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.

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.

Effect of influenza vaccine on warfarin anticoagulation.

Recent studies have indicated that viral infections, influenza vaccination, or drugs that increase interferon synthesis all decrease hepatic drug metabolism. We report a case in which influenza vaccination was temporally related to an increased anticoagulant effect of warfarin. A prospective study evaluating the effect of influenza vaccination on the prothrombin time of eight patients anticoagulated over the long term showed that there was prolongation of prothrombin time of 40%. In a second study, the effect of influenza vaccination on warfarin t1/2 was determined in healthy subjects. No significant effect on warfarin metabolism was observed after vaccination. We conclude that influenza vaccination is associated with increased anticoagulant response in some patients receiving anticoagulants over a long term. This effect appears to be related to some step in the coagulation pathway and not to decreased warfarin metabolism and a subsequent rise in serum concentration.

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.

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.

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.

Clinical pharmacology of dilevalol (I). Comparison of the pharmacokinetic and pharmacodynamic properties of dilevalol and labetalol after a single oral administration in healthy subjects.

Dilevalol (25 mg----50 mg----100 mg) or labetalol (100 mg) was given orally in six healthy subjects. The study was carried out on four occasions with a week interval. Blood samples for plasma drug concentrations were taken for a 24-hour post-drug period. Blood pressure (BP) as well as heart rate (HR) at supine position, during 50 degrees tilting and during a submaximal exercise were measured after each treatment. The mean maximum plasma concentration (Cmax) as well as the mean area under the plasma concentration-time curve (AUC) increased in a dose-dependent manner after dilevalol. These parameters after dilevalol 100 mg were significantly lower than after labetalol 100 mg. No significant differences were observed in the time to maximum concentration (tmax), the distribution half-life (t1/2 alpha) or the elimination half-life (t1/2 beta) between dilevalol and labetalol. There were no significant differences in BP at supine position or during 50 degrees tilting among the dosages. Postural changes in HR during 50 degrees tilting was significantly smaller after dilevalol 100 mg than following labetalol 100 mg. The suppressing effect of dilevalol 100 mg on an increase in HR during a submaximal exercise was significantly greater than during labetalol 100 mg. These data indicate that although plasma drug concentrations are lower after dilevalol than following labetalol, the beta-blocking activity of dilevalol is more potent than labetalol.

Clinical pharmacology of dilevalol (II). The pharmacokinetic, pharmacodynamic, and tolerance studies of dilevalol during repeated administration in healthy subjects.

Dilevalol (50 mg) was given orally twice daily for eight days in six healthy subjects. All parameters were obtained following 1st (on day 1) and 15th (on day 8) dosages. Blood samples for plasma drug concentrations were taken for a 12-hour (after 1st dosage) or a 24-hour (after 15th dosage) post-drug period. Blood pressure (BP) as well as heart rate (HR) at supine position, during 50 degrees tilting and during a submaximal exercise were measured after each dosage. The mean time to maximum concentration (tmax) was faster, and the mean area under the plasma concentration-time curve (AUC) was greater after 15th dosage than following 1st dosage. No significant differences were observed in the maximum plasma concentration (Cmax), the distribution half-life (t 1/2 alpha) or the elimination half-life (t 1/2 beta) between the two dosages. BP at supine position as well as during 50 degrees tilting decreased significantly after each dosage, and did not differ between 1st and 15th dosages. Postural changes in BP or HR during 50 degrees tilting were not induced following 1st or 15th dosage. The suppressing effects (%R) on an increase in HR during a submaximal exercise were significantly larger after 15th dosage than after 1st dosage. A significant correlation was observed between plasma dilevalol concentration and %R in HR. These data indicate that the hypotensive effect of dilevalol is not altered during the repeated administration of the drug for 8 days. However, the beta-blocking activity of dilevalol might be enhanced during the repeated dosages, which is, in part, attributed to dosage-dependent elevation in plasma drug concentrations.

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.

Evaluation of the brainstem with high-resolution CT in cerebellar atrophic processes.

The authors studied the usefulness of computed tomography (CT) for evaluation of the brainstem in cerebellar atrophic processes. Twenty adult subjects without posterior fossa lesion were used for normal CT measurements of the brainstem. The measured values with CT corresponded to those with pneumotomography. Also reviewed were 49 patients with cerebellar atrophy which included spinocerebellar degeneration (25 patients), Shy-Drager syndrome (five), progressive supranuclear palsy (three), chronic phenytoin usage (10), and chronic alcoholism (six). All but the chronic alcoholism group showed atrophy of the brainstem at all locations of measurement when compared with normal controls (p less than 0.05). In addition, the patients with progressive supranuclear palsy had significantly more pronounced midbrain atrophy. In the chronic alcoholism group the measurements of the brachium pontis, the medulla, and the fourth ventricle differed significantly from those of normal controls (p less than 0.05).

Myelopathy due to ossification or calcification of the ligamentum flavum: radiologic and histologic evaluations.

The clinical, radiologic, and histologic features of ossification and calcification of the ligamentum flavum were studied in 18 patients. Ossification (15 patients) usually occurred in the lower thoracic spine in men of various ages, while calcification (three patients) was found exclusively in the cervical region of older women. Histologic examinations of ossification showed mature lamellar bone associated with proliferated cartilage replacing the ligamentum flavum (endochondral ossification). This corresponded well with radiographic and computed tomographic (CT) appearances. In the cases with calcification of the ligamentum flavum, calcification within the degenerated ligamentous fibers was observed on histologic examination, and correlated well with an oval nodular density on radiographs and CT. The two conditions differ in clinical, radiologic, and histologic considerations. Pathomechanisms in the development of these lesions and clinical significance are also discussed.

Occlusion of cerebral artery due to metastasis of chorioepithelioma. Case report.

A case of cerebral metastasis of chorioepithelioma with an occlusion of the angular artery is presented. The occlusion was verified angiographically, surgically, and microscopically.

Anterior decompression for ossification of the posterior longitudinal ligament of the cervical spine.

Anterior decompression and fusion for treating ossification of the posterior longitudinal ligament of the cervical spine was performed in 12 patients. The central part of the vertebral body and the ossified area of the posterior longitudinal ligament were removed by means of a microrongeur and an air drill. The defect was filled with a long bone graft taken from the ilium. The operative results were excellent. Marked improvement of radicular and spinal cord signs was seen in all 12 cases. Three vertebral bodies were fused in one case, four in nine cases, and five in two cases. The highest level of fusion was C-2 and the lowest was T-1. It is considered that any ossification of the ligament below the C-2 level can be removed via an anterior approach as long as no more than five vertebral bodies are involved. Spinal computerized tomography was valuable in providing more detailed information about the stenotic spinal canal and the shape of the ossified ligament.

Role of the posterior hypothalamus in the development of acute brain swelling.

Destructive stereotaxic lesions were made in the posterior hypothalamus, unilaterally or bilaterally, in 26 dogs. In 21 dogs the intracranial pressure (ICP) was maintained in normal range, and in five dogs the ICP was artificially elevated to 300 to 400 mm h2o, preceding the procedures to the hypothlamus. Arterial oxygen and carbon dioxide pressure ((PaO2 and PaCO2) were maintained in the normal range. Before and after each procedure, systemic arterial pressure (SAP) was elevated by intravenous injection of norepinephrine (5 X 10(-3) mg/kg) to determined whether the ICP increases coincidentally with elevation of the systemic arterial pressure. The intracranial pressure/mean arterial pressure ratio of elevation by injection of norepinephrine was not significant regardless of the level of the ICP, or of uni-or bilateral lesions of the hypothalamus. The authors conclude that dysfunction of posterior hypothalamus does not play a specific role in the development of vasomotor paralysis leading to acute brain swelling, under conditons of normal or moderately raised ICP with normal PaCO2 and Pato2 levels.

Computerized tomography findings in dural arteriovenous malformations. Report of three cases.

Changes in the brain parenchyma and cerebrospinal fluid spaces were demonstrated in three cases of dural arteriovenous malformations by computerized tomography (CT). In various combinations, there were 1) vermiform or patchy enhancement after intravenous contrast infusion, 2) abnormal decreased density mainly in the white matter, 3) local mass effect, 4) hydrocephalus, 5) dilatation of the major venous sinus system, and 6) prominent vascular grooving of the skull inner table. Most of these changes were secondary to raised sinus pressure caused by arteriovenous shunt. It was also demonstrated by serial CT that some elements improved after artificial embolization or surgical excision of the malformations.

Atlantoaxial dislocation associated with neurofibromatosis. Report of three cases.

Atlantoaxial dislocation was found in three patients with neurofibromatosis. Roentgenographic findings included marked reduction of sagittal diameter at the C-1 vertebral level, and cervical spine abnormalities associated with mesodermal dysplasia, such as posterior scalloping of the cervical spinal bodies with dural ectasia and vertebral body deformity (vertebral body dysplasia). Although the relationship of the atlas and axis did not change with neck position, all three patients had progressive neurological deficits and were treated by decompressive surgery combined with fusion. The pathogenesis of atlantoaxial dislocation associated with neurofibromatosis is discussed.

Calcification of the ligamentum flavum of the cervical spine. Report of four cases.

Four cases of calcification of the cervical ligamentum flavum are reported, all in women over 60 years of age. Neurological findings were not significantly different from those of other cervical compressive diseases. Among radiological examinations, computerized tomography was the most valuable diagnostic tool. Calcification might have been induced by the degeneration or abnormal nutritional state of the ligamentum flavum. Endocrine abnormalities and inflammatory processes might also have been contributory factors.

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.

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.

Computed tomographic findings of vacuum phenomenon in cervical intervertebral disks.

A gaseous collection in the intervertebral space, or the vacuum phenomenon, was observed on computed tomograms in as many as 30% of the patients with cervical disk disease. The condition most frequently occurred at the level of C5-6. The gas tended to accumulate more often at the marginal region of the intervertebral disks, the level at which marked degeneration was seen; thus, it may be related to cracks within not only the nucleus pulposus, but also in the annulus fibrosus.