Insulin-induced expression of human heat-shock protein gene hsp70

In human hepatoma cell line Hep3B/T2, the human heat-shock-inducible gene hsp70 could be induced by insulin. The dose-dependent insulin effect correlates very well with the dissociation constant of the insulin receptor, indicating that the insulin effect is mediated by the insulin receptor. The expression of hsp70 gene was neither significantly induced by growth factors of epidermal and platelet-derived growth factors, nor by tumor promoter, calcium ionophore, cAMP, and glucocorticoids. These results indicate that the induction of expression of hsp70 gene by insulin is very specific and not cell cycle-related. Furthermore, the insulin-induced expression of hsp70 gene is transient, occurring specifically from 4 to 8 h after insulin addition.     

Enzymes of vitamin B6 degradation. Purification and properties of two N-acetylamidohydrolases

alpha-(N-Acetylaminomethylene)succinic acid hydrolase (Compound A hydrolase, EC 3.5.1-) and alpha-hydroxymethyl-alpha'-(N-acetylaminomethylene)succinic acid hydrolase (Compound B hydrolase, EC 3.5.1-) were purified to homogeneity from Pseudomonas MA-1 and Arthrobacter Cr-7, respectively. The two inducible enzymes catalyze Reactions 1 and 2, respectively, which release the first generally useful anabolic intermediates during growth of these organisms with (formula; see text) pyridoxine as a sole source of carbon and nitrogen. Compound A hydrolase is a monomeric protein of Mr 32,500 with aspartic acid as its NH2-terminal residue. Compound B hydrolase (Mr congruent to 205,000) is a multimer containing probably six identical subunits with glycine as the NH2 terminus. The two enzymes have quite different amino acid analyses, although both are high in Asx and Glx, lack tryptophan, and show similar stabilities to pH and temperature. Compound A hydrolase has a pI of 4.4, a Km of 3.3 microM, and a Vmax of 3.1 mumol X min-1 X mg-1 at pH 6.5 and 25 degrees C; no analogue substrates were found. Compound B hydrolase has a pI of 4.2, a Km of 25 microM, and a Vmax of 3.8 mumol X min-1 X mg-1 at 25 degrees C and pH 7.0; it also hydrolyzes Compound A slowly. Both enzymes are inhibited competitively by di- and tricarboxylic acids, itaconic acid being among the most effective. Sulfite inhibits both enzymes by a time-dependent mechanism not yet understood. The two amidases appear to differ greatly in architecture despite the similarity in properties and in the overall reactions they catalyze.     

Novel extracellular enzymes (ligninases) of Phanerochaete chrysosporium

Abstract 3 New spectrophotometric enzyme assays were developed for the study of microbial lignin-degrading enzymes. The conversion of 2-methoxy-3-phenylbenzoic acid to 2-hydroxy-3-phenylbenzoic acid led to the discovery of an extracellular, aromatic methyl ether demethylase produced by the white-rot fungus Phanerochaete chrysosporium . The conversion of methyl 2-hydroxy-3-phenylbenzoate to 2-hydroxy-3-phenylbenzoic acid allowed the identification of an extracellular, aromatic methyl ester esterase produced by this fungus. The Phanerochaete sp. also excreted an enzyme complex that oxidized 4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one, probably to aliphatic products. All 3 novel enzyme activities were produced together with, and probably comprise a part of, the Phanerochaete ligninolytic enzyme complex. Unlike previously known ligninases, these enzymes did not oxidize 3,4-dimethoxybenzyl alcohol. All 3 were H₂O₂-dependent and were activated by Mn²⁺ ions.     

Inhibition of cell wall-associated enzymes in vitro and in vivo with sugar analogs

Sugar analogs were used to study the inhibition of cell wall-associated glycosidases in vitro and in vivo. For in vitro characterization, cell walls were highly purified from corn (Zea mays L.) root cortical cells and methods were developed to assay enzyme activity in situ. Inhibitor dependence curves, mode of inhibition, and specificity were determined for three sugar analogs. At low concentrations of castanospermine (CAS), 2-acetamido-1,5-imino-1,2,5-trideoxy-d-glucitol, and swainsonine, these inhibitors showed competitive inhibition kinetics with β-glucosidase, β-GIcNAcase, and α-mannosidase, respectively. Swainsonine specifically inhibited α-mannosidase activity, and 2-acetamido-1,5-imino-1,2,5-trideoxy-d-glucitol specifically inhibited β-N-acetyl-hexosamindase activity. However, CAS inhibited a broad spectrum of cell wall-associated enzymes. When the sugar analogs were applied to 2 day old corn seedlings, only CAS caused considerable changes in root growth and development. To ensure that the concentration of inhibitors used in vitro also inhibited enzyme activity in vivo, an in vivo method for measuring cell wall-associated activity was devised.     

HH2A,an immortalized bovine mammary epithelial cell line,expresses the gene encoding mammary derived growth inhibitor (MDGI)

Summary We have established and partially characterized a spontaneously immortalized bovine mammary epithelial cell line, designated HH2a. The cells express the gene encoding for mammary derived growth inhibitor (MDGI) when grown on released collagen gels in the presence of lactogenic hormones. This is the first report of a cell line that expresses MDGI. Immunohistochemical studies showed that HH2a cells contain keratin intermediate filaments and desmosomes. When plated on confluent monolayer of live fibroblasts, HH2a cells extensively contacted with fibroblasts. When embedded in the collagen gels, they rearranged themselves to produce three-dimensional duct-like outgrowths extending into the matrix. The HH2a cell line should be useful in investigations of the roles of cell-cell and cell-extracellular interactions in regulation of breast epithelial cell proliferation, and of the hormonal regulation of MDGI gene expression.     

Subcellular localization of IRS-1 in cell proliferation and differentiation.

The type 1 insulin-like growth factor receptor (IGF-IR) and its docking protein, insulin receptor substrate-1 (IRS-1), play important roles in cell transformation, cell differentiation and aging. IRS-1 and other IRS proteins can, under certain conditions, localize to the nuclei of cells, where they undergo interactions with nuclear and nucleolar proteins. In this study, we confirm and extend these observations, demonstrating that IRS-1 is preferentially nuclear in growing cells. Differentiation and inhibition of ribosomal RNA synthesis cause subcellular redistribution of IRS-1 and other nuclear proteins to the cytoplasm.     

Synthesis and degradation of aflatoxins by Aspergillus parasiticus. II. Comparative toxicity and mutagenicity of aflatoxin B1 and its autolytic breakdown products

A crude mycelial protein extract from a 16-day-old culture of A. parasiticus, on purification, lost 50% of its ability to degrade aflatoxin B1. The addition of hydrogen peroxide increased this activity to 97% of that of the crude extract. Ducklings dosed orally with aflatoxin extracts from 14- and 20-day-old cultures containing 46 micrograms or more of aflatoxin B1 developed enlarged livers, haemorrhaged and died in less than 10 days, giving and LD50 of 17.5 and 17.1 micrograms aflatoxin B1 per 50 g body weight respectively for each extract. When pure aflatoxin B1 was mixed with either the crude or purified mycelial protein extract the aflatoxin B1 level was decreased by 29% as was the toxicity of the mixture. The main breakdown product of aflatoxin B1 was isolated and was shown to have an RF value of 0.34, was non-fluorescent, and was non-toxic for ducklings at oral doses as high as 400 micrograms per 50 g body weight. The mutagenic effect of aflatoxin B1 on Salmonella typhimurium was relative to its concentration. The main breakdown product of aflatoxin B1 was non-mutagenic.     

Comparison of enzyme-linked immunomagnetic chemiluminescence with U.S. Food and Drug Administration's Bacteriological Analytical Manual method for the detection of Escherichia coli O157:H7

Escherichia coli O157:H7, a major foodborne pathogen, has been associated with numerous cases of foodborne illnesses. Rapid methods have been developed for the screening of this pathogen in foods in order to circumvent timely plate culture techniques. Unfortunately, many rapid methods are presumptive and do not claim to confirm the presence of E. coli O157:H7. The previously developed method, enzyme-linked immunomagnetic chemiluminescence (ELIMCL), has been improved upon to allow for fewer incidences of false positives when used to detect E. coli O157:H7 in the presence of mixed cultures. The key feature of this assay is that it combines the highly selective synergism of both anti-O157 and anti-H7 antibodies in the sandwich immunoassay format. This work presents application of a newly semi-automated version of ELIMCL to the detection of E. coli O157:H7 in pristine buffered saline yielding detection limits of approximately 1 x 10(5) to 1 x 10(6) of live cells/mL. ELIMCL was further demonstrated to detect E. coli O157:H7 inoculated into artificially contaminated ground beef at ca. 400 CFU/g after a 5 h enrichment and about 1.5 h assay time for a total detection time of about 6.5 h. Finally, ELIMCL was compared with USFDA's Bacteriological Analytical Manual method for E. coli O157:H7 in a double-blind study. Using McNemar's treatment, the two methods were determined to be statistically similar for the detection of E. coli O157:H7 in ground beef inoculated with mixed cultures of select bacteria.