Dialysis Protocol Research Articles

Suppression of Sulfonylurea- and Glucose-Induced Insulin Secretion In Vitro and In Vivo in Mice Lacking the Chloride Transport Protein ClC-3

Priming of insulin secretory granules for release requires intragranular acidification and depends on vesicular Cl(-)-fluxes, but the identity of the chloride transporter/ion channel involved is unknown. We tested the hypothesis that the chloride transport protein ClC-3 fulfills these actions in pancreatic beta cells. In ClC-3(-/-) mice, insulin secretion evoked by membrane depolarization (high extracellular K(+), sulfonylureas), or glucose was >60% reduced compared to WT animals. This effect was mirrored by a approximately 80% reduction in depolarization-evoked beta cell exocytosis (monitored as increases in cell capacitance) in single ClC-3(-/-) beta cells, as well as a 44% reduction in proton transport across the granule membrane. ClC-3 expression in the insulin granule was demonstrated by immunoblotting, immunostaining, and negative immuno-EM in a high-purification fraction of large dense-core vesicles (LDCVs) obtained by phogrin-EGFP labeling. The data establish the importance of granular Cl(-) fluxes in granule priming and provide direct evidence for the involvement of ClC-3 in the process.

Use of a dialyzable short-chain phospholipid for efficient solubilization and reconstitution of influenza virus envelopes

Virosomes are reconstituted viral envelopes that can serve as vaccines and as vehicles for cellular delivery of various macromolecules. To further advance the use of virosomes, we developed a novel dialysis procedure for the reconstitution of influenza virus membranes that is easily applicable to industrial production and compatible with encapsulation of a variety of compounds. This procedure relies on the use of 1,2-dicaproyl- sn-glycero-3-phosphocholine (DCPC) as a solubilizing agent. DCPC is a short-chain lecithin with detergent-like properties and with a critical micelle concentration of 14 mM. DCPC effectively dissolved the influenza virus membranes after which the nucleocapsids could be removed by ultracentrifugation. The solubilized membrane components were reconstituted either by removal of DCPC by dialysis or by a procedure involving initial dilution of the solubilized membrane components followed by dialysis. Both protocols resulted in removal of 99.9% of DCPC and simultaneous formation of virosomes. Analysis of the virosome preparations by equilibrium sucrose density gradient centrifugation revealed co-migration of phospholipid and protein for virosomes produced by either method. Moreover, both virosome preparations showed morphological and fusogenic characteristics similar to native influenza virus. Size, homogeneity and spike density of the virosomes varied with the two different reconstitution procedures employed. The recovery of viral membrane proteins and phospholipids in the virosomes was found to be higher for the dilution/dialysis procedure than for the simple dialysis protocol. This novel procedure for the production of virosomes is straightforward and robust and allows further exploitation of virosomes as vaccines or as drug delivery vehicles not only in academia, but also in industrial settings.

Expression in Escherichia coli and disulfide bridge mapping of PSC33, an allergenic 2S albumin from peanut

In this work, we describe the expression, purification, and disulfide mapping of the named ‘peanut seed cDNA 33’ (PSC33) peanut allergen. A variant of PSC33 (with N 63, E 64, Q 69 instead of D 63, Q 64, E 69) has been identified in peanut by proteomic analysis of a highly IgE immunoreactive purification fraction. It is 92% homologous to Ara h 6. We raised monoclonal antibodies against PSC33 and amplified it by PCR from peanut leaf genomic DNA. PSC33 was intron-less and the two NEQ and DQE variants of PSC33 were equally amplified. Since expression of the natural PSC33 (DQE) gene was very low in Escherichia coli even with supplementation of rare codon tRNAs, a synthetic gene optimized for expression in E. coli of PSC33 (DQE) was introduced into a pET9-c vector. A high production of protein occurred in the inclusion bodies that was submitted to refolding using an additive-introduced stepwise dialysis protocol which consists in the gradual removal of the denaturing agent guanidine–HCl with controlled introduction of oxidized and reduced glutathione and l-arginine as a chemical chaperone. After reverse phase HPLC purification, 1 mg of pure refolded protein (as assayed by MALDI-TOF mass spectrometry, mouse IgG immunoreactivity and circular dichroism) were obtained with every 100 ml of bacterial culture. Trypsin and CNBr hydrolysis of the protein combined with MALDI-TOF mass spectrometry allowed us to assign disulfide bridges and show that the native and refolded proteins were identical. The four disulfides of canonical 2S albumins were conserved and the two supplementary cysteines of PSC33 were paired together.

Interactions among the three structural motifs of the C-terminal region of human thrombospondin-2.

The C-terminal regions of thrombospondins (TSPs) contain three elements, EGF-like modules (E), a series of Ca(2+)-binding repeats (Ca), and a C-terminal sequence (G). We have looked for interactions among these elements in four recombinant proteins based on human TSP-2: E3CaG-2, CaG-2, E3Ca-2, and Ca-2. When bound Ca(2+) was assayed by atomic absorption spectroscopy or an equilibrium dialysis protocol in which Ca(2+) was removed from the proteins prior to equilibrium dialysis, E3CaG-2 bound 22-27 Ca(2+), CaG-2 bound 17-20 Ca(2+), and E3Ca-2 and Ca-2 bound 14-20 Ca(2+). Approximately 10 of the bound Ca(2+) in E3CaG-2 were exchangeable. The far UV circular dichroism (CD) spectrum of Ca(2+)-replete E3CaG-2 contained a strong negative band at 203 nm attributable to Ca and a less intense negative band at 218 nm attributable to Ca and G. Chelation of Ca(2+) with EDTA shifted the 203 nm band of all four proteins and the 218 nm band of E3CaG-2 and CaG-2 to less negative positions. The apparent EC50 for the far UV CD transition was 0.22 mM Ca(2+) for all proteins, indicating that Ca(2+) binding to Ca is primarily responsible for the CD change. Near UV CD and intrinsic fluorescence revealed that the tryptophan residues in G are sensitive to changes in Ca(2+). Differential scanning calorimetry of the proteins in 2 mM Ca(2+) showed that E3CaG-2 melts with two transitions, 44-51 degrees C and 75-83 degrees C. The lower transition required G, while the higher transition required Ca. Both transitions were stabilized in constructs containing E3. These results indicate that E3, Ca, and G function as a complex structural unit, and that the structures of both Ca and G are influenced by the presence or absence of Ca(2+).

Cloning and functional expression of the mucrosobin protein, a beta-fibrinogenase of Trimeresurus mucrosquamatus (Taiwan Habu).

A venom-specific cDNA encoding for a thrombin-like enzyme designated as mucrosobin has been cloned and sequenced from the cDNA library of the venomous gland of Trimeresurus mucrosquamatus. The full-length cDNA of mucrosobin was assembled by oligonucleotide screening and 5'-rapid amplification of cDNA ends. The amino acid sequence deduced from the cDNA consists of 257 amino acid residues with a putative signal peptide of 24 residues. It is highly homologous to the other thrombin-like enzymes (batroxobin, mucofirase, and calobin), suggesting that it is a serine proteinase with a conserved catalytic triad of His(41), Asp(84) and Ser(179) in the deduced form of mucrosobin protein. Northern blot analysis revealed that the mucrosobin gene encodes an mRNA of 1.5 kb and suggested a tissue-specific expression in the venomous gland. In an effort to study the biological property of mocrosobin, we have expressed the 28-kDa protein as inclusion bodies in Escherichia coli. For analyzing enzymatic activity, the inclusion bodies were solubilized and the recombinant protein was refolded with a two-step dialysis protocol. The refolded recombinant protein exhibited a specific beta-fibrinogenolytic activity. This study offers a possibility of using genetic engineering to acquire a functional snake venom protein with therapeutic potential.

Incremental Initiation of Dialysis: One Center's Experience over a Two-Year Period

This pilot study describes our center's experience with peritoneal dialysis (PD) over the past 2 years using a "healthy start" dialysis protocol with an incremental approach to prescription management. Nonrandomized, prospective pilot study. Single PD unit of a university teaching hospital. Thirteen PD patients who initiated dialysis at our center from April 1997 to June 1999. Patients initiating PD with residual renal Kt/V of 1.0 - 2.0/week were invited to participate. They were given an initial dialysis prescription so that total (residual renal + dialysis) weekly Kt/V exceeded 2.0. The dialysis prescription was "incrementally" increased as residual renal function (RRF) declined. Data collected for all patients included monthly serum chemistries, residual renal weekly Kt/V and creatinine clearance (CCr) at 1- to 2-month intervals, and peritoneal weekly Kt/V and CCr at 3-month intervals and 1 month after each prescription change. To date, we have followed 13 patients on our incremental PD protocol for a total of 159.3 patient-months. Mean serum albumin concentration and mean normalized protein equivalent of nitrogen appearance (nPNA) were stable throughout the study. Mean total Kt/V and CCr remained above the recommended targets of 2.0/wk and 60 L/wk, respectively. Residual renal function declined rather slowly in our PD patients. One patient died from complications of aortic valve surgery and a second died from pneumonia. A third patient died from peritonitis. One patient required a new Tenckhoff catheter after catheter migration. Three patients were temporarily switched to hemodialysis after a hernia repair, a pleural leak, and elective native/transplant nephrectomies, respectively. Two patients were permanently switched to hemodialysis: one after an episode of peritonitis, the second after accidentally damaging his PD catheter. Providing incremental dialysis to maintain adequate total small solute clearance has been technically feasible in our patient population. However, a larger than expected number of complications was seen in our study. Fortunately, complications were easier to manage due to the presence of RRF. Because this study was not designed to compare outcome with that observed after traditional initiation of dialysis, further large-scale studies are needed.

An Amperometric Fructose Biosensor Based on Fructose Dehydrogenase Immobilized in a Membrane Mimetic Layer on Gold

A prototype amperometric fructose biosensor based on membrane-bound fructose dehydrogenase (Gluconobacter sp.) and the coenzyme ubiquinone-6 immobilized in a membrane mimetic layer on a gold electrode has been constructed and tested. A bare gold electrode first was modified through chemisorption of a mixture of octadecyl mercaptan and two short-chain disulfides, 3,3'-dithiodipropionic acid and cystamine dihydrochloride. The membrane-bound enzyme, coenzyme, and additional phospholipid were codeposited through a detergent dialysis protocol. The short-chain modifiers may provide electrostatic interactions with enzyme surface charges, while the alkanethiolate and phospholipids enable hydrophobic interaction with the largely lipophilic, membrane-bound enzyme. At oxidizing potentials, the enzyme electrode responded with catalytic current densities up to 45 μA/cm(2) when exposed to fructose at 10 mM. The sensor exhibited a response time of less than 20 s, a sensitivity of 15 μA/cm(2)·mM and a detection limit of less than 10 μM. Biosensor measurements of d-fructose in apple and orange juice agreed to within a few percent with those made with an enzymatic spectrophotometric assay. The membrane mimetic layer effectively blocked access of interfering ascorbic acid to the electrode surface. Only a 4% positive error was observed in the presence of ascorbic acid at 5% of the fructose concentration (2 mM), which indicates that this construct could be particularly useful for quantitation of fructose in citrus juice.

The peritoneal equilibration test in children

Eight children, aged 15 months to 17 years 9 months, maintained by continuous ambulatory peritoneal dialysis (CAPD)/continuous cycling peritoneal dialysis and nine adults, aged 20-59 years, managed by CAPD were compared using a standardized peritoneal dialysis protocol, the peritoneal equilibration test (PET). The peritoneal glucose concentration tended to equilibrate with the serum glucose more rapidly in children, but the percentage of the glucose load absorbed was not different between the two age groups. There was an inverse trend between the percentage of glucose absorbed and age in children. Peritoneal creatinine clearance scaled to surface area in children was significantly less than that of the adults; however, the clearances became similar when adjusted for body weight. Peritoneal creatinine clearance scaled to surface area bore a positive and significant relationship to age which, when expressed per kilogram body weight, disappeared. Children had a significantly higher dialysate/plasma (D/P) creatinine ratio after the first 2 h of the PET, but this ratio approached unity by 4 h and was not different from adults. The fractional change in the creatinine D/P ratio during the PET was not different between the two age groups. Drain volume adjusted to surface area was significantly less in children than adults. This difference was reversed when drain volume was factored by weight. Similarly drain volume scaled to surface area demonstrated a significant and positive relationship to age, which disappeared when drain volume was expressed per kilogram body weight. Ultrafiltration, whether factored by weight or scaled to surface area, did not differ between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Tyrosine and N-carbamoyl-tyrosine in end-stage renal disease during continuous ambulatory peritoneal dialysis.

Lower-than-normal tyrosine concentrations of unexplained pathogenesis in plasma and intracellular body water have been reported in patients with chronic renal failure. We found a derivative of tyrosine that is not measured by the usual methods of amino-acid analysis because its alpha-amino group is blocked and cannot react to form other derivatives. An in vivo covalent reaction with urea-derived cyanate forms alpha-amino-carbamoyl-tyrosine (N-C-Tyr) in patients with end-stage renal disease. A longitudinal study of patients with end-stage renal disease who were treated with continuous ambulatory peritoneal dialysis shows that plasma that is obtained within 4 hours of the morning meal contains 70.1 +/- 6 mumol/L of tyrosine (mean +/- SEM) and 77.2 +/- 12 mumol/L of N-C-Tyr (mean +/- SEM). Thus there is a molecule of N-C-Tyr for each molecule of tyrosine present. The carbamoylation index or ratio of N-C-Tyr to tyrosine, blood urea nitrogen, episodes of peritonitis, and changes in dialysis protocol were compared. A reduction in the number of peritoneal dialysis exchanges resulted in parallel increases in carbamoylation index and blood urea nitrogen. Altering dialysis by increasing the number of exchanges or adding supplemental hemodialysis resulted in a decrease in the carbamoylation index with a delayed decrease in blood urea nitrogen. We found a significant increase of N-C-Tyr (p = 0.005) and of the carbamoylation index (p = 0.004) during six episodes of peritonitis compared with 10 periods of no peritonitis in two patients who had multiple episodes of peritonitis.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of erythropoietin on parameters of sympathetic nervous activity in patients undergoing chronic haemodialysis.

The effect of erythropoietin (rHu-EPO) on blood pressure and on parameters of sympathetic nervous activity of patients undergoing chronic haemodialysis treatment was evaluated in a controlled and randomized 6-months study. Both groups did not differ significantly at the onset of the study in terms of dialysis protocol, haematological parameters, blood chemistry, weight, blood pressure and parameters of sympathetic nervous activity. Haemoglobin increased from 7.0 +/- 0.3 g dl-1 to 10.0 +/- 0.3 g dl-1 by 6 months on rHu-EPO (n = 11) and remained constant in the control group (n = 11). After 6 months diastolic blood pressure was higher in the treatment group compared with the control group as well as compared with pretreatment values, whereas reactivity to exogenous noradrenaline and platelet alpha 2-adrenoceptor densities ([3H]-yohimbine- and [3H]-UK 14,304-binding sites) were lower. Plasma noradrenaline concentration increased during rHu-EPO compared with pretreatment values, while dry weights were unchanged during the study. The results demonstrate alterations of some parameters of sympathetic nervous activity during rHu-EPO-therapy which were absent in the control group.

Peritoneal Clearances with Different Dialysis Regimens in Children Undergoing Continuous Cycling Peritoneal Dialysis

Peritoneal clearances and dialysate protein losses occurring in paediatric patients undergoing different continuous cycling peritoneal dialysis (CCPD) regimens have not been well defined. We, therefore, evaluated 10 children aged 15.8 +/- 2.5 (SD) years who were maintained on home peritoneal dialysis for 20.5 +/- 10 months. All patients had at least 3 months of CCPD. The patients were admitted to the Clinical Research Center for 48 hours and allocated to five different dialysis protocols. In protocol I, the frequency of exchanges was 10 per 10 hours; in Protocol II it was 5 per 10 hours; and in Protocol III it was 3 per 10 hours. Protocol II D and III D had, in addition, a daytime dwell of one-half the night-time volume. A 1.5% glucose dialysate solution was used for night-time dialysis, and 4.25% glucose dialysate solution for the daytime dwell. The mean inflow dialysate volume per exchange was 36.7 +/- 5.6 ml/kg body weight and was constant in each patient for each study protocol. BUN and creatinine clearances for each protocol were calculated and dialysate protein losses were measured. The data indicate that hourly night-time dialysis (Protocol I) provides best solute clearance. A daytime dwell further enhances the total solute clearance and should be used preferably in anuric patients. Residual urine output contributes significantly to the total solute clearance. Protein losses are maximum with low-frequency exchanges and a daytime dwell. No significant differences in the serum albumin concentrations were found during the different protocols; however, the long-term effect of the protein loss on the nutritional status of the patients requires further evaluation.