Interaction between gastrocnemius muscle weakness and moderate exercise deteriorates joint integrity in rat knee.

The aim of this study was to determine the effect on the knee joint of the interaction between ankle muscle weakness and moderate exercise. Gastrocnemius muscle weakness was induced by intramuscular injection of botulinum toxin type A (BTX) in rats. Low-speed treadmill running (12 m/min for 60 min) was applied for 6 weeks in rats with and without BTX. Untreated animals were used as controls. After BTX injection, the gastrocnemius muscle weakness was confirmed by 3-D motion analysis in kinematic features of the hindlimb during locomotion as an increased maximal dorsiflexion angle during the stance phase. Serum biomarker analysis by enzyme-linked immunosorbent assay revealed that low-speed running decreased the catabolic effect on type II collagen. However, the inhibition of catabolism induced by running exercise was significantly counteracted by BTX injection. In addition, thinning of the cartilage layer and a reduction in the chondrocyte density was also found in the tibial plateau of the knee in the BTX-injected rats after running for 6 weeks. These data suggest that moderate exercise have a positive effect on joint homeostasis. However, ankle muscle weakness may alter the mechanical environment of the knee and impair the integrity of joint cartilage with moderate exercise.

Use of the Japanese health insurance claims database to assess the risk of acute pancreatitis in patients with diabetes: comparison of DPP-4 inhibitors with other oral antidiabetic drugs.

This study was initiated to evaluate the association of acute pancreatitis (AP) with the use of dipeptidyl peptidase-4 (DPP-4) inhibitors among patients with diabetes in Japan. A retrospective cohort study of a large medical and pharmacy claims database was performed to compare the incidence of AP among those receiving DPP-4 inhibitors and those receiving other oral antidiabetic drugs. The incidence of all AP and hospitalizations for AP was similar between the two groups. Previous exposure to DPP-4 inhibitors did not affect occurrence of AP in patients on other oral antidiabetic drugs. The Kaplan-Meier curve for time to AP was similar between the two groups, and was not affected by previous exposure to DPP-4 inhibitors. The Cox proportional hazard models showed the incidence of AP was not significantly higher in those receiving DPP-4 inhibitors. Despite numerous, important limitations related to claims database-based analyses, our results indicate that there is no increased risk of AP with use of DPP-4 inhibitors among patients with diabetes in Japan.

Morphological changes in hind limb muscles elicited by adjuvant-induced arthritis of the rat knee.

We investigated qualitative and quantitative changes in rat hind limb muscles caused by complete Freund's adjuvant (CFA)-induced knee joint pain. One week after CFA injection, muscle atrophy was induced only on the CFA-injected side. Wet weight of the rectus femoris (RF) and soleus (SOL) muscles were significantly decreased by 20% and 19%, respectively. The reduction in cross-sectional areas by CFA was similar for fast and slow muscle fibers in the RF (10% vs 15%, respectively) and SOL muscles (16% vs 16%, respectively). At the light microscopic level, pathological changes were not found in the RF muscles on both sides, although the infiltration of mononuclear cells and muscle regeneration were found in the SOL muscles on CFA-injected and contralateral control sides. On the other hand, electron microscopy revealed degenerative changes in the RF and SOL muscles on the CFA-injected side. Interestingly, sarcomere hypercontraction, indicating overexercise, was observed to a limited extent in the SOL muscles on the control side. In conclusions, knee joint pain can trigger the rapid development of muscle atrophy with degenerative changes not only in thigh but also calf muscles. This indicates that early interventions to inhibit joint pain or inflammation may prevent muscle atrophy.

Alteration in articular cartilage of rat knee joints after spinal cord injury.

OBJECTIVE: Mechanical forces are crucial for the maintenance of the morphologic and functional integrity of articular cartilage. The alteration of the articular cartilage after spinal cord injury (SCI) has been described in relation to a suppression of mechanical forces, since the joint is unloaded and restricted in movement. However, the morphological and biochemical characteristics of the cartilage after SCI are still poorly understood. We identified the localization of cartilage alterations after SCI and verified the influence of mechanical forces on the articular cartilage. METHOD: A total of 32 Wistar rats were used. Sixteen animals underwent an SCI and 16 animals served as control. The articular cartilage of the knee joint was assessed, respectively, at 4, 8, 10, and 12 weeks after intervention by histochemical, histomorphometric, immunohistochemical, and biochemical analyses. RESULTS: Cartilage thickness of spinal cord-injured knees decreased at the tibial and posterior femoral (FP) regions and increased at the anterior femoral (FA) region. Spinal cord injuries decreased the number of chondrocytes at the anterior regions and decreased the cartilage matrix staining only at the tibial regions. Immunolabeling to collagen type II was noted comparably in the superficial layer but noted weakly from the middle to deep layer. Collagen type I existed excessively at the cartilage surface and the pericellular regions. CONCLUSION: Cartilage alterations after SCI would not be explained by only a suppression of mechanical forces by unloading and immobilization, but there may be influences on the cartilage in addition to the change in mechanical forces.

Insulin secretion and insulin sensitivity at different stages of glucose tolerance: a cross-sectional study of Japanese type 2 diabetes.

To evaluate the factors causing glucose intolerance in type 2 diabetes in Japan, insulin secretion and insulin sensitivity were compared across the range of glucose tolerance. Subjects were divided into 3 groups: normal glucose tolerance (NGT), impaired glucose tolerance (IGT), and type 2 diabetes (DM) according to the criteria of the World Health Organization (WHO). We examined insulin secretion and insulin sensitivity using fasting blood glucose and insulin levels and 75 g oral glucose tolerance test (OGTT). We used homeostasis model assessment (HOMA) beta-cell and insulinogenic index (30 minutes) to estimate insulin secretion and HOMA-insulin resistance (IR) and insulin sensitivity index (ISI) composite for insulin sensitivity. Although insulin resistance plays an important role in the development of diabetes in many ethnic populations, the differences in insulin sensitivity between NGT and IGT and between IGT and DM are small in Japanese patients. On the other hand, as glucose intolerance increases, insulin secretion decreases most remarkably both between NGT and IGT and between IGT and DM in Japanese patients. Decreasing insulin secretion and decreasing insulin sensitivity both occur in developing type 2 diabetes in Japanese patients, but decreased basal and early-phase insulin secretion had more pronounced contribution to glucose tolerance than the indices of insulin sensitivity. Japanese type 2 diabetic patients are characterized by a larger decrease in insulin secretion and show less attribution of insulin resistance.

Early events involved in the development of insulin resistance in Zucker fatty rat.

AIM: To clarify the mechanism by which insulin resistance develops in obesity, Zucker fatty rats (ZFR) and lean litter mates (ZLR) were temporally subjected to oral glucose tolerance tests (OGTT) at 6 and 15 weeks of age. METHOD: As candidates for causative factors of insulin resistance, plasma leptin, free fatty acids (FFA) and tumor necrosis factor (TNF)-alpha levels were evaluated. RESULTS: There was no difference in the body weight between the two groups at 6 weeks of age, but ZFR were significantly heavier than ZLR at 15 weeks of age. At 6 weeks of age, blood glucose levels and area under the curve of glucose (AUCg) during OGTT were not significantly different between the two groups, while plasma insulin levels and area under the curve of insulin (AUCi) in the ZFR group were significantly higher than those in the ZLR group. At 15 weeks of age, the blood glucose levels and AUCg as well as plasma insulin levels and AUCi in the ZFR group during OGTT were significantly higher than those in the ZLR group. The ratio of fasting insulin to glucose in the ZFR group was significantly higher than that in the ZLR group at 6 and 15 weeks of age. Peripheral and portal plasma leptin and FFA levels were significantly higher in ZFR than ZLR both at 6 weeks and 15 weeks of age. Meanwhile, at 6 weeks, plasma TNF-alpha levels and expression of TNF-alpha protein in subcutaneous and visceral fat tissues were similar in both groups; however at 15 weeks, these were significantly higher in the ZFR group than the ZLR group. CONCLUSION: These results suggest that FFA rather than TNF-alpha may play an important role in early events involved in the development of insulin resistance and TNF-alpha accelerates insulin resistance together with FFA in the later stage.

Nociceptive stimulation increases NO synthase mRNA and vasopressin heteronuclearRNA in the rat paraventricular nucleus.

Nociceptive stimulation causes neuroendocrine responses such as arginine vasopressin (AVP) release and activation of the hypothalamo-pituitary-adrenal (HPA) axis. We examined the effects of nociceptive stimulation on the expression levels of neuronal nitric oxide synthase (nNOS) mRNA, heteronuclear (hn)RNA for AVP and AVP mRNA in the rat paraventricular nucleus (PVN) and supraoptic nucleus (SON), using in situ hybridization histochemistry. For nociceptive stimulation, formalin (5%) or saline was injected subcutaneously (s.c.) into the bilateral hind paws of rats. The expression of the nNOS gene in the PVN was significantly increased 2 and 6 h after s.c. injection of formalin in comparison with that in untreated and saline injected rats. The expression of the nNOS gene in the SON did not change in the untreated, saline- and formalin-injected rats. The AVP hnRNA in the PVN and SON was also significantly increased 15, 30 min and 2 h after s.c. injection of formalin, though AVP mRNA did not change at any time points that we studied. Plasma concentration of AVP was significantly increased 15 min after s.c. injection of formalin. These results suggest that NO in the PVN may be involved in nociceptive stimulation-induced neuroendocrine responses.

Oral nicotine administration decreases tumor necrosis factor-alpha expression in fat tissues in obese rats.

To investigate the effect of oral nicotine administration on insulin resistance and insulin secretion in an animal model of obesity, Zucker fatty rats were administered nicotine tartrate dihydrate orally through tap water (4.6 mg/kg/d, N group). Plasma nicotine concentrations in N group were 33.67 +/- 10.49 ng/mL. The control (C) group consisted of pair-fed control rats. After 8 weeks of nicotine administration, both groups of rats were administered glucose (2 g/kg) orally in an anesthetized state, and blood was collected for glucose and plasma insulin measurements. The pancreases were isolated and perfused in vitro under pentobarbital anesthesia 1 week after glucose administration. The fat tissues were excised. The levels of tumor necrosis factor (TNF)-alpha protein were assessed using enzyme-linked immunosorbent assay (ELISA) or Western blot analysis. Serum leptin levels were measured using radioimmunoassay (RIA). Blood glucose levels in N group were significantly lower than in C group before and 120 minutes after glucose administration. The insulin secretion from the isolated perfused pancreases of N group appeared to be decreased compared with C group, but there was no significant difference. Histologic examination showed that the mean size of the pancreatic islets in N group was significantly smaller than in C group. The composition ratios of alpha and beta cell mass of the pancreatic islets and fibroelastic tissues were not altered by nicotine administration. Portal TNF-alpha levels were comparable to peripheral levels in both groups. There were no significant differences in peripheral serum levels of TNF-alpha, free fatty acids (FFA), or leptin levels between N and C group. The TNF-alpha levels in visceral fat tissues in N group were significantly lower than those in C group. These results suggest that oral nicotine administration reduces insulin resistance in obese diabetic rats by decreasing production of TNF-alpha in the visceral fat tissues. Decreased islet size may be a secondary phenomenon induced by ameliorated insulin resistance, because the cellularity and fibroelastic tissues were not affected by the nicotine.

Up-regulation of cell surface insulin receptor by protein kinase C-alpha in adrenal chromaffin cells: involvement of transcriptional and translational events.

Our previous study showed that treatment of cultured bovine adrenal chromaffin cells with phorbol 12, 13-dibutyrate (PDBu) or 12-O-tetradecanoylphorbol 13-acetate (TPA) caused a rapid (<15 min) and persistent (>15 h) translocation of both conventional (c) protein kinase C-alpha (PKC-alpha) and novel PKC-epsilon (but not atypical PKC-zeta) from cytosol to membranes, whereas thymeleatoxin (TMX) increased the similar but selective membrane association of only cPKC-alpha. In the present study, chronic (>/=12 h) treatment of chromaffin cells with PDBu raised cell surface (125)I-insulin binding without altering the K(D) value; it developed in a concentration (EC(50) = 1.9 nM)-and time (t(1/2) = 14.6 h)-dependent manner, reaching its maximum 115% increase at 48 h. Either TPA (30 nM) or TMX (EC(50) = 6.4 nM) also increased (125)I-insulin binding by 97 or 88%, whereas the biologically inactive 4alpha-TPA had no effect. The increasing effect of PDBu (30 nM for 24 h) on (125)I-insulin binding was significantly blocked, even when H7, an inhibitor of PKC, was added at 8 h after the initiation of PDBu treatment. Concurrent treatment with brefeldin A, an inhibitor of vesicular transport from the trans-Golgi network, cycloheximide, an inhibitor of protein synthesis, or 5,6-dichlorobenzimidazole riboside, an inhibitor of RNA synthesis, abolished the PDBu-induced increment of (125)I-insulin binding. Western blot analysis, using antibody against the beta-subunit of the insulin receptor, showed that treatment with PDBu (30 nM) or TMX (EC(50) = 2.3 nM) increased levels of insulin receptor precursor (approximately 190 kDa; t(1/2) = 7.1 h) and insulin receptor beta-subunit (t(1/2) = 15.4 h), causing their almost maximum 52 and 59% rises, respectively, at 24 h. Northern blot analysis revealed that PDBu or TMX increased levels of insulin receptor mRNAs by approximately 35% as soon as 3 h, producing its monophasic peak approximately 76% increases at 24 h. All of these increasing effects of PDBu and TMX on (125)I-insulin binding and insulin receptor beta-subunit and insulin receptor mRNA levels were entirely prevented by simultaneous treatment with Gö6976, a selective inhibitor of cPKC. These results suggest that long-term activation of cPKC-alpha up-regulates the density of the cell surface insulin receptor via transcriptional/translational events.

Recent aspects of gestational trophoblastic disease in Japan.

The regional registration in 11 prefectures and one area covering 34% of total Japanese population started in 1974, increasing gradually to 21 prefectures and one area in 1993 covering 48.5% of total Japanese populations, by the Japan Trophoblastic Disease Committee under the auspices of Japan Society of Obstetrics and Gynecology. The results showed marked decreasing trend in incidence of molar pregnancy and choriocarcinoma in Japan. The most frequent antecedent pregnancies of choriocarcinoma has shifted from molar pregnancy in 1974 to term pregnancy in 1993. The Choriocarcinoma Risk Score Table that is in use and of practical significance, differentiating choriocarcinoma from invasive or metastatic mole by reference to simple 8 clinical items with the probability of more than 90% when compared with the histological diagnoses, is also presented.

Development of guanine analyzer to measure activity of guanylate cyclase.

A previous analyzer of adenine compounds by high-performance liquid chromatography was converted for the determination of guanine, its nucleoside and nucleotides by a post-column fluorescence derivatization with phenylglyoxal (PGO) in place of bromoacetoaldehyde. The gel filtration column (Asahipak GS-320H) was used for separation by a mobile phase consisting of 25 mM sodium citrate buffered (pH 4.0)-150 mM NaCl solution and CH3CN (85:15, v/v) containing 15 mM PGO. The separated analytes reacted with flow through PGO in a reaction coil at 90 degrees C into fluorescent derivatives. Those derivatives were detected fluorimetrically, highly selective and quantitatively. The activity of soluble guanylate cyclase (sGC) in the neuroblastoma N1E-115 cell was measured by tracing the peak height of cGMP synthesized from substrate GTP using this guanine analyzer. The sensitivity of the present method was lower than the radioisotope method. However, our modified method was simpler, safer and quicker than the radioisotope method. Furthermore, this method could trace other guanine compounds simultaneously, allowing measurement of guanine metabolizing enzymatic activity. Therefore, it will be useful for screening of effectors on sGC.

123I-MIBG myocardial scintigraphy in diabetic patients: relationship with 201Tl uptake and cardiac autonomic function.

PURPOSE: To investigate the influence of diabetic myocardial damage (suspected myocardial damage; SMD) diagnosed by 201Tl-SPECT and diabetic cardiac autonomic neuropathy (AN) on myocardial MIBG uptake in patients with non-insulin-dependent diabetes mellitus (NIDDM). SUBJECTS AND METHODS: Eighty-seven diabetic patients divided into four subgroups: 23 with SMD (+) AN (+); 19 with SMD (+) AN (-); 27 with SMD (-) AN (+); 18 with SMD (-) AN (-), and 10 controls were studied. Both planar and SPECT images were taken at 30 minutes (early) and 3 hours (delayed) after 123I-MIBG injection. The heart to mediastinum uptake ratio (H/M) and washout ratio of 123I-MIBG (WR) were obtained from both planar images. On SPECT images, the total uptake score (TUS) was obtained by the 5 point score method by dividing the myocardium into 20 segments on visual analysis. Similarly, the difference between the 201Tl image and the 123I-MIBG image in TUS was taken as the difference in the total uptake score (delta TUS) representing cardiac sympathetic denervation without SMD. RESULTS: On both early and delayed planar images, the mean H/M value in the subgroups of diabetic patients was significantly lower in the SMD (+) AN (+) group than in the control group, but among those subgroups, there was statistically significant difference between the SMD (+) AN (+) and SMD (-) AN (-) groups only on the delayed images. Regarding the WR value, there was no statistically significant difference among subjects. On SPECT image analysis, the diabetic subgroup with AN or SMD had statistically significant lower values for TUS than those of the control group. Among diabetics, there was a statistically significant differences between SMD [+] AN [+] and SMD [-] AN [-] on both early and delayed images. Similarly, the SMD [+] AN [-] group also had significantly lower values than those of SMD [-] AN [-] on early images. Regarding delta TUS, there was a statistically significant differences between AN [+] subgroups and controls. Similarly, the mean value for delta TUS was much higher in AN [+] subgroups than in AN [-] subgroups with or without SMD in diabetes mellitus. CONCLUSION: 123I-MIBG myocardial uptake is affected by both SMD and cardiac autonomic neuropathy. Based on the finding that delta TUS was much higher in AN [+] subgroups and there was no statistically significant difference between SMD [+] AN [+] and SMD [-] AN [+] subgroups, a decrease in myocardial 123I-MIBG uptake might progress independently of SMD.

Localization and functional role of synaptotagmin III in insulin secretory vesicles in pancreatic beta-cells.

Pancreatic beta-cells secrete insulin by Ca2+-triggered exocytosis of insulin-containing large dense-core vesicles. Synaptotagmin is a Ca2+/phospholipid-binding protein and is a good candidate for the Ca2+ sensor for exocytosis of synaptic vesicles in neurons. In the present study, we generated a polyclonal antibody against synaptotagmin III, and found that synaptotagmin III immunoreactivity was present at high levels in insulin-containing pancreatic islet cells and insulin-secreting clonal MIN6 cells. In subcellular fractionations of MIN6 cells, synaptotagmin III was recovered in the vesicular fractions containing both insulin and vesicle-associated membrane protein-2 (VAMP-2), but not in synaptophysin-positive fractions. The secretory vesicles immunoprecipitated by anti-VAMP-2 antibody contained synaptotagmin III and insulin. In addition, treatment of streptolysin-O-permeabilized MIN6 cells with anti-synaptotagmin III antibody significantly inhibited Ca2+-triggered insulin secretion. These results indicate that synaptotagmin III is localized in insulin-containing dense-core vesicles in pancreatic beta-cells, and further strongly suggest that synaptotagmin III is the Ca2+ sensor in the exocytosis of insulin secretory vesicles.

[123I-MIBG lung uptake in patients with diabetes mellitus].

The purpose of this study is to clarify the relationship between 123I-MIBG lung uptake and silent myocardial ischemia (SMI), cardiac autonomic neuropathy (AN) or clinical characteristics. For the quantitative analysis, lung to upper mediastinum uptake ratio (L/M) and heart to upper mediastinum uptake ratio (H/M) were obtained from chest planar image. In addition, both lung washout ratio (%WR-L) and heart washout ratio (%WR-H) were calculated from early and delayed images. Each indices were compared in both diabetic and control groups. Mean values of H/M in diabetes with complication were significantly lower than those of control group. Particularly, AN(+)SMI(+) group showed lowest value. Similarly, mean values of %WR-H in diabetes with complication were significantly higher than those of control group and AN(+)SMI(+) group showed highest value. Although mean value of L/M in each diabetic group was significantly higher than that of control group, there was no statistical significance among each diabetes except AN(+)SMI(-) group on early image. Mean value of %WR-L in AN(+) or SMI(+) group was also significantly higher than that of control group, but there was no statistical significance among each diabetic group. The current study suggested that high pulmonary 123I-MIBG uptake in diabetes was independent of the complication of SMI or AN. Pulmonary endothelial dysfunction related with severity of diabetes mellitus was considered to be the most important factor.

Fusion of insulin receptor ectodomains to immunoglobulin constant domains reproduces high-affinity insulin binding in vitro.

A unique feature of the insulin receptor is that it is dimeric in the absence of ligand. Dimerization of two adjacent transmembrane domain (TMD) alpha helices has been shown to be critical in receptor kinase activation. Moreover, previous work has suggested that the TMD is involved in stabilizing the high-affinity binding site; soluble receptors expressed after simple truncation at the ectodomain-TMD junction have reduced affinity for insulin. To further examine this issue, we have replaced the TMD and intracellular domain of the soluble human insulin receptor (HIRs) with constant domains from immunoglobulin Fc and lambda subunits (HIRs-Fc and HIRs-lambda). Studies of receptor biosynthesis and binding characteristics were performed following transient transfection of receptor cDNAs into human embryonal kidney 293 cells. Each hybrid receptor was initially synthesized as a single chain proreceptor, followed by cleavage into alpha- and beta-Fc or beta-lambda subunits. The majority of secreted protein appeared in the cell medium as fully processed heterotetramer. Fc fragments released from HIRs-Fc by papain digestion and analyzed by nonreducing SDS-polyacrylamide gel electrophoresis were dimeric. Furthermore, dissociation constants for both chimeras were similar to those for the full-length holoreceptor (wild-type receptor, Kd1 = 200 pM and Kd2 = 2 nM; HIRs-Fc, Kd1 = 200 pM and Kd2 = 40 nM; and HIRs-lambda, Kd1 = 200 pM and Kd2 = 5 nM). These results extend previous observations that dimerization of the membrane-proximal ectodomain is necessary to maintain an intact high-affinity insulin-binding site.

Alleviation of cisplatin nephrotoxicity: efficacy of local intra-arterial injection concomitant with hemodialysis.

Although cis-diamminedichloroplatinum (cisplatin) is widely used in the treatment of malignant tumors, the dose administered is limited because of side effects such as nephrotoxicity. The plasma levels of non-protein-bound platinum, which can be removed by dialysis, play an important role in the antitumor effects and the appearance of side effects. Selective intra-arterial injection of cisplatin during concomitant hemodialysis (HD) was performed in patients with gynecological malignancies. At a dose of 100 mg, about three times the non-protein-bound platinum excreted in the urine during the same period was removed by HD. The area under the time-concentration curve of plasma total platinum up to 5 h after intra-arterial injection was reduced by 46%. When doses of 200 or 250 mg were administered concomitantly with HD, the maximum plasma levels were suppressed to about the same degree as when 100 mg were injected without concomitant HD. A definite reduction in the incidence of side effects was seen, and a reduction in the severity of nephrotoxicity was also observed when 100 mg were given with HD. No severe side effects were found even when 200 or 250 mg were administered with concomitant HD. The antitumor effects were not reduced or possibly potentiated, but follow-up is still of short duration. These results indicate that chemotherapy with local intra-arterial injections of cisplatin using concomitant HD not only reduces systemic side effects, in particular nephrotoxicity, but also allows for increased doses at the tumor site and can be applied in patients with renal dysfunction.

[A study on the association between glandular dysplasia and cervical squamous neoplasia in surgical specimens].

To clarify the pathogenesis of cervical adenocarcinoma, we studied the biological properties of glandular dysplasia (GD). The coexistence of squamous neoplasia with cervical adenocarcinoma has been demonstrated. We analyzed the incidence of the coexistence of GD with squamous neoplasia in this study. Materials were surgically removed uterine cervix specimens (n = 142), 52 benign disease cases, 19 squamous dysplasias of the uterine cervix, 66 squamous cell carcinomas of the uterine cervix and 5 cervical adenocarcinomas. Diagnosis of GD was based on the general rules for clinical and pathological management of uterine cervical cancer and Ng's criteria (1983). We divided GD into two types: I: endocervical type, and II: endometrioid type. These fell into subtypes, a (mild) and b (severe) based on the observed degrees of cell atypia. 1. GD coexisted in 17.3% of benign disease cases, 15.8% of squamous dysplasias, 24.2% of squamous cell carcinomas and 100% of cervical adenocarcinomas. 2. GD Ib and IIb were not found in any of the benign disease cases, but were present in 15.8% of squamous dysplasias, 13.6% of squamous cell carcinomas and 100% of cervical adenocarcinomas. If GD was defined as only Ib and IIb, GD coexisted with squamous neoplasia in this study. Our results support the theory that both cervical squamous atypia and cervical glandular atypia are derived from reserve cells.

[Clinical usefulness of GnRH agonist therapy for premenopausal women with uterine leiomyoma].

The purpose of this study was to evaluate the tumor volume reducing effect and the frequency with which menopause is induced in premenopausal women with leiomyoma uteri treated with GnRHa buserelin, 900 micrograms/day for 24 weeks. Twenty-six women, whose average age was 49.7 +/- 2.1 years (Mean +/- SD), were enrolled in this study. Uterine and myoma volume were measured by computed tomography (CT) and transvaginal sonography, respectively. Mean uterine and myoma volume had decreased by 33.7% and 39.9%, respectively at 24 weeks of GnRHa therapy. Nine patients were brought to menopause following the treatment. This rate (34.6%) is significantly higher than that of the age matched control group (11.8%), at eighteen months' observation without GnRHa treatment. We conclude that GnRHa treatment for premenopausal women with uterine leiomyoma causes not only temporary ovarian suppression but also has a strong tendency to induce menopause.

Cross-linking of a B25 azidophenylalanine insulin derivative to the carboxyl-terminal region of the alpha-subunit of the insulin receptor. Identification of a new insulin-binding domain in the insulin receptor.

To identify a site within the insulin receptor ectodomain which forms a binding pocket for B25 Phe and is responsible for initiating conformational changes required for high affinity binding of insulin we have used a novel photoreactive insulin, despentapeptide-(B26-B30) [B25 p-azidophenylalanine-alpha-carboxamide] insulin (APC insulin). This derivative has a highly photoreactive azido group incorporated into the aromatic ring of the B25 phenylalanine amide. APC insulin bound to human insulin receptors overexpressed on a transfected Chinese hamster ovary cell line (P3-A) with an apparent potency of 9-fold relative to that of native insulin and stimulated lipogenesis in rat adipocytes with an average potency equal to porcine insulin. Addition of biotin to the B1 Phe amino group to form despentapeptide-(B26-B30) [B1 (6-biotinylamidocaproyl)phenylalanine B25 p-azidophenylalanine-alpha-carboxamide] insulin derivative (Bio-APC insulin) did not adversely affect receptor-binding affinity and provided a convenient ligand for purification of cross-linked complexes. The efficiency of receptor cross-linking with these reagents was high (70%). To identify the site(s) of cross-linking, the insulin receptor in P3-A cells was first metabolically labeled with various individual 3H-labeled amino acids and then photoaffinity labeled with 125I-Bio-APC insulin, isolated, and digested with Lys-C endoproteinase. The resulting cross-linked peptide fragments were separated by streptavidin-affinity chromatography and sequenced. The smallest identified fragment comprised residues 704-718 of the COOH terminus of the alpha-subunit of the insulin receptor. This B25 Phe cross-linked region of the alpha-subunit lies just upstream of the Exon 11-encoded 12-amino acid COOH-terminal region. Aromatic residues in this predicted alpha-helical region may form a binding pocket for B25 Phe to initiate conformational changes required for stabilizing the high affinity binding state.

Selective impairment of the cytoplasmic Ca2+ response to glucose in pancreatic beta cells of streptozocin-induced non-insulin-dependent diabetic rats.

Pancreatic islets from the streptozocin-induced non-insulin-dependent diabetes mellitus (NIDDM) rat model showed a diminished insulin response to 16.7 mmol/L glucose, but the insulin response to arginine remained intact. To evaluate the importance of intracellular calcium concentration ([Ca2+]i) in the diminished insulin response to glucose, the [Ca2+]i of pancreatic beta cells was investigated using fura-2. Glucose produced heterogeneous responses of [Ca2+]i, which were in beta-cell clusters of both the control and NIDDM groups. Many cells showed initial slight decreases of [Ca2+]i, which were followed by gradual and large increments of [Ca2+]i after glucose stimulation of beta cells in the control group. On the other hand, the increase of [Ca2+]i in response to glucose was markedly diminished in beta cells of the NIDDM group compared with controls. The average lag time to [Ca2+]i elevation of beta cells in the NIDDM group was significantly longer than that of the control group. Arginine produced marked increases of [Ca2+]i, in contrast to the effect of glucose stimulation in the NIDDM group. These results suggest that the diminished and delayed [Ca2+]i increases in beta cells of NIDDM rats in response to glucose stimulation are responsible for the selectively impaired insulin response to glucose in the rat model of NIDDM.