Arginine vasopressin neuronal loss results from autophagy-associated cell death in a mouse model for familial neurohypophysial diabetes insipidus.

Familial neurohypophysial diabetes insipidus (FNDI) characterized by progressive polyuria is mostly caused by mutations in the gene encoding neurophysin II (NPII), which is the carrier protein of the antidiuretic hormone, arginine vasopressin (AVP). Although accumulation of mutant NPII in the endoplasmic reticulum (ER) could be toxic for AVP neurons, the precise mechanisms of cell death of AVP neurons, reported in autopsy studies, remain unclear. Here, we subjected FNDI model mice to intermittent water deprivation (WD) in order to promote the phenotypes. Electron microscopic analyses demonstrated that, while aggregates are confined to a certain compartment of the ER in the AVP neurons of FNDI mice with water access ad libitum, they were scattered throughout the dilated ER lumen in the FNDI mice subjected to WD for 4 weeks. It is also demonstrated that phagophores, the autophagosome precursors, emerged in the vicinity of aggregates and engulfed the ER containing scattered aggregates. Immunohistochemical analyses revealed that expression of p62, an adapter protein between ubiquitin and autophagosome, was elicited on autophagosomal membranes in the AVP neurons, suggesting selective autophagy induction at this time point. Treatment of hypothalamic explants of green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) transgenic mice with an ER stressor thapsigargin increased the number of GFP-LC3 puncta, suggesting that ER stress could induce autophagosome formation in the hypothalamus of wild-type mice as well. The cytoplasm of AVP neurons in FNDI mice was occupied with vacuoles in the mice subjected to WD for 12 weeks, when 30-40% of AVP neurons are lost. Our data thus demonstrated that autophagy was induced in the AVP neurons subjected to ER stress in FNDI mice. Although autophagy should primarily be protective for neurons, it is suggested that the organelles including ER were lost over time through autophagy, leading to autophagy-associated cell death of AVP neurons.

A deleted form of human hepatocyte growth factor stimulates hepatic lipogenesis and lipoprotein synthesis in rats.

Here we report the effect of the recombinant human deleted form of hepatocyte growth factor (dHGF) on lipid metabolism in rats. In primary cultured rat hepatocytes, dHGF accelerated incorporation of [(14)C]acetate into cellular lipids in a concentration-dependent manner. dHGF also increased the gene expression and enzyme activity of glucose-6-phosphate dehydrogenase, a rate-limiting enzyme of the pentose phosphate pathway, in hepatocytes. These results suggest that dHGF stimulates hepatocyte lipogenesis through upregulation of the pentose pathway and NADPH formation. Injection of dHGF into normal rats induced elevation of the serum triglyceride, phospholipid and cholesterol levels dose-dependently and in the same time course as the liver growth. dHGF injections stimulated the [(14)C]acetate incorporation into the liver lipids, but not into the adipose tissue nor the small intestine. Serum very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) levels were elevated by dHGF injections. [(14)C]Leucine incorporation into VLDL and LDL was also increased by dHGF injections. In rats with alcohol-induced fatty livers, dHGF treatment markedly diminished the accumulated liver triglyceride, while elevating serum lipid concentrations. The present results indicate that dHGF stimulates exclusively hepatic lipogenesis and increases serum lipoprotein levels in rats.

Tri-iodothyronine and a deleted form of hepatocyte growth factor act synergistically to enhance liver proliferation and enable in vivo retroviral gene transfer via the peripheral venous system.

Retroviral vectors integrate into the target cell genome in a stable manner and therefore offer the potential for permanent correction of the genetic diseases that affect the liver. These vectors, however, usually require cell division to occur in order to allow provirus entry into the nucleus. We have explored clinically acceptable methods to improve the efficiency of retroviral gene transfer to the liver, which avoid the need for liver damage. Tri-iodothyronine (T3) and recombinant hepatocyte growth factor have previously been used to induce hepatocyte proliferation in rat livers and allow in vivo retroviral gene transfer. We investigated the combined effects of these growth factors, with their differing mechanisms of action, on hepatocyte proliferation in vivo and assessed their effectiveness in priming cells for retroviral gene transfer. During the phase of hepatocyte proliferation retrovirus was administered via either the portal or tail vein. Acting synergistically, T3 and a truncated form of recombinant hepatocyte growth factor (dHGF) induced 30% of hepatocytes in normal rat liver to enter DNA synthesis at 24 h. This increased proliferation enabled the liver to be transduced in vivo by retroviral vectors via either the portal or peripheral venous system, achieving transduction efficiencies of 6.9 +/- 1.6% and 4.3 +/- 0.4% respectively. Thus, the liver can be simply and conveniently transduced in vivo with integrating vectors, introduced via the peripheral venous system during a wave of growth factor-induced proliferation, pointing the way to clinically applicable gene transfer techniques.

Sequence analysis of RNA-2 of different isolates of broad bean wilt virus confirms the existence of two distinct species.

The complete nucleotide sequence of RNA-2 from a Japanese isolate IP of broad bean wilt virus (BBWV) was determined. The sequence encodes a single large polyprotein, which contains a putative movement protein and two coat proteins (CPs). The 3'-terminal sequences of RNA-2 were also determined for three other Japanese isolates and two ATCC isolates (PV132 and PV176) of BBWV. The CPs of the four Japanese isolates share 86.8-98.0% amino acid sequences homology with one another and 88.3-96.5% with those reported for the isolate PV131 (BBWV-2). However, they have only 57.9-66.2% homology with those of PV132 and PV176 (BBWV-1).

Effects of the deleted form of hepatocyte growth factor on serum hyaluronate levels in rats with liver cirrhosis.

Effects of the deleted form of hepatocyte growth factor (dHGF) on serum hyaluronate levels, an index for liver cirrhosis, were studied in rats. The levels of serum hyaluronate increased in rats with dimethylnitrosamine- or carbontetrachloride-induced cirrhotic liver with prolongation of prothrombin time, which indicates disorder of liver function. Daily intravenous injection of dHGF reduced the elevated serum hyaluronate levels with improvement of the prolonged prothrombin time. These results suggest that the amelioration of hepatic function disorder by dHGF leads to a reduction of the increased serum hyaluronate levels.

Preventive effects of the deleted form of hepatocyte growth factor against various liver injuries.

The effects of a naturally occurring deleted form of hepatocyte growth factor (HGF) on hepatic disorder were studied in various models of hepatic failure. The pretreatment of rats and mice with the deleted form of HGF prevented the liver injuries and coagulopathy induced by endotoxin, dimethylnitrosamine and acetaminophen and reduced the mortality due to hepatic dysfunction induced by these hepatotoxins. The concurrent administration of the deleted form of HGF also prevented the liver injury and hepatic fibrosis in mice treated with alpha-naphthylisothiocyanate and in rats treated with dimethylnitrosamine. Moreover, the deleted form of HGF normalized the results of the bromosulphalein-clearance test and ameliorated jaundice in rats with periportal cholangiolitic hepatopathy induced by alpha-naphthylisothiocyanate. The deleted form of HGF also reversed the coagulopathy in rats with hepatic disorder induced by dimethylnitrosamine or by 70% resection of cirrhotic liver (induced by carbon tetrachloride). In Long Evans cinnamon rats receiving vehicle, 20 out of 21 animals died within 4 days after the onset of jaundice. After infusion of the deleted form of HGF for 4 days, 7 out of 20 Long-Evans cinnamon rats survived. These results indicate that the deleted form of HGF could have therapeutic potency in patients with severe hepatic failure.

Deleted form of hepatocyte growth factor (dHGF) increases the number of platelets in rats with liver cirrhosis.

The effect of the deleted form of hepatocyte growth factor (dHGF) on thrombopoiesis was studied in rats. When normal rats were injected with dHGF (0.5 mg/kg i.v. twice a day), the number of platelets increased to about 1.5-fold the initial level. In addition, the treatment with dHGF (0.5 mg/kg i.v. twice daily) significantly increased the number of platelets in rats with liver cirrhosis induced by carbon tetrachloride and phenobarbital. When dHGF was given to rats at a dose of 0.05 or 0.5 mg/kg from the beginning of the induction of dimethylnitrosamine liver cirrhosis to day 28, dHGF dose-dependently ameliorated thrombocytopenia and completely prevented it at a dose of 0.5 mg/kg. These results indicate that dHGF may be applicable to the treatment of thrombocytopenia associated with liver cirrhosis.

Identification of casoxin C, an ileum-contracting peptide derived from bovine kappa-casein, as an agonist for C3a receptors.

Casoxin C (Tyr-Ile-Pro-Ile-Gln-Tyr-Val-Leu-Ser-Arg) is a bioactive peptide that was isolated from a tryptic digest of bovine kappa-casein as an anti-opioid peptide in longitudinal strips of guinea pig ileum. Casoxin C also evokes contraction of the ileal strips, and we found that this process was biphasic with rapid and slow components. The contractile profile was very similar to that of human complement C3a(70-77), which is the COOH-terminal octapeptide of C3a and has, although less potent, qualitatively the same biological activities as C3a. Casoxin C also has homology with C3a(70-77). The rapid contraction was mediated by histamine release and the slow contraction was mediated by a prostaglandin E2-like substance, judging from the effects of various pharmacological inhibitors and antagonists on the ileal contraction. Casoxin C had affinity for C3a receptors (IC50 = 40 microM) in the radioreceptor assay. In addition, casoxin C showed phagocyte-stimulating activities. Casoxin C is therefore the first milk-derived peptide identified, that acts through complement C3a receptors.

Antifibrogenic effect of a deletion variant of hepatocyte growth factor on liver fibrosis in rats.

Hepatic fibrosis, which may lead to cirrhosis, is associated with most chronic liver diseases. Current therapies for hepatic fibrosis are, however, generally ineffective. In this report we assessed the efficacy of the treatment of hepatic fibrosis with a naturally occurring deletion variant of hepatocyte growth factor (dHGF). The administration of dHGF increased liver weight and suppressed the increase of hepatic collagen content in rats treated with dimethylnitrosamine (DMN) to induce hepatic fibrosis. Furthermore, dHGF exerted its mitogenic and antifibrogenic activities even after the liver fibrosis had been established with DMN. Northern blot analysis showed that dHGF suppressed the increase of messenger RNA (mRNA) levels of procollagen alpha 2(I), alpha l(III), alpha 1(IV), transforming growth factor beta 1 (TGF-beta1), desmin (a marker of hepatic lipocytes), and alpha-smooth muscle (sm)-actin (a marker of activated hepatic lipocytes). In addition to suppressing the elevated TGF-beta1, mRNA level in hepatic fibrosis, dHGF had a potent ability to decrease TGF-beta1 mRNA level even in a normal liver. Immunohistochemical analysis revealed that desmin-positive cells and alpha-sm-actin-positive cells were increased in the hepatic fibrosis, whereas neither cells were seen in livers of DMN-treated rats given dHGF. We conclude that dHGF prevents and improves the DMN-induced hepatic fibrosis in rats by reducing mRNA levels of procollagens and TGF-beta1, by inhibiting an activation of hepatic lipocytes, and by stimulating liver regeneration. dHGF may be useful for and applicable to the treatment of fibrosis in chronic liver diseases.

Amelioration of disordered hepatic protein synthesis by the deleted form of hepatocyte growth factor in models of liver failure in rats.

Because the liver plays an important role in protein synthesis and cholesterol metabolism and reductions in these functions are observed in almost all hepatic disorders, the effects of the deleted form of hepatocyte growth factor (dHGF) on disordered hepatic protein synthesis were studied in various liver-injured rat models using Wistar male rats. In the 70% hepatectomized rats, plasma clotting time was prolonged and the serum level of total protein and the liver protein content were decreased. The treatment of the animals with dHGF (100-500 micrograms kg-1, i.v., twice daily) ameliorated these parameters at 48 or 72 h. The administration of carbon tetrachloride or D-galactosamine to hepatectomized rats induced a marked prolongation of plasma clotting time and hypoproteinaemia. In the animals treated with dHGF (500 micrograms kg-1, i.v., twice daily) these parameters were rapidly reversed compared with those of control groups. In a hepatocellular necrosis model induced by dimethylnitrosamine, the plasma clotting time was extremely prolonged, and liver protein content, serum total protein, albumin, HDL-cholesterol (as an index of lipoprotein) and plasma lecithin-cholesterol acyltransferase activity severely reduced. In this severely injured model, dHGF (5-500 micrograms kg-1, i.v., twice daily for 28 days) dose-dependently prevented the loss of liver protein content and improved the disordered plasma coagulability and serum protein levels. These results suggest that dHGF is useful for ameliorating the disorders in hepatic functions such as protein synthesis.

[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.

Mixed mesodermal tumor of the ovary: immunohistochemical study with histogenetic consideration.

The clinical, histological and immunohistochemical features of three cases of ovarian mixed mesodermal tumor (MMT) were examined. The epithelial component was serous papillary cystadenocarcinoma in case 1 and 3, and endometrioid adenocarcinoma in case 2. In case 1, undifferentiated adenocarcinoma was also seen. The mesenchymal component was fibrosarcomatous and chondrosarcomatous in case 1 and 2. In case 3, only fibrosarcomatous area was seen. No endometriosis was observed. Immunohistochemically, the epithelial component showed positivity for epithelial membrane antigen in all three cases. S-100 protein was positive in two cases with chondrosarcomatous differentiation. The fibrosarcomatous area showed positivity for vimentin in all three cases. However desmin, myosin and myoglobin were negative. The antibodies thought to be epithelial or mesenchymal markers unexpectedly reacted positively in some cells; for example, EMA was positive in fibrosarcomatous and chondrosarcomatous cells. Therefore, it was speculated that because the undifferentiated tumor cells had a biphasic character, MMT might originate from immature multipotential cells of surface epithelium and subcapsular connective tissue of the ovary.

[MHC class I antigen expression by molar trophoblast].

It is an issue for debate why molar tissues are not rejected by an immunologically potent host, since all genes in complete moles and 2/3 genes in partial moles are considered to be paternally derived. Molar trophoblasts are in direct contact with host cells, and therefore HLA expression by these cells may hold the key to the elucidation of the immunological reaction between molar tissues and the host. It has been reported that villous trophoblasts are negative and extravillous trophoblasts are positive for HLA-A, B,C, but the expressed HLA-A,B,C molecule has been noted to lack their polymorphic determinants. We analyzed the reactivity of two monoclonal antibodies to a monomorphic determinant of HLA-A,B,C (W6/32 and Cappel anti-HLA-A,B,C) with molar trophoblasts, using three uteri containing complete moles and two containing partial moles. The reactivity was examined by an indirect immunoperoxidase method. The staining patterns were almost identical in complete moles and partial moles. Villous trophoblasts showed a negative reaction with both antibodies. On the other hand, extravillous trophoblasts exhibited intense staining for W6/32 and negative staining for Cappel anti-HLA-A,B,C, which may suggest that the expression of a constant region as well as a variant region of HLA-A,B,C molecule is incomplete.

[Cytomorphological reappraisal of choriocarcinoma cells--a proposal of a four cell pattern].

It is generally accepted that choriocarcinoma is composed of two cell types: syncytiotrophoblast (ST)-like cells and cytotrophoblast (CT)-like cells. In normal and molar pregnancy, there is another population of trophoblast in the cell column, the decidua and so forth. They are designated intermediate trophoblast (IT) and multinuclear IT. It remains to be clarified whether IT-like cells and multinuclear IT-like cells are observed in choriocarcinoma. In the present study, choriocarcinoma cells were reappraised cytomorphologically in three cases of uterine choriocarcinoma and additional three cases of metastatic choriocarcinoma. The results were as follows: 1. Choriocarcinoma cells could be classified into four cell types: ST-like cells, CT-like cells, IT-like cells and multinuclear IT-like cells. 2. IT-like cells predominated in metastatic lesions as compared with primary lesions. It seemed that choriocarcinoma shows both differentiations which take place in villous and extravillous trophoblasts of normal and molar pregnancy. IT-like cells were found to infiltrate into vessel walls, which, together with the predominance of IT-like cells in metastatic lesions, suggests that IT-like cells contribute greatly to the hematogenous metastasis in choriocarcinoma. The presence of ST-like cells, CT-like cells and multinuclear IT-like cells in metastatic lesions may indicate a germinative nature of IT-like cells.

Amino acid neurotransmitters in iron-induced epileptic foci of rats.

Injection of iron salts into the rodent cortex has been shown to cause chronic or recurrent seizures. Amino acid levels in the cerebral cortex were examined 1, 3, 6, 9, 24, 48 hours and 4 weeks after an injection of ferric chloride solution to the left sensory motor cortex of rats. Aspartate level decreased 9 and 24 hours after the injections with ferric chloride. No significant change was observed in glutamate level, though glutamine level decreased 3 and 48 hours after the injection. GABA level decreased 6 hours after the injection. On the contrary, alanine and glycine levels increased 1 and 24 hours, and 24 hours after the injection, respectively. These results suggest that these amino acid neurotransmitters are involved in the acute seizure mechanism and in the process of chronic focus formation in the iron-induced epilepsy of rats.

Esophagocardioplasty with gastric patch in the treatment of achalasia.

Result of operative treatment in 196 cases of achalasia was reviewed. The procedures employed include cardiolysis, Heller's extramucosal myotomy, Wendel's cardioplasty, Heyrovsky's esophagocardiostomy, with and without pyloroplasty, cardiac resection and esophagocardioplasty with gastric patch. Follow-up study on 166 cases revealed that the result was classified as good in 99 cases or 59.6 per cent and improved in 46 cases or 27.7 per cent. Overall satisfactory result was obtained in 87.3 per cent. When the result was broken down to groups following three classifications, i.e. according to X-ray, endoscopic and manometric findings, the interesting correlation emerged. Heller's myotomy and esophagocardioplasty with gastric patch gave best results in early stage of achalasia, while in later stage the latter procedure seems to be the operation of choice.