EFFECTS OF ORVUS ES PASTE ON THE MOTILITY AND VIABILITY OF YAK (BOS GRUNNIENS) EPIDIDYMAL AND EJACULATED SPERMATOZOA AFTER FREEZING AND THAWING.

BACKGROUND: The addition of the detergent Orvus ES Paste (OEP) to semen freezing extenders has been observed to improve the post-thaw survival and longevity of spermatozoa from various species but has never been evaluated for yak spermatozoa. OBJECTIVE: This study evaluated the effects of OEP on the post-thaw motility and viability of epididymal and ejaculated yak spermatozoa. MATERIALS AND METHODS: Semen samples were frozen and thawed in semen freezing extender supplemented with 0 %, 0.375 %, 0.75 % or 1.5 % OEP. The motility and viability of frozen-thawed spermatozoa were evaluated before and after 3 h of incubation. RESULTS: The addition of 0.75 % OEP to the freezing extender significantly improved the mean motility and viability values of both the epididymal and ejaculated spermatozoa immediately after thawing, but the beneficial effects on motility disappeared after 3h of incubation. CONCLUSION: Our findings indicate that the addition of 0.75 % OEP is effective for the preservation of yak spermatozoa.

Immunoelectron microscopy of carbonic anhydrase isozyme VI in human submandibular gland: comparison with isozymes I and II.

Carbonic anhydrase VI (CA VI) was purified from human saliva by inhibitor-affinity chromatography, and its distribution was studied in human submandibular gland by the indirect immunoperoxidase technique with a rabbit polyclonal antibody raised against the isozyme. Polyclonal antibodies to human CA I and CA II purified from erythrocytes were also raised and used for immunostaining. SDS-polyacrylamide gel electrophoresis of the purified isozymes revealed a single protein band (CA VI, 42 KD; CA I and CA II, 30 KD). Antibody raised against CA VI did not crossreact with CA I or CA II either by Western or by dot-blotting. However, antibodies against CA I and CA II showed slight crossreaction with each other's antigen by dot-blotting. In a Western blot of purified submandibular gland CA, antibody to CA VI stained the 42 and 30 KD bands, and antibodies to CA I and CA II stained the 30 KD band. The 42 KD but not the 30 KD molecule was cleaved by endo-beta-N-acetylglucosaminidase F, indicating that the former contains N-linked oligosaccharides. Immunostaining for CA VI was seen in the secretory granules and cytosol of serous acinar cells and in the duct luminal contents. Staining specific for CA II was observed in the cytosol of serous acinar and duct epithelial cells. Antibody to CA I reacted only with the walls of small blood vessels. These results suggest that (a) serous acinar cells secrete 42 KD CA VI which functions in the oral cavity and that (b) serous acinar and duct epithelial cells possess cytosolic CA (30 KD CA VI and CA II) which functions in situ.

Flow cytometric determination of active oxygen (hydroperoxide) produced by peripheral blood neutrophils in patients with hematological disorders.

We analyzed active oxygen (hydroperoxide; H2O2) production by peripheral neutrophils in various hematological diseases by flow cytometry. One hundred microliters of heparinized fresh blood was sequentially incubated at 37 degrees C with 2',7'-dichlorofluorescein diacetate and with or without phorbol myristate acetate (PMA). After hemolysis, the pelleted white blood cells were subjected to flow cytometry, and the neutrophil fraction was gated on the cytogram. Production of H2O2 by the fraction was estimated by determining the increase in the relative intensity of fluorescence emitted from the fraction in response to stimulation by PMA. In controlled chronic myelogenous leukemia (CML) (WBC < 1 x 10(10)/1), H2O2 production was normal, while in uncontrolled CML (WBC > or = 1 x 10(10)/1), it was reduced. In myelodysplastic syndrome (MDS), H2O2 production was also reduced, but no significant difference was observed among FAB classification disease types in MDS patients. In untreated acute non-lymphocytic leukemia (ANLL), H2O2 production was reduced, while in the complete remission stage of ANLL, its level was normal, suggesting recovery from normal clones. In aplastic anemia, the H2O2 production level was normal. Steroid therapy might be responsible for the reduction of H2O2 production in non-Hodgkin's lymphoma and multiple myeloma. The production of H2O2 is closely related to the oxygen-dependent bactericidal activity of neutrophils, and, hence, can be utilized as an index to indicate susceptibility to infection. This neutrophil function can be determined easily in ordinary clinical facilities by using flow cytometry, and care should be taken to prevent infection when H2O2 production is reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Histochemical demonstration of carbonic anhydrase activity in the odontogenic cells of the rat incisor.

Aldehyde-fixed, EDTA-demineralized frozen sections of the rat maxillary incisor were histochemically stained for carbonic anhydrase activity, by use of Hansson's method. Intense staining was observed in the odontoblasts, all types of epithelial cells of enamel organ in the maturation zone, cementoblasts, and the cells of the lingual dental sac. Less intense but consistent staining was observed in all types of epithelial cells of odontogenic origin directly facing the pulp and pulp cells adjacent to the odontoblast cell layer in the apical part of the pulp, and was considered due to the carbonic anhydrase-catalyzed reaction. Staining of these cells was completely inhibited by heat pretreatment (120 degrees C, 30 min), 10(-6) mol/L acetazolamide in the incubation medium, incubation by continuous immersion under the liquid surface, and omission of the substrate, NaHCO3. The dentin also exhibited heavy staining which was inhibited by the heat pre-treatment. However, this dentinal staining resisted the inhibition by 10(-3) mol/L acetazolamide and was not inhibited by incubation by continuous immersion or incubation without the substrate NaHCO3. The dentinal staining was thus judged to have been due to non-enzymatic cobalt precipitation.

Esophageal metastasis from prostate cancer: diagnostic use of reverse transcriptase-polymerase chain reaction for prostate-specific antigen.

An extremely rare case of esophageal metastasis from prostate cancer is reported. A 65-year-old man presented with anorexia and back pain. Upper gastrointestinal X-ray fluoroscopy and endoscopy revealed a shallow longitudinal ulcer, with converging mucosal folds, approximately 5 cm above the esophagogastric junction. The histological diagnosis of the biopsied specimen was adenocarcinoma. Blood biochemistry revealed elevated serum prostate-specific antigen (PSA) and gamma-seminoprotein levels. Ultrasonography of the prostate disclosed a hypoechoic lesion in the left lobe, and needle biopsy led to the diagnosis of prostatic adenocarcinoma. Since there was no finding suggestive of a primary lesion, apart from that in the prostate, we conducted reverse transcriptase-polymerase chain reaction (RT-PCR) for PSA. PSA-positive mRNA was demonstrated in the tissue of the esophageal tumor. There are three reports on metastasis to the esophagus from prostate cancer, but this is the first case of esophageal metastasis from prostate cancer without any evidence of metastasis to other organs. The importance of RT-PCR for the diagnosis of primary lesions of metastatic cancer is discussed.

Running-wheel avoidance learning in rats (Rattus norvegicus): effects of contingencies and comparisons of different strains.

In Experiment 1, we showed that active- and passive-avoidance responding in a running wheel was learned because of the avoidance contingency. In Experiment 2, strain differences among four commercially bred rats were assessed in an active-avoidance paradigm. Wistar, Donryu, and Fischer rats learned faster than Sprague-Dawleys. In Experiment 3, learning in a multiple active/passive avoidance schedule was examined, and both components of this task were learned. This multiple schedule was used to investigate strain differences in selectively bred rats in Experiments 4 and 5. Tsukuba low-emotional (TLE) rats responded more than Tsukuba high-emotional (THE) rats in both components. However, discrimination of passive components was better in THE than in TLE rats. Syracuse high-avoidance rats were superior in the active component, whereas Syracuse low-avoidance rats showed superior performance in the passive component.

Texture analysis of histological images of giant cell tumor of bone.

To gain an objective evaluation of histological sections of giant cell tumors of bone (GCT) and osteosarcomas, microscopic pictures were taken and their grey-tone image measured, using a flying spot scanner and computer. Various values of eight parameters expressing certain characteristical brightness distribution patterns were computed, and comparatively examined among the three groups of benign GCT, malignant GCT and osteosarcoma. As a result, some parameters could facilitate differentiation between the histological images of these bone tumors. Especially, "angular second moment (ASM)", "Contrast" and "Coefficient of variation (COV)" were useful even for discrimination between malignant and benign GCT. After factor analysis of the values of these parameters, scores of each factor for a number of histological scene images were plotted on a 2-dimensional factor plane. On this plane, which was considered to be a histological feature plane, cases of benign GCT were separated from those of osteosarcoma. Cases of malignant GCT were distributed between the two groups. These results suggest that this method could be valuable for computer evaluation of histological images of benign GCT and osteosarcomas.

Studies on inhibition of adenosine deaminase by isocoformycin in vitro and in vivo.

Isocoformycin is a structural isomer of coformycin which has been demonstrated to be a potent inhibitor of adenosine deaminase. Isocoformycin showed a weaker inhibition of this enzyme than coformycin; the binding of coformycin to enzyme was irreversible, but isocoformycin inhibition was competitive with substrate. The Ki value of isocoformycin was 4.5 approximately 10 X 10(-8) M. Following intraperitoneal injection of isocoformycin in mice, the adenosine deaminase activity of homogenates of several organs was determined and the following ED50 values (50% inhibition doses) were observed: 29 mg/kg for thymus, 13 mg/kg for spleen, 80 mg/kg for liver and 20 mg/kg for kidney. The inhibition of adenosine deaminase in rabbit blood in vitro was also tested in comparison with coformycin.

Effects of a new adenosine deaminase inhibitor, isocoformycin, on toxicity, antitumor activity and tissue distribution of formycin A and 9-beta-D-arabinofuranosyladenine.

Single intraperitoneal and intravenous injections of isocoformycin at 1,200 mg/kg did not cause the death of mice. Isocoformycin which inhibited adenosine deaminase enhanced significantly the toxicity of formycin A and ara-A at various combination ratios. Isocoformycin potentiated antitumor activity of formycin A and ara-A against L1210 leukemia. Formycin A and ara-A disappeared rapidly from the blood and tissues and could not be found in any tissues even 0.5 hour after a single intraperitoneal injection. However, when used in combination with isocoformycin both were detected in the blood and tissues, especially at high concentration in liver and kidney. These indicate that the deamination of formycin A and ara-A is blocked by isocoformycin in vivo.

Congenital multiple arterioportal fistula: a case report treated with portocaval shunt.

A case of 48-year-old man, with diffuse arterioportal fistula (APF) considered congenital in origin, is described. The patient presented with recurring intractable variceal hemorrhage which was treated by endoscopic ligation or sclerotherapy. In this case, gastroduodenal artery and branches of mesenterial artery were diffusely involved, so these lesions could not be resected. Portocaval shunt between the superior mesenteric vein and the right iliac vein was successfully performed. Follow-up endoscopic examination one year later revealed no evidence of esophageal and gastric varices. To our knowledge, this is the second case in which a patient underwent portocaval shunt successfully for congenital APF.

[A case of amebic colitis cured with multiple cicatricial strictures].

A 53-year-old male was admitted to Gifu Red Cross Hospital with the complaints of diarrhea, abdominal pain, and fever. He had a temperature of 38.4 degrees C and diffuse abdominal tenderness without guarding or rebound. Plain film of the abdomen showed marked dilatation of the transverse colon. Sigmoidoscopy showed multiple aphthoid erosions and pseudomorphic ulcers, and mucosal biopsies demonstrated numerous trophozoites of Entamoeba histolytica. The patient was treated with oral metronidazole with rapid improvement. Barium enema and colonoscopy after improvement showed multiple cicatricial strictures. Although prompt diagnosis and therapy prevented fulminant changes, the patient was cured with multiple cicatricial strictures, a rare complication of amebic colitis. It is important to keep in mind severe amebic colitis in the differential diagnosis of patients with diarrhea and high fever.

[Detection of methicillin-resistant Staphylococcus aureus (MRSA)--relation between respiratory tract and gastro-intestinal tract].

The study was conducted to elucidate the possibility of hospital infection of methicillin-resistant Staphylococcus aureaus (MRSA) through feces. Fecal cultures of 12 inpatients positive for MRSA in the respiratory tract and 11 inpatients negative for MRSA in the respiratory tract were performed from April to October in 1993. Fecal cultures of inpatients positive for MRSA in the respiratory tract were continued until September in 1996, finally a total of 50 cases were enrolled. MRSA was isolated from feces of 7 patients (58.3%) of the 12 patients positive for MRSA in the respiratory tract, while no MRSA was isolated from feces of 11 patients negative for MRSA in the respiratory tract. Twenty-seven patients (54.0%) of 50 patients positive for MRSA in the respiratory tract yielded MRSA in the feces. Twenty-three patients (85.2%) of the 27 patients positive for MRSA in the fecal flora had received H2-blocker, while 11 patients (47.8%) of the 23 patients negative for MRSA in the fecal flora received H2-blocker; these differences were statistically significantly (p < 0.01). These findings suggest the possible role of feces in hospital infection with MRSA and the necessity for careful administration of H2-blocker to patients positive for MRSA in the respiratory tract.

Pulmonary embolization-induced thymus hyperplasia in rabbits.

The effects of pulmonary embolization on the thymus glands of rabbits were studied morphologically, morphometrically and immunohistochemically. Pulmonary embolization was induced by an intravenous injection of 0.4 ml of Sephadex bead suspension (particle size; 150 to 300 microns, about 2,000 per ml). Both mean weight and volume of the thymus of rabbits killed at 2 weeks after embolization, were about 1.5 times more than those in control animals treated with physiologic saline. Histological examinations showed enlargement of the cortex and medulla of the thymus, and the embolized Sephadex beads in the branches of pulmonary arteries of the lung. The area ratios of medulla/cortex, in the embolization group and in control, were not significantly different. The cells with immunohistochemically positive staining of anti-nuclear antigen of monoclonal antibody of Ki-67, were found in both portions of the medulla and cortex. These data suggest that pulmonary embolization in the rabbit induces true thymic hyperplasia. An intravenous injection of India ink into the right highest intercostal artery revealed the distribution of bronchial arteries, which send the branches to the right lobe of the thymus. In 2 out of 4 animals killed 2 weeks after pulmonary embolization, the left lobe of the thymus as well as the right were stained with the injected ink. As it is known that pulmonary vascular obstruction caused a marked increase in the bronchial blood flow, these data suggest that the thymus blood supply from the bronchial arteries increases in the conditions of pulmonary embolization, which might contribute to thymus hyperplasia.

Immunohistochemical localization of two types of carbonic anhydrase isozymes in oncocytoma and oncocytic epithelial cells.

An immunohistochemical study was performed on the localization of carbonic anhydrase isozymes I and II (CA I and CA II, respectively) in oncocytoma and oncocytic epithelial cells. Tumors examined were those deserved to be called oncocytomas such as Warthin's tumor, Hürthle cell tumor of the thyroid gland and renal oncocytoma. In addition, the same immunostaining was done on several pathologic lesions, accompanying moderate number of cells similar in tinctorial character to oncocytes, such as some type of adenoma of the thyroid gland, endometrial hyperplasia, endometriosis and so on. Specific antibodies against CA I and CA II prepared from human erythrocytes were converted into Fab' fragment and labeled with horseradish peroxidase. In each tissue sample, the majority of oncocytes or oncocytic epithelial cells showed a tendency to exhibit an immunostaining reaction for CA I and II intenser than that of adjacent original normal epithelium from which they might derive. To the authors' knowledge, no reports have been appeared on biological relevance of an increased activities of carbonic anhydrase in conjunction with a large number of mitochondria appearing in such oncocytic cells. In the present study, it is revealed that the immunoreactivities of CA isozymes increased in oncocytes as the number of mitochondria increased, suggesting that oncocytes have a high level of carbonic anhydrase for catalyzing the hydration of an increased amount of carbon dioxide generated chiefly via oxidative metabolism by mitochondria, the number of which is notably increased compensatorily for their deficient coupling of oxidative phosphorylation or other metabolic anomaly.

Effects of thyroid hormone and actinomycin D on phosphate uptake of brush-border membrane vesicles from kidney of rachitic rats.

It is generally accepted that 3,5,3'-triiodothyronine (T3) is responsible for most of the known physiological actions of thyroid hormone. Our new rachitogenic diets, deficient in both vitamin D and iodine and low in phosphorus (1.13% calcium and 0.14% phosphorus), induced typical rachitic changes in rats. To know the possible role of T3 on bone and phosphate (Pi) metabolism and its related biochemical and molecular mechanism, weanling male Wistar rats were fed this diet for 28 days, and then treated with 6 micrograms/100g BW of T3 and/or 60 micrograms/100g BW of actinomycin D intraperitoneally. After 2 hours of treatment, renal brush-border membrane vesicles (BBMV) were prepared according to the calcium precipitation method, and their Na(+)-dependent Pi uptake was analyzed by Lineweaver-Burk plots. Km value was not significantly altered by all these treatments. In contrast, Vmax value as a Pi uptake index was significantly increased in T3-treated rats compared with rachitic control. This increase was evident as less as 2 hr after T3 administration, so it was assumed that T3 stimulated the Pi uptake not by mediation of the de novo protein synthesis. However this increase was suppressed by administration of actinomycin D, and then this result suggests that the antirachitic effect of T3 is mediated by somehow de novo protein synthesis. After all, T3 acting as an antirachitic agent, the most likely possibility is that T3 provides favorable conditions for mineralization of bone by improving Pi transport in various types of cells, even under rachitogenic condition, at least of hypophosphatemic type, and correction of phosphate metabolism may be the most essential prerequisite for cure of this type of rickets.

Reverse triiodothyronine nuclear binding in rat brain.

Reverse triiodothyronine (rT3) had been believed biologically inactive, but recently we demonstrated nuclear binding sites for rT3 in human placenta. In this study we examined rT3 binding sites in rat brain. Male Wistar normal rats aged 2, 4 and 9 weeks were killed and the brain was removed for rT3 binding assay. In another series, male Wistar rats weighing about 40g were divided into two groups: group 1 (rickets group), kept on our original rachitogenic diet and group 2, kept on standard diet. After 28 days on either diet they were killed and the brain was removed for assay. Cerebral nuclear protein was extracted with 0.4M KCl buffer. In normal 2 week-old rats specific binding sites for rT3 were detected in all parts of the brain, but at 7 weeks of age the density of the binding sites was decreased and at 9 weeks it is almost 0. Scatchard analysis performed in rachitic rats showed a curvilinear pattern, suggesting two sets of receptors existing in brain tissue; in cerebral cortex one with a association constant (Ka) of 1.07 X 10(8)M-1 and a limited capacity (Bmax) of 0.75 X 10(-15) moles/mg tissue and the other with Ka = 4.93 X 10(6), Bmax = 12.1 X 10(-15), and in thalamus including hypothalamus, one with Ka = 1.00 X 10(8), Bmax = 1.00 X 10(-15) and the other with Ka = 4.57 X 10(6) and Bmax = 18.6 X 10(-15).

Nuclear binding sites for reverse triiodothyronine in human placenta.

It has been generally believed that reverse triiodothyronine (reverse T3, rT3) is biologically inactive. Nevertheless the serum rT3 level is very high in fetal life, when the serum calcium level is higher than in the maternal. For this hypercalcemia in fetal life, there is no convincing evidence that any major calcium regulating hormone is responsible. And the assumption that rT3 may be concerned in active transport of calcium at placenta could be suggested. In this study nuclear protein was isolated from human placenta by extraction with 0.4M KCl buffer and binding studies utilizing radioactive rT3 were carried out. Scatchard analysis presented a curvilinear pattern, suggesting two classes of receptors: One with a association constant (Ka) of 1.14 X 10(8) M-1 and a limited capacity (Bmax) of 32.0 X 10(-15) mol/100 micrograms DNA and the other with Ka = 4.34 X 10(6) and Bmax = 454 X 10(-15). Its relative affinities for several thyroid hormone analogues were calculated. If the affinity for rT3 was assigned l, that for triiodothyronine would be 1/40, thyroxine: 1/63, Triiodothyroacetic acid: 1/18. Sulfhydryl agents affecting these binding characteristics were also studied. The Ka was not substantially changed but the Bmax was notably decreased by dithiothreitol (43%) and 2-mercaptoethanol (26%). In liver nuclei, the binding characteristics of rT3 receptor were analyzed for comparison. In this case only a single class of low affinity-high capacity rT3 binding site with a Ka of 6.69 X 10(6) was detected, a finding which was apparently different from those in placenta.(ABSTRACT TRUNCATED AT 250 WORDS)

[Flow cytometric determination of active oxygen produced by neutrophils in patients with inflammatory diseases].

We investigated active oxygen (H2O2) production by neutrophils in patients with inflammatory diseases such as connective tissue disease and respiratory tract infection. The analysis was done by flow cytometry using as small volume of whole-blood as 100 microliters. H2O2 production as well as C-reactive protein (CRP) were increased in connective tissue diseases, and was decreased after steroid therapy. In a case of neuro-Behçet's disease, changes in H2O2 production was observed in agreement with those in clinical symptoms such as gait disturbance. H2O2 production as well as CRP and neutrophil count were increased in acute respiratory tract infection, but in chronic patients H2O2 production alone was rather reduced. In stages of enhanced H2O2 production, tissue damage or inflammation may exist, the degree of which seems to be well reflected by H2O2 production. During stages of impaired H2O2 production, care should be taken to prevent infections. Using flow cytometry, H2O2 production can be easily determined as one of functions of neutrophils.

[The coefficient of variation of RR intervals (CVRR) on electrocardiogram in patients with essential hypertension with reference to aging, hemodynamics and sympatho-adrenomedullary function].

To evaluate the significance of parasympathetic nerve activity in essential hypertension, we measured the coefficients of variation of RR intervals (CVRR) on electrocardiogram and examined the relationships between CVRR and aging, hemodynamics and sympatho-adrenomedullary function in normotensive subjects (NT) and in patients with essential hypertension (EHT). Mean arterial pressure (MAP), heart rate (HR), plasma noradrenaline concentration (pNA), plasma adrenaline concentration (pAd) and CVRR resting in a supine position were simultaneously measured in 37 NT (33.8 +/- 2.0 years) and 47 mild-to-moderate EHT (51.3 +/- 1.5 years). In both NT and EHT, significantly negative correlations between CVRR and age (NT: r = -0.54, p less than 0.001, EHT: r = -0.41, p less than 0.005) were observed, however, CVRR correlated with neither MAP, HR nor pAd. CVRR tended to correlate negatively with pNA (r = -0.27, p less than 0.1) in NT, unlike in EHT. The mean value of CVRR in EHT (n = 10, age: 38.3 +/- 1.6 years, CVRR: 3.61 +/- 0.37%) was significantly (p less than 0.005) lower than in age-matched NT (n = 10, age: 38.3 +/- 2.5 years, CVRR: 5.76 +/- 0.45%). These results indicate that the parasympathetic tone suggested by CVRR may be related to aging and sympathetic nerve activity, and that parasympathetic function might be impaired in EHT.

[A high risk group of hepatocellular carcinoma in man, with special reference to its clinical significance for the screening of early liver cancer. Gifu Study Group for Early Liver Cancer].

A high risk group of hepatocellular carcinoma (HCC) was statistically established using multiple regression analysis of 331 cases with liver cirrhosis (LC), in which 78 cases later developed HCC. Highly contributing factors to hepatocarcinogenesis were found to be positive HBsAg, age, drinking history, sex (male), history of blood transfusion, history of acute hepatitis (or jaundice) and elevated plasma levels of alpha-fetoprotein. A prospective study was initiated in April, 1985 employing another 122 LC patients to clinically evaluate the significance of the high risk group of HCC. 28 cases with small HCC (less than 3 cm in diameter) were newly found: 4 with chronic hepatitis and 24 with LC, among whom 22 developed from the high risk group (sensitivity 92%, specificity 44%). Three year survival rate of 28 cases thus found was 56%, and causes of death of 9 fatal patients were 4 cancer death, 4 hepatic failure and 1 gastrointestinal bleeding. In conclusion, the high risk group is valuable for the screening of early liver cancers, and treatments of hepatic failure as well as of HCC itself are important to improve the prognosis of HCC patients thus diagnosed.