Identification of cross-reactive and serotype 2-specific neutralization epitopes on VP3 of human rotavirus.

The group A rotaviruses are composed of at least seven serotypes. Serotype specificity is defined mainly by an outer capsid protein, VP7. In contrast, the other surface protein, VP3 (775 amino acids), appears to be associated with both serotype-specific and heterotypic immunity. To identify the cross-reactive and serotype-specific neutralization epitopes on VP3 of human rotavirus, we sequenced the VP3 gene of antigenic mutants resistant to each of seven anti-VP3 neutralizing monoclonal antibodies (N-MAbs) which exhibited heterotypic or serotype 2-specific reactivity, and we defined three distinct neutralization epitopes on VP3. The mutants sustained single amino acid substitutions at position 305, 392, 433, or 439. Amino acid position 305 was critical to epitope I, whereas amino acid position 433 was critical to epitope III. In contrast, epitope II appeared to be more dependent upon conformation and protein folding because both amino acid positions 392 and 439 appeared to be critical. These four positions clustered in a relatively limited area of VP5, the larger of the two cleavage products of VP3. At the positions where amino acid substitutions occurred, there was a correlation between amino acid sequence homology among different serotypes and the reactivity patterns of various viruses with the N-MAbs used for selection of mutants. A synthetic peptide (amino acids 296 to 313) which included the sequence of epitope I reacted with its corresponding N-MAb, suggesting that the region contains a sequential antigenic determinant. These data may prove useful in current efforts to develop vaccines against human rotavirus infection.     

Identification of cognate genes among heterologous strains of group B rotavirus.

The genetic relatedness of group B rotavirus (GBR) strains has previously been documented by hybridization with probes derived from whole genomic sequences, but the relationship of individual genes of heterologous GBR strains has not been evaluated. Definition of cognate GBR genes would facilitate investigation of the determinants of group specificity, serotype identity, and neutralization epitopes. Therefore, we investigated the genetic relatedness of three GBR strains by means of Northern (RNA) blot hybridization with isotopically labeled probes prepared from each of the 11 genes of the IDIR strain of GBR. Under low-stringency conditions, hybridization between each of the IDIR gene probes and genomic RNA from the ADRV strain of GBR was observed. Genomic RNA obtained from a bovine strain of GBR hybridized with 9 of the 11 IDIR gene probes. In most cases, cognate genes of each of the GBR strains appeared to migrate to similar positions following polyacrylamide gel electrophoresis. However, the electropherotype positions of GBR genes 5, 6, and 7 were different for each of the three GBR strains. Identification of these genomic segments among GBR strains should prove helpful in future evaluations of GBR structure and function.     

Water buffalo (Bubalus bubalus Arnee) allotypes: Identification of two specificities controlled by independent genes

Two water buffalo allotypes (Bl and CI) are described, which are located on distinct low molecular weight molecules. Bl is common to water buffalo and cattle. These two markers are inherited in a simple Mendelian manner and controlled by two independent genes.     

ELISA-based method for determining the affinity of bivalent antibodies of two specificities in a mixture

The problem of the evaluation of the affinity for two types of bivalent antibodies in a mixture is considered. It is shown that the binding curve in appropriate coordinates can be used to compose either a system of four equations with four unknowns or a system of two equations with two unknown variables. The numerical solution of these equation systems yields affinity constants for both antibodies and the relationship between concentrations of antibodies studied in the mixture.     

Identification of two novel primate-specific genes in DSCR.

We recently helped to complete the sequence of human chromosome 21 at a very high level of accuracy. Using this sequence we identified two novel genes, designated DSCR9 and DSCR10, in the so-called Down Syndrome Critical Region (DSCR) by computational gene prediction and subsequent cDNA cloning. Both DSCR9 and DSCR10 are expressed preferentially in testis and encode functionally unknown proteins with 149 and 87 amino acid residues, respectively. Zoo blot analysis suggested that both genes are exclusive to primate genomes such as chimpanzee, gorilla, orangutan, crab-eating monkey and African green monkey but are not present in other non-primate mammals including mouse, dog, cat, and chicken. Comparative genomic sequence analysis of DSCR9 and DSCR10 with the corresponding mouse syntenic region confirmed the lack of these genes in the mouse. These results strongly suggest that DSCR9 and DSCR10 have emerged as a new class of gene in the primate lineage during evolution.     

Two genetic arrangements determining flagellar antigen specificities in two diphasic Escherichia coli strains

Abstract Two diphasic Escherichia coli strains, Bi7327-41 and P12b, which spontaneously change their flagellar antigenic characters from H3 to H16, and from H17 to H4, respectively, were investigated. New features of the genetic control of flagellar phase variation in Bi7327-41 and Salmonella were found. Two genes responsible for the alternative flagellar phases H17 and H4 were demonstrated in P12b. They differed from hagA (H16) and hagB (H3), the two flagellin genes of Bi7327-41, in not being alleles of hagB (H3) or sensitive to the hagA (H16)-specific repressor.     

Relation of VP7 amino acid sequence to monoclonal antibody neutralization of rotavirus and rotavirus monotype.

The neutralization epitopes of the VP7 of human rotavirus RV-4 were studied by using five neutralizing mouse monoclonal antibodies to select virus variants resistant to neutralization by each of the antibodies. Antibody resistance patterns and sequence analysis of the RV-4 variants revealed that at least four sites on VP7, located at amino acids 94 (region A), 147 to 148 (region B), 213 (region C), and 291, are involved in neutralization of the human G1 rotavirus RV-4. The A-region site elicited antibody cross-reactive between G types and showed species-restricted immunodominance not related to carbohydrate attachment. The monotype 1b rotavirus M37 lacked this site. The B region contained strain-specific and cross-reactive sites, absent in monotype 1c rotaviruses. The C-region site was present in all G1 rotaviruses tested. Monotype 1a rotaviruses contained all these sites of neutralization. Virus monotype and sensitivity to monoclonal antibody neutralization usually related to the presence of a particular amino acid(s) at or next to the positions at which the mutations were selected in the virus variants.     

Identification of two putative ATP-cassette genes in Encephalitozoon intestinalis.

Currently existing chemotherapeutic compounds are limited and few are effective for treating microsporidiosis. It is possible that resistance of Encephalitozoon to some drugs occurs by efflux mechanisms similar to those previously described for mammalian tumour cells, bacteria or protozoal parasites such as Plasmodium, Leishmania and Entamoeba histolytica. The data in the present study suggest that Encephalitozoon intestinalis contains at least one multidrug resistance gene. We report here two complete sequences EiABC1 and EiABC2, encoding different ATP-binding cassette genes from E. intestinalis, including a P-gp.     

Molecular localization of two cattle allotypic specificities.

McA1 and McA2 are two cattle allotypic specificities carried on high molecular weight glycoproteins and controlled by allelic genes. Results derived from two independent experimental approaches (digestion of McA1 and McA2 antigen preparations with specific glycosidases and inhibition tests of anti McA1 and anti McA2 with simple sugars) showed that mannose is essential in determining the serological specificity of McA1 and glucose of McA2.     

Identification of two hsp90 genes in carp

Two hsp90 cDNA isoforms (hsp90alpha and hsp90beta) were isolated from the common carp (Cyprinus carpio). Gene-specific probes and primers were selected and used in Northern blot hybridization and RT-PCR reactions to measure the basal hsp90 mRNA levels and to follow the inducer-specific expression of the hsp90 genes in different tissues during in vivo studies. The hsp90beta gene is largely constitutively expressed at a fairly high level in all the examined tissues (brain, liver and kidney) and is slightly inducible by an elevated temperature. Hsp90alpha mRNA is present in the brain, but is hardly detectable in the kidney and liver of unstressed animals. In the brain, this gene is greatly upregulated following thermal stress, whereas in the liver and kidney heat shock has only minor effects on its expression. Hsp90alpha, but not hsp90beta, responds to an elevated level of Cd in a dose-, time- and tissue-dependent manner.     

Immunodominance of the VP4 neutralization protein of rotavirus in protective natural infections of young children.

Natural infection by very similar strains of rotavirus during the 1988-1989 rotavirus season in Cincinnati, Ohio, provided complete protection of young children against subsequent rotavirus illnesses for a period of at least 2 years. Using this limited strain variability, we characterized the association between the titers of antibody to either the VP4 or the VP7 neutralization protein and protection against subsequent rotavirus disease. This was done by using reassortants that contained only one of the two rotavirus neutralization proteins of 89-12, a culture-adapted isolate representative of the protective rotavirus strains. The other neutralization protein in these reassortants was derived from a heterologous rotavirus (WC3 or EDIM) to which the infected subjects made little or no neutralizing antibody (titers, < or = 20). The geometric mean titer (GMT) of antibody to 89-12 in convalescent-phase sera from the 21 subjects analyzed was 2,323. The GMT of antibody to a reassortant (strain WC-4) that contained the VP7 protein of 89-12 and VP4 of WC3 was 387. In contrast, the GMT of antibody to a reassortant (strain EDIM-7) that contained the VP4 protein of 89-12 and the VP7 protein of EDIM was 1,078. Thus, the major neutralization response was directed against VP4 rather than VP7, a finding that has important implications for development of appropriate rotavirus vaccines.     

Primary structure of the neutralization antigen of simian rotavirus SA11 as deduced from cDNA sequence.

DNA sequences complementary to the double-stranded RNA coding for the neutralization antigen (glycoprotein VP7) of simian rotavirus SA11 have been cloned. The VP7 gene consists of 1,062 nucleotides, containing an uninterrupted coding sequence of 978 nucleotides which specifies a glycoprotein of 326 amino acids. The significance of a second possible initiation site 30 nucleotides downstream from the first is discussed. Partial amino acid sequence of this glycoprotein showed unequivocally that the cloned segment (segment 9) codes for glycoprotein VP7 of SA11. The resulting amino acid sequence contained only one carbohydrate acceptor site. Possible sites of membrane interaction and antigenic determinants are discussed based on the analysis of the hydrophobicity and hydrophilicity profiles of VP7.     

Identification and specificities of N-terminal acetyltransferases from Saccharomyces cerevisiae.

N-terminal acetylation can occur cotranslationally on the initiator methionine residue or on the penultimate residue if the methionine is cleaved. We investigated the three N-terminal acetyltransferases (NATs), Ard1p/Nat1p, Nat3p and Mak3p. Ard1p and Mak3p are significantly related to each other by amino acid sequence, as is Nat3p, which was uncovered in this study using programming alignment procedures. Mutants deleted in any one of these NAT genes were viable, but some exhibited diminished mating efficiency and reduced growth at 37 degrees C, and on glycerol and NaCl-containing media. The three NATs had the following substrate specificities as determined in vivo by examining acetylation of 14 altered forms of iso-1-cytochrome c and 55 abundant normal proteins in each of the deleted strains: Ard1p/Nat1p, subclasses with Ser-, Ala-, Gly- and Thr-termini; Nat3p, Met-Glu- and Met-Asp- and a subclass of Met-Asn-termini; and Mak3p subclasses with Met-Ile- and Met-Leu-termini. In addition, a special subclass of substrates with Ser-Glu- Phe-, Ala-Glu-Phe- and Gly-Glu-Phe-termini required all three NATs for acetylation.     

Serotypic analysis of VP3 and VP7 neutralization escape mutants of rhesus rotavirus.

Neutralization escape mutants of simian rotaviruses (rhesus rotavirus and SA11) were tested in hemagglutination inhibition and neutralization assays against hyperimmune and infection sera to determine if mutation in an immunodominant epitope could enable neutralization escape. An SA11 mutant with a new glycosylation site at amino acid 211 of VP7 was shown to escape neutralization by hyperimmune but not infection sera.     

Mammalian wax biosynthesis. I. Identification of two fatty acyl-Coenzyme A reductases with different substrate specificities and tissue distributions

The conversion of fatty acids to fatty alcohols is required for the synthesis of wax monoesters and ether lipids. The mammalian enzymes that synthesize fatty alcohols have not been identified. Here, an in silico approach was used to discern two putative reductase enzymes designated FAR1 and FAR2. Expression studies in intact cells showed that FAR1 and FAR2 cDNAs encoded isozymes that reduced fatty acids to fatty alcohols. Fatty acyl-CoA esters were the substrate of FAR1, and the enzyme required NADPH as a cofactor. FAR1 preferred saturated and unsaturated fatty acids of 16 or 18 carbons as substrates, whereas FAR2 preferred saturated fatty acids of 16 or 18 carbons. Confocal light microscopy indicated that FAR1 and FAR2 were localized in the peroxisome. The FAR1 mRNA was detected in many mouse tissues with the highest level found in the preputial gland, a modified sebaceous gland. The FAR2 mRNA was more restricted in distribution and most abundant in the eyelid, which contains wax-laden meibomian glands. Both FAR mRNAs were present in the brain, a tissue rich in ether lipids. The data suggest that fatty alcohol synthesis in mammals is accomplished by two fatty acyl-CoA reductase isozymes that are expressed at high levels in tissues known to synthesize wax monoesters and ether lipids.