Purification, characterization and immunolocalization of fimbrial protein from Porphyromonas (bacteroides) gingivalis.

Rapid and reproducible method is described here for the purification of the 43 kDa fimbrial protein from P. gingivalis by preferential fractionation in the presence of 1% SDS and 0.2M of a bivalent cation at pH 6.5. Homogeneity of the purified 43 kDa was confirmed by SDS-PAGE and Western blot analysis using monoclonal and polyclonal antibodies raised against this protein. Amino acid composition and the amino acid sequence of the first 30 amino acid residues of the purified fimbriae are consistent with the composition and sequence predicted from the cloned gene of the fimbrial subunit. Circular dichroism spectra shows high levels of beta-sheet structure. The purified 43 kDa polymer shows fimbriae-like morphology under the electron microscope. Ultrastructural localization of the 43 kDa protein by the immunogold technique revealed specific labeling of the fimbriae with a diameter of approximately 3.5 to 5.0 nm. Localization of this protein suggest that the 43 kDa component is a fimbrial subunit.     

Molecular characterization of glutamic acid/glutamine-rich secretory proteins from rat submandibular glands

A family of abundant rat submandibular gland secretory proteins has been identified in glandular extracts and characterized. By amino acid analysis these proteins contain approximately 35% glutamic acid and glutamine plus 14% proline. They have therefore been named "Glx-rich proteins" (GRP). Plasmids containing cDNAs for a GRP have been isolated from a cDNA library prepared from rat submandibular gland poly(A)+RNA. The nucleotide sequence of these cDNAs have been determined. Approximately half of the protein coding sequence is composed of a 23-residue tandem repeat which is repeated five times. The first four repeats are highly conserved at both the nucleotide and amino acid level and consist of the prototype sequence: Asn-Gln-Glu-Pro-Pro-Ala-Thr-Ser-Gly-Ser-Glu-Glu-Glu-Gln-Gln-Gln-Gln-Glu- Pro-Thr-Gln-Ala-Glu. The expression of GRP appears to be specific to the submandibular gland. In vitro assays demonstrate that the GRP have a marked affinity for hydroxyapatite. This suggests that GRP may play a role in the formation of the protective acquired pellicle at the saliva-tooth interface.     

Purification and characterization of kallikrein-like serine proteases from rat submandibular glands.

The purification and characterization of kallikrein-like proteases from rat submandibular glands is described. The proteolytic activity of each fraction during purification was monitored on the synthetic substrate N-alpha-tosyl-L-arginine methyl ester (TAME). The purification scheme involved ammonium sulfate precipitation, chromatography on columns of DEAE-Sepharose and Sephadex G-100 and chromatofocusing. Three TAME-hydrolytic activity peaks were eluted from DEAE-Sepharose as unbound fraction (Pool 1), at 125 mM NaCl (Pool 2) and at 250 mM NaCl concentration (Pool 4). Pool 1 further resolved into two protease fractions (1A1 and 1A2), pool 2 into three protease fractions (2A1, 2A2 and 2A3) and pool 4 gave a single major protease peak (4A1) by chromatofocusing on PBE-94. Protease pools 2A2, 2A3, and 4A1 each gave a single band on SDS-polyacrylamide gel electrophoresis with an estimated molecular weight of 34 kDa, 46 kDa and 46 kDa respectively. Pools 1A1, 1A2, 2A1 and 2a2 gave a single precipitin line with anti-rat glandular kallikrein antibodies. 2A3 and 4A1 did not react with these antibodies. Synthetic substrates DL-val-leu-arg-pNA and Bz-pro-phe-arg-pNA, specific for kallikrein-like proteases, were hydrolyzed preferentially by 2A3 and 4A1 but were poor substrates for 1A1, 1A2, 2A1 and 2A2.     

Ethnobotany of the Ratan Mahal Hills,Gujarat, India

Economic Botany - This paper deals with the plants used by aboriginal tribes of Ratan Mahal and surrounding hills. Some of the important food and medicinal plants restricted to these tribes or this...     

Can fossil fuel energy be recovered and used without any CO2 emissions to the atmosphere?

Reviews in Environmental Science and Bio/Technology - The world’s energy system is still dominated by fossil fuels. While there is a rapid reduction in the cost of renewable energy and the...     

Insulin signaling pathway assessment by enhancing antioxidant activity due to morin using in vitro rat skeletal muscle L6 myotubes cells

Molecular Biology Reports - Plant-derived phytochemicals such as flavonoids have been explored to be powerful antioxidants that protect against oxidative stress-related diseases. In the present...     

Binding mode of SARS-CoV-2 fusion peptide to human cellular membrane

Infection of human cells by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) relies on its binding to a specific receptor and subsequent fusion of the viral and host cell membranes. The fusion peptide (FP), a short peptide segment in the spike protein, plays a central role in the initial penetration of the virus into the host cell membrane, followed by the fusion of the two membranes. Here, we use an array of molecular dynamics simulations that take advantage of the highly mobile membrane mimetic model to investigate the interaction of the SARS-CoV2 FP with a lipid bilayer representing mammalian cellular membranes at an atomic level and to characterize the membrane-bound form of the peptide. Six independent systems were generated by changing the initial positioning and orientation of the FP with respect to the membrane, and each system was simulated in five independent replicas, each for 300 ns. In 73% of the simulations, the FP reaches a stable, membrane-bound configuration, in which the peptide deeply penetrated into the membrane. Clustering of the results reveals three major membrane-binding modes (binding modes 1–3), in which binding mode 1 populates over half of the data points. Taking into account the sequence conservation among the viral FPs and the results of mutagenesis studies establishing the role of specific residues in the helical portion of the FP in membrane association, the significant depth of penetration of the whole peptide, and the dense population of the respective cluster, we propose that the most deeply inserted membrane-bound form (binding mode 1) represents more closely the biologically relevant form. Analysis of FP-lipid interactions shows the involvement of specific residues, previously described as the “fusion-active core residues,” in membrane binding. Taken together, the results shed light on a key step involved in SARS-CoV2 infection, with potential implications in designing novel inhibitors.     

Purification and Characterization of Rat Parotid Glycosylated,Basic and Acidic Proline-Rich Proteins

Abstract

A unique family of proline-rich proteins (PRPs) is induced in rats following prolonged isoproterenol treatment. PRPs can be divided into glycosylated (GPRP), basic (BPRP) and acidic (APRP) proline-rich proteins based on their physicochemical characteristics. Inducible rat parotid PRPs were isolated from aqueous extracts of parotid glands of isoproterenol-treated animals by sequential chromatography on columns of DEAE-Sepharose CL-6B, Sephadex G-100 and FPLC on Suprose-12 column. The GPRP showed a single homogeneous band on sodium dodecylpolyacrylamide gel electrophoresis with an estimated molecular weight of approximately 220,000. Compositional analysis of GPRP revealed that this protein contained 19.7% glutamic acid/glutamine, 28.2% proline and 9.5% glycine, and 44% carbohydrate, consisting of fucose (2.81g/100g), mannose (9.78g/100g), galactose (9.29g/100g), N-acetylglucosamine (18.03g/100g) and N-acetylgalactosamine (3.90g/100g). Basic PRPs consisted of a family of proteins with estimated molecular masses ranging from 14–45 kDa. These proteins contained 42.6% proline, 20.65% glutamic acid/glutamine and 21.33% glycine. Acidic PRPs also comprised of a family of metachromatically stained ladder of 40–60 kDa containing 29.1% proline, 21.5% glutamic acid/glutamine and 17.8% glycine. APRP were heavily glycosylated containing N-acetylglucosamine (6.34g/100g), N-acetylgalactosamine (19.04g/100g) and glucuronic acid (38.08g/100g).