Increased insulin demand promotes while pioglitazone prevents pancreatic beta cell apoptosis in Wfs1 knockout mice.

AIMS/HYPOTHESIS: The WFS1 gene encodes an endoplasmic reticulum (ER) membrane-embedded protein called Wolfram syndrome 1 protein, homozygous mutations of which cause selective beta cell loss in humans. The function(s) of this protein and the mechanism by which the mutations of this gene cause beta cell death are still not fully understood. We hypothesised that increased insulin demand as a result of obesity/insulin resistance causes ER stress in pancreatic beta cells, thereby promoting beta cell death. METHODS: We studied the effect of breeding Wfs1 ( -/- ) mice on a C57BL/6J background with mild obesity and insulin resistance, by introducing the agouti lethal yellow mutation (A ( y ) /a). We also treated the mice with pioglitazone. RESULTS: Wfs1 ( -/- ) mice bred on a C57BL/6J background rarely develop overt diabetes by 24 weeks of age, showing only mild beta cell loss. However, Wfs1 ( -/- ) A ( y ) /a mice developed selective beta cell loss and severe insulin-deficient diabetes as early as 8 weeks. This beta cell loss was due to apoptosis. In Wfs1 ( +/+ ) A ( y ) /a islets, levels of ER chaperone immunoglobulin-binding protein (BiP)/78 kDa glucose-regulated protein (GRP78) and phosphorylation of eukaryotic translation initiation factor 2, subunit alpha (eIF2alpha) apparently increased. Levels of both were further increased in Wfs1 ( -/- ) A ( y ) /a murine islets. Electron micrography revealed markedly dilated ERs in Wfs1 (-/-) A ( y ) /a murine beta cells. Interestingly, pioglitazone treatment protected beta cells from apoptosis and almost completely prevented diabetes development. CONCLUSIONS/INTERPRETATION: Wfs1-deficient beta cells are susceptible to ER stress. Increased insulin demand prompts apoptosis in such cells in vivo. Pioglitazone, remarkably, suppresses this process and prevents diabetes. As common WFS1 gene variants have recently been shown to confer a risk of type 2 diabetes, our findings may be relevant to the gradual but progressive loss of beta cells in type 2 diabetes.

Three-dimensional organization of the hepatic microvasculature in hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant systemic fibrovascular dysplasia. Although hepatic vascular shunts are often observed in HHT, the responsible pathological mechanism is unknown. This issue was addressed by performing a 3-dimensional reconstruction study of the hepatic microvasculature of an HHT-involved liver in a 79-year-old woman. Clinical observation revealed high-output congestive heart failure and hepatic encephalopathy due to arteriovenous and portovenous shunts, respectively. Angiography revealed tortuous dilation of hepatic arterial branches and intrahepatic arteriovenous shunts. The 3-dimensional analysis of the autopsy liver revealed focal sinusoidal ectasia, arteriovenous shunts through abnormal direct communications between arterioles and ectatic sinusoids, and portovenous shunts due to frequent and large communications between portal veins and ectatic sinusoids. Type 1 HHT was suggested by the lack of endoglin immunoreactivity in the liver. The 3-dimensional reconstruction study of hepatic microvasculature was successful in identifying the pathological changes responsible for the intrahepatic shunts in HHT.

3-phosphoinositide-dependent protein kinase 1, an Akt1 kinase, is involved in dephosphorylation of Thr-308 of Akt1 in Chinese hamster ovary cells.

To investigate the role of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in the Akt1 phosphorylation state, wild-type (wt) PDK1 and its kinase dead (kd) mutant were expressed using an adenovirus gene transduction system in Chinese hamster ovary cells stably expressing insulin receptor. Immunoblotting using anti-phosphorylated Akt1 antibody revealed Thr-308 already to be maximally phosphorylated at 1 min but completely dephosphorylated at 5 min, with insulin stimulation, whereas insulin-induced Akt1 activation was maintained even after dephosphorylation of Thr-308. Overexpression of wt-PDK1 further increased insulin-stimulated phosphorylation of Thr-308, also followed by rapid dephosphorylation. The insulin-stimulated Akt1 activity was also enhanced by wt-PDK1 expression but was maintained even at 15 min. Thus, phosphorylation of Thr-308 is not essential for maintaining the Akt1 activity once it has been achieved. Interestingly, the insulin-stimulated phosphorylation state of Thr-308 was maintained even at 15 min in cells expressing kd-PDK1, suggesting that kd-PDK1 has a dominant negative effect on dephosphorylation of Thr-308 of Akt1. Calyculin A, an inhibitor of PP1 and PP2A, also prolonged the insulin-stimulated phosphorylation state of Thr-308. In addition, in vitro experiments revealed PP2A, but not PP1, to dephosphorylate completely Thr-308 of Akt1. These findings suggest that a novel pathway involving dephosphorylation of Akt1 at Thr-308 by a phosphatase, possibly PP2A, originally, identified as is regulated downstream from PDK1, an Akt1 kinase.

Electron microscopic observation of established chondrocytes derived from human intervertebral disc hernia (KTN-1) and role of macrophages in spontaneous regression of degenerated tissues.

BACKGROUND CONTEXT: Biological and pathological cell processes during the degeneration of intervertebral discs are as yet poorly understood. PURPOSE: An electron microscope was used to observe disc hernia degeneration at the cellular level as expressed in extruded tissue from a human intervertebral disc and in cultured chondrocytes. The mechanism of spontaneous regression was analyzed in order to investigate the effects of homologous macrophages, and the results of this analysis may be developed into a clinical therapy. STUDY DESIGN/SETTING: Extruded tissue specimens excised during surgery on human intervertebral disc hernia and cultured chondrocytes isolated from the excised tissue were observed by means of electron microscopy. Extracellular matrix metalloproteinase-3 (MMP-3) and its antagonist, tissue inhibitor of metalloproteinases-1 (TIMP-1), were observed by means of immune electron microscopy. Macrophages confirmed by CD68 immunostaining were added to the chondrocyte culture and observed by means of electron microscopy. PATIENT SAMPLE: All control subjects and patients gave written consent to the study. OUTCOME MEASURES: KTN-1 was directly observed without culture, and nuclei degeneration, the development of chromatin granules, changes in the osmotic pressure of the nuclear membrane and rough-surfaced endoplasmic reticulum, and the development of fat droplets were observed. METHODS: Tissues excised during surgery were divided, a part of the tissues were fixed in various fixatives for electron microscopy and immune electron microscopy analysis, and the other part was treated with collagenase. In addition, chondrocytes were isolated and cultured. Human peripheral blood mononuclear cells were separated using the Ficoll method. After culturing the cells, macrophages were collected, added to the chondrocyte culture, and observed under an electron microscope. CD68 positivity of the macrophages was confirmed by CD68 immunostaining. RESULTS: Freshly isolated chondrocytes in the hernia's extruded region differed markedly from cultured chondrocytes. By means of immunoelectron microscopy, MMP-3 and TIMP-1 were localized at the endoplasmic reticulum of the cultured chondrocytes. Infiltration of macrophages among the chondrocytes was observed in the mixed culture. CONCLUSIONS: The tissue extruded from the intervertebral disc showed obvious signs of degeneration, such as changes in osmotic pressure. Macrophages were observed to be the mechanism of spontaneous regression.

Hepatocellular carcinoma arising in an elderly male with primary biliary cirrhosis.

We report the case of an elderly male with asymptomatic primary biliary cirrhosis (PBC) who developed a hepatocellular carcinoma (HCC). The 89-year-old man, who was otherwise healthy, was admitted for investigation of mild hepatic dysfunction, which had been detected during a routine physical check-up. Serum chemistry, positive anti-mitochondrial antibody (M2) and liver biopsy results led to a diagnosis of PBC. Three years later, at age 92, computed tomography (CT) and ultrasound scans of his abdomen revealed a large hepatic tumour, which was confirmed on liver biopsy to be HCC. The tumour ruptured 3 months after diagnosis and the patient was successfully stabilized by coil embolization of his right hepatic artery. We believe that, to date, this is the oldest reported patient to have had interventional radiology for the management of HCC.

Crystal structure of prolyl aminopeptidase from Serratia marcescens.

Prolyl aminopeptidase from Serratia marcescens specifically catalyzes the removal of N-terminal proline residues from peptides. We have solved its three-dimensional structure at 2.3 A resolution by the multiple isomorphous replacement method. The enzyme consists of two contiguous domains. The larger domain shows the general topology of the alpha/beta hydrolase fold, with a central eight-stranded beta-sheet and six helices. The smaller domain consists of six helices. The catalytic triad (Ser113, His296, and Asp268) is located near the large cavity at the interface between the two domains. Cys271, which is sensitive to SH reagents, is located near the catalytic residues, in spite of the fact that the enzyme is a serine peptidase. The specific residues which make up the hydrophobic pocket line the smaller domain, and the specificity of the exo-type enzyme originates from this smaller domain, which blocks the N-terminal of P1 proline.

Early carcinoma of the distal second part of the duodenum treated by wedge resection.

A case of early carcinoma of the distal second part of the duodenum, in a 74-year-old man, is presented. Endoscopic retrograde cholangiopancreatography was performed for diagnosis of a common bile duct stone. During this procedure, small elevated lesions were found incidentally in the distal second part of the duodenum, and the histologic examination of a biopsy specimen showed adenocarcinoma. The lesions were removed by wedge resection, and pathologic examination revealed duodenal carcinoma limited to the lamina propria. Although carcinoma of the duodenum, other than of the ampulla of Vater region, is very rare, it is sometimes possible to detect asymptomatic early tumors. However, this requires careful observation of the entire duodenal mucosa, including that of the distal duodenum, at duodenoscopy.

Roles of the Ser146, Tyr159, and Lys163 residues in the catalytic action of 7alpha-hydroxysteroid dehydrogenase from Escherichia coli.

The Escherichia coli 7alpha-hydroxysteroid dehydrogenase (7alpha-HSDH; EC 1.1.1.159) has been the subject of our studies, including the cloning of its gene, and determination of the crystal structures of its binary and ternary complexes [J. Bacteriol. 173, 2173-2179 (1991); Biochemistry 35, 7715-7730 (1996)]. Through these studies, the Ser146, Tyr159, and Lys163 residues were found to be involved in its catalytic action. In order to clarify the roles of these residues, we constructed six single mutants of 7alpha-HSDH, Tyr159-Phe (Y159F), Tyr159-His (Y159H), Lys163-Arg (K163R), Lys163-Ile (K163I), Ser146-Ala (S146A), and Ser146-His (S146H), by site-directed mutagenesis. These mutants were overexpressed in E. coli WSD, which is a 7alpha-HSDH null strain, and the expressed enzymes were purified to homogeneity. The kinetic constants of the mutant enzymes were determined, and the structures of the Y159F, Y159H, and K163R mutants were analyzed by X-ray crystallography. The Y159F mutant showed no activity, while the Y159H mutant exhibited 13.3% of the wild-type enzyme activity. No remarkable conformational change between the Y159F (or Y159H) and wild-type proteins was detected on X-ray crystallography. On the other hand, the K163I mutant showed just 5.3% of the native enzyme activity, with a 8. 5-fold higher Kd. However, the K163R mutant retained 64% activity, and no remarkable conformational change was detected on X-ray crystallography. In the cases of the S146A and S146H mutants, the activities fairly decreased, with 20.3 and 35.6% of kcat of the wild-type, respectively. The data presented in this paper confirm that Tyr159 acts as a basic catalyst, that Lys163 binds to NAD(H) and lowers the pKa value of Tyr159, and that Ser146 stabilizes the substrate, reaction intermediate and product in catalysis.

Extreme right lobar atrophy of the liver: a rare complication of autoimmune hepatitis.

Lobar atrophy is a rare morphologic change of the liver. We describe a 73-year-old woman with mild liver dysfunction and history of Sjögren's syndrome who had right hepatic lobar atrophy. Serum biochemistry levels were as follows: albumin, 4.5 g/dl; total bilirubin, 1.0 mg/dl; alanine aminotransferase, 25 international units/l; aspartate aminotransferase, 27 international units/l; alkaline phosphatase, 333 international units/l; and gamma-glutamyl transpeptidase, 332 international units/l. Serological data were as follows: rheumatoid factor, 27.9; anti-nuclear antibody, 1:640; and antismooth muscle antibody, 1:80. Viral markers for hepatitis B were all negative. Anti-hepatitis C virus (anti-c-100) was negative. Portal hypertension developed thereafter, and the patient died of hepatic failure at age 76. Postmortem examination revealed autoimmune hepatitis with moderate fibrosis, portal vein thrombus, and complete obstruction of the right hepatic duct due to hepatolithiasis. Terminal hepatic failure resulted from combination of decreased hepatic volume due to the right lobar atrophy, exacerbation of autoimmune hepatitis in the remnant left hepatic lobe, decreased portal venous blood flow due to thrombosis, portal hypertension, and cholangitis with hepatolithiasis. This is the first reported case of hepatic lobar atrophy due to autoimmune hepatitis. From a clinical standpoint, patients with hepatic lobar atrophy, even if asymptomatic, should be followed up with careful attention to progression of liver diseases, portal hypertension, and biliary complications.

A remote conference system for image diagnosis on the World-Wide Web.

OBJECTIVE: The World-Wide Web on the Internet enables an exchange of multimedia information among remote desktop computers. Therefore, a teleradiology system using the Web would allow remote consultation with expert radiologists. Our objective was to establish a Web-based prototype system for image interpretation. CONCLUSION: Our system allows a physician to transmit clinically useful images to an expert radiologist at a different location, who can see them on a Web browser and discuss diagnoses with the physician.

Temperature- and pH-dependent cytotoxic effect of the hemolytic lectin CEL-III from the marine invertebrate Cucumaria echinata on various cell lines.

We investigated the cytotoxicity of CEL-III, one of four Ca2+-dependent galactose/N-acetylgalactosamine (GalNAc)-binding lectins from the marine invertebrate Cucumaria echinata. Among six cell lines tested, MDCK cells showed the highest susceptibility to CEL-III cytotoxicity and its LD50 was estimated to be 53 ng/ml, while no significant cytotoxicity of CEL-III was observed in CHO cells up to 10,000 ng/ml. In the presence of 0.1 M lactose, the cytotoxicity of CEL-III was strongly inhibited. The binding studies using FITC-labeled CEL-III revealed that the amount of CEL-III bound to MDCK cells was about 2-fold greater than that in the case of CHO cells. The cytotoxicity of CEL-III increased with decreasing temperature. The surviving fractions of Vero cells exposed to CEL-III at 4 degrees C were immediately decreased, and more than 90% of exposed cells were killed within 20 min, whereas at 37 degrees C much longer exposure period (more than 10 h) was required to kill 50% of the cells. CEL-III induced the release of carboxyfluorescein (CF) from CF-loaded MDCK cells and this activity was markedly increased at alkaline pH (pH 10) and at lower temperature (4 degrees C). Even in CHO cells, considerable CF release was induced by CEL-III at 4 degrees C and at pH 10 but not at pH 7.5 at both temperatures. In agreement with these results, CHO cells exposed to CEL-III at 4 degrees C and at pH 10 were killed in a dose-dependent manner. These results suggest that CEL-III exhibits cytotoxicity through damaging the plasma membrane by pore-formation in a temperature- and pH-dependent manner. Different susceptibility of each cell line to CEL-III cytotoxicity may be due to differences in the processes leading to pore-formation after binding to cell-surface carbohydrates.

[Clinical characteristics and liver dysfunction in lymphocyte stimulation test-positive elderly].

In order to clarify the characteristics of the side effects of drugs in the elderly, we evaluated clinical features and liver function in 40 lymphocyte stimulation test (LST)-positive elderly. Their ages ranged from 64 to 90 years, with a mean age of 75 years. The major causative agents were antituberculosis agents, antibiotics and antiinflammatory agents, comprising 28%, 22% and 12% of whole drugs, respectively. Liver dysfunction, skin eruptions and fever were the major causes for carrying out LST. The mean latent period was 15 days, and in 80% of the cases, the side effect developed within four weeks after administration of the causative agent. Major clinical symptoms noted during the course were detected in more than the half of the cases. As for liver dysfunction, elevation of GOT, ALP and total bilirubin levels were noted in 76, 63 and 34% of the cases, respectively. These results showed that the hypersensitive side effects of the drugs could appear at any age even in the elderly, and clinical symptoms were often nonspecific and obscure. It was suggested that the presence of mild liver dysfunction and eosinophilia could be helpful markers for the early detection of drug-induced organ dysfunctions as well as careful observation. The possibility of the occurrence of the side effects must be considered on every drug administration. Presence of mild liver dysfunction and eosinophilia may be helpful markers for the early detection of drug-induced organ dysfunction.

Pertussis toxin non-sensitive G protein mediates cholinergic stimulation for secretion of pancreastatin and somatostatin from QGP-1N cells.

To clarify the possible role of a guanine nucleotide-binding protein (G-protein) in the signal transducing system activated by carbachol, actions of carbachol on human pancreastatin producing cell line (QGP-1N) were compared with those of fluoride, a well-known activator of stimulatory (Gs) or inhibitory (Gi) G protein. 10(-5) M of carbachol as well as 20 mM of NaF stimulated secretion of pancreastatin and somatostatin and intracellular Ca2+ mobilization. These secretion and Ca2+ mobilization were not modified by pertussis toxin, an inhibitor of Gi protein. These results suggest that pancreastatin and somatostatin secretions from QGP-1N are regulated by acetylcholine through a muscarinic receptor coupled to the activation of polyphosphoinositide breakdown by a G protein, which appears to be fluoride sensitive but is other than a Gi-like protein.

[Detection of immature granulocytes and atypical and/or abnormal lymphocytes using scatterplots of Coulter STKS].

Scatterplots of the Coulter STKS were studied in order to make better analyses of morphological abnormality of leukocytes. The analytical pattern of a sample failed to completely prevent blood coagulation, especially platelet aggregation, showed the poor separation between lymphocyte and neutrophil populations. On the scatterplot, a small population was occasionally observed in the lower area of normal lymphocyte population with elevation of room temperature, being thought an artificial population. When atypical and/or abnormal lymphocytes increased, the pattern was characterized by the distribution of large lymphoid cells spreading over monocyte population area. The characteristics became clear in proportion to the percentage of large lymphoid cells. To detect immature granulocytes, we introduced a criterion originated in our laboratory. Using the detection criterion, we could obtain the satisfactory results with sensitivity of 85%, specificity of 93%, efficiency of 91% and coefficient correlation (r) of 0.73. It is concluded that the pattern analysis and the detection criterion are useful in the experiments using routine laboratory samples and valuable in clinical implication.

Acetylcholine regulates pancreastatin secretion from the human pancreastatin-producing cell line (QGP-1N).

Studies were made of pancreastatin (PST) secretion from a human PST-producing cell line (QGP-1N) in response to various secretagogues. Cells with immunoreactivity for PST were observed in monolayer cultures of QGP-1N cells. Carbachol stimulated PST secretion and the intracellular Ca2+ mobilization concentration dependently in the range of 10(-6)-10(-4) M. The PST secretion and Ca2+ mobilization induced by carbachol were inhibited by atropine. The calcium ionophore (A23187) stimulated PST secretion. However, cholecystokinin and gastrin-releasing peptide did not stimulate either PST secretion or Ca2+ mobilization. Secretin also did not stimulate PST secretion. The glucose concentration in the culture medium had no effect on PST secretion. These results suggest that PST secretion is mainly regulated by acetylcholine through a muscarinic receptor, and that an increase in intracellular Ca2+ plays an important role in stimulus-secretion coupling in QGP-1N cells.

[Changes in the liver weight in the elderly].

To elucidate the effect of aging on liver weight in the elderly, 582 elderly cases (male 291, female 291), were selected from 2000 elderly autopsied cases on the basis of being free from pathological findings affecting liver weight. Both liver weight and its ratio to body weight decreased with age, and influenced by obesity; the former increased and the latter decreased in obese cases. Analysis according to the degree of obesity also showed decrease of liver weight and is ratio to body weight with age. Comparison between males and females of the same decade revealed the tendency that in females the liver weight was low, while liver weight.body weight ratio was high. The same results were obtained by analysis based on the degree of obesity.

Hemodynamic evaluation of vascular reconstructive surgery for childhood moyamoya disease using single photon emission computed tomography.

To evaluate the efficacy of vascular reconstructive surgery for childhood moyamoya disease, the cerebral blood flow (CBF) in 31 hemispheres of 16 patients was examined by single photon emission computed tomography (SPECT) using the 133Xe inhalation method. Results were divided into two groups; 17 hemispheres with superficial temporal artery-middle cerebral artery (STA-MCA) anastomosis [A(+) group] and 14 hemispheres without anastomosis [A(-) group]. The mean hemispheric CBF (mCBF) and regional CBF (rCBF) in the frontal, temporal, occipital, and basal ganglia regions were calculated. Pre- and postoperative SPECT on the 10 hemispheres of the A(+) group showed an increase in mCBF in 6 hemispheres, the disappearance of the low perfusion area (LPA) in all 5 hemispheres where LPA was present before surgery, and an improvement in rCBF distribution (an increase in rCBF in the frontal and temporal lobes and a decrease in the basal ganglia). This suggests that vascular reconstruction is greatly effective in treating this disease. A comparison between the A(+) group and the A(-) group by postoperative SPECT, as well as the clinical outcomes and the postoperative findings of electroencephalography and angiography, revealed that the A(+) group was superior to the A(-) group in the frequency of LPA (12% and 43%, respectively) and rCBF in the frontal region where STA-MCA anastomosis was usually performed. These results indicate that STA-MCA anastomosis with indirect synangiosis is the most effective treatment of childhood moyamoya disease.