Effects of estriol on cell viability and 1,25-dihydroxyvitamin D3 receptor mRNA expression in cultured human osteoblast-like cells.

It is clinically evident that administration of estriol (E3) increases the bone mass density of the lumbar vertebrae in postmenopausal women, and that combined treatment with estrogen and 1,25-dihydroxyvitamin D3 (VD3) increases femoral neck bone mass density compared with treatment with estrogen alone in postmenopausal osteoporotic women. However, the molecular mechanism whereby treatment with E3 affects osteoblast cell function is still unknown. This study was conducted first to examine the comparative effects of E3 and VD3 on the cell viability of cultured human osteoblast-like cells (HOS) and second to determine whether E3 affects VD3 receptor mRNA expression in HOS. The cell viability and VD3 receptor mRNA expression of cultured HOS were assessed by MTT assay and semi-quantitative reverse transcriptase-polymerase chain reaction with Southern blot analysis, respectively. The treatment with E3 increased the cell viability of cultured HOS compared with untreated control cultures. The increase in cell viability caused by the treatment with E3 was further augmented by the combined treatment with VD3. The addition of either E3 (3.52 x 10(-8) mol/l) or E3 (3.52 x 10(-7) mol/l) to cultured HOS for 24 h resulted in a fourfold and eightfold increase, respectively, in VD3 receptor mRNA expression in HOS, compared with that in untreated control cultures. These results suggest that E3 may up-regulate the cell viability of osteoblast cells, and that the concomitant treatment with E3 and VD3 further augments the cell viability being associated with an E3-induced increase in VD3 receptor mRNA expression in those cells.

Up-regulation by IGF-I of proliferating cell nuclear antigen and Bcl-2 protein expression in human uterine leiomyoma cells.

IGF-I has been reported to play a role in regulating proliferation of human leiomyoma cells. There is, however, little evidence to suggest that IGF-I inhibits apoptosis in the leiomyoma cells. The present study was conducted to elucidate whether IGF-I affects apoptosis and Bcl-2 protein expression, an apoptosis-inhibiting gene product, in cultured leiomyoma cells. In addition, we examined the effect of IGF-I on proliferating cell nuclear antigen (PCNA) expression in cultured leiomyoma cells. Isolated human leiomyoma cells were subcultured in phenol red-free DMEM supplemented with 10% FBS for 120 h and then stepped down to serum-free conditions for an additional 72 h in the absence or presence of graded concentrations of IGF-I (1.0, 10, and 100 ng/ml). The effects of IGF-I on Bcl-2 protein and PCNA expression in cultured leiomyoma cells were assessed by Western immunoblot analysis and immunocytochemical staining, whereas the effects of IGF-I on the cell viability and apoptosis of the cultured cells were determined by 3-(4,5-dimethylatriazol-2-yl)-2,5diphenyltetrasodium bromide (MTT) assay and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling assay, respectively. Immunocytochemical staining demonstrated that IGF-I treatment resulted in the increase in PCNA labeling index in cultured leiomyoma cells in a dose-dependent manner. Immunoblot analysis of proteins extracted from the cultured leiomyoma cells revealed that the addition of IGF-I (10 and 100 ng/ml) significantly increased the expression of 35-kDa immunoreactive PCNA and 26-kDa Bcl-2 protein, compared with those in control cultures. Cell survival and proliferation of cultured leiomyoma cells, assessed by MTT assay, was significantly augmented by IGF-I treatment, compared with those of control cultures. Terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling assay showed that the apoptosis-positive rate of leiomyoma cells treated with IGF-I was significantly decreased, compared with that in control cultures. The present results suggest that IGF-I plays crucial roles in leiomyoma cell growth, not only in promoting the proliferative potential by up-regulation of PCNA expression but also in down-regulating apoptosis by up-regulation of Bcl-2 protein expression in leiomyoma cells.

Association between endometriosis and N-acetyl transferase 2 polymorphisms in a UK population.

The relationship between endometriosis and polymorphisms in the N-acetyl transferase 2 (NAT 2) gene was investigated in a UK population, as this gene has been previously implicated in the aetiology of the disease. Point mutations in the gene result in the variant alleles NAT 2 *5, *6 and *7 from the wild-type NAT 2 *4 allele. Homozygotes for the NAT 2 *4 wild type allele are fast NAT acetylators, while heterozygotes with one wild-type allele and a variant NAT 2 *5, *6 or *7 allele have reduced enzyme activity, and individuals with two variant alleles are slow acetylators. The NAT 2 *4/*6 genotype was significantly more common among affected women (35.2%) than population controls (8.1%; P = 0.0001) or unaffected women (4.2%; P = 0.02). Significantly more affected women (57.4%) were fast acetylators than were population controls (32.3%; P < 0.01) or unaffected women (33.3%; P < 0.05). These data suggest that altered NAT 2 enzyme activity may be a predisposition factor in endometriosis, or that NAT 2 alleles may be in linkage disequilibrium with a susceptibility allele in the same chromosomal region.

Rupture of the spleen as an unusual complication of laparoscopy. A case report.

BACKGROUND: Laparoscopic surgery has many advantages but is not without complications, such as viscus perforation and intraabdominal hemorrhage. CASE: A rare and severe complication, massive abdominal bleeding due to splenic rupture, became symptomatic after an uneventful laparoscopy. Tearing away of delicate peritoneal reflections or small adhesions on the splenic capsule due to induction of pneumoperitoneum can result in sudden rupture and hemorrhage. CONCLUSION: This possibility should be remembered when laparoscopy is necessary in patients with a history of blunt abdominal trauma.

Regulation of circulating levels of IGF-I in pregnant rats: changes in nitrogen balance correspond with changes in serum IGF-I concentrations.

Serum IGF-I concentrations in rats decrease significantly in late pregnancy. To determine if the reduction in serum IGF-I concentrations is attributable to circulating GH or maternal nutritional status, we investigated the effect of treatment with recombinant human GH (rhGH: 100 microgram/rat per day) on IGF-I concentrations during late pregnancy, and evaluated the relationship between maternal nitrogen balance and IGF-I concentrations. Serum IGF-I concentrations and maternal nitrogen balance ((nitrogen intake)-(nitrogen content in faeces and urine)-(nitrogen content in fetus and placenta)) were measured by RIA and the Dumas method. In non-pregnant rats treated with rhGH for 3 days, serum IGF-I concentrations (835.4+/-59.5 ng/ml; P<0.01) were significantly greater than in those animals treated with saline (319.6+/- 95.6 ng/ml). In the pregnant rats, however, there was no significant difference in serum IGF-I between those treated with rhGH (151. 1+/-43.0 ng/ml) and those treated with saline (142.0+/- 39.9 ng/ml) from day 17 to 19 of pregnancy. Maternal nitrogen balance in the pregnant rats increased significantly from day 4 to day 10 of pregnancy (169.5+/-57.4 and 196.1+/- 33.4 mg/day, respectively; P<0. 05) compared with non-pregnant controls (31.9+/-19.9 mg/day) and decreased markedly from day 12 of pregnancy (79.8+/-60.1 mg/day; P<0. 05) onwards, to 14.9+/-47.8 mg/day on day 20 of pregnancy (P<0.01), significantly different from the value on day 10 of pregnancy. The mean difference in maternal nitrogen balance between pregnant and non-pregnant rats was positively correlated (r=0.87, P<0.01) with the mean difference in maternal IGF-I concentrations, using linear regression analysis. These results support the conclusion that the circulating concentration of IGF-I in pregnant rats is associated with the change in nitrogen balance, but not with circulating GH.

Absence of a relationship between endometriosis and the N314D polymorphism of galactose-1-phosphate uridyl transferase in a UK population.

An association between the N314D polymorphism of galactose-1-phosphate uridyl transferase and endometriosis has recently been reported in a North American population. To determine whether such an association exists in the UK population, we genotyped 148 women with sporadic (n = 91) or familial (n = 57) endometriosis, a control population of 95 male blood donors and a control group of 53 women with a normal pelvis at hysterectomy. Heterozygosity for the polymorphism was found in 14.9% (22/148) of affected women, 13.7% (13/95) of male blood donors and 11.3% (6/53) of women with a normal pelvis. There was no statistically significant difference in the frequency of the polymorphism between cases and controls in the UK population, even when the cases were divided into groups of moderate-severe disease, sporadic cases or familial cases. We conclude that the galactose-1-phosphate uridyl transferase N314D polymorphism is unlikely to be associated with endometriosis in the UK population.

[Effect of the GH-PRL superfamily on circulating plasma insulin-like growth factor-1].

To elucidate the effects of growth hormone (GH), prolactin (PRL), and human placental lactogen (hPL) on the regulation of insulin-like growth factor (IGF-1), we compared plasma IGF-1 levels, the pattern of circulating IGF-1-IGF-binding protein complexes (IGF-1 complexes), and unsaturated binding protein (USBP) levels among 1) naturally growing Wistar rats at several developmental stages, 2) rats subcutaneously administered GH, and 3) hypophysectomized rats treated with each of the three hormones. We further evaluated the in vitro secretion of IGF-1 by primary cultured rat hepatocytes, following exposure to the hormones singly or in combination. Plasma IGF-1 and USBP levels were determined by radioimmunoassay and competitive radioassay, respectively. IGF-1 complexes were separated from plasma and culture medium by Sephadex G150 and HPLC gel-chromatography, respectively. The results were as follows. 1) In naturally growing rats, plasma IGF levels were low during fetal life and after birth until 28 days of age, and thereafter increased rapidly to reach an adult level by 35 days. At 35 days, the molecular distribution of IGF-1 switched from an infantile pattern (only 40Kd IGF-1 complex) to an adult form (IGF-1 complexes with both 40Kd and 150Kd proteins). In addition, 150Kd USBP became detectable after 28 days. 2) Administration of GH for 3 days to 13-day-old rats induced 150Kd USBP 9 days earlier than in controls, while plasma IGF-1 levels remained comparable throughout the period examined. 3) In the hypophysectomized rats, plasma IGF-1 levels decreased to approximately one fifth of those in untreated rats, accompanied by the disappearance of 150Kd USBP and 150Kd IGF-1 complex. However, when GH (but not PRL or hPL) was continuously administered for 72 hrs, plasma IGF-1 levels and the circulating profile of IGF-1 complexes were nearly restored to those in control rats. 4) Addition of GH (but not PRL) to the culture medium caused hepatocytes to secrete IGF-1, consisting of only the 40Kd IGF-1 complex. This effect was blocked by the simultaneous addition of hPL with GH. These findings indicate that, of the hormones analyzed, GH is the most important regulator of the plasma IGF-1 concentration and circulating complex forms during the developmental periods in rats, as is also thought to be the case in humans.

[The relationship between the changes in insulin-like growth factor-1 (IGF-1) and the nutritional states evaluated by nitrogen balance in pregnant rats].

The effects of nutrition on serum insulin-like growth factor-1 (IGF-1) concentrations during pregnancy of rats were investigated by using rat cultured hepatocytes in vitro, and by the assessment of nitrogen balance in vivo. IGF-1 concentration was measured by radioimmunoassay, and nitrogen balance was calculated by Pregl-Dumas method. The results were as follows: (1) Cultured rat hepatocytes produced IGF-1 in medium and it was significantly stimulated by the addition of various concentrations of glucose (1.1-4.4 mM) and/or several amino acid concentrations in a dose-related manner. (2) Serum IGF-1 concentrations, which indicated 368.6 +/- 143.8 ng/ml in a non-pregnant fed state, markedly decreased in a fasted state, reaching the levels of 143.8 +/- 30.4 ng/ml after 72 hours fasting. Nitrogen balance in these fasted rats also decreased according to the fasted period. (3) In early pregnancy (Day 0-12), serum IGF-1 concentrations were indistinguishable from those of non-pregnant fed rats. It gradually declined after the 13th day of pregnancy and reached the minimum levels of 77.0 +/- 12.1 ng/ml on the 21st day. On the other hand, mean nitrogen balance which was calculated from the difference of nitrogen retention in the maternal body and that in the fetal body, also decreased after 13 days of pregnancy and reached the levels of 14.9 g/day on the 21st day of pregnancy. These results suggested that IGF-1 concentrations in rat serum and conditioned medium might be regulated by nutritional factors, i.e., glucose and/or several amino acids. The curious profiles of IGF-1 concentrations observed in pregnant rats might be due in part to the effects of nutritional changes between the maternal and fetal body, especially, the changes of protein metabolism represented by the nitrogen balance.

[Immunohistological study for the ras oncogene products in colorectal cancer].

By using anti-NHK-ras and K-ras monoclonal antibodies, paraffin-embedded tissue specimens of 70 cases of colorectal cancers were immunohistologically examined. Correlation between incidence of expression of these oncogenes and either of postoperative survival rate and stage of colorectal cancer were calculated. The positive rate of anti-NHK-ras and K-ras staining were correlated with maximum diameters of tumor, staging, degree of Dukes classification and depth of invasion. Positive and negative rates of between NHK-ras and K-ras staining were well coincident. The cumulative postoperative survival rate of negative cases for anti-K-ras staining showed a significantly higher survival rate than that of the positive cases.

[Immunohistological study on ras and myc oncogene products in pancreatic cancer].

Surgical treatment of the pancreatic cancer is currently unsatisfactory. By using anti-K,H,N-ras monoclonal and anti-myc polyclonal antibodies, paraffin-embedded tissue specimens of the carcinoma in digestive organs were immunohistologically studied. Then, the incidence of expression of these oncogenes and correlative studies of survival rate and disease stage of pancreatic cancer were analyzed. High incidence of expression of ras oncogene in the carcinoma of the pancreas and biliary tract was obtained. Frequency of expression of ras oncogene was correlated to disease stage and T factor for staging the carcinoma of the pancreas. Prognoses of 6 cases of pancreatic cancer without expression of ras oncogene were better than that of pancreatic cancer expressed ras oncogene.