GPR91 deficiency exacerbates allergic contact dermatitis while reducing arthritic disease in mice.

BACKGROUND: Succinate, in addition to its role as an intermediary of the citric acid cycle, acts as an alarmin, initiating and propagating danger signals resulting from tissue injury or inflammatory stimuli. The contribution of this immune sensing pathway to the development of allergic and inflammatory responses is unknown. METHODS: Ear thickness of wild-type (wt) and Sucnr1-deficient (Sucnr1-/- ) mice, sensitized and challenged with oxazolone, was used as a criterion to assess the relevance of SUCNR1/GPR91 expression mediating allergic contact dermatitis (ACD). Results obtained in this system were contrasted with data generated using passive cutaneous anaphylaxis, ovalbumin-induced asthma and arthritis models. RESULTS: We found augmented ACD reactions in Sucnr1-/- mice. This observation correlated with increased mast cell activation in vitro and in vivo. However, exacerbated mast cell activation in Sucnr1-/- mice did not contribute to the enhancement of asthma or arthritis and seemed to be due to alterations during mast cell development as augmented mast cell responses could be recapitulated in wt mast cells differentiated in the absence of succinate. CONCLUSIONS: A deficiency in succinate sensing during mast cell development confers these cells with a hyperactive phenotype. Such a phenomenon does not translate into exacerbation of asthma or mast cell-dependent arthritis. On the contrary, the fact that Sucnr1-/- mice developed reduced arthritic disease, using two different in vivo models, indicates that GPR91 antagonists may have therapeutic potential for the treatment of allergic and autoimmune diseases.

Systemic herpesvirus infection in an Azara's Night Monkey (Aotus azarae).

BACKGROUND: Intra- and inter-species transmission of Human herpesvirus type 1 were noticed. In the present study, the herpesviral infection of a 1-year-old Azara's Night Monkey (Aotus azarae) was investigated. METHODS: Immunohistochemistry and electron microscopy investigations were done. RESULTS: A fatal systemic herpesviral infection was demonstrated. CONCLUSION: The results reveal the susceptibility of Azara's Night Monkey to the Human herpesvirus type 1. Moreover, humans shedding herpes viral particles during the reactivation phase of the infection directly infect the Azara's Night Monkeys.

Peste des petits ruminants in Arabian wildlife.

Recurrence of peste des petits ruminants (PPR) was diagnosed in the United Arabian Emirates in several wild ruminants confirmed by morphological, immunohistochemical, serological and molecular findings. Phylogenetic analysis revealed that the virus strain belongs to lineage IV, which is different to some previously isolated PPR strains from the Arabian Peninsula. This study shows that wild ruminants may play an important epidemiological role as virus source for domestic small ruminants.

The phenylalanyl-tRNA synthetase specifically binds DNA.

The phenylalanyl-tRNA synthetase (FRS) from Thermus thermophilus is modularly composed of several different domains, some of which are not required for aminoacylation. In particular, the enzyme has the structural prerequisites for a DNA-binding protein. We demonstrate by gel retardation and competition experiments that the FRS specifically binds certain DNA sequences of the T. thermophilus genomic DNA. Although the implication of this finding is not yet understood, increasing evidence indicates an alternative function of this enzyme not related to aminoacylation. This might be a fundamental cellular process involved in cell proliferation which is related in bacteria and in humans.

DNA-binding of phenylalanyl-tRNA synthetase is accompanied by loop formation of the double-stranded DNA.

The phenylalanyl-tRNA synthetase (FRS) from Thermus thermophilus has previously been shown to bind DNA. We demonstrate that the "winged" helix-turn-helix motifs in the duplicate domains B5 are the relevant structural elements for this DNA-binding property. By altering particular amino acids in the "wing", the affinity of the FRS to DNA was significantly reduced. Based on experimental data, which indicate that the FRS prefers a certain DNA structure rather than a particular consensus sequence, we propose a novel loop model for the DNA-binding mode of the FRS. In our model we assume that two segments of the same DNA molecule are bound simultaneously by both B5 domains and are aligned in parallel, while the intervening DNA forms a loop. Due to the limited flexibility of the DNA, loop formation is only possible if the respective intervening DNA stretch exceeds a certain length. Several lines of evidence support this model. (1) We demonstrate by gel retardation assays that the DNA requires a minimal number of ca 80 base-pairs to be bound by the FRS. (2) In the presence of the FRS, DNA longer than ca 80 base-pairs has a significantly increased DNase I accessibility. This agrees well with its known preferential cleavage at positions where the minor grove is on the outside of looped-out DNA molecules. (3) The initial cleavage by DNase I of >80 bp long DNA occurs in the middle of the fragment. In a looped molecule this is the position with the highest accessibility to DNase I. The function of the FRS related to DNA binding is still unknown. Since the FRS exists in the nucleus of rapidly growing mammalian cells, and protein-induced DNA bending or looping contributes to several transcription, replication, and recombination systems in both prokaryotes and eukaryotes, it is likely that the FRS, in addition to its aminoacylation function, influences common cellular processes via DNA binding.

NMR spectroscopic evidence for Mn(2+)(Mg(2+)) binding to a precursor-tRNA microhelix near the potential RNase P cleavage site.

The binding of Mg(2+)/Mn(2+) to acceptor stem microhelices as minimal models for precursor-tRNA(Gly) is demonstrated by NMR spectroscopy. From the evaluation of COSY and NOESY spectra, binding sites for Mg(2+)/Mn(2+) can be inferred. In particular, one binding site exists near the ribose moiety of nucleotide -1 at the position of cleavage by RNase P. From comparison with a variant possessing a deoxynucleotide at this position, it is concluded that the 2'-OH group of this nucleotide is indispensable for coordinating the divalent metal ion. Hence, this catalytically important metal ion is "pre-bound" to the precursor-tRNA before complexation with RNase P.

Identification and characterization of the nifH and nifJ promoter regions located on the nif-plasmid pEA3 of Enterobacter agglomerans 333.

Small restriction fragments of the plasmid-borne Enterobacter agglomerans 333 nif region were cloned into a promoter probe plasmid as transcriptional fusions with the lacZ gene. Identification of NifA-dependent promoters was accomplished by using a compatible plasmid which constitutively expresses the Klebsiella pneumoniae nifA gene. beta-Galactosidase assays showed strong activation of the cloned E. agglomerans promoters in Escherichia coli by the heterologous K. pneumoniae nifA gene product. The positions of the promoter fragments on the corresponding restriction map were determined by Southern hybridization. As confirmed by sequencing data, the nifH and nifJ promoters are situated at opposite end-points of the nif gene group and their -24 to -12 nucleotide sequences are similar to the consensus sequence of NtrA-dependent promoters. Also, typical NifA-binding motifs are present in both promoters. The agreement of the promoter proximal regions of nifH and nifJ with the corresponding K pneumoniae sequences is about 80%. Also the upstream regions of these genes are in agreement to some extent.

Domains of phenylalanyl-tRNA synthetase from Thermus thermophilus required for aminoacylation.

The contribution of entire domains or particular amino acid residues of the phenylalanyl-tRNA synthetase (FRS) from Thermus thermophilus to the interaction with tRNA(Phe) was studied. Removal of domain 8 of the beta subunit resulted in drastic reduction of the dissociation constant of the FRS x tRNA(Phe) complex. Neither the removal of arginine 2 of the beta subunit, which makes the only major contact between domains beta1-5 and the tRNA, nor the replacement of the conserved proline 473 by glycine had an influence on the aminoacylation activity of the FRS. Thus, the body comprising domains 1-5 of the beta subunit may not be essential for efficient aminoacylation of tRNA(Phe) by the FRS and rather be involved in other functions.

Participation of the overproduced elongation factor Tu from Thermus thermophilus in protein biosynthesis of Escherichia coli.

The influence of the overproduced elongation factor Tu (EF-Tu) from Thermus thermophilus on the protein biosynthesis in Escherichia coli was investigated both in vivo and in vitro. A kirromycin-resistant E. coli strain became sensitive to this antibiotic upon the expression of the tuf A-gene of T. thermophilus present on a plasmid. In in vitro translation with components of the kirromycin-resistant E. coli strain the poly(Phe) synthesis stopped when minute amounts of the EF-Tu from T. thermophilus were added. Both results indicate the sensitivity of the T. thermophilus EF-Tu to kirromycin and its participation in the polypeptide synthesis of E. coli.

Site-directed mutagenesis of Thermus thermophilus EF-Tu: the substitution of threonine-62 by serine or alanine.

The invariant threonine-62, which occurs in the effector region of all GTP/GDP-binding regulatory proteins, was substituted via site-directed mutagenesis by alanine and serine in the elongation factor Tu from Thermus thermophilus. The altered proteins were overproduced in Escherichia coli, purified and characterized. The EF-Tu T62S variant had similar properties with respect to thermostability, aminoacyl-tRNA binding, GTPase activity and in vitro translation as the wild-type EF-Tu. In contrast, EF-Tu T62A is severely impaired in its ability to sustain polypeptide synthesis and has only very low intrinsic and ribosome-induced GTPase activity. The affinity of aminoacyl-tRNA to the EF-Tu T62A.GTP complex is almost 40 times lower as compared to the native EF-Tu.GTP. These observations are in agreement with the tertiary structure of EF-Tu.GTP, in which threonine-62 is interacting with the Mg2+ ion, gamma-phosphate of GTP and a water molecule, which is presumably involved in the GTP hydrolysis.

Sequence, overproduction and crystallization of aspartyl-tRNA synthetase from Thermus thermophilus. Implications for the structure of prokaryotic aspartyl-tRNA synthetases.

The genes of aspartyl-tRNA synthetase (AspRS) from two Thermus thermophilus strain VK-1 and HB8, have been cloned and sequenced. Their nucleotidic sequences code for the same protein which displays the three characteristic motifs of class II aminoacyl-tRNA synthetases. This enzyme shows 50% identity with Escherichia coli AspRS, over the totality of the chain (580 amino acids). A comparison with the eukaryotic yeast cytoplasmic AspRS indicates the presence in the prokaryotic AspRS of an extra domain between motifs 2 and 3 much larger than in the eukaryotic ones. When its gene is under the control of the tac promoter of the expression vector pKK223-3, the protein is efficiently overexpressed as a thermostable protein in E. coli. It can be further purified to homogeneity using a heat treatment followed by a single anion exchange chromatography. Single crystals of the pure protein, diffracting at least to 2.2 A resolution (space group P2(1)2(1)2(1), a = 61.4 A, b = 156.1 A, c = 177.3 A) are routinely obtained. The same crystals have previously been described as crystals of threonyl-tRNA synthetase [1].

Overproduction of the Thermus thermophilus elongation factor Tu in Escherichia coli.

The elongation factor Tu (EF-Tu) encoded by the tufl gene of the extreme thermophilic bacterium Thermus thermophilus HB8 was expressed under control of the tac promoter from the recombinant plasmid pEFTu-10 in Escherichia coli. Thermophilic EF-Tu-GDP, which amounts to as much as 35% of the cellular protein content, was separated from the E coli EF-Tu-GDP by thermal denaturation at 60 degrees C. The overproduced E coli-born T thermophilus EF-Tu was characterized by: i) recognition through T thermophilus anti-EF-Tu antibodies; ii) analysis of the peptides obtained by cyanogen bromide cleavage; iii) thermostability; iv) guanine nucleotide binding activity in the absence and the presence of elongation factor Ts; and v) ternary complex formation with phenylalanyl-tRNAPhe and GTP.

Organization of the Thermus thermophilus nusA/infB operon and overexpression of the infB gene in Escherichia coli.

The structural gene for translation initiation factor IF2 from Thermus thermophilus was identified on the basis of the N-terminal amino acid sequence of intact T thermophilus IF2 and an internal 25 kDa IF2 fragment. A total of 5135 bp was cloned and sequenced, comprising the open reading frames for p15A, NusA, p10A, IF2, p10B and SecD, which may form an operon. There are pronounced similarities between the operon arrangement and primary sequence of the T thermophilus genes and proteins, respectively, and their counterparts from other organisms. The T thermophilus infB gene was expressed to a high level in E coli. Four hundred milligrams of homogenous T thermophilus IF2 were prepared from 60 g of overproducing cells.

Pavlovian conditioning and multiple chemical sensitivity.

Pavlovian conditioning processes may contribute to some symptoms of multiple chemical sensitivity (MCS). This review summarizes the potential relevance of the literature on conditional taste and olfactory aversions, conditional sensitization, and conditional immunomodulation to understanding MCS. A conditioning-based perspective on MCS suggests novel research and treatment strategies.

Rice burning and asthma hospitalizations, Butte County, California, 1983-1992.

We investigated the association between rice burning and daily asthma hospitalizations in Butte County, California, from 1983 to 1992. Eighty-two percent of planted rice was burned, with a mean of 555 acres burned on days when burning was permitted. For 60% of the days during this period, no rice burning occurred. Peak burning occurred in fall and spring but was not correlated with criteria pollutants. Asthma admissions averaged 0.65/day and peaked in March. In the basic Poisson model with daily asthma hospitalizations as the outcome of interest, burn acreage showed a small but statistically significant elevation of risk for hospitalization per acre of rice burned [relative risk (RR) = 1.0001; 95% confidence interval (CI), 1. 00004-1.0002], after adjusting for maximum daily temperature, seasonal factors, and yearly population. In this model, burn acreage showed a dose-response effect as acreage burned increased. Days with the greatest acreage burned (>499 acres) had the largest risk of hospitalization (RR = 1.23; CI, 1.09-1.39), and days with moderate burning (between 100 and 499 acres) had a slightly lower risk of admission (RR = 1.2; CI, 1.05-1.37). Elevations of air pollutants were not associated with days of increased rice burning; however, rice burn acreage was shown to have a small but statistically significant effect on asthma morbidity in Butte County. This evidence suggests that further limitations on the daily amount of rice straw permitted to be burned should be considered to reduce pulmonary morbidity related to asthma.

Biological monitoring for mercury within a community with soil and fish contamination.

To assess the impact of elevated levels of inorganic mercury in soil and dust and organic mercury in fish, biological monitoring was conducted among Native Americans living next to an inactive mercury mine in Clear Lake, California. Of resident tribal members, 46% (n = 56) participated in biomonitoring. Urine mercury levels are equivalent to background, indicating that soil and dust exposures among study participants are not substantial. The average blood organic mercury level among study participants is 15.6 +/- 8.8 micrograms/l (n = 44), which is higher than levels reported by others among those who do not consume fish (2 micrograms/l). Consistent with results from other studies, a correlation between fish consumption and blood organic mercury is observed (p = 0.03). The margin between observed and established adverse effect levels for adults is examined for blood organic mercury and found to be less than 10-fold for 20% of the study population. Protective public health efforts for the study population and other similarly exposed populations, notably those who consume commercial fish products, are considered.

Genetic analysis of Leishmania mexicana populations from Texas, Latin America, and the Caribbean.

A genetic analysis using enzyme data of 72 Leishmania mexicana isolated from hosts in Texas, Latin America, and the Caribbean is presented. All isolates from each country were combined and considered as local populations. Allomorph (allele determined by electrophoresis) frequencies for 20 enzyme (loci) were calculated and 7 populations (Texas, Mexico, Belize, Guatemala, Ecuador [EC], Venezuela, and the Dominican Republic [DR]) were compared pairwise in the statistic of genetic identity (I) (level of genetic similarity). All populations were found to be genetically similar with a mean I value for all comparisons of 0.890 +/- 0.067. When DR was included as one of the pair compared, I = 0.811 +/- 0.034. Among comparisons that include EC (excluding EC vs. DR), I = 0.875 +/- 0.026. The mean I for the other comparisons was less than 0.9. The data indicate that the DR population is divergent enough from the others that it can be considered at the subspecies/incipient species level of evolutionary divergence; the EC population is, to a lesser extent, distinct from the others, and the other 5 represent geographic populations of 1 widely distributed species. A diagrammatic representation of the allomorphs among the 72 isolates is included. There were some allomorph/geographical (or local) population relationships noted.

Genetic similarity among Central and South American populations of Leishmania (Viannia) braziliensis.

Four populations of Leishmania (Viannia) braziliensis from Central America, Colombia, Peru and Brazil were analyzed and compared for up to 20 enzyme loci. Each of the 180 isolates could be identified as L. braziliensis using combined data from glucose phosphate isomerase and mannose phosphate isomerase. When the most common enzyme band was present at a frequency of < or = 0.95, the populations were polymorphic (more than a single allomorph for an enzyme) for more than 50% of the loci. Included were diagrammatic representations of the enzyme polymorphisms. Comparisons of levels of enzyme polymorphism and of genetic similarity among other Leishmania populations, L. tropica, L. major, L. mexicana, and L. donovani sensu lato, were discussed. The mean +/- SD level of genetic similarity among the four populations was 0.924 +/- 0.036 (range 0.878-0.966), which indicates that L. braziliensis is probably one reproductive population from Mexico in the north to Brazil and Peru in the south.

Biochemical characterization of Trypanosoma spp by isozyme electrophoresis.

Isozyme patterns of 13 enzymes were compared for cultures of Trypanosoma avium, T. vespertilionis, T cruzi and T. rangeli. The isozyme separation was made by cellulose acetate electrophoresis. Each of the species had distinctly migrating isozyme bands for glutamate oxaloacetate transaminase (GOT), isocitrate dehydrogenase (ICD), malic enzyme (ME), 6-phosphogluconate dehydrogenase (6PGD), phosphoglucoisomerase (PGI), phosphoglucomutase (PGM), and malic dehydrogenase (MDH). For other enzymes, two or more species had identically migrating bands. In addition to these interspecific species differences, variability was observed among the strains of T. cruzi and T. rangeli. Among the T. cruzi strains, there were two different isozyme (possibly allozyme) types of the enzymes alanine aminotransferase (ALAT), fructokinase (FK), glucose-6-phosphate dehydrogenase (G6PDH), GOT, MDH and three types of ME. In the T. rangeli isolates two isozyme types for the enzymes ALAT, FK, G6PDH, GOT, ICD, and LDH, were observed. Among the eight strains of T. cruzi studied there were six isozyme types, and among the seven T. rangeli isolates there were four isozyme types. There was an indication that isozyme types were associated with geographical distribution.

Characterization of Leishmania spp. by isozyme electrophoresis.

In this study, isozyme patterns for 14 different enzymes were compared for culture strains of Leishmania braziliensis, L. hertigi, L. mexicana, L. donovani, L. tropica, and L. adleri. The isozyme separation was made by means of cellulose acetate electrophoresis. Each of the species had distinct isozyme patterns for aspartate aminotransferase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and fructokinase. For other enzymes, two or more species had identically migrating bands; however, by using combinations of the other 10 enzymes it was possible to separate any one of the six species. In addition to these interspecific differences the Panama strains of L. braziliensis had two different malic dehydrogenase isozyme patterns; therefore, they fell into two distinct groups. These strains otherwise had identical isozyme patterns.