Adenovirus 36 seropositivity is strongly associated with race and gender, but not obesity, among US military personnel.

BACKGROUND: Although several studies have shown a positive association between evidence of anti-adenovirus 36 (Ad-36) antibodies (Ad-36 exposure) and (1) obesity and (2) serum cholesterol in animals, there is limited research demonstrating this association in humans. There is also limited research on transmission, presentation and demographics of Ad-36 infection. DESIGN: (1) Body mass (body mass index (BMI)), (2) fasting serum cholesterol and triglyceride levels and (3) demographic characteristics were compared between Ad-36 seropositive and seronegative groups. The majority of subjects were matched as cases versus controls on a number of demographic variables. SUBJECTS: A total of 150 obese and 150 lean active-duty military personnel were studied. MEASUREMENTS: Subjects completed a questionnaire regarding demographic and behavioral characteristics. Subject serum samples were tested by serum neutralization assay for the presence of anti-Ad-36 antibodies. RESULTS: In all, 34% of obese and 39% of lean subjects had Ad-36 exposure, an insignificant difference. Serum cholesterol and triglyceride levels were significantly higher among the obese subjects than among the lean, but there were no associations between serum cholesterol and triglyceride levels and Ad-36 exposure. Positive associations were found between Ad-36 exposure and age, race and gender. CONCLUSION: The study stands in contrast to previous work that has shown a positive relationship between Ad-36 exposure and (1) obesity, and (2) levels of serum cholesterol and triglycerides. In this study there was no association in either case. Unanticipated relationships between Ad-36 exposure and age, race and gender were found, and this is the first time that such a link between Ad-36 exposure and demographics has been found.

Development of a novel continuous culture device for experimental evolution of bacterial populations.

The availability of a robust and reliable continuous culture apparatus that eliminates wall growth problems would lead to many applications in the microbial field, including allowing genetically engineered strains to recover high fitness, improving biodegradation strains, and predicting likely antibiotic resistance mechanisms. We describe the design and implementation of a novel automated continuous culture machine that can be used both in time-dependent mode (similar to a chemostat) and turbidostat modes, in which wall growth is circumvented through the use of a long, variably divisible tube of growth medium. This tube can be restricted with clamps to create a mobile growth chamber region in which static portions of the tube and the associated medium are replaced together at equal rates. To functionally test the device as a tool for re-adaptation of engineered strains, we evolved a strain carrying a highly deleterious deletion of Elongation Factor P, a gene involved in translation. In 200 generations over 2 weeks of dilution cycles, the evolved strain improved in generation time by a factor of three, with no contaminations and easy manipulation.

Positive selection of synonymous mutations in vesicular stomatitis virus.

Prevailing evolutionary forces are typically deduced from the pattern of differences in synonymous and non-synonymous mutations, under the assumption of neutrality in the absence of amino acid change. We determined the complete sequence of ten vesicular stomatitis virus populations evolving under positive selection. A significant number of the mutations occurred independently in two or more strains, a process known as parallel evolution, and a substantial fraction of the parallel mutations were silent. Parallel evolution was also identified in non-coding regions. These results indicate that silent mutations can significantly contribute to adaptation in RNA viruses, and relative frequencies of synonymous and non-synonymous substitutions may not be useful to resolve their evolutionary history.

Artificially ambiguous genetic code confers growth yield advantage.

A primitive genetic code is thought to have encoded statistical, ambiguous proteins in which more than one amino acid was inserted at a given codon. The relative vitality of organisms bearing ambiguous proteins and the kinds of pressures that forced development of the highly specific modern genetic code are unknown. Previous work demonstrated that, in the absence of selective pressure, enforced ambiguity in cells leads to death or to sequence reversion to eliminate the ambiguous phenotype. Here, we report the creation of a nonreverting strain of bacteria that produced statistical proteins. Ablating the editing activity of isoleucyl-tRNA synthetase resulted in an ambiguous code in which, through supplementation of a limited supply of isoleucine with an alternative amino acid that was noncoding, the mutant generating statistical proteins was favored over the wild-type isogenic strain. Such organisms harboring statistical proteins could have had an enhanced adaptive capacity and could have played an important role in the early development of living systems.

Size comparisons among integral membrane transport protein homologues in bacteria, Archaea, and Eucarya.

Integral membrane proteins from over 20 ubiquitous families of channels, secondary carriers, and primary active transporters were analyzed for average size differences between homologues from the three domains of life: Bacteria, Archaea, and Eucarya. The results showed that while eucaryotic homologues are consistently larger than their bacterial counterparts, archaeal homologues are significantly smaller. These size differences proved to be due primarily to variations in the sizes of hydrophilic domains localized to the N termini, the C termini, or specific loops between transmembrane alpha-helical spanners, depending on the family. Within the Eucarya domain, plant homologues proved to be substantially smaller than their animal and fungal counterparts. By contrast, extracytoplasmic receptors of ABC-type uptake systems in Archaea proved to be larger on average than those of their bacterial homologues, while cytoplasmic enzymes from different organisms exhibited little or no significant size differences. These observations presumably reflect evolutionary pressure and molecular mechanisms that must have been operative since these groups of organisms diverged from each other.

The microsatellites of Escherichia coli: rapidly evolving repetitive DNAs in a non-pathogenic prokaryote.

We have demonstrated hypervariability of native short-motif repeats (microsatellites) in Escherichia coli. Twenty-five of the longest microsatellites in the E. coli genome were identified. These were analysed for length variability among 22 wild-type (non-mutator) isolates from the E. coli collection of reference (ECOR). Non-coding mononucleotide repeats are consistently polymorphic among these genetically diverse E. coli. Length differences in variable microsatellites allowed all E. coli strains examined to be uniquely differentiated. Phylogenetic analysis of the variable repeats shows ubiquitous homoplasy at the level of divergence represented by the sample set, suggesting that these markers are hypermutable and should prove valuable for the discrimination of closely related strains that are not otherwise genetically differentiable. Genomic analyses suggest that similar markers are also likely to be found in all other prokaryotes.

Evolutionary changes in mutation rates and spectra and their influence on the adaptation of pathogens.

The evolutionary tuning of mutational processes may play a key role in prokaryotic evolution, particularly among pathogens. In this paper we review the evidence that genetic systems controlling the rate and spectrum of heritable mutations have evolved to optimize levels of adaptive variation and rates of genetic change.

Selection against frameshift mutations limits microsatellite expansion in coding DNA.

Microsatellite enrichment is an excess of repetitive sequences characteristic to all studied eukaryotes. It is thought to result from the accumulated effects of replication slippage mutations. Enrichment is commonly measured as the ratio of the observed frequency of microsatellites to the frequency expected to result from random association of nucleotides. We have compared enrichment of specific types of microsatellites in coding sequences with those in noncoding sequences across seven eukaryotic clades. The results reveal consistent differences between coding and noncoding regions, in terms of both the quantity of repetitive DNA and the types present. In noncoding regions, all types of microsatellite (mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeats) are found in excess, and in all cases, these excesses scale in a similar exponential fashion with the length of the microsatellite. This suggests that all types of noncoding repeats are subject to similar mutational and selective processes. Coding repeats, however, appear to be under much stronger and more specific constraints. Tri- and hexanucleotide repeats are found in consistent and significant excess over a wide range of lengths in both coding and noncoding sequences, but other repeat types are much less frequent in coding regions than in noncoding regions. These findings suggest that the differences between coding and noncoding microsatellite frequencies arise from specific selection against frameshift mutations in coding regions resulting from length changes in nontriplet repeats. Furthermore, the excesses of tri- and hexanucleotide coding repeats appear to be controlled primarily by mutation pressure.

Numerous length polymorphisms at short tandem repeats in human cytomegalovirus.

We show the presence of numerous short tandem repeats in the human cytomegalovirus (HCMV) genome and assess their usefulness as molecular markers. The genome is shown to contain at least 24 microsatellite regions that exhibit length polymorphisms. Insertion-deletion polymorphisms at these short tandem repeats are common (80% of repeats examined are polymorphic among two laboratory strains and 10 clinical isolates). This is the first report of widespread microsatellite length polymorphism in a viral genome. Some regions are highly polymorphic: one was revealed by DNA sequencing to contain length variants at five closely linked sites, which combined resulted in 10 variants for this region among the 12 strains and isolates examined. This study not only provides a new molecular marker system for this virus but also extends our understanding of microsatellite polymorphism in two important ways. First, variable-length repeats in HCMV can be considerably shorter than polymorphic repeats previously found in other organisms. Second, highly variable microsatellite repeats are not confined to prokaryotes and eukaryotes, as previously assumed. This variation provides a useful marker system for distinguishing viral isolates, and similar markers are also likely to be found in other large-genome DNA viruses.

Random amplification of polymorphic DNA and microsatellite genotyping of pre- and posttreatment isolates of Candida spp. from human immunodeficiency virus-infected patients on different fluconazole regimens.

Twelve patients infected with the human immunodeficiency virus (HIV) and with CD4 cell counts below 100 cells/microliter received fluconazole daily (200 mg; five patients) or weekly (400 mg; seven patients) for fungal prophylaxis during a 6-month period. Oropharyngeal swabs were taken at regular intervals in order to detect colonization with Candida spp. All yeast isolates were examined with respect to the development over time of fluconazole resistance. Genetic diversity among the strains was assessed in order to discriminate between selection of a resistant subclone and patient recolonization. Genotyping was performed through random amplification of polymorphic DNA (RAPD) analysis. Specific site polymorphisms were assayed by tracking length variability in several microsatellite loci. Finally, to maximize resolution, one of these loci (ERK1) was analyzed by nucleotide sequencing. Although the number of strains analyzed was too small to allow statistical verification, it appeared that when fluconazole was given weekly, a smaller fraction of the strains showed diminished sensitivity than when it was given daily. Genetic analyses allowed three different scenarios to be discerned. Resistance development in an otherwise apparently unchanged strain was seen for 1 of the 12 patients. Clear strain replacement was observed for 3 of the remaining 11 patients. For all other patients minor differences were seen in either the RAPD genotype or the microsatellite allele composition during the course of treatment. In general, microsatellite sequence data is in agreement with data obtained by other methods, but occasionally within-patient heterogeneity is indicated. The present results show that during fluconazole treatment colonizing strains can remain identical, be replaced by clearly different strains, or undergo small changes. Within a patient there may be different levels of intrastrain variation.

Sequence analysis of a compound coding-region microsatellite in Candida albicans resolves homoplasies and provides a high-resolution tool for genotyping.

Sequence diversity at a coding-region microsatellite locus of two diploid Candida species was surveyed. Twenty-one alleles from fourteen strains of Candida albicans and three alleles from two strains of the closely related Candida dubliniensis were sequenced. Results show independent length variation in two contiguous hexanucleotide repeats, one non-contiguous hexanucleotide repeat, and two non-contiguous trinucleotide repeats within a 120 bp coding region. A neighboring, non-repetitive 120 bp region showed no variation. The information density of sequence polymorphisms in this region provides a powerful tool for genotyping microorganisms in epidemiological studies, yielding detailed resolution of closely related strains, and clearly distinguishing the two species studied here. The individual length-variable repeat regions are very short (2-8 repeats), demonstrating that even very short microsatellites can show high levels of length variability when surrounded by similarly repetitive DNA. Extensive homoplasy was discovered among the C. albicans alleles, with the majority of overall length categories consisting of alleles with more than one sequence. Our results show that microsatellite length alone should not be used to assume either sequence identity or identity by descent. Microsatellite length mutations appear to have generated the high degree of both inter- and intraspecific polymorphism seen at the ERK1 locus, and form an island of variability in an otherwise well-conserved gene.

Use of polymorphic short and clustered coding-region microsatellites to distinguish strains of Candida albicans.

We describe the identification of polymorphic microsatellite loci in the pathogenic yeast, Candida albicans. A search for all coding-region microsatellites with more than four repeats that can be found in Candida sequences in GenBank was conducted. Nine such microsatellite sequences consisting of trinucleotide motifs were found. Three of these were perfect microsatellites while the remaining six sequences were found in one imperfect microsatellite and two compound microsatellites. Because of the close proximity of some of these repeats, all could be assayed with six PCR primer pairs. All of these microsatellite sequences were found in five nuclear genes, ZNF1, CCN1, CPH1, EFG1, and MNT2. Except for a single (CTT)5 serine tract, all coded for polyglutamine tracts. Another locus with seven alleles, a region of the ERK1 protein kinase gene, was also examined, and may be a representative of a new class of highly polymorphic "clustered' microsatellites. Such loci, in which several non-contiguous but closely linked microsatellites are clustered together, may be a useful source of DNA polymorphisms in microorganisms in which long microsatellite sequences are unavailable. All seven regions amplified were polymorphic, having between two and seven variable length alleles in the 11 strains of Candida albicans examined. The results of this and similar searches will facilitate epidemiological and evolutionary studies of Candida and other microorganisms.

Systemic reactions to influenza B vaccine.

Standard whole virus influenza vaccine (1974-1976) containing 700 chicken cell agglutinating (CCA) units of type A (Port Chalmers/1/75) or Port Chalmers plus Scotland/840/74) and 500 units of type B (HK/8/73) antigens was found to produce excessive systemic toxicity in adult volunteers. Using experimental monovalent A and B vaccines, most of the observed toxicity was shown to be associated with the B antigen. Injection of 500 CCA units or more of B vaccine was followed within 10-16 hours by malaise and chills in approximately one-third of vaccines. Chills, malaise, and local pain were more common in volunteers lacking prevaccination serum HI antibody than in those in whom this antibody was present. Toxicity was not related to the presence of endotoxin or bacterial contamination of vaccine; it appeared to be "intrinsic" to the viral antigen. The mechanism for the toxicity in man may be the same as the direct pyrogenic effect of influenza antigen for rabbits previously observed by others. Detoxification of the B antigen by prolonged exposure to formalin reduced the side effects of a 500 CCA unit dose to acceptable levels without impairing its antigenicity.

Further studies on the neonatal ferret model of infection and immunity to and attenuation of human parainfluenza viruses.

Earlier we reported on the susceptibility via the intranasal route of inoculation of newborn ferrets to infection by wild and serially egg passaged parainfluenza type 3 virus. The wild type consistently induced fatal infection whereas with serial egg passage, mortality rate decreased while the degree of infectivity remained the same. This attenuation was accompanied by a change in capacity to induce interferon and decreased growth in human and primate cell cultures. The present work reports, similarly, on the susceptibility of newborn ferrets to wild and serially egg passaged parainfluenza types 1 and 2. Wild type infections caused significantly greater mortality than did egg passaged materials, although infectivity rates were approximately the same. Wild and egg passaged strains produced silent infections in adult ferrets and induced specific circulating hemagglutinin inhibiting antibodies. Newborn offspring of dams inoculated with wild or egg passaged viruses resisted challenge with wild virus. The apparent attenuation of the wild type virus with serial egg passage viruses resisted challenge with wild virus. The apparent attenuation of the wild type virus with serial egg passage was correlated with alteration in cell culture host range and capacity to induce interferon. The low and mid level passages of types 1 and 3 were the subject of clinical vaccination trials in man in four age groups: 18-35 years, 14-16 years and 6-12 years. The clinical and serologic results of these studies will be reported elsewhere.