Differential localization of prohormone convertases PC1 and PC2 in two distinct types of secretory granules in rat pituitary gonadotrophs.

Prohormone convertases PC1 and PC2 are endoproteases involved in prohormone cleavage at pairs of basic amino acids. There is a report that prohormone convertase exists in the rat anterior pituitary gonadotrophs, where it had previously been considered that proprotein processing does not take place. In addition to luteinizing hormone and follicle-stimulating hormone, rat pituitary gonadotrophs contain chromogranin A (CgA) and secretogranin II (SgII), two members of the family of granin proteins, which have proteolytic sites in their molecules. In the present study we examined whether there is a close correlation between subcellular localization of prohormone convertases and granin proteins. Ultrathin sections of rat anterior pituitary were immunolabeled with anti-PC1 or -PC2 antisera and then stained with immunogold. Immunogold particles for PC1 were exclusively found in large, lucent secretory granules, whereas those for PC2 were seen in both large, lucent and small, dense granules. The double-immunolabeling also demonstrated colocalization of PC2 and SgII in small, dense granules and of PC1, PC2, and CgA in large, lucent granules. These immunocytochemical results suggest that PC2 may be involved in the proteolytic processing of SgII and that both PC1 and PC2 may be necessary to process CgA.

Immunocytochemical localization of secretory phospholipase A(2)-like protein in the pituitary gland and surrounding tissue of the bullfrog, Rana catesbeiana.

Previously, we obtained a protein that has considerable amino acid sequence homology with secretory phospholipase A(2) (PLA(2)) from a bullfrog pituitary fraction obtained during the purification of thyrotropin (TSH). Subsequently, partial amino acid sequence (N-terminal 45 amino acid residues) analysis revealed this protein to be identical to the N-terminal amino acid sequence of otoconin-22, the major protein of aragonitic otoconia in the Xenopus saccule. In this study we developed an antibody against the N-terminal peptide of the bullfrog protein and applied it for immunocytochemical study of the pituitary and its surrounding tissue. Western blotting analysis showed that this antibody recognizes a 20.4-kD protein that has a molecular mass close to that of otoconin-22. Immunohistochemical reaction with the antibody was not found in any anterior pituitary cells but was intense in the monolayer epithelial cells of the endolymphatic sac surrounding the pituitary gland, which is a major storage site of calcium carbonate in amphibians. An electron microscopic study revealed that the cuboidal cells in the endolymphatic sac contained large, polymorphic secretory granules in their apical cytoplasm. Immunogold particles indicating the presence of a PLA(2)-like protein were observed predominately in these secretory granules. These findings support the view that this PLA(2)-like protein obtained during purification of TSH was derived from the endolymphatic sac adhering to the pituitary and that this protein is a bullfrog otoconin. (J Histochem Cytochem 49:631-637, 2001)

Effectiveness of arterial embolization procedure in uterine cancer patients.

Patients with late stage gynecologic malignancies occasionally develop massive pelvic hemorrhage, and management of the hemorrhage is often difficult. Transcatheter arterial embolization with an absorbable gelatin sponge following the Seldinger method was performed to control hemorrhage in five patients with cancer of the uterine cervix. Pelvic arteriograms of five patients showed no further extravasation and their bleeding ceased. No patients died of pelvic hemorrhage, and all of them eventually died as a result of the original disease within two years of the procedure. As for complications of this procedure, slight fever (3/5) and minimal lumbar pain (2/5) were noticed, which were easily controlled by an indomethacin suppository. Based on these findings, this therapeutic embolization method proved to be useful in the management of massive pelvic hemorrhage in patients with cervical cancer.

Changes in peripheral blood levels and pulse frequencies of GnRH in patients with hypopituitarism.

Pituitary dysfunction occasionally results from brain tumors or the surgical resection of brain tumors. The authors examined two patients with hypogonadotropic secondary amenorrhea, who had undergone surgical removal of brain tumors. Changes in immunoreactive gonadotropin-releasing hormone (GnRH) secretion are of interest in patients with a gonadotropin and gonadal steroid deficit, because both steroid and pituitary feedback systems are altered by tumors or tumor resection. The authors thus measured GnRH, luteinizing hormone, and follicle-stimulating hormone levels every 15 minutes for 4 hours by radioimmunoassay and investigated qualitative and quantitative changes in the pulsatile patterns of these hormones in two hypogonadotropic hypogonadism patients. They also performed similar multiple measurements of GnRH in two normal cycle women in follicular phase and two postmenopausal women. The concentration of plasma GnRH in two hypopituitarism patients was compared with that in two normal cycle women and two postmenopausal women. The study showed that the peripheral blood level of GnRH was significantly lower in two hypopituitarism patients than in both normal cycle and postmenopausal women, and that the pulsatile frequency was not different among these three groups. These findings suggest that alteration of feedback systems results in a decrease in the blood level of GnRH, and that pulses of GnRH maintain normal fluctuation despite the alteration of the hormonal circumstances in two hypogonadotropic hypogonadism patients.

A case of thoracogastropagus diagnosed before delivery.

The subject was a 29-year-old pregnant woman whose fetuses were a conjoined twin. We diagnosed the fetuses as a conjoined twin by ultrasonography at the 12th week of gestation. The patient and her family wanted living infants and hoped that they were separated surgically. We consulted with the doctors of pediatric surgery and cardiovascular surgery departments. At the 18th week of gestation, the fetuses died in utero and they were delivered transvaginally by labor induction. Both infants were female with 430 g in total body weight and both of them were 21 cm in length. Pathological findings were thoracogastro-pagus with a single heart, a single pair of liver and a single small intestine.

Transcervical fallopian tube recanalization under fluoroscopic guidance. The Iwasaki-Hayashi catheter.

This study was designed in order to assess whether the Iwasaki-Hayashi (IH) catheter can be fixed to the uterine cervix easily and successfully during transcervical fallopian tube recanalization (T-FTR) with fluoroscopic guidance, to try T-FTR in special cases, and to investigate the success rate. The study included 21 infertile women with tubal obstruction, diagnosed by hysterosalpingography examined at least twice to exclude tubal spasm. Using the IH catheter, which proved to be very useful, higher therapeutic efficacy could be obtained. A patient with unilateral proximal tubal obstruction became pregnant following natural fertilization in the fallopian tube which had been recanalized by T-FTR. The success rate of recanalization, the pregnancy rate and the take-home-baby rate were 95.2%/patient, 19.0 and 19.0%, respectively.

Blood macrophage colony-stimulating factor and thrombin-antithrombin III complex concentrations in pregnancy and preeclampsia.

Macrophage colony-stimulating factor (M-CSF) is a characteristic cytokine that plays an essential role in placenta maintenance, and thrombin-antithrombin III complex (TAT) is a hemostatic marker that is remarkably altered both in normal pregnancy and in preeclampsia. The present study was designed in order to show various levels of M-CSF and TAT in pregnancies. Peripheral blood was collected from 49 subjects, of whom 31 were normal pregnant women consisting of the four groups (namely 10th, 20th, 30th, and 38th weeks of gestation), 13 were preeclamptic pregnant women (37th week of gestation; mean blood pressure, 158/99 mm Hg), and 5 were nonpregnant controls. We compared blood M-CSF and TAT levels among them. Results showed that blood M-CSF and TAT levels increased significantly with gestational age. Furthermore, the ratio of increase in M-CSF was significantly lower than that in TAT in normal pregnant women compared with controls. In contrast, the ratio of increase in M-CSF was significantly higher than that in TAT in preeclamptic women compared with normal pregnant women. These results concerning the ratio of increase in M-CSF and TAT have not been reported. These findings show that M-CSF level increases significantly in preeclampsia even in its earlier stage, exhibiting a systolic blood pressure of less than 160 mm Hg.

Cisplatin-induced nephrotoxicity and the protective effect of fosfomycin on it as demonstrated by using a crossover study of urinary metabolite levels.

BACKGROUND: Cisplatin induces nephrotoxicity and this study evaluated the protective effect of fosfomycin on it in 11 gynecological cancer patients. METHODS: The N-acetyl-beta-D-glucosaminidase (NAG), beta 2-microglobulin (beta 2MG), creatinine (uCr) and total protein (TP) levels in a 24-hour urine specimen as well as the blood urea nitrogen (BUN) and serum creatinine (sCr) were measured before and after CAPF chemotherapy alone (control) or with fosfomycin. RESULTS: The results were statistically analyzed by using the t-test. NAG, beta 2MG, uCr and TP levels increased significantly after chemotherapy in the control patients, but BUN and sCr levels did not change significantly. The NAG level in the control group was twice as high as in the fosfomycin group 8 days after chemotherapy (p < 0.01). The uCr and TP in control patients increased significantly after chemotherapy when compared to those in patients coad-ministered fosfomycin. There were no significant changes in beta 2MG, BUN and sCr levels. CONCLUSIONS: Cisplatin affected the levels of NAG, beta 2MG, uCr and TP without influencing BUN and sCr levels. Fosfomycin, therefore, may be useful as a supplemental treatment for reducing cisplatin nephrotoxicity, especially proximal tubular damage.

High blood levels of macrophage colony-stimulating factor in preeclampsia.

In pregnancy, the decidual cells produce and secrete large amounts of macrophage colony-stimulating factor (M-CSF). M-CSF stimulates the proliferation and differentiation of trophoblasts. In addition, it stimulates them in a dose-dependent manner to produce certain hormones, such as human chorionic gonadotropin and human placental lactogen. Based on these facts, M-CSF is considered to be an essential cytokine for placental maintenance. Because placental dysfunction may sometimes result from preeclampsia, ascertaining blood M-CSF levels in preeclamptic patients would be of interest. The blood was collected from 33 subjects, of whom 19 were normal pregnant women and 14 were preeclamptic patients. The M-CSF level was determined by the sandwich enzyme-linked immunosorbent assay method using three antibodies. The investigators measured peripheral blood M-CSF levels in preeclamptic subjects and compared them with levels in subjects with normal pregnancies. This study showed that peripheral blood M-CSF levels were significantly higher in preeclamptic patients in the 30th and 38th weeks of pregnancy (P < .005). This is the first report concerning high M-CSF blood levels in preeclamptic patients.

Possible role of protein kinase C in the regulation of intracellular stability of focal adhesion kinase in mouse 3T3 cells.

Effects of various types of protein kinase inhibitor on the adhesion and spreading of BALB/c mouse 3T3 cells and on the phosphorylation and stability of focal adhesion kinase (FAK) in the cells were studied. Inhibitors of protein tyrosine kinases, methyl 2,5-dihydroxycinnamate and herbimycin A, inhibited tyrosine-phosphorylation of FAK and the adhesion of 3T3 cells to fibronectin. Among inhibitors of serine/threonine kinases tested, calphostin C, a specific inhibitor of protein kinase C, inhibited cell spreading rather than cell adhesion, and it induced the decrease of intracellular FAK within 30 min. Inhibitors of tyrosine kinase, A kinase, G kinase, and myosin light chain kinase did not induce such a rapid and specific decrease of FAK. When calphostin C (20 microM) was added to sub-confluent monolayer cultures, serine-phosphorylation of FAK was inhibited by 67% within 2 h, and decrease in the amount of FAK and rounding up of the cells began after 4 h. Label-chase experiments indicated that about 60% of 35S-labeled FAK degraded within 1-2 h after addition of calphostin C to monolayer cultures. These results indicated that serine-phosphorylation of FAK induced by protein kinase C was important in the regulation of metabolic stability of FAK.

Long-term estrogen replacement therapy in female patients with dementia of the Alzheimer type: 7 case reports.

Seven female patients with mild to moderate dementia of the Alzheimer type (DAT) were treated with long-term, low-dose estrogen replacement therapy (ERT) over a period of 5-45 months. Five of the 7 patients were cases who had responded well to short-term ERT with 1.25 mg/day of conjugated equine estrogens (CEE) for 6 weeks. The 7 patients from 56 to 77 years of age received 0.625 mg/day of CEE for 21 days, followed by a pause of 7 days. A 28-day cycle of low-dose ERT was performed repeatedly. In 4 cases, these patients received 5 mg/day of medroxyprogesterone acetate (MPA) during the last 10-12 days of estrogen treatment. Therapeutic efficacy of estrogen was evaluated by psychometric assessments such as the Mini-Mental State Examination (MMSE) and the Hasegawa Dementia Scale (HDS) and a behavior rating scale of the Gottfries-Bråne-Steen geriatric rating scale (GBS). The MMSE and HDS evaluations were performed principally once in 2-4 weeks. In 4 out of the 7 patients, the MMSE and HDS scores were elevated above the pretreatment levels during ERT. The termination of ERT resulted in a decrease in both scores. Furthermore, the GBS scores and daily activities of the same 4 patients were improved during ERT. In these 4 patients cognitive functions were markedly improved throughout the treatment period, while the other 2 patients responded moderately well and another patient did not respond at all. These observations suggest that long-term, low-dose ERT improves cognitive functions, dementia symptoms and daily activities in women with mild to moderate DAT.(ABSTRACT TRUNCATED AT 250 WORDS)

[Changes in monoamine concentrations in developing female rat brains under continuous light].

Norepinephrine (NE), dopamine (DA) and serotonin (5-HT) concentrations in the hypothalamus were measured by HPLC on days 10, 20, 30 and 40 of age in Sprague-Dawley albino female rats after continuously exposed to light (LL). When rats were housed under a lighting schedule (14hr light and 10hr dark: LD), the NE concentration increased linearly. The DA concentration remained lower on days 10, 20 and 30 of age, but a sudden increase in the DA concentration appeared on day 40 of age. An increase in the 5-HT concentration occurred at day 20 of age, and remained at the same level thereafter. LL exerts profound effects on DA and 5-HT concentrations in the hypothalamus; an increase in the 5-HT concentration appeared at day 10 of age, and the peak concentration of DA was reached at day 30 of age. The inference is then that LL exerts effects on the metabolism of 5-HT and DA and promotes the increase in 5-HT and DA concentrations in the hypothalamus, and that the advanced appearance of 5-HT and DA in the hypothalamus seems to correspond with the advancement of puberty onset. Indeed, LL exerted its effects on puberty onset and advanced it.

[Efficacy and endocrinological analysis of combined buserelin-pure FSH-hCG therapy in patients with polycystic ovary syndrome].

The study was designed to investigate the efficacy of combined buserelin-pure FSH-hCG therapy in patients with polycystic ovary syndrome (PCO) and to analyse its underlying hormonal changes. Buserelin was intranasally administered daily to 13 patients (35 cycles) for 4-10 weeks with a mean week of 5.4 +/- 1.6 (SD), followed by concomitant pure FSH administration. Out of the 13 patients, 8 were the ones who had experienced ovarian hyperstimulation syndrome (OHSS) using pure FSH alone. Hormonal analyses were performed on these 8 patients. This combined regimen resulted in a 97.1% ovulation rate (34/35) and a 37.1% incidence rate of OHSS (13/35). In 4 out of the 8 patients who had experienced OHSS, no OHSS was observed in the first cycle of the therapy. Eight patients became pregnant with the therapy. Excluding one patient whose husband had oligozoospermia, 7 patients conceived at the first cycle of the therapy and the pregnancy rate per cycle was 25.0% (7/28). No abortion or multiple pregnancy was observed in any of the 8 cases. Pretreatment with buserelin resulted in significantly decreased serum LH, FSH, LH/FSH ratio, estradiol (E2), testosterone (T), and androstenedione (ASD). Serum E2, T and ASD levels at both preovulatory and midluteal phases were significantly lower with the combined regimen than with pure FSH alone. These results indicate that this combined regimen improves the characteristic endocrine profile of PCO and enables pure FSH to achieve ovulation regularly with a high pregnancy rate, although it does not always inhibit OHSS.

Stimulation of tyrosine- and serine-phosphorylation of focal adhesion kinase in mouse 3T3 cells by fibronectin and fibroblast growth factor.

Phosphorylation of both tyrosine and serine residues of focal adhesion kinase (FAK) was stimulated by the adhesion of BALB/c mouse 3T3 cells to fibronectin, but phosphorylation of threonine was not detectable. Acidic and basic fibroblast growth factors also stimulated the phosphorylation of serine and tyrosine of FAK in cells adhered to poly-L-lysine, but epidermal growth factor and platelet-derived growth factor did not. A fusion protein of fibronectin and basic fibroblast growth factor effectively induced the phosphorylation of FAK. Phosphorylation of FAK in the rat myoblast L-6 cell line, which lacks fibroblast growth factor receptors, was not stimulated by fibroblast growth factors, suggesting that the interaction of fibroblast growth factors with their receptors might cause the phosphorylation of FAK.

Evaluation of estrogen treatment in female patients with dementia of the Alzheimer type.

This study was designed to investigate the therapeutic efficacy of estrogen in female patients with dementia of the Alzheimer type (DAT). Fifteen DAT patients with a mean age of (mean +/- SE) 71.9 +/- 2.4 years were treated with 0.625 mg of conjugated equine estrogens orally twice a day for 6 weeks. Of the 15 DAT patients, 4 were diagnosed as mild, 7 as moderate and 4 as severe. The effects of estrogen on DAT patients were evaluated by psychometric assessments, behavior rating scales, regional cerebral blood flow (rCBF) measurement and quantitative EEG analysis. Psychometric assessments consisted of Mini-Mental State Examination (MMSE) and Hasegawa Dementia Scale (HDS). Dementia syndromes were evaluated by the GBS-Scale (GBSS) and Hamilton Depression Rating Scale (HDRS). During estrogen replacement therapy (ERT), the mean MMSE score (mean +/- SE) increased significantly from 11.6 +/- 1.9 to 13.2 +/- 2.0 at 3 weeks (P < 0.01) and 13.8 +/- 2.0 at 6 weeks (P < 0.001). The mean HDS score increased significantly from 8.6 +/- 2.1 to 11.5 +/- 2.3 at 3 weeks (P < 0.001) and 11.6 +/- 2.6 at 6 weeks (P < 0.01). Significant improvements in the mean scores of the GBSS and HDRS were also observed in the estrogen-treated group, but not in the untreated control group with a mean age of 71.2 +/- 2.5 years (n = 15). The rCBF was measured by single photon emission computed tomography (SPECT). ERT increased the mean rCBF significantly in the lower frontal region (P < 0.01) and primary motor area (P < 0.02) of the right hemisphere. The mean absolute power delta band values in both left and right frontal EEG (Fp1 and Fp2) (P < 0.01) and theta, band values in Fp2 (P < 0.05) decreased significantly during ERT. It is inferred that ERT significantly improves cognitive functions, dementia symptoms, regional cerebral blood flow and EEG activity in female patients with DAT.

[A clinical study on memory function in climacteric and periclimacteric women].

This study was designed to investigate memory function in climacteric and periclimacteric women who lived a normal, ordinary life. Two hundred women treated at the gynecological outpatient clinic of Koshigaya Hospital were divided into 7 groups: groups A(31-35 yr), B(36-40 yr), C(41-45 yr), D(46-50 yr), E(51-55 yr), F(56-60 yr) and G(61-65 yr). Each group consisted of 30 women except group G(n = 20). The memory function of each group was determined and the mean scores for 10 paired hard-associates after three trials of presentation were compared. The mean scores (+/- SD) for groups A and B were 8.0 +/- 2.0 and 8.2 +/- 1.7, respectively, which were not statistically different. The scores for both groups were significantly higher than those for the other groups (p < 0.01). The mean scores for groups C and D were 5.9 +/- 2.1 and 5.6 +/- 2.4, respectively, which were not statistically different. The score for group C was significantly higher than those for groups E(4.5 +/- 2.4), F(4.2 +/- 2.2), and G(3.3 +/- 1.6) (p < 0.05). The score for group D was significantly higher than those for groups F and G(p < 0.05). The score for group E was significantly higher than that for group G(p < 0.01). The decrease in memory function was the greatest in group C. In the climacterium, memory impairment was also observed in group E. The former corresponds to the climacteric commencement age group where cyclic changes in serum estrogen levels decrease or cease, and the latter corresponds to the age group for menopause.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiogenic activity of a fusion protein of the cell-binding domain of fibronectin and basic fibroblast growth factor.

We constructed a fusion protein of the cell-binding domain of human fibronectin and human basic fibroblast growth factor, and prepared a polypeptide with both cell-adhesive activity and growth factor activity. A human gene fragment coding for basic fibroblast growth factor was amplified by the polymerase chain reaction, and introduced into the expression vector pTF7520, which encodes the cell-binding domain of human fibronectin. The resulting plasmid encoded a fusion protein in which basic fibroblast growth factor was added covalently to the C-terminal end of the fibronectin fragment. The fusion protein was expressed in Escherichia coli JM109 cells and purified from the extract by heparin affinity chromatography. The purified fusion protein had cell-adhesive activity toward BALB/c 3T3 cells, and stimulated their DNA synthesis in serum-depleted cultures. The fusion protein gave maximum mitogenic activity at the concentration of 10 nM. The fusion protein adsorbed to culture dishes, or added to collagen gels, stimulated the growth of human umbilical-vein endothelial cells. The fusion protein stimulated the angiogenesis in chorioallantoic membranes of developing chick embryos.

Inhibition of cell adhesion by proteolytic fragments of type V collagen.

Type V collagen inhibits the cell-substratum adhesion of many types of cells. In this study, inhibitory effects of type V collagen on the adhesion of mouse melanoma B16-F10 cells to fibronectin, laminin and vitronectin were investigated. When the culture dishes were coated with a mixture of fibronectin and type V collagen, adhesion of the cells was inhibited by 50% at a fibronectin/collagen molar ratio of 10/1. At a similar molar ratio, adhesion of the cells to laminin was inhibited moderately, but that to vitronectin was not significantly affected. Type V collagen added into culture medium was less effective in inhibiting cell adhesion. The antiadhesive activity of type V collagen was partially retained in the alpha 1 (V) chain of heat-denatured collagen. The alpha 1 (V) chain was split into two large fragments, 90 kDa and 60 kDa, by limited digestion with Staphylococcus aureus V8 proteinase. The 90-kDa fragment, which was derived from the C-terminal half of the alpha 1 (V) chain, inhibited the cell adhesion more profoundly than alpha 1 (V). However, little fibronectin bound to the 90-kDa fragment, while fibronectin bound to the 60-kDa fragment, which was less antiadhesive than the 90-kDa fragment, with the same extent as alpha 1 (V). We therefore concluded that the antiadhesive effect of type V collagen was not due to its specific binding to the fibronectin molecule.

Effects of fibronectin-type V collagen recombinant fusion protein on cell adhesion and cell proliferation.

An expression vector pTF7520-Col-V-In, which encodes a fusion protein of the cell-binding domain of fibronectin (C277) and the insulin- and heparin-binding domain of the alpha 1 chain of human type V collagen, was constructed. E. coli transfected with this plasmid synthesized a 50-kDa fusion protein. This fusion protein, C277-V, was purified from the crude extract by a single step heparin HPLC. Similar amounts of insulin bound to purified C277-V and to the alpha 1 chain of type V collagen as judged by the binding of peroxidase-conjugated insulin. Cell-adhesive activity of C277-V was lower than that of the original fibronectin fragment C274, but similar numbers of cells adhered to both protein substrates when the culture dishes were coated with 1 mM of each protein. Insulin bound to the C277-V substratum stimulated the growth of mouse mammary tumor MTD cells in serum-free culture medium.