Mice lacking the vitamin D receptor exhibit impaired bone formation, uterine hypoplasia and growth retardation after weaning.

1 alpha,25-Dihydroxyvitamin D3[1 alpha,25(OH)2D3], an active form of vitamin D, has roles in many biological phenomena such as calcium homeostasis and bone formation, which are thought to be mediated by the 1 alpha,25(OH)2D3 receptor (VDR), a member of the nuclear hormone receptor superfamily. However, the molecular basis for the actions of 1 alpha,25(OH)2D3 in bone formation, its role during development and VDR genetic polymorphisms for predicting bone mineral density are uncertain. To investigate the functional role of VDR, we generated mice deficient in VDR by gene targeting. We report here that in VDR null mutant mice, no defects in development and growth were observed before weaning, irrespective of reduced expression of vitamin D target genes. After weaning, however, mutants failed to thrive, with appearance of alopoecia, hypocalcaemia and infertility, and bone formation was severely impaired as a typical feature of vitamin D-dependent rickets type II (refs 8, 9). Unlike humans with this disease, most of the null mutant mice died within 15 weeks after birth, and uterine hypoplasia with impaired folliculogenesis was found in female reproductive organs. These defects, such as alopoecia and uterine hypoplasia, were not observed in vitamin D-deficient animals. The findings establish a critical role for VDR in growth, bone formation and female reproduction in the post-weaning stage.

Molecular monitoring and isolation of previously uncultured bacterial strains from the sheep rumen.

To estimate the contribution of uncultured bacterial groups to fiber degradation, we attempted to retrieve both ecological and functional information on uncultured groups in the rumen. Among previously reported uncultured bacteria, fiber-associated groups U2 and U3, belonging to the low-GC Gram-positive bacterial group, were targeted. PCR primers and fluorescence in situ hybridization (FISH) probe targeting 16S rRNA genes or rRNA were designed and used to monitor the distribution of targets. The population size of group U2 in the rumen was as high as 1.87%, while that of group U3 was only 0.03%. Strong fluorescence signals were observed from group U2 cells attached to plant fibers in the rumen. These findings indicate the ecological significance of group U2 in the rumen. We succeeded in enriching group U2 using rumen-incubated rice straw as the inoculum followed by incubation in an appropriate medium with an agent inhibitory for Gram-negative bacteria. Consequently, we successfully isolated two strains, designated B76 and R-25, belonging to group U2. Both strains were Gram-positive short rods or cocci that were 0.5 to 0.8 mum in size. Strain B76 possessed xylanase and alpha-l-arabinofuranosidase activity. In particular, the xylanase activity of strain B76 was higher than that of xylanolytic Butyrivibrio fibrisolvens H17c grown on cellobiose. Strain R-25 showed an alpha-l-arabinofuranosidase activity higher than that of strain B76. These results suggest that strains B76 and R-25 contribute to hemicellulose degradation in the rumen.

Study of edema reduction patterns during the treatment phase of complex decongestive physiotherapy for extremity lymphedema.

Shortening the treatment phase of complex decongestive physiotherapy (CDP) is extremely important both for individual patients and medical economics. In 83 patients with stage II unilateral secondary extremity lymphedema (31 upper extremities and 52 lower extremities), the daily changes in the volume of affected extremities during the treatment phase of CDP were prospectively investigated. For the upper extremity lymphedemas, the biggest change was seen between days 1 (100% residual edema rate) and 2 (46.0 +/- 2.7%; mean +/- SD) of therapy with a 54.0% reduction (p < 0.0001). Between days 2 and 3 (38.0 +/- 2.6%) of therapy, there was an 8.0% reduction (p < 0.05). From days 3 to 6 of therapy, slight changes ranging from 0.2 to 3.2%/day were seen. For the lower extremity lymphedemas, the biggest change was seen between days 1 (100%) and 2 (44.5 +/- 2.1%) of therapy with a 55.5% reduction (p < 0.0001). Between days 2 and 3 (33.5 +/- 2.6%) of therapy, there was an 11.0% reduction (p < 0.001). The daily volume changes from days 4 to 6 were slight, ranging from 0.1 to 1.0%/day. During the treatment phase of CDP, the largest volume changes were seen soon after the start of therapy.

Establishment of a simple and practical procedure applicable to therapeutic angiogenesis.

BACKGROUND: Therapeutic angiogenesis is thought to be beneficial for serious ischemic diseases. This investigation was designed to establish a simple and practical procedure applicable to therapeutic angiogenesis. METHODS AND RESULTS: When cultured skeletal muscle cells were electrically stimulated at a voltage that did not cause their contraction, vascular endothelial growth factor (VEGF) mRNA was augmented at an optimal-frequency stimulation. This increase of VEGF mRNA was derived primarily from transcriptional activation. Electrical stimulation increased the secretion of VEGF protein into the medium. This conditioned medium then augmented the growth of endothelial cells. The effect of electrical stimulation was further confirmed in a rat model of hindlimb ischemia. The tibialis anterior muscle in the ischemic limb was electrically stimulated. The frequency of stimulation was 50 Hz and strength was 0.1 V, which was far below the threshold for muscle contraction. After a 5-day stimulation, there was a significant increase in blood flow within the muscle. Immunohistochemical analysis revealed that VEGF protein was synthesized and capillary density was significantly increased in the stimulated muscle. Rats tolerated this procedure very well, and there was no muscle contraction, muscle injury, or restriction in movement. CONCLUSIONS: We propose this procedure as a simple and practical method of therapeutic angiogenesis.

Thyrotropin-stimulated electrical excitation and its refractoriness in cultured porcine thyroid cells.

This report demonstrates TSH-stimulated electrical excitation in the thyroid and its refractoriness after exposure to TSH. TSH depolarizes the membrane potentials and causes action potentials. TSH-induced electrical activity is characterized by a latent period, rapid depolarization, action potentials (usually two spikes were observed), and then repolarization to the potential level of the silent phase. This TSH-induced electrical excitation is associated with iodide discharge. Previous exposure to TSH induces refractoriness of electrophysiological excitation and iodide discharge to further TSH stimulation.

Effects of iodide on thyroid follicle structure and electrophysiological potentials of cultured thyroid cells.

In cultured porcine thyroid cells, exposure to iodide induces morphological and electrophysiological changes in the cells and suppresses the iodine uptake and organification activities of the cells. NaI affects thyroid structures: after exposure to 10(-7), 10(-6), and 10(-5) M NaI, the follicles first lose their typical roundness, and then the numbers of microvilli decrease. NaI (10(-6) and 10(-5) M) decreases the thyroid electrical membrane potentials. NaI induces suppression of iodine uptake and organification: exposure to 10(-6) and 10(-5) M NaI suppresses subsequently determined iodine uptake and organification. This iodide-induced suppression of iodine uptake and organification may be related to the iodide-induced morphological and electrophysiological changes. The iodide-induced changes and suppression of iodide uptake and organification are reversible. They are observed when thyroid cells are cultured in the presence of TSH.

Isotope cisternography in patients with intracranial hypertension.

Cerebrospinal fluid flow (CSF) was studied using isotope cisternography in 52 patients with increased intracranial pressure (ICP), all of whom showed acute transient rises of ICP, i.e., plateau waves, in their continuous ICP recordings. The patients were assigned to two groups. Group I was comprised of 23 patients without hydrocephalus and high ICP resulting from brain tumors, benign intracranial hypertension, and superior sagittal sinus thrombosis. Group II included 29 patients with either communicating hydrocephalus or high ICP resulting from rupture of intracranial aneurysm. Plateau waves were frequently observed in patients with baseline pressures ranging from 21 to 40 mmHg in both groups. The isotope cisternographic pattern in the Group I patients showed a large accumulation of radioactivity over the cerebral convexities, while that in the Group II patients revealed a complete obstruction of the subarachnoid space over both cerebral convexities. The isotope clearance from the intracranial CSF showed a marked delay in both groups of patients with one exception. The results suggest that, in the limited range of increased ICP caused by delayed CSF absorption, plateau waves are most evident regardless of the isotope cisternographic pattern.

Electrophysiological and morphological cell polarity and iodine metabolism in cultured porcine and human (normal and Graves') thyroid cells.

In cultured porcine and human thyroid cells, electrophysiological and morphological studies showed that cultivation in the presence of TSH, prostaglandin E2 (PGE2) or dibutyryl cyclic AMP (dbcAMP) maintained normal cell polarity with iodine incorporation and organification. Cells cultivated in the absence of these substances had inverted cell polarity and lacked iodide incorporation. In the presence of TSH, PGE2 or dbcAMP, the thyroid cells formed follicles with normal cell polarity and the microvilli pointed toward the follicle lumina. Intracellular resting potentials were -60mV and the electrical potentials in the follicle lumina were negative at -20mV. The transmembrane potential differences (p.d.) between the follicle lumina and the epithelial cells were -40mV and those between the epithelial cells and the culture media -60mV. In the absence of TSH, PGE2 or dbcAMP, the thyroid cells formed 'domes' or hollow spheres with inverted cell polarity and the microvilli pointed toward the culture media. Intracellular resting potentials were -40mV, being less negative than those in the presence of TSH, PGE2 or dbcAMP. The electrical potentials in the 'dome' or hollow sphere cavities were positive at +19mV. The transmembrane p.d. between the culture media and the epithelial cells was -40mV and that between the epithelial cells and the cavities -60mV, indicating that electrophysiologically the cell polarity was inverted in the absence of TSH, PGE2 or dbcAMP. No significant differences in electrophysiology and iodine metabolism were observed between normal and Graves' human thyroid cells in culture.

Localization of mRNAs for novel, atypical as well as conventional protein kinase C (PKC) isoforms in the brain of developing and mature rats.

Using in situ hybridization histochemistry, the localization of mRNAs for 10 isoforms of protein kinase C (PKC) in the rat brain was studied at embryonic and postnatal stages. In the embryonic brain, the gene expression was positive only for PKCepsilon, mu, lambda, and zeta with the former three more evident: The expression for PKCmu and lambda in the ventricular germinal zone and that for PKCepsilon, zeta, and lambda in the mantle zone. In the postnatal brain, the expression for PKCdelta, eta, and theta was detected differentially in a few circumscribed loci such as the thalamus, the habenula, the septum, and the cerebellar granule cells, whereas that for the other isoforms was seen widely in various loci of the gray matter with different intensity. The expression in the cerebellar external granule cell layer was positive only for PKCbeta (betaI and betaII), mu, and lambda with that for PKCbeta confined to its inner zone. There is a general tendency for all PKC isoforms that the expression levels reach at peaks in early postnatal brain and decreases more or less in adult specimens. This is the first report on the spatio-temporal heterogeneity in the gene expression for the whole members of PKC family in the brain throughout development, especially at embryonic days.

Localization of mRNA for Dri 42, subtype 2b of phosphatidic acid phosphatase, in the rat brain during development.

The expression of a gene termed Dri 42, a differentiation-related gene originally identified from intestine and a gene encoding phosphatidic acid phosphatase 2b isoform, was localized in developing and matured rat brains by in situ hybridization histochemical analysis. The gene expression was dominant in the ventricular germinal zone without significant expression in the intermediate, mantle and marginal zones throughout embryonic brain and spinal cord. The dominant expression in the ventricular germinal zone was maintained at P0 and P7, but it markedly decreased at later postnatal stages, while persistently high expression was detected in Bergmann glial cells of the cerebellar Purkinje cell layer throughout the postnatal development.

Ultrastructure of NADPH diaphorase-positive nerve fibers and their terminals in the rat cerebral arterial system.

To investigate how perivascular NO synthase (NOS)-containing nerves in the cerebral arterial system are involved in controlling the cerebral circulation, we observed the ultrastructure of NOS-containing nerve fibers and their terminals by means of nicotinamide adenine dinucleotide hydrogen phosphate-diaphorase (NADPH-d) histochemistry. We also observed the correlation between NADPH-d stained perivascular nerves and the perivascular sympathetic nerves, by means of double staining with NADPH-d histochemistry and tyrosine hydroxylase (TH) immunohistochemistry at the light microscopic level. NADPH-d-positive nerve fibers showed dense distribution mainly in the rostral portion of the circle of Willis and proximal portions of its main branches, where some of the NADPH-d-positive fibers coexisted with TH-positive fibers in a single nerve bundle. NADPH-d-positive nerve fibers were unmyelinated and had close contact with NADPH-d-negative myelinated and unmyelinated nerve fibers in a single nerve bundle, and NADPH-d-positive nerve terminals also existed closely with NADPH-d-negative nerve terminals. The number of NADPH-d-positive nerve terminals and their ratio to all other terminals were significantly higher in the rostral portion of the circle of Willis and the proximal portion of its branches, than the caudal portion of the circle of Willis and the distal portion of its branches. Nerve terminals were observed to locate within 250 nm from the basal lamina of arterial smooth muscle cells in the rostral portion of the circle of Willis and proximal portion of its branching arteries. The present observation confirmed that NOS-containing nerve fibers truly innervate the smooth muscle cells of the arterial wall in the circle of Willis and its main branches. Close contact between NADPH-d-positive and -negative nerve fibers and terminals in these arterial portions may indicate that NOS-containing perivascular nerves may work to modulate the rest of the other perivascular nervous system, such as the sympathetic nerves, to regulate the homeostasis of the arterial tonus.

NOS-positive preganglionic neurons innervate a subpopulation of postganglionic neurons in superior cervical ganglion in rats.

To determine the postganglionic targets of NOS-containing preganglionic neurons, we studied the association of NADPH-diaphorase positive preganglionic fibers and retrogradely labeled postganglionic neurons in the superior cervical ganglion (SCG) in rats. Wheat germ agglutinin-horseradish peroxidase solution was applied to the anterior chamber of the eye, middle cerebral artery, subcutaneous layer of the facial skin, or submucosal layer of the inside of the lip. Two days after tracer application, the rats were perfused with fixative solution. Serial sections of the SCG were stained histochemically for NADPH-diaphorase followed by diaminobenzidine reaction. More than 80% of the labeled postganglionic neurons innervating the structures in the subcutaneous or submucosal layer showed close association with NADPH-diaphorase positive preganglionic nerve terminals; approximately one-third of these labeled neurons were encircled by dense baskets of pericellular terminals. On the other hand, most of the postganglionic neurons innervating the iris (69%) or the cerebral artery (90%) did not show a distinct association with NADPH-diaphorase positive terminals. These results suggest that one of the principal roles of the NOS-containing preganglionic neurons may be in controlling the postganglionic neurons which innervate the structures in the subcutaneous or submucosal layer.

Effect of phenobarbital on adult rat liver cells treated with 3'-methyl-4-dimethylaminoazobenzene in primary culture.

The effect of phenobarbital (PB) on liver cells treated with 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) was studied using primary cultures of normal adult rat liver cells. Following a 1-day attachment period, primary liver cell cultures were treated with 0.24 mM 3'-Me-DAB for 6 days, and then treated with or without PB at 0.75, 1.5 and 3 mM for 19 days. Similarly, control cultures were treated with 0.5% dimethylsulfoxide (DMSO), a solvent for 3'-Me-DAB, for 6 days, and then treated with or without PB in the same way. Each treatment was done on 8 cultures. Chromosome analysis and cytochemical assay for gamma-glutamyltranspeptidase (GGT) activity were carried out on the carcinogen-treated and control cultures between 1 and 2 months after initiation of primary culture. Chromosomal abnormalities were detected in 23 of 32 carcinogen-treated cultures and also in 2 of 28 control cultures tested. However, GGT positive cells were detected only in the carcinogen-treated cultures at a frequency of 22/32. Of the 23 carcinogen-treated cultures with chromosomal abnormalities, 18 contained GGT positive cells. These results show a good correlation between chromosomal abnormality and acquisition of GGT activity at culture dish level. Furthermore, in the carcinogen-treated cultures, PB treatment caused a dose-dependent increase in the number of GGT positive cultures and in the percentage of GGT positive cells in each culture, and also caused a dose-dependent increase in the number of cultures with chromosomal abnormalities.

A novel cytochrome b(o/a)3-type oxidase from Bacillus stearothermophilus catalyzes cytochrome c-551 oxidation.

Gram-positive thermophilic Bacillus species contain cytochrome caa3-type cytochrome c oxidase as their main terminal oxidase in the respiratory chain. To identify alternative oxidases, we isolated several mutants from B. stearothermophilus defective in the caa3-type oxidase activity [Sakamoto, J. et al (1996) FEMS Microbiol. Lett. 143, 151-158]. A novel oxidase was isolated from membrane preparations of one of the mutants, K17. The oxidase was composed of two subunits with molecular masses of 56 and 19 kDa, and contained protoheme IX, heme O, heme A, and Cu in a ratio of 1:0.7:0.2:3. CO difference spectra indicate that the high-spin heme is mainly heme O. These results suggest that the enzyme belongs to the heme-copper oxidase family and is a cytochrome b(o/a)3-type oxidase, whose high-spin heme is mainly heme O and partly heme A. The enzyme oxidized cytochrome c-551, which is a membrane-bound lipoprotein of thermophilic Bacillus. The turnover rate of the activity (Vmax = 190 s[-1]) and its affinity for cytochrome c-551 (Km = 0.15 microM) were much higher than those for yeast and equine heart cytochromes c. The oxidase activity was enhanced by the presence of salts and inhibited by sodium cyanide with a Ki value of 19 microM. The enzyme kinetics suggests that cytochrome c-551 is the natural substrate to this oxidase. Furthermore, the oxidase had similarity to cytochrome ba3-type oxidase from Thermus thermophilus in the subunit composition, partial amino acid sequence, and prosthetic groups, and therefore is suggested to belong to a unique subgroup of the heme-copper oxidase family together with the Thermus enzyme and archaeal oxidases such as Sulfolobus SoxABCD.

Analyses of treadmill locomotion in adult spinal dogs.

The locomotion of the hindlimbs of two adult female spinal dogs, who were able to walk steadily on their hindlimbs 10 months after transection of the spinal cord (T10), and of two normal dogs was analyzed on a treadmill by means of high-speed cinematography and electromyography. With increase in walking speed, the duration of the step cycle was shortened by reduction of the duration of the stance phase, and the stride length was extended mainly by elongation of the transfer distance during the swing phase in both normal and spinal dogs. The patterns of muscle discharges of the hindlimbs in spinal dogs were similar to those in normal dogs. With increase in walking speed, reductions in the burst duration of the extensors were observed in both normal and spinal dogs. These results indicate that spinal dogs can adjust their locomotion speed in the same manner as normal dogs; this supports the theory that a central pattern generator regulating the locomotive activities of the hindlimbs exists in the spinal cord below the transection site.

Tubal pregnancy in a unicornuate uterus with rudimentary horn: a case report.

OBJECTIVE: To report a case of tubal pregnancy in a unicornuate uterus with rudimentary horn on the side of the rudiment and its pathology. DESIGN: Case report. SETTING: University hospital. PATIENT: An 18-year-old woman, primigravida, with tubal pregnancy. INTERVENTION(S): Systemic administration of methotrexate, salpingectomy by laparotomy, and laparoscopic surgery for resection of rudimentary horn. MAIN OUTCOME MEASURE(S): Emergent laparotomy revealed that the intraperitoneal hemorrhage was caused by the rupture of the tubal pregnancy on the same side as the rudimentary horn of the unicornuate uterus. A corpus luteum was found at the ipsilateral ovary of the rudimentary horn. RESULT(S): Image diagnosis and pathological examination of the rudimentary horn revealed that this uterine malformation was a unicornuate uterus with a noncommunicated, noncavitary rudimentary horn, corresponding to class IIc of the American Fertility Society classification of müllerian anomalies. CONCLUSION(S): This is the first report of a tubal pregnancy on the side of the noncommunicating rudimentary horn with the ipsilateral ovary carrying a corpus luteum in a unicornuate uterus.

Bioelectric activity of chick stato-acoustic ganglion cells in culture.

Electrical membrane properties were recorded intracellularly from dissociated embryonic chick stato-acoustic ganglion (S-AG) cells cultivated in vitro for 8 days. Most S-AG cells exhibited the complete action potentials with overshoot by passing current and a half of them fired repetitively during single prolonged stimulation. In some S-AG cells with relatively high input resistances, long-term negative after-potentials were clearly observed at the end of repetitive discharges. The action potential was composed of fast and slow components. The former component was blocked by tetrodtoxin (TTX) and the latter one was markedly suppressed by treatment with MnCl2 or verapamil. These data indicate that the action potential from the chick S-AG perikaryon is produced by both Na and Ca mechanisms together.

Production of secretory component and pathogenesis of gastric cancer in Helicobacter pylori-infected stomach.

The purpose of this study was to examine the production of secretory component (SC) and immunoglobulin A (IgA) in the gastric mucosa with Helicobacter pylori infection and to investigate the influence of immunological reactions on various phases of infection (gastritis, intestinal metaplasia, gastric cancer). Production of SC and IgA was assessed by immunohistochemical staining in (1) endoscopic biopsy samples of H. pylori-eradicated cases (n = 25), and (2) surgically resected stomach tissues of H. pylori-positive gastric cancer cases, intestinal type (IGC, n = 25) and diffuse type (DGC, n = 25). Before eradication therapy, all samples showed positive staining of SC and IgA in epithelial cells, and IgA was also positive in plasma cells in the mucosal layer. H. pylori bacteria were positively stained for SC and IgA. After treatment, the degree of SC and IgA staining in epithelial cells was reduced with successful eradication; but with intestinal metaplasia, SC staining was positive regardless of the results of treatment. In nonmetaplastic mucosa, SC-positive cells were increased in the glandular neck zone to the surface mucosal layer; and the intensity of SC staining was increased in proportion to the degree of mucosal inflammation and IgA-positive cell aggregation. In intestinal metaplasia, SC was positive irrespective of the degree of inflammation. Most cancer foci also showed positive staining of SC, irrespective of histological type. Production of SC and IgA was thought to be a specific reaction against H. pylori infection, occurring from the early to the late stages and not limited to intestinal metaplasia. It was suggested that immunological reactions against H. pylori infection might generally be involved with the pathogenesis of intestinal metaplasia and both histological types of gastric cancer (IGC and DGC).

Association of Helicobacter pylori and diffuse type gastric cancer.

The major purpose of this study was to evaluate the association of Helicobacter pylori and diffuse type gastric cancer (DGC) clinicopathologically (study 1). The second aim was to investigate genetic differences of H. pylori in patients with DGC and intestinal type cancer (IGC) (study 2). The prevalence of H. pylori and the types of histopathological changes were evaluated in resected early gastric cancer (DGC; 25 patients, IGC; 25 patients). Genetic differences of H. pylori in DGC patients (n = 19) and IGC patients (n = 22) were analyzed by polymerase chain reaction (PCR) methods in terms of restriction fragment length polymorphism patterns of the ureB gene and cagA gene positive rates. All patients had evidence of H. pylori infection in the resected stomach, but the positive rate for H. pylori in the area surrounding cancer was 52% (in DGC; 56%, IGC; 48%). But in 40.0% of DGC cases (10/25), mucosal atrophy and intestinal metaplasia were rarely seen in the area surrounding cancer and the positive rate of H. pylori was 80.0% (8/10), in contrast, in 60.0% of IGC cases (15/25), atrophy and metaplasia were progressed and positive rate of H. pylori was 26.7% (4/15) in the area. UreB gene products from 89.5% of DGC cases (17/19) were unable to be digested by Spe I. 31.8% of products from IGC cases (7/22) were also unable to be digested by Spe I, but the positive rate of cagA gene in this group was higher than other groups. The high prevalence of H. pylori infection in DGC patients suggests that H. pylori plays a role in the pathogenesis of DGC, but in the stomach with DGC, it is considered atrophy and intestinal metaplasia are not so implicated in H. pylori, compared with IGC. A genetic specificity of H. pylori in DGC and IGC was indicated by the results, suggesting that H. pylori may play different roles in the pathogenesis of DGC and IGC.