Induction of Ca2+ transport in liposomes by insulin.

The requirement of extracellular Ca2+ for insulin action has been indicated by past studies. With a view to understand the interaction of insulin with Ca2+ in the vicinity of the cell membrane, we have examined the ability of insulin and its constituent polypeptide chains A and B to translocate Ca2+ and Mg2+ across the lipid bilayer in two sets of synthetic liposomes. The first were unilamellar vesicles made of dimyristoylphosphatidylcholine and contained the Ca2+ sensor dye arsenazo III. Peptide-mediated Ca2+ and Mg2+ transport in these vesicles was monitored at 37 degrees C in a neutral buffer containing CaCl2 or MgCl2 using a difference absorbance method. In the second set, multilamellar vesicles of egg lecithin containing trapped fura-2 were employed and the cation transport was followed at 20 degrees C by fluorescence changes in the dye. Control experiments indicated that the hormonal peptides caused no appreciable perturbation of the vesicles leading to leakage of contents or membrane fusion. In both liposome systems, substantial Ca2+ and Mg2+ transport was observed with insulin and the B chain; the A chain was less effective as an ionophore. Quantitative analysis of the transport kinetic data on the B chain showed a 1:1 peptide-Ca2+ complex formed inside the membrane. In light of the available structural data on Ca2+ binding by insulin and insulin receptor, our results suggest the possibility of the hormone interacting with the receptor with the bound Ca2+.

Evaluation of the combination of a bedside D-dimer assay and enzyme-linked immunosorbent soluble fibrin assay in patients with suspected venous thromboembolism.

The objectives of the study were to determine whether the combination of a negative SimpliRED D-dimer assay and a low soluble fibrin result reliably excludes venous thromboembolism, and whether patients with proven venous thromboembolism and a normal SimpliRED D-dimer have evidence of impaired fibrinolysis. The study was a retrospective analysis of a cohort of 262 consecutive patients, 94 patients presenting with suspected deep venous thrombosis and 168 with suspected pulmonary embolism. Fifty-nine patients (22.5%) were classified as venous thromboembolism-positive, 27 with pulmonary embolism, and 32 with deep venous thrombosis. One hundred and fourteen patients (43.5%) had SimpliRED D-dimer and a soluble fibrin result of less than or equal to 2.0 microg/ml; the negative predictive value was 98.2% (95% confidence interval: 93.8-99.8%), and the likelihood ratio was 0.06. Eight patients with proven venous thromboembolism had a negative SimpliRED D-dimer; all had elevated ELISA D-dimer levels and six had elevated soluble fibrin levels. This suggests that patients with venous thromboembolism and a normal SimpliRED result do not have impaired fibrinolysis as a cause of their false-negative result. This study suggests that the combination of SimpliRED and soluble fibrin can be used to exclude venous thromboembolism in over 40% of patients who present with a clinical suspicion of deep venous thrombosis or pulmonary embolism and that the small group of patients with venous thromboembolism and a normal SimpliRED do not have impaired fibrinolysis.

Utility of ultrasonographic venous assessment prior to forearm arteriovenous fistula creation.

AIM: The purpose of this study was to evaluate the clinical utility of Doppler ultrasound (US) prior to native forearm arteriovenous fistula (AVF) creation. MATERIALS AND METHODS: US mapping was carried out pre-operatively to evaluate the major veins and arteries in the appropriate arm. One hundred and 6 patients were identified retrospectively over 2 years with complete clinical and US data. A failed fistula was defined as an inability to provide blood flow to meet adequacy targets by 6 months (urea reduction ratio > or = 65%). RESULTS: Twenty-nine patients (27.4%) had successful forearm AVFs. The mean minimum forearm cephalic vein diameter (CVD) was 2.51 +/- 0.14 and 2.23 +/- 0.06 mm in successful and failed fistulae, respectively (p = 0.04). This result was primarily due to differences observed in women. A receiver operator curve analysis showed that a cutpoint of 2.6 mm for minimum forearm CVD had the greatest predictive value with a likelihood ratio of 3.94 (95% CI: 1.97 - 7.84) for fistula failure. Multivariate logistic regression analysis determined that male gender and minimum forearm CVD were the only significant predictors for fistula success with odds ratios of 3.90 (95% CI: 1.30 - 11.68) and 2.31 (95% CI: 1.00 - 5.43), respectively. The study is limited by the possibility that US results in patients may have lead to an alternative type of access being attempted. CONCLUSIONS: US mapping prior to forearm AVF creation is of modest benefit. Only male gender and minimum forearm CVD were predictive of AVF success.

Calcium binding and translocation properties of glucagon and its fragments.

Earlier studies have indicated that the N- and C-terminal regions of glucagon are functionally and structurally different. We have sought to understand this distinction in terms of the interaction of glucagon and its N- and C-terminal fragments with Ca2+, Mg2+, and Zn2+ in a nonpolar milieu. CD spectral data, in 98% (v/v) trifluoroethanol in water, reveal two binding sites for Ca2+ and Mg2+ and one site for Zn2+ in the intact hormone as well as in the C-terminal 19-29 fragment. The 1-6 fragment did not bind Zn2+ and formed a 2:1 peptide-Ca2+ or -Mg2+ complex. With glucagon and the 19-29 fragment, cation binding caused changes in the peptide's helix content. Fluorescence spectral changes involving Trp-25 in the 19-29 fragment and Trp-25 and Tyr-10 and/or Tyr-13 in glucagon were seen on Ca2+ binding to one of the two sites, while Zn2+ binding produced no change in fluorescence. The spectral data suggest that Ca2+ and Zn2+ binding sites (with Kd in the micromolar range in 98% trifluoroethanol) are distinct and are contained in the C-terminal domain of glucagon. Glucagon and the 19-29 fragment, but not the 1-6 fragment, caused an influx of Ca2+ (as monitored by spectral changes in arsenazo III) in unilamellar vesicles made of dimyristoyllecithin. Leakage of vesicle contents induced by the 19-29 fragment was minimal but was significant (approximately 10%) in the case of glucagon. The transport data suggest an interaction of the C-terminal domain of glucagon with Ca2+ at the lipid-water interface.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of oxytocin with Ca2+: I. CD and fluorescence spectral characterization and comparison with vasopressin.

Extracellular Ca2+ is required for the action of oxytocin and both the hormone and its receptor have binding sites for divalent metal cations. To characterize the cation-bound form of oxytocin, we monitored the binding of Ca2+ and Mg2+ to oxytocin as well as peptides representing its ring and tail regions in trifluoroethanol, a lipid-mimetic solvent, using CD and fluorescence spectroscopy. Binding Ca2+ (Kd approximately 50 microM) caused drastic CD and fluorescence changes leading to a helical conformation. Mg2+ caused CD changes smaller than and opposite to Ca2+. However, the helical structure was enhanced when both Ca2+ and Mg2+ were present together. CD changes in the tail peptide of oxytocin showed its ability to bind Ca2+ and Mg2+ whereas the vasopressin tail peptide did not bind either cation. CD spectral changes on Ca2+ and Mg2+ binding to tocinoic acid (the ring moiety of oxytocin) were much smaller than those of oxytocin. These data suggest that the tail segment of oxytocin potentiates Ca2+ binding by the ring. While vasopressin displayed a CD spectrum similar to that of oxytocin, CD spectra of its cation-bound forms were markedly different from those of oxytocin; the Ca(2+)-induced CD changes in vasopressin were very much smaller and of opposite sign, and Mg(2+)-induced ones significantly larger than in oxytocin. Taken together, our observations bring out the structural differences between oxytocin and vasopressin in the context of their interaction with Ca2+ and Mg2+. This may be relevant to understanding the differences in the bioactive conformations and receptor interactions of the two hormones.

Interaction of gonadotropin-releasing hormone and its agonist analogs with Ca2+ in a nonpolar milieu. Correlation with biopotencies.

Extracellular Ca2+ is necessary for the action of gonadotropin-releasing hormone (GnRH). Assuming that this partly because of the interaction of the hormone with the relatively abundant extracellular Ca2+ in the low dielectric milieu of the bilayer plasma membrane, we studied the interaction of GnRH and five of its agonist analogs with Ca2+ under membrane-mimetic conditions. The peptides used, in increasing order of their reported gonadotropin-releasing activities, were: des-amide GnRH (or GnRH-OH); [Ala6]GnRH; [D-Ala6]GnRH; des-Gly10[D-Ala6,Pro9-NHEt]GnRH and, des-Gly10[D-Trp6,Pro9-NHEt]GnRH. Changes in the far-UV CD and fluorescence spectra of these peptides in trifluoroethanol were used to monitor conformational changes and obtain the Ca2+-binding isotherms. The data show that GnRH and its active analogs contain two Ca2+ binding sites, whereas the inactive analogs have only one. The extent of Ca2+ binding by the agonist peptides paralleled their biological potency ranking. The superactive analog des-Gly10[D-Trp6,Pro9-NHEt]GnRH exhibited the ability to transport Ca2+ ions across large unilamellar vesicles of dimyristoylphosphatidylcholine. Our study shows that significant differences among the GnRH and its analog peptides, suggestive of differences in their conformations, are manifested only in the presence of Ca2+. This observation would provide a basis for understanding GnRH action in terms of the hormone's interaction with Ca2+ in the lipid milieu.

The clinical utility of Doppler ultrasound prior to arteriovenous fistula creation.

Arteriovenous fistula (AVF) is the preferred access for long-term hemodialysis, with superior long-term patency rates; however, early failure rates are significant. Recent evidence has brought into question the preferred site of AVF creation in many patient groups. A preoperative test that could reliably predict the outcome of a proposed AVF would be of great benefit. Doppler ultrasound has been the most extensively studied and widely used test to guide access creation. Accurate and validated measurements of internal vessel diameter, both arterial and venous, and blood flow in the upper extremity are obtainable by Doppler ultrasound. Studies evaluating the utility of Doppler ultrasound prior to AVF creation suggest that vessel size and blood flow are predictive of AVF outcome. An AVF created using a cephalic vein and/or radial artery smaller than 1.5-2.0 mm is likely to fail; such preoperative data may indicate that an upper arm AVF should be the primary access attempted. Further prospective studies are needed to evaluate the utility of Doppler ultrasound.