Chicken oviductal ecto-ATP-diphosphohydrolase. Purification and characterization

An ecto-ATP diphosphohydrolase (ATPDase) was purified to homogeneity from vesiculosomes shed from chicken oviduct. First, the ecto-ATPDase-enriched vesiculosomes were concentrated by filtration, differential centrifugation, and exclusion chromatography. Next, the nonionic detergent, Nonidet P-40, was used to extract the ecto-ATPDase from vesiculosomal membranes, and the solubilized enzyme was further purified by ion exchange (DEAE-Bio-Gel) and lentil-lectin-Sepharose 4B chromatography. In the final stage, immunoaffinity chromatography was utilized to obtain purified ecto-ATPDase. More than 25,000-fold purification was achieved. Specific activity of the purified enzyme was greater than 800 micronol/min/mg of protein with MgATP as the substrate, the highest ever reported for an ATPDase. The enzyme also hydrolyzed other nucleoside triphosphates in the presence of magnesium at similar rates and CaATP and MgADP at lower rates. The molecular mass of the purified glycoprotein was 80 kDa as determined by SDS-polyacrylamide gel electrophoresis and Western blot analysis. Based on its enzymatic properties, the relationship of the chicken oviduct ecto-ATPDase with other reported ATPDases and ecto-ATPases is discussed.     

Embryotoxic doses of vitamin A to rabbits result in low plasma but high embryonic concentrations of all-trans-retinoic acid: risk of vitamin A exposure in humans

Retinoid pharmacokinetics were examined in plasma, placenta and embryos of gestational d 12 rabbits following application of an embryotoxic dosing regimen (10 mg retinyl palmitate/kg body wt per day from gestational d 7 to 12). Vehicle-treated or untreated rabbits served as controls. Physiological concentrations of all-trans-retinoic acid (all-trans-RA) and 13-cis-RA in rabbit plasma (5-8.33 nmol/L) were very close to the endogenous levels in human plasma. In addition, we identified endogenous all-trans-RA, 3,4-didehydroretinol and 3,4-didehydroretinoic acid in rabbit embryo. Following the last retinyl palmitate administration, apparent steady-state concentrations of all retinoids were reached in the examined compartments of rabbits. The major polar retinoid in plasma was 9, 13-di-cis-RA, but its embryonic concentrations were only about 6% of those in plasma. In the embryo, retinol and its esters were found at high concentrations; lower amounts of all-trans-4-oxo-RA and the newly identified 14-hydroxy-4, 14-retro-retinol could also be measured. Embryonic concentrations of all-trans-RA were about 100% higher than endogenous levels. The overall exposure of the embryo to this retinoid was, however, substantial. Embryonic area under the concentration time curve values strongly suggest that the embryotoxicity of the applied dosing regimen is mainly due to the action of all-trans-RA. A very remarkable finding of this study is the marginal increase of plasma concentrations of all-trans-RA over their endogenous levels, which is comparable to the human situation after vitamin A intake. This analogy indicates that high vitamin A intake may be associated with a higher risk for teratogenic effects in humans even in the absence of high elevation of plasma all-trans-RA levels.     

Gestational choriocarcinoma metastatic to the brain treated successfully by stereotactic radiosurgery and chemotherapy

This report analyzes the intracorporal lengths of 236 consecutive organically impotent men who underwent insertion of a penile prosthesis. The average right or left intracorporal length in this series was 20.9 cm, with a standard deviation of 2.2 cm. Intracorporal length ranged from 14-27 cm. This data may be used as reference ranges for penile prosthesis manufacturers, and for Urologists who implant prostheses or perform penile lengthening procedures.     

Electrophysiological dissociation of recency and recognition memory

Event-related brain potentials (ERPs) were recorded during the test phases of two experiments. In experiment 1, subjects first studied two consecutively presented word lists. At test, they were presented with pairs of words, and were required to judge which word had been presented most recently. The test pairs were composed of two previously studied words, one drawn from each list, (Old+Old pairs), one previously studied and one new word (Old+New pairs), or two unstudied words (New+New pairs). At temporo-parietal electrodes, ERPs to Old+Old and Old+New pairs were both reliably more positive-going than those to New+New pairs. At electrode sites overlying prefrontal cortex, ERPs to Old+Old pairs attracting correct recency judgements were more positive, from around 300 ms onwards, than those elicited by the other classes of item, which did not differ from one another. In experiment 2, the test task was changed to one that required discrimination between Old+New items on the one hand, and Old+Old and New+New pairs on the other. ERPs to Old+Old and Old+New pairs once again differed from those to New+New pairs at temporo-parietal sites, but no differences were evident between the ERPs from frontal electrode sites. In line with the evidence from lesion studies, these findings suggest that judgements of relative recency depend upon processes, supported by the prefrontal cortex, additional to those that are necessary for recognition memory. They further suggest that these processes are activated rapidly and selectively in response to pairs of studied items when these must be discriminated on the basis of their relative recency of occurrence.     

The role of DnaJ-like proteins in glucocorticoid receptor.hsp90 heterocomplex assembly by the reconstituted hsp90.p60.hsp70 foldosome complex

The glucocorticoid receptor (GR) is recovered from hormone-free cells in a heterocomplex with the molecular chaperone hsp90, which is required to produce the proper folding state for steroid binding. GR.hsp90 heterocomplexes are formed by a multiprotein system that appears to exist in all eukaryotic cells. Recently, we have reconstituted a receptor.hsp90 heterocomplex assembly system with purified rabbit hsp90 and hsp70 and bacterially expressed human p23 and p60. We have shown that hsp90, p60, and hsp70 form an hsp90.p60. hsp70 complex that converts the GR from a non-steroid binding to a steroid binding form (Dittmar, K. D., and Pratt, W. B. (1997) J. Biol. Chem. 272, 13047-13054). The resulting GR.hsp90 heterocomplex rapidly disassembles unless p23 is present to bind to the ATP-dependent conformation of hsp90 and stabilize its association with the receptor (Dittmar, K. D., Demady, D. R., Stancato, L. F., Krishna, P., and Pratt, W. B. (1997) J. Biol. Chem. 272, 21213-21220). In the current work, we show that the purified rabbit hsp70 utilized in prior studies is contaminated with a small amount of the rabbit DnaJ homolog hsp40. Elimination of the hsp40 from the purified GR.hsp90 assembly system reduces assembly activity, and the activity is restored by addition of the purified yeast DnaJ homolog YDJ-1. hsp40 is a component of the hsp90.p60.hsp70 foldosome complex isolated from reticulocyte lysate with antibody against p60. Under conditions that promote binding of p23 to hsp90 (elevated temperature, ATP, Nonidet P-40, molybdate), a five-membered (p23. hsp90.p60.hsp70.hsp40) complex of chaperone proteins is formed in reticulocyte lysate or from purified proteins. The hsp40-free, purified assembly system has a modest level of assembly activity that is maximally potentiated by YDJ-1 when it is present at about one-twentieth the concentration of hsp70. Although hsp40 is not in the final GR.hsp90 heterocomplex isolated from L cell cytosol, it is in the GR.hsp90 heterocomplex assembled in reticulocyte lysate. We conclude that hsp40 is a component of the multiprotein hsp90-based chaperone system where it potentiates GR.hsp90 heterocomplex assembly.     

Superior mesenteric artery syndrome presenting with acute massive gastric dilatation

A 14-year-old girl presenting with acute massive gastric dilatation secondary to duodenal obstruction by the superior mesenteric artery is described. The diagnosis was facilitated by contrast-enhanced abdominal computerized tomography. She was successfully treated by gastrostomy and subsequent duodenal derotation. This unique presentation of the superior mesenteric artery syndrome is discussed.