Structural Studies of Inositol 1,4,5-Trisphosphate Receptor: COUPLING LIGAND BINDING TO CHANNEL GATING*

The three isoforms of the inositol 1,4,5-trisphosphate receptor (IP₃R) exhibit distinct IP₃ sensitivities and cooperativities in calcium (Ca²⁺) channel function. The determinants underlying this isoform-specific channel gating mechanism have been localized to the N-terminal suppressor region of IP₃R. We determined the 1.9 Å crystal structure of the suppressor domain from type 3 IP₃R (IP₃R3_SUP, amino acids 1–224) and revealed structural features contributing to isoform-specific functionality of IP₃R by comparing it with our previously determined structure of the type 1 suppressor domain (IP₃R1_SUP). The molecular surface known to associate with the ligand binding domain (amino acids 224–604) showed marked differences between IP₃R3_SUP and IP₃R1_SUP. Our NMR and biochemical studies showed that three spatially clustered residues (Glu-20, Tyr-167, and Ser-217 in IP₃R1 and Glu-19, Trp-168, and Ser-218 in IP₃R3) within the N-terminal suppressor domains of IP₃R1_SUP and IP₃R3_SUP interact directly with their respective C-terminal fragments. Together with the accompanying paper (Yamazaki, H., Chan, J., Ikura, M., Michikawa, T., and Mikoshiba, K. (2010) J. Biol. Chem. 285, 36081–36091), we demonstrate that the single aromatic residue in this region (Tyr-167 in IP₃R1 and Trp-168 in IP₃R3) plays a critical role in the coupling between ligand binding and channel gating.     

C-terminal region of human p53 attenuates buffalo p53 N-terminal-specific transactivation of p21 promoter by modulating tetramerization of the protein

Here, we have studied in p53 null H1299 lung carcinoma cells, the dominant-negative effect of human p53 (h-p53) on buffalo p53 (b-p53) induced nuclear transactivation-dependent function. Recently, we have isolated and cloned the full-length cDNA of buffalo p53. Buffalo and human p53 proteins exhibit a high degree of structural and functional similarities. In transiently transfected H1299 cell line b-p53 appeared to be more sensitive to Mdm2-mediated degradation as compared to h-p53, although its ability to transactivate p21 promoter was stronger than that of the human counterpart. This higher transactivation ability of b-p53 was lost in the presence of h-p53 suggesting, a dominant-negative effect of h-p53 on b-p53’s transactivation of p21 promoter. Both human and buffalo p53 proteins could hetero-oligomerize but the b-p53 could tetramerize much faster than the h-p53. A chimeric cDNA construct of human p53 was made where the 1–260 bp N-terminus was replaced with buffalo p53 counterpart and expressed in H1299 cell line. The tetramerization ability of the chimeric p53 protein was comparable to that of h-p53. Properties of b-p53 like stronger p21 transactivation and super sensitivity to Mdm2 mediated degradation were lacking in the chimeric protein. Thus, it is suggested that faster ability of tetramerization as well as higher transactivation property of buffalo p53 is determined by the interplay of N- and C-terminal domains through macromolecular interactions.     

Membrane perturbation through novel cell-penetrating peptides influences intracellular accumulation of imatinib mesylate in CML cells

Chronic myeloid leukemia is a stem cell disease with the presence of Philadelphia chromosome generated through reciprocal translocation of chromosome 9 and 22. The use of first- and second-generation tyrosine kinase inhibitors has been successful to an extent. However, resistance against such drugs is an emerging problem. Apart from several drug-resistant mechanisms, drug influx/efflux ratio appears to be one of the key determinants of therapeutic outcomes. In addition, intracellular accumulation of drug critically depends on cell membrane fluidity and lipid raft dynamics. Previously, we reported two novel cell-penetrating peptides (CPPs), namely, cationic IR15 and anionic SR11 present in tryptic digest of Abrus agglutinin. Here, the potential of IR15 and SR11 to influence intracellular concentration of imatinib has been evaluated. Fluorescent correlation spectroscopy and lifetime imaging were employed to map membrane fluidity and lipid raft distribution following peptide-drug co-administration. Results show that IR15 and SR11 are the two CPPs which can modulate membrane fluidity and lipid raft distribution in K562 cells. Both IR15 and SR11 significantly reduce the viability of CML cells in the presence of imatinib by increasing the intracellular accumulation of the drug.     

Threats to Fish and Fisheries of Kangsabati Reservoir,West Bengal,India

Fishes belonging to the Orders Beloniformes, Cypriniformes, Perciformes, Siluriformes and Synbranchiformes are usually found in the Kangsabati reservoir of West Bengal, India. In recent years among the trapped fishes fishermen failed to get certain fish species which were available to them in the last decade. This prompted us to conduct a survey of ichthyofauna of the said reservoir in respect to the water parameters, keeping in view the anthropogenic activity-induced pollution scenario. It is revealed that the fishes belonging to the species Xenentodon cancila, Nemacheilus savona, Sillaginopsis panijus, Pangasius sutchi, Colisa sota, Mystus cavasius, Mystus seenghala and Mastacembelus armatus are completely absent in the survey area. It is most likely that the eutrophication-induced causes especially, variations in composition and density of plankton as well as the undesirable changes in physical and chemical properties of the water have forced these fishes to migrate elsewhere.     

Detection of Mutations in Tumor Suppressor Genes

Defects in vital genes occur in a high percentage of human diseases, including cancer. Defects could be due to the accumulation of mutations in the genes leading to the production of faulty proteins. Although the biological significance of such mutant proteins still remains in question, recent experiments have demonstrated that genes overproducing faulty proteins are often associated with tumor cell growth. Thep53tumor suppressor gene is the most frequently mutated gene yet identified in human cancer. It is mutated in wide variety of human cancers. Missense mutations are common for thep53gene and are essential for the transforming ability of the oncogene. The wild-typep53gene may directly suppress cell growth or indirectly activate genes that are involved in growth suppression. Thus inactivation of wild-typep53by point mutation may contribute to transformation. Therefore, identification of such mutations have potential clinical implications. Recently, polymerase chain reaction-based advanced molecular techniques had a profound impact on the detection and identification of such mutations. These techniques are sensitive and quantitative tools for the study of the pathogenesis of neoplastic diseases at the single-cell level.     

Characterization of malignant mesenchymal cell line (UISO-RS-3) derived from a human rhabdomyosarcoma and inhibition by pharmacologic doses of estrogen

Summary A new tumor cell line has been established from a malignant pleural effusion in a 28-yr-old female patient with a primary alveolar rhabdomyosarcoma of the left buttock. The in vitro and in vivo growth characteristics, morphologic features, abnormal karyotype, and immunohistochemical staining pattern indicate that this cell line is comprised of primitive malignant mesenchymal cells derived from a human rhabdomyosarcoma. Receptor studies done on tumors grown in male athymic mice revealed a single class of high affinity saturable cytoplasmic estrogen receptor (Bmax 2.6 fm/mg cytosol protein, Kd 0.34 mM). Likewise, sucrose density gradient analysis demonstrated specific low-capacity, high-affinity estradiol binding predominately in the 8S region. Cell growth in monolayer culture and on soft agar in the presence of estradiol was inhibited by pharmacologic concentrations of estradiol in a dose-responsive manner compared with control. We describe a newly characterized malignant mesenchymal cell line derived from an alveolar rhabdomyosarcoma that is inhibited by pharmacologic doses of estradiol in vitro. These findings suggest further investigation into the mechanism(s) of this estrogen-induced inhibition in rhabdomyosarcomas.     

Retinal Pigment Epithelium–Calycal Processes Association in the Catfish Clarias batrachus(Linnaeus, 1758)

Abstract In the duplex retina of the catfish Clarias batrachus(Linnaeus, 1758), the apical processes of the pigment epithelial cells have been found by transmission electron microscopy to be in intimate contact with the calycal processes around the basal portion of the photoreceptor outer segments. It is hypothesized that the retinal pigment epithelium effectively transports synthesized products and metabolites to the photoreceptor inner segments via the anatomical zone of the apical–calycal processes interface in this species.     

The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3’-P and 5’-OH, are processed by mammalian polynucleotide kinase 3’-phosphatase (PNKP), a bifunctional enzyme with 3’-phosphatase and 5’-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14–41 to 55–82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP’s 3’ phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3’-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients’ brain. Finally, long amplicon quantitative PCR analysis of human MJD patients’ brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.     

Anti-apoptotic potential of gymnemic acid phospholipid complex pretreatment in Wistar rats with experimental cardiomyopathy

Cardiomyocyte apoptosis in heart failure has been the topic of research in many recent studies. In the present investigation, the potential cardioprotective effect of gymnemic acid phospholipid complex (GPC) on myocardial apoptosis and cardiac function was studied in doxorubicin (DOX; 30 mg/kg/ip/single dose)-induced cardiomyopathy model in rats. Doxorubicin induced cardiomyopathy was evidenced by significant hemodynamic changes (increased systolic, diastolic, mean arterial pressure and heart rate), decreased heart weight to body weight ratio, increase in serum lactate dehydrogenase (LDH) and Ca2+ levels and decrease in myocardial Na+/K+ ATPase levels along with caspase-3 activation. A marked reduction in glutathione, glutathione peroxidase, glutathione reductase, glutathione-S-transferase, superoxide dismutase and catalase levels along with increase in the levels of thiobarbituric acids (TBARS) were also observed in rat myocardium. In addition, DNA laddering observed on agarose gel electrophoresis and cardiac histopathology study further supplemented myocardial apoptosis. Pre-treatment with GPC significantly reduced DOX-induced cardiac toxicity, including improvement of hemodynamic variables and heart weight to body weight ratio, decreased serum Ca2+ level and LDH levels, myocardial caspase-3 levels, increased Na+/K+ ATPase levels and decreased myocardial TBARS levels and elevated antioxidant enzymes as compared to pathogenic control group. Further, the anti-apoptotic effect of GPC was verified by prevention of internucleosomal DNA laddering on agarose gel electrophoresis and attenuation of histopathological perturbations by doxorubicin. These observations demonstrate that GPC might serve as a cardioprotective formulation in DOX-induced cardiomyopathy in rats.