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L I Pizer

Publications and source records attributed to L I Pizer.

At least 37 records · Page 2Linked to original sources
PubMed1983

Transcription from the BamHI J fragment of herpes simplex virus type 1 (KOS).

RNA transfer experiments (Northern analyses) were used to localize polyadenylated mRNA species made after herpes simplex virus type 1 infection to EcoRI and BamHI fragments and subfragments from the short unique region of the herpes simplex virus type 1 (KOS) genome. Three predominant early mRNAs of 2.5, 1.3, and 0.9 kilobases map in the BamHI J fragment. A detailed restriction map of the BamHI J fragment was constructed.

Cell Line↗
PubMed1983

RNAs transcribed from a 3.6-kilobase SmaI fragment of the short unique region of the herpes simplex virus type 1 genome.

A 3.6-kilobase (kb) SmaI subclone of the BamHI J fragment of herpes simplex virus type 1 (KOS) DNA was utilized to characterize the mRNAs transcribed from the genome segment (0.91 to 0.93 map units) that encodes glycoprotein D mRNA. RNA blotting demonstrated two major RNA species of 2.3 and 1.5 kb. 5' and 3' mapping with 32P-end-labeled DNA fragments indicated that these RNAs are a nested set, each having its own promoter and 3' terminus. Less abundant RNA species with discrete 5' ends were also observed. Precise 5' mapping and sequence data located the initiation sites and demonstrated TATA boxes, CAT boxes, and AC-rich regions in the appropriate positions. 3' mapping located a common end for both mRNAs, but the 2.3-kb mRNA was reduced in size by splicing at a point near the RNA terminus. In vitro runoff transcription experiments confirmed the location of the two promoters and showed that an uninfected cell extract initiated faithfully at both sites. Despite the similarities in DNA structure and the apparent equal efficiency of promoter utilization in vitro, the 2.3-kb mRNA appeared in the cytoplasm early (1 h) after infection, whereas the 1.5-kb mRNA was delayed until 3 h after infection.

Base Sequence↗
PubMed1983

Characterization of a phosphoprotein associated with the SS-B/La nuclear antigen in adenovirus-infected and uninfected KB cells.

We have employed sera from patients with autoimmune disease to characterize the nuclear SS-B/La antigen in uninfected and adenovirus-infected KB cells. A 45,000-dalton phosphorylated polypeptide was specifically precipitated with anti-SS-B sera, and the level of phosphorylation was increased after infection. The increased phosphorylation appears to occur at the same amino acid residues phosphorylated in uninfected cells and results from increased phosphorylating activity rather than from altered enzyme specificity. A competition experiment between infected and uninfected cell extracts indicates that the antigen in the infected cell binds more strongly to SS-B/La antibodies. Fragments of adenovirus-induced virus-associated RNA as well as intact molecules complex with SS-B/La antigen and are immune precipitated with autoimmune sera.

Adenoviridae↗
PubMed1982

Delayed hypersensitivity to herpes simplex virus: murine model.

Cell-mediated immunity has been shown to be clinically important in recovery from herpes simplex virus (HSV) infections. To investigate the role of delayed hypersensitivity (DH) in immunity and protection against HSV, we developed a murine model using the ear-swelling assay. Mice were infected subcutaneously with HSV-1 and ear-challenged, and the swelling was quantified. Significant ear swelling was detected by 3 to 4 days postinfection and peaked at 6 days. The kinetics of development of ear swelling were typical of DH: maximal swelling occurred 24 h post challenge and was diminished by 48 h, and the cellular infiltrate was predominantly mononuclear. Four-hour swelling, indicative of antibody-mediated, immediate-type hypersensitivity, was not detected until 15 days post immunization. The DH response was virus specific and could be transferred to normal recipients with lymph node T cells, but not with B cells or immune serum. This system will provide a useful model for evaluating the protective role of DH in HSV infection and for studying the specificity and interaction of T cells which mediate the response.

Animals↗
PubMed1982

Herpes simplex virus-induced changes in cellular and adenovirus RNA metabolism in an adenovirus type 5-transformed human cell line.

We used the viral transcripts (designated Ad-RNA) that accumulated in the cytoplasm of adenovirus type 5-transformed human embryonic kidney cells (cell line 291-31) as models for cellular RNAs to examine how herpes simplex virus modifies cellular RNA metabolism. Infection of 293-31 cells with herpes simplex virus type 1 strain 17 lead to extensive inhibition of Ad-RNA accumulation by 4 h postinfection. The major part of this inhibition was due to an immediate early or alpha gene function, which reduced the rate of transcription of Ad-RNA within the nuclei of the infected cells. In addition, host polyadenylic acid-containing RNA accumulation and rRNA accumulation were affected, but to a lesser extent and at lower rate than Ad-RNA accumulation. In conjunction with previous data, our experimental data allowed us to propose a general scheme for how herpes simplex virus type 1 alters the metabolism of cellular RNA, the possible mechanisms for these changes, and how they correlate with the regulation of herpes simplex virus gene expression.

Adenoviruses, Human↗
PubMed1982

Herpesvirus glycoprotein synthesis and insertion into plasma membranes.

In the presence of the antibiotic tunicamycin (TM), glycosylation of herpes simplex virus glycoproteins is inhibited and non-glycosylated polypeptides analogous to the glycoproteins are synthesized (Pizer et al., J. Virol. 34:142-153, 1980). The synthesis of viral proteins and DNA occurs in TM-treated cells. By electron microscopy, nucleocapsids can be observed both in the nucleus and the cytoplasm of TM-treated cells; a small number of enveloped virions were observed on the cell surface. Analyses of the proteins in partially purified virus readily detects viral glycoproteins in the control cells, but neither glycoproteins nor nonglycosylated polypeptide analogs were observed in the virus prepared from TM-treated cells. By labeling the surface of infected cells with 125I, viral glycoproteins were detected as soon as 90 min after infection even when protein synthesis was inhibited with cycloheximide and glycosylation was blocked with TM. Labeling the proteins synthesized in infected cells with [35S]methionine showed that the surface glycoproteins detected in the cycloheximide- and TM-treated cells were not synthesized de novo after infection, but were placed on the cell surface by the infecting virus. Studies with metabolic inhibitors and a temperature-sensitive mutant blocked early in the infectious cycle showed that glycoproteins gA/gB and gD were synthesized soon after infection, but that the synthesis of gC was delayed. Under conditions of infection, in which gC and its precursor pgC are not produced, we have been able to observe the relationships between the glycosylated polypeptides that correspond to pgA/pgB and the nonglycosylated analog made in the presence of TM.

Animals↗
PubMed1982

Herpesvirus infection alters the steady-state levels of cellular polyadenylated RNA in polyoma virus-transformed BHK cells.

Polyoma-transformed BHK cells are permissive for the replication of herpes simplex virus type 1. The effect of herpes infection on the steady-state levels of bulk mRNA sequences in these cells was studied by using cDNA to polyadenylated cytoplasmic RNA from uninfected cells. The principal findings were: (i) herpes simplex virus type 1 infection caused a pronounced reduction in the cytoplasmic levels of moderately abundant mRNAs' (ii) after infection, increased amounts of RNA complementary to the cDNA were isolated as part of the nonadenylated cytoplasmic RNA fraction.

Animals↗
PubMed1980

Regulation of phosphoglycerate dehydrogenase levels and effect on serine synthesis in Escherichia coli K-12.

The level of phosphoglycerate dehydrogenase, the first enzyme in the biosynthetic pathway to serine and glycine, has been studied in Escherichia coli grown under different conditions. The enzyme level was not reduced by growth in a medium which contained the end products of the pathway, nor was it elevated when the growth rates was limited by the supply of serine. Elevation of phosphoglycerate dehydrogenase did not occur when charging of tRNA ser was inhibited by serine hydroxamate. However, phosphoglycerate dehydrogenase levels were subject to regulation. Elevated levels of enzyme activity were observed in merodiploids containing multiple copies of the serA gene, and lowered enzyme levels were found in cells grown on carbon sources other than glucose or when certain amino acids were present in the growth medium. The combined effect of these nutritional changes (carbon source and amino acids) reduced the level of phosphoglycerate dehydrogenase to 10 to 12% of that found in wild-type cells and to about 5% of the level in the merodiploids. By using antibody prepared against purified phosphoglycerate dehydrogenase we established that the decrease in enzyme activity reflected decreased amounts of enzyme protein. Constant intracellular concentrations of 3-phosphoglycerate and serine were found in cells with marked differences in phosphoglycerate dehydrogenase activity, indicating that end product inhibition of phosphoglycerate dehydrogenase activity, rather than the amount of the biosynthetic enzymes, is the major factor in regulating the intracellular concentration of serine.

Amino Acids↗
PubMed1980

Characterization of RNA synthesized in isolated nuclei of herpes simplex virus type 1-infected KB cells.

Nuclei isolated from herpes simplex virus (HSV)-infected KB cells actively synthesize HSV RNA in vitro; the RNA can be hybridized with HSV DNA or nuclear RNA from HSV-infected cells. Nascent RNA molecules labeled in vivo with 32PO4 were elongated, utilizing the nuclear system to incorporate Hg-CTP at their 3' ends, and then isolated on an affinity column. Hybridization of isolated nascent RNA molecules showed that greater than 50% of them were HSV specific and that more than 25% were self-complementary.

Carcinoma↗
PubMed1980

Effect of tunicamycin on herpes simplex virus glycoproteins and infectious virus production.

The antibiotic tunicamycin, which blocks the synthesis of glycoproteins, inhibited the production of infectious herpes simplex virus. In the presence of this drug, [14C]glucosamine and [3H]mannose incorporation was reduced in infected cells, whereas total protein synthesis was not affected. Gel electrophoresis of [2-3H]mannose-labeled polypeptides failed to detect glycoprotein D or any of the other herpes simplex virus glycoproteins. By use of specific antisera we demonstrated that in the presence of tunicamycin the normal precursors to viral glycoproteins failed to appear. Instead, lower-molecular-weight polypeptides were found which were antigenically and structurally related to the glycosylated proteins. Evidence is presented to show that blocking the addition of carbohydrate to glycoprotein precursors with tunicamycin results in the disappearance of molecules, possibly due to degradation of the unglycosylated polypeptides. We infer that the added carbohydrate either stabilizes the envelope proteins or provides the proper structure for correct processing of the molecules needed for infectivity.

Glucosamine↗
PubMed1978

Herpes simplex virus replication in pheochromocytoma cell line that responds to nerve growth factor.

Cultured cells of neural origin (PC-12, pheochromocytoma cell line) respond to nerve growth factor (NGF) by extending neurites. These cells whether treated with NGF or not can be infected with herpes simplex virus and produce progeny virus. Viral antigens are detected on the cell surface and fusion of cells to form polykaryocytes takes place. Nucleocapsids are found within the cell nucleus and enveloped virus in a present both in the cytoplasm and extracellular space. Virus was not observed within the neurites but laterations in the neurite microtubular structure occurred after infection.

Animals↗
PubMed1978

Growth dependence of phosphoglyceric acid dehydrogenase activity in cultured rat liver cells.

Rat liver epithelial cells in culture (WIRL-3C) have the enzymes that synthesize serine from 3-phophoglyceric acid. Both phosphoglyceric acid dehydrogenase (PGAD) and serine-phosphate (serine-P) forming activities fluctuate with time after subculture and are higher in growing than confluent cells. This activity pattern was not common for other dehydrogenases in WIRL-3C cells, nor was it common for PGAD activity in other cultured cells. At time of subculture, cells are removed from spent medium, treated with trypsin, and fed fresh medium. None of these parameters causes the rise in activity; in contrast, reduction in cell density and the accompanying stimulation of growth do. PGAD activity decreases when growth is slowed either as the cells progress to the end of the culture cycle, when cells are treated with dexamethasone-phosphate (Dx-P) or dibutyryl cyclic AMP(cAMP) and theophylline or when the serum concentration of the medium is reduced to 0.2%. Under these conditions, decreased PGAD activity is paralleled by a decline in growth and DNA accumulation. PGAD activity in WIRL-3C cells is regulated in a manner closely resembling what has been observed previously in rat liver from the whole animal. The possible use of this system in studying regulation of gene expression in mammalian cells is discussed.

Animals↗
PubMed1978

Herpesvirus infection modifies adenovirus RNA metabolism in adenovirus type 5-transformed cells.

The effect of herpes simplex virus (HSV) infection of mRNA metabolism was examined in a system where the fate of specific RNA sequence can be assayed. Adenovirus type 5-transformed rat embryo cell line 107 synthesizes adenovirus-specific RNA (ad-RNA), which functions in the cytoplasm as mRNA. We have utilized ad-RNA as a model for mRNA metabolism, and in a preliminiary study we characterized ad-RNA in the nucleus and cytoplasm by hybridization to filter-bound adenovirus DNA. The results indicated the as-RNA accumulates in the nucleus and that cytoplasmic polyadenylic acid [poly(A)]-containing ad-RNA turns over with a half-life of a few hours. Pulse-chase experiments confirmed these observations and a half-life of about h was determined for the poly(A)-containing cytoplasmic ad-RNA. A second class of ad-RNA remains in the nucleus, where it turns over with a longer hlaf-life (about 24 h). The infection of 107 cells by HSV was restricted at 37 degree C, giving a burst size of 5 PFU per cell and allowing continued host DNA synthesis. Protein synthesis was inhibited greater than 50% by 7 h after infection, and total RNA synthesis was 50% inhibited by 4 h after infection. During the first 8 h after infection, HSV has little effect on the rate of synthesis of ad-RNA as determined by hybridization of nuclear RNA samples, but,during the same period, HSV inhibits the accumulation of poly(A)-containing ad-RNA in the cytoplasm. The degree of this inhibition increases steadily throughout this period and reaches 60% by 6.5 to 8 h after infection. Nosignificant effect was seen on the accumulation of total cellular poly(A)-containing RNA. It was concluded from these experiments that HSV infection alters the metabolism of ad-RNA so as to prevent the normal appearance of the poly(A)-containing mRNA in the cytoplasm. The result for ad-RNA may not represent the behavior of total cellular poly(A)-containing RNA under conditions where infection is restricted.

Adenoviridae↗
PubMed1977

A conformational change in phosphoglycerate dehydrogenase induced by a shift in pH.

The fluorescence of NADH bound to phosphoglycerate dehydrogenase (3-phosphoglycerate: NAD+ oxidoreductase, EC 1.1.1.95) decreased by 42% between pH 8.5 and 7.0 Serine, an allosteric inhibitor, quenched the fluorescence of enzyme-bound NADH by 29% at pH 8.5, but not at all at pH 7.0. The kinetics of the fluorescence change which occurred when the pH of an enzyme-NADH solution was rapidly shifted from 8.5 to 7.0 was measured using stopped-flow fluorimetry. The kinetics were first order, with a rate constant of 2.83 s-1. This rate constant was similar in magnitude to the rate constants for fluorescence quenching at pH 8.5 by saturating concentrations of serine and glycine, another allosteric inhibitor (Dubrow, R. and Pizer, L.I. (1977) J. Biol. Chem. 252, 1527-1538). These results indicate that the conformation of phosphoglycerate dehydrogenase at pH 7.0 is similar to, but not identical with, the serine-induced conformation at pH 8.5.

Alcohol Oxidoreductases↗
PubMed1977

Transient kinetic and deuterium isotope effect studies on the catalytic mechanism of phosphoglycerate dehydrogenase.

The catalytic mechanism of the phosphoglycerate dehydrogenase reaction in both directions was investigated by studying: (a) pre-steady state transients in reduced coenzyme appearance or disappearance or disappearance and in protein fluorescence; (b) deuterium isotope effects on the transients and on the steady state reactions; and (c) the partial reaction between the enzyme-NADH complex and hydroxypyruvate-P. These studies led to the scheme below for the ternary complex interconversion. E1-NADH-hydroxypyruvate-P(1)equilibriumE2-NADH-hydroxypyruvate-P(2)equilibriumE3-NADH-hydroxypyruvate-P + H+(3)equilibriumE3-NAD+-3-phosphoglycerate(4)equilibriumE4-NAD+-3-phosphoglycerate Steps 1,2, and 4 are ternary complex isomerizations. Step 3 is the hydride transfer. Under steady state conditions isomerization 2 is the rate-determining step in the direction of hydroxypyruvate-P reduction at higher pH values. At lower pH values, the hydride transfer step is also partially rate-determining. The rate-determining step in the direction of 3-phosphoglycerate oxidation occurs subsequent to the hydride transfer step at higher pH values. At lower pH values the rate is determined by both isomerization 4 and the hydride transfer step. Isomerizations 1, 2, and 4 were inhibited by serine, an allosteric inhibitor, indicating that the inactive conformation of the enzyme is incapable of performing any of the steps of the ternary complex interconversion. Phosphoglycerate dehydrogenase corresponds to a V-type allosteric enzyme. When the enzyme-NADH complex was mixed with hydroxypyruvate-P at pH 8.5, a rapid quenching of enzymebound NADH fluorescence occurred. This process was studied under pseudo-first order conditions and shown to be the result of hydroxypyruvate-P binding.

Alcohol Oxidoreductases↗
PubMed1977

Transient kinetic studies on the allosteric transition of phosphoglycerate dehydrogenase.

Stopped flow spectrophotometry was used to investigate the kinetics of the transition of the phosphoglycerate dehydrogenase (3-phosphoglycerate: NAD oxidoreductase, EC 1.1.1.95) reaction from the active to the inhibited rate upon the addition of the physiological inhibitor serine. The transition was characterized by a single first order rate constant (kobs,i) which was independent of enzyme concentration. At pH 8.5, kobs,i increased in a hyperbolic manner with serine concentration from 2 to 8 s-1. The increase in kobs,i occurred at serine concentrations where the steady state inhibition was virtually complete. These results indicate that serine inhibition is an allosteric process involving a conformational change in the enzyme. A model is presented in which serine at low concentrations binds exclusively to the inhibited state of the enzyme and shifts the equilibrium toward that state; at high serine concentrations, serine binds to the active state, facilitating its conversion to the inhibited state. An alternative model, which we favor, proposes two classes of inhibitor binding sites. The kinetics of the fluorescence quenching of enzyme-bound NADH by serine (Sugimoto, E., and Pizer, L.I. (1968) J. Biol. Chem. 243, 2090-2098), measured by stopped flow fluorimetry, was also characterized by a single first order rate constant (kobs,f.q.) which was independent of enzyme concentration. At pH 8.5, kobs,f.q. ranged from 0.4 s-1 at low serine concentrations to 1.1 s-1 at high serine concentrations. These results indicate that the fluorescence quenching induced by serine is a manifestation of a structural change in the enzyme. Enzyme and excess NADH were mixed with substrate and serine in the stopped flow instrument, and enzyme-bound NADH fluorescence was monitored by exciting through the protein at 285 nm. A rapid fluorescence quenching process, which occurred within the mixing time, was followed by a slower fluorescence enhancement process which terminated in a steady state level corresponding to the quenched fluorescence of the enzyme NADH serine complex. The rapid quenching was the result of substrate binding (Dubrow, R., and Pizer, L.I. (1977) J. Biol. Chem. 252, 1539-1551). The fluorescence enhancement was characterized by a single first order rate constant whose value for a given serine concentration corresponded with Kobs,j. This data shows that the quenched state of the enzyme-NADH-complex is the state which is directly responsible for the inhibition of enzyme activity. During catalysis the quenched state is achieved from a different initial conformation, and consequently at a different rate, than in the absence of substrate. kobs,j and kobs,f.q. were also measured using glycine, another inhibitor. The ultraviolet difference spectrum between enzyme and enzyme plus serine was determined and proposed to be the result of the same structural change which is responsible for the fluorescence quenching by serine.

Alcohol Oxidoreductases↗