Lzts1 controls both neuronal delamination and outer radial glial-like cell generation during mammalian cerebral development.

In the developing central nervous system, cell departure from the apical surface is the initial and fundamental step to form the 3D, organized architecture. Both delamination of differentiating cells and repositioning of progenitors to generate outer radial glial cells (oRGs) contribute to mammalian neocortical expansion; however, a comprehensive understanding of their mechanisms is lacking. Here, we demonstrate that Lzts1, a molecule associated with microtubule components, promotes both cell departure events. In neuronally committed cells, Lzts1 functions in apical delamination by altering apical junctional organization. In apical RGs (aRGs), Lzts1 expression is variable, depending on Hes1 expression levels. According to its differential levels, Lzts1 induces diverse RG behaviors: planar division, oblique divisions of aRGs that generate oRGs, and their mitotic somal translocation. Loss-of-function of lzts1 impairs all these cell departure processes. Thus, Lzts1 functions as a master modulator of cellular dynamics, contributing to increasing complexity of the cerebral architecture during evolution.

Clinical characteristics of fulminant-type autoimmune hepatitis: an analysis of eleven cases.

BACKGROUND: Although a few adult cases of fulminant-type autoimmune hepatitis have been reported, their clinical features and prognosis have remained uncertain. AIM: To assess the clinical features and prognosis of patients with fulminant-type autoimmune hepatitis. METHODS: Eleven patients (10%) diagnosed with fulminant-type autoimmune hepatitis in accordance with the 1999 criteria of the International Autoimmune Hepatitis Group were analysed. RESULTS: All 11 patients were female, with a median age of 53 years. Five patients survived without liver transplantation, one received a liver transplantation, and five died without liver transplantation. Nine patients (82%) survived for 2 weeks or more following diagnosis, without liver transplantation. Except for the patient receiving a liver transplantation, serum total bilirubin levels measured during the clinical course were significantly higher in non-survivors than in survivors, although the accompanying serum alanine aminotransferase levels measured for the two groups were similar. Most significantly, serum total bilirubin levels in non-survivors worsened during days 8-15, while levels in survivors improved during the same period. CONCLUSIONS: The short-term prognosis for patients with fulminant-type autoimmune hepatitis may be good. However, patients whose serum total bilirubin levels worsen during days 8-15 should be considered for liver transplantation.

Persistent elevation of serum alanine aminotransferase levels leads to poor survival and hepatocellular carcinoma development in type 1 autoimmune hepatitis.

BACKGROUND: Although the prognosis of type 1 autoimmune hepatitis is generally good with immunosuppressive treatment, the disease progresses in some patients despite the treatment. The prognosis may be determined by the clinical course. AIM: To evaluate the long-term prognosis and assess the predictive factors for a serious event, including the development of hepatocellular carcinoma or death. METHODS: Sixty-nine patients with type 1 autoimmune hepatitis were prospectively followed up regularly, with a median follow-up period of 96 months (49-201 months). RESULTS: During the follow-up period, three patients (4%) developed hepatocellular carcinoma, and two of these three patients died. Another patient died of liver failure. The 10-year survival rate was 98%, and the 10-year hepatocellular carcinoma-free rate was 93%. The four patients experiencing a serious event received higher maintenance doses of corticosteroid during their follow-up periods than those did not. However, serum alanine aminotransferase levels during the follow-up period were higher in these four patients than in the others. CONCLUSIONS: Persistent elevation of serum alanine amniotransferase levels during the follow-up period, rather than factors existing prior to medical treatment is considered to be an important prognostic factor, and it is indicated that poor outcomes may result from the resistance to immunosuppressive treatment.

Effects of propionyl-L-carnitine on cardiac dysfunction in streptozotocin-diabetic rats.

The effects of orally administered propionyl-L-carnitine on cardiac dysfunction in rats with streptozotocin-induced diabetes were investigated. Wistar male rats were divided into four groups: untreated normal, propionyl-L-carnitine (daily for 4 weeks with 3 g/kg orally) -treated normal, untreated diabetic, propionyl-L-carnitine-treated diabetic. Four weeks after streptozotocin administration, plasma lipid levels were increased and myocardial carnitine content was decreased in untreated diabetic rats. These changes were significantly reversed by the propionyl-L-carnitine treatment. Assessment of cardiac function with isolated perfused working hearts revealed a depression of left ventricular developed pressure as well as both maximum positive and negative dP/dt in untreated diabetic as compared with that in normal hearts. Cardiac function at the higher left atrial filling pressures in the propionyl-L-carnitine-treated diabetic rats was improved significantly compared to that in untreated hearts. The data thus suggest that oral administration of propionyl-L-carnitine can reduce abnormalities of cardiac function, correlated with a significant increase in myocardial carnitine content and improved lipid metabolism in terms of lowered plasma lipids.

Clinical and histopathological features of colonic stromal tumor in a child.

Neoplasms of the colonic submucosa are rare in children. Gastrointestinal stromal tumors (GISTs) are undifferentiated tumors, usually diagnosed by immunohistochemistry. We report a 4-year-old girl with a submucosal GIST of the ascending colon, which was detected by computed tomography. Diagnosis after ileocecal resection was established by histology. In addition, sections were examined immunohistochemically, using antibodies against vimentin, desmin, alpha-smooth muscle actin, S100, neuron-specific enolase, c-kit, and CD34. Hematoxylin and eosin-stained sections showed interlacing fascicles with occasional palisades of epithelioid and spindle cells. The tumor cells were positive for vimentin and CD34. To our knowledge, this is the first reported case of colonic stromal tumor in a child.

Higher frequencies of numerical aberrations of chromosome 17 in primary gastric cancers are associated with lymph node metastasis.

We investigated the correlation between the frequency of numerical aberrations of chromosome 17 and clinicopathological features of gastric cancer. The copy number of chromosome 17 was examined with fluorescence in-situ hybridization (FISH) in frozen specimens from 100 primary gastric cancers. Chromosomal numerical aberrations were diagnosed as chromosomal loss (single signal) or gain (triple or more signals), in each cell. The frequency of numerical aberrations of chromosome 17 correlated significantly with the depth of invasion (P < 0.01), lymph node metastasis (P < 0.0001), lymphatic invasion (P < 0.001), and venous invasion (P < 0.01). Numerical aberrations of chromosome 17 were associated with lymph node metastasis in 32 early gastric cancers. Multiple regression analysis identified the depth of invasion and numerical aberrations of chromosome 17 as independent significant determinants of lymph node metastasis. Our findings suggest that alterations in chromosome 17 may be linked with tumor progression in primary gastric cancer. Our results also indicate that numerical aberrations of chromosome 17 detected by FISH provide important information about the malignant potential (in particular, lymph node metastasis) of primary gastric cancer.

Stimulation of muscarinic cholinoceptive neurons in the hippocampus evokes a pressor response with bradycardia.

The injection of neostigmine into the hippocampus of anesthetized rats increased the mean arterial blood pressure (17% of baseline after 60 min injection) and decreased the heart rate (24% of baseline after 60 min injection). These changes were blocked by the co-administration of methylatropine into the hippocampus. Intrahippocampal injection of neostigmine stimulated the secretion of epinephrine and norepinephrine. Adrenodemedullation did not suppress the increase in blood pressure and the decrease in heart rate. It is concluded that the stimulation of muscarinic cholinoceptive neurons in the hippocampus evokes a hypertensive response via an increase in sympathetic drive to the heart and peripheral vasculature, with bradycardia possibly mediated via the parasympathetic system.

Obturator hernia: the usefulness of emergent computed tomography for early diagnosis.

The diagnosis of obturator hernia is difficult, and delayed treatment is associated with serious complications. Recently, computed tomography (CT) has been used successfully for establishing a correct diagnosis. We encountered nine cases of obturator hernia from 1988 to 1997. All the patients were elderly women. In six of them, a correct diagnosis was established by emergent CT scan. In two of the remaining cases, diagnosis could not be established because of unavailability of CT, and the other patient was diagnosed correctly based on clinical features, including a positive Howship-Romberg sign. Surgical repair was performed successfully. One patient died postoperatively because of septicemia associated with anastomotic leakage. Our cases indicate that emergent CT scan is useful for the diagnosis of obturator hernia, particularly in elderly patients presenting with ileus of unknown origin.

[p53 alterations and chromosome 17 aberrations in non-small cell lung cancer].

We studied the relationship between DNA index, numerical abberation of chromosome 17 and alterations of the p53 gene in 23 non-small cell lung cancers. Diploid and aneuploid cells from 23 non-small cell lung cancer with DNA aneuploidy were flow sorted into each cell population using FACStar(plus). They were examined by PCR-SSCP analysis for p53 mutation and by microsatellite analysis for loss of heterozygosity at TP53 locus (17p13.1). They were also analyzed by FISH for copy number of chromosome 17. p53 mutations were found in aneuploid cells from the 11 cases (48%). Among them, 8 cases were informative at TP53 locus, and all showed loss of heterozygosity. Aneuploid cells from 16 cases exhibited gain of chromosome 17 copy number in FISH analysis. DNA index was significantly associated with th mean copy number of chromosome 17, suggesting that the number of chromosome 17 changed with DNA index. There were no associations between p53 mutation and DNA index or mean copy number of chromosome 17. In some cases, we also analyzed structural abberation of short arm of chromosome 17 by FISH using p53-cosmid (17p13.1) and Distal 17p (17p13.3-ptel) probes. The p53 gene located on the increased chromosome 17 was lost due to point mutation or deletion of 17p.

A large-scale Agrobacterium-mediated transformation procedure with a strong positive-negative selection for gene targeting in rice (Oryza sativa L.).

A large-scale transformation procedure handling an adequate number of stable transformants with highly efficient positive-negative selection is a necessary prerequisite to successful gene targeting by homologous recombination, as the integration of a transgene by somatic homologous recombination in higher plants has been reported to be 10(-3) to 10(-5) compared with random integration by non-homologous end joining. We established an efficient and large-scale Agrobacterium-mediated rice transformation protocol that generated around 10(3) stable transformants routinely from 150 seeds and a strong positive-negative selection procedure that resulted in survivors at 10(-2) using the gene for diphtheria toxin A fragment as a negative marker. The established transformation procedure provides a basis for efficient gene targeting in rice.

A type I element composed of the hexamer (ACGTCA) and octamer (CGCGGATC) motifs plays a role(s) in meristematic expression of a wheat histone H3 gene in transgenic rice plants.

Type I element (CCACGTCACCGATCCGCG) is a well-conserved regulatory element found in proximal promoter region of a certain class of plant histone genes, that is composed of two independent cis-acting elements of the hexamer (ACGTCA) and the reverse-oriented octamer (GATCCGCG) motifs. To investigate functional role(s) of the type I element in regulation of a wheat histone H3 gene (TH012) promoter activity in vivo, base substitution mutations were introduced into the element and activities of the mutated promoters were examined in cultured rice cells, and in regenerated roots and anther walls of transgenic rice plants by employing a GUS reporter system. Mutations of each or both of the hexamer and the octamer motifs caused a reduction in the promoter activity in protoplasts transfected transiently or stably transformed calli. The mutation of the octamer motif with or without the mutation of the hexamer motif caused a marked reduction of the promoter activity in the root meristem of transgenic rice although the mutation of the hexamer motif alone caused a weak reduction. In contrast to these results, no effect of the mutations of either the hexamer or the octamer motif was found in the anther wall in which replication-independent activity of the H3 promoter was observed. Our results suggested that the hexamer and the octamer motifs may play important role(s) in regulation of replication-dependent but not of replication-independent expression of the wheat histone H3 gene.

A wheat histone H3 promoter confers cell division-dependent and -independent expression of the gus A gene in transgenic rice plants.

To investigate developmental regulation of wheat histone H3 gene expression, the H3 promoter, which has its upstream sequence to -1711 (relative to the cap site as +1), was fused to the coding region of the gus A gene (-1711H3/GUS) and introduced into a monocot plant, rice. Detailed histochemical analysis revealed two distinct types of GUS expression in transgenic rice plants; one is cell division-dependent found in the apical meristem of shoots and roots and in young leaves, and another is cell division-independent detected in flower tissues including the anther wall and the pistil. In this study, replication-dependent expression occurring in non-dividing cells which undergo endoreduplication could not be discriminated from strict replication-independent expression. The observed expression pattern in different parts of roots suggested that the level of the H3/GUS gene expression is well correlated with activity of cell division in roots. To identify 5' sequences of the H3 promoter necessary for an accurate regulation of the GUS expression, two constructs containing truncated promoters, -908H3/GUS and -185H3/GUS, were analyzed in transiently expressed protoplasts, stably transformed calli and transgenic plants. The results indicated that the region from -909 to -1711 contains the positive cis-acting element(s) and that the proximal promoter region (up to -185) containing the conserved hexamer, octamer and nonamer motifs is sufficient to direct both cell division-dependent and -independent expression. The use of the meristem of roots regenerated from transformed calli for the analysis of cell division-dependent expression of plant genes is discussed.

Somatic hybrids between Brassica oleracea and B. campestris: selection by the use of iodoacetamide inactivation and regeneration ability.

An efficient procedure for obtaining somatic hybrids between B. oleracea and B. campestris has been developed. Hypocotyl protoplasts of B. oleracea were fused with mesophyll protoplasts from three different varieties of B. campestris by the polyethylene glycoldimethylsulfoxide method. The selection of somatic hybrids utilized the inactivation of B. oleracea protoplasts by iodoacetamide (IOA) and the low regeneration ability of B. campestris. The efficiency of recovery of somatic hybrids depended upon the IOA concentration, and when 15 mM IOA was used, 90% of the regenerated plants were found to be hybrid. The somatic hybrids were examined for i) leaf morphology, ii) leucine aminopeptidase (LAP) isozyme and iii) chromosome number. All the hybrids had intermediate leaf morphology and possessed LAP isozymes of both parental species. The chromosome analysis revealed a considerable variation in chromosome number of somatic hybrids, showing the occurrence of multiple fusion and chromosome loss during the culture. Some of the hybrids flowered and set seeds.

Asymmetric somatic hybrids of Brassica: partial transfer of B. campestris genome into B. oleracea by cell fusion.

To examine the possibility of producing asymmetric somatic hybrids of Brassica having a complete genome of one species and a part of the other, we fused inactivated B. oleracea protoplasts with X-irradiated B. campestris protoplasts. The plants obtained were studied with regard to their morphology, isozymes and chromosomes. The morphology of the hybrids was similar to B. oleracea in 9 out of 22 hybrids studied and the rest showed the intermediate phenotype of the parents. Analysis of three isozymes, leucine aminopeptidase, acid phosphatase and esterase indicated that ten hybrids lost B. campestris-specific bands in one or more of the three isozymes examined. The chromosome analysis showed that 90% of the hybrids were aneuploids. In addition, abnormal chromosomes were often found in root tip cells. These results suggested that the hybrids obtained were asymmetric in nature and resulted from elimination of B. campestris chromosomes by X-ray irradiation.

Antisense waxy genes with highly active promoters effectively suppress waxy gene expression in transgenic rice.

To regulate Waxy (Wx) gene expression by introducing antisense genes, we connected the 2.3 kb Wx cDNA having 450 bp of the Wx first intron in reverse orientation to rice Wx and maize alcohol dehydrogenase1 (Adh1) promoters and used these constructs to transform rice plants. Of 10 independent transgenic lines analysed, four lines showed various degrees of reduction in amylose and WAXY (WX) protein levels in the endosperm. In two transgenic lines, complete absence of amylose was observed which made the seeds opaque white like glutinous rice (amylose-deficient waxy (wx) mutant). In one of the transgenic lines, A1 line, the presence of the antisense Wx gene cosegregated with reduction of amylose content in the endosperm. In the same line, a reduction in the level of endogenous Wx mRNA was observed in immature endosperm. Interestingly, this reduction was observed only with mature spliced transcripts but not with unspliced transcripts. Reduced amylose synthesis was also observed in pollen grains of four transgenic lines. These results suggest that integrated antisense Wx gene caused a reduction in amylose synthesis in endosperms and pollen grains of transgenic rice carrying the antisense Wx cDNA. These results indicate that manipulation of starch and other carbohydrates in rice grain is possible using antisense genes.

Proximal promoter region of the wheat histone H3 gene confers S phase-specific gene expression in transformed rice cells.

The cis-regulatory elements that confer cell cycle-dependent expression to the wheat histone H3 gene were investigated in rice cells (Oc strain) transformed with H3/GUS chimeric genes. 5' deletion mutants of the H3 promoter region (from -1711, -908 or -185 to +57 relative to the transcription start site) were joined to the coding sequence of the bacterial beta-glucuronidase (GUS) gene then introduced stably into rice cells. S1 analyses of the RNA from transformed rice cells whose cell cycles had been synchronized by treatment with aphidicolin showed that the steady-state levels of the transcripts from chimeric genes were altered with the change in DNA synthesis and the content of rice H3 mRNA throughout the cell cycle. Even though H3 promoter activity decreased as 5' deletion proceeded, transcripts from the chimeric genes showed increases, as much as 10-fold 1 h after release from the aphidicolin block, which were rapidly lost over the next 4 h. The results suggest that the 242 bp sequence from -185 to +57, which contains the basal promoter region, confers the S phase-specific expression of the H3 gene and that the upstream sequence from position -186 is required for the full activity of this promoter.

Development of a new antithrombogenic continuous ultrafiltration system (ACUS) and its clinical evaluation.

A new totally antithrombogenic continuous ultrafiltration system (ACUS), consisting of polyacrylonitrile-polyethyleneoxide (PAN-PEO) membrane and ionically heparin-bound catheter, tubing, and module header was designed and its performance was confirmed through animal experiments and clinical evaluation. Animal experiments revealed that persistent antithrombogenicity and maintained ultrafiltration without systemic heparinisation were observed only when the three major parts--i.e. (1) the catheter and tubing, (2) the header part of a haemofilter module, and (3) the fibre membrane--were all antithrombogenic and connected to each other without uneven structures. ACUS was clinically applied to 15 oliguric patients with various severe conditions. We found that one filter could function for approximately 26 h without systemic anticoagulation, even in the presence of low blood pressure, and that ACUS did not change platelet function. Thus, ACUS seems to be very suitable for the management of volume overload, especially in patients with severe circulatory problems or bleeding tendencies.

Development of a new antithrombogenic continuous ultrafiltration system.

By interfacing a polyacrylonitrile (PAN)-polyethyleneoxide (PEO) membrane with an ionically heparin-bound catheter, tubing, and module header, a totally antithrombogenic continuous ultrafiltration system (ACUS) was developed and its performance, persistent antithrombogenicity, and well-maintained ultrafiltration level were confirmed through animal experiments. Although the amount of heparin released and accumulated in vitro from those heparinized parts was very low and stable (on the order of 1 x 10(-2) U/cm2/min), partial thromboplastin time evaluated in vivo was not elongated during passage through the ACUS. Extracorporeal circulation time with the ACUS in unheparinized dog model was 458 +/- 302 min (n = 24), whereas those of partially modified (antithrombogenic) system did not exceed 100 min. As compared with that in a conventional continuous arteriovenous hemofiltration system, an extracorporeal circulation with the ACUS in an unheparinized dog model revealed significantly less fluctuation of platelet count, and no adherent platelets were observed on the surface of the PAN-PEO membrane. An ACUS consisting of a PAN-PEO membrane and heparinized parts was thus demonstrated to have good platelet compatibility. An ACUS with a surface area of 0.25 m2 was applied to two patients with acute renal failure. Hemofiltration without systemic heparinization lasted for 44 h per hemofilter, and a stable level of ultrafiltration was maintained. This system seems to be applicable for the clinical management of volume overload, especially in patients with bleeding tendencies or postoperative bleeding.

Long-term results of a new antithrombogenic cardiac wall substitute.

We evaluated the long-term results of a new antithrombogenic cardiac wall substitute which is composed of collagen-coated ultrafine polyester mesh cross-linked by a hydrophilic polyepoxy compound (CUFP), and compared it with glutaraldehyde-treated equine pericardium (GA graft). In an animal study, 20 CUFPs and 18 GA control grafts were implanted as patches in the right ventricular outflow tract in 38 dogs. In the CUFP at 28 days after implantation, a thin neointima, which was almost endothelialized, had been formed. Fibroblasts and vasa vasorum were seen inside both the neointima and the graft wall. At 168 days, transmission electron microscopy revealed smooth muscle-like cells in the neointima, with endothelialization. The CUFP at 486 days had maintained a white, shiny, smooth, thin, and uniform neointima with endothelialization. Neither ulcer nor thrombus formation was seen on the surface, while in the GA graft at 353 days, no endothelialization was noticed in the center and almost no fibroblasts had infiltrated into the graft wall. From these results, the CUFP should be an ideal candidate for cardiac wall substitution.

Mallory-Weiss syndrome with severe bleeding: treatment by endoscopic ligation.

There is no consensus as to the best treatment for Mallory-Weiss tears with severe bleeding. Endoscopic ligation is an inexpensive, readily available, and easily learned technique, in contrast to conventional thermocoagulation or hemoclipping. To evaluate the utility of endoscopic ligation, we performed this technique during emergency endoscopy to treat severe bleeding from Mallory-Weiss tears in four patients in our hospital with continuous active bleeding from Mallory-Weiss tears. The patients were all male with an average age of 40.5 years. Symptoms associated with increased intra-abdominal pressure, including retching and vomiting were reported by all patients. The bleeding points were aspirated and controlled by endoscopic ligation, and complete hemostasis was achieved in all cases. We conclude that endoscopic ligation is easy to perform and may provide an alternative treatment for severe bleeding from Mallory-Weiss tears.