RESUMO
Using phase contrast and fluorescence microscopy we study the influence of the alkylphospholipid, ALP, 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate, ODPC, in giant unilamellar vesicles, GUVs, composed of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), brain sphingomyelin (SM) and cholesterol (Chol). The results show that adding 100µM ODPC (below CMC) to the outer solution of GUVs promotes DOPC membrane disruption over a period of 1h of continuous observation. On the other hand, the presence of SM and Chol in homogeneous fluid lipid bilayers protects the membrane from disruption. Interestingly, by adding 100µM ODPC to GUVs containing DOPC:SM:Chol (1:1:1), which display liquid ordered (Lo)-liquid disordered (Ld) phase coexistence, the domains rapidly disappear in less than 1min of ODPC contact with the membrane. The lipids are subsequently redistributed to liquid domains within a time course of 14-18min, reflecting that the homogenous phase was not thermodynamically stable, followed by rupture of the GUVs. A similar mechanism of action is also observed for perifosine, although to a larger extent. Therefore, the initial stage of lipid raft disruption by both ODPC and perifosine, and maybe other ALPS, by promoting lipid mixing, may be correlated with their toxicity upon neoplastic cells, since selective (dis)association of essential proteins within lipid raft microdomains must take place in the plasma membrane.
Assuntos
Glicerofosfolipídeos/química , Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Microdomínios da Membrana/química , Lipossomas Unilamelares/química , Colesterol/química , Fluidez de Membrana , Microscopia de Fluorescência , Microscopia de Contraste de Fase , Modelos Químicos , Modelos Moleculares , Fosfatidilcolinas/química , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Esfingomielinas/química , TermodinâmicaRESUMO
Hemophilia A is the most common X-linked bleeding disorder; it is caused by deficiency of coagulation factor VIII (FVIII). Replacement therapy with rFVIII produced from human cell line is a major goal for treating hemophilia patients. We prepared a full-length recombinant FVIII (FVIII-FL), using the pMFG-P140K retroviral vector. The IRES DNA fragment was cloned upstream to the P140K gene, providing a 9.34-kb bicistronic vector. FVIII-FL cDNA was then cloned upstream to IRES, resulting in a 16.6-kb construct. In parallel, an eGFP control vector was generated, resulting in a 10.1- kb construct. The 293T cells were transfected with these constructs, generating the 293T-FVIII-FL/P140K and 293T-eGFP/P140K cell lines. In 293T-FVIII-FL/P140K cells, FVIII and P140K mRNAs levels were 4,410 (±931.7)- and 295,400 (±75,769)-fold higher than in virgin cells. In 293T-eGFP/P140K cells, the eGFP and P140K mRNAs levels were 1,501,000 (±493,700)- and 308,000 (±139,300)-fold higher than in virgin cells. The amount of FVIII-FL was 0.2 IU/mL and 45 ng/mL FVIII cells or 4.4 IU/µg protein. These data demonstrate the efficacy of the bicistronic retroviral vector expressing FVIII-FL and MGMT(P140K), showing that it could be used for producing the FVIII-FL protein in a human cell line.
Assuntos
Fator VIII/biossíntese , Vetores Genéticos , Retroviridae/genética , Fator VIII/genética , Ordem dos Genes , Células HEK293 , HumanosRESUMO
10-(Octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC) is an alkylphospholipid that can interact with cell membranes because of its amphiphilic character. We describe here the interaction of ODPC with liposomes and its toxicity to leukemic cells with an ED-50 of 5.4, 5.6 and 2.9 microM for 72 h of treatment for inhibition of proliferation of NB4, U937 and K562 cell lines, respectively, and lack of toxicity to normal hematopoietic progenitor cells at concentrations up to 25 microM. The ED-50 for the non-malignant HEK-293 and primary human umbilical vein endothelial cells (HUVEC) was 63.4 and 60.7 microM, respectively. The critical micellar concentration (CMC) of ODPC was 200 microM. Dynamic light scattering indicated that dipalmitoylphosphatidylcholine (DPPC) liposome size was affected only above the CMC of ODPC. Differential calorimetric scanning (DCS) of liposomes indicated a critical transition temperature (T(c)) of 41.5 degrees C and an enthalpy (H) variation of 7.3 kcal mol(-1). The presence of 25 microM ODPC decreased T(c) and H to 39.3 degrees C and 4.7 kcal mol(-1), respectively. ODPC at 250 microM destabilized the liposomes (36.3 degrees C, 0.46 kcal mol(-1)). Kinetics of 5(6)-carboxyfluorescein (CF) leakage from different liposome systems indicated that the rate and extent of CF release depended on liposome composition and ODPC concentration and that above the CMC it was instantaneous. Overall, the data indicate that ODPC acts on in vitro membrane systems and leukemia cell lines at concentrations below its CMC, suggesting that it does not act as a detergent and that this effect is dependent on membrane composition.
Assuntos
Antineoplásicos/farmacologia , Membrana Celular/efeitos dos fármacos , Leucemia/tratamento farmacológico , Fosfolipídeos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Leucemia/patologia , Lipossomos , Micelas , TermodinâmicaRESUMO
The reduction of circulating neutrophil migration to infection sites is associated with a poor outcome of severe sepsis. alpha-1-Acid glycoprotein (AGP) was isolated from the sera of severely septic patients by HPLC and acrylamide gel electrophoresis and identified by mass spectrometry. Both the isolated protein and commercial AGP inhibited carrageenin-induced neutrophil migration into the rat peritoneal cavity when administered i.v. at a dose of 4.0 microg per rat (95 pmol per rat). Analysis by intravital microscopy demonstrated that both proteins inhibited the rolling and adhesion of leukocytes in the mesenteric microcirculation. The inhibitory activity was blocked by 50 mg/kg aminoguanidine, s.c., and was not demonstrable in inducible nitric oxide synthase (iNOS) knockout mice. Incubation of AGP with neutrophils from healthy subjects induced the production of NO and inhibited the neutrophil chemotaxis by an iNOS/NO/cyclic guanosine 3,5-monophosphate-dependent pathway. In addition, AGP induced the l-selectin shedding by neutrophils. The administration of AGP to rats with mild cecal ligation puncture sepsis inhibited neutrophil migration and reduced 7-day survival from approximately 80% to 20%. These data demonstrate that AGP, an acute-phase protein, inhibits neutrophil migration by an NO-dependent process and suggest that AGP also participates in human sepsis.
Assuntos
Proteínas de Fase Aguda/fisiologia , Migração e Rolagem de Leucócitos , Neutrófilos/imunologia , Orosomucoide/fisiologia , Sepse/imunologia , Proteínas de Fase Aguda/isolamento & purificação , Proteínas de Fase Aguda/farmacologia , Animais , Carragenina/farmacologia , Movimento Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Humanos , Migração e Rolagem de Leucócitos/efeitos dos fármacos , Masculino , Espectrometria de Massas , Neutrófilos/efeitos dos fármacos , Óxido Nítrico , Orosomucoide/isolamento & purificação , Orosomucoide/farmacologia , Ratos , Ratos Wistar , Sepse/sangueRESUMO
Starting with 31 of plasma from dogs in hemorrhagic shock, we have purified the myocardial depressant factor and found that the activity is separate from salts and free amino acids. Moreover, the myocardial depressant factor is present in shock plasma in concentrations of about 1 nmol/ml of plasma. The depressant factor exists as multiple chromatographic forms. The best characterized forms are the anionic forms. A preliminary amino acid composition of the anionic forms has been obtained. These findings should allow more rapid processing of plasma containing high myocardial depressant factor activity to separate the factor and to completely identify this small peptide of great physiologic interest.
Assuntos
Proteínas Sanguíneas , Coração/fisiologia , Choque Hemorrágico/sangue , Aminoácidos/análise , Animais , Pressão Sanguínea , Proteínas Sanguíneas/isolamento & purificação , Proteínas Sanguíneas/fisiologia , Cães , Coração/fisiopatologia , Contração MiocárdicaRESUMO
The complete amino acid sequence of the lectin KM+ from Artocarpus integrifolia (jackfruit), which contains 149 residues/mol, is reported and compared to those of other members of the Moraceae family, particularly that of jacalin, also from jackfruit, with which it shares 52% sequence identity. KM+ presents an acetyl-blocked N-terminus and is not posttranslationally modified by proteolytic cleavage as is the case for jacalin. Rather, it possesses a short, glycine-rich linker that unites the regions homologous to the alpha- and beta-chains of jacalin. The results of homology modeling implicate the linker sequence in sterically impeding rotation of the side chain of Asp141 within the binding site pocket. As a consequence, the aspartic acid is locked into a conformation adequate only for the recognition of equatorial hydroxyl groups on the C4 epimeric center (alpha-D-mannose, alpha-D-glucose, and their derivatives). In contrast, the internal cleavage of the jacalin chain permits free rotation of the homologous aspartic acid, rendering it capable of accepting hydrogen bonds from both possible hydroxyl configurations on C4. We suggest that, together with direct recognition of epimeric hydroxyls and the steric exclusion of disfavored ligands, conformational restriction of the lectin should be considered to be a new mechanism by which selectivity may be built into carbohydrate binding sites. Jacalin and KM+ adopt the beta-prism fold already observed in two unrelated protein families. Despite presenting little or no sequence similarity, an analysis of the beta-prism reveals a canonical feature repeatedly present in all such structures, which is based on six largely hydrophobic residues within a beta-hairpin containing two classic-type beta-bulges. We suggest the term beta-prism motif to describe this feature.
Assuntos
Proteínas de Transporte/química , Manose/metabolismo , Plantas/química , Dobramento de Proteína , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Colectinas , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
A sensitive radioimmunoassay is described for the measurement of plasma concentrations of canine growth hormone (cGH) as low as 0.25 ng/ml. The assay utilizes enzymatically iodinated cGH and the double antibody technique. The mean plasma concentration of growth hormone in the normal dog after overnight fast is 1.75 plus or minus .17 ng/ml. Exogenous cGH was cleared from the plasma of both the normal and hypophysectomized dog with a mean half-life of 25.6 plus or minus 1.0 min and was distributed in a volume equal to 8.9% of the body weight. Insulin hypoglycemia produced a 3- to 5-fold increase in plasma GH in 4 of 6 dogs and arginine infusion failed to produce a statistically significant rise.
Assuntos
Hormônio do Crescimento/sangue , Radioimunoensaio/métodos , Animais , Arginina/farmacologia , Glicemia , Cães , Meia-Vida , Hipofisectomia , Insulina/farmacologia , Radioisótopos do IodoRESUMO
The most sensitive nonradiometric routine assay for angiotensin-converting enzyme (ACE) activity uses fluorometry to detect His-Leu released from Hip-His-Leu. Our results indicate that, in contrast to human serum, rat serum and plasma contain large and variable amounts of dipeptidase activity that lead to a subestimation of the ACE activity measured in 0.1 M potassium phosphate buffer, pH 8.3, containing 0.3 M NaCl, the most commonly used assay for human serum and tissue ACE. We describe and validate an assay for 1 to 10 microL rat and human serum or plasma using 5 mM Hip-His-Leu in 500 microL of 0.4 M sodium borate buffer, pH 8.3, containing 0.9 M NaC1 at 37 degrees C that reduced the subestimation error to less than or equal to 3% (rat serum) and less than or equal to 0.1% (human serum) and increased the ACE activity twofold to threefold. The Km and Vmax are reported for rat serum ACE (Hip-His-Leu) and dipeptidase (His-Leu) in borate buffer and phosphate buffer. Rat serum ACE hydrolysis of Hip-His-Leu measured by fluorometry correlated (r = 0.99, p less than 0.05) with the hydrolysis of angiotensin I measured by high-performance liquid chromatography. A direct method based on amino acid analysis is described for evaluating the dipeptidase error of complex mixtures such as tissue extracts and other physiological fluids. We have found that the assay can be used to measure ACE activity in 25 samples (in duplicate) in 2 hours with small intraassay (2.2%) and interassay (3.9%) coefficients of variation.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Fluorometria/métodos , Peptidil Dipeptidase A/sangue , Renina/sangue , Angiotensina I/sangue , Angiotensina II/sangue , Animais , Cromatografia Líquida de Alta Pressão , Dipeptidases/sangue , Dipeptídeos , Hidrólise , Masculino , Oligopeptídeos , Plasma/enzimologia , Ratos , Ratos EndogâmicosRESUMO
The concentrations of angiotensin converting enzyme (ACE) activity, norepinephrine, and serotonin were measured in microdissected regions of the dog's brainstem and spinal cord. In addition, we determined the in vitro metabolism of 125I-angiotensin I (Ang I) in homogenates of the same brain punch regions. High ACE-specific activity was found in the monoamine-containing regions of the brainstem and in the intermediolateral column of the spinal cord. In brainstem homogenates 125I-Ang I was metabolized to angiotensin II (Ang-[1-8]) and the N-terminal heptapeptide Ang-(1-7). In the presence of MK 422 (50 microM), Ang-(1-7) was still generated, while the production of Ang-(1-8) was inhibited. This study revealed the presence of high ACE activity in monoamine regions of dog brainstem and spinal cord, and showed that the metabolite Ang-(1-7) is the major product generated from Ang I in the presence and absence of ACE inhibition.
Assuntos
Angiotensina I/metabolismo , Tronco Encefálico/metabolismo , Peptidil Dipeptidase A/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Cães , Enalapril/análogos & derivados , Enalapril/farmacologia , Enalaprilato , Norepinefrina/metabolismo , Fragmentos de Peptídeos/metabolismo , Serotonina/metabolismo , Medula Espinal/metabolismoRESUMO
The present study was undertaken to evaluate the participation of the kallikrein-kinin system in the normalization of blood pressure after release of the clip in one-kidney, one clip hypertensive rats ( 1K1C ). Kininogen was determined before and after unclipping by tryptic digestion of denatured rat plasma, and spasmogenic activity was measured with isolated guinea pig ileum. In contrast to human plasma for which bradykinin (BK) is the only trypsin-releasable spasmogenic substance ( TRSS ), rat plasma contains non-BK TRSS (Fractions P1 and P2) as well as BK. Fractions P1 and P2 were separated from BK by SP-Sephadex chromatography. An increase of total TRSS was demonstrated 60 days after clipping and reached a maximum at approximately Day 75, which was two times that of the normotensive controls (NC). The level of total TRSS did not change after unclipping . The increased level of TRSS in the hypertensive state confirmed the observations of other investigators who reported increased kininogen levels but who could not distinguish between BK and non-BK TRSS because bioassays were performed without prior chromatographic separation of the spasmogenic activities. Fractions P1 and P2 were present in the TRSS of both 1K1C and NC plasma, but were two to six times higher in 1K1C and thus probably accounted quantitatively for the increased TRSS in 1K1C . The data suggest that in the hypertensive state there is an alteration in the relative amounts of some plasma proteins that contain non-BK TRSS within their amino acid sequences. Fractions P1 and P2 also contain potentiating peptides and have not yet been purified to homogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Proteínas Sanguíneas/isolamento & purificação , Hipertensão Renovascular/sangue , Tripsina/farmacologia , Animais , Pressão Sanguínea , Bradicinina/sangue , Bradicinina/isolamento & purificação , Cromatografia em Gel , Cobaias , Calicreínas/metabolismo , Rim/metabolismo , Cininogênios/sangue , Cininas/metabolismo , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Ratos , Ratos Endogâmicos , Fatores de TempoRESUMO
Rabbit brain endo-oligopeptidase B inactivates angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) and angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) by hydrolysis of the Pro7-Phe8 peptide bond. The site of hydrolysis was determined in preparative and analytical experiments in which both products were recovered in a molar ratio of 1:1, and the sum of the products plus unhydrolyzed substrate accounted for the starting material. The enzyme has a Km of 6.3 x 10(-5) M for angiotensin II at pH 8.3 and is activated 30-fold with 4.8 mM dithiothreitol. BPP9a ( less than Gln-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro, SQ 20,881) inhibits the inactivation of angiotensin II with an I50 of 5 x 10(-5) M. BPP5a (less than Gln-Lys-Trp-Ala-Pro, SQ 20,475) is less active and D-3-mercapto-2-methylpropanoyl-L-proline (captopril, SQ 14,225) has essentially no activity. These endo-oligopeptidase B in angiotensin I and II metabolism remains to be established.
Assuntos
Angiotensina II/antagonistas & inibidores , Angiotensina I/antagonistas & inibidores , Angiotensinas/antagonistas & inibidores , Encéfalo/enzimologia , Cisteína Endopeptidases , Endopeptidases/metabolismo , Sequência de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina , Animais , Coelhos , Renina/metabolismo , Teprotida/farmacologiaRESUMO
A 2288-bp cDNA sequence encoding dihydrolipoamide dehydrogenase (DLDH; dihydrolipoamide: NAD+ oxido-reductase; EC 1.8.1.4) was obtained by isolating a 1762-bp cDNA clone from a canine skeletal muscle library in the vector, lambda UNIZAP, combined with PCR amplification of the 5' end of the mRNA. The DLDH cDNA sequence contains a 49-bp G+C-rich 5'-untranslated region (UTR), followed by 1527 bp of coding region, and 695 bp of 3'-UTR preceding a 17-bp poly(A) tail. The single open reading frame encodes a precursor DLDH of 509 amino acids (aa) that begins with a 35-aa leader sequence. The 3'-UTR includes six possible polyadenylation signals (three AATAAA, one TATAAA and two AATGAA) and one potential stem-loop region extending from bp 1969-1991. Alignment studies of the canine and human DLDH demonstrate homology within the coding region of 98% at the aa level and 94% at the nt level. Northern blot analysis using the cDNA clone as probe showed wide tissue distribution of the mRNA, with differences in the level of expression among tissues and possible utilization of different polyadenylation sites.
Assuntos
DNA Complementar/genética , Di-Hidrolipoamida Desidrogenase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , Cães , Precursores Enzimáticos/genética , Expressão Gênica , Humanos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Especificidade da EspécieRESUMO
We determined the effect of the extent of protein polymerization on the intestinal hyperplastic adaptation of adult male Wistar rats after 80% resection of the jejunal-ileal segment. Rats received one of four chemically defined solid diets prepared by using casein, two casein hydrolysates of different peptide size distributions, or free amino acids simulating casein and identical in all other components for 12 d, starting 3 d after surgery. Semipaired feeding was used to ensure that the same quantity of food was ingested by each group and as a consequence, nitrogen and energy intakes were reduced to 63% of that obtained with ad libitum feeding of the casein diet to intact rats. No significant differences were demonstrable in food ingestion, weight gain, nitrogen balance, or morphometric data for the remaining jejunal and ileal segments (number of cells/villus, number of cells/crypt, and crypt cell mitosis rate). These data demonstrate that the extent of polymerization of the protein nitrogen source did not affect the hyperplastic adaptative process of the rat. Additional studies in humans are necessary to determine whether intact protein diets can be used first as a nitrogen source in nutritional support of patients with a nonspecific hyperplastic response to surgical resection before the use of expensive hydrolysates and the more expensive amino acid mixtures.
Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Aminoácidos/farmacologia , Caseínas/farmacologia , Colo/cirurgia , Mucosa Intestinal/fisiologia , Adaptação Fisiológica/fisiologia , Aminoácidos/análise , Ração Animal/análise , Animais , Caseínas/análise , Caseínas/metabolismo , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Colo/fisiologia , Hidrólise , Íleo/citologia , Íleo/fisiologia , Íleo/ultraestrutura , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Jejuno/citologia , Jejuno/fisiologia , Jejuno/ultraestrutura , Masculino , Microvilosidades/efeitos dos fármacos , Microvilosidades/fisiologia , Microvilosidades/ultraestrutura , Mitose/efeitos dos fármacos , Mitose/fisiologia , Ratos , Ratos WistarRESUMO
In order to understand angiotensin metabolism in the canine brain, we determined the molecular forms of angiotensin peptides present in the hypothalamus of the dog and carried out measurements of the metabolism of 125I-angiotensin I in homogenates of that tissue. Angiotensin peptides were extracted from canine hypothalamic tissue and quantified by specific radioimmunoassays combined with high-performance liquid chromatography. The major angiotensin peptides detected were angiotensin-(2-7) (391.2 +/- 16.8 pg/g tissue) and angiotensin-(3-7) (864.8 +/- 128.1 pg/g). Angiotensin II immunoreactivity was mainly composed of angiotensin-(3-8) (117.5 +/- 64 pg/g) and trace amounts of angiotensin II and angiotensin III. Angiotensin I immunoreactivity was composed of angiotensin I (52.3 +/- 5.8 pg/g). In separate experiments, addition of 125I-angiotensin I into supernatants (18,000 g for 2 min) of canine hypothalamic homogenates resulted in the accumulation of 125I-angiotensin-(1-7) as the major peptide product (14% of the total 125I-radioactivity) at 2 min. Incubation of the homogenate supernatants with enalaprilat (1 mumol/l), phosphoramidon (10 mumol/l), or ethylenediamine tetraacetic acid (1 mmol/l) did not inhibit the production of 125I-angiotensin-(1-7). In contrast, addition of Z-Pro-Prolinal (1 mumol/l), a specific inhibitor of prolyl endopeptidase, prevented the generation of 125I-angiotensin-(1-7) from 125I-angiotensin I by 47.0 +/- 8.0% (n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Angiotensina II/metabolismo , Endopeptidases/metabolismo , Hipotálamo/metabolismo , Serina Endopeptidases , Angiotensina I/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Cães , Técnicas In Vitro , Radioisótopos do Iodo , Masculino , Fragmentos de Peptídeos/metabolismo , Prolil Oligopeptidases , Radioimunoensaio , Sistema Renina-Angiotensina/fisiologiaRESUMO
Cystinuria is a recessively inherited transport disorder, with at least three mutant alleles (I, II, and III) demonstrable. I/I, II/II, and III/III homozygotes and I/II, I/III, and II/III compound heterozygotes (cystinuric patients) have high urinary concentrations of cystine, lysine, arginine, and ornithine and frequently form cystine stones. +/I heterozygotes (nondetectable) are phenotypically normal, whereas +/II and +/III heterozygotes (detectable) show variable increases in urinary cystine and lysine concentration and at times increases in urinary arginine levels. The objectives of the present study were to determine the frequency of +/II heterozygotes among stone-forming and nonstone-forming individuals from the same region of Brazil and to evaluate the possible relationship between heterozygous cystinuria and urinary lithiasis. When urine samples from 5,150 individuals (5,000 nonstone-forming individuals and 150 stone-forming individuals) were screened by the qualitative cyanide-nitroprusside cystine test, by thin-layer amino acid chromatography, and by quantitative amino acid determination by ion-exchange chromatography, 32 +/II or +/III heterozygotes (26 nonstone-forming and six stone-forming individuals) were detected. The frequency of detectable heterozygotes among the stone-forming individuals (1:25) was significantly higher than that among nonstone-forming individuals (1:104), which provides additional evidence that heterozygosity for +/II and +/III cystinuria is a risk factor in the formation of urinary stones. No significant difference was detected in urinary cystine concentration or in terms of the various characteristics of urolithiasis when stone-forming heterozygotes were compared to nonstone-forming heterozygotes. These data suggest that the tendency towards stone-forming among heterozygotes is probably owing to a complex and multifactorial mechanism.
Assuntos
Cistinúria/genética , Cálculos Urinários/genética , Arginina/urina , Oxalato de Cálcio/metabolismo , Heterozigoto , Humanos , Lisina/urina , Ornitina/urinaRESUMO
We determined the effect of chronic administration of the angiotensin converting enzyme (ACE) inhibitor, enalapril, on the in vivo pulmonary inactivation of bradykinin (BK) and conversion of angiotensin I (Ang I). In addition we assessed whether chronic ACE inhibition influenced the activity of prolylendopeptidase (PEP), which metabolizes Ang I to generate angiotensin-(1-7) (Ang-[1-7]) and inactivates BK. Male Wistar rats were treated orally with enalapril (10 mg/kg once a day) for 7 to 15 days (n = 20) and 21 to 30 days (n = 11). Vehicle-treated rats (7 to 30 days, n = 11) were used as controls. Pulmonary inactivation of BK and conversion of Ang I were determined in conscious enalapril- or vehicle-treated rats before and after intravenous administration of the ACE inhibitor enalaprilat (MK-422, 10 mg/kg). Pulmonary inactivation of BK (%) was determined by comparing equipotent doses of BK injected by the intravenous and intraaortic routes, and Ang I conversion (%) by comparing the pressor effect of Ang I and Ang II injected intravenously. PEP-like activity in plasma and lung homogenates was determined fluorometrically using the synthetic substrate Suc-Gly-Pro-MCA. In control rats, pulmonary BK inactivation averaged 97.6% +/-0.54%. Acute ACE inhibition with MK-422 reduced BK inactivation to 42.0% +/- 2.7%. However, in rats treated chronically with enalapril, BK inactivation was increased as compared with acute ACE inhibition, averaging 58.8% +/- 3.7% at 7 to 15 days and 58.8% +/- 4.5% at 21 to 30 days of treatment. Intravenous administration of MK-422 to the enalapril-treated rats did not return the increased BK inactivation to the level observed during acute ACE inhibition. In contrast, Ang I conversion was significantly reduced from 46.7% +/- 6.5% to 0.9% +/-0.2% by MK-422, and this inhibition remained essentially unchanged during chronic treatment. PEP-like activity in plasma and lung homogenates of control rats was 4.4 +/- 0.3 nmol MCA/min/mL and 11.4 +/- 0.9 nmol MCA/min/mg protein, respectively. After chronic treatment with enalapril there was a progressive increase of PEP-like activity in both plasma and lung, which after 21 to 30 days of treatment averaged 10.7 +/- 1.7 nmol MCA/min/mL and 29.2 +/- 2.8 nmol MCA/min/mg protein, respectively. These data indicate that chronic ACE blockade induces alternative BK-inactivating mechanisms and increases Ang-(1-7)-generating mechanisms.
Assuntos
Angiotensina I/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Bradicinina/metabolismo , Animais , Enalaprilato/farmacologia , Pulmão/metabolismo , Masculino , Peptidil Dipeptidase A/metabolismo , Prolil Oligopeptidases , Ratos , Ratos Wistar , Serina Endopeptidases/metabolismoRESUMO
Phenylketonuria (PKU) is the most common of all aminoacidopathies and is caused by autosomal recessive deficiency of the hepatic phenylalanine hydroxylase system. The diagnosis of PKU should be multifactorial and based on a protein overload test that reveals increased plasma phenylalanine levels during the ingestion of a normal diet, a phenylalanine tolerance test, and in vitro and in vivo activity of the liver enzyme. An individualized diagnosis that characterizes the severity of the disease in each patient provides objective and effective criteria for the dietary treatment of each particular case.
Assuntos
Fenilcetonúrias/terapia , Animais , Dieta , Feminino , Humanos , Fígado/enzimologia , Fenilalanina/sangue , Fenilalanina Hidroxilase/deficiência , Fenilcetonúrias/diagnóstico , Fenilcetonúrias/enzimologia , GravidezRESUMO
Occasional urine samples from 200 stone-forming individuals were screened by the successive application of the cyanide-nitroprusside test, qualitative-semiquantitative thin-layer amino acid chromatography, and quantitative ion-exchange amino acid analysis to determine the frequency of cystinuria in this region of Brazil. Only 1 homozygous cystinuria patient was detected, a lower frequency than 1 to 6 per cent reported in other countries. The patient's family showed a I/I genotype. Since 6 heterozygotes for cystinuria +/II or +/III were also detected, the relative rarity of homozygotes in this sample supports the view of the relatively greater contribution of etiologic factors other than gene frequency to stone formation. The importance of diagnosis based on quantitative amino acid analysis is emphasized because of the different therapeutic and prognostic implications of the homozygote and heterozygote forms of the disease.
Assuntos
Cistinúria/epidemiologia , Cálculos Urinários/etiologia , Aminoácidos/urina , Brasil , Cistinúria/genética , Cistinúria/urina , Feminino , Heterozigoto , Homozigoto , Humanos , Masculino , Cálculos Urinários/urinaRESUMO
A rapid and inexpensive method is described for the enrichment of fetal hemoglobin (HbF) which eliminates the interference of other hemoglobins in the HPLC analysis of gamma chains when HbF is less than or equal to 20-30% of the total Hb. The enrichment procedure, which is carried out on 1 ml hemolysate, is based on the alkaline denaturation of the other Hbs followed by Zn2+ precipitation. Samples are injected into the HPLC apparatus without further treatment. The method was validated by HPLC analysis of hemolysates with high levels of HbF and mixtures prepared by diluting the high HbF hemolysates with adult hemolysates. The relative proportions of gamma chains as well as their chromatographic behavior were unaltered by the HbF enrichment procedure. The method is illustrated by the analysis of hemolysates of normal newborns and of patients with thalassemia, sickle cell diseases and aplastic anemia containing 3.4 to 100% HbF. For the four hemolysates containing greater than 20% HbF, the same quantitative and chromatographic results were obtained by direct analysis and after enrichment. Although reproducible and accurate results were obtained for the enrichment method and HPLC analysis when HbF was greater than or equal to 3%, at lower concentrations the variability of both was unacceptably high, indicating the need for additional or improved methodology for hemolysates containing very low levels of HbF.
Assuntos
Hemoglobina Fetal/isolamento & purificação , Globinas/isolamento & purificação , Adulto , Álcalis , Anemia Aplástica/sangue , Anemia Falciforme/sangue , Cromatografia Líquida de Alta Pressão , Hemólise , Humanos , Recém-Nascido , Conformação Proteica , Desnaturação Proteica , Talassemia/sangueRESUMO
Heterozygotes for cystinuria (types II and III) may be detected on the basis of slight to moderate elevations in urinary cystine, lysine and sometimes arginine. This seems to be a common genetic trait, and there are indications of an increased risk of urinary stone disease. In order to test the sensitivity and reliability of the procedures normally used for screening and diagnosis of heterozygous cystinuria, we studied 32 heterozygotes previously diagnosed by ion-exchange chromatography of urinary amino acids, and 23 healthy individuals. A random urine sample from each subject was analysed using the cyanide-nitroprusside test, thin-layer amino acid chromatography, colorimetric estimations of cystine and lysine, and ion-exchange amino acid chromatography. Thin-layer chromatography provided the highest sensitivity. Thus the frequency of heterozygotes calculated in previous studies, based on screening by the cyanide-nitroprusside test, may be under-estimated. The colorimetric estimations of cystine and lysine provided low sensitivity as screening tests and, compared with the ion-exchange chromatography, were unreliable diagnostic methods.