Biomedical subjects

F Alvarado

Publications and source records attributed to F Alvarado.

At least 19 recordsLinked to original sources

PubMed2006

Sudden deterioration in nonclassical infantile-onset Pompe disease responding to alglucosidase alfa infusion therapy: a case report.

A patient with atypical infantile Pompe disease suffered acute respiratory insufficiency at the age of 8 years which resulted in complete immobilization and dependence on assisted ventilation. Shortly after initiation of enzyme replacement therapy, she regained her mobility and, after 20 months of treatment, she now leads an almost normal life with limited restrictions.

Child↗

PubMed2000

Anticoagulation and minor oral surgery: should the anticoagulation regimen be altered?

PURPOSE: This study was performed to assess the extent of bleeding in anticoagulated patients undergoing minor oral surgery procedures when compared with patients who stop their anticoagulation regimen before surgery and patients who have never been anticoagulated. MATERIALS AND METHODS: Patients on anticoagulant medications undergoing dentoalveolar surgery procedures either stopped their anticoagulation regimen 72 to 96 hours before the planned surgical intervention or continued their regular regimen throughout the time of surgery. Blood loss was measured by weighing sponges used in the procedures, and groups were compared for differences in blood loss. They were also compared with an additional control group that had never been on anticoagulant therapy. RESULTS: There was no difference in blood loss detected among any of the experimental or control groups. No bleeding complications occurred in any anticoagulated patient. CONCLUSIONS: The data suggest that many patients can safely undergo routine outpatient oral surgical procedures without alteration of their regular therapeutic anticoagulation regimens and without additional medical intervention. However, a larger experimental population may be needed to elucidate the appropriateness of this approach to perioperative care.

Analysis of Variance↗

PubMed2000

Direct inhibitory effect of rotavirus NSP4(114-135) peptide on the Na(+)-D-glucose symporter of rabbit intestinal brush border membrane.

The direct effect of a rotavirus nonstructural glycoprotein, NSP4, and certain related peptides on the sodium-coupled transport of D-glucose and of L-leucine was studied by using intestinal brush border membrane vesicles isolated from young rabbits. Kinetic analyses revealed that the NSP4(114-135) peptide, which causes diarrhea in young rodents, is a specific, fully noncompetitive inhibitor of the Na(+)-D-glucose symporter (SGLT1). This interaction involves three peptide-binding sites per carrier unit. In contrast, the Norwalk virus NV(464-483) and mNSP4(131K) peptides, neither of which causes diarrhea, both behave inertly. The NSP4(114-135) and NV(464-483) peptides inhibited Na(+)-L-leucine symport about equally and partially via a different transport mechanism, in that Na(+) behaves as a nonobligatory activator. The selective and strong inhibition caused by the NSP4(114-135) peptide on SGLT1 in vitro suggests that during rotavirus infection in vivo, NSP4 can be one effector directly causing SGLT1 inhibition. This effect, implying a concomitant inhibition of water reabsorption, is postulated to play a mechanistic role in the pathogenesis of rotavirus diarrhea.

Amino Acid Sequence↗

PubMed2000

Rotavirus infection impairs intestinal brush-border membrane Na(+)-solute cotransport activities in young rabbits.

The mechanism of rotavirus diarrhea was investigated by infecting young, specific pathogen-free, New Zealand rabbits with a lapine rotavirus, strain La/RR510. With 4-wk-old animals, virus shedding into the intestinal lumen peaked at 72 h postinfection (hpi), and a mild, watery diarrhea appeared at 124 hpi. No intestinal lesions were seen up to 144 hpi, indicating that diarrhea does not follow mucosal damage but can precede it, as if cell dysfunction were the cause, not the consequence, of the histological lesions. Kinetic analyses with brush-border membrane vesicles isolated from infected rabbits revealed strong inhibition of both Na(+)-D-glucose (SGLT1) and Na(+)-L-leucine symport activities. For both symporters, only maximum velocity decreased with time. The density of phlorizin-binding sites and SGLT1 protein antigen in the membrane remained unaffected, indicating that the virus effect on this symporter is direct. Because SGLT1 supports water reabsorption under physiological conditions, the mechanism of rotavirus diarrhea may involve a generalized inhibition of Na(+)-solute symport systems, hence, of water reabsorption. Massive water loss through the intestine may eventually overwhelm the capacity of the organ for water reabsorption, thereby helping the diarrhea to get established.

Age Factors↗

PubMed1999

Heterogeneity of pig intestinal D-glucose transport systems.

Heterogeneity of intestinal D-glucose transport is demonstrated using pig jejunal brush-border membrane vesicles in the presence of 100/0 (out/in) mM gradients each of NaCl, NaSCN, and KSCN. Two D-glucose transport systems are kinetically distinguished: high-affinity, low-capacity system 1, which is equivalent to the symporter SGLT1; and low-affinity, high-capacity system 2, which is not a member of the SGLT family but is a D-glucose and D-mannose transporter exhibiting no preference for Na(+) over K(+). A nonsaturable D-glucose uptake component has also been detected; uptake of this component takes place at rates 10 times the rate of components characterizing the classical diffusion marker L-glucose. It is also shown that, in this kinetic work, 1) use of D-glucose-contaminated D-sorbitol as an osmotic replacement cannot cause the spurious appearance of nonexistent transport systems and 2) a large range (>/=50 mM) of substrate concentrations is required to correctly fit substrate saturation curves to distinguish between low-affinity transport systems and physical diffusion.

Animals↗

PubMed1998

Prevention of nosocomial infection in a pediatric intensive care unit (PICU) through the use of selective digestive decontamination.

OBJECTIVE: To assess the effectiveness of selective digestive decontamination (SDD) on the control of nosocomial infection (NI) in critically ill pediatric patients. DESIGN: A prospective, randomized, non-blinded and controlled clinical microbiology study. SETTING: The pediatric intensive care unit (PICU) of a tertiary level pediatric university hospital. CRITERIA FOR INCLUSION: Patients 1 month to 14 years old, who underwent some kind of manipulation or instrumentation (mechanical ventilation, vascular cannulation, monitoring of intracranial pressure, thoracic or abdominal drainage, bladder catheterization, peritoneal dialysis, etc.) and/or presented a neurological coma requiring a stay in the PICU of 3 or more days. PATIENTS: Over a period of 2 years, 244 patients met the inclusion criteria; 18 patients were withdrawn because of protocol violation. The treatment group comprised 116 patients and the control group, 110 patients. INTERVENTION: The treatment group received a triple therapy of colimycin, tobramycin and nystatin administered orally or via nasogastric tube every 6 hours. All patients with mechanical ventilation or immune-depression received decontamination treatment of the oropharyngeal cavity with hexitidine (Oraldine 0.5 mg/ml) every 6-8 hours in accordance with the PICU's conventional protocol. METHOD: Up to 10 types of nosocomial infection were diagnosed following criteria of the Centers for Disease Control (CDC). The severity and manipulation of the patients on admission was assessed using the therapeutic intervention scoring system (TISS) and multi-organ system failure scores (MOSF). MEASUREMENTS AND MAIN RESULTS: UNIVARIANT ANALYSIS: SDD did not significantly reduce the incidence of NI, antibiotic use, the length of stay, or mortality; although a small percentage of respiratory and urinary tract infections was detected, catheter-related bacteremia was the most common infection. MULTIVARIANT ANALYSIS: Controlling the risk factors for each child through log regression showed that SDD acted as a protective factor for more than 90% of the sample with respect to the appearance of respiratory and urinary tract infections, reducing the risk of such infections to 1/5 and 1/3, respectively. CONCLUSIONS: SDD was effective in controlling respiratory and urinary tract infections in children admitted to the PICU, but it did not reduce the incidence of other types of nosocomial infection.

Adolescent↗

PubMed1998

Direct inhibitory effect of CCCP on the Cl(-)-H+ symporter of the guinea pig ileal brush-border membrane.

The effect of carbonyl cyanide-m-chlorophenylhydrazone (CCCP) on Cl- uptake across the brush-border membrane (BBM) was quantified using 36Cl and BBM vesicles from guinea pig ileum. CCCP inhibited only partially both the pH gradient-activated Cl- uptake and Cl-/Cl- exchange activities present in these vesicles. In contrast, CCCP had no effect on the initial (2-30 s) decay rate of an imposed proton gradient, as determined using the pH-sensitive fluorophore pyranine. Taken together, these results strongly indicate that the main action of CCCP does not consist of dissipating any imposed pH gradient but rather in inhibiting directly the pH gradient-activated Cl- uptake and Cl-/Cl- exchange activities characterizing the intestinal BBM. Because these two activities can be explained in terms of a single (homogeneous) random, nonobligatory two-site Cl(-)-H+ symporter, in which Cl-/Cl- exchange occurs by counterflow [F. Alvarado and M. Vasseur. Am. J. Physiol. 271 (Cell Physiol. 40): C1612-C1628, 1996], we developed a new, more general three-site symport model that fully explains the Cl- uptake inhibitions caused by CCCP. This new model postulates the existence of a third, allosteric, inhibitory CCCP-binding site separate from either of the two substrate-binding sites of the Cl(-)-H+ symporter, the Cl(-)-binding and the H(+)-binding sites. Finally, we show that, to explain the partial inhibitions observed, it is necessary to postulate that all the substrate-bound carrier complexes, = C-S, I = C-S, A = C-S, and IA = C-S, where C is carrier, I is inhibitor, S is substrate, and A is activator, can form and be translocated.

Animals↗

PubMed1998

Infection and associated risk factors in the immediate postoperative period of pediatric liver transplantation: a study of 176 transplants.

OBJECTIVE: To describe the characteristics of infections occurring in the immediate postoperative period of orthotopic liver transplantation (OLT) in children in a pediatric intensive care unit (PICU) and the associated risk factors. DESIGN: Retrospective cohort study. SETTING: Multidisciplinary 16-bed pediatric intensive care unit in a tertiary university hospital. PATIENTS: One hundred and thirty-three pediatric patients (range 6M to 22 yr) who underwent 176 liver transplantations and were admitted to the pediatric intensive care unit for at least 48 h. RESULTS: A total of 180 infectious episodes (IE) occurred in 78 (59%) patients who underwent 96 (54.5%) liver transplantations (1.35 IE/patient; 1.02 IE/transplantation). The mean stay was 15.4 +/- 1.1 d (mean +/- SD) and 22 patients died in the pediatric intensive care unit. One hundred and thirty-one IE (72.8%) were bacterial, 31 (17.2%) fungal, and 14 (7.8%) mixed bacterial and fungal. 39% of the IE appeared in the first postoperative week and 27% in the second postoperative week. The most common sites of infection were abdomen (48.3%) and blood stream (26.1%). Bacteria, alone or with fungi, were present in 145 IE (1.1 IE/patient). Gram-positive aerobic bacteria (63%) predominated over gram-negative bacteria (54%) and fungi. Fungi were present in 45 IE; 53.4% of the infections occurred in lung (73% yeasts). The risk factors for infection that were identified were related with younger age, lower body weight, longer cold ischemia time, partial graft, and the volume of packed red blood cell transfusions. Infected patients had more postoperative complications and longer stay in the PICU (21 +/- 16 vs. 8 +/- 6 d), but no increase in mortality. The risk of infection increased 2.38 times with partial grafts and 1.1 times with each intraoperative transfusion of 20 ml/kg of packed red blood cells (RBC). CONCLUSIONS: Infection in the immediate postoperative period of pediatric OLT was related with a high morbidity but was not related significantly with increased mortality. The main risk factors for infection in the postoperative period of OLT were related essentially with small recipient size and the inherent complexity of the operation. Routine oropharyngeal decontamination is recommended, as well as early administration of oral nystatin in preoperative intestinal decontamination. The risk of infection increased 2.38 times with partial grafts and 1.1 times with the transfusion of every 20 ml of packed RBC.

Adolescent↗

PubMed1997

pH gradient effects on chloride transport across basolateral membrane vesicles from guinea-pig jejunum.

1. The effects of alkaline-inside pH gradients on 36Cl- uptake were quantified by using brush-border membrane (BBM) and basolateral membrane (BLM) vesicles from guinea-pig jejunum. 2. With BBM vesicles, a pHo/pHi gradient of 5.0/7.5 yielded fast overshoots involving a random, non-obligatory Cl(-)-H+ symport, strongly inhibited by CCCP. In contrast, BLM vesicles responded to similar pH gradients with much smaller, delayed overshoots, unaffected by CCCP. 3. The initial Cl- entry rates into BLM vesicles were a function of each pHo, pHi and delta pH value. They were stimulated by valinomycin in the presence of inward-directed K+ gradients. Short-circuiting the membrane potential with equilibrated K+ and valinomycin inhibited pH gradient-dependent Cl- uptake, but only partially. 4. Taken together, these results indicate that guinea-pig jejunal BLM vesicles possess both Cl- conductance and Cl(-)-H+ symport activities. 5. Even when different, the BBM and the BLM symporters are mechanistically similar. Neither of them involves a Cl(-)-OH- antiport, nor a simultaneous Cl(-)-anion exchange mechanism. Rather, for each membrane, all of these activities (symport, anion exchange) can be explained in terms of a single mobile carrier acting as a random, non-obligatory Cl(-)-H+ symporter where exchange occurs simply by counterflow. Net Cl- translocation via either the ternary (Cl(-)-C-H+) or the binary (Cl(-)-C) complexes accounts, respectively, for the existence of two, operationally distinct, electroneutral and rheogenic components. 6. The BBM symporter appears to involve an AE2 protein, but the molecular identity of the BLM one remains to be established.

Animals↗

PubMed1997

Analysis of costs in a pediatric ICU.

OBJECTIVE: To analyze the actual cost of pediatric intensive care and its different components, particularly the differences between various patient groups, with special reference to the variable cost and the elements included in it. DESIGN: Prospective, observational study. SETTING: Multidisciplinary 12-bed pediatric intensive care unit (PICU) in a tertiary university hospital. PATIENTS: 495 admissions to the unit over 17 consecutive months; 64.2% were medical patients and 35.8% were surgical patients; the mean (SE) stay in the PICU was 6.6 +/- 0.4 days. MEASUREMENTS AND RESULTS: The fixed cost per day per patient was calculated, including the costs of physicians, nurses, auxiliary and other personnel who worked during the study period, and the costs of structural depreciation, maintenance, consumption, and disposable material. The variable cost was individually calculated from the costs of routine procedures and also included expenditure on pharmaceuticals, blood products, biochemical, hematological, and bacteriologic tests, radiology, image diagnosis procedures, and other procedures. The Physiologic Stability Index (PSI) was obtained in the first 24 h after admission. The mean fixed cost per patient per day was u.s. $608, which represents 72% of the total patient cost during this study; 86% of this amount was for personnel (58% for nurses and auxiliary staff). Variable costs came to 28% of the total amount, and were $218 +/- 100 (M +/- SEM) per patient per day. In addition to the costs of their longer stay in the PICU, the daily variable costs of nonsurvivors were higher than those of survivors ($542 +/- 52 vs $179 +/- 7; p < 0.001). We classified the patients into four groups according to their PSI score in the first 24 h; variable daily costs increased (p < 0.05) in all comparisons with the PSI level: group I: < 4 points ($155 +/- 0.5), group II: 5-9 points ($210 +/- 13), group III: 10-14 points ($324 +/- 54), group IV: > 15 points ($480 +/- 42). However, this pattern was not found for all resources: the cost of treatment techniques and biochemical and hematological tests increased, but the consumption of antibiotics, parenteral nutrition, blood products, and bacteriologic tests reached their maximum level in groups I-III and radiology was not significantly influenced by PSI level. CONCLUSIONS: The cost of personnel was the biggest factor in intensive care costs: 62.4% of the total costs. Nonsurvivors generated 3 times the mean variable daily expenditure on survivors and had longer stays in the PICU. The increase in PSI score on the first day was associated with a global increase in variable costs. The cost of treatment techniques significantly increased as the illness became more severe but consumption of antibiotics and parenteral nutrition and use of bacteriologic tests and radiology did not.

Adolescent↗

PubMed1997

Usefulness of gastric intramucosal pH for monitoring hemodynamic complications in critically ill children.

OBJECTIVE: To assess the efficacy of gastric intramucosal pH for the evaluation of tissue perfusion and prediction of hemodynamic complications in critically ill children. DESIGN: Open prospective study without controls. SETTING: Pediatric intensive care unit (ICU) of a tertiary care university pediatric hospital. PATIENTS: Thirty critically ill children (16 boys and 14 girls), age range: 3 months-12 years. MEASUREMENTS AND RESULTS: A tonometry catheter was placed in the stomach of all patients on admission to the pediatric ICU. Simultaneous tonometry and arterial gas measurements were made on admittance and every 6-12 h throughout the study; a total of 202 measurements were made. The catheter was removed after extubation and/or when the patient was hemodynamically stable. Intramucosal pH was calculated using the Henderson-Hasselbalch equation based on the pCO2 of the tonometer and arterial bicarbonate. Intramucosal pH values between 7.30 and 7.45 were considered to be normal. The patient's condition was analyzed using the Pediatric Risk Mortality Score (PRISM). The relations between intramucosal pH and the presence of major hemodynamic complications (cardiopulmonary arrest, shock), minor hemodynamic complications (hypotension, hypovolemia or arrhythmia), death, PRISM score and the duration of the stay in the pediatric ICU were analyzed. Intramucosal pH on admission was 7.48 +/- 0.15 on average (range 7.04-7.68). Five patients (16%) had an intramucosal pH lower than 7.30 on admission; these patients did not have a higher incidence of hemodynamic complications. The 16 patients (53%) who had an intramucosal pH of less than 7.30 at some time during the course of their disease had more hemodynamic complications than the patients who did not have pH lower than 7.30 (p < 0.0001). Every case of cardiopulmonary arrest and shock was related to intramucosal pH of less than 7.30. Patients with major complications (cardiopulmonary arrest and shock) had lower intramucosal pHs than those with minor hemodynamic complications (p = 0.03); similarly, they had low intramucosal pH readings more often than those with minor complications (p = 0.0032). Intramucosal pH values less than 7.30 had a sensitivity of 90% and a specificity of 98% as a predictor of hemodynamic complications. There was no relation between intramucosal pH lower than 7.30 and either PRISM or the duration of the stay in the pediatric ICU. Patients with intramucosal pH less than 7.20 had a higher PRISM than the patients who did not have pH lower than 7.20 (p < 0.05). A patient who died during the study due to cardiopulmonary arrest had prior intramucosal pH measurements of 7.23 and 7.10, and three patients died of late complications after the end of the study. Hemodynamic complications were not detected with arterial pH. Gap pH (arterial pH-intramucosal pH) and standard pH measurements yielded the same results as gastric intramucosal pH. CONCLUSION: Intramucosal pH could provide a useful early indication of hemodynamic complications in critically ill children.

Acid-Base Equilibrium↗

PubMed1997

Solubilized and cleaved VP7, the outer glycoprotein of rotavirus, induces permeabilization of cell membrane vesicles.

It has been previously shown that rotavirus triple-layered particles induce permeabilization of liposomes and membrane vesicles. These effects were mediated by one or both of the solubilized outer-capsid proteins, VP4 and VP7. Permeabilization was dependent on trypsin treatment of the viral particles, suggesting that VP4 was involved. To analyse the respective roles of the outer-capsid proteins in this permeabilization process, we have used membrane vesicles loaded with carboxyfluorescein and virus-like particles derived from insect cells co-expressing various sets of capsid proteins. Virus-like particles containing VP2, VP6 and VP7 (VLP2/6/7) are as efficient in permeabilizing vesicles as triple-layered particles. As with double-layered particles, virus-like particles made of VP2 and VP6 had no effect on vesicle permeabilization. Permeabilization of membrane vesicles required trypsinization of the VP7 solubilized from VLP2/6/7. These results show that solubilized and trypsinized VP7 is able to induce membrane permeabilization, independently of the presence of VP4.

Antigens, Viral↗

PubMed1996

Theoretical and experimental discrimination between Cl(-)-H+ symporters and Cl-/OH- antiporters.

A Cl(-)-H+ symport and a Cl-/OH- antiport cannot be readily distinguished physicochemically, but a kinetic distinction is theoretically possible, because a Cl(-)-H+ symporter involves a two-site carrier whereas a Cl-/OH- antiporter involves a single-site carrier. Accordingly, we have developed kinetic models and equations that we have tested by studying Cl- uptake by isolated guinea pig ileal brush-border membrane vesicles as a function of Cl- or H+ concentration. We conclude that a two-site Cl(-)-H+ symporter with a 1:1 stoichiometry explains the pH-dependent Cl- uptake and Cl-/Cl- exchange activities of the brush-border membrane in terms of a single random nonobligatory mobile carrier where exchange occurs by counterflow. This symport, probably involving an anion exchanger (AE 2) protein, differs, therefore, functionally from the erythrocyte's band 3 AE1, which involves an antiport. The question is whether members of the AE gene family can be functionally diverse, even when their primary structures exhibit up to 50% overall homologies.

Animals↗

PubMed1994

Rotavirus interaction with isolated membrane vesicles.

To gain information about the mechanism of epithelial cell infection by rotavirus, we studied the interaction of bovine rotavirus, RF strain, with isolated membrane vesicles from apical membrane of pig enterocytes. Vesicles were charged with high (quenching) concentrations of either carboxyfluorescein or calcein, and the rate of fluorophore release (dequenching) was monitored as a function of time after mixing with purified virus particles. Purified single-shelled particles and untrypsinized double-shelled ones had no effect. Trypsinized double-shelled virions induced carboxyfluorescein release according to sigmoid curves whose lag period and amplitude were a function of virus concentration and depended on both temperature and pH. The presence of 100 mM salts (Tris Cl, NaCl, or KCl) was required, since there was no reaction in isoosmotic salt-free sorbitol media. Other membrane vesicle preparations such as apical membranes of piglet enterocyte and rat placenta syncytiotrophoblasts, basolateral membranes of pig enterocytes, and the undifferentiated plasma membrane of cultured MA104 cells all gave qualitatively similar responses. Inhibition by a specific monoclonal antibody suggests that the active species causing carboxyfluorescein release is VP5*. Ca2+ (1 mM), but not Mg2+, inhibited the reaction. In situ solubilization of the outer capsid of trypsinized double-shelled particles changed release kinetics from sigmoidal to hyperbolic and was not inhibited by Ca2+. Our results indicate that membrane destabilization caused by trypsinized outer capsid proteins of rotavirus leads to fluorophore release. From the data presented here, a hypothetical model of the interaction of the various states of the viral particles with the membrane lipid phase is proposed. Membrane permeabilization induced by rotavirus may be related to the mechanism of entry of the virus into the host cell.

Animals↗

PubMed1994

Rheogenic Cl- conductance and Cl(-)-Cl(-)-exchange activities in guinea pig jejunal basolateral membrane vesicles.

We describe a method for the simultaneous purification of apical (brush-border membrane) and basolateral membrane (BLM) vesicles from the same sample of guinea pig jejunum. We applied functional tests to demonstrate the absence of reciprocal cross contamination between the two vesicle preparations. By using the BLM vesicles and a rapid filtration technique, we quantified 36Cl uptake under conditions of equilibrated pH (pHout = pHin = 7.5). The presence of 200 mM cis of either Na+ or K+, or an equimolar mixture of both, significantly increased the initial Cl- entry rate. In the presence of K+, valinomycin further increased Cl- uptake, but no Cl- uphill transport was ever observed under any of the conditions. All the increases were abolished by voltage clamping, indicating that the alkali-metal ions act by creating an inside-positive membrane potential capable of stimulating a Cl(-)-conductance pathway. In the absence of K+, BLM vesicle preloading to obtain a [Cl-]out/[Cl-]in = 16/200 mM gradient (delta Cl-) resulted in a 500% increase in the initial 36Cl- entry rate, accompanied by a transient Cl- accumulation, with an overshoot at approximately 5 min. In the presence of both a positive-inside electrical gradient (delta psi) and a delta Cl-, the initial Cl- uptake rate was increased by 800%, indicating that the effects of delta psi and of delta Cl- are additive. The delta Cl- effect was blocked, but only partially, by short-circuiting the membrane potential with equilibrated K+ and valinomycin, thus indicating that it has both rheogenic and electroneutral components. We conclude that Cl- influx across the guinea pig intestinal BLM involves a Cl(-)-conductance pathway plus a distinct Cl(-)-Cl(-)-exchange system, exhibiting both electroneutral and rheogenic components. Alternatively, the possibility can also be entertained that the conductance and the exchange pathways share a common molecular basis, e.g., a nonobligatory Cl(-)-Cl- exchanger or rheogenic uniport.

Animals↗

PubMed1993

Action of robenidine on the intestinal transport and digestion of nutrients in rabbit.

Robenidine is an anticoccidial guanidine used as an additive in rabbit fodder. Because its action is restricted to the small intestine, the present work addresses the question whether robenidine affects the growth of the animals, sugar and amino acid intestinal transport and membrane-bound intestinal digestion. For this purpose we have determined the intestinal transport of the substrates, and the enzymatic activity of neutral aminopeptidase and sucrase. We have found that robenidine diminishes the tissue accumulation of L-leucine and D-galactose at long incubation times, and increases the transepithelial mucosal to serosal flux of both substrates. These results suggest that robenidine may stimulate the enterocyte basolateral membrane flux of sugars and neutral amino acids. These results have been corroborated by means of isolated brush border and basolateral membrane vesicles. Apart from these effects, robenidine has also been shown to increase the enzymatic activity of neutral aminopeptidase and sucrase and thus resulting in a better digestion of nutrients.

Aminopeptidases↗

PubMed1993

Buffer-dependent pH sensitivity of the fluorescent chloride-indicator dye SPQ.

The fluorescence intensity of 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) in an N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) 2-(N-morpholino)ethanesulfonic acid (MES)-Tris(hydroxymethyl)aminomethane buffer, pH 7.0, decreased as a function of Cl- concentration and/or gluconate concentration, as expected. Contrary to expectation, however, the fluorescence intensity progressively increased as the pH decreased, independently of the presence of gluconate. Consequently, the modulation of SPQ fluorescence by commonly used buffers was investigated as a function of pH. Titration curves demonstrated SPQ quenching and yielded pK values characteristic of each buffer. from here, pH-independent Stern-Volmer constants, KQbase, were calculated for each of the morpholine derivatives, MES and 3-(N-morpholino)-2-hydroxypropanesulfonic acid. In contrast, HEPES and piperazine-N,N'-bis(2-ethanesulfonic acid), which are piperazine derivatives, exhibited an additional pH-independent "molecular" quenching constant KmQ throughout the pH range 3-10. To study chloride fluxes, therefore, what counts is the apparent Cl-Stern-Volmer constant KappCl, which is a function of both pH and buffer composition. Equations describing these relationships are presented. In conclusion, unless both pH and the buffer composition are taken into account, SPQ is unsuitable for studying the concomitant transmembrane fluxes of Cl- and H+.

Alkanesulfonates↗

PubMed1992

Heterogeneity of L-alanine transport systems in brush-border membrane vesicles from rat placenta during late gestation.

The placental uptake of L-alanine was studied by using purified brush-border membrane vesicles from rat trophoblasts. Saturation curves were carried out at 37 degrees C in buffers containing 100 mM (zero-trans)-NaSCN, -NaCl, -KSCN, -KCl, or -N-methyl-D-glucamine gluconate. The uncorrected uptake results were fitted by non-linear regression analysis to an equation involving one diffusional component either one or two saturable Michaelian transport terms. In the presence of NaCl, two distinct L-alanine transport systems were distinguished, named respectively System 1 (S-1; Vm1 about 760 pmol/s per mg of protein; KT1 = 0.5 mM) and System 2 (S-2; Vm2 about 1700 pmol/s per mg; KT2 = 9 mM). In contrast, in the presence of K+ (KCl = KSCN) or in the absence of any alkali-metal ions (N-methyl-D-glucamine gluconate), only one saturable system was apparent, which we identify as S-2. When Na+ is present, S-1, but not S-2, appears to be rheogenic, since its maximal transport capacity significantly increases in the presence of an inside-negative membrane potential, created either by replacing Cl- with the permeant anion thiocyanate (NaSCN > NaCl) or by applying an appropriate K+ gradient and valinomycin. alpha-(Methylamino)isobutyrate (methyl-AIB) appears to be a substrate of S-1, but not of S-2. For reasons that remain to be explained, however, methyl-AIB inhibits S-2. We conclude that S-1 represents a truly Na(+)-dependent mechanism, where Na+ behaves as an obligatory activator, whereas S-2 cannot discriminate between Na+ and K+, although its activity is higher in the presence of alkali-metal ions than in their absence (Na+ = K+ > N-methyl-D-glucammonium ion). S-2 appears to be fully developed 2 days before birth, whereas S-1 undergoes a capacity-type activation between days 19.5 and 21.5 of gestation, i.e. its apparent Vmax. nearly doubles, whereas its KT remains constant.

Alanine↗