Matrix metalloproteinase 2 improves the transplanted adipocyte survival in mice.

BACKGROUND: Fat tissue is a common material for autologous transplantation in plastic and reconstructive surgery. Basic fibroblast growth factor (bFGF) ameliorates the fat graft survival. A transplantation model has shown the gene expression of matrix metalloproteinases (MMPs) to increase in adipocytes. The aim of this study is to investigate the role of MMPs in the amelioration of survival by bFGF. MATERIALS AND METHODS: 3T3-L1 adipocytes were incubated with or without 10 microg mL(-1) bFGF for 8 h in the presence or absence of the MMP inhibitor GM6001, vascular endothelial growth factor (VEGF), MMP-2 or anti-bFGF antibody to study the effect of bFGF on MMP-2 mRNA expression, MMP-2 activity, fat accumulation or 2-deoxyglucose uptake. Collagen sheets containing l x l0(7) adipocytes with or without bFGF in the presence or absence of GM6001 were subcutaneously transplanted into mice, and the appearance, histology, mRNA expression and fat accumulation of the grafts were analysed 4 weeks after transplantation. RESULTS: The MMP-2 expression was drastically induced by bFGF among MMPs in 3T3-L1 adipocytes. MMP-2 accelerated fat accumulation, peroxisome proliferator-activated receptor gamma (PPAR gamma) mRNA expression, and glucose uptake to an extent similar to those induced by bFGF, respectively. The bFGF-induced increases were inhibited by the blocking of MMP-2. The transplantation of adipocytes into mice showed that bFGF ameliorates the appearance and fat accumulation, as well as mRNA expression in grafts. These effects were almost or partly inhibited by a MMP blockade. CONCLUSIONS: MMP-2 may be involved in the mechanism by which bFGF ameliorates the survival of fat grafts.

In vivo transfer of hepatocyte growth factor gene accelerates proliferation of hepatic oval cells in a 2-acetylaminofluorene/partial hepatectomy model in rats.

To clarify the effect of hepatocyte growth factor (HGF) on proliferation of hepatic oval cells, we transferred HGF gene into liver of the Solt-Farber rat model. Male Fisher 344 rats were infected with a recombinant adenovirus carrying the cDNA for HGF (pAxCAHGF) from tail vein. HGF mRNA showed its peak at 4 days, and diminished thereafter. The total and proliferating cell nuclear antigen-positive hepatic oval cells were significantly elevated in HGF-transferred rats, in which stem cell factor and c-kit mRNA increased at each time point. Our results suggest that in vivo transfer of the HGF gene into liver accelerates proliferation of hepatic oval cells in the Solt-Farber model in rats.

Human chorionic gonadotropin beta-core fragment is present in the human placenta.

The presence of hCG beta-core fragment (beta-core) in the human placenta has been controversial. To clarify its presence in the villous tissue, first, we developed an enzyme immunoassay which is highly specific for beta-core. Then, we investigated the presence of beta-core immunoreactivity in the supernatants of placental organ culture and those of primary culture of trophoblasts as well as in the placental extracts at different stages of gestation. The immunoreactivity of beta-core was demonstrated in each sample, and the amount was at least 5% of intact hCG immunoreactivity. Immunohistochemical analysis of the placental tissue showed localization of beta-core immunoreactivity to the syncytiotrophoblast. Western blot analysis of the supernatants of organ culture as well as urine samples of pregnant women showed two major bands with molecular weight of approximately 15000. On the other hand, beta-core immunoreactivity in the pregnant sera ranged from 0.1 to 0.19% of intact hCG immunoreactivity. These results suggest the presence and possible secretion of beta-core fragment in the human placenta, and its immunoreactivity in the serum is likely to be reduced due to its short half life in the maternal blood.

Fatal sepsis associated with acute pancreatitis caused by Moraxella catarrhalis in a child.

We describe a 4-year-old boy with Cornelia de Lange syndrome who died of septic shock caused by Moraxella catarrhalis bacteremia. At autopsy there was evidence of acute hemorrhagic pancreatitis with abscesses. Gram-negative diplococci were seen histologically in the abscesses and pancreatic ducts.

Rapid increase in plasma nitrite concentration following intravenous administration of nipradilol.

Nipradilol, a beta-adrenoceptor antagonist with vasodilator activity, has a structure which contains a NO2 group. In anesthetized dogs, the time course of the systemic vasodilation and plasma nitrite (NO2-) concentration was studied following intravenous administration of nipradilol (1 mg/kg). A fall in systemic vascular resistance was observed at 1 min, which was rapidly followed by a significant increase in the plasma NO2- concentration. It is indicated that nipradilol exerts systemic vasodilatation via nitric oxide (NO) release in vivo.

High forskolin production in hairy roots of Coleus forskohlii.

Hairy roots of Coleus forskohlii were induced by infection with the Agrobacterium rhizogenes MAFF 03-01724 strain. Growth and forskolin production of two hairy root clones cultured in various liquid media were examined. Hairy root clone B9 grew well in woody plant liquid medium and showed a high forskolin yield (ca. 1.3 mg/ 100 ml flask) after 5 weeks of culture. The time course of growth and forskolin production of the clone B9 cultured in woody plant liquid medium was also examined. Rapid growth started at week 2 and continued until week 5. The highest forskolin yield (ca. 1.6 mg/100 ml flask) was obtained at week 5. Productivity was much higher than that previously reported.

[A case of aortoduodenal fistula following radiotherapy of retroperitoneal metastatic disease].

A new case of aortoduodenal fistula was added to the five cases previously reported in the literature, in which malignancy and/or its treatments could be implicated. This 67 year-old woman, six years previously had been placed on a therapy including irradiation on the pelvis for cancer of uterine cervix. For this time she underwent a radiotherapy completed in a total dose of 55.6 Gy combined with hyperthermia and chemotherapy for retroperitoneal metastatic disease with excellent response. Three months later she had hematemesis followed by melena and deteriorated to hemorrhagic shock. Emergent aortography detected contrast extravasation from the aorta with subsequent opacification of the duodenum, and immediate intraaortic balloon occlusion was done, but she died soon thereafter. Postmortem examination revealed the fistula from the aorta just above the bifurcation to a 2 by 1.5 cm. area of the posterior wall of the third portion of the duodenum. Accentuated arteriosclerosis in locally irradiated portion of the aorta, obstruction of small arteries from organized thrombus and hyaline necrosis in the wall of the fistulous tract were defined without evidence of tumor invasion. Based upon the findings of the patient reported herein, radiation might be another possible etiologic factor in aortoduodenal fistula, as well as tumor invasion per se.

NAD(P)H-dependent 6'-deoxychalcone synthase activity in Glycyrrhiza echinata cells induced by yeast extract.

The crude extract prepared from Glycyrrhiza echinata cells treated with yeast extract catalyzed the formation of liquiritigenin (5-deoxyflavanone) and isoliquiritigenin (6'-deoxychalcone) in addition to naringenin (5-hydroxyflavanone) when incubated with 4-coumaroyl-CoA and malonyl-CoA in the presence of high concentrations (0.1 mM or higher) of NADPH. Incubation without NADPH, or with low concentrations (0.01 mM or lower), gave only naringenin as a reaction product. With NADH (1 mM), the major product was naringenin accompanied by a small quantity of liquiritigenin. The initial product of the assay with 1 mM NADPH was isoliquiritigenin, indicating a reaction catalyzed by 6'-deoxychalcone synthase (DOCS). Subsequent formation of liquiritigenin was attributed to the presence of chalcone isomerase in the crude extract. The results constitute the first demonstration in vitro of DOCS activity which, in G. echinata cells and other leguminous plants, is involved in the biosynthesis of retrochalcone and 5-deoxyisoflavonoid-derived phytoalexins.

Stimulation of chalcone synthase activity by yeast extract in cultured Glycyrrhiza echinata cells and 5-deoxyflavanone formation by isolated protoplasts.

Chalcone synthase activity catalyzing the formation of naringenin (5-hydroxyflavanone) was detected in cell suspension cultures of Glycyrrhiza echinata. This activity rapidly increased by treatment of the cells with yeast extract, while non-treated cells showed a constant low activity. Isolated G. echinata protoplasts accumulated retrochalcone (echinatin) and its biosynthetic intermediate (licodione) during 24 h of culture. When the protoplasts were incubated with [(14)C(U)]phenylalanine, liquiritigenin (5-deoxyflavanone) was transiently labeled, indicating the induction of 6'-deoxychalcone synthase. The formation of liquiritigenin, in addition to naringenin, was observed when the crude extracts from the protoplasts were assaved for CHS activity.

Effect of rebamipide on acetic acid-induced gastric ulcer in rats: involvement of hepatocyte growth factor.

BACKGROUND: Rebamipide is used clinically as an anti-ulcer agent, especially in Japan. The major mechanisms of rebamipide include prostaglandin induction and free radical scavenging. Since prostaglandins are inducers of hepatocyte growth factor (HGF), we examined the effect of rebamipide on the expression of HGF, c-met, cyclooxygenase-2 (Cox-2) and subtype of the prostaglandin E2 receptor (EP2) in acetic acid-induced gastric ulcer, a model of human ulcer. METHODS: Ninety-six male Fisher rats were used in the experiments. Gastric ulcers were produced by injecting 50 microl of 20% acetic acid into subserosa of the border between the fundic and antral gland areas. The rats of the rebamipide group were fed a diet containing 60 mg kg(-1) day(-1) rebamipide and killed on days 10, 30, 60, 90, 120 and 150 after ulceration. Reverse transcription polymerase chain reaction of HGF, c-met, Cox-2 and EP2 gene and immunohistochemistry of proliferating cell nuclear antigen (PCNA) were performed. RESULTS: In the rebamipide group, gastric ulcer index was significantly smaller than in the control group at each time-point except at 10 days (P < 0.05, each); up-regulation of HGF, c-met, Cox-2 and EP2 mRNA was also observed. The mRNA level of HGF was significantly correlated with that of Cox-2 and EP2 (P < 0.05, each). The PCNA-labelled epithelial cells in the rebamipide group were also greater than in the control group on days 10, 30, 90 and 120 (P < 0.05, each). CONCLUSION: The study suggests that rebamipide has anti-ulcerative effects on gastric mucosal cells via up-regulation of HGF, c-met, Cox-2 and EP2.

Immobilization of ultra-thin layer of monoclonal antibody on glass surface.

When preparing an affinity column and a biosensor, it is desirable to immobilize a unimolecular layer of pure protein on a matrix. In this work, we tried to immobilize a monoclonal antibody on a surface of a glass test-tube as a model, to confirm the stability of this ultra-thin layer by an enzyme immunoassay, and to estimate the thickness of the layer on a slide glass by Fourier transform infrared reflection spectrometry. A new test-tube was washed and dried. The tube was filled with 5% 3-aminopropyltriethoxysilane. The 3-aminopropylsilylated surface was treated with glutaraldehyde and 5.6.10(-2) mg/ml solution of a normal mouse monoclonal antibody. The Schiff base between glutaraldehyde and the antibody was further reduced with 7.9.10(-3)% NaBH4. The tube was washed with 0.05% Tween 20 to block non-specific binding. The antibody immobilized on the surface was measured by an enzyme immunoassay based on a reaction of anti-mouse immunoglobulin G labelled with alkaline phosphatase, with which p-nitrophenol was produced from p-nitrophenylphosphate as a substrate. Meanwhile, various amounts of the antibody were immobilized on slide glasses in the same manner. The antibody on each surface was measured by Fourier transform infrared reflection spectrometry. The antibody immobilized under the final conditions was detectable by the enzyme immunoassay, and stable at 4 degrees C for ten days. The antibody on the slide glass was a unimolecular layer, as judged from the Fourier transform infrared spectra referred to -CONH- band semiquantitatively. Thus, we found the optimal conditions for immobilizing an ultra-thin layer of the monoclonal antibody on the glass surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Systemic management of cerebral edema based on a new concept in severe head injury patients.

Cerebral hypothermia treatment of critical brain injury patients was studied based on the management and control of cerebral thermo-pooling, synaptic excitation, hypermetabolic demand, and the systemic critical condition of the metabolic reserve. The initial pathophysiological changes after trauma included a progressive increase in brain tissue temperature. Such cerebral thermo-pooling, which reached a maximum of 43.8 degrees C, can change or damage the vascular proteins directly. The brain tissue temperature was influenced by four factors: 1. the cerebral metabolism, 2. the systemic excess energy metabolism, 3. the CPP that carries the systemic energy to the brain tissue, and 4. the cerebral blood flow that leads to washout of brain tissue temperature. Mild cerebral hypothermia (32-33 degrees C) managed by the whole body compartment cooling technique in the critical conditions of diffuse brain injury patients (GCS < 4) produced a good recovery in 8 of 10 patients. Continuous monitoring of the jugular venous oxygen saturation and BTT/TMT was effective for evaluating cerebral ischemia and oxygen metabolic disturbances even during cerebral hypothermia treatment.

Reconstruction of adactyly for 4th and 5th toes: the study of foot pressure.

Analysis of foot pressures in cases of reconstructed adactyly of the lateral toes is presented. Two cases with adactyly of the 4th and 5th toes in which new toes were created by cross-knee tubed pedicle flap were chosen and foot pressures of both the operated foot and the contralateral normal foot were measured postoperatively. The results did not indicate any functional disturbance after this method of reconstruction.

Regulation of retrochalcone biosynthesis: Activity changes of O-methyltransferases in the yeast extract-induced Glycyrrhiza echinata cells.

Three O-methyltransferases which catalyze S-adenosyl-L-methionine (SAM)-dependent O-methylation of licodione (LMT), flavone/flavonol (FMT), and caffeic acid (CMT) were separated from the callus culture of Glycyrrhiza echinata, and characteristic differences between their pH optima and Mg(2+) requirement for activity were demonstrated. The activity of LMT, which is involved in retrochalcone (echinatin) biosynthesis, but not of FMT or CMT, was found to be stimulated when suspension-cultured G. echinata cells were treated with yeast extract (YE), which causes rapid production of echinatin in the cells. Cycloheximide suppressed both the YE-induced echinatin formation and LMT enhancement. The results indicate a selective induction of retrochalcone pathway in Glycyrrhiza cells in response to stress.

A factor XI deficiency associated with a nonsense mutation (Trp501stop) in the catalytic domain.

We identified a novel mutation in an asymptomatic 65-year-old Japanese man with severe factor XI deficiency. Sequence analysis after polymerase chain reaction single-stranded conformation polymorphism (PCR-SSCP) analysis of his factor XI gene revealed a G-->A transition in codon 501 of exon 13, resulting in a substitution of Trp501 (TGG) by a stop codon (TAG) in the catalytic domain. This mutation abolished a FokI restriction site. The PCR product from normal subjects was digested with FokI and yielded two fragments, one of 223 bp and one of 47 bp. The PCR product from the patient gave a single 270-bp fragment, demonstrating possible homozygosity.

Red blood cell status in alcoholic and non-alcoholic liver disease.

Macrocytosis is most commonly associated with vitamin B(12) and folic acid deficiency, followed by alcoholism, liver disease, and other pathologic conditions. We studied the red cell and vitamin status in 423 consecutive patients with various liver diseases, including 31 with acute viral hepatitis (AVH), 105 with chronic hepatitis (CH), and 134 with alcoholic liver disease (ALD), who consisted of 84 with non-cirrhotic alcoholic liver disease (NCALD) and 50 with alcoholic liver cirrhosis (ALC), 60 with non-alcoholic liver cirrhosis (NALC), and 93 with hepatocellular carcinoma (HCC). The mean corpuscular volume (MCV) and red cell distribution width (RDW) were significantly higher in patients with ALD and NALC, and among them macrocytosis occurred more frequently in patients with ALC. Macrocytic anemia was mostly found in cirrhotic patients, in which the Child-Pugh score was closely related to the development of macrocytic anemia. In ALD, the MCV was significantly correlated with the estimated alcohol consumption and inversely correlated with the serum folic acid level, which, however, was often maintained within the normal range in patients with macrocytic ALC. After abstinence from alcohol, the MCV and RDW were reduced significantly and were associated with an increasing serum folic acid level. This suggests that macrocytic anemia was a common feature of alcoholic and non-alcoholic liver cirrhosis and that alcohol abuse and folic acid deficiency play a secondary role in macrocytosis.

Severe exacerbation of hepatitis after short-term corticosteroid therapy in a patients with "latent" chronic hepatitis B.

We present a case of severe exacerbation of hepatitis after short-term corticosteroid therapy for chronic inflammatory demyelinating polyneuropathy (CIPD) with "latent" chronic hepatitis B showing no HBV-related antigens and antibodies. After corticosteroid pulse therapy for CIPD, the patient had severe exacerbation of hepatitis twice. Although she did not show any hepatitis B virus (HBV)-related antigens or antibodies, sequences of HBV were detected in serum and liver by a nested polymerase chain reaction. A sequence analysis of HBV at the second exacerbation showed that the G-to-A point mutation at nucleotide 1896 that converted codon 28 from tryptophan (TGG) to a stop codon (TAG) in the precore region resulted in amino acid change, which has been frequently observed in fulminant hepatitis and severe hepatitis in Japan.

Occult hepatitis B virus infection in HBs antigen-negative hepatocellular carcinoma in a Japanese population: involvement of HBx and p53.

Hepatitis B virus (HBV) genome was reported to be detected in serum or liver tissues in hepatocellular carcinoma (HCC) patients negative for hepatitis B surface antigen (HBsAg). Hepatitis B x (HBx) and p53 protein were reported to play an important role in HBV-related hepatocarcinogenesis. To clarify latent HBV infection in HBsAg- and anti-hepatitis C virus (anti-HCV)-negative HCC in a Japanese population and involvement of HBx and p53 protein in these patients, we performed the sensitive and specific nested polymerase chain reaction (PCR) and immunohistochemical analysis. Of 1,024 HCC patients we saw between 1974 and 1998, 66 (6.4%) were negative for HBsAg and anti-HCV. Serum DNA was amplified by nested PCR by using specific primers of surface (S), core (C) and X regions in 26 patients negative for HBsAg and anti-HCV. Eighteen (69%) patients were positive for either S, C, or X region and the results of PCR were confirmed by Southern blotting. Of 18 PCR-positive patients, 3 were positive for anti-HBs and 9 were positive for anti-HBc, however, one was negative for any HBV markers. In HBsAg-negative and PCR-positive patients, the positive rates of expression of HBx and p53 were 8/13 (62%) and 7/13 (54%), being comparable to those in HBsAg-positive HCC patients. The results of the present study suggest that high prevalence of HBV infection is observed in HBsAg-negative HCC in a Japanese population and expression of HBx and p53 is consistent with a role, in these patients, for the transforming ability of these proteins.