RESUMO
Various studies indicate a relationship between increased oxidative stress and hypertension, resulting in increased DNA damage and consequent excretion of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). The aim of this study was to compare urinary 8-oxodG levels in African and Caucasian men and to investigate the association between ambulatory blood pressure (BP) and pulse pressure (PP) with 8-oxodG in these groups. We included 98 African and 92 Caucasian men in the study and determined their ambulatory BP and PP. Biochemical analyses included, urinary 8-oxodG, reactive oxygen species (ROS) (measured as serum peroxides), ferric reducing antioxidant power (FRAP), total glutathione (GSH), glutathione peroxidase (GPx) and glutathione reductase (GR) activity. The African men had significantly higher systolic (SBP) and diastolic blood pressure (DBP) (both p < 0.001). Assessment of the oxidative stress markers indicated significantly lower 8-oxodG levels (p < 0.001) in the African group. The African men also had significantly higher ROS (p = 0.002) with concomitant lower FRAP (p < 0.001), while their GSH levels (p = 0.013) and GR activity (p < 0.001) were significantly higher. Single and partial regression analyses indicated a negative association between urinary 8-oxodG levels with SBP, DBP and PP only in African men. These associations were confirmed in multiple regression analyses (SBP: R(2) = 0.41; ß = -0.25; p = 0.002, DBP: R(2) = 0.30; ß = -0.21; p = 0.022, PP: R(2) = 0.30; ß = -0.19; p = 0.03). Our results revealed significantly lower urinary 8-oxodG in African men, accompanied by a negative association with BP and PP. We propose that this may indicate a dose-response relationship in which increased oxidative stress may play a central role in the up-regulation of antioxidant defence and DNA repair mechanisms.
Assuntos
População Negra/estatística & dados numéricos , Monitorização Ambulatorial da Pressão Arterial , Desoxiguanosina/análogos & derivados , Espécies Reativas de Oxigênio/metabolismo , População Branca/estatística & dados numéricos , 8-Hidroxi-2'-Desoxiguanosina , Adulto , Desoxiguanosina/urina , Exercício Físico/fisiologia , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Methylation of DNA in eukaryotic cells, global as well as gene-specific, is affected by endogenous and endogenous factors. In this paper, it is reported that deviations in DNA methylation and expression of genes involved in DNA repair and the cell cycle are affected in 143B cultured cells containing an expression vector. Global DNA methylation analysis with cytosine-extension assay revealed a decreased global DNA methylation in the presence of the expression vector. Less promoter-specific methylation, as measured by bisulfite-MS PCR, was observed for MGMT and p16INK4a in vector-containing cells. Comet assay investigations revealed a negative effect on the DNA repair capacity of both BER and NER in Complex III compromised cells. This was reflected in the down-regulation of hOGG1 and ERCC1 expression. The results presented in this paper support the existence of a strong relationship between impaired mitochondrial function and deviations in DNA methylation and extend this relationship to impaired DNA repair.
Assuntos
Metilação de DNA , Reparo do DNA , Células Eucarióticas/metabolismo , Linhagem Celular , Inibidor p16 de Quinase Dependente de Ciclina/genética , Metilases de Modificação do DNA/genética , Epigênese Genética , Regulação da Expressão Gênica , Regiões Promotoras GenéticasRESUMO
The autosomal recessive disorder, hereditary tyrosinemia type 1 (HT1), is caused by a defective fumarylacetoacetate hydrolase enzyme. Consequently intermediate metabolites such as fumarylacetoacetate, succinylacetone and p-hydroxyphenylpyruvic acid accumulate. Characteristic to HT1 is the development of hepatocellular carcinoma, irrespective of dietary intervention or pharmacological treatment. Carcinogenesis may occur through a chromosomal instability mutator phenotype or a microsatellite instability phenotype, and deficient DNA repair may be a contributing factor thereof. The purpose of this study was to investigate the expression of DNA repair proteins, and the possible occurrence of microsatellite instability in HT1. Gene expression analyses show low expression of hOGG1 and ERCC1 in HT1 patient lymphocytes. Results from microsatellite instability analyses show allelic imbalance on chromosome 7 of the fah(-/-) mouse genome, and instability of the D2S123, D5S346 and (possibly) D17S250 microsatellite markers, in HT1 patient lymphocytes.
Assuntos
Reparo do DNA/genética , Instabilidade Genômica/genética , Instabilidade de Microssatélites , Tirosinemias/genética , Animais , Pré-Escolar , DNA Glicosilases/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Lactente , Linfócitos/metabolismo , CamundongosRESUMO
Hereditary tyrosinemia type 1 is an autosomal recessive metabolic disorder, which is caused by a defective fumarylacetoacetate hydrolase enzyme, and consequently metabolites such as succinylacetone and p-hydroxyphenylpyruvate accumulate. We used a modified comet assay to determine the effect of these metabolites on base- and nucleotide excision repair pathways. Our results indicate that the metabolites affected the repair mechanisms differently, since the metabolites had a bigger detrimental effect on BER than on NER.
Assuntos
Reparo do DNA , Heptanoatos/metabolismo , Ácidos Fenilpirúvicos/metabolismo , Tirosinemias/genética , Tirosinemias/metabolismo , Linhagem Celular , Ensaio Cometa , Humanos , Peróxido de Hidrogênio/toxicidadeRESUMO
We investigated the protective effect of pravastatin, simvastatin and atorvastatin on striatal DNA damage as a potential method to conserve and protect the nigrostriatal neurons. C57Bl/J6 mice were treated with combinations of a statin and the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). DNA damage, DNA sensitivity to H(2)O(2)in vitro, and DNA repair capacity was measured using the single cell gel electrophoresis (comet) assay. MPTP treatment increased DNA damage in the striatum. Contrary to expectation, the statins studied here did not protect DNA against H(2)O(2) induced damage but did in fact cause DNA damage. Treatment with simvastatin showed a significant increase in levels of DNA damage (p < or = 0.0018) and DNA damage induced in vitro with H(2)O(2) was significantly increased by pravastatin (p < or = 0.0001). DNA repair and repair capacity was slightly increased by simvastatin, significantly increased by pravastatin (p=0.0093), but slightly decreased by atorvastatin. In the MPTP treated groups, pre-treated with statins, pravastatin (p < or =0.0036) and simvastatin (p < or = 0.021), increased the level of DNA damage, while atorvastatin did not exhibit a significant effect. All three statins, administered prior to MPTP, offered slight protection against H(2)O(2) induced DNA damage and simvastatin and atorvastatin decreased the DNA repair capacity of the cells insignificantly. Treatment with statins, under the experimental conditions used, increased baseline levels of DNA damage, but DNA repair processes were left intact because the amount of repair also increased. Oxidative damage, however, largely exceeded the extent of DNA repair of mice treated with both the statins and MPTP.
Assuntos
Dano ao DNA/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Intoxicação por MPTP/prevenção & controle , Neostriado/efeitos dos fármacos , Fármacos Neuroprotetores , Animais , Ensaio Cometa , Reparo do DNA/efeitos dos fármacos , Intoxicação por MPTP/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacosRESUMO
Petrol attendants are exposed to petrol volatile organic compounds (VOCs) which may have genotoxic and carcinogenic effects. The single-cell gel electrophoresis assay (comet assay) is a method highly sensitive to DNA damage induced by environmental and occupational exposure to carcinogenic and mutagenic agents. The aim of this study was to evaluate the level of exposure of petrol attendants to petrol VOCs and also to determine their effect on DNA damage and repair in lymphocytes of African petrol attendants. The exposed group consisted of 20 subjects, randomly selected from three petrol stations. A control group of 20 unexposed subjects was also chosen and matched for age and smoking habits with the exposed group. Sorbent tubes were used to assess personal exposure of petrol attendants. The comet assay was used to investigate the basal DNA damage and repair capacity in isolated lymphocytes of petrol attendants and unexposed subjects. Blood samples were taken from the petrol attendants at the end of their 8-h working shift and also from the unexposed subjects. The petrol attendants were found to be exposed to levels of petrol VOCs lower than the South African occupational exposure limit for constituent chemicals. A significant relationship was found between the volume of petrol sold during the shift and the average concentrations of benzene, toluene and the total VOCs measured. However, relative humidity had a negative correlation with the average concentrations of benzene, toluene, xylene and the total VOCs. Significantly higher basal DNA damage was observed with the exposed group compared to the unexposed group. The period of exposure influenced the level of DNA damage and the calculated repair capacity. Smoking and age had a significant influence on the level of DNA damage. DNA repair capacity was delayed in smokers of both exposed and unexposed group.
Assuntos
Poluentes Ocupacionais do Ar/toxicidade , Dano ao DNA , Gasolina/toxicidade , Linfócitos/efeitos dos fármacos , Exposição Ocupacional/efeitos adversos , Adulto , Poluentes Ocupacionais do Ar/análise , Estudos de Casos e Controles , Ensaio Cometa/métodos , Reparo do DNA , Monitoramento Ambiental/métodos , Humanos , Masculino , Exposição Ocupacional/análise , Projetos Piloto , África do SulRESUMO
BACKGROUND: Genetic variation in the beta2 (ADRB2) and beta3 (ADRB3) adrenergic receptor genes are associated with obesity and insulin resistance. To further elucidate the role of these genes in the pathophysiology of obesity the present study investigated associations between certain polymorphisms in ADRB2 and ADRB3 and parameters of carbohydrate and lipid metabolism in a population of African origin. MATERIAL AND METHODS: Data of 102 black South African women obtained in the POWIRS (Profile of Obese Women with the Insulin Resistance Syndrome) study were used. Endpoint measurements included several anthropometric variables, resting blood pressure, plasma glucose, insulin, free fatty acids (FFA), ghrelin, leptin and lipids, and insulin resistance as estimated by the homeostasis model assessment (HOMA-IR) index. Polymorphisms were analyzed via PCR based methods. RESULTS: The percentage body fat was significantly lower (p< or =0.05) and the FFA significantly higher (p< or =0.05) in lean subjects (BMI< or =25 kg/m2) with the Glu27 variant allele compared to subjects with the Gln27 wildtype allele of the ADRB2 gene. In contrast, the variant allele of the ADRB2 gene was significantly positive associated (p< or =0.05) with the HOMA-IR-index in overweight black African women (BMI>25 kg/m2). No significant differences in parameters of the metabolic syndrome were apparent between subjects with the wildtype and variant alleles in the ADRB3 gene. CONCLUSION: The presence of the Glu27 and Arg64 polymorphisms of the ADRB2 and ADRB3 genes are not directly related to indices of the metabolic syndrome.
Assuntos
Síndrome Metabólica/genética , Polimorfismo Genético , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 3/genética , Tecido Adiposo/anatomia & histologia , Adulto , População Negra , Pressão Sanguínea , Índice de Massa Corporal , Ácidos Graxos não Esterificados/sangue , Feminino , Humanos , Leptina/sangue , MagrezaRESUMO
We present the first VP2-gene phylogenetic analysis of African horsesickness (AHS) viruses within a serotype. Thirteen AHSV 7 isolates were obtained from cases that occurred in South Africa during 1998-1999, and three were historical AHSV 7 isolates. The goals were to start a database of isolates of known location and time of isolation and to determine if we could identify the origin of an AHS outbreak in the surveillance area in the Western Cape. We prepared full-length cDNA copies of the VP2-genes of the isolates. Nucleic acid sequence data of a 786 bp region was used to characterize the genetic relationships between the isolates. The nucleic acid identities between the isolates ranged from 95.5 to 100%. Isolates from common geographical regions grouped together. Characterization of field isolates revealed the presence of two AHSV 7 lineages in South Africa during this period. The grouping of the viruses into two clades accurately reflected the geographical groupings of the isolates. The average nucleic acid divergence between the clades was 4.3%. Within the clades the divergence was 0.5 and 0.1%, respectively. The data suggests that the AHS outbreak in the Western Cape could have been an incursion from the Kwazulu Natal Province.
Assuntos
Vírus da Doença Equina Africana/classificação , Doença Equina Africana/epidemiologia , Proteínas do Capsídeo/genética , Surtos de Doenças , Filogenia , Doença Equina Africana/virologia , Vírus da Doença Equina Africana/genética , Animais , Doenças dos Cavalos/epidemiologia , Doenças dos Cavalos/virologia , Cavalos , Epidemiologia Molecular , Polimorfismo de Fragmento de Restrição , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Sorotipagem , África do Sul/epidemiologiaRESUMO
The outer capsid protein VP2 of African horsesickness virus (AHSV) is a major protective antigen. We have cloned full-length VP2 genes from the reference strains of each of the nine AHSV serotypes. Baculovirus recombinants expressing the cloned VP2 genes of serotypes 1, 2, 4, 6, 7 and 8 were constructed, confirming that they all have full open reading frames. This work completes the cloning and expression of the first full set of AHSV VP2 genes. The clones of VP2 genes of serotypes 1, 2, 5, 7 and 8 were sequenced and their amino acid sequences were deduced. Our sequencing data, together with that of the published VP2 genes of serotypes 3, 4, 6 and 9, were used to generate the first complete sequence analysis of all the (sero)types for a species of the Orbivirus genus. Multiple alignment of the VP2 protein sequences showed that homology between all nine AHSV serotypes varied between 47.6 % and 71.4 %, indicating that VP2 is the most variable AHSV protein. Phylogenetic analysis grouped together the AHSV VP2s of serotypes that cross-react serologically. Low identity between serotypes was demonstrated for specific regions within the VP2 amino acid sequences that have been shown to be antigenic and play a role in virus neutralization. The data presented here impact on the development of new vaccines, the identification and characterization of antigenic regions, the development of more rapid molecular methods for serotype identification and the generation of comprehensive databases to support the diagnosis, epidemiology and surveillance of AHS.
Assuntos
Vírus da Doença Equina Africana/classificação , Sequência de Aminoácidos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Orbivirus/classificação , Doença Equina Africana/virologia , Vírus da Doença Equina Africana/genética , Vírus da Doença Equina Africana/metabolismo , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Proteínas do Capsídeo/metabolismo , Clonagem Molecular , Cavalos , Camundongos , Dados de Sequência Molecular , Orbivirus/genética , Filogenia , Recombinação Genética , Alinhamento de Sequência , Análise de Sequência de DNA , SorotipagemRESUMO
Orthostatic reflexes were examined at 375 m and after 60 min of exposure in a hypobaric chamber at 3660 m using a 20-min 70 degrees head-up tilt (HUT) test. Mean arterial blood pressure, R wave-R wave interval (RRI), and mean cerebral blood flow velocity (MFV) were examined with coarse-graining spectral analysis. Of 14 subjects, 7 at 375 m and 12 at 3660 m were presyncopal. Immediately on arrival to high altitude, breathing frequency and MFV increased, and endtidal PCO2, RRI, RRI complexity, and the parasympathetic nervous system indicator decreased. MFV was similar in HUT at both altitudes. The sympathetic nervous system indicator increased with tilt at 3660 m, whereas parasympathetic nervous system indicator decreased with tilt at both altitudes. Multiple regression analysis of supine variables from either 375 or 3660 m and the time to presyncope at 3660 m indicated that, after 1 h of exposure, increased presyncope at altitude was the result of 1). ineffective peripheral vasoconstriction, despite increased cardiac sympathetic nervous system activity with HUT, and 2). insufficient cerebral perfusion owing to cerebral vasoconstriction as the result of hypoxic hyperventilation-induced hypocapnia.
Assuntos
Adaptação Fisiológica , Altitude , Hipotensão Ortostática/etiologia , Hipotensão Ortostática/fisiopatologia , Adulto , Barorreflexo , Velocidade do Fluxo Sanguíneo , Pressão Sanguínea , Circulação Cerebrovascular , Feminino , Frequência Cardíaca , Humanos , Hiperventilação/etiologia , Hiperventilação/fisiopatologia , Incidência , Masculino , Respiração , Síncope/epidemiologia , Síncope/fisiopatologia , Teste da Mesa InclinadaAssuntos
Defeitos Congênitos da Glicosilação/sangue , Defeitos Congênitos da Glicosilação/diagnóstico , Cegueira/etiologia , Defeitos Congênitos da Glicosilação/classificação , Defeitos Congênitos da Glicosilação/complicações , Feminino , Humanos , Lactente , Deficiência Intelectual/etiologia , Focalização Isoelétrica , Convulsões/etiologia , África do Sul , Transferrina/metabolismoRESUMO
The effect of the effluent from a chicken meat-processing plant on the antibiotic-resistant bacterial profile was investigated in an almost closed water reticulation system. Of the 273 faecal coliform isolates 256 (93%) were resistant to one or more of the eight antibiotics tested. The most prevalent isolates were for the beta-lactam antibiotics ampicillin and cephalothin followed by the sulphonamides sulphatriad and cotrimoxazole. Eleven different resistance patterns were identified with a single pattern, comprising of ampicillin-, cephalothin-, streptomycin-, sulphatriad-, cotrimoxazole- and tetracyclin-resistant isolates, dominating the meat-processing effluent. An apparent correlation was observed between the specific use of certain antibiotics and the prevalence of the corresponding resistant bacterial isolates. The drugs used to treat the occasional infections, belonging to the beta-lactam and sulphonamide group of antibiotics, seemed to have a more pronounced effect on the antibiotic-resistant bacterial profile in the primary water source than those drugs used as feed additives, oxytetracyclin and the aminoglycoside flavomycin.
Assuntos
Antibacterianos/farmacologia , Microbiologia Ambiental , Bactérias Gram-Negativas/efeitos dos fármacos , Esgotos/microbiologia , Ampicilina/farmacologia , Cefalosporinas/farmacologia , Cefalotina/farmacologia , Resistência Microbiana a Medicamentos , Indústria Alimentícia , Bactérias Gram-Negativas/isolamento & purificação , Testes de Sensibilidade Microbiana , Penicilinas/farmacologia , África do Sul , Sulfonamidas/farmacologia , Tetraciclinas/farmacologia , Combinação Trimetoprima e Sulfametoxazol/farmacologiaRESUMO
We have identified a novel transcription factor TGP (TG Binding Protein) that binds to the consensus sequence 5'-TGTGGGGTGG-3' in the promoter and intron of the human pro-alpha1(I) collagen gene. This recognition sequence, or sequences closely resembling these sequences, was also identified in the pro-alpha1(I) and pro-alpha2(I) collagen genes of other species. Competition experiments revealed that TGP is related to but distinguishable from the Ap4/5 family of transcription factors and that it can be separated from Ap4/5 according to size.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Pró-Colágeno/genética , Regiões Promotoras Genéticas/genética , Elementos de Resposta/genética , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Ligação Competitiva , Linhagem Celular , Sequência Consenso/genética , DNA/genética , DNA/metabolismo , Pegada de DNA , Proteínas de Ligação a DNA/química , Elementos Facilitadores Genéticos/genética , Humanos , Íntrons/genética , Peso Molecular , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Especificidade da Espécie , Especificidade por Substrato , Transativadores/química , Transativadores/metabolismo , Fatores de Transcrição/químicaRESUMO
In the pro-alpha1(I) collagen gene a number of cis-regulatory elements, which interact with a variety of trans-acting factors, are present in the promoter and first intron. We have undertaken a comprehensive study of Sp1, Ap1, and Ap2 binding in the region spanning -442 to +1697 nt. DNase I footprinting analysis revealed these factors bind with varying affinities to some of the potential sites: Sp1 binds to 16 of 34 potential sites, Ap2 binds to 22 of 40 potential binding sites, and Ap1 binds to its only potential site. The Sp1 sites were mostly clustered in the intron region, while the Ap2 sites were clustered in the promoter region. Transmission electron microscopic analysis of DNA-protein complexes not only confirmed these results, but also clearly showed that heterologous and/or homologous protein-protein interactions between Sp1 and/or Ap2 bring the promoter and intron in contact with each other, with the resulting looping out of the intervening DNA. This strongly suggests that the DNA-looping model is an explanation for the orientation preference of the enhancing element in the first intron as these interactions possibly create an optimum environment for the binding of the rest of the transcriptional machinery.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Pró-Colágeno/genética , Elementos de Resposta/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação , Linhagem Celular , DNA/química , DNA/genética , DNA/ultraestrutura , Pegada de DNA , Sondas de DNA/química , Sondas de DNA/genética , Sondas de DNA/metabolismo , Sondas de DNA/ultraestrutura , Proteínas de Ligação a DNA/ultraestrutura , Desoxirribonuclease I/metabolismo , Regulação da Expressão Gênica , Humanos , Íntrons/genética , Microscopia Eletrônica , Modelos Genéticos , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas/genética , Ligação Proteica , Deleção de Sequência/genética , Fator de Transcrição Sp1/ultraestrutura , Termodinâmica , Fator de Transcrição AP-2 , Fatores de Transcrição/ultraestruturaRESUMO
The conjugation of benzoyl-CoA with the aliphatic and acidic amino acids by glycine N-acyltransferase, as well as the amides of the latter group, was investigated. Bovine and human liver benzoyl-amino acid conjugation were investigated using electrospray ionization tandem mass spectrometry (ESI-MS-MS). Bovine glycine N-acyltransferase catalyzed conjugation of benzoyl-CoA with Gly (Km(Gly) = 6.2 mM), Asn (Km(Asn) = 129 mM), Gln (Km(Gln) = 353 mM), Ala (Km(Ala) = 1573 mM), Glu (Km(Glu) = 1148 mM) as well as Ser in a sequential mechanism. In the case of the human form, conjugation with Gly (Km(Gly) = 6.4 mM), Ala (Km(Ala) = 997 mM), and Glu was detected. The presence of these alternative conjugates did not inhibit bovine glycine N-acyltransferase activity significantly. Considering the relatively low levels at which these conjugates are formed, it is unlikely that they will have a significant contribution to acyl-amino acid conjugation under normal conditions in vivo. However, their cumulative contribution to acyl-amino acid conjugation under metabolic disease states may prove to have a useful contribution to detoxification of elevated acyl-CoAs.
Assuntos
Aciltransferases/metabolismo , Alanina/metabolismo , Ácido Glutâmico/metabolismo , Glicina/metabolismo , Serina/metabolismo , Aciltransferases/isolamento & purificação , Animais , Bovinos , Humanos , CinéticaRESUMO
In this study, we monitored the changes in arterial blood pressure continuously in two groups of Caucasian men during normal passive orthostasis as well as reversed passive orthostasis. Group A consisted of a group of 23 younger men (16 +/- 0.5 years) and group B consisted of 21 older men (62.9 +/- 2.7 years). The normal passive orthostatic test and the reversed passive orthostatic test were used to induce blood pressure changes. We found that the temporary and initial changes in blood pressure during the normal and reversed orthostatic tests were significantly lower in the older group. Heart rate increases were also lower in the older group. These findings could be explained in terms of a reduced compliance of the thin walled venous blood vessels in the elderly.
Assuntos
Envelhecimento/fisiologia , Pressão Sanguínea/fisiologia , Fenômenos Fisiológicos Cardiovasculares , Postura/fisiologia , Adolescente , Artérias/fisiologia , Barorreflexo/fisiologia , Complacência (Medida de Distensibilidade) , Humanos , Hipotensão Ortostática/fisiopatologia , Masculino , Pessoa de Meia-Idade , Pressorreceptores/fisiologia , Teste da Mesa InclinadaRESUMO
Carnitine palmitoyltransferase I (CPT I) is one of the enzymes associated with normal mitochondrial membrane transport of certain metabolites. The importance of the enzyme in normal energy production is well illustrated during fasting conditions when a large flux of long-chain fatty acids must be transported over the mitochondrial membrane to undergo beta-oxidation. Up to now CPT I activity has been assayed in various tissues, including liver, leukocytes, platelets, and fibroblasts by the use of an isotope exchange forward assay which measures the rate of palmitoyl-l-[methyl-3H]carnitine formation from palmitoyl-CoA and l-[methyl-3H]carnitine. We have developed an electrospray ionization mass spectrometric method for detecting palmitoylcarnitine formation from palmitoyl-CoA and carnitine, thus avoiding the use of radiolabeled isotopes. In this assay, time-dependent conversion of free carnitine by CPT I to palmitoylcarnitine is measured quantitatively, relative to isotopically labelled palmitoylcarnitine, by parent ion monitoring of fragment ion m/z 85. The specific activity of CPT I in fibroblasts and leukocytes compared well with the activity determined with the isotope exchange method, however, the combination of high sensitivity and selectivity of tandem mass spectrometry along with the environment-friendly nature of the electrospray method makes it an ideal technique to measure CPT I activity.
Assuntos
Carnitina O-Palmitoiltransferase/metabolismo , Leucócitos/enzimologia , Células Cultivadas , Ativação Enzimática , Fibroblastos/enzimologia , Humanos , Espectrometria de Massas/métodos , Palmitoil Coenzima A/metabolismo , Palmitoilcarnitina/metabolismoRESUMO
Head-down tilted bedrest is a ground-based microgravity simulation model. Since in this position the influence of chief external determinants of circadian blood pressure variation, i.e. activity and posture, are reduced, it may reveal endogenous oscillatory factors. The effects of 42 days of 6 degrees head-down tilt on the circadian profiles of continuous finger blood pressure, heart rate, stroke volume, cardiac output and total peripheral resistance were analysed. In seven healthy volunteers (25-31 years) twelve 22 h Portapres registrations were performed: two in an ambulatory baseline period, eight during 42 days of head-down tilt, and two during recovery. Stroke volume was estimated by a pulse contour method ('Modelflow') from the finger arterial blood pressure tracing. Head-down tilt rapidly reduced circadian BP variation, especially for diastolic blood pressure. No effect of long-term head-down tilt on blood pressure level was observed. The day-night difference in heart rate was essentially unaffected. Cardiac output was maintained through an increase of heart rate and simultaneous decline of stroke volume. Our observations confirm the overriding importance of physical activity and orthostatic load on the diurnal variation of BP. The time-frame of the changes in stroke volume and heart rate during head-down tilt might point to a contribution of other factors besides a reduction of circulating blood volume affecting cardiovascular performance under these conditions.
Assuntos
Pressão Sanguínea/fisiologia , Ritmo Circadiano/fisiologia , Decúbito Inclinado com Rebaixamento da Cabeça/fisiologia , Hemodinâmica/fisiologia , Adulto , Osso e Ossos/metabolismo , Metabolismo Energético/fisiologia , Dedos/irrigação sanguínea , Frequência Cardíaca/fisiologia , Humanos , Sistema Imunitário/fisiologia , Masculino , Músculos/fisiologia , Volume Sistólico/fisiologia , Fatores de Tempo , Resistência Vascular/fisiologia , Ausência de PesoRESUMO
The effect of the presence of the regions 5' and 3' of the hIL-5 gene on the expression of recombinant hIL-5 in CHO-cells was investigated. The 3.2 kb hIL-5 gene-fragment was cloned and four different dexamethasone-inducible rhIL-5 mammalian expression-vectors were constructed, each containing a different part of the gene-fragment. Results indicated that deletion of the region 5' to the gene increases production three-fold whereas a four-fold decrease in production was observed with deletion of the region 3' to the gene. Deletion of both regions increased rhIL-5 production by only one and a half-fold. These results indicate that there are elements in a 928 bp region 3' to the gene, including the 3'-NTR, that have an enhancing or stabilizing effect on expression of hIL-5 in CHO-cells.