Expression cloning and characterization of a renal electrogenic Na+/HCO3- cotransporter

Bicarbonate transporters are the principal regulators of pH in animal cells, and play a vital role in acid-base movement in the stomach, pancreas, intestine, kidney, reproductive system and central nervous system. The functional family of HCO3- transporters includes Cl- -HCO3- exchangers, three Na+/HCO3- cotransporters, a K+/HCO3- cotransporter, and a Na+-driven Cl- -HCO3- exchanger. Molecular information is sparse on HCO3- transporters, apart from Cl- -HCO3- exchangers ('anion exchangers'), whose complementary DNAs were cloned several years ago. Attempts to clone other HCO3- transporters, based on binding of inhibitors, protein purification or homology with anion exchangers, have so far been unsuccessful. Here we monitor the intracellular pH and membrane voltage in Xenopus oocytes to follow the expression of the most electrogenic transporter known: the renal 1:3 electrogenic Na+/HCO3- cotransporter from the salamander Ambystoma tigrinum. We now report the successful cloning and characterization of a cDNA encoding a cation-coupled HCO3- transporter. The encoded protein is 1,035 amino acids long with several potential membrane-spanning domains. We show that when it is expressed in Xenopus oocytes, this protein is electrogenic, Na+ and HCO3- dependent, and blocked by the anion-transport inhibitor DIDS, and conclude that it is the renal electrogenic sodium bicarbonate cotransporter (NBC).     

Dollars may not buy as many QALYs as we think: a problem with defining quality-of-life adjustments

The scale of health state quality that should be used to compute quality-adjusted life years (QALYs) ranges from 0 (death) to 1.0 (excellent health); this is called the "Q" scale. But many cost-utility analyses (CUAs) in the literature use the upper anchor of the scale to denote only the absence of the particular health condition under investigation, and weight the disease state proportional to this endpoint; these are called "q" scales. Computations using q-scale health-state weights ignore the fact that the average patient is still subject to chronic and acute conditions comorbid with the condition being analyzed; the absence of a particular condition is not in general the same as excellent health, i.e., the Q scale is longer than a q scale. CUAs based on q scales yield "qALYs." Incremental $/qALY ratios are generally lower than $/QALY ratios; in the example presented, $/qALY must be inflated by about 15% to yield $/QALY. Other CUAs correctly weight disease states using the Q scale, but erroneously assign a quality weight of 1.0 to absence of the disease in the CUA computations. The results of such analyses are called "NP-QALYs," as the correction factor to compute QALYs is not a simple proportional adjustment. The authors suggest that analysis doing cost-utility analyses without access to primary data from treated patients use average age-specific health-related quality-of-life weights from population-based studies to represent the state of not having a particular disease. Consumers of CUAs should closely examine the nature of the QALYs in any published analyses before making decisions based on their results.     

Stress fracture of the sternum: an unusual injury?

Stress fracture of the sternum is a rare condition which presents as acute anterior chest pain after repetitive upper-body exercise. Two case reports are presented and it is postulated that this is an often underdiagnosed condition which should be considered in the differential diagnosis of acute chest pain in the athlete. Awareness of the injury together with meticulous clinical examination supported by good quality radiographs or isotope bone scan may lead to an increase in the diagnosis of this injury.     

Pharmacokinetics of dolasetron after oral and intravenous administration of dolasetron mesylate in healthy volunteers and patients with hepatic dysfunction

In previous studies, dolasetron was shown to have both renal and hepatic elimination mechanisms. This study was conducted to determine the impact of varying degrees of hepatic dysfunction on the pharmacokinetics and safety of dolasetron and its reduced metabolites. Seventeen adults were studied: six healthy volunteers (group I), seven patients with mild hepatic impairment (Child-Pugh class A; group II), and four patients with moderate to severe hepatic impairment (Child-Pugh class B or C1; group III). Single 150-mg doses of dolasetron mesylate were administered intravenously and orally, with a 7-day washout period separating treatments. After intravenous administration, no differences were observed between healthy volunteers and patients with hepatic impairment in maximum plasma concentration (Cmax), areas under the plasma concentration-time curve (AUC), or elimination half-life (t1/2) of intact dolasetron. No significant differences were found in Cmax, AUC, or apparent clearance (C(lapp)) of hydrodolasetron, the primary metabolite of dolasetron. The mean t1/2 increased from 6.87 hours in group I to 11.69 hours in group III. After oral administration, C(lapp) of hydrodolasetron decreased by 42%, and Cmax increased by 18% in patients with moderate to severe hepatic impairment. There were less changes in patients with mildly hepatic impairment. Total percentage of dose excreted as metabolites was similar for healthy volunteers and patients with hepatic impairment, although urinary metabolite profiles differed slightly. Dolasetron was well tolerated and there were no apparent differences in adverse effects between groups or treatments. Because hepatic impairment did not influence Cl(app) of hydrodolasetron after intravenous administration, and the range of plasma concentrations of hydrodolasetron after oral administration was not different from those observed in healthy volunteers, dosage adjustments are not recommended for patients with hepatic disease and normal renal function.     

Sonographic detection of focal changes in the liver hilus in patients receiving corticosteroid therapy

OBJECTIVE: While diffuse deposition of fat may occur with corticosteroid (CS) administration both in the liver and in other organs, comparatively little is known about focal changes in the liver under corticosteroid medication. Therefore, we evaluated pattern and extent of focal hepatic steatosis by ultrasound (US) in patients receiving corticosteroids. SUBJECTS AND METHODS: 93 patients with known inflammatory bowel disease (IBD) received corticosteroids during a period of at least six weeks prior to the ultrasound examination and 28 IBD-patients had no corticosteroids within the last three years. 13 additional patients received corticosteroids for other reasons than IBD for > 1 year. 80 healthy volunteers served as controls. Focal changes of the liver as assessed by high resolution ultrasound (Acuson 128, 3.5 and 5 MHz) were defined as areas of brighter echogenicity compared to the general aspect of the liver. The size of the hyperechoic areas was documented (photoprint). RESULTS: 40/93 IBD-patients with corticosteroids (43%) had definite areas of brighter echos in the hilus region of the liver. In IBD-patients without corticosteroids only one patient showed a focal brighter echogenicity, whereas in the non-IBD group with corticosteroids 8/13 had focal lesions (62%). In the control group only four healthy subjects showed brighter areas (5%). CONCLUSION: Bright focal areas in the liver hilus occur in > 40% of IBD-patients during corticosteroid medication. This phenomenon occurs in IBD-patients as frequently and as intense as in other patients with longstanding corticosteroid therapy. There is a hilar area of the liver with typical size and location which reacts to corticosteroid administration with hyperechoic reflexes at ultrasound investigation. This is important to know when it comes to the differential diagnosis of focal changes.     

Reproducibility of polypeptide spot positions in two-dimensional gels run using carrier ampholytes in the isoelectric focusing dimension

The reproducibility of complex protein patterns in two-dimensional (2-D) gels run with carrier ampholytes in the first dimension has been investigated. Two different laboratories collaborated in the study and 18 or 19 gels were run in each laboratory for comparison. The electrophoresis chemicals, running devices, and samples were standardized in both labs. The resulting 37 gels were scanned with a charge-coupled device (CCD) camera and spots were located, counted, quantified, and matched using a commercially available image analysis system. Subsequently, the reproducibility of spot position was determined. To perform the statistical analysis, the test gels were initially each matched to a master reference gel. Next, three sets of 12 gels (the image analysis software database could analyze only 12 gels at a time) were analyzed and the isoelectric point (pI) and molecular weight (M(r)) positional variation of all the spots that matched across the gels in each set was determined. The resulting statistical analysis indicates very high reproducibility of the carrier ampholyte technique.     

Mechanism-based inactivation of human liver cytochrome P450 2A6 by 8-methoxypsoralen

The P450 2A6 catalyzed 7-hydroxylation of coumarin proceeded with a mean Km of 0.40 (+/-0.13) microM and Vmax of 6.34 nmol/nmol P450/min (36-fold variation) in microsomal preparations from a panel of 12 human livers. Substrate depletion was avoided during the kinetic determinations. 8-Methoxypsoralen (8-MOP) is a potent mechanism-based inactivator of human liver P450 2A6 and reconstituted purified recombinant P450 2A6 based on the following evidence: 1) 8-MOP causes time, concentration, and NADPH-dependent loss of P450 2A6 activity that is not reversed by potassium ferricyanide or extensive dialysis, 2) loss of P450 2A6 activity is associated with a loss of spectrally observable P450, 3) addition of nucleophiles or reactive oxygen scavengers to the incubations does not prevent inactivation of P450 2A6, and 4) 8-MOP-dependent P450 2A6 inactivation is inhibited (concentration dependent) by the addition of a competitive inhibitor (pilocarpine). Inactivation is selective for P450 2A6 at low concentrations of 8-MOP (2.5 microM) after short incubation time periods (3 min) and was characterized by a KI of 0.8 and 1.9 microM in a reconstituted and microsomal system, respectively, and a kinact of 1 min-1 and 2 min-1 in a reconstituted and microsomal system, respectively. A substrate depletion partition ratio of 21 was calculated for the inactivation of recombinant P450 2A6. Potency and selectivity suggest that 8-MOP could be a useful tool in vitro for evaluating P450 2A6 activity in various enzyme preparations.     

Dynamic endocrine testing. The Mayo Clinic model

An endocrine testing center (ETC) is a universal requirement for the practice of endocrinology. Modifications of the Mayo Clinic model for an ETC should be applicable to most endocrine practices. Key components of an ETC include a centralized testing area, registered nurse-physician team, detailed testing protocols, and patient education programs.     

Cleavage of single strand RNA adjacent to RNA-DNA duplex regions by Escherichia coli RNase H1

RNase H1 from Escherichia coli cleaves single strand RNA extending 3' from an RNA-DNA duplex. Substrates consisting of a 25-mer RNA annealed to complementary DNA ranging in length from 9-17 nucleotides were designed to create overhanging single strand RNA regions extending 5' and 3' from the RNA-DNA duplex. Digestion of single strand RNA was observed exclusively within the 3' overhang region and not the 5' overhang region. RNase H digestion of the 3' overhang region resulted in digestion products with 5'-phosphate and 3'-hydroxyl termini. The number of single strand RNA residues cleaved by RNase H is influenced by the sequence of the single strand RNA immediately adjacent to the RNA-DNA duplex and appears to be a function of the stacking properties of the RNA residues adjacent to the RNA-DNA duplex. RNase H digestion of the 3' overhang region was not observed for a substrate that contained a 2'-methoxy antisense strand. The introduction of 3 deoxynucleotides at the 5' terminus of the 2'-methoxy antisense oligonucleotide resulted in cleavage. These results offer additional insights into the binding directionality of RNase H with respect to the heteroduplex substrate.