Optimizing production of in vivo-matured oocytes from superstimulated Holstein cows for in vitro production of embryos using X-sorted sperm.

The present study aimed to establish an efficient system for the production of female embryos from dairy cows by in vitro fertilization (IVF) using X-sorted sperm and in vivo-matured oocytes collected by ovum pick up (OPU). Nonlactating Holstein cows (n = 36) were administered a controlled intravaginal progesterone-releasing (controlled internal drug release) device (d 0), underwent dominant follicle ablation (DFA) or ovulation by administration of 100 µg of GnRH on d 5, and were superstimulated with FSH and PGF2α, following standard procedures. Controlled internal drug release devices were removed on the evening of d 8 or on the morning of d 9, depending on the experiment. For LH surge induction, 200 µg of GnRH was administered on the morning of d 10 (0 h). In experiment 1, the peak (48.1%) of ovulating follicles was detected at 29 to 32 h after GnRH injection (0 h), and the range in the timing of the initiation of ovulation was less by timing from GnRH administration (30.0 ± 2.8h) rather than by timing the onset of estrus (32.7 ± 4.7h). Only 0.9% of total ovulated follicles were recorded before 26 h after GnRH injection. Therefore, OPU was carried out at 26 h and IVF occurred at 30 h after GnRH in experiments 2 and 3. In experiment 2, 83.3 ± 10.8% of oocytes with expanded cumulus cells had extruded the first polar body at 30 h after GnRH injection. The aim of experiment 3 was to compare the effect of either DFA or GnRH-induced LH surge before superstimulation on the efficiency of embryo production by IVF following superstimulation. Progesterone concentrations from d 10 to 12 in the DFA group were lower than those in the GnRH group. A greater proportion of recovered oocytes with expanded cumulus cells from ≥ 8-mm follicles was observed in the DFA group than in the GnRH group (95.9 and 77.4%, respectively). Blastocyst rates in the DFA and GnRH groups (58.0 and 52.8%, respectively) did not differ from those of oocytes collected from nonstimulated OPU and matured in vitro (49.9%). However, the proportion of high-quality blastocysts was higher in the DFA group compared with the GnRH group (54.9 vs. 21.5%). Our results demonstrate that high rates of good-quality blastocysts can be produced by IVF with X-sorted frozen sperm using in vivo-matured oocytes collected by OPU from cows after DFA and superstimulation combined with ovulation induction.

Roles of TT virus infection in various types of chronic hepatitis.

An unenveloped single-stranded virus, which might be a causative agent for posttransfusion non-A-G hepatitis, was recently found and named "TT virus" (TTV). There is still controversy over the role of TTV in chronic hepatitis. Therefore, we have examined the prevalence of TTV in various types of chronic hepatitis in Japan. TTV DNA was detected in 11 of 40 patients (27.5%) with non-B, non-C chronic hepatitis, 13 of 46 patients (28.3%) with type B chronic hepatitis, 21 of 55 patients (38.2%) with type C chronic hepatitis, and 41 of 131 subjects (31.3%) with normal liver function tests. The positivity rate for TTV DNA tended to increase with age. The detection rate did not differ statistically between non-B, non-C chronic hepatitis and type B or type C chronic hepatitis, or normal subjects. The distribution of TTV genotypes was not significantly different among them. Clinical characteristics of the chronic illness were similar for patients with or without TTV in all hepatitis groups. The etiologic role of TTV in chronic hepatitis is not confirmed from the statistical and clinical standpoint.

Effect of long-term administration of JTP-2942, a novel thyrotropin-releasing hormone analogue, on neurological outcome, local cerebral blood flow and glucose utilization in a rat focal cerebral ischemia.

The effect of JTP-2942, a novel thyrotropin-releasing hormone analogue on neurological examination, local cerebral blood flow (l-CBF) and local cerebral glucose utilization (l-CGU) were examined when JTP-2942 was administered for 4 weeks after 1 week reperfusion following ischemia in a rat middle cerebral artery (MCA) occlusion. Left middle cerebral artery ischemia was induced for 90 min followed by reperfusion. JTP-2942 (0.03 or 0.003 mg/kg) or saline (vehicle) were administered for 4 weeks after 1 week ischemia, and then the drug was withdrawn. Neurological symptoms and motor disturbance based on inclined plane test were measured once a week after 1 week ischemia. l-CBF and l-CGU were measured by quantitative autoradiographic technique after 6 weeks ischemia. The adjacent sections subjected to l-CBF or l-CGU measurement were stained with Hematoxylin-Eosin, and the infarction volume was measured. JTP-2942 (0.03 mg/kg) significantly ameliorated neurological symptoms and motor disturbance at 5 weeks after ischemia as compared with vehicle, and then after completion of drug administration, amelioration effect continued. JTP-2942 (0.03 mg/kg) also significantly ameliorated the reduced l-CBF and l-CGU in the peri-infarcted areas such as the frontal cortex, motor cortex and medial caudate-putamen. No significant differences were noted in the infarction volume among MCA occlusion rats. This indicates that activating reduced metabolic turnover associated with synaptic connection changes or the activation of compensation mechanisms may result in improvement of neurological symptoms and motor disturbances. It is therefore expected that JTP-2942 may be a possible therapeutic agent for motor disturbance during the subacute or chronic cerebral infarction.

Emergence of YMDD motif mutants of hepatitis B virus during lamivudine treatment of immunocompetent type B hepatitis patients.

Lamivudine is an effective antiviral agent for the treatment of chronic type B hepatitis. Recent studies have shown the appearance of lamivudine resistant viruses with mutations at the tyrosine-methionine-aspartate-aspartate (YMDD) motif of the viral polymerase in hepatitis B virus (HBV) infected patients who received orthotopic liver transplantation. In order to confirm the appearance of such mutant HBV in immunocompetent patients, the HBV sequences in and around the YMDD motif of HBV DNA polymerase were examined in the sera from 16 lamivudine treated and 10 untreated control patients. Approximately 200 bases including the YMDD motif of HBV DNA polymerase were amplified by polymerase chain reaction (PCR) and sequenced directly by an automated sequencer. Of the 16 patients receiving lamivudine, mutant viruses with mutations in the YMDD motif were found in 3 of 8 patients treated with lamivudine for 52 weeks. However, this mutation was not found in any of the 8 patients treated for 32 weeks or a shorter period. Mutant viruses appeared after 40 weeks of treatment and were undetectable within 12 weeks after the cessation of the treatment. Such mutant viruses were not detected in any of the 10 untreated patients. This study confirms the emergence of YMDD mutant viruses during long-term lamivudine treatment in immunocompetent type B hepatitis patients. The results from this study suggest the need for combination therapies to reduce the levels of such mutant viruses in some patients.

[TTV infection in healthy subjects].

Recently, a novel hepatitis related virus was described and named as TTV. The positive rates of TTV DNA in healthy subjects to far reported ranged from 1% to 62%, therefore, there must be large differences in the prevalence of TTV in healthy subjects from country to country. However, detailed data are lacking in many reports, care should be taken in analyzing them. Generally, elderly subjects tend to have increased positivity in the same area. Sanitary environment may influence the positive rate. In addition the distribution of genotype may influence the positive rate. Although the high prevalence of TTV in healthy subjects may indicate the TTV as non-hepatopathogenic virus, further studies will be needed before drawing the conclusion.

The role of TT virus infection in acute viral hepatitis.

Recently, transfusion-transmitted virus (TTV) was discovered to be a potential causative agent for non-A-E hepatitis. Little is known about the relation between TTV and the clinical courses of various types of acute viral hepatitis. One hundred twenty-five patients with acute viral hepatitis who were admitted to the Chiba University Hospital between 1984 and 1998 and 100 persons with normal liver function tests were tested for the presence of TTV in their sera. Serum samples were tested for TTV DNA and genotype by polymerase chain reaction (PCR). TTV DNA was detected in 15 of 35 patients (43%) with non-A-E hepatitis, 14 of 33 patients (42%) with hepatitis C, 8 of 28 patients (29%) with hepatitis A, 7 of 29 patients (24%) with hepatitis B, and 37 of 100 subjects with normal liver function tests (37%). The detection rate did not differ statistically between non-A-E hepatitis and hepatitis A, B, C, or controls. The distribution of TTV genotypes was similar in non-A-E, A, B, C types, and controls. The clinical characteristics of the acute illnesses were similar for patients with or without TTV in hepatitis non-A-E, A, B, or C. Although TTV was detected frequently in non-A-E acute hepatitis, no etiologic role for TTV could be established.

Expression of p53 and p21WAF1/CIP1 proteins in gastric and esophageal cancers: comparison with mutations of the p53 gene.

The p53 gene has been shown to be commonly mutated in various human cancers, and mutant p53 can act as a dominant oncogene. The intact p53 protein is also known to induce the cyclin-dependent kinase inhibitor p21WAF1/CIP1 and is implicated in cell cycle arrest. We investigated p53 gene alterations in gastric adenocarcinoma and esophageal squamous cell carcinoma to elucidate the association of the nuclear accumulation of the p53 protein and/or p21WAF1/CIP1 protein. Abnormalities of the tumor suppressor gene p53 protein and the expression of p21WAF1/CIP1 protein were analyzed by immunohistochemical techniques in 32 cases of gastric adenocarcinoma and 15 cases of esophageal squamous cell carcinoma. Twenty cases of gastric cancer and five cases of esophageal cancer were also analyzed for p53 gene mutation by polymerase chain reaction and direct nucleotide sequencing. Overexpression of p53 protein was found in 13/32 (41%) of gastric cancers and 5/15 (33%) of esophageal cancers. We found immunodetectable p53 in 10/14 cases with mutations and in none of 11 cases without mutations in gastric and esophageal cancers. Hence, immunohistochemical and genetic analyses gave concordant results in 84% of 25 cases, revealing a good correlation between immunostaining of p53 and missense mutation of the p53 gene. p53 immunostaining was not observed in cases with frameshift or splicing mutation. The expression of p21WAF1/CIP1 protein was found in 9/32 (29%) of gastric cancers and 4/15 (27%) of esophageal cancers and in 2/14 (14%) cases with alteration of the p53 gene and in 5/11 (45%) without. These results suggest that abnormalities of p53 may be closely associated with the pathogenesis of gastric adenocarcinoma and esophageal squamous cell carcinoma and that the immunoreactivity of p53 protein is a general indicator of the tumors with altered p53 function. The expression of p21WAF1/CIP1 protein was suppressed in the neoplastic tissues with and without p53 gene alteration.

Marked improvement of diabetic diarrhea with the somatostatin analogue octreotide.

Somatostatin and its long-acting analogue octreotide have been used in various diarrheal disorders, including neoplastic and nonneoplastic diseases of the gastrointestinal tract. In two insulin-dependent diabetic patients with autonomic neuropathy and chronic steatorrheic diarrhea refractory to conventional medications, subcutaneous administration of octreotide markedly improved the volume and frequency of stools in both patients. This change was accompanied by a clear improvement in their rapid gastrointestinal tract transit times. The treatment also greatly improved their orthostatic hypotension. No adverse effects of octreotide were observed after treatment for 7 months in one patient and 2 months in the other.

[The effect of alacepril on insulin sensitivity in patients with essential hypertension].

The aim of this study was to determine the effect of angiotensin-converting-enzyme inhibitor, alacepril, on insulin sensitivity in patients with essential hypertension (EHT). Ten patients (5 men and 5 women) with EHT (3 with mild diabetes and 2 with borderline glucose tolerance) participated. We measured insulin sensitivity using the two-hour euglycemic-hyperinsulinemic clamp technique and plasma glucose and insulin responses to a 75 g oral glucose tolerance test (75 g OGTT) before and after 6-8 weeks of treatment with alacepril (dose, 50 mg/day). Glucose infusion rate (GIR) during the last 30 min of the clamp study increased from 5.83 +/- 0.70 to 6.59 +/- 0.65 mg per kilogram of body weight per minute (P < 0.05) after treatment with alacepril. The insulin-sensitivity index, which was calculated by dividing the GIR by the mean insulin concentration during the same period of the clamp, also increased from 5.91 +/- 0.66 to 7.20 +/- 0.90 (P < 0.05) after treatment with alacepril. Plasma glucose responses to a 75 g OGTT were changed from diabetic pattern to borderline pattern in two patients and from borderline pattern to normal pattern in one patient after treatment with alacepril. Body weight did not significantly change throughout the study in any of the patients studied. Our study demonstrated that alacepril significantly improves insulin sensitivity in patients with EHT.

Seroepidemiological survey for antibody to Borrelia burgdorferi in cows.

Antibody to Borrelia burgdorferi was examined in 380 healthy and 38 clinical cases of cows from Hokkaido and Shizuoka in Japan. In healthy animals, IgG and IgM antibody to B. burgdorferi HO14 strain were found in 44 cows (14.6%) and 24 cows (8.0%) from Hokkaido. In contrast, antibody-positive case was not observed except for only 1 case which was IgM positive (1/79: 1.3%) in cows from Shizuoka. Mean antibody levels of healthy animals in Hokkaido and Shizuoka were 0.651 and 0.263 (IgG antibody to HO14 strain), 0.642 and 0.169 (IgG to HP3 strain), 0.613 and 0.367 (IgM to HO14 strain) and 0.582 and 0.286 (IgM to HP3 strain). The differences of the antibody levels between cows from Hokkaido and Shizuoka were significant. Seasonal difference was found in seropositive cows from Hokkaido. The rate of seropositive cows was high in summer (23.4% in June and 11.8% in July) but low in winter (0% in January and February). The pattern was discussed to be associated with activation of ticks. One of 4 cows with arthritis showed significantly higher IgG antibody level than that of healthy cows and cows with some disease, although the serum was collected from Shizuoka where antibody-positive animals for B. burgdorferi were rare among healthy cows. This high IgG antibody may suggest that the arthritis of such cows was caused by infection with B. burgdorferi. Two of 7 cows with unclassified abortion showed positive antibody reaction in Hokkaido. These cases, however, may not be related to the B. burgdorferi infection because the positive rate was similar to those of healthy cows in the same season.

Disopyramide induces insulin secretion and plasma glucose diminution: studies using the in situ canine pancreas.

The mechanism of disopyramide-induced hypoglycemia, a life-threatening complication in the antiarrhythmic drug treatment, is still controversial. To elucidate this, we have evaluated plasma insulin (IRI) and glucagon (IRG) responses in the pancreatic vein (PV) of the in situ pancreas as well as responses of plasma IRI, IRG, and glucose in the femoral artery (FA) to disopyramide phosphate administration in anesthetized dogs. First, infusion of disopyramide at a dose of 50 mg for ten minutes directly into the pancreatic artery, but not the vehicle, increased significantly plasma IRI concentration in the PV (P less than .05 or less), where the IRI response started within three minutes and reached a peak of 2.8-fold preinfusion value at 30 minutes after starting the infusion (n = 7). Plasma IRI concentration in the FA also increased slightly but significantly (P less than .05). Plasma IRG concentration in the PV initially decreased significantly (P less than .05 or less) and in the FA at one point (P less than .05) during the infusion, and then increased significantly after cessation of the infusion, showing a peak of 1.9-fold preinfusion value at 60 minutes in the PV and the FA (P less than .05). Plasma glucose concentration in the FA decreased slowly and significantly after the infusion (P less than .05 or less) and fell by 16% of the baseline value at 60 minutes (P less than .05). Second, serum disopyramide concentration of 13.7 +/- 2.8 micrograms/mL at ten minutes, which corresponds to a twofold to threefold concentration of the human therapeutic level (n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)