Identification and characterization of membralin, a novel tumor-associated gene, in ovarian carcinoma

Identification of new genes in cancer is the key to understand the molecular basis of tumor development as well as provide potential diagnostic markers and therapeutic targets. A novel gene, membralin (GeneBank accession number: DQ005958), was cloned from a human ovarian cancer cell line. Human membralin is unique and does not share significant sequence homology with other human genes, only membralins of other species. The gene contains 11 exons which encode at least two spliced variants in human cancer. The long form of membralin (membralin-1) comprises all 11 exons, encoding a protein of 620-amino acids long and the short form of membralin (membralin-3) contains all exons except for exon 10, encoding a protein of 408 amino acids. Expression of different membralin isoforms depends on tissue type. The long form, membralin-1, is expressed in ovarian and colorectal carcinomas but not in breast or pancreatic carcinomas, which express only the short splice form, membralin-3. Membralin-1-GFP fusion protein demonstrates exclusive cytoplasmic localization. Based on quantitative real-time PCR, in situ hybridization and Western blot analysis, membralin was highly expressed in ovarian serous carcinomas as compared to ovarian surface epithelium (P<0.001). Ovarian carcinomas in effusions demonstrated a significantly higher level of membralin expression than in solid tumors (P<0.001). In conclusion, these findings represent the first characterization of human membralin and suggest that membralin is a novel tumor-associated marker in ovarian serous carcinomas.     

Glycogen synthase kinase-3 plays a crucial role in tau exon 10 splicing and intranuclear distribution of SC35. Implications for Alzheimer's disease

Tauopathies, including Alzheimer's disease, are neurodegenerative disorders in which tau protein accumulates as a consequence of alterations in its metabolism. At least three different types of alterations have been described; in some cases, an aberrant mRNA splicing of tau exon 10 occurs; in other cases, the disorder is a consequence of missense mutations and, in most cases, aberrant tau hyperphosphorylation takes place. Glycogen synthase kinase-3 (GSK-3) has emerged as a key kinase that is able to interact with several proteins involved in the etiology of Alzheimer's disease and other tauopathies. Here, we have evaluated whether GSK-3 is also able to modulate tau-mRNA splicing. Our data demonstrate that GSK-3 inhibition in cultured neurons affects tau splicing resulting in an increase in tau mRNA containing exon 10. Pre-mRNA splicing is catalyzed by a multimolecular complex including members of the serine/arginine-rich (SR) family of splicing factors. Immunofluorescence studies showed that after GSK-3 inhibition, SC35, a member of the SR family, is redistributed and enriched in nuclear speckles and colocalizes with the kinase. Furthermore, immunoprecipitated SC35 is phosphorylated by recombinant GSK-3beta. Phosphorylation of a peptide from the SR domain by GSK-3 revealed that the peptide needs to be prephosphorylated, suggesting the involvement of a priming kinase. Our results demonstrate that GSK-3 plays a crucial role in tau exon 10 splicing, raising the possibility that GSK3 could contribute to tauopathies via aberrant tau splicing.     

Structural basis for isozyme-specific regulation of electron transfer in nitric-oxide synthase

Three nitric-oxide synthase (NOS) isozymes play crucial, but distinct, roles in neurotransmission, vascular homeostasis, and host defense, by catalyzing Ca(2+)/calmodulin-triggered NO synthesis. Here, we address current questions regarding NOS activity and regulation by combining mutagenesis and biochemistry with crystal structure determination of a fully assembled, electron-supplying, neuronal NOS reductase dimer. By integrating these results, we structurally elucidate the unique mechanisms for isozyme-specific regulation of electron transfer in NOS. Our discovery of the autoinhibitory helix, its placement between domains, and striking similarities with canonical calmodulin-binding motifs, support new mechanisms for NOS inhibition. NADPH, isozyme-specific residue Arg(1400), and the C-terminal tail synergistically repress NOS activity by locking the FMN binding domain in an electron-accepting position. Our analyses suggest that calmodulin binding or C-terminal tail phosphorylation frees a large scale swinging motion of the entire FMN domain to deliver electrons to the catalytic module in the holoenzyme.     

sll1981, an acetolactate synthase homologue of Synechocystis sp. PCC6803, functions as l-myo-inositol 1-phosphate synthase

l-myo-inositol 1-phosphate synthase (EC 5.5.1.4; MIPS) catalyzes the first rate limiting conversion of d-glucose 6-phosphate to l-myo-inositol 1-phosphate in the inositol biosynthetic pathway. In an earlier communication we have reported two forms of MIPS in Synechocystis sp. PCC6803 (Chatterjee et al. in Planta 218:989–998, 2004). One of the forms with a ~50 kDa subunit has been found to be coded by an as yet unassigned ORF, sll1722. In the present study we have purified the second isoform of MIPS as a ~65 kDa protein from the crude extract of Synechocystis sp. PCC6803 to apparent homogeneity and biochemically characterized. MALDI-TOF analysis of the 65 kDa protein led to its identification as acetolactate synthase large subunit (EC 2.2.1.6; ALS), the putatively assigned ORF sll1981 of Synechocystis sp. PCC6803. The PCR amplified ~1.6 kb product of sll1981 was found to functionally complement the yeast inositol auxotroph, FY250 and could be expressed as an immunoreactive ~65 kDa MIPS protein in the natural inositol auxotroph, Schizosaccharomyces pombe. In vitro MIPS activity and cross reactivity against MIPS antibody of purified recombinant sll1981 further consolidated its identity as the second probable MIPS gene in Synechocystis sp. PCC6803. Sequence comparison along with available crystal structure analysis of the yeast MIPS reveals conservation of several amino acids in sll1981 essential for substrate and co-factor binding. Comparison with other prokaryotic and eukaryotic MIPS sequences and phylogenetic analysis, however, revealed that like sll1722, sll1981 is quite divergent from others. It is probable that sll1981 may code for a bifunctional enzyme protein having conserved domains for both MIPS and acetolactate synthase (ALS) activities.Anirban Chatterjee and Krishnarup Ghosh Dastidar contributed equally.     

Introgression of a novel salt-tolerant L-myo-inositol 1-phosphate synthase from Porteresia coarctata (Roxb.) Tateoka (PcINO1) confers salt tolerance to evolutionary diverse organisms

We have previously demonstrated that introgression of PcINO1 gene from Porteresia coarctata (Roxb.) Tateoka, coding for a novel salt-tolerant L-myo-inositol 1-phosphate synthase (MIPS) protein, confers salt tolerance to transgenic tobacco plants (Majee, M., Maitra, S., Dastidar, K.G., Pattnaik, S., Chatterjee, A., Hait, N.C., Das, K.P. and Majumder, A.L. (2004) A novel salt-tolerant L-myo-inositol-1-phosphate synthase from Porteresia coarctata (Roxb.) Tateoka, a halophytic wild rice: molecular cloning, bacterial overexpression, characterization, and functional introgression into tobacco-conferring salt-tolerance phenotype. J. Biol. Chem. 279, 28539-28552). In this communication we have shown that functional introgression of the PcINO1 gene confers salt-tolerance to evolutionary diverse organisms from prokaryotes to eukaryotes including crop plants albeit to a variable extent. A direct correlation between unabated increased synthesis of inositol under salinity stress by the PcINO1 gene product and salt tolerance has been demonstrated for all the systems pointing towards the universality of the application across evolutionary divergent taxa.     

Influence of altered photoperiods on serum melatonin and its receptors (MT1 and MT2) in the brain, retina, and ovary in carp Catla catla

Daily variation in melatonin receptor (MT1 and MT2) density in three specific tissues-brain, retina, and ovary-and its temporal relationship with serum melatonin were evaluated for the first time in a freshwater teleost, the carp Catla catla, under natural as well as altered photoperiods in different reproductive phases of the annual cycle. Cosinor analysis was used to determine rhythmic features of the serum melatonin and receptors (MT1 and MT2) in different tissues. In each photoperiodic group, irrespective of season, the daily minimum serum melatonin level was noted at midday. However, the daily peak value of melatonin varied in relation to both photo-schedules and reproductive phases. Under natural photoperiods (NPs; duration varied with seasons) and short photoperiods (SPs; light [L]:dark [D] 8:16), it occurred in the late dark phase during the preparatory phase, and at midnight in the remaining parts of the annual cycle. On the other hand, in each reproductive phase, compared to corresponding NP carp, the daily melatonin peak under long photoperiods (LPs; L:D 16:8) exhibited a phase delay of ～2-3?h (occurring during the late dark phase). The melatonin levels at each sampling point were highest during the postspawning phase and lowest during the spawning phase, irrespective of the photoperiodic history of the fish. In each tissue, Western blot analysis revealed a band at ～37?kDa and a band at ～36?kDa corresponding to the molecular weights of native MT1 and MT2 receptor proteins, respectively, with the band intensity of MT1 always being higher than that of a 36-kDa protein. The content of both melatonin receptor proteins varied significantly according to the studied tissue (being highest in the retina, intermediate in the brain, and lowest in the ovary), time in the daily cycle (peak at midnight and fall at midday), and reproductive phase in the annual cycle (highest in the spawning phase and lowest in the postspawning phase). Remarkably, no significant effects of altered photoperiod were detected on any rhythm parameters of either MT1 or MT2 in any of the studied tissues. Collectively, the results of the present study suggest a role of photoperiod in determining daily and seasonal profiles of serum melatonin, but not its receptor proteins, on the ovary or on any nongonad tissues in carp.     

The reaction of HOCl and cyanocobalamin: corrin destruction and the liberation of cyanogen chloride

Overproduction of hypochlorous acid (HOCl) has been associated with the development of a variety of disorders such as inflammation, heart disease, pulmonary fibrosis, and cancer through its ability to modify various biomolecules. HOCl is a potent oxidant generated by the myeloperoxidase-hydrogen peroxide-chloride system. Recently, we have provided evidence to support the important link between higher levels of HOCl and heme destruction and free iron release from hemoglobin and RBCs. Our current findings extend this work and show the ability of HOCl to mediate the destruction of metal-ion derivatives of tetrapyrrole macrocyclic rings, such as cyanocobalamin (Cobl), a common pharmacological form of vitamin B12. Cyanocobalamin is a water-soluble vitamin that plays an essential role as an enzyme cofactor and antioxidant, modulating nucleic acid metabolism and gene regulation. It is widely used as a therapeutic agent and supplement, because of its efficacy and stability. In this report, we demonstrate that although Cobl can be an excellent antioxidant, exposure to high levels of HOCl can overcome the beneficial effects of Cobl and generate proinflammatory reaction products. Our rapid kinetic, HPLC, and mass spectrometric analyses showed that HOCl can mediate corrin ring destruction and liberate cyanogen chloride (CNCl) through a mechanism that initially involves α-axial ligand replacement in Cobl to form a chlorinated derivative, hydrolysis, and cleavage of the phosphonucleotide moiety. Additionally, it can liberate free Co, which can perpetuate metal-ion-induced oxidant stress. Taken together, these results are the first report of the generation of toxic molecular products through the interaction of Cobl with HOCl.     

Catalytic mechanism and substrate specificity of HIF prolyl hydroxylases

This review describes the catalytic mechanism, substrate specificity, and structural peculiarities of alpha-ketoglutarate dependent nonheme iron dioxygenases catalyzing prolyl hydroxylation of hypoxia-inducible factor (HIF). Distinct localization and regulation of three isoforms of HIF prolyl hydroxylases suggest their different roles in cells. The recent identification of novel substrates other than HIF, namely β2-adrenergic receptor and the large subunit of RNA polymerase II, places these enzymes in the focus of drug development efforts aimed at development of isoform-specific inhibitors. The challenges and prospects of designing isoform-specific inhibitors are discussed.