HLA genes in Uros from Titikaka Lake, Peru: origin and relationship with other Amerindians and worldwide populations.

Uros population from the Titikaka Lake live in about 42 floating reed ('totora') islands in front of Puno City (Peru) at a 4000 m high altiplano. They present both an mtDNA and a human leucocyte antigen (HLA) profile different from the surrounding populations: mtDNA A2 haplogroup is common to Uros and Amazon forest lowland Amerindians. HLA genetic distances between populations have been calculated and neighbour-joining dendrograms and correspondence analyses were carried out. Approximately 15 006 HLA chromosomes from worldwide populations have been used for comparisons. Only eight HLA-A alleles have been found, three of them accounting for most of the frequencies. The same phenomenon is seen for HLA-B, HLA-DRB1 and HLA-DQB1 alleles: a few alleles (3, 4 and 3, respectively) are present in most individuals. The presence of HLA-B*4801 and HLA-DRB1*0901 alleles in a relatively high frequency (although not the most frequent alleles found) is a characteristic shared with Asians and some populations from the Andean altiplano. Three specific Uros haplotypes have been found among the most frequent ones: HLA-A*680102-B*3505-DRB1*0403-DQB1*0302; HLA-A*2402-B*1504-DRB1*1402-DQB1*0301; and HLA-A*2402-B*4801-DRB1*0403-DQB1*0302. The present study suggests that Uros may have been one of the first populations from the shores of the Titikaka Lake coming from the Amazonian forest, which might have given rise to other later differentiated ethnic group (i.e. Aymaras). Uros HLA profile is also useful to study genetic epidemiology of diseases linked to HLA and to construct a future transplant waiting list by adding up regional lists in order to get a bigger pool for transplanting with better HLA matching.

The peopling of Madeira Archipelago (Portugal) according to HLA genes.

The Madeira-Porto Santo Archipelago was officially colonized in 1420 by Portuguese settlers. Its importance in Columbus' information for the American discovery and for slave traffic across the Atlantic is unquestionable. Thus, a complex peopling may have given rise to a present-day high admixture of ethnicities according to HLA genes. A sample of 173 healthy unrelated Madeirans was analysed and compared with 6986 HLA chromosomes from other worldwide populations. Genetic distances, neighbour-joining dendrograms and correspondence analyses were used for comparisons. Southern European, North African (including Canary Islands), Jewish and Mediterranean typical HLA alleles were found and genetic distances from Madeirans to these populations were the closest ones. In addition A*24-B*65-DRB1*0102-DQB1*0501 and A*68-B*08-DRB1*0301-DQB1*0201 haplotypes were newly found in Madeira and not found in any other population. Jewish-Armenian-Middle East haplotype (A*33-B*65-DRB1*0102-DQB1*0501) is one of the most common haplotypes; this haplotype is also present in Spaniards and North Africans. Quantitatively, Portuguese, North Africans (Algerians), Spaniards and Canary Islanders (in this order) are the most important parental populations to Madeirans. Results are discussed on the basis of the recorded historical peopling which does not show a noticeable African gene input in present-day Madeiran population according to our data; one of the closest related populations found is the Canary Islanders, suggesting that Guanche (Canary Islands first inhabitants) slaves gene flow is still noticed at present, both in Madeira and in Canary Islands populations.

A novel allele, HLA-G*010114, with a non-coding DNA base change in exon 2.

The non-classical human leucocyte antigen (HLA) class I locus, HLA-G, shows a low protein polymorphism and a more varied DNA (eight proteins and 28 alleles). HLA-G DNA polymorphism accounts mainly for changes at third codon bases of the protein coding exons; this does not imply amino acid change in most cases. This relatively high HLA-G DNA polymorphism in comparison with their protein polymorphism suggests that evolutionary forces are acting upon HLA-G for invariance. This may be related to the immunotolerogenic function postulated for HLA-G.

HLA-E polymorphism in Amerindians from Mexico (Mazatecans), Colombia (Wayu) and Chile (Mapuches): evolution of MHC-E gene.

Human leukocyte antigen (HLA)-E is a nonclassical class I (Ib) gene with a restricted polymorphism. Only eight DNA alleles and three proteins of this gene have been described and their frequencies analyzed in Caucasian, Oriental, Asian Indian, and Negroid populations. In the present study, HLA-E polymorphism has been analyzed in six Amerindian and Mestizo populations from North and South America and compared with previously described populations. HLA-E*0101 is the most frequent allele found in all populations except in Afrocolombian and Wayu Amerindians, in which blood group analyses show a high admixture with Caucasian and African populations. Mazatecan and Mapuche (two Amerindian groups from North and South America, respectively) presented similar HLA-E frequencies, whereas Wayu Indians are more similar to the Afrocolombian population. The Mexican and Colombian Mestizo show similar allele frequencies to Amerindians with high frequencies of HLA-E*0101 and HLA-E*010302 alleles. Also, frequencies in Negroids and Asian Indians present a similar distribution of HLA-E alleles. These data are in agreement with worldwide restricted polymorphism of HLA-E because no new allele was detected in the six populations studied. The allelic frequencies show differences among Caucasian, Oriental, Mestizo and Indian populations. Ape major histocompatibility complex-E allelism is also very restricted: common chimpanzee (one allele); bonobo (two alleles); gorilla (two alleles); orangutan (one allele); rhesus monkey (eight alleles); cynomolgus monkey (two alleles); and green monkey (two alleles).

HLA-G polymorphism and evolution.

Six proteins, one null allele and 22 human leukocyte antigen (HLA)-G alleles were found in humans. Bonobo, chimpanzee and gorilla only show one allele and orangutan shows five alleles. All Cercopithecus alleles show stop codons at position 164 (Macaca mulatta with seven DNA alleles, Macaca fascicularis with seven DNA alleles and Cercopithecus aethiops with three DNA alleles). Cotton-top tamarin New World monkeys showed 20 DNA and protein alleles; the major histocompatibility complex (MHC)-G New World sequences seem to be closer to MHC-E and lack typical MHC-G primates intron 2-specific deletion. This seems to suggest that MHC-G genes in New World primates are not orthologous and that their function may be similar to that of classical presenting MHC genes.

HLA Genes in Mayos Population from Northeast Mexico.

HLA class I and class II alleles have been studied in 60 unrelated people belonging to Mayos ethnic group, which lives in the Mexican Pacific Sinaloa State. Mayos HLA profile was compared to other Amerindians and worldwide populations' profile. A total of 14,896 chromosomes were used for comparisons. Genetic distances between populations, Neigbour-Joining dendrograms and correspondence analyses were performed to determine the genetic relationship among population. The new specific Mayo HLA haplotypes found are: HLA-A*02-B*35-DRB1*1406-DQB1*0301; HLA-A*02-B*48-DRB1*0404-DQB1*0302; HLA-A*24-B*51-DRB1*0407-DQB1*0302 and HLA-A*02-B*08-DRB1*0407-DQB1*0302. However, the typical Meso American HLADRB1*0407 represents a 40% of all DRB1 alleles. While common HLA characteristics are found in Amerindian distant ethnic groups, still new group specific HLA haplotypes are being found, suggesting that a common founder effect (i.e. high DRB1*0407) is noticed. Moreover, new HLA haplotypes are almost certainly appearing along time probably due to specific pathogen (?) selection for diversity. Mayo language is close to the Tarahumara one (another geographically close group); notwithstanding both groups are not genetically close according to our results, showing again the different evolution of genes and languages, which do not correlate. Finally, Sinaloa is one of the Mexican States in which more European genes are found. However, the results presented in this paper, where no European HLA genes are seen in Mayos, should have a bearing in establishing transplant programs and in HLA and disease studies.

DNA topoisomerase I from parasitic protozoa: a potential target for chemotherapy.

The growing occurrence of drug resistant strains of unicellular prokaryotic parasites, along with insecticide-resistant vectors, are the factors contributing to the increased prevalence of tropical diseases in underdeveloped and developing countries, where they are endemic. Malaria, cryptosporidiosis, African and American trypanosomiasis and leishmaniasis threaten human beings, both for the high mortality rates involved and the economic loss resulting from morbidity. Due to the fact that effective immunoprophylaxis is not available at present; preventive sanitary measures and pharmacological approaches are the only sources to control the undesirable effects of such diseases. Current anti-parasitic chemotherapy is expensive, has undesirable side effects or, in many patients, is only marginally effective. Under this point of view molecular biology techniques and drug discovery must walk together in order to find new targets for chemotherapy intervention. The identification of DNA topoisomerases as a promising drug target is based on the clinical success of camptothecin derivatives as anticancer agents. The recent detection of substantial differences between trypanosome and leishmania DNA topoisomerase IB with respect to their homologues in mammals has provided a new lead in the study of the structural determinants that can be effectively targeted. The present report is an up to date review of the new findings on type IB DNA topoisomerase in unicellular parasites and the role of these enzymes as targets for therapeutic agents.

Aumento de la incidencia de neumonía de posible origen bacteriano en el último trienio / Increased frequency of pneumonia of possible bacterial etiology during the last three years

Objetivo: Estudiar a los pacientes ingresados por neumonía en el último trienio, considerando sólo la neumonía definida previamente como típica o bacteriana. Métodos: Se estudiaron retrospectivamente todas las neumonías típicas adquiridas en la comunidad ingresadas durante los años 1998, 1999 y 2000. Se consideró como neumonía típica o de origen bacteriano aquella que cumplía 3 de 5 criterios: Fiebre >39 °C y de presentación brusca, dolor de costado o equivalentes (dolor abdominal o meningismo); auscultación de condensación, radiología de consolidación; leucocitosis >-12.000 mm3 con neutrofilia >-6.000 mm. Resultados: Se diagnosticaron un total de 271 neumonías, 75 en 1998, 85 en 1999 y 111 en 2000. La incidencia por 100.000 niños al año <5 años de nuestra Area Sanitaria era de 87,9, 98,6 y 126,3, respectivamente, y el porcentaje de ingresos por neumonía típica en relación con el número total de ingresos al año en la Unidad era de 5,8, 6,8 y 8 por ciento, respectivamente. Se realizó hemocultivo en 155 pacientes, que era positivo en 8 casos (5 por ciento): Streptococcus pneumoniae (n= 6), Haemophilus in fluenzae (n= 1) y Streptococcus pyogenes (n= 1): En la radiología, el 93,4 por ciento de los pacientes tenía una consolidación y el 6,6 por ciento presentaba un infiltrado. Se diagnóstico un derrame pleural en el 10 por ciento de los casos. Conclusiones: Existe un aumento de la frecuencia de niños ingresados en nuestro hospital por neumonía de características bacterianas en el último trienio. Consideramos que este aumento es debido, fundamentalmente, a neumonías causadas por neumococo (AU)

Changes in glutathione-redox balance induced by hexachlorocyclohexane and lindane in CHO-K1 cells.

1. The basal cytotoxic effect of the organochlorine pesticides hexachlorocyclohexane and lindane on CHO-K1 cultures was assessed at fractions of their lethal doses as determined by the neutral red incorporation (NRI) assay (NRI(6.25), NRI(12.5) and NRI(25)). The sulphur-redox cycle enzymes glutathione peroxidase, glutathione reductase and glutathione S-transferase, and total and oxidized glutathione were evaluated at several points during the standard growth curve of the cultures. 2. After incubation with each compound for 24 h, both glutathione peroxidase and reductase showed a substantial increase at the lowest exposure doses (NRI(6.25))--more significantly for lindane than for 1,2,3,4,5,6-hexachlorocyclohexane (HCH)--and dropped at higher doses of both compounds. The reduced and oxidized glutathione content was greatly diminished at the lower exposures, whereas the total glutathione content was higher at NRI(12.5) values. 3. Changes in cell membrane integrity were assessed for a wide range of pesticide concentrations with the lactate dehydrogenase release assay and lipid peroxidation. Membrane leakage and peroxide production were significantly enhanced at concentrations of HCH 50 microg ml(-1), although this effect was not significant at lindane concentrations < 200 microg ml(-1). 4. Lipid peroxidation increased with exposure to HCH at concentrations as low as NRI(6.25), whereas in the case of lindane, this increase was only significant at doses of NRI(25) and above.

Alterations of the glutathione-redox balance induced by metals in CHO-K1 cells.

The effects of cadmium (Cd(2+)), mercury (Hg(2+)), lead (Pb(2+)), copper (Cu(2+)) and nickel (Ni(2+)) on the glutathione (GSH)-redox cycle were assessed in CHO-K1 by the neutral red uptake inhibition (NR) assay (NR(6.25), NR(12.5) and NR(25)). Mercury proved to be the most and lead the least toxic of the metals tested. The effects on GSH content and intracellular specific activities of enzymes involved in the GSH-redox balance were measured after a 24-h exposure. Total GSH content increased significantly in cultures exposed to the lowest metal concentration assayed (NR(6.25)), but fell to below control values when exposed to concentrations equivalent to NR(25). Oxidised glutathione content dropped significantly at NR(6.25), while somewhat higher values were obtained for cultures exposed to higher doses. Glutathione peroxidase (Gpx) activities were 1.2-, 1.5-, 1.6-, 2.0- and 2.5-fold higher than untreated controls for cadmium, copper, mercury, nickel and lead, respectively, at concentrations equivalent to NR(6.25). Gpx activity declined at metal concentrations equivalent to NR(12.5) and NR(25). Glutathione reductase activity remained almost unchanged except at low doses of mercury, nickel and lead. Glutathione-S-transferase activity decreased at rising metal concentrations. The results suggest that a homeostatic defence mechanism was activated when cells were exposed to doses equivalent to NR(6.25) while the ability of the cells to respond weakened as the dose increased. A close relationship was also observed between metal cytotoxicity, total GSH content and the dissociation energy of the sulphur-metal bonds. These facts confirm the involvement of antioxidant defence mechanisms in the toxic action of these ions.

Determinación de residuos de siete insecticidas organofosforados en frutas mediante cromatografía de gases con detector de nitrógenofósforo y confirmación por espectrometría de masas / Residue analysis of seven organophosphorous insecticides in fruit by GC/NPD and confirmation by mass spectrometry

Se ha estudiado la presencia de residuos de siete insecticidas organofosforados en peras y manzanas adquiridas en la provincia de León. Se ha utilizado un sistema de extracción para matrices acuosas y determinación analítica mediante cromatografía de gases (GC) y detección con un detector de nitrógeno-fósforo (NPD). Las muestras positivas se confirmaron mediante cromatografía de gases y espectrometría de masas (GC/MS). Los insecticidas analizados mediante una técnica de extracción de multiresiduos fueron: diclorvós, diazinón, metilparatión, metil-pirimifós, paratión, malatión y fentión. Se han analizado 54 muestras obtenidas en la cesta de la compra de la ciudad de León, 28 manzanas (13 de la variedad Reineta y 15 de la variedad Golden) y 26 peras (15 de la variedad Conferencia y 11 de la variedad Blanquilla), de las que aparecieron 6 (11 por ciento) unidades (1 manzana de la variedad Reineta, 3 peras de la variedad Conferencia y 2 peras de la variedad Blanquilla) contaminadas con diazinón. Ninguna de las muestras sobrepasó el límite máximo de residuos (LMR) establecido para este compuesto (0.5 ppm) por el RD 280/94 en productos vegetales. Los datos de consumo medio por habitante de Castilla y León de peras y manzanas nos han permitido conocer la ingesta diaria estimada (IDE) del diazinón (rango 0,004 0,045 µg/kg/día), que al compararlo con la ingesta diaria admisible (IDA) (2 µg/kg/día), permite estimar un margen de seguridad comprendido entre 44 y 500 (AU)

Cyclodiene organochlorine insecticide-induced alterations in the sulfur-redox cycle in CHO-K1 cells.

The effect of the cyclodiene organochlorine pesticides aldrin, dieldrin and endosulfan was assessed on CHO-K1 cultures at fractions of their lethal doses, determined by the neutral red (NRI) incorporation assay (NRI6.25, NRI12.5 and NRI25). Glutathione peroxidase, reductase and S-transferase, and total and oxidised glutathione were evaluated along the standard growth curve of the cultures. After a 24-h incubation with each insecticide, glutathione peroxidase incurred a large increase, while glutathione reductase and S-transferase activities were slightly higher than untreated controls. Unlike oxidised glutathione, the content of total glutathione declined significantly after exposure to cyclodiene insecticides. Changes in cell membrane integrity were assessed by the lactate dehydrogenase (LDH) release assay and lipid peroxidation for a wide range of pesticide concentrations. Membrane leakage and peroxide production were significantly enhanced at concentrations of aldrin and as low as 12.5 microg/ml, whereas dieldrin and endosulfan increased membrane fragility at much higher concentrations.

Efecto del Salbutamol a dosis dopantes en los depósitos grasos de ratas entrenadas en condiciones aeróbicas / Effect of aerobic exercise and salbutamol treatment on fatty depots in wistar rats

El objeto de este trabajo ha sido probar si la reducción en los depósitos de grasa en ratas tratadas simultáneamente con salbutamol y condiciones de entrenamienot físico era debida al ejercicio o al tratamiento con agonistas adrenérgicos. Las ratas fueron tratadas con salbutamol a dos dosis distintas: terapéutica (16mg/kf peso corporal, dos veces al día), y de dopaje (3 mg/kg de peso corporal, dos veces al día), y los animales fueron entrenados siguiendo un protocolo aeróbico durante el experimento (90 días). algunos animales fueron tratados con propanolol (10mg/kg peso corporal, dos veces al día, 30 minutos antes del tratamiento con salbutamol), un Beta-antagonista no específico. Los niveles de grasa perirrenal decayeron sustancialmente sin camios en el peso corporal. Esta reducción en los depósitos de grasa ocurrió tanto por el entrenamiento como por el tratamiento con salbutamol, pero no fue revertido cuando se administró propanolol. La reducción en los depósitos de grasa en ratas fue una consecuencia del ejercicio sin implicación del sistema adrenérgico. De la misma manera, se observó una disminución significativa de los niveles plasmáticos de ácidos grasos y triglicéridos sanguíneos como consecuencia de la administración del salbutamol a las dosis de dopaje, que no pudo ser revertida por propranolol (AU)

Muscle and serum changes with salbutamol administration in aerobically exercised rats.

Treatment of experimental animals subjected to 90 days physical training programme plus repeated doses of salbutamol, a beta-adrenergic agonist, administered under two different regimes: therapeutic (16 microg/kg body weight, twice a day) and doping (3 mg/kg body weight, twice a day), caused a marked increase in size of skeletal (soleus, gastrocnemius and plantaris) leg muscles. Adrenergic involvement of salbutamol-linked hypertrophy was demonstrated by co-administration of the non-specific beta-adrenergic antagonist D,L-propranolol (10 mg/kg body weight twice a day). The salbutamol-induced muscle hypertrophy was associated with an early increase in creatine phosphokinase (CK) and its myocardial isozyme (CKmb), without significant changes in lactate dehydrogenase (LDH), alanine aminotransferase (AAT) and aspartate aminotransferase (DAT). The induction of muscle-injury biomarkers was completely abolished by co-administration of propranolol, thus suggesting the adrenergic involvement of these alterations.

Alterations on polyamine content and glutathione metabolism induced by different concentrations of paraquat in CHO-K1 cells.

The effect of herbicide paraquat has been assessed on CHO-K1 cultures at different concentrations. Glutathione peroxidase, reductase and S-transferase, as well as total and oxidized glutathione, were evaluated along the standard growth curve of the cultures. Paraquat was then administered during mid-log phase at concentrations that produced a calculated lethality of 6.25%, 12.5% and 25%, using the lysosomal dye assay, neutral red. After 24hr of incubation with paraquat, glutathione peroxidase suffered a large dose-response increase, unlike glutathione reductase and S-transferase, the activities of which were lower than untreated controls. The profile of total glutathione content was similar to that found for glutathione peroxidase, increasing with the administered doses of the herbicide. Polyamine content has been also studied at the same concentrations of paraquat, showing that intracellular spermidine and spermine pools were negatively affected with paraquat in a dose-response manner, unlike putrescine, which maintained elevated pools at the three concentrations assayed.

Early alterations of polyamine metabolism induced after acute administration of clenbuterol in mouse heart.

An acute treatment of mice with clenbuterol, a beta-adrenergic agonist, produced a marked increase of polyamines levels in heart, particularly during the early phase of administration of the drug. A single dose of 1.5 mg/kg caused as much as a 10 fold induction in activity of ornithine decarboxylase (ODC) and 3 to 4 fold increase in levels of putrescine, spermidine and spermine in mouse heart. Maximum changes were observed 3 to 4 hours post-administration of clenbuterol. This treatment did not produce any change in S-adenosylmethionine decarboxylase activity. The induction of cardiac ODC by clenbuterol was also dose dependent with a peak at about 5 micromol/kg. Co-administration of difluoromethylornithine, an irreversible inhibitor of ODC, or propranolol, a nonspecific beta-antagonist, with clenbuterol completely prevented the induction of ODC activity as well as the increase in polyamine levels in heart. However, pretreatment with alprenolol or metoprolol, the specific beta1 and beta2-antagonists, respectively, produced only partial prevention. The cardiac ODC from controls as well as clenbuterol treated mice exhibited similar affinity (Km) for its substrate, ornithine, while maximum enzyme activity (Vmax) was about 14 fold higher in clenbuterol treated mouse heart than in the control. Clenbuterol produced no change in the level of specific ODC mRNA or the protein, but the enzyme from the drug-treated mouse heart was considerably more stable than the control. Pretreatment of mice with either cycloheximide or actinomycin D followed by administration of clenbuterol could not prevent the induction in ODC activity suggesting that de novo biosynthesis of the enzyme protein or ODC mRNA was not responsible for induction of ODC activity. Post-translational changes in ODC may be responsible for an early increase of ODC activity due to clenbuterol treatment.

S-adenosylmethionine synthesis in Leishmania infantum promastigotes.

Methionine adenosyltransferase (MAT), S -adenosylmethionine (AdoMet), and S -adenosylhomocysteine (AdoHcy), have been analysed at different time-points during the growth curve of Leishmania infantum. MAT activity and AdoMet content peaked in the lag and early log phases, whereas higher levels of AdoHcy were found in stationary phase cells. MAT activity of cell extracts displayed hyperbolic kinetics for both its substrates, l -methionine and ATP, with km values of 35 microm and 5 m m, respectively. MAT has an absolute requirement for divalent cations, and is dependent on sulfydryl protective agents. Unlike other sources, L. infantum MAT activity seems to be transcriptionally regulated, with an accumulation of MAT-mRNA during rapid growth periods of promastigotes.

Plasma and muscle polyamine levels in aerobically exercised rats treated with salbutamol.

The induction of hypertrophy of cardiac and skeletal muscles has been studied after treatment with two different salbutamol dosages, therapeutic and doping. Treatment of rats subjected to a physical training schedule with repeated doses (16 microg kg(-1) per day or 3 mg kg(-1) per day) of salbutamol, a specific beta-adrenergic agonist, induced a marked increase in both skeletal and heart-muscle weight, whereas total body weight did not change significantly. Adrenergic involvement of salbutamol-linked muscle hypertrophy was demonstrated by co-administration of the non-specific beta-adrenergic antagonist, propranolol (20 mg kg(-1) per day). Salbutamol-induced muscle hypertrophy was associated with an increase in serum, skeletal-muscle and heart levels of the naturally occurring polyamines putrescine, spermidine and spermine. These observations suggest the involvement of polyamines in muscle hypertrophy and the possible role of blood polyamines as exposure biomarkers in beta-adrenergic-muscle hypertrophy.