Localization of melatonin receptor 1 in mouse retina and its role in the circadian regulation of the electroretinogram and dopamine levels

Melatonin modulates many important functions within the eye by interacting with a family of G-protein-coupled receptors that are negatively coupled with adenylate cyclase. In the mouse, Melatonin Receptors type 1 (MT(1)) mRNAs have been localized to photoreceptors, inner retinal neurons, and ganglion cells, thus suggesting that MT(1) receptors may play an important role in retinal physiology. Indeed, we have recently reported that absence of the MT(1) receptors has a dramatic effect on the regulation of the daily rhythm in visual processing, and on retinal cell viability during aging. We have also shown that removal of MT(1) receptors leads to a small (3-4 mmHg) increase in the level of the intraocular pressure during the night and to a significant loss (25-30%) in the number of cells within the retinal ganglion cell layer during aging. In the present study we investigated the cellular distribution in the C3H/f(+/+) mouse retina of MT(1) receptors using a newly developed MT(1) receptor antibody, and then we determined the role that MT(1) signaling plays in the circadian regulation of the mouse electroretinogram, and in the retinal dopaminergic system. Our data indicate that MT(1) receptor immunoreactivity is present in many retinal cell types, and in particular, on rod and cone photoreceptors and on intrinsically photosensitive ganglion cells (ipRGCs). MT(1) signaling is necessary for the circadian rhythm in the photopic ERG, but not for the circadian rhythm in the retinal dopaminergic system. Finally our data suggest that the circadian regulation of dopamine turnover does not drive the photopic ERG rhythm.     

Normalization of Tumor Microenvironment by Neem Leaf Glycoprotein Potentiates Effector T Cell Functions and Therapeutically Intervenes in the Growth of Mouse Sarcoma

We have observed restriction of the murine sarcoma growth by therapeutic intervention of neem leaf glycoprotein (NLGP). In order to evaluate the mechanism of tumor growth restriction, here, we have analyzed tumor microenvironment (TME) from sarcoma bearing mice with NLGP therapy (NLGP-TME, in comparison to PBS-TME). Analysis of cytokine milieu within TME revealed IL-10, TGFβ, IL-6 rich type 2 characters was switched to type 1 microenvironment with dominance of IFNγ secretion within NLGP-TME. Proportion of CD8⁺ T cells was increased within NLGP-TME and these T cells were protected from TME-induced anergy by NLGP, as indicated by higher expression of pNFAT and inhibit related downstream signaling. Moreover, low expression of FasR⁺ cells within CD8⁺ T cell population denotes prevention from activation induced cell death. Using CFSE as a probe, better migration of T cells was noted within TME from NLGP treated mice than PBS cohort. CD8⁺ T cells isolated from NLGP-TME exhibited greater cytotoxicity to sarcoma cells in vitro and these cells show higher expression of cytotoxicity related molecules, perforin and granzyme B. Adoptive transfer of NLGP-TME exposed T cells, but not PBS-TME exposed cells in mice, is able to significantly inhibit the growth of sarcoma in vivo. Such tumor growth inhibition by NLGP-TME exposed T cells was not observed when mice were depleted for CD8⁺ T cells. Accumulated evidences strongly suggest NLGP mediated normalization of TME allows T cells to perform optimally to inhibit the tumor growth.     

Comparative kinomics of Plasmodium organisms: unity in diversity

Phosphorylation by protein kinases is a very common and crucial process in many signal transduction pathways in eukaryotes. This review describes comparative protein kinase analysis of two apicomplexa Plasmodium falciparum (3D7 strain) and Plasmodium yoelii yoelii (17XNL strain) which are causative agents of malaria in human and African rat respectively. Sensitive bioinformatics techniques enable identification of 82 and 60 putative protein kinases in P. falciparum and P. yoelii yoelii respectively and these sequences could be classified into known subfamilies of protein kinases. The most populated kinase subfamilies in both the plasmodium species correspond to CAMK and CMGC groups. Analysis of domain architectures enables detection of uncommon domain organization in kinases of both the organisms such as kinase domain tethered to EF hands as well as PH domain. Components of MAPK signaling pathway is not well conserved in plasmodium organisms. Such observations suggest that plasmodium protein kinases are highly divergent from other eukaryotes. A transmembrane kinase with 6 membrane spanning segments in P. falciparum seems to have no orthologue in P. yoelii yoelii. 19 P. falciparum kinases have been found to cluster separately from P. yoelii yoelii kinases and hence these kinases are unique to P. falciparum genome. Only 28 orthologous pairs of kinases seem to be present between these two plasmodium organisms. Comparative kinome analysis of two plasmodium species has thus provided clues to the function of many protein kinases based upon their classification and domain organization and also implicate marked differences even between two plasmodium organisms.     

Automethylation Activities within the Mixed Lineage Leukemia-1 (MLL1) Core Complex Reveal Evidence Supporting a “Two-active Site” Model for Multiple Histone H3 Lysine 4 Methylation

The mixed lineage leukemia-1 (MLL1) core complex predominantly catalyzes mono- and dimethylation of histone H3 at lysine 4 (H3K4) and is frequently altered in aggressive acute leukemias. The molecular mechanisms that account for conversion of mono- to dimethyl H3K4 (H3K4me1,2) are not well understood. In this investigation, we report that the suppressor of variegation, enhancer of zeste, trithorax (SET) domains from human MLL1 and Drosophila Trithorax undergo robust intramolecular automethylation reactions at an evolutionarily conserved cysteine residue in the active site, which is inhibited by unmodified histone H3. The location of the automethylation in the SET-I subdomain indicates that the MLL1 SET domain possesses significantly more conformational plasticity in solution than suggested by its crystal structure. We also report that MLL1 methylates Ash2L in the absence of histone H3, but only when assembled within a complex with WDR5 and RbBP5, suggesting a restraint for the architectural arrangement of subunits within the complex. Using MLL1 and Ash2L automethylation reactions as probes for histone binding, we observed that both automethylation reactions are significantly inhibited by stoichiometric amounts of unmethylated histone H3, but not by histones previously mono-, di-, or trimethylated at H3K4. These results suggest that the H3K4me1 intermediate does not significantly bind to the MLL1 SET domain during the dimethylation reaction. Consistent with this hypothesis, we demonstrate that the MLL1 core complex assembled with a catalytically inactive SET domain variant preferentially catalyzes H3K4 dimethylation using the H3K4me1 substrate. Taken together, these results are consistent with a “two-active site” model for multiple H3K4 methylation by the MLL1 core complex.     

Identification of novel single nucleotide polymorphisms in the DGAT1 gene of buffaloes by PCR-SSCP

Diacylglycerol O-acyltransferase 1 (DGAT1) is a microsomal enzyme that catalyzes the final step of triglyceride synthesis. The DGAT1 gene is a strong functional candidate for determining milk fat content in cattle. In this work, we used PCR-SSCP (polymerase chain reaction-single-strand conformation polymorphism) and DNA sequencing to examine polymorphism in the region spanning exon 7 to exon 9 of the DGAT1 gene in Murrah and Pandharpuri buffaloes. Three alleles (A, B and C) and four novel single-nucleotide polymorphisms were identified in the buffalo DGAT1 gene. The frequencies of the alleles differed between the two buffalo breeds, with allele C being present in Murrah but not in Pandharpuri buffalo. The allele variation detected in this work may influence DGAT1 expression and function. The results described here could be useful in examining the association between the DGAT1 gene and milk traits in buffalo.     

Functional diversity of human protein kinase splice variants marks significant expansion of human kinome

Background

The low pH environment of the human stomach is lethal for most microorganisms; but not Escherichia coli, which can tolerate extreme acid stress. Acid resistance in E. coli is hierarchically controlled by numerous regulators among which are small noncoding RNAs (sncRNA).

Results

In this study, we individually deleted seventy-nine sncRNA genes from the E. coli K12-MG1655 chromosome, and established a single-sncRNA gene knockout library. By systematically screening the sncRNA mutant library, we show that the sncRNA GcvB is a novel regulator of acid resistance in E. coli. We demonstrate that GcvB enhances the ability of E. coli to survive low pH by upregulating the levels of the alternate sigma factor RpoS.

Conclusion

GcvB positively regulates acid resistance by affecting RpoS expression. These data advance our understanding of the sncRNA regulatory network involved in modulating acid resistance in E. coli.     

Effects of congenital stationary night blindness type 2 mutations R508Q and L1364H on Cav1.4 L-type Ca2+ channel function and expression

At least 48 mutations in the CACNA1F gene encoding retinal Ca(v)1.4 L-type Ca(2+) channels have been linked to X-linked recessive congenital stationary night blindness type 2 (CSNB2). A large number of these are missense mutations encoding full-length alpha1-subunits that can potentially form functional channels. We have previously shown that such missense mutations can confer their phenotype by different pathological mechanisms, such as complete lack of alpha1 subunit protein expression or dramatic changes in channel gating. Here we investigated the functional consequences of CSNB2 missense mutations R508Q and L1364H. We found no (R508Q) or only minor (L1364H) changes in the gating properties of both mutants after heterologous expression in Xenopus laevis oocytes (at 20 degrees C). However, both mutants resulted in altered expression density of Ca(v)1.4 currents. When expressed in the mammalian cell line tsA-201, the current amplitude of L1364H channels was reduced when cells were grown at 30 degrees C and both mutations affected total alpha1 protein expression. This effect was temperature dependent. Our data provide evidence that, in contrast to previously characterized CSNB2 missense mutations, the clinical phenotype of R508Q and L1364H is unlikely to be explained by changes in channel gating. Instead, these mutations affect the protein expression of Ca(v)1.4 Ca(2+) channels.