Purification and partial characterization of candidate antidiuretic hormone water channel proteins of M(r) 55,000 and 53,000 from toad urinary bladder

Antidiuretic hormone (ADH) increases toad bladder granular cell apical membrane osmotic water permeability (Pf) by insertion of cytoplasmic vesicles containing water channels into the apical membrane. Termination of ADH stimulation results in endocytosis of water channel-containing membrane. In previous work, we have purified water channel-containing vesicles and demonstrated that they contain 12 major protein bands when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On the basis of vectorial labeling studies of granular cells and purified vesicles, we have proposed previously that vesicle proteins of 55, 53, and 17 kDa are ADH water channel components. In this report, we have purified and analyzed these three proteins using a combination of SDS-PAGE, peptide mapping, amino acid composition, and amino-terminal analyses. The 55- and 53-kDa proteins are distinct protein species possessing a high degree of structural similarity. Both possess a large content of cysteine. The 17-kDa protein appears to be a proteolytic fragment of the 53-kDa protein. None of these three proteins is phosphorylated or contains large amounts of covalently linked carbohydrate. ADH-elicited Pf is inhibited by the organic mercurial reagent fluorescein mercuric acetate (FMA). Exposure of water channel-containing vesicles to FMA labels selectively four vesicle proteins of 92, 55, 53, and 29 kDa while reducing vesicle Pf by 82%. The combination of FMA and 2-mercaptoethanol or exposure to another mercurial reagent, n-ethylmaleimide, does not inhibit vesicle Pf. Together, these data provide additional evidence for the role of the 55- and 53-kDa proteins as components of the ADH water channel. These candidate ADH water channel proteins are distinct from a 28-kDa candidate water channel protein (CHIP 28) isolated recently from human erythrocyte membranes and kidney proximal tubule by Agre and co-workers (Preston, G. M., Carroll, T. P., Guggino, W. B., and Agre, P. (1992) Science 256, 385-387).     

Modulation of cytochrome P-450-dependent monooxygenases, glutathione and glutathione S-transferase in rat liver by geniposide from Gardenia jasminoides

Geniposide is an iridoid glycoside extracted from the fruits of Gardenia jasminoides, which are used as a food colorant and as a traditional Chinese medicine for treatment of hepatic and inflammatory diseases. The effects of geniposide and G. jasminoides fruit crude extract on liver cytochrome P-450 (P-450)-dependent monooxygenases, glutathione and glutathione S-transferase were investigated using rats treated orally with the iridoid glycoside (0.1 g/kg body weight/day) or the fruit crude extract (2 g/kg/day) for 4 days. The treatments decreased serum urea nitrogen level but increased liver to body weight ratio, total hepatic glutathione content and hepatic cytosolic glutathione S-transferase activity. Treatments with geniposide and G. jasminoides decreased P-450 content, benzo[a]pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, and erythromycin N-demethylation activities in liver microsomes without affecting aniline hydroxylation activity. The natural products had no effect on glutathione content and monooxygenase activities in kidney microsomes. Immunoblotting analyses of liver microsomal proteins using mouse monoclonal antibody 2-13-1 to rat P4503A1/2 revealed that geniposide and G. jasminoides crude extract decreased the intensity of a P4503A-immunorelated protein. Protein blots probed with mouse monoclonal antibody 1-12-3 to rat P4501A1 and rabbit polyclonal antibody against human P4502E1 showed that both treatments had little or no effect on P4501A and 2E proteins. The present findings demonstrate that geniposide from G. jasminoides has the ability to inhibit a P4503A monooxygenase and increase glutathione content in rat liver.     

Prolongation of allograft survival by 1,25-dihydroxyvitamin D3

BACKGROUND: 1,25-Dihydroxyvitamin D3, the hormonal form of vitamin D, is now believed to play a significant role in the immune responses, both in vitro and in vivo, preventing the development of several autoimmune diseases. These studies suggest that 1,25-dihydroxyvitamin D3 may be effective in prolonging allograph survival. METHODS: To test the hypothesis that 1,25-dihydroxyvitamin D3 would prolong allograft survival, neonatal heart grafts were transplanted to allogeneic recipients receiving either 19-nor-1,25-dihydroxyvitamin D2 (200 ng/day) or 1,25-dihydroxyvitamin D3 (50 ng/mouse/day) orally through the diet. The efficacy of 1,25-dihydroxyvitamin D3 in prolonging graft survival in a vascularized model was determined by heterotopic ACI to Lewis heart transplants. RESULTS: The provision of exogenous 1,25-dihydroxyvitamin D3 or an analog, 19-nor-1,25-dihydroxyvitamin D2, to mice markedly prolonged the survival of neonatal mouse heart allografts. Similar results were obtained with a vascularized heterotopic heart transplant model in rats. Cyclosporine at a maximum 25 mg/kg dose for mice proved less effective than 1,25-dihydroxyvitamin D3. Graft survival in mice differing at class I and class II loci (B10.A(4R) --> C57BL/10) increased from 13.0+/-1.1 days to 51.0+/-5.6 days and was significantly better than cyclosporine monotherapy (33.2+/-3.6). Rat heart survival in a high responder strain combination (ACI --> Lewis) increased from 6.2+/-0.3 to 25.2+/-2.8 days. The increased survival of the transplants brought about with 1,25-dihydroxyvitamin D3 was not accompanied by hypercalcemia in rats. CONCLUSION: These results suggest that 1,25-dihydroxyvitamin D3 can be used as an effective agent in preventing graft rejection.     

Effect of the hydrolysis method on the determination of the amino acid composition of proteins

Acetabular reconstruction in both primary and revision hip arthroplasty often requires reconstruction of deficient acetabular bone stock. The exact role of allografts remains controversial. Published results of structural allografting are presented. Recent literature supports the use of segmental allografts for reconstruction of large segmental and combined defects.     

Effect of alcohol consumption on adult and aged bone: a histomorphometric study of the rat animal model

The adult and aged skeleton exist in a time when osteoporosis and age-related bone loss is at a maximum, and it is modified by lifestyle factors such as alcohol. To determine the effect of life-long alcohol consumption on the adult and aged rat model, 4-week-old female Sprague-Dawley rats were divided into three diet groups. Alcohol-treated animals were fed a modified Lieber-DeCarli diet ad libitum containing 35% ethanol-derived calories, whereas the pair-fed animals (weight-matched to ethanol rats) received an isocaloric liquid diet in which maltose-dextrin substituted calories supplied by ethanol. Chow animals were fed a standard rat chow ad libitum. Proximal tibiae were removed and prepared for histomorphometric analysis after 3, 6, 9, 12, or 18 months on the diets. Previous studies, with young animals, showed that chronic alcohol consumption during the age of bone development reduced bone volume and trabecular number in cancellous bone. The present study demonstrates that these reductions last throughout life. The rate of bone formation is reduced in alcohol-fed animals, but most bone cell parameters are relative normal, except for wall thickness, indicating a reduced osteoblast activity.     

Magnetic studies of the trinuclear center in laccase and ascorbate oxidase approached by EPR spectroscopy and magnetic susceptibility measurements

The trinuclear centers in Rhus vernicifera laccase and Cucumis sativus ascorbate oxidase have been studied by EPR spectroscopy and magnetic susceptibility measurements over the wide range of 5 K to 300 K. The EPR spectra showed that type II copper receives increasing tetrahedral distortion with raising temperature. Magnetic susceptibilities of laccase showed that both of type I and type II coppers are almost fully paramagnetic since the antiferromagnetic interaction between type III coppers is extremely strong from 5 K to 300 K. On the other hand, the effective magnetic moment of ascorbate oxidase is contributed by ca. 1.7 Cu2+ even below ca. 100 K, since type II Cu is partly in the reduced form. The effective magnetic moment continuously increased with raising temperature because the antiferromagnetic interaction between type III coppers is not as strong as in the case of laccase. The simulation of the SQUID measurement results suggested that the conformational change of the ascorbate oxidase molecule caused the temperature dependence of the antiferromagnetic interaction. The type II Cu EPR signals in laccase and ascorbate oxidase were conspicuously broadened with raising temperature because of the increasing contribution of the triplet state by type III Cu's and/or of the rapid relaxation which finally led to only ca. 30% detection of the type II Cu signals at room temperature. The stepwise binding of azide to the trinuclear center made one of type III Cu's to be EPR detectable. SQUID measurements indicated that only one Cu in the trinuclear center is paramagnetic and other two Cu's are antiferromagnetically coupled for both of the one- and two-azide bound forms. The binding mode of azide to the trinuclear center was discussed based on some models.     

Interkeratinocyte adherens junctions: immunocytochemical visualization of cell-cell junctional structures, distinct from desmosomes, in human epidermis

The intramedullary localization of schwannomas is rare, corresponding to 0.3% of all intraspinal tumors. The authors report the case of a 52-year-old white female patient that presented with symptoms of spinal compression by the presence of an intramedullary schwannoma at the level C4-C6. There were no symptoms of neurofibromatosis, entity frequently related to the lesion. The magnetic resonance imaging examination and the per-operatory biopsy were decisive factors in planning and executing the treatment, by establishing the characteristics, location and diagnosis of the lesion. Its delimitation and posterior location have facilitated total surgical exeresis. The transoperatively histopathologic examination allowed adequate surgical procedure. The Schwann cell is not found normally in the central nervous system and its presence in this site has been subject of many theories exposed in this paper, which proposes comprehensive review of the clinical aspects, imaging diagnosis, pathology, differential diagnosis and treatment of schwannomas. It is probable that, with the advances verified in the available diagnostic methods, a greater number of these lesions may be diagnosed in the future.