Biomedical subjects
Publications and source records attributed to F Cimino.
A group of 463 general surgery patients received topical applications of lincomycin and gentamycin, which significantly reduced the incidence of infected wounds compared to the untreated control group. This confirmed the prophylactic efficacy of topical antibiotic treatment which is also less dangerous than parenteral administration.
A specific, sensitive, and rapid method to measure pseudouridine in human blood serum is described. The method is based on the following steps: (i) deproteinization of serum samples by filtration on membrane cones or by acetonitrile; (ii) purification of nucleosides and concentration of the sample by affinity chromatography on phenylboronate gel followed by lyophilization; and (iii) separation of nucleosides and their quantitation by reverse-phase high-performance liquid chromatography. The pseudouridine mean value in 30 normal subjects was 2.52 +/- 0.28 nmol/ml. The procedure also allows the identification of inosine, uridine, guanosine, and adenosine. Nevertheless, the presence in human blood serum of enzymatic activities which convert adenosine to inosine and cytidine to uridine prevents the precise quantitation of these nucleosides. All the compounds were identified by comparing their retention times and absorbance ratios (A280/A254) with those of pure compounds, as well as by cochromatography.
A human cDNA library was constructed using M13 derivative vectors. The simple and rapid procedures for sequencing single-stranded DNA by the dideoxy chain termination method allowed a screening of individual clones directly by DNA sequence analysis. Some of these clones were identified as coding for: serum albumin, alpha1-antitrypsin, retinol-binding protein, prothrombin, haptoglobin, and metallothionein. Furthermore, a clone coding for aldolase B was tentatively identified on the basis of high sequence homology with rabbit muscle aldolase.
The catabolism of nucleic acids, particularly tRNA, produces a variety of modified nucleosides which are not reutilized by mammalian cells. Investigation of these compounds in body fluids, mainly urine, has recently provided evidence of altered metabolic situations in tumor-bearing patients. The factors involved in the alterations of modified nucleosides formation are connected with altered tRNA-modifying enzymes and/or altered turnover of subpopulations of tRNA. A common pattern in tumor cells or tissues is the presence of isoaccepting tRNA species containing aberrant nucleoside modifications. Several modified nucleosides have been detected and quantitated by HPLC analysis of the urine of normal subjects and cancer patients. Results obtained, in the authors' laboratory, among others, indicate a possible correlation between urinary excretion of these compounds and the course of the disease, with implications for the follow-up of therapeutic treatment. Particular reference should be made to psi, which appears to be a suitable marker for monitoring these subjects. The data from the authors' laboratory also show that the analysis of modified nucleosides in blood may be considered a useful tool in the search for proper markers associated with the cancer status. In this respect psi is suggested as a biochemical indicator for cancer patients.
The tRNA modifying enzyme, S-adenosylmethionine:tRNA(guanine-7-)-methyltransferase, has been extensively purified from Salmonella typhimurium. A rapid and efficient purification method using phosphocellulose chromatography followed by ammonium sulfate precipitation and Sephadex G-100 gel filtration is described. The enzyme appears to be a single polypeptide chain with a molecular weight of approximately 25 000--30 000 daltons. The Km for S-adenosylmethionine and for undermethylated tRNA is 53 microM and 3.4 microM, respectively. The methylation reaction is dependent on added monovalent or divalent cations; 5 mM spermidine, 3 mM MgCl2 and 1 mM spermine are the most effective. The enzyme, though not homogeneous, is free from contaminating ribonucleases and other tRNA methyltransferases.
A new method to measure pseudouridine (psi rd) in blood serum has been used to study patients with cancer of breast, lung, colon, and ovaries, and patients with hepatomas, non-Hodgkin lymphomas, and acute lymphoblastic leukemias, at different stages. The results indicate: (1) a significant (p less than 0.01) difference between the psi rd serum level of the cancer patient group and the normal control group; (2) that the increase of the level of serum psi rd is correlated both to the stage of the disease and to the spreading of the tumor tissue; (3) a correlation between the response to therapy and the behaviour of psi rd serum levels.
Purified tRNA genes are expressed when microinjected into the nucleus of X.laevis oocytes. In this paper we describe a method to assay the capacity to be aminoacylated of the tRNA transcribed in the frog oocytes. The method exploits the radiochemical purity of the transcript and relies on the binding of aminoacyl-tRNA but not of uncharged tRNA to purified elongation factor EF-Tu. We also present some preliminary results on several single point mutants of tRNAPro from Caenorhabditis elegans. We show that nucleotide 73 of tRNAPro can be substituted by any other nucleotide without loss of acceptor activity. A double mutant, causing transition from G45G46 to A45A46 has lost acceptor activity. Also inactive is a mutant carrying an insertion of a single base in the anticodon loop.
Explore the source record for details and available documents.
A fast method for a single-step fractionation of a number of tRNA methyltransferases from Salmonella typhimurium is described. The method basically consists of ion-exchange chromatography on a phosphocellulose column and permits the separation of the enzymes forming mt6A, m1G, m5U, m7G. The enzyme fractions appear sufficiently purified to allow the estimation of some molecular and kinetic properties. The apparent KM for adenosylmethionine range between 1.5 to 3.2 X 10(-5) M, whereas KM for undermethylated tRNA range between 3.1 X 10(-5) M to 3.1 X 10(-4) M. Glycerol gradient determination indicates the following Mr for the native proteins: 25 X 10(3), 40 X 10(3), 50 X 10(3) and 65 X 10(3) for m7G-, mt6A-, m1G- and m5U-forming enzymes, respectively. A complete analysis of methylated nucleosides formed in vivo in S. typhimurium has been obtained: it also allowed us to infer the pattern of the various tRNA methyltransferases for this prokaryote. The tRNA methyltransferase forming mt6A has been isolated for the first time from any type of cell.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Treatment of unadenylylated glutamine synthetase from Escherichia coli with tetranitromethane or with N-acetylimidazole produces alterations in catalytic parameters that are similar to alterations caused by the physiologically important process of adenylylation. All three modification reactions lead to a change in divalent ion requirement for biosynthetic activity; the unmodified enzyme requires Mg(2+) for activity, whereas the modified enzymes exhibit increased activity with Mn(2+). The gamma-glutamyl transferase activity of the modified enzyme is more sensitive to feedback inhibition by tryptophan, histidine, CTP, and AMP, and to inhibition by Mg(2+) or to inactivation by 5 M urea. Finally, the pH optimum for the unmodified enzyme is 7.9, while the modified enzymes are more active at pH 6.8. Since treatment of the enzyme with N-acetylimidazole results in a decrease in absorbancy at 278 mmu and treatment with tetranitromethane causes an increase in absorbancy at 428 mmu, the effects of these reagents are probably due to modification of certain tyrosyl groups on the enzyme. However, other evidence indicates that the tyrosyl residues which are susceptible to adenylylation in the adenylyltransferase-catalyzed reaction are not involved in the acetylation or nitration reactions.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.