Use of RAPD fingerprinting for delineating populations of hilsa shad Tenualosa ilisha (Hamilton, 1822)

RAPD was used to delineate the hilsa populations sampled from the Ganga, Yamuna, Hooghly, and Narmada Rivers at six different locations. Six degenerate primers were used to generate the fragment patterns from the samples collected. All primers were highly polymorphic and generated high numbers of amplification products. Nei's genetic distances were calculated between locations. The overall average genetic distance among all the six locations was 0.295. The Fst value within the Ganga was 0.469 and within the Hooghly it was 0.546. The overall Fst value for the six populations analyzed was 0.590. The UPGMA dendrogram clustered the hilsa into two distinct clusters: Ganga and Yamuna populations and the Hooghly and Narmada populations.     

Modeling the bound conformation of Pemphigus Vulgaris-associated peptides to MHC Class II DR and DQ Alleles

Background

Pemphigus vulgaris (PV) is a severe autoimmune blistering disorder characterized by the presence of pathogenic autoantibodies directed against desmoglein-3 (Dsg3), involving specific DR4 and DR6 alleles in Caucasians and DQ5 allele in Asians. The development of sequence-based predictive algorithms to identify potential Dsg3 epitopes has encountered limited success due to the paucity of PV-associated allele-specific peptides as training data.

Results

In this work we constructed atomic models of ten PV associated, non-associated and protective alleles. Nine previously identified stimulatory Dsg3 peptides, Dsg3 96–112, Dsg3 191–205, Dsg3 206–220, Dsg3 252–266, Dsg3 342–356, Dsg3 380–394, Dsg3 763–777, Dsg3 810–824 and Dsg3 963–977, were docked into the binding groove of each model to analyze the structural aspects of allele-specific binding.

Conclusion

Our docking simulations are entirely consistent with functional data obtained from in vitro competitive binding assays and T cell proliferation studies in DR4 and DR6 PV patients. Our findings ascertain that DRB1*0402 plays a crucial role in the selection of specific self-peptides in DR4 PV. DRB1*0402 and DQB1*0503 do not necessarily share the same core residues, indicating that both alleles may have different binding specificities. In addition, our results lend credence to the hypothesis that the alleles DQB1*0201 and *0202 play a protective role by binding Dsg3 peptides with greater affinity than the susceptible alleles, allowing for efficient deletion of autoreactive T cells.     

Fat and Wingless signaling oppositely regulate epithelial cell-cell adhesion and distal wing development in Drosophila

Development of organ-specific size and shape demands tight coordination between tissue growth and cell-cell adhesion. Dynamic regulation of cell adhesion proteins thus plays an important role during organogenesis. In Drosophila, the homophilic cell adhesion protein DE-Cadherin (DE-Cad) regulates epithelial cell-cell adhesion at adherens junctions (AJs). Here, we show that along the proximodistal (PD) axis of the developing wing epithelium, apical cell shapes and expression of DE-Cad are graded in response to Wingless (Wg), a morphogen secreted from the dorsoventral (DV) organizer in distal wing, suggesting a PD gradient of cell-cell adhesion. The Fat (Ft) tumor suppressor, by contrast, represses DE-Cad expression. In genetic tests, ft behaves as a suppressor of Wg signaling. Cytoplasmic pool of beta-catenin/Arm, the intracellular transducer of Wg signaling, is negatively correlated with the activity of Ft. Moreover, unlike that of Wg, signaling by Ft negatively regulates the expression of Distalless (Dll) and Vestigial (Vg). Finally, we show that Ft intersects Wnt/Wg signaling, downstream of the Wg ligand. Fat and Wg signaling thus exert opposing regulation to coordinate cell-cell adhesion and patterning along the PD axis of Drosophila wing.     

Gene expression profiles in HPV-immortalized human cervical cells treated with the nicotine-derived carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

Human papillomavirus (HPV) infection is an established etiological factor for cervical cancer. Epidemiological studies suggest that smoking in combination with HPV infection plays a significant role in the etiology of this disease. We have previously shown that the tobacco carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is present in human cervical mucus. Here, we hypothesized that treatment of HPV-16-immortalized human ectocervical cells (Ecto1/E6E7) with NNK would alter the expression of genes involved in cellular transformation. Ecto1/E6E7 cells were treated with water (vehicle control) alone or with 1 μM, 10 μM, and 100 μM of NNK in water for 12 weeks. The colony-forming efficiency increased following NNK treatment; the maximum effect was observed after 12 weeks with 100 μM NNK. Microarray analysis revealed that, independent of the dose of NNK, expression of 30 genes was significantly altered; 22 of these genes showed a dose-response pattern. Genes identified are categorized as immune response (LTB4R), RNA surveillance pathway (SMG1), metabolism (ALDH7A1), genes frequently expressed in later stages of neoplastic development (MT1F), DNA binding (HIST3H3 and CHD1L), and protein biosynthesis (UBA52). Selected genes were confirmed by qRT-PCR. Western blot analysis indicates that phosphorylation of histone 3 at serine 10, a marker of cellular transformation, was up-regulated in cells treated with NNK. This is the first study showing that NNK can alter gene expression that may, in part, account for transformation of HPV-immortalized human cervical cells. The results support previous epidemiological observations that, in addition to HPV, tobacco smoking also plays an important role in the development of cervical cancer.     

Leishmania donovani vs immunity: T-cells sensitized from Leishmania of one donor may modulate their cytokines pattern on re-stimulation with Leishmania from different donor in visceral leishmaniasis

Antimony resistance is frequently encountered during treatment of visceral leishmaniasis (VL) and the differences are well characterized by inadequate IFN-γ dominant type-1 protection mechanisms. The part played by Leishmania parasites derived from antimony treated patients in the outcome of an immune response largely remains to be investigated. In the present study we observed that macrophages of BALB/c mice infected with antimony non-responder (SAG-NR) isolates had a greater amastigote burden than antimony responder (SAG-R) isolates. Later it was observed that antigen from SAG-NR and R L. donovani isolates elicit different cytokine responses in peritoneal blood mononuclear cells (PBMCs) from patients with VL. The production of IFN-γ by T-cells in VL patients increased in response to Leishmania derived from responder patients but this response within same T-cells was lower when sensitized from Leishmania from a non-responder VL patient. On the other hand, IL-4 and IL-10 expression was increased when primed with parasites from non-responder VL source. Such a differential pattern of cytokine expression by the same T-cell population produced to Leishmania from different donors, needs further exploration.     

Structural Evidence of Substrate Specificity in Mammalian Peroxidases: STRUCTURE OF THE THIOCYANATE COMPLEX WITH LACTOPEROXIDASE AND ITS INTERACTIONS AT 2.4 ? RESOLUTION*S?

The crystal structure of the complex of lactoperoxidase (LPO) with its physiological substrate thiocyanate (SCN^–) has been determined at 2.4Å resolution. It revealed that the SCN^– ion is bound to LPO in the distal heme cavity. The observed orientation of the SCN^– ion shows that the sulfur atom is closer to the heme iron than the nitrogen atom. The nitrogen atom of SCN^– forms a hydrogen bond with a water (Wat) molecule at position 6′. This water molecule is stabilized by two hydrogen bonds with Gln⁴²³ N^ε2 and Phe⁴²² oxygen. In contrast, the placement of the SCN^– ion in the structure of myeloperoxidase (MPO) occurs with an opposite orientation, in which the nitrogen atom is closer to the heme iron than the sulfur atom. The site corresponding to the positions of Gln⁴²³, Phe⁴²² oxygen, and Wat⁶′ in LPO is occupied primarily by the side chain of Phe⁴⁰⁷ in MPO due to an entirely different conformation of the loop corresponding to the segment Arg⁴¹⁸–Phe⁴³¹ of LPO. This arrangement in MPO does not favor a similar orientation of the SCN^– ion. The orientation of the catalytic product OSCN^– as reported in the structure of LPO·OSCN^– is similar to the orientation of SCN^– in the structure of LPO·SCN^–. Similarly, in the structure of LPO·SCN^–·CN^–, in which CN^– binds at Wat¹, the position and orientation of the SCN^– ion are also identical to that observed in the structure of LPO·SCN.Lactoperoxidase (LPO⁴; EC 1.11.1.7) is a Fe³⁺ heme enzyme that belongs to the mammalian peroxidase family (1). The family of mammalian peroxidases comprises lactoperoxidase (2), eosinophil peroxidase (3), thyroid peroxidase (4), and myeloperoxidase (MPO) (5). LPO, eosinophil peroxidase, and MPO are responsible for antimicrobial function and innate immune responses (6–8), whereas thyroid peroxidase plays a key role in thyroid hormone biosynthesis (9). These peroxidases are different from plant and fungal peroxidases because unlike plant and fungal enzymes, the prosthetic heme group in mammalian peroxidases is covalently linked to the protein (10). There are also several striking structural and functional differences among the mammalian peroxidases (11). The heme group in MPO is attached to the protein via three covalent linkages (12), whereas LPO (12, 13), eosinophil peroxidase (12), and thyroid peroxidase (12) contain only two ester linkages. These covalent and various non-covalent linkages contribute differentially to the high stability of the heme core as well as for the peculiar values of their redox potentials (2, 14). Furthermore, MPO consists of two disulfide-linked protein chains, whereas LPO, eosinophil peroxidase, and thyroid peroxidase are single chain proteins, although their chain lengths differ greatly. In addition, their sequences contain several critical amino acid differences that may also contribute to the variations in the stereochemical environments of the substrate-binding sites. As a consequence of these differences, the mammalian enzymes oxidize various inorganic ions such as SCN^–, Br^–, Cl^–, and I^– with differing specificities and potencies. Biochemical studies have shown that LPO catalyzes preferentially the conversion of SCN^– to OSCN^– (15, 16), whereas MPO uses halides (17, 18) with a preference for chloride ion as the substrate. The preferences of eosinophil peroxidase and thyroid peroxidase are bromide and iodide, respectively. However, the stereochemical basis of the reported preferences for the substrates by mammalian heme peroxidases is still unclear. So far, the structures of only two mammalian enzymes, MPO and LPO, have been determined (12, 13). It is of considerable importance to identify the structural parameters that are responsible for the subtle specificities. In the present work, we have attempted to address this question through the new crystal structures of LPO complexes with SCN^– ions using goat, bovine, and buffalo lactoperoxidases. Because the overall structures of complexes of SCN^– with LPO from all three species were found to be identical, the structure of the complex of buffalo LPO with SCN^– and the ternary complex with SCN^– and CN^– will be discussed here, and buffalo LPO will be termed hereafter as LPO. To highlight the factors pertaining to binding specificity of SCN^–, a comparison of the structures of LPO·SCN^– and MPO·SCN^– has also been made, revealing many valuable differences pertaining to the observed orientations of the common substrate, SCN^– ion, when bound at the substrate-binding site in the distal heme cavity of the two structures. The structures of LPO·SCN^– and MPO·SCN^– clearly show that the bound SCN^– ions are present in the distal heme cavity of two enzymes with opposite orientations. In the structure of LPO·SCN^–, the sulfur atom is closer to the heme iron than the nitrogen atom, whereas in that of MPO·SCN^–, the nitrogen atom is closer to the heme iron than the sulfur atom. As a result of this, the interactions of the SCN^– ion in the distal site of two proteins differ drastically. Gln⁴²³, a conserved water (Wat) molecule at position 6′, and a well aligned carbonyl oxygen of Phe⁴²² in the proximity of the substrate-binding site in LPO against a protruding Phe⁴⁰⁷ in MPO seem to play the key roles in inducing the observed orientations of SCN^– ions in LPO and MPO. The structure of LPO·SCN^– has also been compared with the structure of its ternary complex with SCN^– and CN^– ions.     

Autoacetylation of the MYST Lysine Acetyltransferase MOF Protein

The MYST family of histone acetyltransferases (HATs) plays critical roles in diverse cellular processes, such as the epigenetic regulation of gene expression. Lysine autoacetylation of the MYST HATs has recently received considerable attention. Nonetheless, the mechanism and function of the autoacetylation process are not well defined. To better understand the biochemical mechanism of MYST autoacetylation and the impact of autoacetylation on the cognate histone acetylation, we carried out detailed analyses of males-absent-on-the-first (MOF), a key member of the MYST family. A number of mutant MOF proteins were produced with point mutations at several key residues near the active site of the enzyme. Autoradiography and immunoblotting data showed that mutation of these residues affects the autoacetylation activity and HAT activity of MOF by various degrees demonstrating that MOF activity is highly sensitive to the chemical changes in those residues. We produced MOF protein in the deacetylated form by using a nonspecific lysine deacetylase. Interestingly, both the autoacetylation activity and the histone acetylation activity of the deacetylated MOF were found to be very close to that of wild-type MOF, suggesting that autoacetylation of MOF only marginally modulates the enzymatic activity. Also, we found that the autoacetylation rates of MOF and deacetylated MOF were much slower than the cognate substrate acetylation. Thus, autoacetylation does not seem to contribute to the intrinsic enzymatic activity in a significant manner. These data provide new insights into the mechanism and function of MYST HAT autoacetylation.