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M A Parniak

Publications and source records attributed to M A Parniak.

At least 73 records · Page 4Linked to original sources
PubMed1992

Variable tRNA content in HIV-1IIIB.

Low molecular weight RNA in HIV-1 is found in three size classes resembling 7S RNA, 5S RNA, and tRNA. The 2-dimensional polyacrylamide gel electrophoresis (2D PAGE) patterns of tRNA found in HIV-1 have been determined in virus produced in five different cell types: H9, UHC1 (a U937-derived clone), UHC8 (an RT(-) derivative of U937), HeLa, and COS. The presence of the putative primer tRNA for reverse transcriptase, tRNA(Lys,3), has also been determined either by hybridization with a tRNA(Lys,3)-specific DNA probe or by a comparison of the electrophoretic mobility of viral tRNA species with purified human tRNA(Lys,3). Our results indicate the following: 1) The number of tRNA species found in infectious HIV-1IIIB produced in different cell types varies, according to cell type, from greater than 20 to 4, indicating that only 4 or less tRNA species are required for the viral infectious life cycle. 2) There are 1-3 tRNA species tightly associated to the viral genomic RNA, depending upon the cell type producing the virus. 3) The putative primer tRNA, tRNA(Lys,3), is detected with the tRNA(Lys,3)-specific hybridization probe in the tRNA of HIV-1 produced in H9 cells, and the tightly associated tRNA species in this virus has the same electrophoretic mobility in 1-D PAGE as purified tRNA(Lys,3). On the other hand, we cannot detect tRNA(Lys,3) in the tRNA of HIV-1 produced in HeLa cells, and the tightly associated tRNA found in this virus does not migrate with the same electrophoretic mobility as tRNA(Lys,3).

Animals↗
PubMed1992

Mechanism of inhibition of HIV-1 infection in vitro by purified extract of Prunella vulgaris.

Crude extracts of four Chinese herbs, Arctium lappa, Astragalus membranaceus, Andrographis paniculata, and Prunella vulgaris, were assessed in several tissue culture lines for anti-HIV activity and for cytotoxicity. One extract, obtained from P. vulgaris, was able to significantly inhibit HIV-1 replication with relatively low cytotoxicity. The active factor was purified using sequential precipitations with ethanol and n-butanol, followed by reverse-phase and gel permeation high-performance liquid chromatographic separations. The active component was anionic with a molecular weight of approximately 10 kDa. The purified extract inhibited HIV-1 replication in the lymphoid cell line MT-4, in the monocytoid cell line U937, and in peripheral blood mononuclear cells at effective concentrations of 6, 30, and 12.5 micrograms/ml, respectively. Pretreatment of uninfected cells with the extract prior to viral exposure did not prevent HIV-1 infection. By contrast, preincubation of HIV-1 with the purified extract dramatically decreased infectiousness. The purified extract was also able to block cell-to-cell transmission of HIV-1, prevented syncytium formation, and interfered with the ability of both HIV-1 and purified gp120 to bind to CD4. PCR analysis confirmed the absence of HIV-1 proviral DNA in cells exposed to virus in the presence of the extract. These results suggest that the purified extract antagonizes HIV-1 infection of susceptible cells by preventing viral attachment to the CD4 receptor.

Astragalus propinquus↗
PubMed1992

Isolation and fractionation of retroviral tRNAs.

Previous studies concerning the analysis of retroviral tRNA populations involved intracellular metabolic labeling of RNA, followed by the isolation of viral RNA and lengthy sucrose gradient centrifugation for the separation of tRNAs found in various viral compartments. A more rapid, convenient, and safer method for achieving similar aims is described. Isolated total viral RNA is end-labeled in vitro, and tRNA subgroups are fractionated using commercial Nucleobond AX-20 mini columns. 2-D PAGE analysis of mouse mammary tumor virus tRNA fractionated in this way yields gel patterns similar to those obtained with previously described methods.

Chromatography↗
PubMed1992

In vitro selection of variants of human immunodeficiency virus type 1 resistant to 3'-azido-3'-deoxythymidine and 2',3'-dideoxyinosine.

Drug-resistant variants of human immunodeficiency virus type 1 (HIV-1) have been isolated by in vitro selection. MT-4 cells were infected with either a laboratory strain (HIV-IIIB) or a clinical isolate (no. 187) of HIV-1 and maintained in medium containing subeffective concentrations of the drugs 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyinosine (ddI). By gradually increasing the drug concentration in the culture medium during propagation of the virus on fresh MT-4 cells, we were able to isolate variants of HIV-IIIB and clinical isolate 187 which showed up to 100-fold increases in resistance to the drugs. The drug resistance phenotypes remained stable after propagation of the variants in the absence of drug pressure for over 2 months. However, variants resistant to one drug showed little or no cross-resistance to the other, suggesting that the genetic bases for resistance to the compounds differed. Genotypic analysis of these nucleoside-resistant variants by polymerase chain reaction (PCR) with primer pairs previously shown to correspond to mutations responsible for resistance to AZT was also carried out. A heterogeneity of genotypes was observed, with known mutations at pol codons 70 and 215 occurring in most of the AZT-resistant variants generated from either HIV-IIIB or clinical strain 187. However, mutations in codons 67 and 219 were less frequently detected, and none of these changes were observed in each of four variants resistant to ddI. Cloning and sequencing studies of the reverse transcriptase coding region of two of the isolates were also performed and confirmed the PCR data that had been obtained. In addition to previously described mutation sites responsible for resistance to AZT, an HIV-IIIB-resistant variant was shown to be mutated at positions 108 (Val----Ala) and 135 (Ile----Thr), while a resistant variant of strain 187 was mutated at positions 50 (Ile----Val) and 135 (Ile----Val).

Base Sequence↗
PubMed1992

Novel mutation in the human immunodeficiency virus type 1 reverse transcriptase gene that encodes cross-resistance to 2',3'-dideoxyinosine and 2',3'-dideoxycytidine.

We have used the technique of in vitro selection to generate variants of human immunodeficiency virus type 1 (HIV-1) that are resistant to 2',3'-dideoxyinosine (ddI) and cross-resistant to 2',3'-dideoxycytidine (ddC). The complete reverse transcriptase (RT)-coding regions, plus portions of flanking sequences, of viruses possessing a ddI-resistant phenotype were cloned and sequenced by polymerase chain reaction (PCR)-based methods. We observed that several of these viruses possessed mutations at amino acid sites 184 (Met-->Val; ATG-->GTG) and 294 (Pro-->Ser; CCA-->TCA). These mutations were introduced in the pol gene of infectious, cloned HXB2-D DNA by site-directed mutagenesis. Viral replication assays confirmed the importance of site 184 with regard to resistance to ddI. The recombinant viruses thus generated displayed more than fivefold-greater resistance to ddI than parental HXB2-D did. Moreover, more than fivefold-greater resistance to ddC was also documented; however, the recombinant viruses continued to be inhibited by zidovudine (AZT). No resistance to ddI, ddC, or AZT was introduced by inclusion of mutation site 294 in the pol gene of HXB2-D. PCR analysis performed on viral samples obtained from patients receiving long-term ddI therapy confirmed the presence of mutation site 184 in five of seven cases tested. In three of these five positive cases, the wild-type codon was also detected, indicating that mixtures of viral quasispecies were apparently present. Viruses possessing a ddI resistance phenotype were isolated from both subjects whose viruses contained only the mutated rather than wild-type codon at position 184 as well as from a third individual, whose viruses appeared to be mostly of the mutated variety.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Generation of nucleoside-resistant variants of HIV-1 by in vitro selection in the presence of AZT or DDI but no by combinations.

Several laboratories have shown that AZT-resistant variants of HIV-1 can be isolated from patients who have received prolonged therapy with this drug. Our laboratory has now been able to generate HIV-1 variants resistant to both AZT and ddI, in tissue culture, by using step-wise increases in the concentrations of each of these compounds over a 10-week period. This work has been performed by culturing wild-type clinical strains of HIV-1 as well as the HIV-3b laboratory strain of this virus under such conditions. The ID50 values obtained for the resistant viruses thus generated vary between 50-100 times above those of the parental wild-type strains in each case. Furthermore, we have identified several new mutation sites in the HIV-1 pol gene that are responsible for the observed resistance to AZT and ddI. We have not succeeded, however, in generating drug-resistant strains of HIV-1, under conditions in which several compounds or anti-viral agents were simultaneously present during the in vitro selection process. Combinations of drugs which failed to yield drug-resistant variants included AZT plus ddI, AZT plus alpha-interferon, and ddI plus alpha-interferon. These findings indicate that HIV drug resistance is less likely to occur in tissue culture when combinations of drugs are used, and provide rationale for the development of combination clinical trials for treatment of HIV-associated disease.

Amino Acid Sequence↗
PubMed1991

Incorporation of tRNA into normal and mutant HIV-1.

During retroviral assembly, tRNAs are incorporated into the virion, one of which serves as a primer for the reverse transcription reaction. Using two dimensional polyacrylamide gel electrophoresis, we have studied the patterns of tRNAs incorporated into HIV-1 (3B) produced either in the lymphoid cell line H-9 or in the monocytic cell line U937. We have also examined viral tRNA patterns incorporated in a non-infectious, mutant virion which lacks pol gene products and processed gag protein. Our results lead to the following conclusions: 1) tRNA incorporated into HIV-1 is a select subpopulation of the host-cell's tRNA. 2) The type of tRNA incorporated into the virion is dependent upon cell type. 3) There can be multiple species of tRNA of similar mobilities tightly associated to the viral genome. 4) The packaging of putative primer tRNA into virions requires either the synthesis of pol gene products, the processing of gag proteins, or both, while the incorporation of non-primer tRNAs does not.

Cell Line↗
PubMed1991

Alterations in cell tetrahydrobiopterin levels may regulate queuine hypomodification of tRNA during differentiation of murine erythroleukemia cells.

The base at the first anticodon ("wobble") position of certain eukaryotic tRNA species is either guanine or the hypermodified base queuine. These tRNA species are synthesized with guanine in the wobble position (tRNAG); this guanine can then be replaced with queuine by the action of the enzyme tRNA-guanine ribosyltransferase. In the present report, we show that tRNAG levels increased in response to the induction of erythroid differentiation of murine erythroleukemia (MEL) cells. We also found that tRNA-guanine ribosyltransferase was significantly inhibited by tetrahydrobiopterin. MEL cells showed a transient threefold increase in tetrahydrobiopterin levels 6 to 12 h after exposure of the cells to inducers such as DMSO or tetramethylurea. The increase in tetrahydrobiopterin preceded the increase in tRNAG which in turn preceded the appearance of phenotypic changes characteristic of differentiation. By contrast, a mutant MEL cell line unable to differentiate in response to inducers showed no change in the level of tetrahydrobiopterin or of tRNAG upon exposure to DMSO. N-acetylserotonin, a well-characterized inhibitor of tetrahydrobiopterin synthesis, prevented the DMSO-mediated increase in tetrahydrobiopterin in normal MEL cells. N-acetylserotonin also inhibited the increase in tRNAG levels and the appearance of phenotypic differentiation in these cells.

Animals↗
PubMed1991

Biological comparison of wild-type and zidovudine-resistant isolates of human immunodeficiency virus type 1 from the same subjects: susceptibility and resistance to other drugs.

We used a viral endpoint dilution assay to show changes in the proportion of zidovudine (azidothymidine; AZT)-resistant viruses within a heterogeneous mixture of human immunodeficiency virus type 1 (HIV-1) quasispecies isolated from patients on long-term AZT therapy. Several HIV-1 isolates, which could replicate in 10 microM AZT, were susceptible to both 2',3'-dideoxycytidine and a novel cytosine analog BCH-189, in which a sulfur atom replaces the 3' carbon of the pentose ring. In certain instances, cross-resistance was seen with 3'-didehydro-2',3'-dideoxythymidine. Although most strains of AZT-resistant HIV-1 displayed reduced susceptibility to 3'-azido-2',3'-dideoxyuridine, two strains were identified for which this was not the case.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Interaction with a monoclonal antibody alters the expression of co-operativity by phenylalanine hydroxylase from rat liver.

Phenylalanine hydroxylase purified from rat liver shows positive co-operativity in response to variations in phenylalanine concentration when assayed with the naturally occurring cofactor tetrahydrobiopterin. In addition, preincubation of phenylalanine hydroxylase with phenylalanine results in a substantial activation of the tetrahydrobiopterin-dependent activity of the enzyme. The monoclonal antibody PH-1 binds to phenylalanine hydroxylase only after the enzyme has been preincubated with phenylalanine and is therefore assumed to recognize a conformational epitope associated with substrate-level activation of the hydroxylase. Under these conditions, PH-1 inhibits the activity of phenylalanine hydroxylase; however, at maximal binding of PH-1 the enzyme is still 2-3 fold activated relative to the native enzyme. The inhibition by PH-1 is non-competitive with respect to tetrahydropterin cofactor. This suggests that PH-1 does not bind to an epitope at the active site of the hydroxylase. Upon maximal binding of PH-1, the positive co-operativity normally expressed by phenylalanine hydroxylase with respect to variations in phenylalanine concentration is abolished. The monoclonal antibody may therefore interact with phenylalanine hydroxylase at or near the regulatory or activator-binding site for phenylalanine on the enzyme molecule.

Animals↗
PubMed1989

Glucocorticoid stimulation of tetrahydrobiopterin levels and phenylalanine hydroxylase activity in rat hepatoma cells.

Incubation of H4-II-E-C3 rat hepatoma cells with either hydrocortisone or dexamethasone resulted in 3- to 5-fold increases in the levels of both phenylalanine hydroxylase and its essential cofactor, tetrahydrobiopterin. Maximum elevation of phenylalanine hydroxylase was noted after 24 h of incubation, whereas significant increases in tetrahydrobiopterin were found only after 48 h exposure of the cells to glucocorticoids. Removal of hormone from the culture medium resulted in rapid loss of cell tetrahydrobiopterin, but a much slower decline in the level of phenylalanine hydroxylase. Thus, although the levels of both phenylalanine hydroxylase and tetrahydrobiopterin in rat hepatoma cells are regulated by glucocorticoids, this regulation is apparently not strictly coordinated. Nevertheless, control of cellular tetrahydrobiopterin levels may be an important regulator of hepatic phenylalanine catabolism since significant increases in the ability of intact rat liver cells to hydroxylate phenylalanine were observed only after 48 h exposure to glucocorticoids, in correlation with increases in cell tetrahydrobiopterin content.

Animals↗
PubMed1988

Enhancement of glycogen concentrations in primary cultures of rat hepatocytes exposed to glucose and fructose.

Glycogen synthesis in isolated hepatocytes can occur from glucose both by a direct mechanism and by an indirect process in which glucose is first metabolized to C3 intermediates before use for glycogenesis via gluconeogenesis. We studied the incorporation into glycogen of glucose and the gluconeogenic substrate, fructose, in primary cultures of hepatocytes from fasted rats. In the presence of insulin, both glucose and fructose promoted net deposition of glycogen; however, fructose carbon was incorporated into glycogen to a greater extent than that from glucose. When glucose and fructose were administered simultaneously, the glycogenic utilization of glucose was stimulated 2-3-fold, and that of fructose was increased by about 50%. At constant hexose concentrations, the total incorporation of carbon, and the total accumulation of glycogen mass, from glucose and fructose when present together exceeded that from either substrate alone. Fructose did not change the relative proportion of glucose carbon incorporated into glycogen via the indirect (gluconeogenic) mechanism. The synergism of glucose and fructose in glycogen synthesis in isolated rat hepatocytes in primary culture appears to result from a decrease in the rate of degradation of newly deposited glycogen, owing to (i) decreased amount of phosphorylase a mediated by glucose and (ii) noncovalent inhibition of residual phosphorylase activity by some intermediate arising from the metabolism of fructose, presumably fructose 1-phosphate.

Animals↗
PubMed1988

Effect of alkaline pH on the activity of rat liver phenylalanine hydroxylase.

The pH optimum of rat liver phenylalanine hydroxylase is dependent on the structure of the cofactor employed and on the state of activation of the enzyme. The tetrahydrobiopterin-dependent activity of native phenylalanine hydroxylase has a pH optimum of about 8.5. In contrast, the 6,7-dimethyltetrahydropterin-dependent activity is highest at pH 7.0. Activation of phenylalanine hydroxylase either by preincubation with phenylalanine or by limited proteolysis results in a shift of the pH optimum of the tetrahydrobiopterin-dependent activity to pH 7.0. Activation of the enzyme has no effect on the optimal pH of the 6,7-dimethyltetrahydropterin-dependent activity. The different pH optimum of the tetrahydrobiopterin-dependent activity of native phenylalanine hydroxylase is due to a change in the properties of the enzyme when the pH is increased from pH 7 to 9.5. Phenylalanine hydroxylase at alkaline pH appears to be in an altered conformation that is very similar to that of the enzyme which has been activated by preincubation with phenylalanine as determined by changes in the intrinsic protein fluorescence spectrum of the enzyme. Furthermore, phenylalanine hydroxylase which has been preincubated at an alkaline pH in the absence of phenylalanine and subsequently assayed at pH 7.0 in the presence of phenylalanine shows an increase in tetrahydrobiopterin-dependent activity similar to that exhibited by the enzyme which has been activated by preincubation with phenylalanine at neutral pH. Activation of the enzyme also occurs when m-tyrosine or tryptophan replace phenylalanine in the assay mixture. The predominant cause of the increase in activity of the enzyme immediately following preincubation at alkaline pH appears to be the increase in the rate of activation by the amino acid substrate. However, in the absence of substrate activation, phenylalanine hydroxylase preincubated at alkaline pH displays an approximately 2-fold greater intrinsic activity than the native enzyme.

Animals↗
PubMed1988

Time-dependent alterations in the pathway of glucose incorporation into glycogen in primary cultures of rat hepatocytes following glucose starvation.

Isolated rat hepatocytes maintained in primary culture were able to use glucose for glycogen synthesis by both direct and indirect mechanisms. Cells that had been isolated from fed animals and then cultured in the absence of glucose, but in the presence of gluconeogenic substrates such as pyruvate and amino acids, had decreased glycogen contents compared with similar cells that had been cultured in the presence of glucose. Upon reexposure to glucose, the glucose-starved cells showed time-dependent changes in the preferred pathway for the use of glucose for glycogen synthesis. These changes were noted either in the absence or presence of insulin; however, net accumulation of glycogen was observed only in the presence of the hormone.

Animals↗
PubMed1985

Studies on the primary structure of rat liver phenylalanine hydroxylase.

The primary structure of phenylalanine hydroxylase purified from rat liver was investigated with high speed gel filtration chromatography, cyanogen bromide cleavage and end group analyses of polypeptides derived from the enzyme. On gel filtration in the presence of 6M guanidine hydrochloride, the enzyme gave a single peak corresponding to a molecular weight of 52,000. In the same system the enzyme that had been cleaved with cyanogen bromide gave two peptides (CB1, Mr = 32,800 and CB2, Mr = 20,400). Sequence studies showed that the alignment of these two peptides was CB1 - CB2. Furthermore, in experiments using 32P phosphorylated enzyme, the site of phosphorylation by cAMP-dependent protein kinase was found to be located on the CB1 peptide. The NH2-terminus of this enzyme, which was found to be blocked, was shown to be N-acetylalanine. By both carboxypeptidase A digestion and hydrazinolysis, the carboxyl terminus was identified as serine. These data indicate that the phenylalanine hydroxylase molecule from rat liver is composed of subunits which are homogenous or, at least, very similar in their primary structure.

Amino Acids↗
PubMed1984

Spectroscopic investigation of ligand interaction with hepatic phenylalanine hydroxylase: evidence for a conformational change associated with activation.

We have examined the interaction of phenylalanine hydroxylase with phenylalanine, tetrahydropterin cofactors, and an activating phospholipid, lysophosphatidylcholine. Incubation of native phenylalanine hydroxylase with phenylalanine or lysophosphatidylcholine results in an increase in the fluorescence emission of the enzyme at 360 nm, which closely parallels the increase in tetrahydrobiopterin-dependent activity observed under these conditions. The presence of tetrahydrobiopterin in the absence of phenylalanine results in quenching of the enzyme fluorescence emission; this quenching exhibits a sharp end point at about 1 mol of tetrahydrobiopterin bound/mol of enzyme subunit. The binding of tetrahydrobiopterin under these conditions is unexpectedly tight, with an estimated KD of 10-20 nM, while in the presence of lysophosphatidylcholine, the KD is increased to about 25 microM. Quenching experiments with sodium iodide indicate greater exposure of tryptophan residues in the phenylalanine-activated enzyme. The ultraviolet difference spectrum of phenylalanine hydroxylase in the presence of phenylalanine exhibits a peak at 238 nm, which correlates with the fluorescence increase and activation, as well as additional changes in the aromatic region, which do not correlate well with activation. Phenylalanine does not alter the far-ultraviolet circular dichroism spectrum of phenylalanine hydroxylase. In contrast, lysophosphatidylcholine appears to induce a dramatic change in enzyme secondary structure upon activation. These results suggest that activation of phenylalanine hydroxylase results in a conformation change and the exposure of buried tryptophan(s) and possibly a cysteine residue.

Animals↗
PubMed1984

The interaction of aromatic amino acids with rat liver phenylalanine hydroxylase.

We have examined the interaction of hepatic phenylalanine hydroxylase with the phenylalanine analogs, tryptophan and the diastereomers of 3-phenylserine (beta-hydroxyphenylalanine). Both isomers of phenylserine are substrates for native phenylalanine hydroxylase at pH 6.8 and 25 degrees C, when activity is measured with the use of the dihydropteridine reductase assay coupled with NADH in the presence of the synthetic cofactor, 6-methyl-5,6,7,8-tetrahydropterin. However, while erythro-phenylserine exhibits simple Michaelis-Menten kinetics (Km = 1.2 mM, Vmax = 1.2 mumol/min X min) under these conditions, the threo isomer exhibits strong positive cooperativity (S0.5 = 4.8 mM Vmax = 1.4 mumol/min X mg, nH = 3). Tryptophan also exhibits cooperativity under these conditions (S0.5 = 5 mM, Vmax = 1 mumol/min X mg, nH = 3). The presence of 1 mM lysolecithin results in a hyperbolic response of phenylalanine hydroxylase to tryptophan (Km = 4 mM, Vmax = 1 mumol/min X mg) and threo-phenylserine (Km = 2 mM, Vmax = 1.4 mumol/min X mg). erythro-Phenylserine is a substrate for native phenylalanine hydroxylase in the presence of the natural cofactor, L-erythro-tetrahydrobiopterin (BH4) (Km = 2 mM, Vmax 0.05 mumol/min X mg, nH = 2). Preincubation of phenylalanine hydroxylase with erythro-phenylserine results in a 26-fold increase in activity upon subsequent assay with BH4 and erythro-phenylserine, and hyperbolic kinetic plots are observed. In contrast, both threo-phenylserine and tryptophan exhibit negligible activity in the presence of BH4 unless the enzyme has been activated. The product of the reaction of phenylalanine hydroxylase with either isomer of phenylserine was identified as the corresponding p-hydroxyphenylserine by reaction with sodium periodate and nitrosonaphthol. With erythro-phenylserine, the hydroxylation reaction is tightly coupled (i.e. 1 mol of hydroxyphenylserine is formed for every mole of tetrahydropterin cofactor consumed), while with threo-phenylserine and tryptophan the reaction is largely uncoupled (i.e. more cofactor consumed than product formed). Erythro-phenylserine is a good activator, when preincubated with phenylalanine hydroxylase (A0.5 = 0.2 mM), with a potency about one-third that of phenylalanine (A0.5 = 0.06 mM), while threo-phenylserine (A0.5 = 6 mM) and tryptophan (A0.5 approximately 10 mM) are very poor activators. Addition of 4 mM tryptophan or threo-phenylserine or 0.2 mM erythro-phenylserine to assay mixtures containing BH4 and phenylalanine results in a dramatic increase in the hydroxylation at low concentrations of phenylalanine.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗