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D C Barker

Publications and source records attributed to D C Barker.

At least 37 records · Page 2Linked to original sources
PubMed1999

Polymerase chain reaction-based identification of New World Leishmania species complexes by specific kDNA probes.

Here we define a new approach for the detection and characterisation of Leishmania complexes by polymerase chain reaction (PCR) and specific hybridisation. The first step consists of PCR amplification of kDNA minicircles using general kinetoplastid primers, which generate a polymorphic multi-banding pattern for all Leishmania species and other Kinetoplastidae. The second step is the identification of the Leishmania species complexes by hybridisation of the PCR products with specific kDNA probes. Polymorphic PCR-products from a genetically diverse set of Leishmania species were analysed by electrophoresis and the banding patterns compared with multi-locus enzyme electrophoresis (MLEE) data. The banding patterns produced by Leishmania species were very heterogeneous, making kDNA-PCR useful for determining closely related strains and for fingerprinting individual strains. The degree of kDNA-PCR and MLEE polymorphism was compared using UPGMA dendrograms. Three complex-specific probes were generated from major PCR bands of reference stocks belonging to the Leishmania mexicana, Leishmania donovani and Leishmania braziliensis complexes, and hybridisation of these probes to membrane-bound PCR products could reliably identify the strain to a complex level. A combination of kDNA-PCR fingerprinting and hybridisation with kDNA probes was found to be useful for both sensitive detection and direct identification of Leishmania species complexes.

Animals↗
PubMed1999

The ATPase subunit 6 gene sequence predicts that RNA editing is conserved between lizard- and human-infecting Leishmania.

Here we investigate the similarities in the kinetoplastid RNA editing process between human- and lizard-infecting Leishmania species. We present the sequence of the maxicircle-encoded ATPase subunit 6 gene from L. (V.) panamensis, L. (L.) mexicana and L. (L.) donovani species of human-infecting Leishmania. These represent the first available sequences of this gene from Leishmania species other than the lizard-infecting L. tarentolae. The gene sequences are highly conserved, both over the edited and unedited parts of the gene, implying that the RNA editing process is likely to be highly conserved between Leishmania species. Indeed, the first editing domain is absolutely conserved in all three Leishmania species studied and L. tarentolae. A phylogeny based on part of the ATPase subunit 6 gene placed the lizard-infecting Leishmania within the monophyletic Leishmania genus, supporting previous data which suggest that lizard- and human-infecting Leishmania species are closely related.

Adenosine Triphosphatases↗
PubMed1999

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.

Animals↗
PubMed1999

Kala-azar in a high transmission focus: an ethnic and geographic dimension.

In 1994-1996, we studied a group of 58 game wardens stationed in an area known to be highly endemic for visceral leishmaniasis (kala-azar) for evidence of infection with Leishmania donovani. Leishmania DNA was detected by the polymerase chain reaction in the peripheral blood of cases of active kala-azar, former patients with visceral leishmaniasis, patients, and asymptomatic subjects. Using the cloned antigen rk39, antibodies were detected in 44.2% of the game wardens while leishmanin skin test result was positive in 77% of our sample. It was shown that certain tribes from northern Sudan were more likely to develop subclinical infections, while those of the Baria tribe from southern Sudan and those of the Nuba tribe from western Sudan were more likely to develop visceral leishmaniasis. Whether this is due to genetic factors or previous exposure to Leishmania parasites remains to be elucidated.

Animals↗
PubMed1998

Identification of Mexican Leishmania species by analysis of PCR amplified DNA.

Leishmania parasites isolated into culture from patients with LCL or DCL from four different Mexican states were characterised using polymerase chain reaction (PCR), hybridisation with specific probes, and isoenzymes. PCR of the parasites showed that 10 of 11 of those isolates were members of the mexicana complex. This was confirmed in seven cases by isoenzymes. Restriction enzyme digests of PCR products of Mexican isolates showed the isolates to be different from the L.(L.) mexicana reference strain BEL21. Two (C2 and AM) of the isolates were shown to be a possible mixed infection between mexicana and braziliensis complex members. With a second set of samples from different patients from Campeche state, PCR of 14 biopsies indicated the presence of braziliensis complex members in six of the samples. The results showed that most of our isolates of Leishmania which come from the states of Tabasco and Veracruz are members of the Leishmania mexicana complex, but they seem to be different from the L.(L.) mexicana BEL21 reference strain. By hybridisation most of the biopsies (seven out of 14) from Campeche belong to the L. braziliensis complex and two out of 14 to L. mexicana complex and three out of 14 hybridised with both complexes, and two biopsies were negative. In Campeche, which is very close to Tabasco state and has border with Guatemala, we found members of the L. mexicana and L. braziliensis complexes.

Animals↗
PubMed1998

Characterisation of Leishmania telomeres reveals unusual telomeric repeats and conserved telomere-associated sequence.

Characterisation of the telomeres of Leishmania is important for understanding many aspects of the parasitic life of this primitive protozoan and for the completion of the physical map and sequencing of the genome. After sequencing more than 300 telomere-derived clones from Leishmania braziliensis and Leishmania major, a conserved 100 bp sequence was identified immediately adjacent to the telomere at the chromosome end and was named LCTAS (Leishmania conserved telomere-associated sequence). The LCTAS contains two conserved sequence boxes, and is present in all Leishmania species studied. The organisation of the LCTAS in the telomeric region differs between L. braziliensis and L. major: in L. major the LCTASs are tandemly repeated, while in L. braziliensis the LCTAS is present as a single copy per end. Two additional TASs with 1.6 kb and 274 bp repeat structures, which are apparently different to LCTAS, were isolated and mapped onto a L. braziliensis 250 kb multicopy minichromosome and the L. major chromosome 1, respectively. An unusual feature in L. braziliensis is that the telomeric repeats are often comprised of a novel tandem repeat CCCTAACCCGTGGA. A 'slippage' mechanism for LCTAS formation is proposed in this study as an alternative way for the synthesis and maintenance of telomeres and subtelomere regions.

Animals↗
PubMed1998

Analysis of the genomic organisation of a small chromosome of Leishmania braziliensis M2903 reveals two genes encoding GTP-binding proteins, one of which belongs to a new G-protein family and is an antigen.

Leishmania braziliensis M2903 contains a highly amplified small chromosome. This work is aimed at resolving its structural organization and determining whether this unusual chromosome contains specific genes encoding proteins with important functions in disease pathology or drug resistance. Our results show that the M2903 250-kb small chromosome contains LD1 sequences and has an inverted repeat structure. The LD1 sequences and two cDNAs (cDNA2 and cDNA53) were mapped on a cosmid contig, and the two cDNAs and the corresponding genomic fragments from the small chromosome were sequenced. The gene encoding cDNA2 predicts a putative GTP-binding protein with homology to other GTP-binding proteins only in the G-1 domain region; however, four other conserved motifs can be recognized. Sequence similarity to cDNA53 is located in at least five chromosomes, and its small chromosome copy is a pseudogene. An open reading frame downstream of the cDNA53 pseudogene predicts another GTP-binding protein that belongs to a new G-protein family with an unusual conserved GTP-binding domain and a newly characterized conserved sequence motif. A portion of this GTP-binding protein gene was studied previously in L. aethiopica as a recombinant antigen that reacts with human antibodies.

Amino Acid Sequence↗
PubMed1998

Leishmania braziliensis: characterisation of a complex specific subtelomeric repeat sequence and its use in the detection of parasites.

A 1.6-kb tandem repeat sequence had previously been identified in the subtelomeric region of mini- and megabase chromosomes from Leishmania braziliensis. Southern hybridisation was used to demonstrate that the repeat is complex specific. The sequence was characterised in strains representing four species of the L. braziliensis complex. This data allowed an assessment of the evolutionary relationship of the four species. PCR primers targeted to the repeat amplify only DNA from species of the L. braziliensis complex. Titration assays indicate that a minimum of 50 fg of parasite DNA can be detected by PCR alone. Southern hybridisation increases the limit of detection to 5 fg. Interspecies variation in the repeat sequence enabled restriction enzyme digestion of PCR products to distinguish individual species within the L. braziliensis complex.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

The genomic fingerprinting of the coding region of the beta-tubulin gene in Leishmania identification.

We have demonstrated the polymorphism of the beta-tubulin gene region in Leishmania and its value in the identification of the parasite. In this work we have shown that the coding region of the gene has sufficient variation to accurately discriminate these parasites at the subgenus level. Nevertheless, intrasubgenus diversity, for particular restriction enzymes, was found in New World Leishmania belonging to the Leishmania subgenus. For instance, differences were found between mexicana and amazonensis strains. A unique pattern at the species level was found in particular species of both subgenera, e.g. L. (L.) major strain P and L. (L.) tropica belonging to the Leishmania subgenus, and L. (V.) panamensis strain LS94 from the Viannia subgenus. Particular endonucleases are diagnostic in Leishmania species discrimination as in the case of PvuII for the mexicana and amazonensis. This variation evidenced in the beta-tubulin gene region of Leishmania also occurred in other Kinetoplastida e.g. Trypanosoma cruzi, Leptomonas spp. and Crithidia spp. Moreover, these organisms showed a different genomic fingerprinting for the beta-tubulin gene among them and also Leishmania. Thus, the polymorphism of the coding region of the beta-tubulin gene can be used as a molecular marker for the identification of Leishmania.

Animals↗
PubMed1998

Cardiovascular and Valsalva responses during parabolic flight.

We investigated the integrated cardiovascular responses of 15 human subjects to the acute gravitational changes (micro- and hypergravity portions) of parabolic flight. Measurements were made with subjects quietly seated and while subjects performed controlled Valsalva maneuvers. During quiet, seated, parabolic flight, mean arterial pressure increased during the transition into microgravity but decreased as microgravity was sustained. The decrease in mean arterial pressure was accompanied by immediate reflexive increases in heart rate but by absent (or later-than-expected) reflexive increases in total vascular resistance. Mean arterial pressure responses in Valsalva phases IIl, III, and IV were accentuated in hypergravity relative to microgravity (P < 0.01, P < 0.01, and P < 0. 05, respectively), but accentuations differed qualitatively and quantitatively from those induced by a supine-to-seated postural change in 1 G. This study is the first systematic evaluation of temporal and Valsalva-related changes in cardiovascular parameters during parabolic flight. Results suggest that arterial baroreflex control of vascular resistance may be modified by alterations of cardiopulmonary, vestibular, and/or other receptor activity.

Adult↗
PubMed1998

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.

Adolescent↗
PubMed1997

Genomic DNA repeat from Leishmania (Viannia) braziliensis (Venezuelan strain) containing simple repeats and microsatellites.

In this paper the Leishmania (Viannia) braziliensis complex is defined as containing all species of the actual subgenus Viannia. Organisms of the L. (V) braziliensis complex are the causative agents of localized human cutaneous and mucocutaneous leishmaniasis in South America, much of Central America and some ares of North America. In our search for better species and subspecies diagnostic probes we focused our research on repetitive DNA, since it provides a greater number of target sites for hybridization. In this work we report the isolation and sequencing of a 1.8 kb DNA region, LbJ38, which is probably tandemly repeated or dispersed at least 4 times along one chromosome and is naturally present in L. (V) braziliensis genomic DNA. This region contains microsatellites and simple repeat DNA sequences and was isolated by screening a genomic DNA cosmid library with complex- and species-specific probes. No homology was found with other Leishmania microsatellite or repetitive DNA. The utility of this repetitive sequence and primers derived from it in the identification of L. (V) braziliensis is demonstrated. As far as we are aware, this is the first report of sequence characterized repetitive microsatellite and GC rich simple repeat DNA from the nuclear genome of New World Leishmania.

Animals↗
PubMed1997

Factors associated with the perception that debt influences physicians' specialty choices.

PURPOSE: To investigate the responses of individual physicians to educational debt. METHOD: Data on 5,175 physicians were taken from the 1991 Robert Wood Johnson Foundation Survey of Young Physicians, a nationally representative survey of physicians under age 45 who had had two to ten years of practice experience as of 1991. The physicians' overall perceptions about the extents to which debt had been an important determinant of specialty choice were explored using multivariate logistic regression analyses. RESULTS: Only 3.2% of the physicians indicated that debt had had a major influence on their specialty choices. About half (56%) of those who felt that debt had been a major influence indicated that they had foregone some training because of their debt levels. Controlling for debt level, the physicians who had had children during medical school and those whose parents had less education and lower incomes were more likely to say that debt had been an influence (p < .05). An examination of the specialties that the physicians reported having foregone because of debt indicated that these physicians had reacted to debt in different ways--some had chosen more specialized fields while others had chosen more generalized fields. CONCLUSION: While the overall effect of debt was small, some individuals were influenced by debt in a variety of ways. Paying attention to the effects of debt on this small population may improve training for some physicians and help better target programs that attempt to influence physicians by alleviating debt.

Adult↗
PubMed1997

Diagnosis of visceral leishmaniasis by the polymerase chain reaction using blood, bone marrow and lymph node samples from patients from the Sudan.

We have evaluated the sensitivity of the polymerase chain reaction (PCR) as a diagnostic tool for Leishmania donovani using blood, bone marrow and lymph node samples from Sudanese patients with a confirmed infection. Forty patients were diagnosed by microscopic examination of bone marrow or lymph node samples. The PCR was able to detect parasite DNA in 37 out of 40 blood samples. In bone marrow and lymph node samples, the PCR was able to detect parasite DNA in all 7 and 6 samples, respectively. We suggest that the PCR should be considered as a valuable and sensitive tool for the diagnosis of L. donovani infection. However, if PCR diagnosis is to supplement or even replace microscopic diagnosis in developing countries, a large number of patients with no apparent signs of infection and patients with other diseases have to be tested in order to evaluate its true potential.

Animals↗