Poliovirus induces an early impairment of mitochondrial function by inhibiting succinate dehydrogenase activity.

Poliovirus infection of COS-1 and T47D cells caused a rapid decrease in total cell respiration, and this was attributed to an inhibition of mitochondrial respiration. The stimulation of mitochondrial respiration by pyruvate plus malate or succinate was impaired in saponin-permeabilised cells. However, this inhibition could be overcome by the addition of N,N,N',N'-tetramethyl-1, 4-phenylenediamine and ascorbate. The activity of succinate dehydrogenase was impaired in parallel with the inhibition of mitochondrial respiration during poliovirus infection. This shows that mitochondrial function is profoundly altered during poliovirus infection and that this occurs primarily through inhibition of electron flow at complex II of the mitochondrial respiratory chain.

Purification, crystallization and preliminary X-ray analysis of human recombinant cytosolic serine hydroxymethyltransferase.

As an enzyme of the thymidylate synthase cycle, serine hydroxymethyltransferase (SHMT) has a key role in nucleotide biosynthesis. Elevated activities of SHMT have been correlated with the increased demand for nucleotide biosynthesis in tumors of human and rodent origin, making this enzyme a novel target for cancer chemotherapy. Here the purification and crystallization of recombinant human cytosolic SHMT are reported. Crystals belong to space group P6222 or P6422 with cell parameters a = b = 155.0, c = 235.5 A and diffract to at least 3.0 A resolution.

Characterisation of a human serine hydroxymethyltransferase pseudogene and its localisation to 1p32.3-33.

The conversion of serine and tetrahydrofolate to glycine and 5,10 methylene tetrahydrofolate by serine hydroxymethyltransferase (SHMT, EC 2.1.2.1) is the major route for the provision of one-carbon units for biosynthetic reactions. SHMT cDNAs have been cloned from both rabbit and man, and a human mitochondrial SHMT gene sequence has recently been reported. We have isolated phage clones containing human genomic sequences homologous to cytosolic SHMT and have found these to contain a processed pseudogene (SHMT-ps1) with a 90% identity to cloned SHMT cDNAs. SHMT-ps1 contains 2335 nt that are homologous to SHMT but it has an additional leader sequence of 203 nt of unknown origin. The complete SHMT-ps1 sequence of 2538 nt is bounded by two 16 nt direct repeats that are characteristic of retroelement insertion sites. Two phage clones containing SHMT-ps1 have been mapped by fluorescence in situ hybridisation to 1p32.3-33. In addition, an SHMT CDNA clone hybridized to the same region and to 17p11.2-12. The latter is consistent with a previous localisation of the gene for cytosolic SHMT.

Detection of the 5' region of the rubella virus genome in clinical samples by polymerase chain reaction.

BACKGROUND: Maternal rubella infection in early pregnancy has a high probability of causing congenital rubella infection. In some cases it may be difficult to establish the risk of congenital infection and polymerase chain reaction (PCR) based techniques are therefore being applied to prenatal diagnosis. OBJECTIVES: To investigate whether the non-structural region of the rubella virus (RV) genome can be detected in clinical samples using PCR, thereby providing a prenatal assay independent of those currently used to detect the structural protein coding region. STUDY DESIGN: Oligonucleotide primers coding for RV nucleotides 1-17 and 541-558 from the non-structural protein coding region of the RV genome were used in a reverse transcription polymerase chain reaction (NS RT-PCR) to amplify 558 nucleotides of RV cDNA. Amplification of RV specific sequences was confirmed by Southern hybridization. RESULTS: The specificity of the assay was confirmed by the detection of RV RNA from both wild-type and vaccine strains of RV, pharyngeal swabs from two adult males with acute rubella and products of conception from three women with serologically confirmed primary rubella in pregnancy. RV RNA was not detected in uninfected HEL and Vero cells or peripheral blood mononuclear cells. The results were concordant with those of an RT-PCR directed to the E1 protein coding region and with virus isolation. CONCLUSIONS: Detection of the non-structural coding region of the RV genome in clinical samples suggests that NS RT-PCR could be used as a confirmatory assay for prenatal diagnosis of congenital rubella, and that it will be of value for the identification of nucleotide changes in the 5' region of the RV genome.

Sequence variation in 5' termini of rubella virus genomes: changes affecting structure of the 5' proximal stem-loop.

Variation within a 523 nucleotide region proximal to the 5' terminus of seven rubella virus strains has been analysed. Compared to the Therien strain twenty sites of nucleotide variation have been identified, three of which are in the 5' untranslated region. Individual strains have between three and nine nucleotide differences, only three of which result in amino acid substitutions. TO-336 has a serine for threonine at amino acid (aa) 42 and CM arginine for histidine at aa 159. RA27/3 has arginine for lysine at aa 3 and serine for threonine at aa 42. Nucleotide differences which affect a stem-loop structure reported to be important for binding of host cell proteins have been identified.

Comparison of the genomes of the wild-type French viscerotropic strain of yellow fever virus with its vaccine derivative French neurotropic vaccine.

The French neurotropic vaccine, or FNV, was used extensively in Africa to control yellow fever (YF). Although efficacious, the vaccine caused an unacceptable rate of post-vaccinal complications in children and was subsequently replaced by the 17D vaccine. Here we report that the genomes of the wild-type YF virus French viscerotropic virus and its attenuated vaccine derivative, FNV virus from the Institut Pasteur, Paris, (FNV-IP) differ by 77 nucleotides encoding 35 amino acid substitutions. Comparison of FNV-IP and three other isolates of FNV with other YF vaccine strains (17D-204 and 17DD derived from wild-type strain Asibi) revealed that during the two attenuation processes two common nucleotide changes arose that encode two amino acid substitutions: one is in the membrane protein at amino acid 35 (M-35), the other in non-structural (NS) protein 4B at NS4B-95. These common substitutions may be important in the process of attenuation of viscerotropic disease for humans and monkeys, and/or may be involved in loss of mosquito competence of the vaccine viruses.

Translational control of mammalian serine hydroxymethyltransferase expression.

We have investigated the role of an open reading frame upstream of and overlapping that coding for rabbit cytosolic serine hydroxymethyltransferase in the post-transcriptional regulation of enzyme expression. Elimination of the AUG codon initiating translation of the upstream open reading frame by site-directed mutagenesis (to an AUC codon) was assessed by a transfection assay in COS-1 cells. The mutation relieves translational repression with a 100-fold increase in expressed serine hydroxymethyltransferase activity compared to control-transfected cells and in the absence of any increase in the level of serine hydroxymethyltransferase mRNA. The upstream open reading frame may play a regulatory role in changing levels of expression of serine hydroxymethyltransferase through a translational control mechanism.

Effect of chewing gums on plaque pH after a sucrose challenge.

The objective of this study was to determine whether sugarless chewing gums sweetened with different sweeteners differ in their ability to reduce an acidogenic response from a 10 percent sucrose-rinse challenge. Five commercially available chewing gums and two control regimens ("no gum" or paraffin) were tested using a plaque pH telemetry system. The gums were sweetened with sucrose, high-intensity sweeteners (aspartame, saccharin, or acesulfame-K), or a polyol (xylitol). Using a seven-period randomized block design, eight adult panelists were challenged with a 10 percent sucrose solution and then randomly used one of the test regimens during each of the seven test sessions. Each two-hour test session was divided into five periods: resting baseline (five minutes); sucrose rinse challenge (two minutes); postsucrose challenge (ten minutes); gum chewing (ten minutes); post gum chewing (ninety-three minutes). The factors analyzed were: the area of the curve (pH X Time) below pH 5.5, the minimum plaque pH attained, the changes in plaque pH over relevant intervals, and the length of time the plaque pH remained below pH 5.5. The various response variables showed a similar pattern of statistically significant differences. All of the sugarless gums were effective in significantly increasing plaque pH and in reducing the area under the curve after the sucrose challenge compared with "no gum" treatment. No statistically significant differences were noted among the sugarless gums. The response to sucrose gum was intermediate between sugarless gums and "no gum" but was not statistically different from "no gum" or three of the sugarless gums.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of rubella virus E1 fusion proteins for detection of rubella virus antibodies.

Two glutathione S-transferase fusion proteins containing 44 (p1503) and 75 (p1509) amino acid residues of the rubella virus E1 glycoprotein were expressed in Escherichia coli with the aim of producing a recombinant rubella virus antigen for use in serological assays. p1503 contained three neutralizing and hemagglutinating epitopes (G. M. Terry, L. M. Ho-Terry, P. Londesborough, and K. R. Rees, Arch. Virol. 98:189-197, 1988); p1509 contained the putative neutralization domain described by Mitchell et al. (L. A. Mitchell, T. Zhang, M. Ho, D. Decarie, A. Tingle, M. Zrein, and M. Lacroix, J. Clin. Microbiol. 30:1841-1847, 1992) in addition to the three epitopes present in p1503. Both fusion proteins were soluble and affinity purified on glutathione-Sepharose 4B. In Western blots (immunoblots), p1503 and p1509 reacted with human sera containing rubella virus-specific immunoglobulin G. When used as antigens in indirect enzyme immunoassays to detect rubella virus-specific immunoglobulin G, p1503 correctly identified the rubella virus antibody status of 43 (76.8%) and p1509 correctly identified that of 48 (85.7%) of 56 serum samples received for routine rubella virus antibody screening. The results obtained with p1509 compare well with those obtained with a latex agglutination assay.

Genetic engineering of hybridoma glutamine metabolism.

The murine hybridoma PQXB1/2 cannot be adapted to grow in culture media containing < 0.5 mM glutamine. Transformants selected following electroporation of PQXB1/2 cells with vectors containing a Chinese hamster glutamine synthetase (GS) cDNA under the control of the SV40 early promoter also failed to grow in the absence of glutamine in the culture medium. PQXB1/2 cells have, however, been transformed to glutamine independence following electroporation with a vector containing this glutamine synthetase cDNA under the control of the human cytomegalovirus immediate early promoter. In these cells, sufficient active glutamine synthetase was expressed from one vector per cell to enable growth in glutamine-free media. The specific activity of glutamine synthetase in two transformed cell lines producing parental levels of antibody was increased by 128 and 152%, respectively (0.57 and 0.63 mumol min-1 per 10(6) cells in transformants compared with parental levels of 0.25 mumol min-1 per 10(6) cells). This reprogramming of glutamine synthetase expression and glutamine metabolism is important for developing strategies to deal with ammonia toxicity and the production of cell lines with improved metabolic processes.

Acidogenicity of high-intensity sweeteners and polyols.

PURPOSE: To evaluate the plaque pH responses to sucrose, fructose, xylitol, sorbitol, acesulfame-K, aspartame, and saccharin at equal sweetness levels (equivalent to 10% sucrose) and water using an indwelling plaque pH telemetry system. MATERIALS AND METHODS: Eight adult panelists used each sweetener once in a Latin square study design. Plaque was allowed to accumulate for 3-6 days before each challenge period, and the panelists fasted for 12 hours prior to the 2-minute test rinse. Plaque pH was monitored for a 2-hour period after the rinse exposure. The parameters examined were area of the curves under pH 5.5 (pH X Time), pH changes from baseline, lowest pH attained and time below pH 5.5. RESULTS: The the high-intensity sweeteners (aspartame, saccharin and acesulfame-K) and the polyols (sorbitol and xylitol) were all non-acidogenic and were not significantly different from each other while both sucrose and fructose were highly acidogenic.

Enhancement of monoclonal antibody yield by hybridoma fed-batch culture, resulting in extended maintenance of viable cell population.

Hybridoma batch cultures were extended using feed formulations based on nutrient consumption measured during different batch culture phases when (a) growth but negligible antibody production was taking place; (b) maximum antibody production rate and declining viable cell growth rate were observed. Strategy (a) was the more successful (2.8-fold compared with 1.8-fold antibody titer increase) and maintained cell viability for longer. Analysis of the effects of omitting individual amino acids yielded results which were consistent with those from the feeding experiment.

Recombinant rubella E1 fusion proteins for antibody screening and diagnosis.

BACKGROUND: Until rubella is eradicated there will be a continuing need for rubella antibody surveillance. Antigen production using recombinant DNA technology may be a viable alternative to traditional techniques of producing antigens for enzyme immunoassays (EIAs). OBJECTIVES: To investigate the potential of bacterial fusion proteins containing rubella E1 protein sequences for use in EIAs to detect rubella antibodies. STUDY DESIGN: Purified bacterial fusion proteins containing rubella E1 sequences to be used as antigens in EIAs and compared to 'traditional' assays using virus derived antigens for rubella antibody screening. RESULTS: cDNA clones coding for the complete rubella E1 protein sequence and subfragments of E1 were modified for expression as carboxy terminal fusions with either beta-galactosidase or glutathione-S-transferase. beta-galactosidase fusions with the complete E1 coding sequence or amino acids 201 to 307, which contain known epitopes, resulted in the production of predominantly insoluble fusion proteins unsuitable for use in EIA. Nine glutathione-S-transferase-E1 fusion proteins were produced with individual fusion proteins exhibiting varying properties with regard to the levels of protein produced, apparent stability, solubility and the potential for affinity purification using glutathione agarose. Reduction of the E1 component to only 44 amino acids containing three B-cell epitopes (Terry et al., 1988) produced an abundant soluble GST-E1 fusion protein (3.5 mug/ml), which could be affinity purified using glutathione agarose. This fusion protein has been successfully used in EIA to detect rubella antibodies. CONCLUSIONS: We have shown that GST-E1 fusions have potential as antigens in tests for rubella antibodies.