Differential effects of amino acid substitutions in the beta-sheet floor and alpha-2 helix of HLA-A2 on recognition by alloreactive viral peptide-specific cytotoxic T lymphocytes

Crystallographic studies of the HLA-A2 molecule have led to the assignment of a putative peptide binding site that consists of a groove with a beta-pleated sheet floor bordered by two alpha-helices. A CTL-defined variant of HLA-A2, termed HLA-A2.2F, differs from the common A2.1 molecule by three amino acids: a Leu to Trp substitution at position 156 in the alpha-2 helix, a Val to Leu substitution at position 95 in the beta-sheet floor of the groove, and a Gln to Arg substitution at position 43 in a loop outside of the groove. Another HLA-A2 variant, termed CLA, has a single Phe to Tyr substitution at position 9 that is sterically located adjacent to position 95 in the beta-sheet floor of the groove. We have determined which of the amino acid substitutions at positions 9, 43, 95, or 156 could individually affect recognition by panels of A2.1 allospecific and A2.1-restricted influenza viral matrix peptide-specific CTL lines, using a panel of site-directed mutants and CLA. Recognition by allospecific CTL lines was generally unaffected by any one of the amino acid substitutions, but was eliminated by the double substitution at positions 95 and 156. Allorecognition by some CTL lines was eliminated by a single substitution at position 9 or 95. In contrast, recognition by A2.1-restricted matrix peptide specific CTL was totally eliminated by a single substitution at position 9 or 156. The substitution at position 43 in a loop away from the peptide binding groove had no effect on allorecognition or matrix peptide recognition. These results indicate that amino acid residues in the floor or alpha-2 helical wall of the peptide binding groove of the HLA-A2 molecule can differentially affect allorecognition and viral peptide recognition.     

Cellulose and lignin degradation in forest soils: Response to moisture,temperature, and acidity

The concentration of lignin in plant tissue is a major factor controlling organic matter degradation rates in forest ecosystems. Microbial biomass and lignin and cellulose decomposition were measured for six weeks in forest soil microcosms in order to determine the influence of pH, moisture, and temperature on organic matter decomposition. Microbial biomass was determined by chloroform fumigation; lignin and cellulose decomposition were measured radiometrically. The experiment was designed as a Latin square with soils of pH of 4.5, 5.5, and 6.5 adjusted to 20, 40, or 60% moisture content, and incubated at temperatures of 4, 12, or 24°C. Microbial biomass and lignin and cellulose decomposition were not significantly affected by soil acidity. Microbial biomass was greater at higher soil moisture contents. Lignin and cellulose decomposition significantly increased at higher soil temperatures and moisture contents. Soil moisture was more important in affecting microbial biomass than either soil temperature or soil pH.     

Ethylene receptor expression is regulated during fruit ripening, flower senescence and abscission

Using theArabidopsis ethylene receptorETR1 as a probe, we have isolated a tomato homologue (tETR) from a ripening cDNA library. The predicted amino acid sequence is 70% identical toETR1 and homologous to a variety of bacterial two component response regulators over the histidine kinase domain. Sequencing of four separate cDNAs indicates that tETR lacks the carboxyl terminal response domain and is identical to that encoded by the tomatoNever ripe gene. Ribonuclease protection showed tETR mRNA was undetectable in unripe fruit or pre-senescent flowers, increased in abundance during the early stages of ripening, flower senescence, and in abscission zones, and was greatly reduced in fruit of ripening mutants deficient in ethylene synthesis or response. These results suggest that changes in ethylene sensitivity are mediated by modulation of receptor levels during development.     

15N resonance assignments of oxidized and reducedChromatium vinosum high-potential iron protein

The¹⁵N resonances in reduced and oxidizedChromatium vinosum high-potential iron protein have been assigned by use of¹H-¹H COSY spectra and¹H-¹⁵N HMQC, HMQC-COSY, and HMQC-NOESY spectra. Unambiguous assignment of 70 of 85 backbone¹⁵N resonances in the reduced protein and 62 of 85 resonances in the oxidized protein are made, as are 12 of 21 side-chain¹⁵N resonances.     

Common mutations in the phosphofructokinase-M gene in Ashkenazi Jewish patients with glycogenesis VII--and their population frequency.

Phosphofructokinase (PFK) catalyzes the rate-limiting step of glycolysis. Deficiency of the muscle enzyme is manifested by exercise intolerance and a compensated hemolytic anemia. Case reports of this autosomal recessive disease suggest a predominance in Ashkenazi Jews in the United States. We have explored the genetic basis for this illness in nine affected families and surveyed the normal Ashkenazi population for the mutations we have found. Genomic DNA was amplified using PCR, and denaturing gradient-gel electrophoresis was used to localize exons with possible mutations. The polymorphic exons were sequenced or digested with restriction enzymes. A previously described splicing mutation, delta 5, accounted for 11 (61%) of 18 abnormal alleles in the nine families. A single base deletion leading to a frameshift mutation in exon 22 (delta C-22) was found in six of seven alleles. A third mutation, resulting in a nonconservative amino acid substitution in exon 4, accounted for the remaining allele. Thus, three mutations could account for all illness in this group, and two mutations could account for 17 of 18 alleles. In screening 250 normal Ashkenazi individuals for all three mutations, we found only one delta 5 allele. Clinical data revealed no correlation between the particular mutations and symptoms, but male patients were more symptomatic than females, and only males had frank hemolysis and hyperuricemia. Because PFK deficiency in Ashkenazi Jews is caused by a limited number of mutations, screening genomic DNA from peripheral blood for the described mutations in this population should enable rapid diagnosis without muscle biopsy.     

Characterization of Inorganic Biocarriers That Moderate System Upsets during Fixed-Film Biotreatment Processes

Inorganic matrices were developed for fixed-film bioreactors affording protection to microorganisms and preventing loss of bioreactor productivity during system upsets. These biocarriers, designated Type-Z, contain ion-exchange properties and possess high porosity and a high level of surface area, which provide a suitable medium for microbial colonization. Viable cell populations of 10⁹/g were attainable, and scanning electron micrographs revealed extensive external colonization and moderate internal colonization with aerobic microorganisms. Laboratory-scale bioreactors were established with various biocarriers and colonized with Pseudomonas aeruginosa, and comparative studies were performed. The data indicated that bioreactors containing the Type-Z biocarriers were more proficient at removing phenol (1,000 ppm) than bioreactors established with Flexirings (plastic) and Celite R635 (diatomaceous earth) biocarriers. More significantly, these biocarriers were shown to moderate system upsets that affect operation of full-scale biotreatment processes. For example, subjecting the Type-Z bioreactor to an influent phenol feed at pH 2 for periods of 24 h did not decrease the effluent pH or reactor performance. In contrast, bioreactors containing either Celite or Flexirings demonstrated an effluent pH drop to ~2.5 and a reduction in reactor performance by 75 to 82%. The Celite reactor recovered after 5 days, whereas the bioreactors containing Flexirings did not recover. Similar advantages were noted during either nutrient or oxygen deprivation experiments as well as alkali and organic system shocks. The available data suggest that Type-Z biocarriers represent an immobilization medium that provides an amenable environment for microbial growth and has the potential for improving the reliability of fixed-film biotreatment processes.     

Antibody to herpes simplex virus type 2 as serological marker of sexual lifestyle in populations.

OBJECTIVES--To examine the epidemiology of antibody to herpes simplex virus type 2 and to assess its suitability as a serological marker of sexual behaviour in populations with high and low prevalences. DESIGN--Cross sectional survey. SETTING--Department of genitourinary medicine and blood donation centre in central London. SUBJECTS--Representative sample of 869 patients attending department between November 1990 and December 1991, and 1494 consecutive blood donors attending for donation between February and April 1992. METHOD--Participants had a blood sample taken for antibody testing with a novel type specific assay and completed a questionnaire. RESULTS--Prevalence of antibody differed significantly between the two groups (188/833 (22.7%) clinic attenders; 102/1347 (7.6%) blood donors). In both populations antibody was strongly associated with sex, sexual orientation, years of sexual activity, number of lifetime sexual partners, and past infection with sexually transmitted diseases after other factors were controlled for. Only 130 (45%) of all those with antibody had symptoms suggestive of genital herpes, and 79 (27.4%) had had genital herpes diagnosed. Of those without antibody to herpes simplex viruses type 1 and 2, 8.0% reported genital blisters or sores and 1.1% had had genital herpes diagnosed by a doctor. CONCLUSIONS--The strong relation between herpes simplex virus type 2 and sexual lifestyle suggests that the presence of antibody to the virus may be suitable for use as an objective, serological marker of patterns of sexual behaviour in different populations. These data show that only a minority of those infected with herpes simplex virus type 2 have a diagnosis of genital herpes or express clinical symptoms, making serological determinants of infection essential for epidemiological studies.     

Reclassification of Thiobacillus thyasiris as Thiomicrospira thyasirae comb. nov., an organism exhibiting pleomorphism in response to environmental conditions

The halotolerant, facultatively chemolithoautotrophic, gramnegative sulphur-oxidizing organism isolated from the gills of Thyasira flexuosa is shown to exhibit pleomorphism consistent with its being a member of the genus Thiomicrospira and not a Thiobacillus as originally classified. Its distinctive morphology, DNA base composition, and similarity of 16S rRNA sequences to that of Thiomicrospira sp. strain L-12 (which differs only in detail from the type species Thiomicrospira pelophila), lead us to reclassify this strain as Thiomicrospira thyasirae DSM5322.