Repair of closely opposed cyclobutyl pyrimidine dimers in UV-sensitive human diploid fibroblasts

An enzyme-sensitive site assay has been used to examine the fate of closely opposed pyrimidine dimers (bifilar enzyme-sensitive sites) in fibroblasts from individuals afflicted with various genetic disorders that confer increased cellular sensitivity to UV radiation. The disappearance of bifilar enzyme-sensitive sites was found to be normal in cells from individuals with Fanconi's anemia, Cockayne's syndrome, dyskeratosis congenita and the variant form of xeroderma pigmentosum. The rate of bifilar enzyme-sensitive site removal in XP cells assigned to complementation group C was reduced by an amount similar to that observed for the repair of isolated dimers. Our results indicate that the initiation of repair at closely opposed dimers is slow in XP-C cells but normal in all other cells examined.     

Purification and characterization of a novel enzyme, N-carbamoylsarcosine amidohydrolase, from Pseudomonas putida 77

N-Carbamoylsarcosine amidohydrolase, a novel enzyme involved in the microbial degradation of creatinine in Pseudomonas putida 77, was purified 27-fold to homogeneity with a 63% overall recovery through simple purification procedures including successive ammonium sulfate fractionation, DEAE-cellulose chromatography, and crystallization. The relative molecular mass of the native enzyme estimated by the ultracentrifugal equilibrium method is 102,000 +/- 5000, and the subunit Mr is 27,000. The Km and Vm values for N-carbamoylsarcosine are 3.2 mM and 1.75 units/mg protein, respectively. Ammonia, carbon dioxide, and sarcosine were formed stoichiometrically from N-carbamoylsarcosine through the action of the purified enzyme preparation. N-Carbamoyl amino acids with a methyl group or hydrogen atom on the amino-N atom and possessing glycine, D-alanine, or one of their derivatives as an amino acid moiety served well as substrates for N-carbamoylsarcosine amidohydrolase. N-Carbamoylsarcosine, N-methyl-N-carbamoyl-D-alanine, N-carbamoylglycine, and N-carbamoyl-D-alanine were hydrolyzed at relative rates of 100, 12.8, 9.8, and 7.3, respectively, by the enzyme. N-Carbamoyl derivatives of D-tryptophan, D-phenylalanine, and those of some other amino acids including D-phenylglycine and p-hydroxy-D-phenylglycine were also hydrolyzed by the enzyme. For the L-isomers of all N-carbamoyl amino acids tested there was no production of ammonia, carbon dioxide, or the corresponding amino acids due to the action of the enzyme. Cupric, mercuric, and silver ions inhibited the enzyme strongly, and some thiol reagents were also found to be inhibitory.     

Reaction kinetics of cephalexin synthesizing enzyme from Xanthomonas citri

In enzymatic synthesis of cephalexin from D-alpha-phenylglycine methyl ester (PGM) and 7-amino-3-deacetoxy-cephalosporanic acid (7-ADCA) using alpha-acylamino-beta-lactam acylhydrolase from Xanthomonas citri, it was found that this enzyme catalyzes all three reactions including PGM hydrolysis, cephalexin synthesis, and cephalexin hydrolysis. Based on our experimental results, a mechanistic kinetic model for cephalexin synthesizing enzyme system having acyl-enzyme intermediate was proposed. From this kinetic model, the reaction rate equations for three reactions were derived, and the kinetic parameters were evaluated. A good agreement between the simulation results and the experimental results was found.     

Oesophageal peristalsis in the cat: the role of central innervation assessed by transient vagal blockade

Studies were performed on five cats to assess the role of extrinsic vagal innervation in the control of peristalsis in the smooth muscle oesophagus. Transient vagal nerve blockade was accomplished by cooling the cervical vagosympathetic nerve trunks previously isolated in skin loops on each side of the neck. Peristalsis throughout the body of the oesophagus was monitored using a continuously perfused multilumen manometry tube. Striated and smooth muscle portions of the esophagus were delineated by abolishing smooth muscle activity with atropine. Secondary peristalsis was assessed by intra-oesophageal balloon distension studies. The threshold volume for balloon-induced secondary peristalsis was lower in the smooth muscle oesophagus. Unilateral vagal blockade reduced the incidence of primary and secondary peristalsis in the striated muscle oesophagus but not in the smooth muscle oesophagus. Bilateral vagal nerve blockade abolished primary swallow-induced peristalsis and secondary peristalsis in both the smooth and striated muscle cat oesophagus. Administration of cholinergic agents or adrenergic blocking agents failed to restore secondary peristalsis in the smooth muscle oesophagus during vagal cooling. We conclude that connections to the central nervous system via the vagal nerve trunks are required for normal secondary as well as primary peristalsis in both the smooth and striated muscle portions of the cat oesophagus.     

Purification and characterization of a bone metalloproteinase that degrades gelatin and types IV and V collagen

A third metalloendopeptidase activity, gelatinase, has been completely separated from the collagenase and proteoglycanase activities of rabbit bone culture medium. Although the proteinase could not be purified to homogeneity in large amounts, it was possible to obtain accurate molecular weight values and activity after electrophoresis on non-reduced SDS/polyacrylamide gels. The latent form had an Mr of 65 000 which could be activated with 4-aminophenylmercuric acetate, APMA, to a form of Mr 61 000; under reducing conditions the latent and active forms had Mr of 72 000 and 65 000, respectively. Trypsin was a very poor activator of the latent enzyme. Gelatinase degraded gelatins derived from the interstitial collagens and it also had low activity on native types IV and V collagen and on insoluble elastin. Gelatinase acted synergistically with collagenase in degrading insoluble interstitial collagen. The specific mammalian tissue inhibitor of metalloproteinases inhibited gelatinase by forming a stable inactive complex. Comparison of the properties of gelatinase with those of collagenase and proteoglycanase suggest that the three proteinases form a family which together are capable of degrading all the major macromolecules of connective tissue matrices.     

Preparation of multilamellar vesicles of defined size-distribution by solvent-spherule evaporation

A novel method of preparing multilamellar vesicles is described. The process involves dispersing in aqueous solutions small spherules of volatile hydrophobic solvents in which amphipathic lipids are dissolved. The lipids form vesicles when the solvents are evaporated in the proper manner. The resulting vesicles have been characterized morphologically with microscopy and electron microscopy. The method yields multilamellar vesicles with a defined size distribution which can be adjusted by varying the duration of mechanical agitation of the spherules and by varying the concentration of amphipathic lipids in the solvents. This is the first fundamentally new method of multilamellar vesicle preparation since Bangham's report in 1965 (Bangham, A.D., Standish, M.M. and Watkins, J.C. (1965) J. Mol. Biol. 13, 238-252).     

Effect of sulfhydryl-disulfide state on protein phosphorylation: phosphorylation of bovine serum albumin

Bovine serum albumin (BSA) was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase under general protein phosphorylation conditions. The optimal pH for this phosphorylation was 9.0. The K0.5 (the concentration required for 50% of maximal phosphorylation) for BSA at pH 7.5 was 15 microM. One mole of phosphate was incorporated per mole of BSA, and only one phosphopeptide fragment was obtained after extensive proteolysis with trypsin. BSA phosphorylation required dithiothreitol or GSH, but GSH was only one-fiftieth as effective as dithiothreitol. GSSG counteracted the effect of dithiothreitol and GSH. Phosphorylation increased in a time-dependent and dithiothreitol concentration-dependent manner when BSA was preincubated with dithiothreitol. The increase in the incorporation of 32P correlated with the appearance of up to six free sulfhydryl groups. The effect of dithiothreitol on BSA appeared reversible, since reoxidation of reduced BSA decreased its susceptibility to phosphorylation. These experiments showed that this in vitro phosphorylation is dependent on the sulfhydryl-disulfide state of BSA. The possible implications of the sulfhydryl-disulfide state of proteins in the regulation of phosphorylation are discussed.     

Synthesis of the transferrin receptor by cultures of embryonic chicken spinal neurons

We have purified a glycoprotein from chicken sciatic nerves, sciatin, which has pronounced trophic effects on avian skeletal muscle cells in culture. Recent studies have shown that sciatin is identical to the iron-transport protein, transferrin, in terms of its physicochemical structure, immunological reactivity, and biological activity. To determine whether transferrin is synthesized and released by neuronal tissue, we incubated cultures of dissociated chicken spinal neurons in a medium free of L-leucine containing either L-3H-amino acids or L-[14C]leucine and immunoprecipitated transferrin with highly specific antibodies. The radiolabeled protein precipitated by rabbit heteroclonal, goat heteroclonal, or mouse monoclonal antitransferrin antibodies increased in specific activity in a linear manner for at least 30 min. Synthesis of this protein was abolished by the presence of puromycin (20 micrograms/ml) or cycloheximide (10(-5) M). The disappearance of the radiolabeled protein from cells was linear with a half-life (t 1/2) of 8-10 h. When immunoprecipitates were separated by SDS gel electrophoresis, a prominent band corresponding to transferrin (Mr 84,000) was visualized by staining with Coomassie Blue. However, when such gels were fluorographed, no radioactivity was apparent in the transferrin region of the gel although a prominent radioactive band was visualized at an Mr of 56,000. The protein of Mr 56,000 was not simply a degradation product of transferrin because this particular protein band was not generated by incubating radiolabeled transferrin with unlabeled neuronal homogenates. The protein of Mr 56,000 was purified from embryonic chicken brain and spinal cord by immunoabsorption chromatography on mouse monoclonal antitransferrin IgG conjugated to Sepharose 4B followed by affinity chromatography on immobilized transferrin. The purified protein bound radioiodinated transferrin and was precipitated by rabbit anti-chicken transferrin-receptor antibodies. Furthermore, this receptor protein was found to be localized on the plasma membrane of dorsal root ganglion neurons by immunocytochemistry using the peroxidase-antiperoxidase technique, and by blocking experiments, which showed that antitransferrin receptor IgG could inhibit the binding of fluorescein-conjugated transferrin at 4 degrees C to cultured neurons in vitro. From these data, we conclude that transferrin is not synthesized by cultures of chicken spinal cord neurons, but that the receptor for transferrin is synthesized by these cultures and is precipitated by antitransferrin antibodies as an antigen-receptor complex.     

Sequence and expression of a human type II mesothelial keratin

Using mRNA from cultured human mesothelial cells, we constructed bacterial plasmids and lambda phage vectors that contained cDNA sequences specific for the keratins expressed in these cells. A cloned cDNA encoding keratin K7 (55 kD) was identified by positive hybrid selection. Southern Blot analysis indicated that this sequence is represented only once in the human genome, and Northern Blot analysis demonstrated that the gene encoding K7 is expressed in abundance in cultured bronchial and mesothelial cells, but only weakly in cultured epidermal cells and not at all in liver, colon, or exocervical tissue. The predicted amino acid sequence of this keratin has revealed a striking difference between this keratin and the type II keratins expressed in epidermal cells: whereas all of the epidermal type II keratins thus far sequenced have long nonhelical termini rich in glycine and serine, this mesothelial type II keratin has amino and carboxy terminal regions that are unusually short and lack the inexact repeats of glycine and serine residues.