Effects of skyrin, a receptor-selective glucagon antagonist, in rat and human hepatocytes.

Peptidic glucagon antagonists have been shown to lower blood glucose levels in diabetic models (1-3), but attempts to identify small molecular weight glucagon receptor-binding antagonists have met with little success. Skyrin, a fungal bisanthroquinone, exhibits functional glucagon antagonism by uncoupling the glucagon receptor from adenylate cyclase activation in rat liver membranes (1). We have examined the effects of skyrin on cells transfected with the human glucagon receptor and on isolated rat and human hepatocytes. The skyrin used was isolated from Talaromyces wortmanni American Type Culture Collection 10517. In rat hepatocytes, skyrin (30 micromol/l) inhibited glucagon-stimulated cAMP production (53%) and glucose output (IC50 56 micromol/l). There was no detectable effect on epinephrine or glucagon-like peptide 1 (GLP-1) stimulation of these parameters, which demonstrates skyrin's selective activity. Skyrin was also evaluated in primary cultures of human hepatocytes. Unlike cell lines, which are largely unresponsive to glucagon, primary human hepatocytes exhibited glucagon-dependent cAMP production for 14 days in culture (EC50 10 nmol/l). Skyrin (10 micromol/l) markedly reduced glucagon-stimulated cAMP production (55%) and glycogenolysis (27%) in human hepatocytes. The inhibition of glucagon stimulation was a specific property displayed by skyrin and oxyskyrin but not shared by other bisanthroquinones. Skyrin is the first small molecular weight nonpeptidic agent demonstrated to interfere with the coupling of glucagon to adenylate cyclase independent of binding to the glucagon receptor. The data presented in this study indicate that functional uncoupling of the human glucagon receptor from cAMP production results in metabolic effects that could reduce hepatocyte glucose production and hence alleviate diabetic hyperglycemia.

Enhancement of plasminogen binding to U937 cells and fibrin by complestatin.

Plasminogen binds to endothelial and blood cells as well as to fibrin, where the zymogen is efficiently activated and protected from inhibition by alpha 2-antiplasmin. In the present study we have found that complestatin, a peptide-like metabolite of a streptomyces, enhances binding of plasminogen to cells and fibrin. Complestatin, at concentrations ranging from 1 to 5 microM, doubled 125I-plasminogen binding to U937 cells both in the absence and presence of lipoprotein(a), a putative physiological competitor of plasminogen. The binding of 125I-plasminogen in the presence of complestatin was abolished by epsilon-aminocaproic acid, suggesting that the lysine binding site(s) of the plasminogen molecule are involved in the binding. Equilibrium binding analyses indicated that complestatin increased the maximum binding of 125I-plasminogen to U937 cells without affecting the binding affinity. Complestatin was also effective in increasing 125I-plasminogen binding to fibrin, causing 2-fold elevation of the binding at approximately 1 microM. Along with the potentiation of plasminogen binding, complestatin enhanced plasmin formation, and thereby increased fibrinolysis. These results would provide a biochemical basis for a pharmacological stimulation of endogenous fibrinolysis through a promotion of plasminogen binding to cells and fibrin.

Adaptation to sustained high plasma vasopressin in water and electrolyte homeostasis in the rat transgenic for the metallothionein-vasopressin fusion gene.

Prolonged exposure of tissues to a receptor agonist often leads to adaptive changes that limit the subsequent responsiveness of the tissue to the same agonist. Recently, we have generated rats transgenic for the metallothionein I-human arginine vasopressin (AVP) fusion gene (Tg), which produced high plasma AVP with relatively preserved renal water excretion, suggesting that there might be adaptive mechanism(s) for maintaining water and electrolyte homeostasis against chronic AVP oversecretion from the earliest stage of life. In this study, to investigate whether down-regulation of AVP V2 receptor (V2R), which could possibly be caused by long-standing high plasma AVP, participates in this adaptive mechanism(s), non-peptidic V2R antagonist OPC31260 was administered to reverse the down-regulation, and water loading was performed after V2R antagonist treatment had been withdrawn. Additionally, to confirm the down-regulation, Northern blotting analysis for V2R mRNA was carried out. Tg rats showed slightly decreased urine volume and water intake with an equivalent plasma [Na(+)] level (Tg 140.4 +/- 0.6 mEq/l; control 139.3 +/- 0.6 mEq/l) under basal conditions. After water loading using a liquid diet containing zinc, which stimulates the promoter region in the transgene, the urine increase showed only limited suppression with a dramatically increased plasma AVP level and mild hyponatremia (135.8 +/- 1.8 mEq/l) in Tg rats. When diet containing OPC31260 had been provided for 4 days until the day before the start of water loading, antidiuresis and hyponatremia (125.4 +/- 1.mEq/l) were significantly potentiated. V2R mRNA expression in kidney was significantly less in Tg rats than in control rats under basal conditions, and this suppression was restored by OPC31260 treatment to levels comparable with those of control rats. These results suggest that long-standing high plasma AVP causes V2R down-regulation, and it may play an important role in the adaptive mechanism(s) for maintaining water and electrolyte homeostasis in chronically AVP-overexpressing rats.

Overexpression of vasopressin in the rat transgenic for the metallothionein-vasopressin fusion gene.

Arginine vasopressin (AVP) is a major antidiuretic hormone, the overproduction of which causes diluting hyponatremia in humans and is called the syndrome of inappropriate antidiuresis (SIAD). To study physiological changes resulting from AVP overproduction and to develop an animal model of hyponatremia, the human AVP gene was expressed under the control of the metallothionein promoter in transgenic (Tg) rats. Analyses of AVP immunoreactivity (irAVP) in the tissues revealed that the transgene is expressed mainly in the central nervous system. Gel filtration showed that irAVP in the brain and plasma was properly processed AVP. AVP purified from the brains of both Tg and control rats also exerted equal bioactivity to generate cAMP in LLC-PK1 cells. The founder rats did not show any physical or anatomical abnormalities. Under basal conditions, Tg rats had high plasma AVP levels (Tg 13.8 +/- 2.5 pg/ml; control 2.7 +/- 1.2 pg/ml; n=6 in both groups; means +/- S.E.M.), decreased urine volume, and normal plasma [Na(+)]. Hypertonic saline injected i.p. did not affect AVP secretion in Tg rats. In response to a zinc-supplemented liquid diet, plasma AVP decreased in control rats, but increased in Tg rats (Tg 32.7 +/- 2.7 pg/ml; control 1.0+/-0.1 pg/ml; n=6), resulting in hyponatremia (Tg 135.2 +/- 2.5 mEq/l; control 140.8 +/- 0.4 mEq/l; n=6). To our knowledge, this is the first transgenic animal to show diluting hyponatremia. This transgenic rat may therefore provide a useful model in which to investigate various physiological alterations resulting from the oversecretion of AVP which involve SIAD, stress response, behavior, and blood pressure.

Polyamine regulation of the rat pro-opiomelanocortin gene expression in AtT-20 cells.

Polyamines are a ubiquitous group of amines that play diverse biological roles. In the anterior pituitary, intracellular polyamine levels are reported to show diurnal changes, although the biological significance remains to be elucidated. In this study, we examined the effects of polyamines on the transcriptional activity of the rat pro-opiomelanocortin (POMC) gene using AtT20PL, a clone of the AtT20 cell line in which an approximately 0.7 kb of the rat POMC 5' promoter-luciferase fusion gene was stably incorporated. The results showed that three representative polyamines (putrescine, spermidine and spermine) all stimulated POMC promoter activity in a time- and dose-related manner, spermine showing the most potent effect (maximum approximate three-fold increase). This effect was not observed under treatment with actinomycin D, suggesting the effect of polyamine at the transcriptional level. On the other hand, methylglyoxal bis (guanylhydrazone), an inhibitor of polyamine synthesis, showed the opposite effect, further supporting the positive role of intracellular polyamines. Taken together, our findings suggest that polyamines are involved in the regulation of POMC gene expression (especially in terms of diurnal changes) in corticotroph cells. The precise molecular mechanisms of polyamine effects await further research.

Enhancement of plasminogen binding and fibrinolysis by chloropeptin I.

Plasminogen is a zymogen of the fibrinolytic serine protease, plasmin. Plasminogen binds, through its lysine binding sites in the kringle domain, to blood and vascular cells or fibrin, where it is efficiently activated and exerts fibrinolytic activity (1,2). We have recently found that complestatin, a peptide-like metabolite of streptomyces (3,4), enhances plasminogen binding to U937 cells and fibrin, thus potentiating fibrinolysis (5). In the present study, complestatin was found to be converted by an acid treatment to a more active isomer in enhancing plasminogen binding to U937 cells. This isomer was identified to be chloropeptin I, which was recently isolated from a culture of Streptomyces sp. by Matsuzaki et al. as an inhibitor of gp 120-CD4 binding (6). The present paper deals with the stimulation of fibrinolysis by chloropeptin I.

[An MRI and SPECT study of frozen gait without other manifestations of parkinsonism in the elderly].

The authors have sometimes experienced cases of frozen gait without any other manifestations of parkinsonisms in the elderly, so we examined these cases using MR imaging and SPECT imaging. The group with frozen gait (4 cases, aged 78 to 82 years) which failed to respond to L-dopa therapy, had no limb-kinetic apraxia or frontal signs, but did exhibit "kinésie paradoxale". The clinical symptoms of case 4 were consistent with so-called "pure akinesia". CT findings in this group failed to elucidate the pathogenesis of frozen gait. MR imaging of all of the cases except for the case of pure akinesia (case 4) revealed a high signal intensity areas in subcortical lesions (especially the frontal area). Case 4 had only a few spotty high-signal intensity areas. We then compared 6 cases which have the same lesions (on MRI) as cases 1-3 without frozen gait in cases 1-3 using 123I-IMP SPECT. Relatively low accumulation of radionuclide in the frontal area was observed in these cases. Case 4 had a slightly low perfusion area in the frontal lobe. However in the patient treated with L-threo-DOPS, accumulation of radionuclide in the frontal area increased slightly after L-threo-DOPS therapy. It appears that one of the reasons for frozen gait in the elderly is incomplete infarct of the subcortical white matter with a low perfusion area in the frontal cortex. Furthermore, in some cases of frozen gait it is difficult to distinguish between cases with white matter disorders and cases of pure akinesia on the basis of clinical symptoms and CT alone.

[Test production of 60Co sealed sources of high specific activity (author's transl)].

To establish the production technique of 60Co sources with high specific activity, test production was started in 1967 by using JRR-3 and JMTR reactors. Two kinds of cobalt targets, a wafer type (diameter 8.0 mmxthickness 2.3 mm, 1.1g) and a pellet type (diameter 1.0 mmxlength 1.0mm, 6.9 mg) were used. The former was irradiated at a maximum nvt of 4.4x10(20) neutrons per cm2, yielding a maximum specific activity of 8.5 Ci per gram, and the latter at 2.2x10(21) neutrons per cm2, yielding a maximum specific activity of 68.1 Ci per gram, and the total activity of 60Co sources amounted to 4.1 kCi. After the 60Co sources were enclosed in stainless steel capsules, these sealed sources were submitted to wipe and immersion tests to examine surface contamination and leakage. Through the test production, could be obtained much important knowledge such as irradiation method, handling of highly active materials, sources assemblage and remote welding.

Epithelioid malignant schwannoma. A case report.

A case of epithelioid malignant schwannoma (EMS) is reported. The tumor arose in the left radial nerve at the axillary fossa of a 65-year-old male. A few months after resection of the primary axillary tumor, several intrapulmonary metastases appeared. Microscopically, the primary tumor showed highly cellular areas of polygonal or rounded cells, resembling lymphoma or melanoma, while the metastatic tumors revealed cord formation or rows, resembling carcinoma. Immunohistochemical studies showed that some of these tumor cells contained S-100 protein. Ultrastructurally, these tumor cells revealed delicate cytoplasmic projections, which contained bundles of microfilaments. However, the tumor cells did not have melanosomes. Varying amounts of basal lamina material surrounded the tumor cells. From the above features, we obtained a correct diagnosis of EMS.

Development of ergometer attachment for power and maximum anaerobic power measurement in swimming.

The ergometer can be a versatile means of measurement if attachments are developed for special purposes or if attachment is developed for multi-uses. In this study, an ergometer attachment for the measurement of power was designed and the measurement of power and the maximum anaerobic power in swimming was tested. A rotation drum was attached to one pedal of an ergometer. The rotation of this drum was synchronized with the rotation of the pedal. One end of a wire for a traction by a swimmer was connected to the drum. The other end of the wire was attached to a belt around the waist of a swimmer. The swimmer swam at full strength, thus causing the drum to rotate. The rotational velocity of the drum was detected as voltage by a magnetic permanent motor and transformed to wire tractional velocity; this velocity was equal to swimming velocity. The wire tension (= load) was controlled by a load adjusting lever of the ergometer. This wire tension was equal to the load which was added to the swimmer. The power calculation was based on a curved regression equation approximated from the load and the velocity. This equation was shown as follows; (P + a) (v + b) = (P0 + a)b or its development (P + a)v = b(P0 - P) and provided that P: force or load, v: swimming velocity, P0: maximum tractional force, a and b: constants. This ergometer attachment made it possible to measure and evaluate the power and the maximum anaerobic power in swimming with ease and at low cost. Measurement and evaluation are easily performed using the system, which is just one example of the possible applications of the ergometer.

[A family with multiple endocrine neoplasia type I presenting prolactinoma, Zollinger-Ellison syndrome and hyperparathyroidism].

A family of multiple endocrine neoplasia type I with five confirmed cases in three generations is described. All of them have primary hyperparathyroidism in common. The propositus is 51 year-old male. After a year of symptoms of gastroduodenal ulcer, he was found to have elevated levels of serum gastrin and PTH. The serial imaging studies revealed a tumor in pancreatic head, and Zollinger-Ellison syndrome was diagnosed. The gastrin level was reduced into normal range after extirpation of the tumor, but post surgical elevation of Calcium put the patient under parathyroidectomy, which normalized serum PTH and Calcium levels. His two sisters (I and II), the mother of them, and the daughter of sister I, had neither signs nor symptoms until family study showed hypercalcemia in all. Sister I is a 54 year-old female with enlarged parathyroid. The hyperparathyroidism is of chemical type, but no other endocrinological abnormality is found. The Calcium level decreased after parathyroidectomy. Sister II is a 56 year-old female. The only sign was galactorrhea. Serum PTH and Calcium decreased after parathyroidectomy. The prolactinoma was diagnosed by the increased prolactin levels and enhanced mass lesion in sella turcica. Her serum prolactin levels is now within normal range since she is on bromocryptine. The mother of the above three siblings and the daughter of the sister I are now under further study.

Involvement of upstream open reading frames in regulation of rat V(1b) vasopressin receptor expression.

The V(1b) vasopressin receptor, expressed mainly in the corticotroph of the anterior pituitary, mediates the stimulatory effect of vasopressin on ACTH release. To clarify the regulation of receptor expression, we cloned, sequenced (up to approximately 5 kb from the translation start site), and characterized the 5'-flanking region of the rat V(1b) receptor gene. We identified the transcription start site by amplification of cDNA ends and found a new intron within the 5'-untranslated region (5'-UTR) by comparing the sequence with that of cDNA. We then confirmed that the obtained promoter indeed has transcriptional activity by use of the luciferase reporter in AtT-20 mouse corticotroph cells. Interestingly, there were five short upstream open reading frames (uORFs) located within the 5'-UTR that were found to suppress V(1b) expression. Subsequent mutational analyses showed that the two downstream uORFs have an inhibitory effect on expression in both homologous and heterologous contexts. Furthermore, the inhibition did not accompany a parallel decrease in mRNA, suggesting that the suppressive effect occurs at a level downstream of transcription. Taken together, our data strongly suggest that the expression of the V(1b) receptor is regulated at the posttranscriptional as well as transcriptional level through uORFs within the 5'-UTR region of the mRNA. Whether the uORF-mediated regulation of V(1b) expression is functionally linked to any intracellular and/or extracellular factor(s) awaits further research.