RESUMO
BACKGROUND: Hyperlipidemia animal models have been established, but complete gene expression profiles of the transition from normal lipid levels have not been obtained. Miniature pigs are useful model animals for gene expression studies on dietary-induced hyperlipidemia because they have a similar anatomy and digestive physiology to humans, and blood samples can be obtained from them repeatedly. METHODOLOGY: Two typical dietary treatments were used for dietary-induced hyperlipidemia models, by using specific pathogen-free (SPF) Clawn miniature pigs. One was a high-fat and high-cholesterol diet (HFCD) and the other was a high-fat, high-cholesterol, and high-sucrose diet (HFCSD). Microarray analyses were conducted from whole blood samples during the dietary period and from white blood cells at the end of the dietary period to evaluate the transition of expression profiles of the two dietary models. PRINCIPAL FINDINGS: Variations in whole blood gene expression intensity within the HFCD or the HFCSD group were in the same range as the controls provide with normal diet at all periods. This indicates uniformity of dietary-induced hyperlipidemia for our dietary protocols. Gene ontology- (GO) based functional analyses revealed that characteristics of the common changes between HFCD and HFCSD were involved in inflammatory responses and reproduction. The correlation coefficient between whole blood and white blood cell expression profiles at 27 weeks with the HFCSD diet was significantly lower than that of the control and HFCD diet groups. This may be due to the effects of RNA originating from the tissues and/or organs. CONCLUSIONS: No statistically significant differences in fasting plasma lipids and glucose levels between the HFCD and HFCSD groups were observed. However, blood RNA analyses revealed different characteristics corresponding to the dietary protocols. In this study, whole blood RNA analyses proved to be a useful tool to evaluate transitions in dietary-induced hyperlipidemia gene expression profiles in miniature pigs.
Assuntos
Dieta , Hiperlipidemias/genética , Análise de Sequência com Séries de Oligonucleotídeos , Transcriptoma , Animais , Glicemia , Peso Corporal , Colesterol/sangue , Análise por Conglomerados , Regulação da Expressão Gênica , Hiperlipidemias/etiologia , Hiperlipidemias/metabolismo , Modelos Animais , Anotação de Sequência Molecular , Suínos , Porco Miniatura , Triglicerídeos/sangueRESUMO
BACKGROUND: Carnosine is a dipeptide that improves exercise performance. The carnosine synthesis mechanism through carnosine and ß-alanine ingestion remains unclear. Therefore, we investigated the tissue distribution of carnosine synthase, ATP-grasp domain-containing protein-1 (ATPGD1) mRNA, and ATPGD1 and carnosine specific dipeptidase (CN1) gene expression profiles in mice that were given carnosine or ß-alanine orally. METHODS: ddY mice (7-week-old) were randomly divided into three groups (n = 6 to 8 animals per group) and were orally given 2 g/kg body weight of carnosine, ß-alanine, or water. After 15, 30, 60, 120, 180, or 360 min of treatment, the tissues (brain, blood, liver, kidneys, olfactory bulbs, hindleg muscles) were collected. The obtained tissues measured the expression of ATPGD1 and CN1 genes using quantitative PCR methods. RESULTS: The ATPGD1 gene was expressed in muscle and to a lesser extent in brain. The expression of ATPGD1 in the vastus lateralis muscle increased significantly at 180 min (P = 0.023) after carnosine ingestion and 60 (P = 0.023) and 180 min (P = 0.025) after ß-alanine ingestion. Moreover, the carnosine group showed a significantly increased renal expression of the CN1 gene 60 min after ingestion (P = 0.0015). CONCLUSIONS: The ATPGD1 gene showed high expression levels in brain and muscle. The ß-alanine or carnosine administration significantly increased ATPGD1 and CN1 expression in mice.
RESUMO
A Manduca sexta (tobacco hornworm) cysteine protease inhibitor, MsCPI, purified from larval hemolymph has an apparent molecular mass of 11.5 kDa, whereas the size of the mRNA is very large (â¼9 kilobases). MsCPI cDNA consists of a 9,273 nucleotides that encode a polypeptide of 2,676 amino acids, which includes nine tandemly repeated MsCPI domains, four cystatin-like domains and one procathepsin F-like domain. The procathepsin F-like domain protein was expressed in Escherichia coli and processed to its active mature form by incubation with pepsin. The mature enzyme hydrolyzed Z-Leu-Arg-MCA, Z-Phe-Arg-MCA and Boc-Val-Leu-Lys-MCA rapidly, whereas hydrolysis of Suc-Leu-Tyr-MCA and Z-Arg-Arg-MCA was very slow. The protease was strongly inhibited by MsCPI, egg-white cystatin and sunflower cystatin with K(i) values in the nanomolar range. When the MsCPI tandem protein linked to two MsCPI domains was treated with proteases, it was degraded by the cathepsin F-like protease. However, tryptic digestion converted the MsCPI tandem protein to an active inhibitory form. These data support the hypothesis that the mature MsCPI protein is produced from the MsCPI precursor protein by trypsin-like proteases. The resulting mature MsCPI protein probably plays a role in the regulation of the activity of endogenous cysteine proteases.
Assuntos
Clonagem Molecular , Cistatinas/genética , Cisteína Proteases/genética , Inibidores de Cisteína Proteinase/genética , Proteínas de Insetos/genética , Manduca/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Catepsina F/química , Catepsina F/genética , Cistatinas/química , Cistatinas/metabolismo , Cisteína Proteases/química , Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/metabolismo , DNA Complementar/genética , Genes de Insetos , Hemolinfa/química , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Larva/enzimologia , Larva/genética , Manduca/enzimologia , Manduca/metabolismo , Dados de Sequência Molecular , Precursores de Proteínas/genéticaRESUMO
An Apios americana trypsin inhibitor, AATI, was purified from Apios tubers by chromatography on DEAE Cellulofine A-500 and Sephadex G-50. The molecular mass of AATI was determined to be 6,437 Da by matrix-assisted laser desorption and ionization time-of-flight mass spectrometer (MALDI-TOF-MS). It showed strong inhibitory activity toward serine proteases, and the inhibition constants toward trypsin and chymotrypsin were 3.0 x 10(-9) M and 1.0 x 10(-6) M respectively. The inhibitory activity was not affected by heating at 80 degrees C for 2 h or by incubation at a wide range of pH values, suggesting that AATI has remarkable heat-stability and pH-stability. AATI cDNA consists of 552 nucleotides, and includes an open reading frame encoding a protein of 116 amino acids. The results of N-terminal amino acid sequencing of AATI and MALDI-TOF-MS analysis suggested that the deduced amino acid sequence had 50 and seven extra amino acids at the N- and C-termini respectively. Thus the mature AATI protein consists of 59 amino acid residues. Comparison of the amino acid sequence with those of the trypsin inhibitors from plants suggests that AATI belongs to the Bowman-Birk family and that it contains two possible reactive sites toward trypsin at Lys62 and Arg88.
Assuntos
Fabaceae/genética , Inibidor da Tripsina de Soja de Bowman-Birk/química , Inibidor da Tripsina de Soja de Bowman-Birk/genética , Sequência de Aminoácidos , Sequência de Bases , Cromatografia , Cromatografia em Gel , Clonagem Molecular , Primers do DNA , Estabilidade de Medicamentos , Fabaceae/química , Dados de Sequência Molecular , Peso Molecular , Inibidores de Proteases/química , Inibidores de Proteases/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Termodinâmica , Inibidor da Tripsina de Soja de Bowman-Birk/isolamento & purificaçãoRESUMO
A cysteine protease inhibitor (CPI) with an apparent molecular mass of 11.5kDa was purified from larval hemolymph of the tobacco hornworm (Manduca sexta) by gel filtration on Sephadex G-50 followed by hydrophobic and ion-exchange column chromatographies. The purified cysteine proteinase inhibitor, denoted as MsCPI, strongly inhibited the plant cysteine protease, papain, with a K(i) value of 5.5 x 10(-9)M. Nucleotide sequence analysis of a partial cDNA encoding MsCPI indicated that MsCPI consists of 105 amino acid residues in a sequence that is similar to sarcocystatin A from Sarcophaga peregrina. However, northern blotting and PCR analyses using the specific primers of MsCPI suggested that the mRNA encoding MsCPI had a size of more than 12 kilobases, which included at least six tandemly repeated MsCPI segments. MsCPI was expressed in Escherichia coli and the recombinant protein effectively inhibited cysteine proteases from plants as well as from animals such as cathepsins B (K(i), 6.8 nM), H (3.0 nM), and L (0.87 nM). There was no inhibition exhibited toward trypsin, chymotrypsin, subtilisin, pepsin or themolysin.