricted CD4+ T cell recognition of human melanoma cells following treatment with bryostatin-1

The majority of melanoma cells express detectable levels of HLA class II proteins, and an increased threshold of cell surface class II is crucial for the stimulation of CD4+ T cells. Bryostatin-1, a protein kinase C (PKC) activator, has been considered as a potent chemotherapeutic agent in a variety of in vitro tumor models. Little is known about the role of bryostatin-1 in HLA class II Ag presentation and immune activation in malignant tumors, especially in melanoma. In this study, we show that bryostatin-1 treatment enhances CD4+ T cell recognition of melanoma cells in the context of HLA class II molecules. We also show that bryostatin-1 treatment of melanoma cells increases class II protein levels by upregulating the class II transactivator (CIITA) gene. Flow cytometry and confocal microscopic analyses revealed that bryostatin-1 treatment upregulated the expression of costimulatory molecules (CD80 and CD86) in melanoma cells, which could prolong the interaction of immune cells and tumors. Bryostatin-1 also induced cellular differentiation in melanoma cells, and reduced tumorigenic factors such as pro-cathepsins and matrix-metalloproteinase-9. These data suggest that bryostatin-1 could be used as a chemo-immunotherapeutic agent for reducing tumorigenic potential of melanoma cells while enhancing CD4+ T cell recognition to prevent tumor recurrence.