Effect of 5-aminolevulinic acid on the expression of carcinogenesis-related proteins in cultured primary hepatocytes.

Acute intermittent porphyria (AIP) is a heme pathway disorder caused by a decrease in the activity and synthesis of porphobilinogen deaminase. Thus, the first heme precursor 5-aminolevulinic acid (ALA) accumulates in the liver. Reactive oxygen species (ROS) resulting from ALA oxidation may be correlated to a higher incidence of hepatocellular carcinoma (HCC) in AIP patients. However, the molecular mechanisms of this relationship have not been thoroughly elucidated to date. In this study, we investigated the effect of increasing levels of ALA on the expression of proteins related to DNA repair, oxidative stress, apoptosis, proliferation and lipid metabolism. Primary rat hepatocytes were isolated by the collagenase perfusion method, lipoperoxidation was evaluated by a TBA fluorimetric assay and Western blotting was used to assess protein abundance. The data showed that ALA treatment promoted a dose-dependent increase of p53 expression, downregulation of Bcl-2, HMG-CoA reductase and OGG1 and an increase in lipoperoxidation. There was no alteration in the expression of the transcription factor NF-κB, catalase and superoxide dismutase. ALA oxidation products induced protein regulation patterns, suggesting the interconnection of cellular processes, such as the intrinsic pathway of apoptosis, redox homeostasis, cell proliferation, lipid metabolism and DNA repair. This study helps to elucidate the molecular mechanisms of hepatotoxicity mediated by ALA pro-oxidant effects and supports the hypothesis that ALA accumulation correlates with a higher incidence of hepatic carcinogenic events.

The effects of high hydrostatic pressure at subzero temperature on the quality of ready-to-eat cured beef carpaccio.

We compared the application of high hydrostatic pressure (HHP) on unfrozen carpaccio (HHP at 20 °C) and on previously-frozen carpaccio (HHP at -30 °C). HHP at 20 °C changed the color. The pressure increase from 400 to 650 MPa and the time increment from 1 to 5 min at 400 MPa increased L* and b*. a* decreased only with 650 MPa for 5 min at 20 °C. The prior freezing of the carpaccio and the HHP at -30 °C minimized the effect of the HHP on the color and did not change the shear force, but increased expressible moisture as compared to the untreated carpaccio. HHP at 20 °C was more effective in reducing the counts of microorganisms (aerobic total count at 30 °C, Enterobacteriaceae, psychrotrophs viable at 6.5 °C and lactic acid bacteria) than HHP at -30 º C. With HHP at 20 °C, we observed a significant effect of pressure and time on the reduction of the counts.

Effect of the addition of conventional additives and whey proteins concentrates on technological parameters, physicochemical properties, microstructure and sensory attributes of sous vide cooked beef muscles.

Beef muscles submitted to four enhancement treatments (1.88% whey protein concentrate (WPC)+1.25% sodium chloride (NaCl); 1.88% modified whey protein concentrate (MWPC)+1.25%NaCl; 0.25% sodium tripolyphosphate (STPP)+1.25%NaCl; 1.25%NaCl) and a control treatment (non-injected muscles) were sous vide cooked. Muscles with STPP+NaCl presented a significantly higher total yield (106.5%) in comparison to those with WPC/MWPC+NaCl (94.7% and 92.9%, respectively), NaCl alone (84.8%) or controls (72.1%). Muscles with STPP+NaCl presented significantly lower shear force values than control ones; also, WPC/MWPC+NaCl added muscles presented similar values than those from the other treatments. After cooking, muscles with STPP+NaCl or WPC/MWPC+NaCl depicted compacted and uniform microstructures. Muscles with STPP+NaCl showed a pink colour, meanwhile other treatment muscles presented colours between pinkish-grey and grey-brown. STPP+NaCl added samples presented the highest values of global tenderness and juiciness. The addition of STPP+NaCl had a better performance than WPC/MWPC+NaCl. However, the addition of WPC/MWPC+NaCl improved total yield in comparison to NaCl added or control ones.

Activation of kinin B receptor triggers differentiation of cultured human keratinocytes.

BACKGROUND: Keratinocyte life span is modulated by receptors that control proliferation and differentiation, key processes during cutaneous tissue repair. The kinin B(1) receptor (B(1)R) has been reported in normal and pathological human skin, but so far there is no information about its role in keratinocyte biology. OBJECTIVES: To determine the consequence of kinin B(1)R stimulation on tyrosine phosphorylation, a key signalling mechanism involved in keratinocyte proliferation and differentiation. METHODS: Subconfluent primary cultures of human keratinocytes were used to investigate tyrosine phosphorylation, epidermal growth factor receptor (EGFR) transactivation, cell proliferation and keratinocyte differentiation. Cell proliferation was assessed by measuring bromodeoxyuridine incorporation whereas assessment of cell differentiation was based on the expression of filaggrin, cytokeratin 10 (CK10) and involucrin. RESULTS: The major proteins phosphorylated, after B(1)R stimulation, were of molecular mass 170, 125, 89 and 70 kDa. The 170- and 125-kDa proteins were identified as EGFR and p125(FAK), respectively. Phosphorylation was greatly reduced by GF109203X and by overexposure of keratinocytes to phorbol 12-myristate 13-acetate, indicating the participation of protein kinase C. B(1)R stimulation did not increase [Ca(2+)]i, but triggered EGFR transactivation, an event that involved phosphorylation of Tyr(845), Tyr(992) and Tyr(1068) of EGFR. B(1)R stimulation did not elicit keratinocyte proliferation, but triggered cell differentiation, visualized as an increase of filaggrin, CK10 and involucrin. Blockade of EGFR tyrosine kinase by AG1478, before B(1)R stimulation, produced an additional increase in filaggrin expression. CONCLUSIONS: The kinin B(1)R may contribute to keratinocyte differentiation and migration by triggering specific tyrosine signalling pathways or by interacting with the ErbB receptor family.

Effect of salt addition on sous vide cooked whole beef muscles from Argentina.

Sodium chloride (NaCl, 0-1.4%) and sodium tripolyphosphate (STPP, 0-0.5%) were added to Semitendinosus muscles and submitted to sous vide cooking at different temperatures (55-75°C). The effects of these three factors on pH, cooking loss, instrumental colour parameters, protein solubilization and distribution, and micro- and ultra-structure were evaluated. Quadratic surface responses equations were obtained from data (pH, cooking loss and colour parameters) as a function of the salts concentrations and cooking temperature. Both salts - alone or in combination - successfully reduced cooking loss. The best results were obtained for the combinations 0.25%STPP+1.20%NaCl and 0.25%STPP+0.70%NaCl, and temperatures between 60 and 65°C. Under these conditions, cooking loss was reduced close to 0%. pH was only dependent on STPP concentration, with a threshold concentration value of 0.25%. Temperature increment and NaCl addition produced a redness reduction. STPP incorporation recovered partially this parameter in comparison to non-added samples. Microscopy and SDS-PAGE results support the effect of the selected combinations of factors, suggesting that both salts together induced protein solubilization and gelation upon heating.

Effect of salt addition on the thermal behavior of proteins of bovine meat from Argentina.

Research was undertaken to investigate how the addition of sodium chloride (NaCl) and/or sodium tripolyphosphate (TPP) to sous vide cooked meat pieces produces an increase in water holding capacity (WHC). Semitendinosus muscles were injected to obtain tissue final concentrations of 0.70% NaCl, 0.25% TPP, 0.70% NaCl+0.25% TPP, and 1.20% NaCl+0.25% TPP. SDS-PAGE analysis showed increased protein solubilization in those treatments which included NaCl. Thermal analysis of whole muscles and isolated myofibrils showed the destabilizing effect of NaCl and a global stabilizing effect of TPP. Both salts together induced a destabilizing global effect, where TPP assisted NaCl in breaking the meat structure. It is suggested that the WHC increments are related to conformational changes in myofibrillar proteins and to the weakening of myofibrillar structure by the removal of myofibrillar proteins.

Optimization of whey protein concentrate and sodium chloride concentrations and cooking temperature of sous vide cooked whole-muscle beef from Argentina.

Response surface methodology was used to optimize the effect of cooking temperature (CT: 65-75°C) and the incorporation of whey protein concentrate (WPC: 0-3.5%) and sodium chloride (NaCl: 0-2.5%) on technological, physical and sensory characteristics of cooked whole-muscle beef. Post-injection weight loss diminished when NaCl concentration increased. Moreover, the increment of both additives produced a reduction of cooking loss. An opposite effect was observed with the increment of CT. As it was expected, a total yield improvement was achieved by increasing both ingredients and diminishing CT. Equivalent yields are achieved complementing both ingredients, meaning that if one ingredient concentration is reduced the other has to be increased. Shear force values were not affected by the studied factors. Instead, lightness was reduced by their increment. At 65°C, injected muscles had lower flavour and odour scores than control. At all CT analyzed, the incorporated brines improved juiciness and tenderness-related attributes. Present results recommend the use of a CT of 70°C and maxima WPC and NaCl concentrations of 2.6% and 1.9%, respectively.

Evolution of physiological and haematological parameters with training load in elite male road cyclists: a longitudinal study.

AIM: The aim of this study was to describe and evaluate physiological parameters as a control tool for the monitoring of training in a group of elite cyclists during one season of training. METHODS: The study is divided into two periods (winter or ''volume'' mesocycle and spring or ''intensity'' mesocycle) between the tests that they carried out in the laboratory, consisting of a ramp test to exhaustion (work load increases 25 W X min(-1)) and a maximal lactate steady state (MLSS) test on a cycle ergometer. Macronutrients and hematological variables were recorded during the test periods as were the volume and the intensity of training sessions during the whole period of the study. RESULTS: The physiological data were similar to those previously reported for professional cyclists (approximately 450 Watts, approximately 78 mL x kg(-1) x min(-1)) and the values for the MLSS also agree with previous studies (approximately 250 Watts). Subjects improved the first ventilatory threshold (VT(1)) (approximately 52% to approximately 60% VO(2max)) and the second ventilatory threshold (VT(2)) (approximately 82% to approximately 87% VO(2max)) after the first period of training even though its low intensity focused on the performance of VT(1) (77% training in ''zone 1'', under VT(1)). The MLSS improved after the first period (approximately 225 to approximately 250 Watts) and remained high in the second (approximately 255 Watts). High levels of creatine kinase (approximately 230 U x L(-1)) and urea (37 mg x L(-1)) were found, also a decrease in hemoglobin values (approximately 15.4 to approximately 14.7g x dL(-1)). CONCLUSION: The high level reached by the subjects after the first period of training suggests that two effort tests could be enough to plan training. On the other hand, the decrease in some red blood cell and nutrition parameters suggests that there should be greater control over them during the season.

Effect of whey protein concentrate and sodium chloride addition plus tumbling procedures on technological parameters, physical properties and visual appearance of sous vide cooked beef.

Beef muscles cooked by the sous vide system were evaluated for the effects of pre-injection tumbling, brine addition and post-injection tumbling on technological parameters, physical properties, visual appearance and tissue microstructure. The muscles were injected at 120% (over original weight) with a brine formulated to give a concentration of 3.5% whey protein concentrate and 0.7% sodium chloride on an injected raw product basis. Pre-injection tumbling did not affect most of the evaluated parameters. Brine addition reduced significantly the cooking and total weight losses. Total weight loss was 7.2% for injected muscles, and significantly higher (28.2%) for non-injected ones. Brine incorporation increased pH and reduced shear force values of cooked muscles. Extended post-injection tumbling (5rpm-10h) improved brine distribution and visual appearance, and also diminished the shear force values of cooked muscles. However, this treatment increased the weight losses of post-injection tumbling and cooking-pasteurization stages.

Relevamiento del adulto mayor en el área programática del Hospital Argerich (GCBA) / Survey of the greater adult in the programmatic area of the Argerich Hospital (GCBA)

Los problemas de salud más frecuentes fueron: Hipertensión arterial: 46 por ciento. Incontinencia urinaria: 30 por ciento. Enfermedad coronaria: 15 por ciento. Caídas: 15 por ciento. Obesidad: 12 por ciento. Problemas de salud no referidos por los pacientes: Incontinencia urinaria: 30 por ciento. Caídas: 37 por ciento. Constipación: 7 por ciento. Trastornos prostáticos: 7 por ciento. Elevada proporción de pacientes con Polofarmacia: Pudiendo ocasionar Caídas e Institucionalizaciones. La gran mayoría eran independientes para las actividades instrumentales de la vida diaria, dada su condición de ambulatorios. A partir de este relevamiento se sugieren nuevas líneas de trabajo: Corroborar la adherencia de los hipertensos a la medicación. Evaluar indicaciones de medicaciones y su utilidad terapéutica. Un elevado porcentaje de los pacientes evaluados no tenían médico de referencia. Actualmente continúan ingresando pacientes en el protocolo.

Relevamiento del adulto mayor en el área programática del Hospital Argerich (GCBA) / Survey of the greater adult in the programmatic area of the Argerich Hospital (GCBA)

Los problemas de salud más frecuentes fueron: Hipertensión arterial: 46 por ciento. Incontinencia urinaria: 30 por ciento. Enfermedad coronaria: 15 por ciento. Caídas: 15 por ciento. Obesidad: 12 por ciento. Problemas de salud no referidos por los pacientes: Incontinencia urinaria: 30 por ciento. Caídas: 37 por ciento. Constipación: 7 por ciento. Trastornos prostáticos: 7 por ciento. Elevada proporción de pacientes con Polofarmacia: Pudiendo ocasionar Caídas e Institucionalizaciones. La gran mayoría eran independientes para las actividades instrumentales de la vida diaria, dada su condición de ambulatorios. A partir de este relevamiento se sugieren nuevas líneas de trabajo: Corroborar la adherencia de los hipertensos a la medicación. Evaluar indicaciones de medicaciones y su utilidad terapéutica. Un elevado porcentaje de los pacientes evaluados no tenían médico de referencia. Actualmente continúan ingresando pacientes en el protocolo. (AU)

Polarized expression of the GFP-tagged rat V(1a) vasopressin receptor.

We investigated the targeting of the V(1a) receptor fused with the green fluorescence protein (V(1a)R-GFP) in polarized MDCK cells. Cells expressing V(1a)R-GFP displayed binding to vasopressin (AVP) and AVP-induced calcium responses, similar to cells expressing the wild-type V1a receptor. Interestingly, as with the wild-type V(1a)R, V(1a)R-GFP is preferentially distributed in the basolateral side of MDCK cells as monitored by confocal microscopy. Furthermore, AVP induced internalization of GFP-tagged receptors. Therefore, the GFP-tagged V(1a) receptor retains all the sorting signals of the wild-type receptor and offers an excellent system to elucidate the mechanisms of cell trafficking of V(1a) receptors.

Effect of calcium chloride marination on bovine Cutaneus trunci muscle.

The aim of this investigation was to determine the possibility of using calcium chloride solution in tough muscles (Cutaneus trunci) to reduce the aging period required to increase tenderness, without introducing undesirable flavors. Muscles were marinated in 0.25 M CaCl(2) solution for 2 h and after that aged for 0, 1, 2, 3, 4, 5 and 7 days. Tenderness was evaluated by the myofibrillar fragmentation index (MFI), Warner Bratzler shear force (WBS) and sensory panel evaluation. MFI values showed significant differences between treated and control samples aged for 1, 2 and 3 days (P<0.05). MFI values of treated samples aged 3 days were similar to those obtained for the control samples but aged seven days. WBS values were not significantly different between samples. Consumer panelists preferred treated samples aged 3 days to the control ones aged 7 days. It was concluded that calcium chloride treatment can be used in Cutaneus trunci muscle to reduce the aging time required to increase tenderness.

Hydrolysis of kininogens by degranulated human neutrophils and analysis of bradykinin as chemotactic factor for cells isolated from peripheral blood.

Human neutrophils play a pivotal role in acute inflammation including the regulation of vascular permeability. We have examined the capacity of neutrophil enzymes to hydrolyse human kininogens in vitro and have also explored the potentiality of bradykinin to induce chemotactic migration on neutrophils isolated from peripheral blood. Isolated neutrophils were stimulated with either f-Met-Leu-Phe, thrombin or silica particles coated with human IgG. Neutrophil enzymes obtained by degranulation produced, after 45 min of incubation with high and low molecular weight kininogens, the complete transformation of both proteins in polypeptides ranging from 20 to less than 10 kDa in molecular mass. Supernatants obtained from nonstimulated neutrophils did not modify the molecular size of kininogens. The assay used to test the chemoattractant capacity of synthetic bradykinin on human neutrophils showed that this peptide has no chemotactic activity on cells isolated from healthy subjects. Our results show that stimulation of human neutrophils with opsonized silica, thrombin and the chemotactic peptide f-Met-Leu-Phe induces release of kininogen-hydrolyzing enzymes from these cells.

Vasopressin stimulates tyrosine phosphorylation by activation of PKC in the rat smooth muscle cell line, A-10.

Arginine vasopressin (AVP)-induced tyrosine phosphorylation was studied in a rat smooth muscle cell line, A-10, by western blotting, using a monoclonal antibody against phosphotyrosine. AVP stimulated the phosphorylation of several cellular proteins of molecular mass 60-130 kDa in a time- and dose-dependent manner. Phosphorylation was mediated largely by V(1)receptor subtype since it was inhibited by selective V(1)antagonist and was only partially elicited by the V(2)agonist, desmopressin. Heterotrimeric G-proteins seemed to be involved in the phosphorylation mechanism because fluoraluminates, an activator of heterotrimeric G-proteins (and thus an uncoupler of the receptor-G-protein interaction) inhibited the AVP-induced phosphorylation. The protein kinase C (PKC) inhibitors: staurosporine, H7 and GF109203X are able to block the AVP-stimulated phosphorylation. The last of these has been shown to be one of the most selective inhibitors of PKC. These results indicate that PKC is upstream of the phosphorylation, a motion which is supported by the fact that the AVP-stimulated phosphorylation was downregulated by phorbol esters.

Vasopressin and bradykinin receptors in the kidney: implications for tubular function.

Vasopressin and bradykinin are two of the most important peptides in regulating vascular tone, water, and ionic balance in the body, and thus they play a key role in controlling blood pressure. In addition to being a potent vasoconstrictor, Vasopressin also has an antidiuretic activity in the kidney, whereas kinins regulate renal blood flow in addition to their vasodilatory and natriuretic activity. We review here the primary evidence for the localization of the vasopressin and kinin receptors and their role in ionic and water regulation in the kidney.

Immunolocalization of V1 vasopressin receptors in the rat kidney using anti-receptor antibodies.

By using immunocytochemical techniques we have been able to localize the V1 vasopressin receptor in the rat kidney. Immunoblotting using an antiserum raised against an affinity-purified vasopressin receptor showed a 55,000 daltons protein band that has a molecular mass similar to that of the liver V1 vasopressin receptor, as demonstrated by cross-linking studies. Immunoblotting of the antibody showed a band of 55,000 daltons in A-10 cells, which contains the V1 subtype, whereas it did not stain LLC-PK1 cells, which possess the V2 subtype, showing that the antibody recognizes the V1 vasopressin receptor. The immunostaining of kidney sections with this antiserum showed a strong reaction of the connecting tubules and cortical and medullary collecting ducts. The immunostaining pattern of connecting tubule and collecting duct cells was different, that is, the former showed a staining of both the apical and basal plasma membrane but also in the cytoplasm, whereas the latter showed a strong reaction mainly in the basolateral membrane. Immunostaining of consecutive serial sections with an antiserum raised against tissue kallikrein, an enzyme present exclusively in connecting tubules, and with the anti-receptor serum allowed us to show, for the first time, the presence of the vasopressin receptor in the connecting tubule cells and their absence in intercalated cells, the other cell type present in connecting tubules. These findings support experiments carried in the eighties on the release of renal tissue kallikrein by AVP.

Renal immunocytochemical distribution and pharmacological properties of the dual angiotensin II/AVP receptor.

We have recently characterized a novel angiotensin II/vasopressin (Ang II/AVP) dual receptor coupled to adenylate cyclase and responding with equal sensitivity to Ang II and AVP. To gain insight into putative renal physiological roles of the dual Ang II/AVP receptor, we determined its pharmacological binding properties and renal immunocytochemical distribution. The effective displacement of [3H]AVP by [1-deamino-Val14,D-Arg8]-vasopressin (DVDAVP), a specific antidiuretic AVP analogue, supports a V2-type AVP receptor characteristic of the Ang II/AVP receptor. Displacement of 125I-Ang II by losartan but not by PD 123319 defines the Ang II/AVP receptor as a novel AT1 receptor isoform coupled to adenylate cyclase, in contrast to prototype Ca(2+)-mobilizing AT1 receptors. Neither Ang II nor AVP displace each other, corroborating the predicted discrete binding domains for Ang II and AVP but presenting an enigma for the dissection of putative Ang II- and AVP-specific hierarchical roles of the dual Ang II/AVP receptor. The renal cytolocalization of the Ang II/AVP receptor to the outer medullary thick ascending limb tubules and inner medullary collecting ducts is consistent with the well-established AVP stimulation of sodium and water reabsorption in these tubules. These data suggest that the Ang II/AVP receptor might provide the molecular basis for the observed similar stimulatory effects of Ang II and AVP on renal tubular sodium and fluid reabsorption at physiological hormone concentrations.

Ontogeny of bradykinin B2 receptors in the rat kidney: implications for segmental nephron maturation.

Kinins modulate renal function, yet their role in the developing kidney is largely unknown. To explore the developmental role of the kallikrein-kinin system, we examined the postnatal ontogeny and intrarenal localization of B2 receptors in the rat. Northern blot analysis and RT-PCR documented the expression of B2 receptor mRNA in the kidney and extrarenal tissues of fetal, neonatal and adult animals. The abundance of B2 receptor mRNA is 10- to 30-fold higher in neonatal than adult tissues in the following order: kidney > heart > aorta > lung > brain. Receptor autoradiography revealed a gradual shift in the localization of bradykinin binding sites from the outer cortex in the newborn to the outer medulla in weanling and maturing rats. The almost complete displacement of [125I]tyr(zero)-bradykinin by HOE-140 indicates that the majority of kinin receptors in the developing kidney belong to the B2 type. Immunolocalization studies using antipeptide antibodies directed against various portions of the receptor revealed that B2 receptors are first expressed on the luminal aspect of the upper limb of S-shaped bodies and differentiating cortical collecting ducts. In marked contrast, the metanephric mesenchyme, pretubular aggregates and glomeruli display weak or no B2 receptor immunoreactivity. Following completion of nephrogenesis, B2 receptor expression shifts to both luminal and basolateral aspects of connecting tubules and collecting ducts. The results demonstrate that bradykinin B2 receptor gene expression is activated in the developing kidney and cardiovascular system. The spatially restricted expression of B2 receptors in the differentiating epithelium of the distal nephron, the site of kinin formation, supports the hypothesis that kinins are paracrine modulators of segmental nephron maturation.