RESUMO
Although many studies have been carried out in order to understand the implication of copper (Cu) in the pathogenesis of multiple sclerosis (MS), the exact role that this metal plays in the disease is not still clear. Because of the lack of information in this subject, the present study compared the serum and cerebrospinal (CSF) levels of copper in MS patients in respect to a control group, matched for age and sex, finding a significant increase of metal concentrations, in both biological fluids of MS subjects. To confirm the possible impairment of Cu metabolism, we analyzed ceruloplasmin (Cp) level and activity, seeing as this protein is an established peripheral marker in diseases associated with Cu imbalance. By comparing these two parameters between control and MS subjects, we found an increase of Cp levels, associated with a decrease in Cp activity, in the second group. By analysing these data, free copper levels were calculated, significantly increased in serum of MS subjects; the increase in free copper could be one of the predisposing factors responsible for the Cu altered levels in CSF of MS patients. At the same time, this alteration could be attributable to the inability to incorporate Cu by Cp, probably due to the high oxidative environment found in serum of MS patients. Overall, all these copper alterations may play a role in MS pathogenesis.
Assuntos
Ceruloplasmina/líquido cefalorraquidiano , Cobre/sangue , Cobre/líquido cefalorraquidiano , Esclerose Múltipla/sangue , Esclerose Múltipla/líquido cefalorraquidiano , Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Surface-enhanced Raman spectroscopy (SERS) allows a new insight into the analysis of cell physiology. In this work, the difficulty of producing suitable substrates that, besides permitting the amplification of the Raman signal, do not interact with the biological material causing alteration, has been overcome by a combined method of hydrothermal green synthesis and thermal annealing. The SERS analysis of the cell membrane has been performed with special attention to the cellular prion protein PrP(C). In addition, SERS has also been used to reveal the prion protein-Cu(II) interaction in four different cell models (B104, SH-SY5Y, GN11, HeLa), expressing PrP(C) at different levels. A significant implication of the current work consists of the intriguing possibility of revealing and quantifying prion protein expression in complex biological samples by a cheap SERS-based method, replacing the expensive and time-consuming immuno-assay systems commonly employed.
Assuntos
Misturas Complexas/análise , Perfilação da Expressão Gênica/métodos , Proteínas PrPC/análise , Proteínas PrPC/metabolismo , Análise Espectral Raman/métodos , Células HeLa , HumanosRESUMO
Molecular flexibility and rigidity are required to determine the function and specificity of protein molecules. Some psychrophilic enzymes demonstrate a higher catalytic efficiency at low temperatures, compared to the efficiency demonstrated by their meso/thermophilic homologous. The emerging picture suggests that such enzymes have an improved flexibility of the structural catalytic components, whereas other protein regions far from functional sites may be even more rigid than those of their mesophilic counterparts. To gain a deeper insight in the analysis of the activity-flexibility/rigidity relationship in protein structure, psychrophilic carbonic anhydrase of the Antarctic teleost Chionodraco hamatus has been compared with carbonic anhydrase II of Bos taurus through fluorescence studies, three-dimensional modeling, and activity analyses. Data demonstrated that the cold-adapted enzyme exhibits an increased catalytic efficiency at low and moderate temperatures and, more interestingly, a local flexibility in the region that controls the correct folding of the catalytic architecture, as well as a rigidity in the hydrophobic core. The opposite result was observed in the mesophilic counterpart. These results suggest a clear relationship between the activity and the presence of flexible and rigid protein substructures that may be useful in rational molecular and drug design of a class of enzymes playing a key role in pathologic processes.
Assuntos
Anidrases Carbônicas/química , Sequência de Aminoácidos , Animais , Bovinos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Luz , Modelos Moleculares , Dados de Sequência Molecular , Perciformes , Maleabilidade , Conformação Proteica , Espalhamento de Radiação , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Software , Espectrometria de Fluorescência , Temperatura , TermodinâmicaRESUMO
Physiological and health related responses to dietary inclusion of genetically modified (GM) full-fat soybean meal (Roundup Ready; GM-soy) and maize (MON810 Bt-maize; GM-maize), as well as non-parental, untransformed lines (nGM-soy and nGM-maize D2), were evaluated in farmed Atlantic salmon (Salmo salar L.) parr during the first 8 months of feeding. Significant effects of dietary GM presence were only found in intestinal Na+-dependent d-glucose uptake and SGLT1 protein level in the region pyloric caeca in which the highest values were found in the GM-soy, intermediate in the nGM-soy, and lowest in the standard FM fed groups. Data from this study confirm that GM soybeans (RRS) and maize (MON810) at inclusion levels of about 6% appear to be as safe as commercially available nGM soy and maize in diets for Atlantic salmon parr. Results from studies with higher inclusion levels and with non-modified, isogenic or near-isogenic parental lines as control groups are pending.
Assuntos
Ração Animal , Digestão/fisiologia , Alimentos Geneticamente Modificados , Glycine max , Sistema Imunitário/efeitos dos fármacos , Salmo salar/crescimento & desenvolvimento , Zea mays , Animais , Carboidratos da Dieta , Gorduras na Dieta , Proteínas Alimentares , Imunoglobulina M/sangue , Imunoglobulina M/efeitos dos fármacos , Salmo salar/imunologia , Glycine max/genética , Zea mays/genéticaRESUMO
Classical Hodgkin lymphoma (cHL) is a malignancy with complex pathogenesis. The hallmark of the disease is the presence of large mononucleated Hodgkin and bi- or multinucleated Reed/Sternberg (H/RS) cells. The origin of HRS cells in cHL is controversial as these cells show the coexpression of markers of several lineages. Using a proteomic approach, we compared the protein expression profile of cHL models of T- and B-cell derivation to find proteins differentially expressed in these cell lines. A total of 67 proteins were found differentially expressed between the two cell lines including metabolic proteins and proteins involved in the regulation of the cytoskeleton and/or cell migration, which were further validated by western blotting. Additionally, the expression of selected B- and T-cell antigens was also assessed by flow cytometry to reveal significant differences in the expression of different surface markers. Bioinformatics analysis was then applied to our dataset to find enriched pathways and networks, and to identify possible key regulators. In the present study, a proteomic approach was used to compare the protein expression profiles of two cHL cell lines. The identified proteins and/or networks, many of which not previously related to cHL, may be important to better define the pathogenesis of the disease, to identify novel diagnostic markers, and to design new therapeutic strategies.
Assuntos
Doença de Hodgkin/metabolismo , Proteoma , Proteômica , Linhagem Celular Tumoral , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Doença de Hodgkin/genética , Humanos , Modelos Biológicos , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Proteômica/métodosRESUMO
Bortezomib (bort) has improved overall survival in patients with multiple myeloma (MM), but the majority of them develop drug resistance. In this study, we demonstrate that bone marrow (BM) fibroblasts (cancer-associated fibroblasts; CAFs) from bort-resistant patients are insensitive to bort and protect the RPMI8226 and patients' plasma cells against bort-induced apoptosis. Bort triggers CAFs to produce high levels of interleukin (IL)-6, IL-8, insulin-like growth factor (IGF)-1 and transforming growth factor (TGF) ß. Proteomic studies on CAFs demonstrate that bort resistance parallels activation of oxidative stress and pro-survival autophagy. Indeed, bort induces reactive oxygen species in bort-resistant CAFs and activates autophagy by increasing light chain 3 protein (LC3)-II and inhibiting p62 and phospho-mammalian target of rapamycin. The small-interfering RNA knockdown of Atg7, and treatment with 3-methyladenine, restores bort sensitivity in bort-resistant CAFs and produces cytotoxicity in plasma cells co-cultured with CAFs. In the syngeneic 5T33 MM model, bort-treatment induces the expansion of LC3-II(+) CAFs. TGFß mediates bort-induced autophagy, and its blockade by LY2109761, a selective TßRI/II inhibitor, reduces the expression of p-Smad2/3 and LC3-II and induces apoptosis in bort-resistant CAFs. A combination of bort and LY2109761 synergistically induces apoptosis of RPMI8226 co-cultured with bort-resistant CAFs. These data define a key role for CAFs in bort resistance of plasma cells and provide the basis for a novel targeted therapeutic approach.
Assuntos
Antineoplásicos/farmacologia , Bortezomib/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Mieloma Múltiplo/tratamento farmacológico , Pirazóis/farmacologia , Pirróis/farmacologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Idoso , Idoso de 80 Anos ou mais , Animais , Autofagia/efeitos dos fármacos , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Combinação de Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Mieloma Múltiplo/genética , Mieloma Múltiplo/mortalidade , Mieloma Múltiplo/patologia , Plasmócitos/efeitos dos fármacos , Plasmócitos/metabolismo , Plasmócitos/patologia , Cultura Primária de Células , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Análise de Sobrevida , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Monitoring the fluorescence quenching of the pH-sensitive dye Acridine orange, proton accumulation in the presence of an inside-negative transmembrane potential was measured in eel (Anguilla anguilla) intestinal brush-border membrane vesicles. It was demonstrated that the proton accumulation was specifically increased by the presence of the dipeptide glycyl-glycine in the extravesicular space, showing saturation kinetics at increasing dipeptide concentrations and was specifically inhibited by diethylpyrocarbonate. Data reported suggest the presence of an electrical-potential-dependent H+/glycyl-glycine cotransport system in the eel intestinal brush-border membrane vesicles.
Assuntos
Glicilglicina/metabolismo , Mucosa Intestinal/metabolismo , Microvilosidades/metabolismo , Laranja de Acridina , Animais , Enguias , Concentração de Íons de Hidrogênio , Cinética , Prótons , Espectrometria de FluorescênciaRESUMO
The mechanism of Na+/L-proline cotransport, present on brush-border membrane (BBM) vesicles of the European eel intestine, was studied. Initial cotransport rates, depending on increasing proline and Na+ concentrations in the extravesicular medium (zero-trans conditions), were measured by monitoring the decay of an inside-negative membrane potential, i.e. the fluorescence quenching of the voltage-sensitive cyanine dye 3,3'-diethylthiacarbocyanine iodide (DiS-C2(5)). By simultaneously estimating the substrate-dependent Na(+)-influx (with the fluorescent dye) and the Na(+)-dependent [3H]substrate influx, it was concluded that proline was cotransported with 1 Na+ ion and glucose with 2 Na+ ions. The kinetics of proline/Na+ cotransport were then investigated. Graphical analysis excluded a ping-pong mechanism. Under rapid equilibrium assumptions, by fitting model equations to rate values it was possible to exclude the random and the ordered Na+/proline mechanisms. Therefore, in eel intestinal BBM vesicles, the mechanism of proline/Na+ cotransport is ordered and prolineout binds to the carrier prior to Na+out.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Proteínas de Transporte/metabolismo , Mucosa Intestinal/metabolismo , Prolina/metabolismo , Sódio/metabolismo , Anguilla , Animais , Transporte Biológico , Glucose/metabolismo , Técnicas In Vitro , Cinética , Potenciais da Membrana , Microvilosidades/metabolismoRESUMO
In this paper we have tested two different procedures (the "three-step" and the "four-step" procedures) for the covalent immobilization of glutamate dehydrogenase (GDH) onto silicon supports. Atomic force microscopy (AFM), Fourier-transform infrared spectroscopy (FT-IR), fluorescence spectroscopy and an enzymatic assay were used to probe the structure and activity of the immobilized enzyme. Our results demonstrate that coupling through the "three-step" procedure does not significantly affect either the fold pattern or the activity of the enzyme, suggesting that this method could be ideally suited to the development of high quality monolayers for use in enzyme-based planar biosensors.
Assuntos
Técnicas Biossensoriais/instrumentação , Enzimas Imobilizadas , Glutamato Desidrogenase , Silício , Microscopia de Força Atômica , NAD/metabolismo , Oxirredução , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The monoclonal antibody 6313/G2 raised against the mammalian type I (AT1) angiotensin II (Ang II) receptor (Ang II-R) also recognises a component in teleost (eel) tissue preparations that binds radiolabelled Ang II, and has an isoelectric point (pI) of 6.5 and molecular mass of 75 kDa. Immunohistochemical analysis using this antibody showed specific binding sites in eel intestine, kidney, gill and liver sections. The same antibody was used here to evaluate the presence and distribution of Ang II-R in target tissues of the Antarctic teleost icefish (Chionodraco hamatus). Immunocytochemistry of intestine and gill sections showed that the antibody bound to uniformly distributed intracellular sites and cell surface membranes in absorptive cells in the intestine and chloride and pavement cells in the gills. It also stained endothelium and both the longitudinal and circular layers of smooth muscle cells in the intestine. In the kidney, only the tubules in the trunk stained positively while the head (atubular part of the kidney) was negative. In kidney tubules, in contrast with other tissues, the receptor was most concentrated in the cytoplasm underlying the basolateral membranes, with somewhat weaker staining beneath the apical cell membrane. Immunoblotting identified a single component from trunk kidney preparations that focused at pI 5.9 in isoelectric focusing gels and showed a molecular mass of 75 kDa in SDS-polyacrylamide gels. The data suggest that, as in other teleosts, Ang II has a physiological role in the icefish.
Assuntos
Angiotensina II , Peixes/metabolismo , Brânquias/química , Intestinos/química , Túbulos Renais/química , Receptores de Angiotensina/análise , Animais , Citoplasma/química , Eletroforese em Gel de Poliacrilamida , Imuno-Histoquímica , Focalização IsoelétricaRESUMO
This paper describes the formation of glutamate dehydrogenase monolayers on silicon dioxide, and their characterization by means of physical techniques, i.e., fluorescence spectroscopy and Fourier-transform infrared spectroscopy. Detailed investigations of the intrinsic stability of native proteins in solution were carried out to elucidate the occurrence of conformational changes induced by the immobilization procedure. The enzyme monolayers were deposited on SiO2 after preexposing silicon surfaces to 3-aminopropyltriethoxysilane and reacting the silylated surfaces with glutaric dialdehyde. The optical characterization demonstrates that the immobilization does not interfere with the fold pattern of the native enzyme. In addition, fluorescence spectroscopy, thermal denaturation, and quenching studies performed on the enzyme in solution well describe the folding and unfolding properties of glutamate dehydrogenase. The photophysical studies reported here are relevant for nanobioelectronics applications requiring protein immobilization on a chip.
Assuntos
Glutamato Desidrogenase/química , Dióxido de Silício/química , Silício/química , Fenômenos Biofísicos , Biofísica , Luz , Propilaminas , Conformação Proteica , Espalhamento de Radiação , Silanos/química , Espectrometria de Fluorescência , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , Fatores de Tempo , Triptofano/químicaRESUMO
The current therapy for ovarian cancer has advanced from alkylating agents, to a combination of carboplatinum and paclitaxel offering increased survival. Although most patients respond to this first-line therapy, initially, the majority of these patients relapse within 2 years. The mechanisms responsible for acquired drug resistance in ovarian cancer have been elucidated only in part. They include i) enhanced drug export, ii) activation/inhibition of intracellular signalling pathways, iii) molecular alterations in tubulin isotype composition. A better understanding of these mechanisms is needed, in order to develop new approaches, aimed at overcoming resistance to anticancer agents, and to reveal the complexity of causes, which contribute to drug resistance. In this review we offer an updated overview of proteomic studies on the molecular mechanisms of paclitaxel resistance. These proteomic studies also identify potential targets for modulating drug resistance, that could be predictive of response to chemotherapy in ovarian carcinomas.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Paclitaxel/uso terapêutico , Proteômica/métodos , Biomarcadores Tumorais/análise , Feminino , Humanos , Tubulina (Proteína)/metabolismoRESUMO
This cross-sectional study investigated with two-dimensional gel electrophoresis coupled to MALDI-TOF and MRI the relationship between PBMCs protein expression profile and whole-brain atrophy in 16 unselected RR-MS IFN-treated patients compared with 6 RR IFN-untreated and 12 matched healthy control subjects. Grey/white matter fraction, T1/T2 lesion load and clinical variables were considered too. Twenty six proteins showed significant differential expression among RR IFN-treated patients and control samples. Four of these (IN35, GANAB, PP1B, SEPT2) resulted correlated with clinical and MRI findings in RR IFN-treated MS patients. Future clinical applications remain to be validated by other techniques and confirmed by a larger study.
Assuntos
Atrofia/patologia , Encéfalo/patologia , Leucócitos Mononucleares/metabolismo , Esclerose Múltipla Recidivante-Remitente/metabolismo , Esclerose Múltipla Recidivante-Remitente/patologia , Adulto , Idoso , Atrofia/fisiopatologia , Encéfalo/fisiopatologia , Estudos Transversais , Progressão da Doença , Eletroforese em Gel Bidimensional , Feminino , Humanos , Leucócitos Mononucleares/imunologia , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/imunologia , Fibras Nervosas Mielinizadas/patologia , Monoéster Fosfórico Hidrolases/análise , Monoéster Fosfórico Hidrolases/metabolismo , Projetos Piloto , Valor Preditivo dos Testes , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Adulto JovemRESUMO
Intestinal nutrient absorption in fish adapted to low temperature was investigated by isolating, with a Mg(2+)-precipitation procedure, brush-border membrane vesicles (BBMVs) from intestines of the Antarctic teleost Trematomus bernacchii. In particular, D-glucose transport was analyzed by measuring both 1) fluorescence changes of the electrical potential-sensitive dye 3,3'-diethylthiadicarbocyanine iodide [DiS-C2(5)] and 2) intravesicular uptake of D-[14C]glucose. Results demonstrated that transport of D-glucose across intestinal BBMs of the Antarctic fish is stimulated by the presence of a transmembrane Na+ gradient (out > in) and was specifically inhibited by phloridzin. Furthermore, Na(+)-dependent D-glucose uptake was strongly enhanced by the presence of an electrical potential (inside-negative) across the membrane. There was a marked difference in temperature dependence of Na(+)-sugar cotransport between the Antarctic fish and a temperate fish, such as the European yellow eel., Na(+)-dependent D-glucose uptake in T. bernacchii intestinal BBMV reached its maximal rate at -2-0 degree C (close to fish living temperature) and was exponentially inactivated by incubation at higher temperatures. Kinetic analysis of D-glucose influx indicated the presence of a single Na(+)-dependent carrier process (apparent maximal carrier-mediated influx = 0.233 +/- 0.009 nmol.mg protein-1.min-1; apparent half-saturation constant for carrier-mediated influx = 0.157 +/- 0.026 mmol/l) and a nonsaturable transfer component (apparent diffusional permeability of membrane to the sugar = 0.233 +/- 0.016 microliter.mg protein-1.min-1). The Na(+)-dependent carrier-mediated mechanism was specific for sugars, since it was partially inhibited by the presence in the extravesicular medium of other monosaccharides, but not by ascorbic acid or amino acids of different types. These data suggest that in the intestine of Antarctic fish luminal D-glucose transport takes place by a specific Na(+)-dependent electrogenic secondary active transport working well at subzero temperatures.
Assuntos
Peixes/metabolismo , Mucosa Intestinal/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Anguilla/metabolismo , Fenômenos Fisiológicos da Nutrição Animal , Animais , Regiões Antárticas , Ligação Competitiva , Transporte Biológico , Metabolismo dos Carboidratos , Glucose/farmacocinética , Intestinos/enzimologia , Cinética , Microvilosidades/enzimologia , Microvilosidades/metabolismo , Sódio/fisiologia , Transportador 1 de Glucose-Sódio , Especificidade por Substrato , TemperaturaRESUMO
L-[3H]lysine uptake was measured in brush-border membrane vesicles prepared from intestinal mucosa of the European eel Anguilla anguilla. Lysine uptake occurred via 1) a nonsaturable component with an apparent diffusional permeability (P) of 0.58 microliter.mg protein-1.min-1,2) a Na-dependent transport system [half-saturation constant (Kapp) 0.16 mM, maximal transport rate (Jmax) 3.57 nmol.mg protein-1.min-1]; 3) a Na-independent transport system (Kapp 0.17 mM, Jmax 2.77 nmol.mg protein-1.min-1). Both carrier-mediated processes were accelerated by the presence of an intravesicular negative membrane potential. Hill analysis of L-lysine influx, over a wide range of external Na concentrations, resulted in a Hill coefficient (n) of approximately 2, suggesting that two or more Na ions may be associated with amino acid transport. The inhibition of lysine uptake by other amino acids was studied. Na-dependent lysine uptake was competitively inhibited by proline [inhibitory constant (Ki) approximately 2 mM] and may occur by a system specific for cationic amino acids. Na-independent lysine uptake was competitively inhibited by alanine (Ki approximately 1 mM) and may occur by a classic L system.
Assuntos
Aminoácidos/farmacologia , Mucosa Intestinal/metabolismo , Lisina/farmacocinética , Animais , Transporte Biológico , Interações Medicamentosas , Enguias , Intestinos/fisiologia , Íons , Cinética , Potenciais da Membrana , Microvilosidades/metabolismo , Concentração Osmolar , Sódio/farmacologiaRESUMO
Transport of L-ascorbate by intestinal brush-border membrane vesicles of European eel Anguilla anguilla was stimulated by a transmembrane Na gradient (out > in) but not by a similarly directed gradient of K. Under short-circuited membrane potential conditions, a kinetic analysis of L-ascorbate influx indicated the presence of a single Na-dependent carrier process (Kapp = 0.75 +/- 0.07 mM and Jmax = 0.33 +/- 0.03 nmol.mg protein-1.min-1) and a nonsaturable transfer component with an apparent diffusional permeability (P) of 0.27 +/- 0.02 microliter.mg protein-1.min-1. D-Isoascorbate was a competitive inhibitor of L-ascorbate influx exhibiting a Ki of 8.21 +/- 0.63 mM. The electrogenic nature of +Na-L-ascorbate cotransport was confirmed by a stimulatory effect of an inside-negative membrane potential on vitamin uptake. Hill analysis of L-ascorbate influx over a wide range of external Na concentrations suggested a 2 Na-to-1 L-ascorbate binding ratio. Results indicate that the vitamin L-ascorbate is transported across fish intestinal brush-border membranes by an electrogenic Na-dependent carrier process in conjunction with more than one sodium ion.
Assuntos
Ácido Ascórbico/farmacocinética , Enguias/metabolismo , Mucosa Intestinal/metabolismo , Animais , Ácido Ascórbico/antagonistas & inibidores , Transporte Biológico/efeitos dos fármacos , Cátions/farmacologia , Intestinos/fisiologia , Potenciais da Membrana , Microvilosidades/metabolismo , Modelos Biológicos , Concentração Osmolar , Sódio/farmacocinética , Sódio/farmacologia , Fatores de TempoRESUMO
Carbonic anhydrase (CA) activity was measured in blood, intestine, kidney and gill of two Antarctic teleosts, the haemoglobinless Chionodraco hamatus and the red-blooded Trematomus bernacchii, and of the temperate teleost Anguilla anguilla. In all species, the highest CA activity was in the gills, with the greatest activity in C. hamatus. CA activity in the blood was highest in A. anguilla, but none was detected in the blood of C. hamatus despite the presence of plasma CA inhibitors. The enzyme was present but its activity was low in the intestine and kidney of all three species. The existence of very high CA activity in C. hamatus gills compared with the red-blooded species was investigated further by isolating and characterising the branchial cytosolic CA isoforms. The turnover rate of the C. hamatus isoform was significantly higher than that of T. bernacchii and A. anguilla. The isoforms from both the Antarctic species exhibited lower apparent K(m) (K(m,app)) and heat stability than those from A. anguilla. Sensitivity to sulphonamides was similar in all species and was within the range of the mammalian CA II isoform. The branchial CA isoforms of C. hamatus, T. bernacchii and A. anguilla displayed relative molecular masses of 28.9, 29.9 and 31.2 kDa, respectively. The results suggest that the hemoglobinless teleost possesses a different branchial cytosolic CA isoform from that of red-blooded teleosts.
Assuntos
Anguilla/metabolismo , Anidrases Carbônicas/análise , Peixes/metabolismo , Animais , Regiões Antárticas , Anidrases Carbônicas/sangue , Brânquias/enzimologia , Hemoglobinas/análise , Intestinos/enzimologia , Rim/enzimologiaRESUMO
Brush-border membrane vesicles (BBMV) were prepared from European eel (Anguilla anguilla) intestinal epithelium by a magnesium-ethylene glycolbis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) precipitation technique. Amino acid transport by these purified vesicle preparations was investigated using either radiolabeled substrates or the voltage-sensitive fluorescent dye 3,3'-diethylthiadicarbocyanine iodide [DiSC2(5)]. All amino acids tested exhibited carrier-mediated, Na+-dependent and Na+-independent transfer processes plus diffusion. The only exceptions were glutamic acid and proline, which displayed Na+ dependency and diffusion but did not appear to be transported by Na+-independent agencies. Carrier-mediated transport kinetic constants (Kapp and Jmax) for several amino acids are reported. Cis-inhibition experiments suggested the presence of at least four distinct Na+-dependent transport systems in eel intestinal BBMV: 1) an anionic transport process for glutamic and aspartic acids; 2) a cationic mechanism for lysine and arginine; 3) a relatively specific neutral amino acid carrier for proline and alpha-(methylamino)isobutyric acid; and 4) a nonspecific neutral amino acid system for most other substrates of this group. This scheme for carnivorous fish intestine most closely approximates that reported for mammalian gut with minor dissimilarities that may relate to metabolic differences or specific dietary requirements of the two vertebrate groups.
Assuntos
Aminoácidos/metabolismo , Enguias/metabolismo , Mucosa Intestinal/metabolismo , Animais , Transporte Biológico , Fenômenos Biomecânicos , Técnicas In Vitro , Intestinos/ultraestrutura , Microvilosidades/metabolismo , Sódio/farmacologiaRESUMO
H(+)/peptide cotransport in brush-border membrane vesicles (BBMVs) from eel (Anguilla anguilla) intestine was studied by measuring d-[(3)H]-phenylalanyl-l-alanine uptake and by monitoring peptide-dependent intravesicular acidification using the pH-sensitive dye Acridine Orange. d-[(3)H]-phenylalanyl-l-alanine influx was greatly stimulated by an inside-negative membrane potential and enhanced by an inwardly directed H(+) gradient. In parallel, vesicular H(+) influx was significantly increased in the presence of extravesicular d-phenylalanyl-l-alanine or a series of glycyl and l-prolyl peptides. H(+)/peptide cotransport displayed saturable kinetics involving a single carrier system with apparent substrate affinities of 0.9-2.6 mmol l(-1) depending on the particular peptide. All substrates tested competed with this system. Pre-incubation of BBMVs with dipeptides prevented diethylpyrocarbonate inhibition of transport activity, suggesting that the substrates mask histidine residues involved in the catalytic function of the transporter. Using human PepT1-specific primers, a reverse transcription-polymerase chain reaction (RT-PCR) signal was detected in eel intestine. Our results suggest that, in eel intestine, a brush-border membrane 'low-affinity'-type H(+)/peptide cotransport system is present that shares kinetic features with the mammalian intestinal PepT1-type transporters.
Assuntos
Anguilla/metabolismo , Caderinas , Proteínas de Transporte/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de Membrana Transportadoras , Simportadores , Anguilla/genética , Animais , Sequência de Bases , Transporte Biológico Ativo , Proteínas de Transporte/genética , Primers do DNA/genética , Dipeptídeos/metabolismo , Humanos , Cinética , Microvilosidades/metabolismo , Transportador 1 de Peptídeos , Peptídeos/metabolismo , PrótonsRESUMO
In both herbivorous tilapia (Oreochromis mossambicus) and carnivorous rockfish (Sebastes caurinus) intestinal and pyloric cecal brush-border membrane vesicles (BBMV), [14C]glycylsarcosine ([14C]Gly-Sar) uptake was stimulated by a transmembrane proton gradient. A transmembrane K(+)-diffusion potential (inside negative) stimulated [14C]Gly-Sar uptake above that observed with short-circuited vesicles, whereas an inwardly directed Na+ gradient in both fishes had no effect on peptide uptake. In tilapia, [14C]Gly-Sar influx occurred by the combination of 1) a high-affinity, saturable, proton gradient-dependent carrier system [Kt [concentration that equals one-half of maximum influx (Jmax)] = 0.56 +/- 0.08 mM; Jmax = 1,945.0 +/- 174.6 pmol.mg protein-1.10 s-1]; 2) a low-affinity, nonsaturable (within 1-10 mM), proton gradient-dependent carrier system (nonsaturable carrier-mediated transport component = 4,514.0 +/- 28.1 pmol.mg protein-1.10 s-1.mM-1); and 3) a diffusional component accounting for < 10% of total influx within the concentration range tested. Influx (10 s) of 1-10 mM [14C]Gly-Sar in tilapia intestine was significantly (P < 0.01) inhibited by 10 mM diethylpyrocarbonate, a specific inhibitor of proton-coupled peptide transport systems. [14C]Gly-Sar influx into tilapia BBMV showed cis-inhibition and trans-stimulation by Gly-Pro, suggesting that [14C]Gly-Sar and Gly-Pro shared the same mucosal peptide transporter in fish. These observations strongly suggest that intestinal transport of peptides in herbivorous and carnivorous fishes is proton gradient dependent, electrogenic, sodium independent, and qualitatively resembles the peptide transport paradigm proposed for mammals.