(+/-)-cis-2-methylspiro[1,3-oxathiolane-5,3'-quinuclidine] hydrochloride, hemihydrate (SNI-2011, cevimeline hydrochloride) induces saliva and tear secretions in rats and mice: the role of muscarinic acetylcholine receptors.

We investigated effects of (+/-)-cis-2-methylspiro[1,3-oxathiolane-5,3'-quinuclidine] hydrochloride, hemihydrate (SNI-2011, cevimeline hydrochloride), a rigid analogue of acetylcholine, on saliva and tear secretions in rats and mice to evaluate its therapeutical efficacy for xerostomia and xerophthalmia in patients with Sjogren's syndrome and X-ray exposure in the head and neck. Intraduodenal administrations of SNI-2011 increased saliva secretion in a dose-dependent manner at doses ranging from 3 to 30 mg/kg in normal rats and mice, two strains of autoimmune disease mice and X-irradiated saliva secretion defective rats. The salivation elicited by SNI-2011 was completely inhibited by atropine. A similar atropine-sensitive response was observed in tear secretion. In rat submandibular/sublingual gland membranes, [3H]quinuclidinyl benzilate (QNB) binding was saturable, and Scatchard plot analysis revealed a single population of binding sites with a Kd of 22 pM and a maximal binding capacity of 60 fmol/mg protein. The competitive inhibition curve of the [3H]QNB binding by SNI-2011 was obtained, and its dissociation constant value calculated from IC50 was 1-2 microM. These results suggest that SNI-2011 increases saliva and tear secretions through a direct stimulation to muscarinic receptors in salivary and lacrimal glands, and they suggest that SNI-2011 should be beneficial to patients with Sjögren's syndrome and X-ray exposure in the head and neck.

Long-lasting salivation induced by a novel muscarinic receptor agonist SNI-2011 in rats and dogs.

The sialogogic effect of SNI-2011, a novel muscarinic receptor agonist, (+/-)-cis-2-methylspilo [1,3-oxathiolane-5,3'-quinuclidine] hydrochloride, hemihydrate, was compared with that of pilocarpine hydrochloride in a dose range in which the two muscarinic agonists exhibited approximately similar efficacy in eliciting salivation. Pilocarpine (0.66-2.0 mg/kg, i.d.) induced a marked but short-lasting salivation in rats, whereas the salivation induced by SNI-2011 (20-60 mg/kg, i.d.) lasted 1.4- to 1.8-fold longer. In dogs, the sialogogic effect of SNI-2011(1-3 mg/kg, i.v.) also lasted about 2-fold longer than that of pilocarpine (0.1-0.3 mg/kg, i.v.). The plasma SNI-2011 level that caused salivation at a rate of 0.4 ml/min was about 100 ng/ml and higher rates of salivation (over 0.4 ml/min) induced by 1 mg/kg SNI-2011 lasted for about 90 min in dogs. The plasma pilocarpine level that caused salivation at a rate of 0.4 ml/min was about 25 ng/ml and the higher rate of salivation (over 0.4 ml/min) induced by 0.1 mg/kg pilocarpine lasted only for 20 min in dogs. Effective plasma levels of SNI-2011 persisted longer than those of pilocarpine. These results indicate that SNI-2011 may be useful in the treatment of xerostomia because of its long-lasting sialogogic action.

Serum levels of dehydroepiandrosterone sulfate in patients with asymptomatic cortisol producing adrenal adenoma: comparison with adrenal Cushing's syndrome and non-functional adrenal tumor.

The reported number of adrenal incidentalomas has been increasing because of wider application of imaging techniques. Patients with asymptomatic cortisol producing adrenal adenoma (ASCA) which secretes cortisol without clinical evidence of Cushing's syndrome has been more frequently observed than previously assumed, and they have a risk of adrenal insufficiency after adrenalectomy. Therefore patients with incidentalomas should be screened for cortisol overproduction. The aim of this study is to discover an easy screening test to uncover ASCA. We investigated the hormone profiles of 4 patients with ASCA in comparison with 11 patients with non-functional adrenal tumor and 10 patients with adrenal Cushing's syndrome. We also investigated the expression of dehydroepiandrosterone sulfotransferase (DHEA-ST) in surgically removed attached non-neoplastic adrenal tissues by immunostaining, which was considered to represent the degree of suppression of the hypothalamo-pituitary-adrenal axis. Serum dehydroepiandrosterone sulfate (DHEA-S) levels of all the patients with ASCA and adrenal Cushing's syndrome were lower than those of healthy subjects of corresponding age, but they were within the normal range in the patients with non-functional adrenal tumors. The serum DHEA-S level reflects the degree of suppression of the normal adrenal gland by cortisol hypersecretion from adrenal tumors. But the serum level of DHEA-S decreases with age, and because the normal range of serum DHEA-S is low in elderly subjects, we should be careful to evaluate the level of DHEA-S in elderly patients with adrenal Cushing's syndrome or ASCA. The immunohistochemical study showed DHEA-ST expression was noticeably suppressed in the adjacent adrenal cortex in ASCA and adrenal Cushing's syndrome. The decreased expression of DHEA-ST may reflect autonomous neoplastic cortisol secretion and subsequent ACTH suppression in ASCA and adrenal Cushing's syndrome. A single measurement of plasma ACTH or measurement of ACTH response to corticotropin-releasing hormone was not enough to screen for ASCA because of the wide variation among the cases. Dexamethasone suppression test is essential in identifying ASCA and also a single determination of serum DHEA-S is easy and may be useful for the screening of ASCA in adrenal incidentalomas in young and middle aged subjects, and is especially useful for outpatients.

The 3'-terminal exon of the family of steroid and phenol sulfotransferase genes is spliced at the N-terminal glycine of the universally conserved GXXGXXK motif that forms the sulfonate donor binding site.

The guinea pig estrogen sulfotransferase gene has been cloned and compared to three other cloned steroid and phenol sulfotransferase genes (human estrogen sulfotransferase, human phenol sulfotransferase, and guinea pig 3 alpha-hydroxysteroid sulfotransferase). The four sulfotransferase genes demonstrate a common outstanding feature: the splice sites for their 3'-terminal exons are identically located. That is, the 3'-terminal exon splice sites involve a glycine that constitutes the N-terminal glycine of an invariably conserved GXXGXXK motif present in all steroid and phenol sulfotransferases for which primary structures are known. This consistency strongly suggests that all steroid and phenol sulfotransferase genes will be similarly spliced. The GXXGXXK motif forms the active binding site for the universal sulfonate donor 3'-phosphoadenosine 5'-phosphosulfate. Amino acid sequence alignment of 19 cloned steroid and phenol sulfotransferases starting with the GXXGXXK motif indicates that the 3'-terminal exon for each steroid and phenol sulfotransferase gene encodes a similarly sized C-terminal fragment of the protein. Interestingly, on further analysis of the alignment, three distinct amino acid sequence patterns emerge. The presence of the conserved functional GXXGXXK motif suggests that the protein domains encoded by steroid and phenol sulfotransferase 3'-terminal exons have evolved from a common ancestor. Furthermore, it is hypothesized that during the course of evolution, the 3'-terminal exon further diverged into at least three sulfotransferase subdivisions: a phenol or aryl group, an estrogen or phenolic steroid group, and a neutral steroid group.

Characterization of guinea pig estrogen sulfotransferase expressed by Chinese hamster ovary cell-K1 stable transfectants.

Estrogen sulfotransferase (EST) activity expressed by Chinese hamster ovary (CHO)-K1 cells stably transfected with a plasmid containing a guinea pig EST complementary DNA insert was subjected to biochemical characterization, and the EST protein was further examined by nondenaturing isoelectric focusing and immunoblot analysis. CHO-K1 cells transfected with the same plasmid without the EST complementary DNA insert as well as untransfected CHO-K1 cells did not demonstrate either EST activity or the presence of an immunologically related protein. The EST expressed by the stably transfected CHO-K1 cells was found to manifest Michaelis-Menten kinetics and would use only estrogenic steroids as substrates, whereas other forms of steroids, such as pregnenolone, dehydroepiandrosterone, cortisol, and testosterone, were not acted on. When 17 beta-estradiol was used as a substrate, sulfonation occurred exclusively at the 3 position; 17-sulfonate was not formed. Thus, the expressed EST acted selectively on the 3-hydroxyl group of phenolic steroids. The apparent Km values for estrone, 17 beta-estradiol, and estriol were 60, 70, and 40 nM, respectively. The maximum velocity (Vmax) determinations for estrone and 17 beta-estradiol were equivalent, whereas the Vmax for estriol was reduced by 33%. Of the three estrogens, only 17 beta-estradiol caused substrate inhibition at a high concentration. Steroid sulfonation requires 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as the active sulfonate donor, and the Km value for PAPS was 1.2 microM. In steroid sulfotransferase reactions, two products are formed: the sulfonated steroid product and the desulfonated cofactor, 3'-phosphoadenosine-5'-phosphate (PAP). The sulfonation of 17 beta-estradiol was inhibited by PAP in a dose-dependent manner. In addition, the Km for PAPS was increased by PAP, whereas the Vmax was unaffected, indicating competitive inhibition (Ki, approximately 0.52 microM). The EST protein expressed by the CHO-K1 cell stable transfectants demonstrated a mol wt of 34 kilodaltons, as determined by sodium dodecyl sulfate-gel electrophoresis. Additionally, when the expressed EST protein was subjected to isoelectric focusing, it was found to consist of multiple charge isoforms. These findings are comparable to what has been previously reported for native guinea pig adrenocortical EST. Furthermore, the charge isoform pattern that was demonstrated for the expressed EST was similar to the pattern observed for the native protein.

Malignant exophthalmos associated with multiple myeloma.

We report a patient with malignant exophthalmos associated with multiple myeloma which showed no evidence of direct orbital involvement of plasma cells. This exophthalmos had similarities with Graves' ophthalmopathy, but the patient had no detectable autoimmune thyroid diseases. Plasmapheresis was effective not only for the treatment of heart and renal failure due to the myeloma kidney but also for the malignant exophthalmos. As the serum monoclonal IgG level was decreased by plasmapheresis, the improvement of proptosis, visual acuity, and hypertrophy of the extraocular muscle as measured by magnetic resonance imaging were observed. It is suggested that humoral factors removed by plasmapheresis might be involved in the pathogenesis of this nonendocrine exophthalmos.

Cryopreservation of porcine blastocysts by vitrification.

The objective of this study was to assess the possibility of cryopreservation of porcine expanded and hatched blastocysts by vitrification. The following four types of vitrification solutions were applied in this study: (i) EFT, a mixture of 7.2 M ethylene glycol, 0.003 M ficoll, and 0.3 M trehalose; (ii) DAP213, 2 M dimethyl sulfoxide, 1 M acetamide, and 3 M propylene glycol; (iii) DAP213-T, DAP213 supplemented with 0.3 M trehalose; and (iv) EPT, 4 M ethylene glycol, 3.1 M propylene glycol and 0.3 M trehalose. The embryos collected on Days 5 to 7 (Day 0 = onset of estrus) were allocated to eight experimental groups according to the types of vitrification solution and the developmental stage. In the toxicity test, the embryos were equilibrated in the respective vitrification solutions either in a single step or in four steps and then transferred to 1 M sucrose in a single step at 22 degrees C without cooling. As a whole, the viabilities of embryos equilibrated in the solutions were lower than those of control embryos. The stepwise equilibration was superior to a single-step equilibration in the in vitro survival of, especially, expanded blastocysts after dilution. No significant difference was observed between the vitrification solutions for the four-step method. In the vitrification test, the embryos were equilibrated in the solutions by the four-step method, loaded into a 0.25-ml plastic straw, and plunged into liquid nitrogen. Although viable embryos were obtained after warming from all of the combinations except the hatched blastocyst-EPT group, viabilities were further reduced by cooling (range of reduction rates: 60 to 100%). The possible cause of low survival after warming is also discussed concerning the cryophysical properties of vitrification solutions.

Cryoprotective effect of egg yolk in cryopreservation of porcine embryos.

The objective of the present study was to assess the effect of egg yolk supplementation in solutions for either cooling or freezing of porcine embryos. In Experiment 1, porcine embryos collected on Day 3 to 7 (Day 0 = onset of estrus) were allocated to 12 experimental groups according to their developmental stages (four-cell to compact morula, early blastocyst to blastocyst, expanded blastocyst, and hatched blastocyst) and the concentrations of egg yolk (0, 5, and 10% (v/v)) for cooling to 0 degrees C. Whereas no embryos ranging from four-cell to blastocyst survived, the 10-min exposure to 0 degrees C despite egg yolk supplementation, expanded blastocysts and hatched blastocysts allocated to egg yolk-supplemented groups survived cooling to 0 degrees C. In Experiment 2, porcine expanded and hatched blastocysts collected on Day 6 or 7 were assigned to six experimental groups according to their developmental stages and the type of cryoprotectant used (10% glycerol, 11% Me2SO, and 10% propylene glycol) for freezing at -196 degrees C. All freezing solutions contained either 5 or 10% egg yolk. The embryos were equilibrated with one of the freezing solutions, cooled from 25 to -7 degrees C at 1 degree C/min, seeded at that temperature, then cooled to -36 degrees C at 0.3 degree C/min, and finally plunged into liquid nitrogen for storage. The frozen embryos were thawed by immersion in 37 degrees C water. After the cryoprotectants were removed by dilution, the embryos were cultured for 48 h to evaluate their viability.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypersensitivity to thromboxane A2 in cholesterol-rich human platelets.

The effect of changes in platelet membrane cholesterol content on thromboxane A2 (TXA2)-induced platelet activation was studied. Concentrations of 9,11-epithio-11,12-methano-TXA2 (STA2), a stable analogue of TXA2 which can cause half-maximal aggregation and release of [14C]serotonin in cholesterol-rich platelets were significantly lower than those in cholesterol-normal platelets. STA2-induced increase in cytosolic calcium concentration and [32P]phosphatidic acid formation in cholesterol-rich platelets were significantly greater than those in cholesterol-normal platelets. The maximal concentration of binding site (Bmax) for SQ29548 was significantly increased in cholesterol-rich platelets compared with cholesterol-normal platelets, while the equilibrium dissociation rate constant (Kd) for SQ29548 did not differ between cholesterol-rich and cholesterol-normal platelets. The present study suggested that sensitivity to TXA2 was increased by the incorporation of cholesterol into platelet membrane and that the cause of hypersensitivity to TXA2 in cholesterol-rich platelets may be partly explained by an increase in binding capacity for TXA2.

Ultrasonic observation of postpartum uterine involution in the cow.

To learn about the progress of postpartum uterine involution in the dairy cow, the authors wanted to know whether the ultrasonic linear scanner is applicable for this purpose. The uterus was observed by the transducer, which was inserted into the rectum. Clear cross-sectional images of the endometrium were obtained in this way; however, those of the myometrium and the perimetrium were not very clear. Observation on the progress of uterine involution was started on Day 8 postpartum and continued to Day 43 postpartum. The diameter and area of the uterine horn and the endometrium in cross-sectional ultrasonograph images of the endometrium were estimated using a Magiscan 2 image analyzer. The relationship between the estimated dimensions of the uterine horn and postpartum days were satisfactorily fitted into polynomial regressions. Using the ultrasonic linear scanner, it was confirmed that uterine involution was completed at approximately 40 d postpartum. The applicability of the ultrasonic linear scanner for diagnosis of uterine involution in the postpartum cow is demonstrated by the results of the present study.

Identification of the globin chain and the amino acid residue responsible for polymerization of bullfrog (Rana catesbeiana) hemoglobin with iodo[14C]acetamide.

The bullfrog (Rana catesbeiana) major hemoglobin dissociates into its constituent globin chains (alpha and beta) which are separated by Sulfopropyl-Sephadex C-25 column chromatography after alkylation with iodo[14C]acetamide. Each globin chain has two cysteine residues and those of the beta-globin chain in the tetramer are preferentially alkylated with iodoacetamide.