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K M Munir

Publications and source records attributed to K M Munir.

18 recordsLinked to original sources
PubMed1994

Herpes thymidine kinase mutants with altered catalytic efficiencies obtained by random sequence selection.

We have obtained 190 active Herpes simplex virus type 1 thymidine kinase mutants by substituting a 33 nucleotide sequence with 20% degeneracy for a portion of the nucleotide sequence that encodes the putative thymidine binding site [K.M. Munir, D.C. French, D.K. Dube and L.A. Loeb (1992) J. Biol. Chem., 167, 6584-6589]. In order to classify these mutants with respect to thymidine kinase activity we determined the ability of Escherichia coli harboring these mutants to form colonies in the presence of varying concentrations of thymidine. Escherichia coli harboring one of the mutant enzymes was able to form colonies at a concentration of thymidine lower than did the wild type. It was able to phosphorylate thymidine more rapidly than the wild type both in vivo and in vitro. The increased thymidine kinase activity was manifested by (i) a 42% enhanced uptake of [methyl-3H]thymidine into E. coli, (ii) a 2.4 times higher rate of [methyl-3H]thymidine incorporation into acid-insoluble material and (iii) a 5-fold increase in the kcat of the purified enzyme compared to the wild type. Herpes thymidine kinase purified from other mutants that formed colonies at higher thymidine concentrations than that of the wild type exhibited a decrease in kcat. The kcat of one of these mutant thymidine kinases was 10(-4) of that of the wild type enzyme. This study demonstrates that a spectrum of mutant enzymes with different catalytic properties can be obtained by selection from a plasmid with random sequence substitutions and this can be done in the absence of rational protein design.

Amino Acid Sequence↗
PubMed1993

Selection of new biologically active molecules from random nucleotide sequences.

Genetic diversity can be achieved in vitro by inserting random nucleotide (nt) sequences into cloned genes. In the case of enzymes, subsequent genetic complementation can be used to select for new mutants that exhibit different substrate specificities, altered catalytic activities, or altered temperature sensitivities. Using this technique, one can also analyze the contribution of different amino acid residues to the structure and function of enzyme. Selecting biologically active DNA sequences from large random populations provides a new method for identifying nt sequences with unique functions. Analogous random sequence selection techniques have been applied to determine the consensus sequence of the Escherichia coli promoters, DNA and RNA sequences that bind specific protein(s), DNA regulatory sequences, ribozyme(s) and ligand-specific RNA(s). In this manuscript, we will consider recent data obtained in our laboratory as a result of inserting random sequences into the putative nucleoside-binding site of herpes simplex virus type 1 (HSV-1) thymidine kinase (TK). We have obtained over 2000 new mutant HSV-1 TKs, some of which are stable at higher temperatures or have altered substrate specificity and/or catalytic rates when compared to those of the wild-type enzyme.

Base Sequence↗
PubMed1993

Thymidine kinase mutants obtained by random sequence selection.

Knowledge of the catalytic properties and structural information regarding the amino acid residues that comprise the active site of an enzyme allows one, in principle, to use site-specific mutagenesis to construct genes that encode enzymes with altered functions. However, such information about most enzymes is not known and the effects of specific amino acid substitutions are not generally predictable. An alternative approach is to substitute random nucleotides for key codons in a gene and to use genetic selection to identify new and interesting enzyme variants. We describe here the construction, selection, and characterization of herpes simplex virus type 1 thymidine kinase mutants either with different catalytic properties or with enhanced thermostability. From a library containing 2 x 10(6) plasmid-encoded herpes thymidine kinase genes, each with a different nucleotide sequence at the putative nucleoside binding site, we obtained 1540 active mutants. Using this library and one previously constructed, we identified by secondary selection Escherichia coli harboring thymidine kinase mutant clones that were unable to grow in the presence of concentrations of 3'-azido-3'-deoxythymidine (AZT) that permits colony formation by E. coli harboring the wild-type plasmid. Two of the mutant enzymes exhibited a reduced Km for AZT, one of which displayed a higher catalytic efficiency for AZT over thymidine relative to that of the wild type. We also identified one mutant with enhanced thermostability. These mutants may have clinical potential as the promise of gene therapy is increasingly becoming a reality.

Amino Acid Sequence↗
PubMed1992

Permissible amino acid substitutions within the putative nucleoside binding site of herpes simplex virus type 1 encoded thymidine kinase established by random sequence mutagenesis [corrected].

We determined the essentiality of all amino acid replacements within an 11-codon sequence in the putative nucleoside-binding site of thymidine kinase encoded by herpes simplex virus type 1. This involved partial randomization of 11 codons in the gene to create a degenerate library, followed by genetic complementation using a tk- Escherichia coli strain and selection of unnatural active enzymes. We produced and tested 53,000 variants; of which 190 were found to be biologically active. Sequence analyses of functional variants revealed a high degree of flexibility in accommodating different types of amino acid substitutions in this region. However, no replacement was tolerated at proline-173, whereas tyrosine-172 could be replaced by only phenylalanine. To further define permissible substitutions at specified positions, we constructed a library with randomization at only four test codons. We produced and tested 600,000 variants; of which only 5 were active. Again proline-173 was conserved, and only tyrosine and phenylalanine were found at position 172. The identification of these conserved amino acids should provide important insights into the understanding of the structural basis of catalysis by this enzyme.

Amino Acid Sequence↗
PubMed1991

Artificial mutants generated by the insertion of random oligonucleotides into the putative nucleoside binding site of the HSV-1 thymidine kinase gene.

We have obtained 42 active artificial mutants of HSV-1 thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) by replacing codons 166 and 167 with random nucleotide sequences. Codons 166 and 167 are within the putative nucleoside binding site in the HSV-1 tk gene. The spectrum of active mutations indicates that neither Ile166 nor Ala167 is absolutely required for thymidine kinase activity. Each of these amino acids can be replaced by some but not all of the 19 other amino acids. The active mutants can be classified as high activity or low activity on two bases: (1) growth of Escherichia coli KY895 (a strain lacking thymidine kinase activity) in the presence of thymidine and (2) uptake of thymidine by this strain, when harboring plasmids with the random insertions. E. coli KY895 harboring high-activity plasmids or wild-type plasmids can grow in the presence of low amounts of thymidine (less than 1 microgram/mL), but are unable to grow in the presence of high amounts of thymidine. On the other hand, E. coli KY895 harboring low-activity plasmids can grow at a high concentration of thymidine (greater than 50 microgram/mL) in the media. The high-activity plasmids also have an enhanced [3H]dT uptake. The amounts of thymidine kinase activity in vitro in unfractionated extracts do not correlate with either growth at low thymidine concentration or the rate of thymidine uptake. Heat inactivation studies indicate that the mutant enzymes are without exception more temperature-sensitive than the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Normal hepatocytes exhibiting histone H3 with antibody accessible sites that are cryptic in carcinogen-altered hepatocytes.

A Mr 14,000 polypeptide (p14), identified as liver fatty acid binding protein, in normal liver cytosol was shown previously to be the principal target of the carcinogen, N-2-fluorenylacetamide (2-acetylaminofluorene), early during hepatic carcinogenesis in rats. Immunohistochemical analyses using rabbit antiserum against pure p14/liver fatty acid binding protein revealed marked increases in the levels of the protein in cytoplasm specifically during mitosis in normal and regenerating hepatocytes, and throughout the cell cycle in hyperplastic and malignant hepatocytes brought about by carcinogen, N-2-fluorenylacetamide (2-acetylaminofluorene) or 3'-methyl-4-dimethylaminoazobenzene. Present also in normal hepatocytes was a nuclear antigen that was not detected in the hyperplastic hepatocytes, benign hepatocytic adenomas, and hepatocellular carcinomas produced by these carcinogens. The nuclear antigen was discerned to be a Mr 17,000 polypeptide (p17) in extracts of normal liver nuclei and nucleosomes. In the present study, the p17 was purified by high-performance liquid chromatography and identified as being the three variants of histone H3, based on common molecular size, amino acid composition, electrophoretic migration in Triton-acetic acid-urea gels, and Western blot and histochemical reactions using affinity-purified antibodies. The histone H3 of all tested organs reacted specifically with the antiserum in Western blots following sodium dodecyl sulfate gel electrophoresis. In contrast, in a survey of 23 normal rat organs, nuclei of virtually only hepatocytes were reactive immunohistochemically. In view of the exceptional immunohistochemical reactivity of nuclei of normal hepatocytes, attributable to accessible histone H3, and the lack of such reaction in carcinogen-altered hepatocytes, the collected evidence indicates that normal hepatocytes contain uniquely available histone H3 sites that become cryptic during the chemical carcinogenesis.

Animals↗
PubMed1988

Late target protein of the carcinogen N-2-fluorenylacetamide in rat liver.

In previous studies, administration of a radioactive tracer dose of the liver carcinogen, N-2-fluorenylacetamide (2-acetylaminofluorene; FAA), to normal or carcinogen-fed rats led to the presence of one or two principal labeled carcinogen: protein complexes in liver cytosol. The early target Mr 14,000 protein of the carcinogen in normal rats was identified as being liver fatty acid-binding protein and was associated in hepatocytes with normal mitosis and the cell proliferation brought about by either of the two liver carcinogens, FAA or 3'-methyl-4-dimethylaminoazobenzene. Continued ingestion of any of the three hepatocarcinogens, FAA, 3'-methyl-4-dimethylaminoazobenzene, or ethionine, resulted in the progressive loss of the early radioactive complex and the concurrent gain in liver cytosol of the late carcinogen: protein complex (Mr approximately 150,000) formed by the tracer dose of FAA. Present attempts to extract FAA derivatives from the late carcinogen: protein complex with organic solvents indicated that virtually all of the carcinogen was apparently covalently bound to the resultant denatured protein. It is unknown whether the covalent interaction occurred in vivo or as an accompaniment of the protein denaturation associated with the solvent extractions. In support of a possible noncovalent interaction, treatment of the unextracted complex with sodium dodecyl sulfate, urea, and beta-mercaptoethanol followed by electrophoresis readily dissociated the majority of the bound carcinogen. The late carcinogen: protein complex was shown to contain a 55 kDa subunit (p55), which was purified to homogeneity according to molecular size. The subunit is a relatively basic polypeptide with a pI of 8.4 to 8.6. In Western blots using rabbit immunoglobulins against the p55, the late target protein was found to be present at low concentrations in liver cytosols of normal rats, and was induced to relatively high levels by ingestion of the carcinogen for 3 to 5 weeks. The induction of high levels of the late target protein explains in part the progressive elevation in content of the late carcinogen: protein complex in rat liver during carcinogenesis by FAA. The isolated p55 was susceptible to a spontaneous stepwise breakdown, resulting in a ladder of decreasing molecular sizes with an average unit difference of 5.8 kDa per step over six size intervals. The p55 subunit was detected in nonhepatic organs of normal rats, but unlike levels in liver, the levels there were not affected by ingestion of the carcinogen.(ABSTRACT TRUNCATED AT 400 WORDS)

2-Acetylaminofluorene↗
PubMed1984

Deviations in ornithine-related metabolism during hexachlorocyclohexane-induced hepatocarcinogenesis in mice: evidence for conversion of glutamate to ornithine.

Chronic administration of technical-grade hexachlorocyclohexane in Swiss male mice resulted in necrosis, and later in adenomatous nodules and hepatocellular carcinomas at 3, 7, and 10 months, respectively, after initiation of the experiment. A definite pattern of changes were observed of arginase, ornithine transaminase, ornithine carbamoyltransferase activities, and metabolites related to ornithine. Conversion of glutamate to ornithine correlated well with the decreased glutamate and constant ornithine in liver of mice fed hexachlorocyclohexane for 7 months.

Animals↗
PubMed1984

Comparative formation of chlorophenol metabolites from hexachlorocyclohexane in mouse and rat in vivo and in vitro.

Comparative formation of chlorophenol metabolites were evaluated in hexachlorocyclohexane (HCH) fed mouse and rat livers by h.p.l.c. No significant species differences were noted in the formation of chlorophenol metabolites studied both in vivo and in vitro. The results indicate that the marked species difference that is observed in tumor induction between mice and rats by HCH may not be due to the different rate of formation of chlorophenol metabolites.

Animals↗
PubMed1984

Effect of hexachlorocyclohexane on diethylnitrosamine-induced hepatocarcinogenesis in rat and its failure to promote skin tumors on dimethylbenz[a]anthracene initiation in mouse.

The promoting action of hexachlorocyclohexane (HCH) was evaluated in female Wistar rats by counting the gamma-glutamyltranspeptidase-positive foci which appeared in the liver after diethylnitrosamine (DENA) initiation. Liver tumor promotion by HCH was further confirmed in a long-term tumorigenicity study after DENA pretreatment. It seems likely that HCH is specific for liver tumor promotion since it failed to promote dimethylbenz[a]anthracene-initiated skin tumors in mice.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1984

Induction of oral mucosal tumors in hamsters and rats treated with methyl(acetoxymethyl)nitrosamine.

An oral tumor model has been developed in inbred Syrian golden hamsters by continuous applications every 2 weeks of methyl(acetoxymethyl)nitrosamine [(DMN-OAC) CAS: 56856-83-8; methylnitrosaminomethyl ester acetic acid] at 2 mg/kg body weight alone or by a single application of DMN-OAC followed by continuous twice weekly applications of 12-O-tetradecanoylphorbol 13-acetate (TPA) (1 microgram/animal). Similar studies were done in the W rat buccal mucosa. In the hamsters treated continuously with DMN-OAC, 100% of the tumors were observed in the cheek pouch; none were observed at other sites. In contrast, in the rats treated similarly, only a 67% tumor incidence was observed, of which only 42% were oral tumors. A promoter effect of TPA was observed in hamster cheek pouch tumors induced by DMN-OAC, whereas rat oral mucosa did not respond to TPA treatment.

Animals↗
PubMed1983

Hexachlorocyclohexane-induced tumorigenicity in mice under different experimental conditions.

Continuous administration of 500 ppm hexachlorocyclohexane (HCH) in the diet induced 100% liver tumors in mice, but none were observed in rats and hamsters treated with the same dose of HCH. In Swiss male mice, tumor incidence at earlier ages was greater than that observed in corresponding age groups of Swiss female mice. It was further observed that in Swiss females, sex hormones retarded the tumorigenic effect of HCH; in BALB/c mice a similar phenomenon was not observed.

Animals↗
PubMed1983

Influence of estradiol on the benzo(a)pyrene hydroxylase activity induced by hexachlorocyclohexane.

Basal BP-hydroxylase activity was measured in male Swiss mice from the age of 3 weeks to 20 months. Maximal enzyme activity was at the age of 5 months. Comparison of the inducibility of BP-hydroxylase by HCH was also investigated in male and female mice of different ages. Male mice showed higher induction of BP-hydroxylase by HCH than females of the same ages. Sterilization of female mice enhanced enzyme induction. Estradiol exhibited competitive inhibition of BP-hydroxylase activity. After treatment with HCH for 8 months, female mice had a lower tumour incidence than males, and this paralleled a lower induction of BP-hydroxylase.

Aging↗
PubMedDate not supplied

Developmental psychiatry: is there any other kind?

This paper describes the importance of the developmental perspective in psychiatry and addresses the lack of a developmental focus in the DSM-based descriptive empirical model. Although the publication of DSM-III in 1980 represented a "breakthrough" in psychiatry, the revisions of its diagnostic framework over the subsequent two decades have not adapted to the rapidly evolving changes in the field. In this paper we argue that, like once-grand theories, the breakthroughs in the diagnostic framework need to transform. The developmental perspective provides an interdisciplinary and conceptual framework linking facts and theories. It is inherent in different aspects of psychiatry and readily accommodates the descriptive-empirical model by means of inclusive concepts borrowed from developmental psychopathology and psychobiology. It also makes important contributions to a process-oriented approach to measurement. Developmentally operationalized and multidimensional constructs stand to broaden psychiatric domains beyond diagnosable disorders. This argues for preventive and early treatment interventions for a variety of mild, subthreshold, or delayed symptoms of various conditions, based on an understanding of the causal mechanisms and developmental processes involved.

Child Psychiatry↗