Functional analysis of titin/connectin N2-B mutations found in cardiomyopathy

Hypertrophic cardiomyopathy and dilated cardiomyopathy are two major clinical phenotypes of “idiopathic” cardiomyopathy. Recent molecular genetic analyses have now revealed that “idiopathic” cardiomyopathy is caused by mutations in genes for sarcomere components. We have recently reported several mutations in titin/connectin gene found in patients with hypertrophic cardiomyopathy or dilated cardiomyopathy. A hypertrophic cardiomyopathy-associated titin/connectin mutation (Arg740Leu) was found to increase the binding to actinin, while other dilated cardiomyopathy-associated titin/connectin mutations (Ala743Val and Val54Met) decreased the binding to actinin and Tcap/telethonin, respectively. We also reported several other mutations in the N2-B region of titin/connectin found in hypertrophic cardiomyopathy and dilated cardiomyopathy. Since the N2-B region expresses only in the heart, it was speculated that functional alterations due to the mutations cause cardiomyopathies. In this study, we investigated the functional changes caused by the N2-B region mutations by using yeast-two-hybrid assays. It was revealed that a hypertrophic cardiomyopathy-associated mutation (Ser3799Tyr) increased the binding to FHL2 protein, whereas a dilated cardiomyopathy-associated mutation (Gln4053ter) decreased the binding. In addition, another TTN mutation (Arg25618Gln) at the is2 region was found in familial DCM. Because FHL2 protein is known to tether metabolic enzymes to N2-B and is2 regions of titin/connectin, these observations suggest that altered recruitment of metabolic enzymes to the sarcomere may play a role in the pathogenesis of cardiomyopathies.     

EXPRESSION OF MUC APOMUCINS IN NORMAL PANCREAS AND PANCREATIC TUMOURS

The epithelial expression of apomucins MUC1, MUC2, MUC3, and MUC5/6 was examined in normal pancreas and in pancreatic lesions, using immunohistochemical methods. In normal pancreas ( n =5), MUC1 apomucin was expressed in ducts and some acini, but there was no expression of MUC2, MUC3, or MUC5/6. In chronic pancreatitis ( n =5), MUC1 apomucin was expressed, but expression of the other apomucins was not noted. However, mucous hyperplastic foci of pancreatic ducts expressed MUC5/6 apomucin in 2/5 cases (40 per cent). In intraductal papillary-mucinous neoplasm (IPMN) of the pancreas ( n =9), MUC1, MUC2, MUC3, and MUC5/6 apomucins were expressed in 8/9 (89 per cent), 0/9 (0 per cent), 4/9 (44 per cent), and 9/9 (100 per cent) cases, respectively. In pancreatic mucinous cystadenoma ( n =8), MUC1, MUC2, MUC3, and MUC5/6 apomucins were expressed in 7/8 (88 per cent), 0/8 (0 per cent), (25 per cent), and 3/8 (38 per cent) cases, respectively. In invasive ductal adenocarcinoma of the pancreas ( n =25), expression of MUC1, MUC2, MUC3, and MUC5/6 apomucins was found in 25/25 (100 per cent), 1/25 (4 per cent), 20/25 (80 per cent), and 24/25 (96 per cent) cases, respectively. Atypical mucous duct hyperplasia near cancer cells consistently expressed MUC1 apomucin and occasionally expressed MUC3 and MUC5/6. In positive cases, MUC1 apomucin expression was noted in the cell membrane facing the ductal or neoplastic lumina, while expression of MUC2, MUC3, and MUC5/6 apomucins was found in the cytoplasm. These results suggest that MUC3 and MUC5/6 apomucins newly emerge during the neoplastic transformation of pancreatic mucinous cystadenoma and IPMN and during pancreatic ductal carcinogenesis, while MUC1 apomucin remains positive and MUC2 apomucin remains almost negative during neoplastic transformation.     

Effects of human neonatal serum on DNA synthesis in suckling and adult rat hepatocytes in primary culture

The effect of human neonatal serum on DNA synthesis in suckling and adult rat hepatocytes in primary culture was investigated to characterize growth regulating factors of the liver in neonates and to confirm whether the stimulatory factor is human hepatocyte growth factor (hHGF). Neonatal serum stimulated DNA synthesis of both adult and suckling rat hepatocytes. The stimulatory effect was dose-dependent up to 20% in volume. The molecular weight of the stimulatory substance in neonatal serum was between 12 500 and 25 000, as estimated by gel filtration. Its activity was stable after heating at 56°C for 20 min, but was lost after heating at 90°C for 30 s, and easily passed through S- or heparin-Sepharose columns. The concentration of hHGF quantified by ELISA was too low to stimulate DNA synthesis in vitro. Biological and biochemical properties of the growth stimulatory activity in neonatal serum differed from that of hHGF. The presence of other growth factors in human neonatal serum for suckling and adult hepatocytes was suggested.     

Successful Endoscopic Treatment of Diffuse Antral Vascular Ectasia and Early Gastric Carcinoma in an Aged Patient

Diffuse antral vascular ectasia and a lla type early gastric cancer were detected in an 83-year-old male patient through an endoscopy during treatment for iron deficiency anemia. Monopolar electrocoagulation was effective for eradication of red spots which represented ectatic vessels. Five sessions of the therapy resulted in the ablation of almost all of the vascular lesions, negative fecal occult blood tests and improvement of his anemia. The lla lesion was endoscopically resected from the posterior wall of the upper body. Histologic examination demonstrated well differentiated adenocarcinoma confined to the mucosal layer. Both the vascular ectasia and carcinoma did not show any sign of recurrence after a year and a half.     

Effect of manoalide on apoptosis induced by deprivation of growth factors in vascular endothelial cells

研究ｍａｎｏａｌｉｄｅ对除去生长因子（ａＦＧＦ和血清）而诱导的血管内皮细胞凋亡的影响．方法：通过细胞形态观察,ＤＮＡ凝胶电泳及荧光显微术等方法确定ｍａｎｏａｌｉｄｅ对细胞凋亡的抑制或促进作用．结果：向去除ａＦＧＦ和血清的培养液中加低浓度的ｍａｎｏａｌｉｄｅ（１－４μｍｏｌ·Ｌ－１）,培养细胞４８ｈ,细胞的脱壁和ＤＮＡ片断化受到抑制;ｍａｎｏａｌｉｄｅ浓度为７μｍｏｌ·Ｌ－１时,促进细胞脱壁和ＤＮＡ片断化．结论：低浓度的ｍａｎｏａｌｉｄｅ（２μｍｏｌ·Ｌ－１）抑制血管内皮细胞凋亡,而较高浓度的ｍａｎｏａｌｉｄｅ（７μｍｏｌ·Ｌ－１）促进该细胞凋亡．     

Recording of electroencephalograms and electrocardiograms during daytime sleep in trained canines: Preparation of the sleeping dogs

Abstract  Although respiration in trained canines is well investigated, the process of preparing dogs has not been described in any great detail. Moreover, their daytime patterns of sleep and wakefulness during 1 or 2 h of electroencephalogram (EEG) and electrocardiogram (ECG) recordings are not clear. Therefore, we describe the process of selecting and training dogs, in which we recorded EEG and ECG in the laboratory. First, 14 of 1242 dogs dealt with over a 1 year period were chosen. They were trained for 2 h to lie quietly and to sleep in the laboratory; this training procedure was repeated 152 times. Three dogs were then selected and a permanent tracheostomy was performed in one. Finally, EEG and ECG were recorded with the bipolar fine needle electrodes; respiration was recorded simultaneously through a tube inserted to a tracheostomy in one dog. Wakefulness, slow wave sleep (SWS) and rapid eye movement (REM) sleep (REMS) were identified according to the EEG pattern and on the basis of the behavioral criteria. Recordings were performed 12 or 13 times in each dog. Complete sleep cycles, including wakefulness, SWS and REMS in this sequence, were observed 3.9–4.1 times. The mean duration of SWS was 2.2–4.4 min and that of REMS was 3.5–4.6 min. The REMS latency was 33.9–41.8 min. Fluctuation of heart rate with respiration, termed respiratory sinus arrhythmia, was noted in the ECG. Heart beat increased with inspiration and decreased with expiration. The present study demonstrates how to select and train sleeping dogs and shows their undisturbed daytime sleep and wakefulness patterns.     

The microbial flora from root canals and periodontal pockets of non-vital teeth associated with advanced periodontitis

Microflora from root canals and periodontal pockets of periodontally affected teeth were compared in order to elucidate the as yet unknown relationship between pulpal and periodontal disease. Caries-free teeth affected with advanced periodontitis and diagnosed as clinically dead by electric pulp testing were selected. The root canals and periodontal pockets were sampled, and the bacterial flora examined by both culture and interference microscopy. The results indicated that the aerobe/anaerobe ratio in the periodontal pocket was 0.23, while it was 0.0022 in the root canal, the large predominance of obligate anaerobes reflecting the anaerobic environment found in the root canal. Morphological classification obtained from interference microscopy showed similar proportions of morphotypes in the two sites. Results of anaerobic culture demonstrated a significantly higher rate of detection of facultative Streptococcus bacteria in the periodontal pocket than in the root canal. The predominant bacterial species common to both regions were Streptococcus, Peptostreptococcus, Eubacterium, Bacteroides, and Fusobacterium for obligate anaerobes. As for facultative anaerobes, Actinomyces and Streptococcus were detected predominantly in the periodontal pocket. The occurrence of micro-organisms common to both sites in this study suggests that the periodontal pocket may be a possible source of root canal infections.     

Cyclic peptides

In order to investigate the role of the L-2-hydroxy-3-methylbutanoic acid residue at position 2 of AM-toxin I (cyclic tetradepsipeptide) on necrotic activity for apple leaves, two analogs, [L-lactic acid²] AM-toxin I and [L-2-hydroxy-4-methylpentanoic acid²] AM-toxin I, were synthesized by the conventional method for peptide synthesis. The toxic activity of the former analog was much weaker than that of the natural AM-toxin I and the latter analog. CD spectra of AM-toxin I and these analogs were correlated to those toxic activities, indicating that bulkiness of the side chain at position 2 is important for the maintenance of active conformation and the induction of necrotic activity.     

Novel technique for correcting penile curvature with severe hypospadias; Ventral lengthening with tunica vaginalis flap patching

The adequate correction of penile curvature is essential for successful hypospadias surgery. We describe a novel technique to correct severe penile curvature with a tunica vaginalis flap on the penile ventrum. We applied a tunica vaginalis flap to lengthen the ventral aspect of the tunica albuginea in two boys with significant curvature and proximal hypospadias. Tunica vaginalis flap patching to the ventral aspect of the penis is safe and technically feasible. If penile curvature is severe or the penis is small in hypospadiac patients, lengthening the ventral aspect using a tunica vaginalis flap is likely to expand instead of dorsal plication or ventral graft.