Showing papers by "Public Health Research Institute published in 1994"

Tyrosine phosphorylated p91 binds to a single element in the ISGF2/IRF-1 promoter to mediate induction by IFN alpha and IFN gamma, and is likely to autoregulate the p91 gene.

Abstract: ISGF2 was initially identified, purified and cloned as an interferon-alpha (IFN alpha) induced transcription factor that binds to the IFN-stimulated response element (ISRE) of IFN alpha/beta-stimulated genes (ISGs). It was reported to be transcriptionally regulated by several cytokines including IFN alpha and IFN gamma. IFN alpha and IFN gamma inducibility is mediated by a single element: a high affinity, nearly palindromic version of the IFN gamma activation site (GAS). The ISGF2 GAS is bound specifically by p91, which was previously identified as a subunit of the ISG activator ISGF3, and shown to mediate IFN gamma induction of the GBP gene via a GAS. Tyrosine phosphorylation and DNA binding activity of p91 parallel transcription of ISGF2 in response to IFN alpha and/or IFN gamma, consistent with induction mediated by only a GAS. Transcription of the genes that encode p91 and p113, another subunit of ISGF3, is activated only by IFN alpha. This result suggests induction mediated by an ISRE, and implies autoregulation, requiring the products of both genes. Specificity of the ISRE is the basis for the previous conclusion. In contrast, it appears likely that the ISGF2 GAS, and p91 or related factors, also mediate induction of ISGF2 by IL-6 and prolactin. Convergence of signalling pathways from at least four cytokines on this single site would thus be a key aspect of a general role for ISGF2 in cellular growth control.

A novel dodecadepsipeptide, cereulide, isolated from Bacillus cereus causes vacuole formation in HEp-2 cells

Abstract: A HEp-2 cell-vacuolation factor was extracted and purified from the culture supernatant of a Bacillus cereus strain which caused emetic-syndrome food poisoning. The final preparation was chemically pure, and the toxin was named as cereulide. Mass spectrometry, NMR studies and chemical degradation revealed that the cereulide is a cyclic dodecadepsipeptide, (D-O-Leu-D-Ala- L-O-Val-L-Val)3, which is closely related to the potassium ionophore, valinomycin.

Discontinuous mechanism of transcription elongation

Abstract: During transcription elongation, three flexibly connected parts of RNA polymerase of Escherichia coli advance along the template so that the front-end domain is followed by the catalytic site which in turn is followed by the RNA product binding site. The advancing enzyme was found to maintain the same conformation throughout extended segments of the transcribed region. However, when the polymerase traveled across certain DNA sites that seemed to briefly anchor the front-end domain, cyclic shifting of the three parts, accompanied by buildup and relief of internal strain, was observed. Thus, elongation proceeded in alternating laps of monotonous and inchworm-like movement with the flexible RNA polymerase configuration being subject to direct sequence control.

p53 Gene Abnormalities Are Closely Related to Hepatoviral Infections and Occur at a Late Stage of Hepatocarcinogenesis

Abstract: Hepatocellular carcinoma (HCC) accumulates a mutation of the p53 gene with a common substitution of nucleotide in a particular site. It is hypothesized that infection of hepatitis B virus (HBV) or exposure to aflatoxins could induce it. In Japan, the concentration of aflatoxins in the environment is low; however, infection of HBV and/or hepatitis C virus (HCV) is frequently seen in patients with HCC. The purpose of our studies was to determine whether these hepatoviral factors influence p53 alterations. In our results, p53 abnormalities, which were composed of loss of heterozygosity (LOH) and/or point mutation, were shown in 39% of patients. We postulated that they occurred at late stages in tumor growth based on the following two results. LOH analysis on p53 showed that most of the tumor nodule consisted of two phenotypes, LOH and non-LOH cancer cells. The p53 abnormalities correlated with the grade of cancer cell atypia which advanced with tumor growth. HBV and HCV infections were identified by polymerase chain reaction using DNA extracted from cancerous and noncancerous regions of the liver. By these methods, the patients who had been infected with either HBV or HCV showed an incidence of p53 abnormalities (45%) higher than those infected by neither (13%). However, the detection rate of these viruses was lower in the HCC region (33%) than that in the noncancerous region (56%) in cases with mutated p53 . The low rate of HCV detection (22%) in the HCC region with altered p53 was attributable to these different viral detection rates. There was a difference in pattern of p53 mutational changes in patients depending upon whether they were infected by HBV or by HCV. Two of three HBV-infected patients had a transversional change of nucleotide at the G:C site to T:A. However, in cases with HCV, four of eight patients had a transitional change of nucleotide of p53 . These results showed that HBV and HCV infections affect carcinogenic pathways causing p53 abnormalities independently. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Oligonucleotide arrays: new concepts and possibilities.

Abstract: Advances in solid–phase oligonucleotide synthesis and hybridization techniques have led to an incipient technology based on the use of oligonucleotide arrays. The inclusion of a large number of oligonucleotide probes within a single array greatly reduces the cost of their synthesis and allows thousands of hybridizations to be carried out simultaneously. The range of potential applications of oligonucleotide arrays was expanded by the realization that nucleic acids can be sequenced by hybridizing them to all possible oligonucleotides of a given length. Additional possibilities are offered by novel types of oligonucleotide arrays that are capable of parallel sorting, isolating, and manipulating thousands, and even millions, of nucleic acid species. Fields, such as site–directed mutagenesis, protein engineering, and recombinant DNA technology, would benefit from using these arrays. Further, these approaches could enable the analysis of entire genomes by preparing ordered fragment libraries, and by sequencing complex pools of nucleic acids, in a novel approach that provides long–range sequence information by generating nested nucleic acids and then surveying the oigonucleotides contained in the nested strands. This would allow large diploid genomes to be sequenced directly in a completely automated procedure that does not require fragment cloning or chromosome mapping.

Clustering of class I HLA molecules on the surfaces of activated and transformed human cells.

Abstract: We surveyed cells for the clusters of class I HLA molecules, HLA-I, which we have previously found on JY lymphoblasts. Two fluorescence techniques, fluorescence resonance energy transfer and electron exchange quenching, detected clustered HLA-I molecules on activated normal B and T cells, on cells of B and T lymphoblast lines, and on transformed fibroblasts. No HLA-I clusters were detectable in the surfaces of resting B or T cells or normal fibroblasts. HLA clustering correlates perfectly with the presence of the HC-10 epitope of beta 2-microglobulin (beta 2m)-free heavy chains at the cell surface although not with the amount of this epitope expressed. Clustering was reversed by exogenous beta 2m, but this did not change the amount of HC-10 bound. This suggests that a form of beta 2m-free heavy chain in equilibrium with both native HLA molecules and fully denatured HC-10-positive heavy chains is involved in HLA-I cluster formation.

Epithelial wound healing in the denervated cornea

Abstract: We studied the epithelial healing of denervated corneas in New Zealand albino rabbits with their left trigeminal ganglia surgically amputated. On the 14th day after amputation, the corneas were keratectomized (in 8.5 mm diameter) and documentation of the healing process began. We calculated the epithelial healing rate using simple regression analysis. We observed a mean healing rate of 0.463 +/- 0.059 mm2/hr (mean +/- SE) in the denervated corneas, compared to 0.609 +/- 0.031 mm2/hr in the control corneas; a statistically significant difference of P < 0.001. We performed scanning electron microscopic observation (SEM) at three points; before keratectomy, 48 hrs after keratectomy, and 14 days after keratectomy. SEM observation revealed that, in contrast to the control corneas, the surface of the epithelial cells in denervated corneas appeared rough with numerous exfoliating cells observed. This indicates that the epithelial cells might attach only weakly to the floor in denervated corneas. Transmission electron microscopic observation (TEM) performed at 48 hrs and 14 days after keratectomy also supports this finding. For example, the intercellular space is widened and fewer desmosomes are observed in denervated corneas. Using immunohistochemistry, the surface of the wound bed was covered with fibronectin in a similar fashion to the control. In the late stage, the denervated corneas demonstrated spontaneous epithelial breakdown with 83% of them having persistent epithelial defects. Epithelial healing in the control corneas displayed no abnormal signs. On the 14th day after keratectomy, these eyes were enucleated for immunohistochemistry using bromodeoxyuridine (Brd U) to observe dividing cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The regulation of competence transcription factor synthesis constitutes a critical control point in the regulation of competence in Bacillus subtilis.

Abstract: comK, which encodes the competence transcription factor, is itself transcriptionally activated at the transition from exponential growth to stationary phase in Bacillus subtilis. MecA, a negative regulator of competence, also inhibits comK transcription when overexpressed, and a mecA null mutation results in comK overexpression. Although null mutations in mecA, as well as in another gene, mecB, are known to bypass the requirements for nearly all of the competence regulatory genes, the comK requirement is not suppressed by mecA inactivation. Various competence regulatory genes (comA, srfA, degU, abrB, sin, and spo0A) are shown to be required for the expression of comK. srfA transcription is shown to occur equally in cells destined for competence and those destined not to become competent. In contrast, comK transcription is restricted to the presumptive competent cells. These and other results are combined to describe a regulatory pathway for competence.

Presentation of native epitopes in the V1/V2 and V3 regions of human immunodeficiency virus type 1 gp120 by fusion glycoproteins containing isolated gp120 domains.

Abstract: The immune response to viral glycoproteins is often directed against conformation- and/or glycosylation-dependent structures; synthetic peptides and bacterially expressed proteins are inadequate probes for the mapping of such epitopes. This report describes a retroviral vector system that presents such native epitopes on chimeric glycoproteins in which protein fragments of interest are fused to the C terminus of the N-terminal domain of the murine leukemia virus surface protein, gp70. The system was used to express two disulfide-bonded domains from gp120, the surface protein of human immunodeficiency virus type 1 (HIV-1), that include potent neutralization epitopes. The resulting fusion glycoproteins were synthesized at high levels and were efficiently transported and secreted. A fusion protein containing the HXB2 V1/V2 domain was recognized by an HIVIIIB-infected patient serum as well as by 17 of 36 HIV-1 seropositive hemophiliac, homosexual male and intravenous drug user patient sera. Many of these HIV+ human sera reacted with V1/V2 domains from several HIV-1 clones expressed in fusion glycoproteins, indicating the presence of cross-reactive antibodies against epitopes in the V1/V2 domain. Recognition of gp(1-263):V1/V2HXB2 by the HIVIIIB-infected human patient serum was largely blocked by synthetic peptides matching V1 but not V2 sequences, while recognition of this construct by a broadly cross-reactive hemophiliac patient serum was not blocked by individual V1 or V2 peptides or by mixtures of these peptides. A construct containing the V3 domain of the IIIB strain of HIV-1, gp(1-263):V3HXB2, was recognized by sera from a human and a chimpanzee that had been infected by HIVIIIB but not by sera from hemophiliac patients who had been infected with HIV-1 of MN-like V3 serotype. The reactive sera had significantly higher titers when assayed against gp(1-263):V3HXB2 than when assayed against matching V3 peptides. Immunoprecipitation of this fusion glycoprotein by the human serum was only partially blocked by V3 peptide, indicating that this infected individual produced antibodies against epitopes in V3 that were expressed on the fusion glycoprotein but not by synthetic peptides. These data demonstrated that the chimeric glycoproteins described here effectively present native epitopes present in the V1/V2 and V3 domains of gp120 and provide efficient methods for detection of antibodies directed against native epitopes in these regions and for characterization of such epitopes.

Fungal plasma membrane proton pumps as promising new antifungal targets

Abstract: Fungi are widely dispersed in nature and frequently appear as pathogens in the animal and plant kingdoms. The incidence of opportunistic fungal infections in humans has increased due to the human immunodeficiency virus and the application of modern medical approaches that subvert natural protective barriers to infection. Also, fungal blights continue to threaten crops worldwide. As a result, new antifungal agents are needed to address these critical problems. Existing antifungals can be used to effectively treat most cases of topical infection caused by the opportunistic pathogen Candida albicans, which is the principal agent of nosocomially acquired fungal infections. However, life-threatening, disseminated Candida infections are treated with more modest success. Existing antifungals can be toxic or ineffective because of natural resistance or even induced resistance. This limited efficacy largely reflects the restricted range of cellular targets considered during the development of current antifungals. The advancement of highly selective fungicidal reagents requires the recognition of new essential cellular targets. The fungal plasma-membrane proton pump is a high-abundance essential enzyme with a number of well-understood molecular properties that should facilitate the development of new antifungals. The proton pump is important for intracellular pH regulation and the maintenance of electrochemical proton gradients needed for nutrient uptake. It is a member of the P-type class of ion-transport enzymes, which are present in nearly all external cellular membranes. Typical P-type enzymes such as the Na+,K(+)-ATPase and H+,K(+)-ATPase are well established as specific targets for surface-active cardiac glycosides and anti-ulcer therapeutics. The development of new classes of selective antifungals targeted to the proton pump will require exploitation of the well-characterized genetic, kinetic, topological, regulatory, and drug-interaction features of the fungal enzyme that discriminate it from related host P-type enzymes. New antifungal drugs of this type should be relevant to the control of fungal pathogens of medical and agricultural importance and may be applicable to the control of intracellular parasites that also depend on closely related proton pumps for survival.

Method of sorting a mixture of nucleic acid strands on a binary array

Abstract: A method of sorting mixtures of nucleic acid strands comprising hybridizing the strands to an array of immobilized oligonucleotides, each of which includes a constant segment adjacent to a variable segment. The constant segment of the immobilized oligonucleotides can be made complementary to the ends of strands obtained by digesting a double-stranded nucleic acid with a restriction enzyme and restoring the restriction sites, thereby permitting the sorting of strands according to their variable sequences adjacent to their constant terminal restored restriction sites.

Regulation of competence-specific gene expression by Mec-mediated protein-protein interaction in Bacillus subtilis

Abstract: The expression of competence genes in Bacillus subtilis is controlled by a signal transduction cascade which increases the expression of a competence transcription factor (CTF, encoded by comK) during the transition from exponential growth to stationary phase. The transcription of CTF (ComK) is decreased by the product of the mecA gene, and this inhibition is relieved in response to an unknown signal received from upstream in the regulatory pathway. Inactivation of either mecA or another gene, mecB, results in overproduction of ComK. We show here that the concentration of MecA protein does not vary markedly with culture medium, as a function of growth stage, or in competent and noncompetent cells. We also show that MecA can interact directly with ComK. Finally, evidence is presented suggesting that MecB functions prior to MecA in the signaling pathway. A model is discussed which involves the sequestration of ComK by MecA binding and the release of the transcription factor when an appropriate signal is relayed to MecA by MecB.

Pf1 virus structure : helical coat protein and DNA with paraxial phosphates

Abstract: The helical path of the DNA in filamentous bacteriophage Pf1 was deduced from different kinds of existing structural information, including results from x-ray fiber diffraction. The DNA has the same pitch, 16 angstroms, as the surrounding helix of protein subunits; the rise and rotation per nucleotides are 6.1 angstroms and 132 degrees, respectively; and the phosphates are 2.5 angstroms from the axis. The DNA in Pf1 is, therefore, the most extended and twisted DNA structure known. On the basis of the DNA structure and extensive additional information about the protein, a model of the virion is proposed. In the model, the DNA bases reach out, into the protein, and the lysine and arginine side chains reach in, between the DNA bases, to stabilize the paraxial phosphate charges; the conformation of the protein subunit is a combination of alpha and 3(10) helices.

RifR mutations in the beginning of the Escherichia coli rpoB gene

Abstract: In Escherichia coli, mutations conferring rifampicin (Rif) resistance map to the rpoB gene, which encodes the 1342-amino acid β subunit of RNA polymerase. Almost all sequenced RifR mutations occur within the Rif region, encompassing rpoB codons 500–575. A strong RifR mutation lying outside the Rif region, which changed Val146 to Phe was previously reported, but was not recovered in subsequent studies. Here, we used site-directed mutagenesis followed by selection on Rif to search for RifR mutations in the evolutionarily conserved segment of rpoB around codon 146. Strong RifR mutations were obtained when Val146 was mutated, and several weak RifR mutations were also isolated near position 146. The results define a new, N-terminal cluster of RifR mutations, in addition to the classical central Rif region.

A novel, glycan-dependent epitope in the V2 domain of human immunodeficiency virus type 1 gp120 is recognized by a highly potent, neutralizing chimpanzee monoclonal antibody.

Abstract: An anti-gp120 monoclonal antibody (MAb), C108G (gamma 1, kappa), was isolated from a chimpanzee that had been infected with strain IIIB of human immunodeficiency virus type 1 (HIV-1IIIB) and subsequently immunized with the recombinant glycoprotein rgp160MN. This MAb is specific for the IIIB strain of HIV-1 and related clones and exhibits very potent neutralization of these viruses; e.g., 100% neutralization of approximately 8 x 10(3) infectious units of HXB2 was achieved with 125 ng of C108G per ml. Commensurate with this potent neutralizing activity, the apparent affinity of C108G for rgp160LAI was very high, i.e., approximately 3 x 10(10) liters/mol. The C108G epitope was not destroyed by reduction of gp120 disulfide bonds but was profoundly disrupted by removal of N-linked sugars from gp120. Despite the importance of a glycan(s) in forming the C108G epitope, specific binding of C108G to synthetic peptides overlapping in amino acids 162 to 169 of the V2 region was detected, albeit with an affinity approximately 2,000-fold lower than that of C108G's binding to glycosylated envelope protein. This epitope mapping correlated with results of competition assays using MAbs of known epitope specificities. To our knowledge, this is the first description of an anti-V2 MAb raised in response to HIV-1 infection. Its potent neutralizing activity and epitope specificity indicate that the V2 domain of gp120 may be an effective target of the protective immune response and, therefore, potentially an important component of HIV vaccines.

Topology of the RNA polymerase active center probed by chimeric rifampicin-nucleotide compounds

Abstract: Spatial organization of the binding sites for the priming substrate, the template DNA, and the transcription inhibitor rifampicin (Rif) in Escherichia coli RNA polymerase (EC 2.7.7.6) was probed with chimeric compounds in which Rif is covalently attached to a ribonucleotide. The compounds bind to RNA polymerase in bifunctional manner and serve as substrates for RNA chain extension, yielding chains up to 8 nucleotides in length, with Rif linked to their 5' termini. These products act as potent inhibitors of normal transcription. Using the linker between the two ligands as ruler, we determined the distance between the sites for Rif and the priming nucleotide to be approximately 15 A. A reactive side group placed in the linker next to Rif crosslinks to the template strand of DNA at the -2 or -3 position of the promoter. Thus, bound Rif is juxtaposed to DNA immediately upstream of the start site, suggesting that Rif plugs the channel leading RNA out of the active center.

Mutation of the putative nucleotide binding site of the Bacillus subtilis membrane protein ComFA abolishes the uptake of DNA during transformation.

Abstract: ComFA is a membrane protein required for the uptake of transforming DNA following its binding to the Bacillus subtilis competent-cell surface. ComFA, which resembles members of the DEAD family of ATP-driven helicases, contains sequences similar to those found in many ATP-binding proteins and thought to represent the ATP-binding sites of these proteins. We have suggested that ComFA may function as a DNA translocase and/or helicase, using the energy of ATP hydrolysis to mediate the uptake of DNA. As a partial test of this hypothesis, we have introduced mutations into highly conserved glycyl and lysyl residues of the putative ATP-binding site, located, respectively, at positions 151 and 152, and determined the effects of these alterations on in vivo function. A substitution of the conserved lysyl by a glutamyl residue (K152E) and a double G151R-K152N mutation each resulted in a nearly 1,000-fold decrease in transformability, equivalent to that observed in a ComFA null mutant. A K152N mutation caused a partial loss-of-function phenotype. These effects were manifested at the level of DNA uptake; no marked effects on the final levels of DNA binding were noted. When either the K152E mutant allele or the G151R-K152N double mutant allele was combined in single copy with wild-type comFA, a dominant negative phenotype expressed on the level of DNA uptake was observed, suggesting that ComFA acts in a complex with other proteins, with additional molecules of ComFA, or with both. Images

Immunobiologic and biochemical properties of mutants of toxic shock syndrome toxin-1.

Abstract: Toxic shock syndrome (TSS) is a multisystem illness caused mainly by Staphylococcus aureus producing TSS toxin-1 (TSST-1). A variant of TSST-1 has been isolated from ovine mastitis S. aureus. This toxin, TSST-ovine (TSST-O) is only weakly T cell mitogenic, is nonpyrogenic, does not enhance endotoxin shock, and does not cause TSS in the miniosmotic pump model. The sequence of the ovine gene (tstO) differs from the TSST-1 gene (tstH) by 14 nucleotides that change seven amino acids in the mature protein of which two are in the C-terminal half. A gene fusion containing half of both tstH and tstO was made and cloned into S. aureus. The fusion protein contained the two C-terminal amino acid differences that are in TSST-O at residues 132 and 140. The fusion protein was not T cell mitogenic and did not elicit TSS in two rabbit models. Additional experiments used mutagenesis to change the lysine residue at position 132 of TSST-O to glutamate (TSST-OK132E), as exists in TSST-1, and to change the lysine residue of the human-ovine fusion at position 132 to glutamate (TSST-11140T). Both mutants were pyrogenic, enhanced endotoxin shock, and caused TSS in the miniosmotic pump model. However, the proteins were only partially T cell mitogenic. The restoration of lethality of TSST-O and the human-ovine fusion by changing the lysine to glutamate, as exists in TSST-1, indicates that residue 132 is important in lethality. The failure to regenerate complete T cell mitogenicity of the same mutants indicates that residues 132 and 140 are important for that activity.

Sensitive nucleic acid sandwich hybridization assays and kits

Abstract: Nucleic acid sandwich hybridization assays are provided that incorporate one or a combination of background reduction steps. Those steps include use of a separate capture probe and separation from immobilized capture probes by cleavage and isolation. A very sensitive assay for RNA targets includes both of those steps, plus RNA binary probes, and RNA-directed RNA ligase and amplification by an RNA-directed RNA polymerase. Kits of reagents for performing assays according to this invention are also provided.

Inhibitory effect of topical application of polymerized ferulic acid, a synthetic lignin, on tumor promotion in mouse skin two-stage tumorigenesis

Abstract: A dehydrogenation polymer of ferulic acid (DHP-FA), a synthetic lignin, was evaluated for its ability to inhibit tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) in the 7,12-dimethylbenz[a]anthracene-treated skin of female ICR mice. Topical application of DHP-FA inhibits TPA-induced tumor promotion, whereas a monomeric ferulic acid does not show the inhibitory effect.