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Plasmid transformation of Streptomyces venezuelae: modified procedures used to introduce the gene(s) for p-aminobenzoate synthase.

Sucrose was unsuitable as an osmotic stabilizer in buffer solutions and media used for transformation of Streptomyces venezuelae ISP5230. Its replacement with NaCl, together with other modifications in the procedure, allowed efficient formation and regeneration of protoplasts but did not support transformation of S. venezuelae ISP5230 by vectors pIJ41 and pIJ941. With pIJ702, transformants with a low plasmid-copy-number and altered growth characteristics were obtained. Both pIJ702 and pIJ941, but not pIJ41, transformed S. venezuelae 13s; when pIJ941 was used, the plasmid in 18 of 20 transformants contained a deletion in the region reported to code for replication and transfer. The modified plasmid transformed S. venezuelae ISP5230 efficiently and was used to introduce a fragment of DNA from the pab locus of the wild-type into a Cml-1 mutant of ISP5230 blocked in chloramphenicol formation. Transformants that overproduced p-aminobenzoic acid were obtained but they remained blocked in chloramphenicol production; thus, the cloned pab fragment did not contain genes able to complement the cml-1 mutation. The results also suggest that the Cml-1 phenotype is not due to a defective reaction common to the biosynthesis of p-aminobenzoic acid and chloramphenicol.

4-Aminobenzoic Acid

Studies of arboviruses in Southwestern Venezuela: I. Isolations of Venezuelan and Eastern Equine Encephalitis viruses from sentinel hamsters in the Catatumbo region.

The purpose of this report is to describe isolations of Venezuelan (VEE) and Eastern (EEE) Equine Encephalitis virus made in the lowland moist tropical forest of the Catatumbo region on the southwestern part of the State of Zulia, Venezuela. We have isolated four strains of EEEV from sentinel hamsters exposed at Caño Mocho and Madre Vieja sites in 1973 and 1974, and three strains of EEEV in Hacienda (Hda.) Las Nubes in 1975. Both viruses were recovered during silent interepidemic periods and we believe these viruses are maintained in this region in sylvatic conditions. The recovered virus strains were detected within 24 to 48 hours, both in SMB and Vero Cell monolayer cultures and the sentinel hamsters yielded virus infectivities up to 10(4) PFU ml. Our VEEV isolate (IVIC PAn 23645-47), recovered during the silent interepizodemic period had an elution profile on the hydroxylapatite column corresponding to that of a I-D (VEEV-3880) or a I-E (VEEV-63A216) 'enzootic' subtype. However, considering other in vitro criteria (KHI; HA pH 5.8-6.0; small plaque size in Vero monolayers with suitable overlay media), this later and other previous isolates had some very distinct properties of the 'epizootic' strains. Thus, the evidence suggests that in Venezuela the VEEV cycle in nature is maintained either by the so called 'enzootic' and/or 'epizootic' virus types, or the virus population of the isolates have particular in vitro properties which do not correlate to the virulence markers in vivo. We consider this important question must be further clarified, and in addition, the isolation of three strains of EEEV are reported; this is the first report of the presence of this virus in Venezuela. Although the EEEV isolates may be of the South American type, they must be considered as potentially dangerous in the case of outbreaks.

Animals

[Onychomyosis caused by Trichopyton soudanense. 1st isolation in Venezuela].

Trichophyton soudanense is a dermatophyte wich is of highest incidence in Africa. Cases of tinea produced by this fungus have been described in England, Switzerland, Germany, Australia, the United States and Brazil. This investigation reports the first isolation of this fungus in Venezuela, from a lesion of the nail in a female patient who has always resided in Venezuela. The identification of this fungus was based on macroscopic and microscopic morphological characteristics; biological properties, scare or no growth when the only nitrogen source in the culture medium is provided by potassium or ammonium nitrate, and the production of dark pigment when inoculated in Lowenstein-Jensen medium. Experimental innoculation in animals produced an endothrix parasitism of the hair. Reference is made ot the incidence of the most frequently occuring dermatophytes in Venezuela.

Adult

[Selenium content in the blood cattle from Venezuela. I. Central and Portugueste zones].

Whole blood concentrations of selenium were determined in 776 dairy cows and calves from 28 farms located in three States (Aragua, Carabobo and Guárico) of the central zone of Venezuela and in Portuguesa State. Selenium levels largely varied according to both the geographic distribution and animals breed. Cattle from Guárico State showed the lowest average concentration (0.21 ppm) and those from Portuguesa the highest one (0.67 ppm). A group of animals from Turén (a District of Portuguesa State) showed the highest average concentration (1.64 ppm, range 1.02 +/- to 3.24 ppm). Concentrations of this magnitude in animals under grazing conditions could be related to high levels of selenium either in the soil or in the pasture. It is concluded that such animals are localized inaseleniferous zone (Turén). It must be noted that Jaffé et al. (op. cit.) found high levels of selenium in sesame seeds (Sesamum indicum) from this area and in blood and urine samples of children from Villa Bruzual, a little town of Turén. The results of this wok significantly differ from those obtained in other countries (of Europe and North America), where animals are managed under conditions very different from those of the tropical areas like Venezuela. The role this factor is discussed. This is the first report on blood levels in large animals of economical importance in Venezuela.

Analysis of Variance

Chloramphenicol resistance in Streptomyces: cloning and characterization of a chloramphenicol hydrolase gene from Streptomyces venezuelae.

A 6.5 kb DNA fragment containing a chloramphenicol-resistance gene of Streptomyces venezuelae ISP5230 was cloned in Streptomyces lividans M252 using the high-copy-number plasmid vector pIJ702. The gene was located within a 2.4 kb KpnI-SstI fragment of the cloned DNA and encoded an enzyme (chloramphenicol hydrolase) that catalysed removal of the dichloroacetyl moiety from the antibiotic. The deacylated product, p-nitrophenylserinol, was metabolized to p-nitrobenzyl alcohol and other compounds by enzymes present in S. lividans M252. Examination of the genomic DNA from several sources using the cloned 6.5 kb SstI fragment from S. venezuelae ISP5230 as a probe showed a hybridizing region in the DNA from S. venezuelae 13s but none in the DNA from another chloramphenicol producer, Streptomyces phaeochromogenes NRRLB 3559. The resistance phenotype was not expressed when the 6.5 kb SstI fragment or a subfragment was subcloned behind the lac-promoter of plasmid pTZ18R in Escherichia coli.

Amidohydrolases

Sporulation of Streptomyces venezuelae in submerged cultures.

Shaken cultures of Streptomyces venezuelae ISP5230 in minimal medium with galactose and ammonium sulphate as carbon and nitrogen sources, respectively, showed extensive sporulation after 72 h incubation at 37 degrees C. The spores formed in these cultures resembled aerial spores in their characteristics. The ability of the spores to withstand lysozyme treatment was used to monitor the progress of sporulation in cultures and to determine the physiological requirements for sporulation. In media containing ammonium sulphate as the nitrogen source, galactose was the best of six carbon sources tested. With galactose S. venezuelae ISP5230 sporulated when supplied with any of several nitrogen sources; however, an excess of nitrogen source was inhibitory. In cultures containing galactose and ammonium sulphate, sporulation was suppressed by a peptone supplement. The onset of sporulation was accompanied by a drop in intracellular GTP content. When decoyinine, an inhibitor of GMP synthase, was added to a medium containing starch and ammonium sulphate, a slight increase in sporulation was seen after 2 d. The suppression of sporulation by peptone in liquid or agar cultures was not reversed by addition of decoyinine. A hypersporulating mutant of S. venezuelae ISP5230 was altered in its ability to assimilate sugars. In cultures containing glucose the mutant sporulated more profusely than did the wild-type and did not acidify the medium to the same extent. However, the suppressive effect of glucose on sporulation was not merely a secondary result of acid accumulation.

Adenosine

Zoonotic cutaneous leishmaniasis due to Leishmania (Viannia) braziliensis associated with domestic animals in Venezuela and Brazil.

After outbreaks of cutaneous leishmaniasis in Solano State, Venezuela, 5% of the population had parasitized ulcers while after similar outbreaks in Mesquita, Rio de Janeiro State, Brazil, 9% had the disease. In these foci children, including some under six years of age, were affected. There was no significant difference in the occurrence of the disease according to sex or type of employment. In Solano, 3% of dogs and 28% of donkeys had parasitized lesions, while in Mesquita these indices were 19.8% and 30.8% respectively. The parasite from man, dogs and equines was identified as Leishmania (Viannia) braziliensis, by zymodeme and serodeme characterization. In these foci there is evidence suggesting that leishmaniasis is a zoonosis, possibly with equines and dogs as reservoirs, although both a wild enzootic cycle and the role of man as a source of infection can not be ruled out. Transmission is assumed to occur peridomestically by sandfly vectors such as Lutzomyia panamensis in Venezuela and Lutzomyia intermedia in Brazil. Information about the origin of these foci suggests that infected equines may be an important factor in the dissemination of the parasite in a peridomestic situation where these sandflies are abundant.

Adolescent

Field evaluation of an exoantigen-containing Babesia vaccine in Venezuela.

Bovine babesiosis is endemic in Venezuela, causing significant losses in highly susceptible imported cattle. Current immunoprophylactic methods include the less desirable use of live parasites. Inactivated vaccines derived from exoantigen-containing supernatant fluids of in vitro Babesia bovis and B. bigemina cultures have been developed and constitute a major improvement in vaccine safety, stability and ease of handling. Vaccination trials conducted under field conditions provide the final evaluation of a culture-derived B. bovis-B. bigemina vaccine. During a 5-year period, approximately 8,000 cattle were vaccinated and 16 clinical trials carried out in 7 states of Venezuela. Clinical, serologic and parasitologic data were collected monthly from 10% of the animals over a 2-year period. Data were also collected from a similar number of nonvaccinated control cattle. Analysis of results from these trials demonstrated a reduction in the incidence of clinical disease among vaccinated animals and complete protection against mortality caused by babesiosis. Vaccine efficacy was measured calculating the incidence rates of disease and mortality among vaccinated and nonvaccinated cattle. Use of this inactivated vaccine offers the best combination of safety, potency and efficacy for the effective immunoprophylactic control of bovine babesiosis.

Animals

Cryptosporidiosis among patients with acquired immunodeficiency syndrome in Zulia State, Venezuela.

We studied the prevalence of Cryptosporidium in 29 patients with acquired immunodeficiency syndrome (AIDS) from Zulia State, Venezuela. They ranged in age from five months to 46 years. Two were children and 27 were adults, of which six were women. Of the 21 men, 66.6% reported homosexual behavior. Three stool samples from each patient were examined, and modified Ziehl-Neelsen carbolfuchsin staining of formalinether stool concentrates was used to identify Cryptosporidium oocysts. To detect the presence of other intestinal parasites, direct wet mounts and iron-hematoxylin-stained smears were examined. Cryptosporidium was found in 12 (41.3%) of the patients and was identified as a single parasitic infection in seven of the 12 patients (58.3%). Other pathogenic parasites encountered were Giardia lamblia (3 of 12, 25%), Entamoeba histolytica (1 of 12, 8.3%), Ascaris lumbricoides, Trichuris trichiura, and Strongyloides stercoralis (each 1 of 12, 8.3%). Blastocystis hominis, an organism with an uncertain taxonomic position and pathogenicity, was observed in three of 12 patients (25%). An inflammatory exudate was observed in 10 of 12 patients infected with Cryptosporidium. Most of the patients with this infection presented with chronic watery diarrhea and weight loss. Our results suggest that Cryptosporidium is very common in AIDS patients with diarrhea in Venezuela. However, the role of this parasite as an enteropathogen in these patients is uncertain.

AIDS-Related Opportunistic Infections

Brucella suis infections in collared peccaries in Venezuela.

A bacteriologic and serologic study was conducted on two ranches in the states of Apure and Guarico, Venezuela for brucellosis in collared peccaries (Tayassu tajacu). One hundred thirty-nine peccaries were necropsied and tissues were cultured. Forty-three isolations of Brucella suis biovar 1, were made from lymph nodes and spleens of 25 males and 18 females. Antibody to Brucella sp. was detected in sera from 122 animals by the rapid plate agglutination, standard tube agglutination, 2-mer-captoethanol, rivanol, complement fixation and card tests. Young animals had infection and reactor rates nearly as high as the older animals indicating most were infected at a relative early age. Results suggest that this species may transmit brucellosis when living with domestic animals. This is the first report of B. suis biovar 1 from collared peccaries in Venezuela.

Age Factors

IgM antibodies to native phenolic glycolipid-I in contacts of leprosy patients in Venezuela: epidemiological observations and a prospective study of the risk of leprosy.

In a randomized, double-blind vaccine trial in Venezuela, about 29,000 contacts of leprosy patients have been vaccinated with either a mixture of heat-killed Mycobacterium leprae and BCG or BCG alone, and are being re-surveyed annually to detect new cases of leprosy. All contacts had a serum sample collected at the time of entry into the trial, and 13,020 of these sera have been analyzed for antibodies to phenolic glycolipid-I (PGL-I). Antibody levels have been related to various characteristics of the contacts and to their risk of developing leprosy in the following 4 years. A strong association was found between PGL-I antibody level and the risk of developing leprosy, in spite of possible modification of the incidence rate induced by vaccination. Antibody levels were higher in females than in males, and declined progressively with age. Household contacts had higher levels than did non-household contacts, and levels were higher in individuals from the state in Venezuela which has the highest incidence of the disease. No substantial differences were found in antibody levels between contacts of multibacillary and paucibacillary patients, which may in part reflect the influence of treatment, and there was no clear association with the presence of BCG or lepromin scars or with skin-test responses to PPD and leprosy soluble antigen. The assay of antibodies to PGL-I seems unlikely to provide a sensitive or specific test for infection with M. leprae, and measuring PGL-I antibody levels as a screening procedure to identify those at high risk of developing leprosy is unlikely to be particularly useful in most leprosy control programs. Such assays may be useful for the epidemiological monitoring of changes in the intensity of infection with M. leprae in a community and for the study of carefully defined groups of contacts during some phases of control programs.

Adolescent

A genetic study of the malaria vector Anopheles nuneztovari from western Venezuela.

A photomap of larval polytene chromosomes of Anopheles nuneztovari (population B) from western Venezuela is presented. Samples of An. nuneztovari B taken from 4 study sites in the states of Barinas and Táchira comprise a single panmictic population, which is chromosomally identical to the cytotype identified by Kitzmiller et al. (1973) from western Venezuela and Colombia. The frequency of polymorphism 2La has increased significantly in the 16 years that have elapsed since the 1973 study. In addition, inversion 2La does not conform to Hardy-Weinberg equilibrium. These 2 changes are most likely due to environmental alterations which have resulted in the heterozygous condition becoming more locally adaptive.

Animals

Soft contact lens damage: a one-year study in Caracas, Venezuela.

We reviewed 424 requests for soft contact lens cleaning that had been sent to a service laboratory in Caracas, Venezuela in the course of a year. We believe that this sample represents a cross-section of problem lenses in a tropical country (Venezuela). Among the cases for which data were available, there were more extended wear lenses than daily wear lenses sent for cleaning. A majority of the lens wearers were females and/or over age 20. The wearers were experienced handling their lenses. A relatively high percentage of them (52 of 184; 28%) used homemade solutions (unpreserved saline, distilled water, tap water, and/or baby shampoo) for the care and cleaning of their lenses. Although the reasons for referral were similar to those reported from temperate zones, we found a much higher incidence of combined deposits (protein-lipid-inorganic) than reported in studies carried out in other countries. Fungal invasion seemed more prevalent in our area than in temperate zones. From our results, the need for better education of wearers is obvious. In addition, special measures for the control of fungal growth may be required locally. The high incidence of this type of growth may be partly related to tropical conditions that favor the dispersal of fungal spores.

Adolescent

[Program for the production of mollusks in Venezuela].

This paper discusses an integrated project in Venezuela, planned and designed to ascertain the best procedures required for production of large quantities of shellfish, including: selection and maintenance of pollution-free growing areas; the best harvesting methods; and the safest and most economical marketing procedures. The major problems involved in producing safe, wholesome and nutritious shellfish are primarily those of sanitation and adequate preservation of the foods until they finally reach the consumer. Therefore, it is essential that these products be produced and handled in a sanitary manner on a continuous basis. There are numerous codes of recommended handling practices for shellfish, most developed only after many technical conferences were organized to evaluate all of the available scientific evidence. Therefore, for producing safe shellfish in Venezuela, this publication recommends the adoption of these sanitary and handling practices, designed and tested by the faculty and staff personnel of Universidad Simón Bolívar.

Animals

Molecular cloning of the genes for anthranilate synthetase from Streptomyces venezuelae ISP 5230.

Fragments of genomic DNA from Streptomyces venezuelae ISP5230 were cloned in the Escherichia coli expression vector pTZ18R and the plasmids were used to transform E. coli JA194 (trpE). The transformants included a prototrophic strain containing a recombinant plasmid, pDQ181, with an approximately 6.8-kb insert. Subcloning located the trpE-complementing DNA in a 2.4-kb segment. Transformation of E. coli ED23 (lacking both trpE and trpG functions) with plasmids containing the 2.4-kb DNA segment gave prototrophic strains exhibiting both the ASI and ASII activities of anthranilate synthetase. The results indicated that trpE and trpG are clustered in S. venezuelae. Regions hybridizing to the pDQ181 insert were present in the genomic DNA of other streptomycetes.

Anthranilate Synthase

Purification, properties and immunological detection of a bromoperoxidase-catalase from Streptomyces venezuelae and from a chloramphenicol-nonproducing mutant.

A new bromoperoxidase-catalase was purified from the chloramphenicol-producing actinomycete Streptomyces venezuelae ISP 5230. The homogeneous enzyme showed brominating activity, catalase activity and a very low peroxidase activity. The spectral properties and pH dependence of the catalase activity showed similarities to conventional catalases. In contrast to other haem-bromoperoxidases, the bromoperoxidase-catalase was stable when treated with an ethanol/chloroform mixture. Gel filtration gave an estimated Mr of 127,000-136,000. SDS-PAGE showed a single band corresponding in mobility to a species with an Mr of 61,000. The pI was estimated to be 4.5. The bromoperoxidase-catalase was not present in active form in a mutant of S. venezuelae ISP 5230, blocked in the chlorination step of chloramphenicol biosynthesis. However, an inactive species of the enzyme was detected in crude extracts of the mutant by using antibodies. From these results it is concluded that this bromoperoxidase participates in the chlorination step during chloramphenicol biosynthesis.

Catalase

Defining the networks that connect RNase III and RNase J-mediated regulation of primary and specialized metabolism in Streptomyces venezuelae.

UNLABELLED: RNA metabolism involves coordinating RNA synthesis with RNA processing and degradation. Ribonucleases play fundamental roles within the cell, contributing to the cleavage, modification, and degradation of RNA molecules, with these actions ensuring appropriate gene regulation and cellular homeostasis. Here, we employed RNA sequencing to explore the impact of RNase III and RNase J on the transcriptome of Streptomyces venezuelae. Differential expression analysis comparing wild-type and RNase mutant strains at distinct developmental stages revealed significant changes in transcript abundance, particularly in pathways related to multicellular development, nutrient acquisition, and specialized metabolism. Both RNase mutants exhibited dysregulation of the BldD regulon, including altered expression of many cyclic-di-GMP-associated enzymes. We also observed precocious chloramphenicol production in these RNase mutants and found that in the RNase III mutant, this was associated with PhoP-mediated regulation. We further found that RNase III directly targeted members of the PhoP regulon, suggesting a link between RNA metabolism and a regulator that bridges primary and specialized metabolism. We connected RNase J function with translation through the observation that RNase J directly targets multiple ribosomal protein transcripts for degradation. These findings establish distinct but complementary roles for RNase III and RNase J in coordinating the gene expression dynamics critical for S. venezuelae development and specialized metabolism. IMPORTANCE: RNA processing and metabolism are mediated by ribonucleases and are fundamental processes in all cells. In the morphologically complex and metabolically sophisticated Streptomyces bacteria, RNase III and RNase J influence both development and metabolism through poorly understood mechanisms. Here, we show that both ribonucleases are required for the proper expression of the BldD developmental pathway and contribute to the control of chloramphenicol production, with an interesting connection to phosphate regulation for RNase III. Additionally, we show that both RNases have the potential to impact translation through distinct mechanisms and can function cooperatively in degrading specific transcripts. This study advances our understanding of RNases in Streptomyces biology by providing insight into distinct contributions made by these enzymes and the intriguing interplay between them.

Streptomyces