Anaerobic Chemolithotrophic Growth of the Haloalkaliphilic Bacterium Strain MLMS-1 by Disproportionation of Monothioarsenate.

A novel chemolithotrophic metabolism based on a mixed arsenic-sulfur species has been discovered for the anaerobic deltaproteobacterium, strain MLMS-1, a haloalkaliphile isolated from Mono Lake, California, U.S. Strain MLMS-1 is the first reported obligate arsenate-respiring chemoautotroph which grows by coupling arsenate reduction to arsenite with the oxidation of sulfide to sulfate. In that pathway the formation of a mixed arsenic-sulfur species was reported. That species was assumed to be monothioarsenite ([H2As(III)S(-II)O2](-)), formed as an intermediate by abiotic reaction of arsenite with sulfide. We now report that this species is monothioarsenate ([HAs(V)S(-II)O3](2-)) as revealed by X-ray absorption spectroscopy. Monothioarsenate forms by abiotic reaction of arsenite with zerovalent sulfur. Monothioarsenate is kinetically stable under a wide range of pH and redox conditions. However, it was metabolized rapidly by strain MLMS-1 when incubated with arsenate. Incubations using monothioarsenate confirmed that strain MLMS-1 was able to grow (µ = 0.017 h(-1)) on this substrate via a disproportionation reaction by oxidizing the thio-group-sulfur (S(-II)) to zerovalent sulfur or sulfate while concurrently reducing the central arsenic atom (As(V)) to arsenite. Monothioarsenate disproportionation could be widespread in nature beyond the already studied arsenic and sulfide rich hot springs and soda lakes where it was discovered.

A kinetoplast DNA minicircle probe (B18) specific for the Leishmania subgenus Viannia.

This paper reports the development, sequence and specificity of probe B18, which hybridizes only to the kinetoplast deoxyribonucleic acid (kDNA) minicircle of Leishmania species of the subgenus Viannia. This probe was developed in 1985 and has been used extensively since, on whole kDNA, Leishmania dot-blots and kDNA minicircles amplified by the polymerase chain reaction.

Leishmania (Viannia) guyanensis: a new minicircle class exclusive to this specie isolated from a DNA cosmid library useful for taxonomic purposes.

A new minicircle class exclusive to this specie isolated from a DNA cosmid library useful for taxonomic purposes. Experimental Parasitology 94, 143-149. In this paper we describe a new minicircle class exclusive to Leishmania (Viannia) guyanensis. The minicircle class was obtained with the aid of a total DNA cosmid library. The library was screened with an EcoRI fragment isolated from L. (V.) guyanensis (M4147). After screening seven clones were selected which showed strong hybridisation. Clones were digested and hybridised with the same probe. After hybridisation only one clone containing the desired fragment was positive. The fragment sized around 1000 bp was subcloned into pBluescript for sequencing. Sequence analysis using the GCG programme showed no substantial homology with any sequences previously reported, apart from the expected homology with the conserved region of Leishmania kDNA sequences. The probe hybridised strongly only to L. (V.) guyanensis kDNA after medium stringency washing.

Kinetoplast minicircle DNA from Leishmania (Viannia) lainsoni.

To investigate the phenomenon that PCR of Leishmania (V.) lainsoni minicircles using primers B1 and B2 gives anomalous small-sized products, unlike all other members of the Leishmania Viannia subgenus, cloned kDNA minicircles from L. (Viannia) lainsoni were sequenced using fluorescent dye terminator reactions. The sequence of L. (V.) lainsoni where the primer B2 would be expected to bind, was different from the other members of the L. Viannia subgenus, matching in only 7 out of 19 bases with the sequence of L. (V.) braziliensis at this position. The sequence obtained from the cloned minicircles enabled the design of a new primer which, when combined with B1, allowed the amplification of full sized minicircles in L. (V.) lainsoni, but not other members of the L. Viannia subgenus. Comparison of the sequence obtained for Leishmania (V.) lainsoni with other Leishmania minicircle DNA confirms that Leishmania (V.) lainsoni is more similar to members of the L. Viannia subgenus than to other Leishmania, but is distinctly different.

Geographic distribution and clinical description of leishmaniasis cases in Peru.

Studies were conducted from 1986 through 1993 to further define the geographic distribution and relative importance of different species of Leishmania as a cause of leishmaniasis in Peru. Patients with a clinical diagnosis of cutaneous and/or mucosal or diffuse cutaneous leishmaniasis were enrolled at the Naval Medical Research Institute Detachment (NAMRID) Laboratory in Lima, the Tropical Disease Clinic at San Marcos University Daniel A. Carrión, the Central Military Hospital, and a Ministry of Health hospital in Cusco, Peru. Clinical features, lesion aspirates, and biopsy tissue were obtained from each patient. All specimens were collected and assayed separately, including multiple specimens from some of the same patients for Leishmania parasites by inoculating aliquots of either aspirates or biopsy tissue suspensions onto Senekji's blood agar medium. Stocks of Leishmania isolates were used to prepare promastigotes to produce extracts for identifying the Leishmania species by the cellulose acetate electrophoresis enzyme technique. A total of 351 isolates of Leishmania were obtained from 350 patients who were infected primarily in the low and high jungle of at least 15 different Departments of Peru. Of the 351 isolates, 79% were identified as L. (V.) braziliensis, 7% as L. (V.) guyanensis, 10% as L. (V.) peruviana, 2% as L. (V.) lainsoni, and 1.7% as L. (L.) amazonensis. The clinical form of disease varied depending on the species of Leishmania, with L. (V.) braziliensis being associated most frequently with cutaneous, mucosal ulcers and mixed cutaneous and mucosal disease, and L. (V) peruviana, L. (V.) guyanensis, L. (V.) lainsoni with cutaneous lesions. Leishmania (L.) amazonensis was isolated from six patients, three with cutaneous lesions, one with mucosal lesions, and two with diffuse cutaneous lesions. Among all of the leishmaniasis cases, males were affected more frequently, and cases occurred among patients less than 10 to more than 51 years of age. These data further defined the geographic distribution and the relative frequency of Leishmania species associated with different clinical forms of leishmaniasis in Peru.

Detection of Leishmania (Viannia) braziliensis complex in wild mammals from Colombian coffee plantations by PCR and DNA hybridization.

The small mammal fauna of coffee plantations in SW Colombia was surveyed to determine which of the species present were infected with parasites of the Leishmania (Viannia) braziliensis complex and might therefore act as reservoirs of human cutaneous leishmaniasis. Fifty animals of seven different species were captured. Tissue samples were taken from the ears of specimens from each of the seven species. Thirty three samples were analysed by polymerase chain reaction (PCR) using oligonucleotide primers directed against conserved regions of L. (V) braziliensis complex kinetoplast DNA. Three of the samples (two from mouse opossums Micoureus demerarae, and one from a pygmy rice at Microryzomys minutus) gave positive results based on PCR analysis. When the samples were subjected to DNA hybridization (dot blot) analysis using the B18 (L. (V.) braziliensis complex-specific) probe, a total of ten specimens belonging to six species (the opossums M. demerarae and Didelphis marsupilalis, the rodents Melanomys caliginosus, Mi. minutus and Rattus rattus, and a rabbit Sylvilagus brasiliensis) gave positive results, indicating that all these animals had flies of species occurring in the same habitat by allowing them to feed on infected animals.

Development of PCR assays to detect genetic variation amongst equine herpesvirus-1 isolates as an aid to epidemiological investigation.

A search for variable restriction sites has been carried out for equine herpesvirus-1 (EHV-1) in an attempt to develop markers which can be used to group epidemiologically related viruses into groups, and to learn more about the dynamics of EHV-1 disease. Crude viral DNA extracts of EHV-1, prepared by Hirt extraction, were digested with AluI, HaeIII, or RsaI, and Southern blotted following electrophoresis. DNA fingerprints, produced by probing the Southern blots with the EHV-1 EcoR1-I fragment, separated 56 isolates into 16 groups. The variable sites within the EcoR1-I fragment were mapped approximately using fragments from within EcoR1-I, and the precise location of the variable sites determined from the DNA sequence of this fragment. Oligonucleotide primers flanking the variable sites were synthesized, and used in PCR assays to detect variable fragments. The AluI variable fragment was found to result from the presence or absence of a single AluI site. In contrast, the variable bands seen with HaeIII and RsaI, resulted from variation in the copy number of two tandemly repeated sequences, one of which had not previously been recognized. In addition, HaeIII digests of EHV-1 isolates probed with the glycoprotein B (gB) gene of EHV-1 also separated isolates into two groups. The variable HaeIII site was mapped towards the 5'-end of the gB gene and a PCR assay established. The distribution of the variable AluI site within the EcoR1-I fragment and the HaeIII site within the gB gene were estimated on a large number of clinical isolates using PCR on unpurified viral tissue culture medium. Both sites had a good distribution and together with additional variable sites should provide the basis for the rapid DNA fingerprinting of EHV-1 isolates.

Structure of an interferon-alpha 2 gene expressed in the bovine conceptus early in gestation.

Genomic and cDNA clones for bovine trophoblast interferon (IFN) have been isolated by probing a bovine genomic library and a bovine embryonic (day-18 post coitus) cDNA library respectively with the ovine trophoblast IFN cDNA. The two DNA sequences were identical; sequence analysis demonstrated 80% identity between the amino acid sequence of bovine trophoblast IFN and ovine trophoblast IFN, and 70% identity with a previously identified bovine IFN-alpha 2. Southern blotting of bovine genomic DNA indicated the presence of a minimum of three trophoblast IFN genes. Primer extension analysis identified the transcription start site in the 5' flanking region of the bovine IFN gene. Computer-aided analysis of the 5' flanking sequence demonstrated a similarity with that of bovine IFN-alpha 2 and the existence of a possible viral induction sequence.

Sheep antiluteolytic interferon: cDNA sequence and analysis of mRNA levels.

A cloned cDNA has been isolated by probing a sheep blastocyst cDNA library using a synthetic oligonucleotide representing the N-terminal amino acid sequence of the antiluteolytic protein, ovine trophoblast protein-1. Sequence analysis of the cDNA confirms the 70% homology between the antiluteolysin and the interferon-alpha family of proteins; however, the sequence reported here differs at several points from previously reported amino acid and cDNA sequences for the antiluteolysin. In-vitro translation of day-16 poly(A)+ RNA indicated that antiluteolysin mRNA is a major constituent of total mRNA at this stage of blastocyst development, and Northern blotting confirmed that antiluteolysin mRNA production occurred between days 13 and 22 after oestrus. This is consistent with the stage at which embryonic extracts are antiluteolytic on administration in vivo. These and other data confirm that the ovine trophoblast antiluteolysin is an interferon, and suggest that at least five isoforms of this protein may exist.

Interferon sequence homology and receptor binding activity of ovine trophoblast antiluteolytic protein.

Sequencing of the 40 N-terminal amino acids of the blastocyst protein responsible for blocking corpus luteum regression in early pregnancy in sheep revealed a 37% homology with human alpha-interferon; 28% of the remaining amino acid changes were conservative. 125I-Labelled human alpha-interferon bound to membrane receptors from sheep uteri with an approximate Kd of 4 x 10(-11) M; binding was inhibited by unlabelled alpha-interferon or purified blastocyst antiluteolytic protein. The blastocyst antiluteolytic protein therefore closely resembles the interferon-alpha family of antiviral proteins.