Annotation of hypothetical proteins orthologous in Pongo abelii and Sus scrofa

A hypothetical protein is predicted to be expressed from an open reading frame without known experimental evidence of translation. They constitute a substantial fraction of proteomes. Domain extraction from these hypothetical sequences helps to search for protein coding genes for protein structural and functional annotation. We describe the analysis of prediction data in a sequence dataset of hypothetical protein orthologs of Pongo abelii (orangutan) and Sus scrofa (pig). It should be noted that these orangutan-pig orthologs are also non-homologous to human proteins. These predicted data find application in the genome wide annotation of proteins in poorly understood genomes.

Abbreviations

PDB - Protein Data Bank, DEG - Database of Essential Genes, CDD - Conserved Domain Database, IUCN - International Union for Conservation of Nature.     

Spectroscopic and molecular docking studies on chlorambucil interaction with DNA

Chlorambucil (CMB) is an anticancer drug used for the treatment of variety of cancers. Structural and conformational changes associated with DNA after binding with CMB were explored using spectroscopic techniques to get insight into the mechanism of action of CMB at molecular level. Different molar ratios of CMB-DNA complex were prepared with constant DNA concentration under physiological conditions. FTIR spectroscopy, UV-visible spectroscopy, CD spectroscopy and molecular docking studies were employed to determine the binding site and binding constant of CMB with DNA. The results show CMB binds DNA through nitrogenous bases (thymine, guanine and cytosine). The binding constant was calculated to be 1.3×10(3)M(-1), which suggests weak binding of CMB with DNA double helix. FTIR and CD results show that CMB do not disturb native B-conformation of DNA and it continues to remain in its B conformation even at higher concentrations of CMB. The molecular docking results are in corroboration with our experimental results and provides structural insight into the interaction site.     

Zn2+-dependent Activation of the Trk Signaling Pathway Induces Phosphorylation of the Brain-enriched Tyrosine Phosphatase STEP: MOLECULAR BASIS FOR ZN2+-INDUCED ERK MAPK ACTIVATION*

Excessive release of Zn²⁺ in the brain is implicated in the progression of acute brain injuries. Although several signaling cascades have been reported to be involved in Zn²⁺-induced neurotoxicity, a potential contribution of tyrosine phosphatases in this process has not been well explored. Here we show that exposure to high concentrations of Zn²⁺ led to a progressive increase in phosphorylation of the striatal-enriched phosphatase (STEP), a component of the excitotoxic-signaling pathway that plays a role in neuroprotection. Zn²⁺-mediated phosphorylation of STEP₆₁ at multiple sites (hyperphosphorylation) was induced by the up-regulation of brain-derived neurotropic factor (BDNF), tropomyosin receptor kinase (Trk) signaling, and activation of cAMP-dependent PKA (protein kinase A). Mutational studies further show that differential phosphorylation of STEP₆₁ at the PKA sites, Ser-160 and Ser-221 regulates the affinity of STEP₆₁ toward its substrates. Consistent with these findings we also show that BDNF/Trk/PKA mediated signaling is required for Zn²⁺-induced phosphorylation of extracellular regulated kinase 2 (ERK2), a substrate of STEP that is involved in Zn²⁺-dependent neurotoxicity. The strong correlation between the temporal profile of STEP₆₁ hyperphosphorylation and ERK2 phosphorylation indicates that loss of function of STEP₆₁ through phosphorylation is necessary for maintaining sustained ERK2 phosphorylation. This interpretation is further supported by the findings that deletion of the STEP gene led to a rapid and sustained increase in ERK2 phosphorylation within minutes of exposure to Zn²⁺. The study provides further insight into the mechanisms of regulation of STEP₆₁ and also offers a molecular basis for the Zn²⁺-induced sustained activation of ERK2.     

Infrared spectroscopic analysis of tumor pathology

Infrared spectra of normal and malignant breast tissues were measured in the 600 cm(-1) to 4000 cm(-1) region. The measured spectroscopic features which are the spectroscopic fingerprints of the tissues contain the vital information about the malignant and normal tissues. Fourier Transform Infrared (FTIR) data on 25 cases of infiterating ductal carcinoma of breast with different grades of malignancy from patients of different age groups were analyzed. The samples were taken from the tumor sections of the tissue removed during surgery. Infrared spectra demonstrate significant spectral differences between the normal and the cancerous breast tissues. In particular changes in frequency and intensity in the spectra of protein, nucleic acid and glycogen vibrational modes as well as the band intensity ratios for lipid/proteins, protein/nucleic acids, protein/glycogen were observed. This allows to make a qualitative and semi quantitative evaluation of the changes in proliferation activity from normal to diseased tissue. It was evident that the sample to sample or patient to patient variations were small and the spectral differences between normal and diseased tissues were reproducible. The findings establish a framework for additional studies, which may enable us to establish a relation of the diseased state with its infrared spectra.     

Intranasal immunization with recombinant toxin-coregulated pilus and cholera toxin B subunit protects rabbits against Vibrio cholerae O1 challenge

Intranasal immunization, a noninvasive method of vaccination, has been found to be effective in inducing systemic and mucosal immune responses. The present study was aimed at investigating the efficacy of intranasal immunization in inducing mucosal immunity in experimental cholera by subunit recombinant protein vaccines from Vibrio cholerae O1. The structural genes encoding toxin-coregulated pilus A (TcpA) and B subunit of cholera toxin (CtxB) from V. cholerae O1 were cloned and expressed in Escherichia coli . Rabbits were immunized intranasally with purified TcpA and CtxB alone or a mixture of TcpA and CtxB. Immunization with TcpA and CtxB alone conferred, respectively, 41.1% and 70.5% protection against V. cholerae challenge, whereas immunization with a mixture of both antigens conferred complete (100%) protection, as assayed in the rabbit ileal loop model. Serum titers of immunoglobulin G (IgG) antibodies to TcpA and CtxB, and anti-TcpA- and anti-CtxB-specific sIgA in intestinal lavage of vaccinated animals were found to be significantly elevated compared with unimmunized controls. Vibriocidal antibodies were detected at remarkable levels in rabbits receiving TcpA antigen and their titers correlated with protection. Thus, mucosal codelivery of pertinent cholera toxoids provides enhanced protection against experimental cholera.     

Poly-β-hydroxybutyrate accumulation in cyanobacteria under photoautotrophy

Poly-β-hydroxybutyrate (PHB) is a biodegradable and biocompatible polymer that has immense potential in the field of environmental, agricultural and biomedical sciences. An alternative host system has been explored in this study for low-cost production. Examination of 25 cyanobacterial species from 19 different genera for photoautrophic production of polyhydroxyalkanoates (PHAs) under batch culture demonstrated that 20 species were poly-β-hydroxybutyrate (PHB) accumulators, while others were found to be negative. Presence of PHB was confirmed by UV-spectroscopy, (1)H-NMR spectroscopy and GC-MS analysis. Accumulation of PHB in cyanobacteria was found to be species specific. The PHB extracted from Nostoc muscorum exhibited comparable material properties with the commercial PHB, thus advocating its potential applications in various fields.     

Fluorescence polarization of trypsin digested photosystem II membranes

Fluorescence polarization of photosystem II particles treated with trypsin and incubated with high salt-medium (2M NaCl) was investigated. The presence of atrazine and TMPD in normal and salt-washed particles induced a decrease in the polarization ratios. Similar results were obtained at low concentrations of trypsin. On the basis of our observations we suggest that the presence of these perturbing agents causes a reorganisation of the membrane components and alters pigment-pigment and pigment-protein interactions. The results of fluorescence polarization demonstrate trypsin entry into the membrane after the digestion of the peripheral proteins.Abbreviations Chl chlorophyll - Mes 2-(N-morpholino)-ethanesulfonic acid - OEC oxygen evolving complex - PS II photosystem II - TMPD N, N, N - N tetramethyl-p-phenyl-enediamine - DPH 1, 6-diphenyl-1, 3, 5-hexatriene