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R K Bhat

Publications and source records attributed to R K Bhat.

4 recordsLinked to original sources
PubMed2003

German cockroach extract increases bronchial epithelial cell interleukin-8 expression.

BACKGROUND: Cockroach exposure has been recognized as a common trigger for asthma. While dust mite and Aspergillus fumigatus aeroallergens have been noted to have direct effects on airway epithelium, direct effects of cockroach proteins have not been determined. OBJECTIVE: The purpose of this study was to investigate whether cockroach extract has a direct pro-inflammatory effect on airway epithelium. METHODS: We examined the effect of crude German cockroach (Blattella germanica) extract on IL-8 expression in a human bronchial epithelial cell line (16HBE14o-cells) and primary human bronchial epithelial cells. Transcription from the IL-8 promoter and protein abundance were determined by reporter assay and enzyme-linked immunosorbent assay (ELISA), respectively. Endotoxin levels in the crude cockroach extracts were determined using the Limulus Amebocyte Lysate assay. Protease activity was assessed using Azocoll as a substrate. RESULTS: We found that crude cockroach extract induced a synergistic increase in TNF-alpha-induced transcription from the IL-8 promoter. The synergistic effect was observed with as little as 0.3 micro g/mL of crude cockroach extract, while larger concentrations (30 micro g/mL) approximately doubled TNF-alpha-induced IL-8 promoter activity. Similar effects of cockroach extract on IL-8 protein abundance were observed in both 16HBE14o- and primary human bronchial epithelial cells. Addition of endotoxin at concentrations found in the cockroach extract had no effect on TNF-alpha-mediated IL-8 expression. The serine protease inhibitors aprotinin and phenylmethylsulphonyl fluoride abolished cockroach-induced synergy, while the cysteine protease inhibitors E64 and leupeptin had little effect. Measurement of protease activity using Azocoll as a substrate confirmed the presence of protease activity in cockroach extracts. Addition of recombinant Bla g 2, Bla g 3 and Bla g 5 had no effect on TNF-alpha-induced IL-8 promoter activation. Finally, cockroach extract also increased TNF-alpha-induced transcription from the IL-6 promoter. CONCLUSIONS: German cockroach extract contains novel serine protease activity which has a direct pro-inflammatory effect on airway epithelial cells

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PubMed1975

A light scattering investigation of the propylurea dissociation of human hemoglobin A.

The subunit dissociation of human hemoglobin A by propylurea in several liganded and chemically modified states was investigated by light scattering molecular weight methods. The dissociation data were analyzed by means of the equation developed in our earlier studies: deltaF degrees D EQUALS TO DELTAF degrees D, W - 2N'RTKB[D], where deltaF degrees D and deltaf degreees D,W represent the free energy of dissociation of hemoglobin tetramers into half-molecules consisting of alpha beta dimers in the presence and in the absence of propylurea, KB is the binding constant of the urea to the average peptide unit, [D] is its concentration, and N' is the number of amino acid sites exposed per half-molecule on dissociation. It is found that the dissociation of oxyhemoglobin, cyanmethemoglobin, and N-ethylmaleimide oxyhemoglobin is characterized by essentially the same N' value of 15 to 21 plus or minus 3, that are close to the 19 amino acid residues per surface which comprise the smaller alpha beta contact area, seen in the X-ray crystallographic model of horse hemoglobin of Perutz and coworkers. Due to the very low degree of dissociation of deoxyhemoglobin, only a very approximate estimate of N' of about the same order of magnitude could be obtained for this form of the protein. In contrast, a significantly lower value of N' was obtained with bis(maleimidomethyl) ether modified oxyhemoglobin of 8 plus or minus 3, that is cross-linked at cysteine residue F9 (93)beta and histidine residue FG4 (97) beta in the same beta chains. Our results suggest that alterations caused by the presence of the cross-linking reagent reflect both the loss in amino acid residues that can interact with the urea at the blocked segments of the polypeptide chains in the dissociated state of hemoglobin and the changes in accessibility of some of the amino acid residues perturbed by the introduction of the reagent in the parent tetrameric form.

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