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M K Ray

Publications and source records attributed to M K Ray.

At least 37 records · Page 2Linked to original sources
PubMed1996

Transcriptional regulation of a mouse Clara cell-specific protein (mCC10) gene by the NKx transcription factor family members thyroid transciption factor 1 and cardiac muscle-specific homeobox protein (CSX).

This report defines the elements between bp -800 and -166 that regulate the quantitative level of mouse CC10 (mCC10) transcription in the lungs. The elements in this promoter domain are the response elements for the NKx2.1 homeobox protein, thyroid transcription factor 1 (TTF1). DNase I footprint analysis identified five binding sites for TTF1 between bp -800 and - 166. These sites are located at bp -344 to -335, - 282 to -273, -268 to -263, -258 to -249, and - 199 to - 190. In addition to these enhancer elements, two TTF1 binding sites were identified in the proximal promoter region (bp - 166 to + 1), at bp -74 to -69 and -49 to -39. An identical footprint of the mCC10 promoter region was also observed with another member of the NKx family, NKx 2.5, the cardiac muscle-specific homeobox protein (CSX). Deletion and linker-scanner mutational analyses of the TTF1 binding sites in the mCC10 distal promoter region with transient cotransfection into CV1 cells with either TTF1 or CSX identified the site located between bp -282 and -273 as the major regulator of CC10 expression, with minor regulation by sites at bp -344 to -335 and -258 to -249. The importance of the NKx binding site at bp -282 to -273 was verified in vivo. Transgenic mice generated with the human growth hormone gene fused to 800 bp of the mCC10 promoter containing a mutation in the TTF1 binding site at bp -282 to -273 showed a reduction in transgene expression equal to that of the mice generated with only 166 bp of 5'-flanking DNA. This report emphasizes the importance of TTF1 or related factors as major regulators of pulmonary gene expression and demonstrates the potential of NKx proteins to bind and activate heterologous target genes.

Animals↗
PubMed1996

Immunohistochemical localization of mouse Clara cell 10-KD protein using antibodies raised against the recombinant protein.

To investigate the developmental regulation of the mouse Clara cell 10-KD protein (mCC10), we raised an antibody against the recombinant mCC10 protein. The coding region for the mature mCC10 protein was placed in frame with the glutathione-S-transferase gene in the pGEX2-T bacterial expression vector. The GST-mCC10 fusion protein was expressed in E. coli DH5 alpha cells. The fusion protein was purified and eluted using glutathione-Sepharose beads. The GST-mCC10 fusion protein was injected into rabbits to raise antibodies. The rabbit anti-mCC10 antibody was tested by immunoblot analysis using both purified protein as well as extracts of lung, liver, and uterus. The antibodies produced were used in immunohistochemistry and immunoelectron microscopy to detect the cellular localization of this protein in the above organs. This anti-mCC10 antibody will be useful for future investigation of the developmental biology of the lung.

Animals↗
PubMed1995

LEC18, a dominant Chinese hamster ovary glycosylation mutant synthesizes N-linked carbohydrates with a novel core structure.

The dominant Chinese hamster ovary cell glycosylation mutant, LEC18, was selected for resistance to pea lectin (Pisum sativum agglutinin (PSA)). Lectin binding studies show that LEC18 cells express altered cell surface carbohydrates with markedly reduced binding to 125I-PSA and increased binding to 125I-labeled Datura stramonium agglutinin (DSA) compared with parental cells. Desialylated [3H]Glc-labeled LEC18 cellular glycopeptides that did not bind to concanavalin A-Sepharose exhibited an increased proportion of species that were bound to DSA-agarose. Most of these glycopeptides bound to ricin-agarose and were unique to LEC18 cells. This fraction was purified from approximately 10(10) cells and shown by 1H NMR spectroscopy and methylation linkage analysis to contain novel N-linked structures. Digestion of these glycopeptides with mixtures of beta-D-galactosidases and N-acetyl-beta-D-glucosaminidases gave core glycopeptides that, in contrast to cores from parental cells, were mainly not bound to concanavalin A-Sepharose or to PSA-agarose. 1H NMR spectroscopy, matrix-assisted laser desorption ionization/time of flight mass spectrometry, electrospray mass spectrometry, and collision-activated dissociation mass spectrometry showed that the LEC18 core glycopeptides contained a new GlcNAc residue that substitutes the core GlcNAc residues. Methylation linkage analysis of the parent compound provided evidence that the GlcNAc is linked at O-6 to give the following novel, N-linked core structure. [formula: see text]

Animals↗
PubMed1995

cis-acting elements involved in the regulation of mouse Clara cell-specific 10-kDa protein gene. In vitro and in vivo analysis.

Transient transfection and murine germ line gene transfer analysis was used to determine the regions of DNA necessary to confer the appropriate level and cell specificity of the expression of the gene coding for the murine Clara cell 10-kDa protein, mCC10. To identify the cis-acting elements involved in the regulation of mCC10 gene, different lengths of the 5'-flanking sequence were ligated to the bacterial chloramphenicol acetyltransferase gene for transient transfection to H441 cells (human lung adenocarcinoma cell line). The corresponding sequences were also fused to the human growth hormone gene and transferred to the murine genome for an in vivo analysis of mCC10 promoter activity. The results of the transient transfection analysis identified the region from -166 to -124 of the 5'-flanking region of the mCC10 gene as necessary for the expression of this gene in H441 cells. The transgenic mouse analysis confirmed that the 166 base pairs of 5'-flanking DNA was sufficient to confer cell-specific expression. However, the transgenic mouse analysis also showed that, to achieve the full quantitative level of transgene (human growth hormone) expression, regions between -803 and -166 base pairs of the 5'-flanking sequences are required for maximum expression of mCC10 gene promoter activity.

Animals↗
PubMed1994

Occurrence and expression of cspA, a cold shock gene, in Antarctic psychrotrophic bacteria.

The homologue of cold shock gene cspA of Escherichia coli was detected in various isolates of Antarctic psychrotrophs representing both Gram-positive and Gram-negative bacteria. The Northern hybridization study indicated that the transcript size of cspA in the psychrotrophic Gram-positive bacterium Arthrobacter protophormiae and Gram-negative Pseudomonas fluorescens was similar to that of E. coli and that the cspA homologues in these two psychrotrophs were expressed constitutively at a low level both at 4 degrees C and 22 degrees C. In P. fluorescens, the expression of cspA mRNA was inducible after shift of temperature from 22 to 4 degrees C and the maximum level of induction occurred after 1 h which correlated with the time-lag required for growth of the culture after temperature shift.

Antarctic Regions↗
PubMed1994

Phosphorylation of membrane proteins in response to temperature in an Antarctic Pseudomonas syringae.

Temperature-dependent phosphorylation and dephosphorylation of membrane proteins was studied in vitro in a number of psychrotrophic Antarctic bacteria which grow between 0 and 30 degrees C. One of them, a Pseudomonas syringae isolate, was studied in detail and was found to have three membrane proteins of molecular mass 30, 65 and 85 kDa which were phosphorylated differently in response to low and high temperatures. The 65 kDa protein was phosphorylated only at lower temperatures (between 0 and 15 degrees C). The 30 kDa protein was phosphorylated more at higher temperatures and was possibly a histidine kinase. This protein was present in all the psychrotrophic Pseudomonas species studied and in Sphingobacterium antarcticus. A possible role for these proteins in sensing environmental temperature is proposed.

Adenosine Triphosphate↗
PubMed1994

Phosphorylation of lipopolysaccharides in the Antarctic psychrotroph Pseudomonas syringae: a possible role in temperature adaptation.

Phosphorylation of lipopolysaccharide (LPS) from a psychrotrophic bacterium, Pseudomonas syringae, from Antarctica was studied by using sucrose gradient-separated membrane fractions. The bacterium was found to possess an LPS kinase which could phosphorylate more LPS postsynthetically at higher temperatures. The phosphorylation was low at a lower temperature and was also found to occur in vivo. After phosphorylation of LPS in vitro, it was found that the major part of the radioactivity (> 85%) was associated with the core oligosaccharide region of the LPS. The phosphate groups of this region are probably involved in the binding of metal ions, which could be removed by EDTA. The cells grown at the lower temperature probably contained fewer divalent cations because of the smaller amount of phosphate and thereby were more sensitive to EDTA. The cells were also more sensitive to cationic antibiotics at the lower temperature. A possible role of this differential phosphorylation of LPS in modulating the function of the outer membrane as a permeability barrier in the psychrotroph is discussed.

Adaptation, Physiological↗
PubMed1993

Cloning and characterization of the mouse Clara cell specific 10 kDa protein gene: comparison of the 5'-flanking region with the human rat and rabbit gene.

The mouse Clara Cell 10 kiloDalton (kDa) protein (mCC10) cDNA was used to isolate a recombinant phage containing the mCC10 gene sequence in a 14 kilobase (kb) insert from a mouse genomic library. A total of 7.7 kb of this clone was sequenced. The sequenced region included: 3.3 kb of 5'-flanking region, 4.2 kb intragenic sequence and 0.2 kb of DNA flanking the 3' end of the gene. Computer assisted sequence analysis identified potential cis acting response elements for the glucocorticoid receptor, hepatocyte nuclear factor (HNF3) and octamer (Oct1) binding protein. The presence of B1 murine repetitive sequence also has been identified in a similar position reported in rat CC10 5'-flanking sequence. As with the rat CC10, the mCC10 5'-flanking region also contains deletions of a 2.1 kb and a 0.3 kb sequence present in the rabbit uteroglobin gene, these regions are reported to contain a cluster of glucocorticoid/progesterone receptor binding sites and estrogen receptor binding sites, respectively.

Animals↗
PubMed1993

Characteristics of the eukaryotic initiation factor 2 associated 67-kDa polypeptide.

A eukaryotic initiation factor 2 (eIF-2) associated 67-kDa polypeptide (p67) protects the eIF-2 alpha-subunit from eIF-2 kinase(s) catalyzed phosphorylation, and this promotes protein synthesis in the presence of active eIF-2 kinase(s), [Datta, B., et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3324-3328]. This report presents the results of studies related to characteristics of p67 action and the mechanism of p67:eIF-2 interaction: (1) p67 antibodies inhibited protein synthesis in hemin-supplemented rabbit reticulocyte lysates, and such inhibition was reversed by preincubation of the antibodies, specifically with p67. (2) p67 inhibited HRI- and dsI-catalyzed phosphorylations of the eIF-2 alpha-subunit and histones, but it did not inhibit casein kinase catalyzed phosphorylation of the eIF-2 beta-subunit. (3) p67 bound specifically to the eIF-2 gamma-subunit. p67 co-immunoprecipitated with the eIF-2 subunits when a p67/eIF-2 mixture was treated with p67 or eIF-2 subunit antibodies and protein A agarose. However, when eIF-2 was preincubated specifically with the eIF-2 gamma-subunit antibodies, subsequent co-immunoprecipitation of p67 with the eIF-2 subunits was completely inhibited. Similarly, preincubation of p67 and p67 antibodies prevented subsequent p67 binding to eIF-2. Preincubation of eIF-2, with either eIF-2 alpha- or beta-subunit antibodies, had no effect on p67 co-immunoprecipitation with the eIF-2 subunits. (4) p67:eIF-2 interaction is necessary for p67 activity to protect the eIF-2 alpha-subunit from eIF-2 kinase(s) catalyzed phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Casein Kinases↗
PubMed1992

The eukaryotic initiation factor 2-associated 67-kDa polypeptide (p67) plays a critical role in regulation of protein synthesis initiation in animal cells.

The eukaryotic initiation factor 2 (eIF-2)-associated 67-kDa polypeptide (p67) isolated from reticulocyte lysate protects the eIF-2 alpha subunit from eIF-2 kinase-catalyzed phosphorylation and promotes protein synthesis in the presence of active eIF-2 kinases. We have now studied the roles of p67 and eIF-2 kinases in regulation of protein synthesis using several animal cell lysates and an animal cell line (KRC-7) in culture under various growth conditions. The results are as follows. (i) Both p67 and eIF-2 kinase(s) are present in active forms in all animal cells under normal growth conditions and p67 protects the eIF-2 alpha subunit from eIF-2 kinase-catalyzed phosphorylation, thus promoting protein synthesis in the presence of active eIF-2 kinases. (ii) In heme-deficient reticulocyte lysates and in serum-starved KRC-7 cells in culture, p67 is deglycosylated and subsequently degraded. This leads to eIF-2 kinase-catalyzed eIF-2 alpha-subunit phosphorylation and thus to protein synthesis inhibition. (iii) Addition of a mitogen (namely, phorbol 12-myristate 13-acetate) to serum-starved KRC-7 cells in culture induces an increase of p67 and thus increases protein synthesis. These results suggest the following conclusions. (i) Protein synthesis inhibition in a heme-deficient reticulocyte lysate is not due to the activation of an eIF-2 kinase (heme-regulated inhibitor), as is generally believed, but is due to degradation of p67. The heme-regulated inhibitor is present in an active form and possibly in equal amounts in both heme-deficient and heme-supplemented reticulocyte lysates but cannot phosphorylate eIF-2 alpha subunit because of the presence of p67. (ii) p67 is essential for protein synthesis as it protects the eIF-2 alpha subunit from eIF-2 kinase-catalyzed phosphorylation and promotes protein synthesis in the presence of one or more active eIF-2 kinases present in all animal cells. (iii) p67 is both degradable and inducible. Only the p67 level correlates directly with the protein synthesis activity of the cell, indicating that p67 is a critical factor in protein synthesis regulation in animal cells.

Animals↗
PubMed1992

Extracellular protease from the antarctic yeast Candida humicola.

The psychrotrophic, dimorphic yeast Candida humicola, isolated from Antarctic soil, secretes an acidic protease into the medium. The secretion of this protease by C. humicola was found to be dependent on the composition of the medium. In YPD or yeast nitrogen base medium containing either amino acids or ammonium sulfate as the nitrogen source, the activity of the protease in the medium was low (basal level). However, when yeast nitrogen base medium was depleted of amino acids or ammonium sulfate and supplemented with proteins, the activity of the enzyme increased. The secretion of the enzyme was greater during exponential growth at low temperatures than during growth at higher temperatures. The purified protease had a molecular mass of 36,000 Da and was inhibited by pepstatin, iodoacetamide, and sodium dodecyl sulfate. Despite the prevalent cold temperatures in Antarctica, this extracellular protease of the psychrotrophic yeast C. humicola was active at temperatures ranging from 0 to 45 degrees C, with an optimum activity at 37 degrees C.

Adaptation, Physiological↗
PubMed1991

A novel glycosylation phenotype expressed by Lec23, a Chinese hamster ovary mutant deficient in alpha-glucosidase I.

Lec23 Chinese hamster ovary (CHO) cells have been shown to possess a unique lectin resistance phenotype and genotype compared with previously isolated CHO glycosylation mutants (Stanley, P., Sallustio, S., Krag, S. S., and Dunn, B. (1990) Somatic Cell Mol. Genet. 16, 211-223). In this paper, a biochemical basis for the lec23 mutation is identified. The carbohydrates associated with the G glycoprotein of vesicular stomatitis virus (VSV) grown in Lec23 cells (Lec23/VSV) were found to possess predominantly oligomannosyl carbohydrates that bound strongly to concanavalin A-Sepharose, eluted 3 sugar eq beyond a Man9GlcNAc marker oligosaccharide on ion suppression high pressure liquid chromatography, and were susceptible to digestion with jack bean alpha-mannosidase. Monosaccharide analyses revealed that the oligomannosyl carbohydrates contained glucose, indicating a defect in alpha-glucosidase activity. This was confirmed by further structural characterization of the Lec23/VSV oligomannosyl carbohydrates using purified rat mammary gland alpha-glucosidase I, jack bean alpha-mannosidase, and 1H NMR spectroscopy at 500 MHz. [3H]Glucose-labeled Glc3Man9GlcNAc was prepared from CHO/VSV labeled with [3H]galactose in the presence of the processing inhibitors castanospermine and deoxymannojirimycin. Subsequently, [3H]Glc2Man9GlcNAc was prepared by purified alpha-glucosidase I digestion of [3H]Glc3Man9GlcNAc. When these oligosaccharides were used as alpha-glucosidase substrates it was revealed that Lec23 cells are specifically defective in alpha-glucosidase I, a deficiency not previously identified among mammalian cell glycosylation mutants.

1-Deoxynojirimycin↗
PubMed1989

Glycosylation of eukaryotic peptide chain initiation factor 2 (eIF-2)-associated 67-kDa polypeptide (p67) and its possible role in the inhibition of eIF-2 kinase-catalyzed phosphorylation of the eIF-2 alpha-subunit.

We have reported previously that a 67-kDa polypeptide (p67) present in reticulocyte lysates protects the alpha-subunit of reticulocyte eukaryotic peptide chain initiation factor 2 (eIF-2) from phosphorylation by an eIF-2 kinase, heme-regulated protein synthesis inhibitor (Datta, B., Chakrabarti, D., Roy, A.L., and Gupta, N. K. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3324-3328). We now present evidence that this p67 contains multiple O-linked N-acetylglucosamine (GlcNAc) residues, and these glycosyl residues may be required for p67 activity to protect the eIF-2 alpha-subunit from eIF-2 kinase phosphorylation. Our results are as follows. 1) p67 binds specifically to wheat germ agglutinin, and such binding is completely inhibited in the presence of 0.2 M GlcNAc. 2) The binding of p67 to wheat germ agglutinin leads to complete loss of p67 activity to protect the eIF-2 alpha-subunit from eIF-2 kinase phosphorylation. 3) p67 accepts 10-12 [3H]galactose molecules from UDP-[3H]galactose in the presence of galactosyltransferase. This radioactivity is resistant to endo-beta-N-acetylglucosamine F (+ peptide:N-glycosidase F) treatment but is completely lost when the 3H-labeled p67 is treated with sodium borohydride in mild alkali (beta-elimination reaction). These results suggest that p67 contains terminal GlcNAc moieties O-linked to the protein. 4) Upon hexosaminidase treatment, p67 reaction product migrated as a lower molecular mass (Mr approximately 65 kDa) protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 5) A monoclonal antibody (D1) against p67 has been isolated. D1 apparently recognizes a specific GlcNAc-containing peptide epitope in p67 and does not react with hexosaminidase-treated p67. These results suggest that p67 activity in the cell may also be regulated post-transcriptionally by glycosylation of p67 protein.

Animals↗
PubMed1989

Gastrointestinal polyposis syndromes.

Well recognized cutaneointestinal syndromes in which colonic polyps are a constant and defining feature include Gardner's syndrome (familial adenomatous polyposis), Peutz-Jeghers syndrome, and Cronkhite-Canada syndrome. Colonic polyps are also found with increased frequency in Cowden's disease, the Muir-Torre syndrome, and neurofibromatosis. The Ruvalcaba-Myhre-Smith syndrome is a newly defined entity in this category. The relationship between acrochordons and colonic polyps remains a controversial issue, awaiting additional studies.

Humans↗
PubMed1988

DNA sequence analysis of the Olir2-76 and Ossr1-92 alleles of the Oli-2 region of the yeast Saccharomyces cerevisiae. Analysis of related amino-acid substitutions and protein-antibiotic interaction.

Petite deletion mapping helped to generate a fine-structure genetic map of the Oli-2 region of the mitochondrial genome of Saccharomyces cerevisiae. Here we report the DNA sequence analysis of the Oli-2 region from two drug-resistant alleles (Olir2-76 and Ossr1-92) which are located in the gene for subunit-6 of mitochondrial ATPase, in agreement with their genetic locations on the mitochondrial genome. An analysis of the corresponding amino-acid substitutions is also presented in the context of protein-antibiotic interactions.

Adenosine Triphosphatases↗
PubMed1988

Role of iron in the enhancement by Agrobacterium tumefaciens infection in mice.

Microorganisms require iron for their growth and usually compete with their host for available iron from the system. Iron supplementation to host causes an increase of available iron both to host and to potential microbial invaders and favours the latter more than the former as the bacteria release siderophores which are responsible for iron transport mechanism. In view of this observation a study was done to deal with the distribution of storage and injected iron given as an overload within a physiological pool, taking mice as the host, with a correlation to its utilization by Agrobacterium tumefaciens and with bacterial growth and multiplication. The results obtained help in understanding the host--parasite relationships, regarding bacterial virulence and infection and the growth-promoting effect of iron, as iron promoted the development and progression of serum-exposed A. tumefaciens in mice.

Animals↗