Showing papers by "University of Medicine and Dentistry of New Jersey published in 1998"

Molecular Genetics of the RNA Polymerase II General Transcriptional Machinery

Abstract: Transcription initiation by RNA polymerase II (RNA pol II) requires interaction between cis-acting promoter elements and trans-acting factors. The eukaryotic promoter consists of core elements, which include the TATA box and other DNA sequences that define transcription start sites, and regulatory elements, which either enhance or repress transcription in a gene-specific manner. The core promoter is the site for assembly of the transcription preinitiation complex, which includes RNA pol II and the general transcription fctors TBP, TFIIB, TFIIE, TFIIF, and TFIIH. Regulatory elements bind gene-specific factors, which affect the rate of transcription by interacting, either directly or indirectly, with components of the general transcriptional machinery. A third class of transcription factors, termed coactivators, is not required for basal transcription in vitro but often mediates activation by a broad spectrum of activators. Accordingly, coactivators are neither gene-specific nor general transcription factors, although gene-specific coactivators have been described in metazoan systems. Transcriptional repressors include both gene-specific and general factors. Similar to coactivators, general transcriptional repressors affect the expression of a broad spectrum of genes yet do not repress all genes. General repressors either act through the core transcriptional machinery or are histone related and presumably affect chromatin function. This review focuses on the global effectors of RNA polymerase II transcription in yeast, including the general transcription factors, the coactivators, and the general repressors. Emphasis is placed on the role that yeast genetics has played in identifying these factors and their associated functions.

Indentation Damage and Mechanical Properties of Human Enamel and Dentin

Abstract: Understanding the mechanical properties of human teeth is important to clinical tooth preparation and to the development of "tooth-like" restorative materials. Previous studies have focused on the macroscopic fracture behavior of enamel and dentin. In the present study, we performed indentation studies to understand the microfracture and deformation and the microcrackmicrostructure interactions of teeth. It was hypothesized that crack propagation would be influenced by enamel rods and the dentino-enamel junction (DEJ), and the mechanical properties would be influenced by enamel rod orientation and tooth-to-tooth variation. Twenty-eight human third molars were used for the measurement of hardness, fracture toughness, elastic modulus, and energy absorbed during indentation. We examined the effect of enamel rod orientation by propagating cracks in the occlusal surface, and in the axial section in directions parallel and perpendicular to the occlusal surface. The results showed that the cracks in the enamel a...

Biological Properties of Recombinant α-Interferons: 40th Anniversary of the Discovery of Interferons

Abstract: IFNs were first described as potent antiviral agents 40 years ago, and recombinant IFN-alpha2a and IFN-alpha2b were approved for the treatment of hairy cell leukemia just 11 years ago Today, alpha-IFNs are approved worldwide for the treatment of a variety of malignancies and virologic diseases Although the exact mechanism of action of IFN-alpha in the treatment of such diseases is not fully understood, many advances have been made in the characterization of the physicochemical and diverse biological properties of this highly pleiotropic cytokine Here we review recent developments in our understanding of the antiviral and immunoregulatory properties of IFN-alpha, the nature of the multisubunit IFN-alpha receptor, and the molecular mechanisms of signal transduction Where available, we have included comparative data on recombinant alpha-IFNs derived from both naturally occurring and nonnaturally occurring synthetic genes We also review clinical data and data on the side effects and antigenicity of different sources of recombinant alpha-IFNs in humans These latter topics are of clinical interest, because they may potentially affect the efficacy of these various products Hopefully, what is already known about IFN will prompt further exploration into the mechanism(s) of action of IFN-alpha and thus deliver new applications for this prototypic cytokine, whose full therapeutic potential is yet to be realized

Molecular beacon sequence analysis for detecting drug resistance in Mycobacterium tuberculosis

Abstract: We developed a new approach to DNA sequence analysis that uses fluorogenic reporter molecules--molecular beacons--and demonstrated their ability to discriminate alleles in real-time PCR assays of genomic DNA. A set of overlapping molecular beacons was used to analyze an 81-bp region of the Mycobacterium tuberculosis rpoB gene for mutations that confer resistance to the antibiotic rifampin. In a blinded study of 52 rifampin-resistant and 23 rifampin-susceptible clinical isolates, this method correctly detected mutations in all of the resistant strains and in none of the susceptible strains. The assay was carried out entirely in sealed PCR tubes and was simple to perform and interpret. This approach can be used to analyze any DNA sequence of moderate length with single base pair accuracy.

Brain-derived neurotrophic factor modulates hippocampal synaptic transmission by increasing N-methyl-d-aspartic acid receptor activity

Abstract: Neurotrophins (NTs) have recently been found to regulate synaptic transmission in the hippocampus. Whole-cell and single-channel recordings from cultured hippocampal neurons revealed a mechanism responsible for enhanced synaptic strength. Specifically, brain-derived neurotrophic factor augmented glutamate-evoked, but not acetylcholine-evoked, currents 3-fold and increased N-methyl-d-aspartic acid (NMDA) receptor open probability. Activation of trkB NT receptors was critical, as glutamate currents were not affected by nerve growth factor or NT-3, and increased open probability was prevented by the tyrosine kinase inhibitor K-252a. In addition, the NMDA receptor antagonist MK-801 blocked brain-derived neurotrophic factor enhancement of synaptic transmission, further suggesting that NTs modulate synaptic efficacy via changes in NMDA receptor function.

New core promoter element in RNA polymerase II-dependent transcription: sequence-specific DNA binding by transcription factor IIB

Abstract: A sequence element located immediately upstream of the TATA element, and having the consensus sequence 5'-G/C-G/C-G/A-C-G-C-C-3', affects the ability of transcription factor IIB to enter transcription complexes and support transcription initiation. The sequence element is recognized directly by the transcription factor IIB. Recognition involves alpha-helices 4' and 5' of IIB, which comprise a helix-turn-helix DNA-binding motif. These observations establish that transcription initiation involves a fourth core promoter element, the IIB recognition element (BRE), in addition to the TATA element, the initiator element, and the downstream promoter element, and involves a second sequence-specific general transcription factor, IIB, in addition to transcription factor IID.

Perioperative Blood Transfusion and Postoperative Mortality

Abstract: Context.—The risks of blood transfusion have been studied extensively but the benefits and the hemoglobin concentration at which patients should receive a transfusion have not.Objective.—To determine the effect of perioperative transfusion on 30- and 90-day postoperative mortality.Design.—Retrospective cohort study.Setting.—A total of 20 US hospitals between 1983 and 1993.Participants.—A total of 8787 consecutive hip fracture patients, aged 60 years or older, who underwent surgical repair.Main Outcome Measures.—Primary outcome was 30-day postoperative mortality; secondary outcome was 90-day postoperative mortality. The "trigger" hemoglobin level was defined as the lowest hemoglobin level prior to the first transfusion during the time period or, for patients in the nontranfused group, as the lowest hemoglobin level during the time period.Results.—Overall 30-day mortality was 4.6% (n=402; 95% confidence interval [CI], 4.1%-5.0%); overall 90-day mortality was 9.0% (n=788; 95% CI, 8.4%-9.6%). A total of 42% of patients (n=3699) received a postoperative transfusion. Among patients with trigger hemoglobin levels between 80 and 100 g/L (8.0 and 10.0 g/dL), 55.6% received a transfusion, while 90.5% of patients with hemoglobin levels less than 80 g/L (8.0 g/dL) received postoperative transfusions. Postoperative transfusion did not influence 30- or 90-day mortality after adjusting for trigger hemoglobin level, cardiovascular disease, and other risk factors for death: for 30-day mortality, the adjusted odds ratio (OR) was 0.96 (95% CI, 0.74-1.26); for 90-day mortality, the adjusted hazard ratio was 1.08 (95% CI, 0.90-1.29). Similarly, 30-day mortality after surgery did not differ between those who received a preoperative transfusion and those who did not (adjusted OR, 1.23; 95% CI, 0.81-1.89).Conclusions.—Perioperative transfusion in patients with hemoglobin levels 80 g/L (8.0 g/dL) or higher did not appear to influence the risk of 30- or 90-day mortality in this elderly population. At hemoglobin concentrations of less than 80 g/L (8.0 g/dL), 90.5% of patients received a transfusion, precluding further analysis of the association of transfusion and mortality.

Gut-derived mesenteric lymph but not portal blood increases endothelial cell permeability and promotes lung injury after hemorrhagic shock.

Abstract: Objective To determine whether gut-derived factors leading to organ injury and increased endothelial cell permeability would be present in the mesenteric lymph at higher levels than in the portal blood of rats subjected to hemorrhagic shock. This hypothesis was tested by examining the effect of portal blood plasma and mesenteric lymph on endothelial cell monolayers and the interruption of mesenteric lymph flow on shock-induced lung injury. Background Data The absence of detectable bacteremia or endotoxemia in the portal blood of trauma victims casts doubt on the role of the gut in the generation of multiple organ failure. Nevertheless, previous experimental work has clearly documented the connection between shock and gut injury as well as the concept of gut-induced sepsis and distant organ failure. One explanation for this apparent paradox would be that gut-derived inflammatory factors are reaching the lung and systemic circulation via the gut lymphatics rather than the portal circulation. Methods Human umbilical vein endothelial cell monolayers, grown in two-compartment systems, were exposed to media, sham-shock, or postshock portal blood plasma or lymph, and permeability to rhodamine (10K) was measured. Sprague-Dawley rats were subjected to 90 minutes of sham or actual shock and shock plus lymphatic division (before and after shock). Lung permeability, pulmonary myeloperoxidase levels, alveolar apoptosis, and bronchoalveolar fluid protein content were used to quantitate lung injury. Results Postshock lymph increased endothelial cell monolayer permeability but not postshock plasma, sham-shock lymph/plasma, or medium. Lymphatic division before hemorrhagic shock prevented shock-induced increases in lung permeability to Evans blue dye and alveolar apoptosis and reduced pulmonary MPO levels. In contrast, division of the mesenteric lymphatics at the end of the shock period but before reperfusion ameliorated but failed to prevent increased lung permeability, alveolar apoptosis, and MPO accumulation. Conclusions Gut barrier failure after hemorrhagic shock may be involved in the pathogenesis of shock-induced distant organ injury via gut-derived factors carried in the mesenteric lymph rather than the portal circulation.

Integrating cytosolic calcium signals into mitochondrial metabolic responses

Abstract: Stimulation of hepatocytes with vasopressin evokes increases in cytosolic free Ca2+ ([Ca2+]c) that are relayed into the mitochondria, where the resulting mitochondrial Ca2+ ([Ca2+]m) increase regulates intramitochondrial Ca2+-sensitive targets. To understand how mitochondria integrate the [Ca2+]c signals into a final metabolic response, we stimulated hepatocytes with high vasopressin doses that generate a sustained increase in [Ca2+]c. This elicited a synchronous, single spike of [Ca2+]m and consequent NAD(P)H formation, which could be related to changes in the activity state of pyruvate dehydrogenase (PDH) measured in parallel. The vasopressin-induced [Ca2+]m spike evoked a transient increase in NAD(P)H that persisted longer than the [Ca2+]m increase. In contrast, PDH activity increased biphasically, with an initial rapid phase accompanying the rise in [Ca2+]m, followed by a sustained secondary activation phase associated with a decline in cellular ATP. The decline of NAD(P)H in the face of elevated PDH activity occurred as a result of respiratory chain activation, which was also manifest in a calcium-dependent increase in the membrane potential and pH gradient components of the proton motive force (PMF). This is the first direct demonstration that Ca2+-mobilizing hormones increase the PMF in intact cells. Thus, Ca2+ plays an important role in signal transduction from cytosol to mitochondria, with a single [Ca2+]m spike evoking a complex series of changes to activate mitochondrial oxidative metabolism.

A vaccine consisting of recombinant Borrelia burgdorferi outer-surface protein A to prevent lyme disease

Abstract: Background Lyme disease is a multisystem inflammatory disease caused by infection with the tick-borne spirochete Borrelia burgdorferi and is the most common vector-borne infection in the United States. We assessed the efficacy of a recombinant vaccine consisting of outer-surface protein A (OspA) without adjuvant in subjects at risk for Lyme disease. Methods For this double-blind trial, 10,305 subjects 18 years of age or older were recruited at 14 sites in areas of the United States where Lyme disease was endemic; the subjects were randomly assigned to receive either placebo (5149 subjects) or 30 μg of OspA vaccine (5156 subjects). The first two injections were administered 1 month apart, and 7515 subjects also received a booster dose at 12 months. The subjects were observed for two seasons during which the risk of transmission of Lyme disease was high. The primary end point was the number of new clinically and serologically confirmed cases of Lyme disease. Results The efficacy of the vaccine was 68 percen...

Requirement of RSF and FACT for Transcription of Chromatin Templates in Vitro

Abstract: Transcription of naked DNA in vitro requires the general transcription factors and RNA polymerase II. However, this minimal set of factors is not sufficient for transcription when the DNA template is packaged into chromatin. Here, a factor that facilitates activator-dependent transcription initiation on chromatin templates was purified. This factor, remodeling and spacing factor (RSF), has adenosine triphosphate-dependent nucleosome-remodeling and spacing activities. Polymerases that initiate transcription with RSF can only extend their transcripts in the presence of FACT (facilitates chromatin transcription). Thus, the minimal factor requirements for activator-dependent transcription on chromatin templates in vitro have been defined.

Production of Vascular Endothelial Growth Factor by Murine Macrophages: Regulation by Hypoxia, Lactate, and the Inducible Nitric Oxide Synthase Pathway

Abstract: Murine thioglycolate-induced peritoneal macrophages (MPMs) and the murine RAW264.7 macrophage-like cell line (RAW cells) constitutively produce vascular endothelial growth factor (VEGF). VEGF production is increased under hypoxic conditions or after cell activation with interferon-gamma (IFNgamma) and endotoxin (lipopolysaccharide, LPS). In contrast, tumor necrosis factor-alpha is produced only by IFNgamma/LPS-activated cells. Lactate (25 mmol/L) does not increase VEGF production by these cells. However, hypoxia, lactate, and IFNgamma/LPS-activated MPMs express angiogenic activity, whereas normoxic, nonactivated MPMs do not. Lack of angiogenic activity is not due to an antiangiogenic factor(s) in the medium of these cells. Angiogenic activity produced by hypoxia and lactate-treated MPMs is neutralized by anti-VEGF antibody, which also neutralizes most of the angiogenic activity produced by IFNgamma/LPS-activated MPMs. The inducible nitric oxide synthase inhibitors Ng-nitro-L-arginine-methyl ester (1.5 mmol/L) and aminoguanidine (1 mmol/L) block production of angiogenic activity by MPMs and RAW cells. In RAW cells, Ng-nitro-L-arginine-methyl ester and AG block IFNgamma/LPS-activated, but not constitutive, VEGF production, whereas in MPMs, neither constitutive nor IFNgamma/LPS-activated VEGF synthesis is affected. Synthesis of tumor necrosis factor-alpha is also unaffected. In contrast to normoxic, nonactivated MPMs, inducible nitric oxide synthase-inhibited, IFNgamma/LPS-activated MPMs produce an antiangiogenic factor(s). We conclude that VEGF is a major contributor to macrophage-derived angiogenic activity, and that activation by hypoxia, lactate, or IFNgamma/LPS switches macrophage-derived VEGF from a nonangiogenic to an angiogenic state. This switch may involve a posttranslational modification of VEGF, possibly by the process of ADP-ribosylation. ADP-ribosylation by MPM cytosolic extracts or by cholera toxin switches rVEGF165 from an angiogenic to a nonangiogenic state. In IFNgamma/LPS-activated MPMs, the inducible nitric oxide synthase-dependent pathway also regulates the expression of an antiangiogenic factor(s) that antagonizes the bioactivity of VEGF and provides an additional regulatory pathway controlling the angiogenic phenotype of macrophages.

A Human RNA Polymerase II Complex Containing Factors That Modify Chromatin Structure

Abstract: We have isolated a human RNA polymerase II complex that contains chromatin structure remodeling activity and histone acetyltransferase activity. This complex contains the Srb proteins, the Swi-Snf complex, and the histone acetyltransferases CBP and PCAF in addition to RNA polymerase II. Notably, the general transcription factors are absent from this complex. The complex was purified by two different methods: conventional chromatography and affinity chromatography using antibodies directed against CDK8, the human homolog of the yeast Srb10 protein. Protein interaction studies demonstrate a direct interaction between RNA polymerase II and the histone acetyltransferases p300 and PCAF. Importantly, p300 interacts specifically with the nonphosphorylated, initiation-competent form of RNA polymerase II. In contrast, PCAF interacts with the elongation-competent, phosphorylated form of RNA polymerase II.

Accumulation of chlorpyrifos on residential surfaces and toys accessible to children

Abstract: Quantitative examination of major pathways and routes of exposure to pesticides is essential for determining human risk. The current study was conducted in two apartments and examines the accumulat...

Clinical and Biologic Activity of an Estrogenic Herbal Combination (PC-SPES) in Prostate Cancer

Abstract: Background Herbal mixtures are popular alternatives to demonstrated therapies. PC-SPES, a commercially available combination of eight herbs, is used as a nonestrogenic treatment for cancer of the prostate. Since other herbal medicines have estrogenic effects in vitro, we tested the estrogenic activity of PC-SPES in yeast and mice and in men with prostate cancer. Methods We measured the estrogenic activity of PC-SPES with transcriptional-activation assays in yeast and a biologic assay in mice. We assessed the clinical activity of PC-SPES in eight patients with hormone-sensitive prostate cancer by measuring serum prostate-specific antigen and testosterone concentrations during and after treatment. Results In complementary yeast assays, a 1:200 dilution of an ethanol extract of PC-SPES had estrogenic activity similar to that of 1 nM estradiol, and in ovariectomized CD-1 mice, the herbal mixture increased uterine weights substantially. In six of six men with prostate cancer, PC-SPES decreased serum testostero...

NMR structure of the histidine kinase domain of the E. coli osmosensor EnvZ

Abstract: Bacteria live in capricious environments, in which they must continuously sense external conditions in order to adjust their shape, motility and physiology1. The histidine–aspartate phosphorelay signal-transduction system (also known as the two-component system) is important in cellular adaptation to environmental changes in both prokaryotes and lower eukaryotes2,3. In this system, protein histidine kinases function as sensors and signal transducers. The Escherichia coli osmosensor, EnvZ, is a transmembrane protein with histidine kinase activity in its cytoplasmic region2. The cytoplasmic region contains two functional domains4: domain A (residues 223–289) contains the conserved histidine residue (H243), a site of autophosphorylation as well as transphosphorylation to the conserved D55 residue of response regulator OmpR, whereas domain B (residues 290–450) encloses several highly conserved regions (G1, G2, F and N boxes) and is able to phosphorylate H243. Here we present the solution structure of domain B, the catalytic core of EnvZ. This core has a novel protein kinase structure, distinct from the serine/threonine/tyrosine kinase fold, with unanticipated similarities to both heat-shock protein 90 and DNA gyrase B.

Infant and Maternal Outcomes in the Pregnancies of Asthmatic Women

Abstract: We examined the relationship between infant and maternal outcomes and asthma complicating pregnancy, using historical cohort analysis of singleton live deliveries in New Jersey hospitals between 1989 and 1992 (n = 447,963). Subject mother-infant dyads were identified from linked birth certificate and maternal and newborn hospital claims data. Women with an International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) diagnosis code (493) for asthma (n = 2,289) were compared with a fourfold larger randomly selected control sample (n = 9,156) from the remaining pool of women. After controlling for the effects of important confounding variables, maternal asthma was associated with the following adverse infant outcomes: preterm infant (odds ratio [OR] = 1.36; 95% confidence interval [CI], 1.18 to 1.55), low birth weight (OR = 1. 32; 95% CI, 1.10 to 1.58), small-for-gestational age (OR = 1.26; 95% CI, 1.10 to 1.45), congenital anomalies (OR = 1.37; 95% CI, 1.12 to 1.68), and increased infant hospital length of stay (OR = 1.44; 95% CI, 1.25 to 1.65). The adverse maternal outcomes associated with maternal asthma were: pre-eclampsia (OR = 2.18; 95% CI, 1.68 to 2. 83), placenta previa (OR = 1.71; 95% CI, 1.05 to 2.79), cesarean delivery (OR = 1.62; 95% CI, 1.46 to 1.80), and increased maternal hospital length of stay (OR = 1.86; 95% CI, 1.60 to 2.15). The results emphasize the need for maternal asthma to be added to the list of conditions that increase the risk of adverse pregnancy outcomes.

Regulation of Translational Initiation during Cellular Responses to Stress

Abstract: Chemicals and conditions that damage proteins, promote protein misfolding, or inhibit protein processing trigger the onset of protective homeostatic mechanisms resulting in "stress responses" in mammalian cells. Included in these responses are an acute inhibition of mRNA translation at the initiation step, a subsequent induction of various protein chaperones, and the recovery of mRNA translation. Separate, but closely related, stress response systems exist for the endoplasmic reticulum (ER), relating to the induction of specific "glucose-regulated proteins" (GRPs), and for the cytoplasm, pertaining to the induction of the "heat shock proteins" (HSPs). Activators of the ER stress response system, including Ca(2+)-mobilizing and thiol-reducing agents, are discussed and compared to activators of the cytoplasmic stress system, such as arsenite, heavy metal cations, and oxidants. An emerging integrative literature is reviewed that relates protein chaperones associated with cellular stress response systems to the coordinate regulation of translational initiation and protein processing. Background information is presented describing the roles of protein chaperones in the ER and cytoplasmic stress response systems and the relationships of chaperones and protein processing to the regulation of mRNA translation. The role of chaperones in regulating eIF-2 alpha kinase activities, eIF-2 cycling, and ribosomal loading on mRNA is emphasized. The putative role of GRP78 in coupling rates of translation to processing is modeled, and functional relationships between the HSP and GRP chaperone systems are discussed.

Multiple Pathways of Neuronal Death Induced by DNA-Damaging Agents, NGF Deprivation, and Oxidative Stress

Abstract: Here, we compare the pathways by which DNA-damaging agents, NGF deprivation, and superoxide dismutase 1 (SOD1) depletion evoke apoptosis of sympathetic neurons. Previous work raised the hypothesis that cell cycle signaling plays a required role in neuronal apoptosis elicited by NGF deprivation and the DNA-damaging agent camptothecin. To test this hypothesis, we extended our investigation of DNA-damaging agents to cytosine arabinoside (AraC) and UV irradiation. As with NGF deprivation and camptothecin treatment, the cyclin-dependent kinase inhibitors flavopiridol and olomoucine protected neurons from apoptosis induced by AraC and UV treatment. These observations support the model that camptothecin, AraC, and UV treatment cause DNA damage, which leads to apoptosis by a mechanism that, as in the case of NGF deprivation, includes activation of cell cycle components. Flavopiridol and olomoucine, however, had no effect on death induced by SOD1 depletion, suggesting that CDKs do not play a role in this paradigm of neuronal death. To compare further the mechanisms of death evoked by NGF withdrawal, SOD1 depletion, and DNA-damaging agents, we investigated their responses to inhibitors of cysteine aspartases, elements of apoptotic pathways. The V-ICEinh and BAF, two peptide inhibitors of cysteine aspartases, protected neurons in all three death paradigms. In contrast, the cysteine aspartase inhibitory peptide zVAD-fmk conferred protection from NGF withdrawal and SOD1 depletion, but not DNA-damaging agents, whereas acYVAD-cmk protected only from SOD1 depletion. Taken together, these findings indicate that three different apoptotic stimuli activate separate pathways of death in the same neuron type.

Exposing Pluralistic Ignorance to Reduce Alcohol Use Among College Students1

Abstract: Research has shown that students' beliefs about alcohol use are characterized by pluralistic ignorance: The majority of students believe that their peers are uniformly more comfortable with campus alcohol practices than they are. The present study examines the effects of educating students about pluralistic ignorance on their drinking behavior. Entering students (freshmen) participated in either a peer-oriented discussion, which focused on pluralistic ignorance, or an individual-oriented discussion, which focused on decision making in a drinking situation. Four to 6 months later, students in the peer-oriented condition reported drinking significantly less than did students in the individual-oriented condition. Additional results suggest that the peer-oriented discussion reduced the prescriptive strength of the drinking norm. The implications of these results for models of social influence and for the representation of peer opinion are discussed.