Correlation between vascular and cellular responses to atrial natriuretic peptide and to sodium nitroprusside

Atrial natriuretic peptide (ANP) and sodium nitroprusside (SNP) increase cGMP in vascular smooth muscle cells and act as vasodilators in some, but not all, blood vessels. In this present study, we attempted to correlate the ability of these two agents to dilate blood vessels with the ability to increase cGMP in cultured vascular smooth muscle cells. In the isolated guinea pig heart, SNP dose-dependently increased coronary flow while ANP was ineffective. In smooth muscle cells cultured from the coronary system, SNP increased intracellular cGMP in a dose-dependent manner while ANP had no effect on cGMP in these cells. In isolated guinea-pig thoracic aorta, precontracted with K+, both ANP and SNP produced relaxation and ANP was the more potent. In smooth muscle cells cultured from the aorta, ANP and SNP increased cGMP and the potency relationship was similar to the intact vessel. These results support the view that phenotypic properties of vascular smooth muscle cells can account for differences in the response of blood vessels to vasodilators.     

Maturation of the posterolateral spinal fusion and its effect on load-sharing of spinal instrumentation. An in vivo sheep model

We investigated the temporal relationship among the biomechanical, radiographic, and histological properties of a posterolateral spinal fusion mass to elucidate the changes in load-sharing of the spinal instrumentation and that of the fusion mass throughout the healing process. Destabilization of the posterior spinal column and transpedicular screw fixation at the segments between the third and fourth and the fifth and sixth lumbar vertebrae was performed in twenty-four sheep. A posterolateral spinal arthrodesis with use of autologous corticocancellous bone graft was done randomly at one of the two segments; the other segment (without bone graft) served as the instrumented control. Six animals each were killed at four, eight, twelve, and sixteen weeks postoperatively. Biomechanical testing showed that the posterolateral fusion mass had increased mechanical stiffness after the fourth week. The strain on the hardware, measured with use of rods instrumented with strain-gauges, decreased significantly (p < 0.01) beginning at eight weeks. Radiographically, three independent observations of each of the six animals at each time-period showed that, although all of the fusion masses were considered solid unions at sixteen weeks, bridging of trabecular bone was noted during only ten of eighteen observations at twelve weeks, three of eighteen observations at eight weeks, and none of eighteen observations at four weeks. Computerized tomography and histomorphometric analyses demonstrated that mineralization in the fusion mass increased in a linear fashion even after eight weeks. Histologically, the fusion mass consisted predominantly of woven bone at eight weeks; thereafter, it was gradually trabeculated. CLINICAL RELEVANCE: We found a great discrepancy between biomechanical stability and histological maturation of the posterolateral fusion mass. The biomechanical properties of a stable spinal fusion preceded the radiographic appearance of a solid fusion by at least eight weeks, suggesting that immature woven bone provided substantial stiffness to the fusion mass. The spinal instrumentation was subjected predominantly to bending stress rather than to axial stress, and the load-sharing of the spinal instrumentation decreased concurrently with the development of the spinal fusion.     

Effect of vitamin A supplementation on diarrhoea and acute lower-respiratory-tract infections in young children in Brazil

A beneficial effect of periodic vitamin A supplementation on childhood mortality has been demonstrated, but the effect on morbidity is less clear. We investigated the effect of vitamin A supplementation on diarrhoea and acute lower-respiratory-tract infections (ALRI) in children from northeastern Brazil in a randomised, double-blind, placebo-controlled community trial. 1240 children aged 6-48 months were assigned vitamin A or placebo every 4 months for 1 year. They were followed up at home three times a week, and data about the occurrence and severity of diarrhoea and ALRI were collected. Any child with cough and respiratory rate above 40 breaths per min was visited by a paediatrician. The overall incidence of diarrhoea episodes was significantly lower in the vitamin-A-supplemented group than in the placebo group (18.42 vs 19.58 x 10(-3) child-days; rate ratio 0.94 [95% Cl 0.90-0.98]). The benefit of supplementation was greater as regards severe episodes of diarrhoea; the incidence was 20% lower in the vitamin A group than in the placebo group (rate ratio 0.80 [0.65-0.98]). With the standard definition of diarrhoea (> or = 3 liquid or semi-liquid stools in 24 h) the effect of vitamin A on mean daily prevalence did not reach significance, but as the definition of diarrhoea was made more stringent (increasing number of stools per day), a significant benefit became apparent, reaching for diarrhoea with 6 or more liquid or semi-liquid stools in 24 h a 23% lower prevalence. We found no effect of vitamin A supplementation on the incidence of ALRI. The reduction in severity of diarrhoea may be the most important factor in the lowering of mortality by vitamin A supplementation.     

Use of the ARPE-19 cell line as a model of RPE polarity: basolateral secretion of FGF5

PURPOSE: To determine the polarity of fibroblast growth factor 5 (FGF5) secretions from retinal pigment epithelium (RPE) cells and to examine the viability and utility of the ARPE-19 cell line as a model for the study of RPE polarity. METHODS: Influenza infection and adenovirus-mediated gene transfer were used to deliver and express genes encoding influenza hemagglutinin (HA), p75-NTR (a neurotrophin receptor), low-density lipoprotein (LDL) receptor (LDLR), and FGF5 in confluent monolayers of ARPE-19 cells. The localization of HA, p75-NTR, and LDLR was determined by confocal microscopy. Domain selective biotinylation assays were used to quantitatively determine the polarities of p75-NTR and LDLR. The secretion of FGF5 into the apical and basal media of ARPE-19 cultures was examined by immunoblot analysis of conditioned media. RESULTS: Hemagglutinin and p75-NTR were found to be localized on the apical surface of infected and transduced ARPE-19 cells. In contrast, LDLR was associated preferentially with the basolateral membrane of ARPE-19 cells. Biotinylation studies indicated that 84% of p75-NTR was present on the apical surface, and 79% of LDLR was basolaterally polarized. Over the course of 6 hours, more than 90% of the total secreted FGF5 protein accumulated in the basolateral media. CONCLUSIONS: ARPE-19 cells exhibit a polarized distribution of cell surface markers when examined by either confocal microscopy or surface-labeling assays. This indicates that the ARPE-19 cell line is a valid model for studies of RPE cell polarity. FGF5, a secreted protein normally produced by RPE cells, is accumulated preferentially in the basal media after only 6 hours, suggesting that it is vectorially secreted from the basolateral surface of ARPE-19 cells.     

Antibodies against transforming growth factor-beta 1 suppress intimal hyperplasia in a rat model

Intimal hyperplasia is induced by therapeutic vascular interventions and often results in clinically important narrowing of the vascular lumen. Examination of the role of TGF-beta 1 in a rat carotid artery injury model confirmed the presence of a previously reported increase in TGF-beta 1 mRNA in the media of injured arteries. Administration of neutralizing anti- TGF-beta 1 antibodies significantly (P < 0.05) reduced the size of the intimal lesions that developed after carotid balloon injury. A control antibody had no effect. The intimal/medial area ratio was also reduced in the anti-TGF-beta 1 group relative to controls (P < 0.01). Immunohistochemical staining showed that two TGF-beta 1-induced extracellular matrix components, EDA + fibronectin and versican, were greatly increased in the untreated neointimal lesions, but were almost completely absent from the lesions of the anti-TGF-beta 1-treated animals. We conclude that TGF-beta 1 is causally involved in the development of intimal hyperplasia, and that anti-TGF-beta 1 agents may be useful in achieving at least partial control of this condition.     

Glucose plus insulin infusion improves cerebral outcome after asphyxial cardiac arrest

Hyperglycemia before ischemia worsens cerebral outcome. The aim of this study was to determine the cerebral effects of giving glucose with or without insulin after asphyxial cardiac arrest. Rats underwent 8 min of asphyxial cardiac arrest. After arrest, Group 1 received NaCl; Group 2, insulin; Group 3, glucose; and Group 4, glucose plus insulin, all intravenously. Neurological deficit (ND) scores were 14+/-10%, 22+/-12%, 12+/-10% and 2+/-2% in Groups 1-4, respectively, 72 h after reperfusion. Overall histological damage (HD) scores were 4, 2, 3 and 1, respectively. Group 4 fared significantly better than group 1 on both scores. Glucose after asphyxial cardiac arrest in rats produces no increased brain damage while glucose plus insulin improves cerebral outcome.     

Temperature-regulated expression of the tac/lacl system for overproduction of a fungal xylanase in Escherichia coli

Temperature-regulated expression of recombinant proteins in the tac promoter (Ptac) system was investigated. Expression levels of fungal xylanase and cellulase from N. patriciarum in E. coli strains containing the natural lacI gene under the control of the Ptac markedly increased with increasing cultivation temperature in the absence of a chemical inducer. The specific activities (units per milligram protein of crude enzyme) of the fungal xylanase and cellulase produced from recombinant E. coli strain pop2136 grown at 42 degrees C were about 4.5 times higher than those of the cells grown at 23 degrees C and were even slightly higher when compared with cells grown in the presence of the inducer isopropyl beta-D-thiogalactopyranoside. The xylanase expression level in the temperature-regulated Ptac system was about 35% of total cellular protein. However, this system can not be applied to E. coli strains containing lacIq, which confers over production of the lac repressor, for high-level expression of recombinant proteins. In comparison with the lambda PL system, the Ptac-based xylanase plasmid in E. coli pop2136 gave a considerably higher specific activity of the xylanase than did the best lambda PL-based construct using the same thermal induction procedure. The high-level expression of the xylanase using the temperature-regulated Ptac system was also obtained in 10-litre fermentation studies using a fed-batch process. These results unambiguously demonstrated that the temperature-modulated Ptac system can be used for overproduction of some non-toxic recombinant proteins.     

Breakfasts high in monoglyceride or triglyceride: no differential effect on appetite or energy intake

OBJECTIVE: To test whether isoenergetic isoenergetically-dense doses of dietary 1-monoglyceride or triglyceride differentially influence appetite and meal-to-meal energy intake in man. DESIGN: Six men and six women were each studied twice in a 2 d protocol. On day 1 (maintenance day) they were fed a medium fat (MF) maintenance diet (MF: 40% fat, 47% carbohydrate and 13% protein by energy) calculated at 1.6 x resting metabolic rate (RMR). On day 2 (manipulation day) at 08.30 h subjects consumed a high-fat breakfast designed to contain 80-85% of RMR, composition 10% protein, 56% fat and 34% carbohydrate by energy, with 65% of energy for fat derived as either 1-monoglyceride or triglyceride. Food and energy intake were monitored at lunch (given at 12.30pm) and throughout the remainder of the day. During this time subjects had ad libitum access to isoenergetic, isoenergetically dense MF (40:47:13) foods (550kJ/100g), until 22.30pm). Subjective hunger and satiety were tracked hourly, during waking hours. RESULTS: There was no significant effect of fat type on food or energy intake at lunch or during the ad libitum period. There was no diet effect on subjective hunger (F(1,10)0.00; P= 0.975) in the inter-meal periods of morning or afternoon, nor during the whole day. Subjects found both diets to be similarly pleasant (F(1,61)0.84;P= 0.364). Men and women responded similarly, except that men ate more on all occasions than women. CONCLUSIONS: This study suggests that when a large dose of l-monoglyceride or triglyceride is incorporated into a breakfast meal, it behaves in a manner that is very similar to triglyceride in terms of the effects on hunger, appetite or feeding behaviour.