Structural requirements for the inhibitory action of 12,13-epoxytrichothecenes on protein synthesis in eukaryotes

1. The inhibitory actions of ten trichothecene antibiotics were investigated, in reticulocyte cell-free systems synthesizing protein in vitro, by studying polyribosome profiles and kinetics of amino acid incorporation in the presence or absence of the drugs. 2. The modes of action observed were critically dependent on the drug concentrations used, but the antibiotics tested could be divided into four distinct groups, each exerting a characteristic inhibitory response. 3. The inhibitory action observed in every case was controlled by the chemical structure of the individual trichothecene and in particular was closely related to the nature of the substituent groups present on C-3, C-4, C-8 and C-15 of the molecule.     

An IgG human monoclonal antibody that reacts with HIV-1/GP120, inhibits virus binding to cells, and neutralizes infection.

A human mAb (HmAb) termed F105 was obtained by fusion of antibody-producing EBV-transformed cells with the HMMA2.11TG/O cell line. F105 is an IgG1 kappa antibody that binds to the surfaces of cells infected with all HIV-1 strains tested: MN, RF, IIIB, and SF2, but not uninfected cells. The HmAb immunoprecipitates GP120 from all four strains. F105 does not react with denatured GP120 on Western blots, but does react with viral lysates and purified GP120 dotted onto nitrocellulose filter paper under nondenaturing conditions. rGP120 from SF2 and soluble rCD4 inhibit antibody binding to infected cells in a dose-dependent manner. F105 inhibits the binding of free, infectious virions to uninfected HT-H9 cells with 50% of maximal (100%) inhibition at approximately 1 microgram/ml. F105 inhibits infection of HT-H9 cells by 100 tissue culture infective dose 50% units of MN and IIIB strains with 50% inhibition at concentrations of HmAb readily achievable in man. It appears that the F105 HmAb reacts with a conformationally defined epitope on HIV-1/GP120 that is exposed on the free virion and is important for binding to the cell surface by the virion. The epitope, which is immunogenic in humans, appears to be within, or topographically near, the CD4-binding site. F105 and the F105 epitope are potentially useful in therapy and in the design of peptide or anti-Id based vaccines; monitoring of the expression of the Id may prove useful in evaluating immune responses in infected individuals or vaccinated volunteers.     

Effects of serotonin1-like receptor agonists on autonomic neurotransmission.

Serotonin1A receptor agonists, 8-hydroxy-2-(di-n-propylamino)tetralin and 10-methyl-11-hydroxyaporphine, inhibited electrical stimulation-induced contraction of the guinea-pig ileum. These agonists also inhibited the pressor and tachycardiac responses to low frequency (0.25 Hz) but not to high frequency (2.0 Hz) electrical stimulation of the sympathetic nervous system in pithed rats. Serotonin1B receptor agonist RU 24969 inhibited pressor and tachycardiac responses to both low and high frequencies of stimulation in pithed rats. In the cat nictitating membrane, serotonin1A receptor agonists did not alter contractions elicited by electrical stimulation (0.1-3.0 Hz). Serotonin not only contracted the cat nictitating membrane but also facilitated contractile responses to low frequency (0.1-1.0 Hz) stimulation. The contractile effect of serotonin in the cat nictitating membrane was blunted by bretylium, methysergide, and ketanserin, but not by metoclopramide. The facilitatory effect of serotonin was antagonized by methysergide, but not by ketanserin, pindolol, propranolol, or metoclopramide. These results suggest that serotonin1A receptors modulate autonomic neurotransmission in the guinea-pig ileum and pithed rats, but not in the cat nictitating membrane. Serotonin contracts the cat nictitating mebrane via serotonin2 subtypes, while facilitating stimulated contractile responses through the serotonin1-like receptors.     

Coordinate regulation of glycogen metabolism in the yeast Saccharomyces cerevisiae. Induction of glycogen branching enzyme.

The yeast glycogen branching enzyme (EC 2.4.1.18) is shown to be induced in batch culture simultaneously with the onset of intracellular glycogen accumulation. The branching enzyme structural gene (GLC3) has been cloned. Its predicted amino acid sequence is very similar to procaryotic branching enzymes. Northern analysis indicates that GLC3 mRNA abundance increases in late exponential growth phase coincident with glycogen accumulation. Disruption of the branching enzyme structural gene establishes that branching enzyme activity is an absolute requirement for maximal glycogen synthesis.     

Immunogenetics of collagen-induced arthritis in rats. Both MHC and non-MHC gene products determine the epitope specificity of immune response to bovine and chick type II collagens.

Seven inbred, RT1-congenic rat strains were immunized with native bovine (BII), porcine (PII), or chick (CII) type II collagen and observed for onset, incidence, and severity of arthritis. Clinical results were compared with IgG reactive with native rat type II collagen (RII) and the purified, renatured cyanogen-bromide peptides of BII, CII, or RII. Immunodominant responses to CB11, CB9,7, and CB12 of RII were identified. Secondary responses to CB8 and CB10 also occurred. Reproducible patterns of peptide reactivity were defined in each strain and reflected both RT1 and non-RT1 genotypes plus the species of immunizing collagen. BN non-RT1 gene products moderated clinical arthritis but increased the levels of reactivity to CB11 in three strains carrying RT1l,n,av1 haplotypes. WF (RT1u) rats were susceptible to collagen-induced arthritis (CIA) and developed very high levels of autoantibodies with dominant responses to rat CB11 after CII injections and to rat CB11 and CB9,7 after BII injections. DA (RT1av1) rats developed the most severe arthritis but had only moderate (total) levels of anti-RII IgG: a broad response to CB11, CB10, and CB9,7 after CII injections but predominantly to CB12 and CB9,7 after BII injections. Three RT1n strains--DA.1N(BN), WF.1N(MAXX), and BN--were resistant to BII-induced CIA but developed mild arthritis after immunization with CII. After BII: BN IgG reacted with CB9-7, CB11, and CB12; DA.1N and WF.1N IgG reacted with CB9,7 and CB12. After CII: BN IgG reacted broadly with CB11, CB9-7, CB12, and CB8; WF.1N IgG reacted to CB9-7, CB11, CB8, and CB12; DA.1N IgG reacted with CB8, CB11, and CB9-7. Thus, selective induction of CIA in BN, WF.1N, and DA.1N rats by CII correlated with serum IgG reactivity to rat CB11, but overall strain results identified no single cyanogen-bromide peptide as expressing the sole "arthritogenic" epitope in CIA.     

Cloning and expression of Candida albicans ADE2 and proteinase genes on a replicative plasmid in C. albicans and in Saccharomyces cerevisiae.

Summary A plasmid vector (denoted pRC2312) was constructed, which replicates autonomously in Escherichia coli, Saccharomyces cerevisiae and Candida albicans. It contains LEU2, URA3 and an autonomously replicating sequence (ARS) from C. albicans for selection and replication in yeasts, and bla (ampicillin resistance) and ori for selection and replication in E. coli. S. cerevisiae AH22 (Leu^–) was transformed by pRC2312 to Leu^– at a frequency of 1.41 × 10⁵ colonies per g DNA. Transformation of C. albicans SGY-243 (Ura-) to Ura⁺ with pRC2312 resulted in smaller transformant colonies at a frequency of 5.42 × 10³ per g DNA where the plasmid replicated autonomously in transformed cells, and larger transformant colonies at a frequency of 32 per g DNA, in which plasmid integrated into the genome. Plasmid copy number in yeasts was determined by a DNA hybridization method and was estimated to be 15±3 per haploid genome in S. cerevisiae and 2–3 per genome in C. albicans replicative transformants. Multiple tandem integration occurred in integrative transformants and copy number of the integrated sequence was estimated to be 7–12 per diploid genome. The C. albicans ADE2 gene was ligated into plasmid pRC2312 and the construct transformed Ade^– strains of both C. albicans and S. cerevisiae to Ade⁺. The vector pRC2312 was also used to clone a fragment of C. albicans genomic DNA containing an aspartic proteinase gene. C. albicans transformants harboring this plasmid showed a two-fold increase in aspartic proteinase activity. However S. cerevisiae transformants showed no such increase in proteinase activity, suggesting the gene was not expressed in S. cerevisiae.     

Complete nucleotide sequence and gene organization of plasmid NTP16.

We have determined the complete nucleotide sequence of the 8.3-kb multicopy plasmid NTP16 and produced a functional map of its gene organization. Sixty percent of the plasmid DNA comprises transposon-derived sequences; in the remaining 3320 bp, we have identified three protein coding regions. NTP16 has a ColE1-type replication system, a cis-acting stability locus and a mobilization system comprising an oriT site and one mobilization protein. The roles of the other two protein products of this plasmid are unknown, but they are possibly involved in the plasmid incompatibility system.     

Characterization of the opa (class 5) gene family of Neisseria meningitidis

Class 5 outer membrane proteins of Neisseria meningitidis show both phase- and antigenic variation of expression. The proteins are encoded by a family of opa genes that share a conserved framework interspersed with three variable regions, designated the semivariable (SV) region and hypervariable regions 1 (HV1) and 2 (HV2). In this study, we determined the number and DNA sequence of all of the opa genes of meningococcal strain FAM18, to assess the structural and antigenic variability in the family of proteins made by one strain. Pulsed field electrophoresis and Southern blotting showed that there are four opa genes in the FAM18 chromosome, and that they are not tightly clustered. DNA sequence analysis of the four cloned genes showed a modest degree of diversity in the SV region and more extensive differences in the HV1 and HV2 regions. There were four versions of HV1 and three versions of HV2 among the four genes. Each of the FAM18 opa loci contained a gene with a unique combination of SV, HV1, and HV2 sequences. We used lambda gt11 cloning and synthetic peptides to demonstrate that HV2 sequences completely encode the epitopes for two monoclonal antibodies specific for different class 5 proteins of FAM18.     

Use of thionitrobenzoic acid to characterize the stability of nitric oxide in aqueous solutions and in porcine aortic endothelial cell suspensions

Nitric oxide is an important vasodilator which can be biologically produced from leukocytes and endothelial cells. However, it is highly unstable, which is an obstacle to detection and quantitation. We have exploited the reactivity of nitric oxide with thiols to establish an assay based on oxidation of thionitrobenzoic acid (TNB). The oxidation of thionitrobenzoic acid and the reaction with oxygen, which was measured by employing an oxygen electrode, were examined after the addition of nitric oxide solutions. The inhibition of aggregation of human platelets after challenge with 2.5 microM adenosine diphosphate was also investigated. These studies show the following properties of nitric oxide in aqueous solutions. (i) Nitric oxide is highly reactive to oxygen. (ii) Thiols react with a labile, highly reactive nitric oxide-oxygen product. (iii) Medium with very low oxygen content increases the life span of nitric oxide in aqueous solution. We also used the nitric oxide quantitation using TNB to study the metabolism of nitric oxide by porcine aortic endothelial cells and the results show that nitric oxide added to these cells in low oxygen content solution is stable. From these studies, we conclude that deoxygenated solutions stabilize nitric oxide. An important consequence of low oxygen content at localized tissue sites may be to augment biological effects mediated by nitric oxide.