RESUMO
OBJECTIVE: To analyze the incidents related to patient safety (IRSP) and their risk factors during in-hospital transfer (IHT) of critical patients after the application of a protocol, and to evaluate safety during transfer using quality indicators. DESIGN: A prospective, observational and non-intervention cohort study was carried out. SETTING: A 10-bed multipurpose Intensive Care Unit (ICU) of a second level university hospital. PATIENTS: All IHTs of critical patients in the ICU for diagnostic tests and to the operating room between March 2011 and March 2017 were included in the study. MAIN MEASUREMENTS: Demographic variables, patient severity, transfer priority, moment of the day, reason and type of transfer team. Pre-transport checklist items and IRSP were collected. A biannual analysis was made of quality indicators designed for IHT. RESULTS: A total of 805 transfers were registered, mostly of an urgent nature (53.7%) and for diagnostic tests (77%). In turn, 112 transfers (13.9%) presented some type of IRSP; 54% related to the equipment and 30% related to team and organization. Adverse events occurred in 19 (2.4%) transfers. Risk factors identified in the multivariate analysis were mechanical ventilation and the transport team. The evolution of the indicators related to transport was significantly favorable. CONCLUSIONS: After the application of an IHT protocol, IRSP are low. The main risk factor is invasive mechanical ventilation. The experience of the team performing IHT influences the detection of a greater number of incidents.
Assuntos
Unidades de Terapia Intensiva , Segurança do Paciente , Estudos de Coortes , Hospitais , Humanos , Estudos ProspectivosRESUMO
OBJECTIVES: To assess diagnosis and therapeutic decisions-making by General Practitioners (GP) using ApnealinkTM® (AL) in patients with high suspicion of obstructive sleep apnea (OSA), in comparison with conclusions of Hospital Sleep Unit (HSU) specialists based on home respiratory polygraphy (PGR) results. METHODS: This study involved patients previously selected by HSU for sleep testing by PGR. After it, patients were offered to complete AL test. PGR was checked at HSU; AL was checked by hemoglobin desaturation index of 4% (4% ODI), (4% AL) and 3% (3% ODI) patients with positive test to proceed with CPAP; and those with negative test for further testing. Automatically adjusted 4% AL, was considered valid as it was demonstrated to be equivalent to manual AL. Results were compared by automatically adjusted 3%AL against PGR results. RESULTS: 48 patients were collected. 43 had AL valid test, 45 had PGR valid study, and 41 had both valid test. 27 patients (62,8%) had positive 4% AL (OR 5,51, p < 0,05), that showed AHI ≥ 15/h at 3% AL test; and 19 patients (42,2%) had a positive PGR test. 31 (72%) patients had a positive 3% AL. AL had shown to be a good screening method of SAHS. CONCLUSIONS: There is equivalence between the decisions of GP and HSU. AL is a good diagnostic tool and screening method for OSA in primary care when it is used in patients with high suspicion of moderate-severe OSA.
Assuntos
Clínicos Gerais , Síndromes da Apneia do Sono , Apneia Obstrutiva do Sono , Humanos , Oximetria , Polissonografia , Atenção Primária à Saúde , Síndromes da Apneia do Sono/diagnóstico , Apneia Obstrutiva do Sono/diagnóstico , Apneia Obstrutiva do Sono/terapiaRESUMO
OBJECTIVE: To analyze the incidents related to patient safety (IRSP) and their risk factors during in-hospital transfer (IHT) of critical patients after the application of a protocol, and to evaluate safety during transfer using quality indicators. DESIGN: A prospective, observational and non-intervention cohort study was carried out. SETTING: A 10-bed multipurpose Intensive Care Unit (ICU) of a second level university hospital. PATIENTS: All IHTs of critical patients in the ICU for diagnostic tests and to the operating room between March 2011 and March 2017 were included in the study. MAIN MEASUREMENTS: Demographic variables, patient severity, transfer priority, moment of the day, reason and type of transfer team. Pre-transport checklist items and IRSP were collected. A biannual analysis was made of quality indicators designed for IHT. RESULTS: A total of 805 transfers were registered, mostly of an urgent nature (53.7%) and for diagnostic tests (77%). In turn, 112 transfers (13.9%) presented some type of IRSP; 54% related to the equipment and 30% related to team and organization. Adverse events occurred in 19 (2.4%) transfers. Risk factors identified in the multivariate analysis were mechanical ventilation and the transport team. The evolution of the indicators related to transport was significantly favorable. CONCLUSIONS: After the application of an IHT protocol, IRSP are low. The main risk factor is invasive mechanical ventilation. The experience of the team performing IHT influences the detection of a greater number of incidents.
RESUMO
Potent stem/progenitor cells have been isolated from normal human dental pulps, termed dental pulp stem cells (DPSCs). However, no study has described the presence of stem cell populations in human dental pulp from the third molar with embryonic phenotypes. The dental pulp tissue was cultured in media with the presence of LIF, EGF, and PDGF. In the present study, we describe a new population of pluripotent stem cells that were isolated from dental pulp (DPPSC). These cells are SSEA-4(+), Oct4(+), Nanog(+), FLK-1(+), HNF3beta(+), Nestin(+), Sox2(+), Lin28(+), c-Myc(+), CD13(+), CD105(+), CD3(-), CD45(-), CD90(low), CD29(+), CD73(low), STRO-1(low) and CD146(-). We have investigated by SEM analysis and q-RT-PCR the capacity of DPPSCs to 3D differentiate in vitro using the Cell Carrier 3D glass scaffold into tissues that have similar characteristics to embryonic mesoderm and endoderm layers. These data would support the use of these cells, which are derived from an easily accessible source and can be used in future regeneration protocols for many tissue types that differentiate from the three embryonic layers.
Assuntos
Polpa Dentária/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Dente Serotino , Adolescente , Adulto , Biomarcadores/metabolismo , Diferenciação Celular , Células Cultivadas , Polpa Dentária/metabolismo , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/ultraestrutura , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Alicerces Teciduais , Adulto JovemRESUMO
3-Hydroxy-3-methylglutaric aciduria is a rare autosomal recessive genetic disorder that affects ketogenesis and leucine metabolism. The disease is caused by mutations in the gene coding for 3-hydroxy-3-methylglutaryl-coenzyme A lyase (HL). To date 26 different mutations have been described. A (betaalpha)(8) TIM barrel structure has been proposed for the protein, and almost all missense mutations identified so far localize in the beta sheets that define the inside cavity. We report an Italian patient who bears homozygously a novel HL mutation, c.608G > A (p. G203E) in beta sheet six. A structural model of the mutated protein suggests that glutamic acid 203 impedes catalysis by blocking the entrance to the inner cavity of the enzyme. Loss of functionality has been confirmed in expression studies in E. coli, which demonstrate that the G203E mutation completely abolishes enzyme activity. Beta sheet six and beta sheet two are the two protein regions that accumulate most missense mutations, indicating their importance in enzyme functionality. A model for the mechanism of enzyme function is proposed.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Meglutol/urina , Mutação de Sentido Incorreto , Oxo-Ácido-Liases/química , Oxo-Ácido-Liases/genética , Sequência de Aminoácidos , Animais , Criança , Análise Mutacional de DNA , Humanos , Masculino , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Homologia de Sequência de AminoácidosRESUMO
Mitochondrial HMG-CoA synthase deficiency is an inherited metabolic disorder caused by a defect in the enzyme that regulates the formation of ketone bodies. Patients present with hypoketotic hypoglycaemia, encephalopathy and hepatomegaly, usually precipitated by an intercurrent infection or prolonged fasting. The diagnosis may easily be missed as previously reported results of routine metabolic investigations, urinary organic acids and plasma acylcarnitines may be nonspecific or normal, and a high index of suspicion is required to proceed to further confirmatory tests. We describe a further acute case in which the combination of urinary organic acids, low free carnitine and changes in the plasma acylcarnitine profile on carnitine supplementation were very suggestive of a defect in ketone synthesis. The diagnosis of mitochondrial HMG-CoA synthase deficiency was confirmed on genotyping, revealing two novel mutations: c.614G > A (R188H) and c.971T > C (M307T). A further sibling, in whom the diagnosis had not been made acutely, was also found to be affected. The possible effects of these mutations on enzyme activity are discussed.
Assuntos
Hidroximetilglutaril-CoA Sintase/deficiência , Erros Inatos do Metabolismo/diagnóstico , Doenças Mitocondriais/diagnóstico , Carnitina/análogos & derivados , Carnitina/sangue , Carnitina/farmacologia , Análise Mutacional de DNA , Genótipo , Heterozigoto , Humanos , Lactente , Masculino , MutaçãoRESUMO
3-Hydroxy-3-methylglutaric aciduria is a human autosomal recessive metabolic disorder that usually appears within the first year of life. The causes of this aciduria are lethal mutations in the gene encoding for 3-hydroxy-3-methylglutaryl coenzyme A lyase (HL). HL is a mitochondrial matrix enzyme that catalyzes the last step of ketogenesis and leucine catabolism. This gene has been mapped to chromosome 1 at locus 1pter-p33 and its genomic organisation comprises 9 exons whose sizes vary between 64-678 bp. The human cDNA sequence was reported in 1993 with the first genetic study of two Acadian-French Canadian siblings. To date, 24 mutations in 36 patients have been described; most of them are single-base substitutions causing amino acid replacements and a variety of splicing defects. In the population studied two mutations appear predominant: g.122GA (8 patients and 15 alleles) frequent in Saudi Arabia, and g.109GT (6 patients and 12 alleles), prevalent in Spain. At least seven mutations are clustered in the second half of exon 2 affecting aminoacids E37, R41 and D42 and conforming a possible hot spot. The genotype-phenotype correlation is difficult to establish since the probands received different treatments, and the onset of an acute episode frequently depends on external factors such as fasting or acute illness.
Assuntos
Meglutol/urina , Mutação , Oxo-Ácido-Liases/deficiência , Oxo-Ácido-Liases/genética , Processamento Alternativo , Cromossomos Humanos Par 1 , Análise Mutacional de DNA , Éxons , Genótipo , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/urina , Leucina/metabolismo , Mitocôndrias/enzimologia , Fenótipo , Polimorfismo GenéticoRESUMO
Deficiency of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHMGS) is a recessive disorder of ketogenesis that has been previously diagnosed in two children with hypoglycaemic hypoketotic coma during fasting periods. Here, we report the results of molecular investigations in a third patient affected by this disease. Sequencing of the entire coding region of the HMGCS2 gene revealed two missense mutations, G212R and R500H. Mendelian inheritance was confirmed by the analysis of parental samples and neither of the mutations was found on 200 control chromosomes. Functional relevance was confirmed by in vitro expression studies in cytosolic HMGS-deficient cells. Whereas wild-type cDNA of the HMGCS2 gene reverted the auxotrophy for mevalonate, the cDNAs of the mutants did not. The disease may be recognised by specific clinical and biochemical features but it is difficult to confirm enzymatically since the gene is expressed only in liver and testis. Molecular studies may facilitate or confirm future diagnoses in affected patients.
Assuntos
Hidroximetilglutaril-CoA Sintase/deficiência , Hidroximetilglutaril-CoA Sintase/genética , Mitocôndrias/enzimologia , Animais , Sequência de Bases , Células CHO , Cricetinae , Análise Mutacional de DNA , Primers do DNA/genética , DNA Complementar/genética , Feminino , Expressão Gênica , Genes Recessivos , Humanos , Técnicas In Vitro , Lactente , Masculino , Mutagênese Sítio-Dirigida , Mutação de Sentido IncorretoRESUMO
Blattella germanica has two cytosolic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase genes, HMG-CoA synthase-1 and -2. HMG-CoA synthase-1 gene shows several features of processed genes (retroposons): it contains no introns but has a short direct-repeat sequence (ATTATTATT) at both ends. An atypical feature is the presence at both ends of the gene of short inverse repeats flanked by direct repeats. There is neither a TATA box nor a CAAT box in the 5' region. Comparative analysis with other species suggests that the HMG-CoA synthase-1 gene derives from HMG-CoA synthase-2. Cultured embryonic B. germanica UM-BGE-1 cells express HMG-CoA synthase-1 but not HMG-CoA synthase-2, suggesting that the intron-less gene is functional. In addition, it can complement MEV-1 cell line, which is auxotrophic for mevalonate. We show that compactin and mevalonate do not significantly affect the mRNA levels of HMG-CoA synthase-1 in UM-BGE-1 cells. Compactin induces a 6.7-fold increase in HMG-CoA reductase activity, which is restored to normal levels by mevalonate. HMG-CoA synthase activity is not modified by either of these effectors, suggesting that the mevalonate pathway in this insect cell line is regulated by post-transcriptional mechanisms affecting HMG-CoA reductase but not HMG-CoA synthase.
Assuntos
Baratas/genética , Genes de Insetos , Proteínas de Insetos/genética , Lovastatina/análogos & derivados , Retroelementos , Animais , Sequência de Bases , Baratas/citologia , Baratas/enzimologia , Regulação Enzimológica da Expressão Gênica , Hidroximetilglutaril-CoA Redutases/biossíntese , Hidroximetilglutaril-CoA Redutases/genética , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Hidroximetilglutaril-CoA Sintase/biossíntese , Hidroximetilglutaril-CoA Sintase/genética , Íntrons , Lovastatina/farmacologia , Ácido Mevalônico/metabolismo , Dados de Sequência Molecular , Especificidade da EspécieRESUMO
Two novel point mutations in the 3-hydroxy-3-methylglutaryl coenzyme A lyase gene were found in a French patient with double heterozygous 3-hydroxy-3-methylglutaric aciduria. Amplification by reverse transcriptase-polymerase chain reaction of the mRNA using five different pairs of oligonucleotides produced no differences in the fragments amplified with respect to the control. Single-strand conformation polymorphism analysis showed that only one amplified fragment was different in the patient vs. control. Sequencing of the amplified fragments showed two missense point mutations, A698G and T788C, each of them mixed with the wild-type sequence. These mutations produced the changes H233R and L263P, leading to changes in the enzyme activity, which was largely abolished. The father and one brother of the proband were heterozygous for the L263P mutation and the mother and one daughter were heterozygous for the H233R mutation, which confirms the double-heterozygous character of the patient. Another sibling was free of the mutations. An enzymatic restriction analysis has been proposed to screen the occurrence of these two mutations in future patients.
Assuntos
Alelos , Meglutol/urina , Oxo-Ácido-Liases/genética , Mutação Puntual , Sequência de Aminoácidos , Pré-Escolar , Análise Mutacional de DNA , Feminino , Humanos , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/urina , Oxo-Ácido-Liases/deficiência , Linhagem , Polimorfismo Conformacional de Fita Simples , Mapeamento por Restrição , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
3-Hydroxy-3-methylglutaric aciduria is a rare recessive monogenic disorder that affects ketogenesis and the catabolism of L-leucine. We report the biochemical and molecular characterization of a mutation in the 3-hydroxy-3-methylglutaryl coenzyme A lyase gene in four new probands, three Spanish and one Turkish, affected by 3-hydroxy-3-methylglutaric aciduria, all homozygous for the nonsense mutation Glu37Ter, which was reported by our group in two probands of Portuguese and Moroccan origin (15). In addition to the aberrant mRNAs found in the two previous probands, a novel species of mature HL mRNA was observed in the patients studied here, since a new cDNA, skipped in exons 2 and 3, was obtained from the mRNAs by reverse-transcription PCR (RT-PCR). Thus, three mRNA species were produced in aberrant splicings as a result of this nonsense mutation: (i) one of the expected size that contains the premature stop codon UAA, (ii) another with a deletion of 84 bp corresponding to the whole of exon 2, and (iii) a new species found now, with a deletion of 192 bp corresponding to skipping of the whole of exons 2 and 3, whose translation product led to the loss of seven amino acids in the leader peptide and 57 amino acids in the terminal domain of the mature enzyme. The association of a nonsense mutation with the skipping of the exon that contains it, plus the following exon, is an unusual finding not seen previously in HL deficiencies. The mutation described here shows the highest incidence (> 37%) of total HL deficiencies reported.
Assuntos
Éxons/genética , Meglutol/urina , Mutação , Oxo-Ácido-Liases/genética , Pré-Escolar , Humanos , Lactente , Masculino , Região do MediterrâneoRESUMO
A novel two-base deletion in the 3-hydroxy-3-methylglutaryl coenzyme A lyase (HL) gene was found in a Spanish patient with homozygous 3-hydroxy-3-methylglutaric aciduria. Amplification by RT-PCR of the mRNAs showed that the gene was transcribed into three different mRNAs. One showed the complete deletion of exons 5 and 6 located between nucleotides 348 and 561 of the HL cDNA. The second transcript showed deletion of exon 6 only, and the third contained a two-base deletion CT in exon 6, corresponding to nucleotides 504 and 505 of the HL cDNA. These aberrant mRNAs are predicted to encode three abnormal HMG-CoA lyase proteins; the first (from skipped exons 5 and 6) lacks 71 amino acids, which represents 24% of the mature protein; the second, (from the skipping of exon 6, producing a frameshift) contains only 192 amino acids, the last 26 of which are missense amino acids preceding a stop codon; the third contains only 175 amino acids, the last 7 of which are missense. Northern blot analysis showed that the HL mRNA levels of the patient were 4% of the control. PCR quantitative analysis indicated that the mRNA lacking exons 5 and 6 was the most abundant, representing 88% of the total. The other two mRNAs represented 8% and 4%, respectively. In the genomic DNA only one CT deletion was found at positions +7 and +8 at beginning of exon 6. No mutations were observed in the splice donor, splice acceptor, or pyrimidine-rich sequences of the intronic regions flanking exons 5 and 6. All three aberrant mRNAs resulted only from the deletion of nucleotides CT. We suggest that this deletion may affect the interaction between the small nuclear ribonucleoproteins (snRNPs) and exon 6, and that, as a result, the abnormal splicing of the pre-mRNA produces two different aberrant transcripts.
Assuntos
Meglutol/urina , Erros Inatos do Metabolismo/enzimologia , Oxo-Ácido-Liases/genética , Deleção de Sequência , Adolescente , Sequência de Aminoácidos , Northern Blotting , Southern Blotting , Feminino , Fibroblastos , Mutação da Fase de Leitura , Humanos , Erros Inatos do Metabolismo/genética , Dados de Sequência Molecular , Oxo-Ácido-Liases/química , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , Splicing de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Transcrição GênicaRESUMO
A novel nonsense mutation associated with the skipping of constitutive exon 2 of the 3-hydroxy-3-methylglutaryl-CoA lyase gene was found in two patients, from Portugal and Morocco, with 3-hydroxy-3-methylglutaric acidemia. By reverse transcriptase PCR and single-strand conformational polymorphism a G-T transversion was located, at nucleotide 109, of the 3-hydroxy-3-methylglutaryl-CoA lyase cDNA, within exon 2. Two mRNAs were produced as a result of this nonsense mutation: one of the expected size that contains the premature stop codon UAA, and the other with a deletion of 84 bp corresponding to the whole of exon 2. This deletion produced the loss of the last seven amino acids of the leader peptide and the first 21 amino acids of the mature protein. The nonsense mutation was found in a purine-rich GGAAG sequence, which is equal to, or similar to, others reported to be exonic splicing enhancers (ESE). We suggest that the nonsense mutation may affect a possible ESE on exon 2, which would hinder the splice site selection and facilitate an aberrant splice with the skipping of this exon. Determination by quantitative PCR shows that the ratio of mRNA with the nonsense mutation to the mRNA with the deletion is approx. 3:1.
Assuntos
Éxons , Mutagênese , Oxo-Ácido-Liases/deficiência , Oxo-Ácido-Liases/genética , Sequência de Aminoácidos , Sequência de Bases , Feminino , Humanos , Lactente , Íntrons , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita SimplesRESUMO
A novel point mutation in the 3-hydroxy-3methyl-glutaryl coenzyme A lyase gene was found in a Turkish patient with homozygous 3-hydroxy-3-methylglutaric acidemia. Amplification by RT-PCR of the mRNA using a six different pairs of oligonucleotides produced no differences in four of the fragments amplified with respect to the control, but generated two fragments of different size. One was representative of a deletion of 126 bp and the other of an insertion of 78 bp. These abnormal mRNAs resulted from a G-->C transversion at the nucleotide +1 of an intron, which changed the invariant GT dinucleotide of the 5' donor splice site. This was associated with the occurrence of an alternative splicing, which led to the skipping of the whole exon of 126 bp, and also with the activation of one cryptic donor splice site in the same intron. These aberrant spliced mRNAs are predicted to encode two abnormal HMG-CoA lyase proteins: the first results in a protein with an internal deletion of 42 amino acids, whose enzyme activity is largely abolished, as the catalytic site was completely removed; the second contains 17 missense amino acids that precede a stop codon. Northern blot analysis showed that the overall content of these aberrantly spliced mRNAs in proband fibroblasts was the same as that found in control fibroblasts. However, hardly any transcript was observed corresponding to the inserted mutated mRNA when it was examined by a specific probe. To quantify the relative proportion of the two mRNAs, a quantitative RT-PCR (the DNA-mimic PCR reaction) was carried out. Results show that the proportion of the inserted mRNAs with respect to the deleted mRNA is only 1.2%. The father, mother, and two brothers of the proband were heterozygous in the G-->C mutation in the +1 nucleotide of the intron considered, while the two alleles of another brother were free of the mutation.
Assuntos
Oxo-Ácido-Liases/genética , Splicing de RNA , RNA Mensageiro/metabolismo , Sequência de Bases , Southern Blotting , Análise Mutacional de DNA , Éxons , Humanos , Recém-Nascido , Íntrons , Masculino , Dados de Sequência Molecular , Mutação Puntual , Reação em Cadeia da Polimerase , Deleção de SequênciaRESUMO
Levels of mRNA for the two 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthases, (HMG-S1 and HMG-S2), and for HMG-CoA reductase (HMG-R) of Blattella germanica were analyzed in the fat body during the first gonadotrophic cycle. HMG-S2 and HMG-R showed the highest mRNA levels on day 0 and decreased thereafter, whereas HMG-S1, showed faint expression. Western blot using specific antibodies for HMG-S1 and HMG-S2 showed no detectable levels for HMG-S1 but a clear pattern for HMG-S2. Both results point to a very limited role for HMG-CoA synthase-1 in B. germanica fat body that the functional enzyme in this organ is HMG-CoA synthase-2. HMG-CoA reductase and synthase proteins shared a cyclic pattern (maximum levels at day 4 and minimum levels on days 0 and 8), which was coincident with the pattern of activity. The delay between gene transcription and protein synthesis suggests a finely regulated translation mechanism. Moreover, the pattern of mevalonate synthesis parallels that of vitellogenin production, suggesting a coordinate mechanism between the mevalonate pathway and the production of vitellogenin.
Assuntos
Baratas/enzimologia , Hidroximetilglutaril-CoA Redutases/metabolismo , Hidroximetilglutaril-CoA Sintase/metabolismo , Vitelogênese/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos , Corpo Adiposo/enzimologia , Feminino , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Sintase/genética , Hidroximetilglutaril-CoA-Redutases NADP-Dependentes , Dados de Sequência Molecular , RNA Mensageiro , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The isoprenoid pathway leads to various essential non-sterol products in insects. These end products have a crucial role in growth, differentiation, sexual maturation, and reproduction. 3-Hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA) synthase (EC 4.1.3.5.) has generally been considered one of the committed steps of the pathway. We had previously reported the cloning of a cytosolic HMG-CoA synthase cDNA in Blattella germanica; we have now isolated and characterized a new cDNA clone for HMG-CoA synthase in this insect. Analysis of this 1716-base pair cDNA reveals a deduced protein of 455 residues with a molecular mass of 51,424 Da. The two HMG-CoA synthases have 69% identical amino acid residues, and both lack an N-terminal leader peptide to target the protein into mitochondria. This HMG-CoA synthase cDNA can revert the Chinese hamster ovary-K1-derived cell line, Mev-1, which is a defective mutant for HMG-CoA synthase. Both HMG-CoA synthase genes are expressed differently throughout development. Analysis of adult tissues shows higher expression in ovary and fat body. The expression of HMG-CoA synthase (EC 4.1.3.5.) and reductase (EC 1.1.1.34) genes during the gonadotrophic cycle in B. germanica shows that the three genes of the isoprenoid pathway are developmentally regulated in the ovary.
Assuntos
Baratas/genética , Regulação Enzimológica da Expressão Gênica , Genes de Insetos , Hidroximetilglutaril-CoA Sintase/biossíntese , Hidroximetilglutaril-CoA Sintase/genética , Ovário/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Baratas/enzimologia , Feminino , Hidroximetilglutaril-CoA Redutases/biossíntese , Hidroximetilglutaril-CoA Redutases/metabolismo , Hidroximetilglutaril-CoA Sintase/metabolismo , Dados de Sequência Molecular , Ovário/fisiologia , RNA Mensageiro/metabolismo , Mapeamento por RestriçãoRESUMO
We have determined the levels of mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase under different metabolic situations to examine its potential role as a regulatory protein in the ketogenic pathway. We used specific antibodies directed against a peptide of the amino acid sequence of the protein as deduced from the cDNA sequence. The amount of mitochondrial HMG-CoA synthase protein rapidly increased in response to cyclic AMP, dexamethasone, starvation, fat feeding, and diabetes, whereas it was decreased by insulin and refeeding. Insulin was also able to counteract the increase in mitochondrial HMG-CoA synthase levels observed under the diabetic condition. Furthermore, the finding that quantitative changes in HMG-CoA synthase protein were less marked than those in the corresponding mRNA in starved and diabetic rats suggests either translational control or increased degradation of either mRNA or protein. All these results indicate that mitochondrial HMG-CoA synthase is a regulatory element in the ketogenic process.
Assuntos
Diabetes Mellitus Experimental/enzimologia , Gorduras na Dieta , Hidroximetilglutaril-CoA Sintase/metabolismo , Mitocôndrias Hepáticas/enzimologia , Animais , Western Blotting , Bucladesina/farmacologia , Dexametasona/farmacologia , Ingestão de Alimentos , Jejum , Hidroximetilglutaril-CoA Sintase/biossíntese , Hidroximetilglutaril-CoA Sintase/isolamento & purificação , Insulina/farmacologia , Cinética , Mitocôndrias Hepáticas/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , InaniçãoRESUMO
We have explored the role of mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase in regulating ketogenesis. We had previously cloned the cDNA for mitochondrial HMG-CoA synthase and have now studied the regulation in vivo of the expression of this gene in rat liver. The amount of processed mitochondrial HMG-CoA synthase mRNA is rapidly changed in response to cyclic AMP, insulin, dexamethasone and refeeding, and is greatly increased by starvation, fat feeding and diabetes. We conclude that one point of ketogenic control is exercised at the level of genetic expression of mitochondrial HMG-CoA synthase.