Topological photoaffinity labeling of the rabbit ileal Na+/bile-salt-cotransport system

For the investigation of the topology of the rabbit ileal Na+/bile-salt-cotransport system, composed of a 93-kDa integral membrane protein and a peripheral 14-kDa bile-acid-binding protein (ILBP), we have synthesized photolabile dimeric bile-salt-transport inhibitors (photoblockers), G1-X-G2, where two bile acid moieties (G1 and G2) are tethered together via a spacer, X, and where one of the two bile acid moieties carries a photoactivatable group. These photoblockers specifically interact with the ileal Na+/bile-salt-cotransport system as demonstrated by a concentration-dependent inhibition of [3H]cholyltaurine uptake by rabbit ileal brush-border membrane vesicles and by inhibition of photolabeling of the 93-kDa and 14-kDa bile-salt-binding proteins by 7,7-azo and 3,3-azo derivatives of cholyltaurine. Ileal bile-salt uptake was specifically inhibited by the photoblockers, which were not taken up themselves by the small intestine as demonstrated by in vivo ileal perfusion. Dependent on the photoblocker used several polypeptides in the molecular-mass range of 14-130 kDa were labeled. The cytoplasmically attached 14-kDa ILBP was significantly labeled only by inhibitors that are photoactivatable in bile acid moiety G1, suggesting that during binding and translocation of a bile-salt molecule by the ileal bile-salt-transport system the steroid nucleus gets access to the cytoplasmic site of the ileal brush-border membrane first. Photoaffinity labeling in the frozen state with the transportable 3,3-azo and 7,7-azo derivatives of cholyltaurine revealed a time-dependent increase in the extent of labeling of the 14-kDa and 93-kDa proteins, suggesting a labeling of these proteins from the cytoplasmic site of the ileal brush-border membrane. By photoaffinity labeling in the frozen state with the various photoblockers time-dependent changes in the extent of photoaffinity labeling of bile-salt-binding proteins were observed, demonstrating the possibility of topological analysis of the rabbit ileal Na+/bile-salt-cotransport system.     

Clinical experience with temporary vena caval filters

PURPOSE: To look at the benefits and complications of different vena caval filters inserted prophylactically. Three temporarily implantable caval filter systems were used in 67 patients. MATERIALS AND METHODS: Twelve Cook filters (six transjugular, six transfemoral), 11 Angiocor filters (one transjugular, 10 transbrachial), and 44 Antheor filters (three transjugular, four transfemoral, 37 transbrachial) were successfully implanted. In known iliac vein or caval thrombosis, the prophylactic filters were placed during thrombolytic therapy in 46 cases, surgery in 17 cases, thrombosis in pregnancy in three cases, and high-dose heparinization without lysis in one case. RESULTS: One patient had a fatal pulmonary embolism during treatment; seven thrombi were detected in the filter. Other complications were caused either by the underlying therapy alone (one fatal outcome of abdominal aorta aneurysmal surgery, two cases of cerebral hemorrhage, two cases of retroperitoneal hematomas, two cases of streptokinase fever reactions, one compartment syndrome, two cases of macrohematuria), by the combination of therapy and caval filter implantation (three cases of groin hematomas, three cases of arm hematomas), or by filter implantation alone (two cases of subclavian vein thrombosis, one catheter infection, one dislocation, one air embolism, one basket rupture). The bleeding complications were related to the aggressive thrombolytic therapy and would have occurred without filter implantation. CONCLUSION: Because temporary caval filters have no long-term complications per se, their use seems sensible as long as there are stringent indications, including the presence of iliac vein or caval thrombosis and risk of thrombus mobilization. The Antheor filter system was the most convenient system for implantation.     

Effects of calcium supplementation and lactation on iron status

Calcium has been shown to inhibit iron absorption. The consequences of chronic calcium supplementation on iron status are unclear, however. As part of a randomized calcium-supplementation trial in lactating and nonlactating women in the postpartum period, we determined whether long-term calcium supplementation and lactation status affected iron stores as measured by serum ferritin concentrations. Subjects (95 lactating and 92 nonlactating) were enrolled at approximately 6 mo postpartum and then randomly assigned to receive either 500 mg Ca as calcium carbonate or a placebo twice daily with meals for 6 mo. Lactating women weaned their infants approximately 2 mo after enrollment (ie, approximately 8 mo postpartum). Calcium supplementation had no effect on serum ferritin concentrations. At the end of the study, geometric mean serum ferritin concentrations were 28.4 microg/L in the calcium-supplemented group and 27.5 microg/L in the placebo group (P > 0.5). Lactation status was significantly related to serum ferritin concentrations. At baseline, serum ferritin concentrations were higher in lactating women than in nonlactating women (47.7 compared with 31.5 microg/L, P < 0.001). In lactating women, serum ferritin concentrations decreased by a mean of 17 microg/L after weaning. By 12 mo postpartum, mean serum ferritin concentrations in women who were previously lactating were not significantly higher than those of nonlactating women (30.5 compared with 25.5 microg/L). These findings provide reassurance that long-term calcium supplementation does not impair iron stores. Furthermore, lactation status should be considered when assessing iron nutriture of women and determinants of iron status in populations.     

Separation and characterization of Na+,K(+)-ATPase containing vesicles

Na+,K(+)-ATPase was reconstituted in vesicles prepared by a dialysis method. Ion-exchange chromatography was used to obtain well characterized fractions from the inhomogeneous vesicle preparation. Lipid and protein content was determined by optical methods during the elution process. It was possible to separate fractions with distinct enzymatic and transport activities. A protocol was set up, which allowed to calculate the average number of 5-IAF labeled ion pumps per vesicle in the different fractions. The dependence of the number of protein molecules per vesicle was studied as function of the initial protein concentration added to the lipid solution before dialysis. The transport activity disappears completely at very low protein concentrations (3.3 micrograms protein per mg lipid). This observation is in favor of the proposal discussed in the literature, that the heterodimer (alpha beta)2 is the transport-active form of the Na+,K(+)-ATPase. The presented method can be applied to all reconstituted vesicle preparations in which the proteins can be labeled quantitatively with a fluorescence dye.     

Lipid and lipoprotein profiles in children with familial hypercholesterolaemia: effects of therapy

In 71 children with familial hypercholesterolaemia the effect of dietary and/or medical treatment was evaluated. Initial total cholesterol and low density lipoprotein (LDL)-cholesterol levels were significantly lower in children who were consecutively treated by diet (Step-One-Diet) than in those who received additional medication. By dietary treatment, the median total cholesterol level (236.5 mg/dl; range 210-510 mg/dl) was reduced by 7.4% and the median LDL-cholesterol level (162 mg/dl; range 126-423 mg/dl) by 9.9%. By dietary and medical therapy, the median total cholesterol level (330 mg/dl; range 270-424 mg/dl) was reduced by 29.7% and the median LDL-cholesterol level (263 mg/dl; 192-333 mg/dl) by 25.9%. High density lipoprotein (HDL)-cholesterol and HDL 3 remained unchanged. HDL 2 showed a significant decrease of 15.6% up to 27 mg/dl (13-42 mg/dl) on medical treatment. Apolipoprotein A I levels did not change during therapy. Initial apolipoprotein B levels were significantly higher in children who were treated by diet and medication and were reduced by 28.9% by combined therapy. In 28 patients (39.4%) an excess of lipoprotein (a) was detected. Regarding the apolipoprotein E phenotype, 32.2% of the patients carried the risk gene epsilon4 in a hetero- or homozygous form. CONCLUSION: Early dietary and/or medical treatment in hypercholesterolaemic children significantly ameliorates the lipoprotein status. The pretherapy lipoprotein status seems to prognosticate the effectiveness of therapy.     

Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P

We evaluated the effects of nitric oxide (NO) generators and endogenous production of NO elicited by substance P (SP) in the angiogenesis process. Angiogenesis was monitored in the rabbit cornea in vivo and in vitro by measuring the growth and migration of endothelial cells isolated from coronary postcapillary venules. The angiogenesis promoted in the rabbit cornea by [Sar9]-SP-sulfone, a stable and selective agonist for the tachykinin NK1 receptor, and by prostaglandin E1 (PGE1), was potentiated by sodium nitroprusside (SNP). Conversely, the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME), given systemically, inhibited angiogenesis elicited by [Sar9]-SP-sulfone and by PGE1. Endothelial cells exposed to SNP exhibited an increase in thymidine incorporation and in total cell number. Exposure of the cells to NO generating drugs, such as SNP, isosorbide dinitrate, and glyceryl trinitrate, produced a dose-dependent increase in endothelial cell migration. Capillary endothelial cell proliferation and migration produced by SP were abolished by pretreatment with the NO synthase inhibitors N omega-mono-methyl-L-arginine (L-NMMA), N omega-nitro-L-arginine (L-NNA), and L-NAME. Exposure of the cells to SP activated the calcium-dependent NO synthase. Angiogenesis and endothelial cell growth and migration induced by basic fibroblast growth factor were not affected by NO synthase inhibitors. These data indicate that NO production induced by vasoactive agents, such as SP, functions as an autocrine regulator of the microvascular events necessary for neovascularization and mediates angiogenesis.     

Characterization by 1H NMR of a C32S,C35S double mutant of Escherichia coli thioredoxin confirms its resemblance to the reduced wild-type protein

A mutant of Escherichia coli thioredoxin containing serine residues in place of the two active-site cysteines, termed C32S,C35S, previously shown to be partially able to substitute for reduced thioredoxin in certain phage systems, has been characterized by 1H NMR spectroscopy at pH values between 5.5 and 10. The 1H NMR spectrum of the mutant at pH 5.5 is very similar to that of the wild-type protein in either the reduced or oxidized state. Chemical shift changes in the vicinity of the active site serines indicate that the nearby hydrophobic pocket is somewhat changed, probably as a result of the replacement of the cysteine thiols with the smaller, more hydrophilic hydroxyl side chains and a change in the preferred chi 1 angles of the side chains. Although the pattern of amide protons persistent in 2H2O differs only slightly between the two forms of the wild-type protein, the pattern observed for the C32S,C35S mutant shows characteristic features that correspond closely with those of the reduced wild-type protein rather than with the oxidized form. The pH dependence of the mutant protein shows a single group titrating close to the active site with a pKa of 8.3, which we assign to the buried carboxyl group of Asp 26 by analogy with the behavior of wild-type thioredoxin. The pKa is significantly higher for the mutant protein, consistent with an increase in the hydrophobicity of the pocket where the carboxyl is buried, probably due to repacking caused by the removal of the cysteine thiols and the placement of the serine hydroxyls in positions where they interact better with solvent. The results demonstrate that the solution behavior of the mutant protein is similar in many ways to that of reduced wild-type thioredoxin, explaining its partial activity in the two essential roles of reduced thioredoxin as a subunit of phage T7 DNA polymerase and in the assembly of filamentous phage.