Sotaro Mushiake

The Rab8 GTPase regulates apical protein localization in intestinal cells

A number of proteins are known to be involved in apical/basolateral transport of proteins in polarized epithelial cells. The small GTP-binding protein Rab8 was thought to regulate basolateral transport in polarized kidney epithelial cells through the AP1B-complex-mediated pathway. However, the role of Rab8 (Rab8A) in cell polarity in vivo remains unknown. Here we show that Rab8 is responsible for the localization of apical proteins in intestinal epithelial cells. We found that apical peptidases and transporters localized to lysosomes in the small intestine of Rab8-deficient mice. Their mislocalization and degradation in lysosomes led to a marked reduction in the absorption rate of nutrients in the small intestine, and ultimately to death. Ultrastructurally, a shortening of apical microvilli, an increased number of enlarged lysosomes, and microvillus inclusions in the enterocytes were also observed. One microvillus inclusion disease patient who shows an identical phenotype to Rab8-deficient mice expresses a reduced amount of RAB8 (RAB8A; NM_005370). Our results demonstrate that Rab8 is necessary for the proper localization of apical proteins and the absorption and digestion of various nutrients in the small intestine.

A blocking peptide for transforming growth factor-β1 activation prevents hepatic fibrosis in vivo

BACKGROUND/AIMS Thrombospondin-1 is a major activator of transforming growth factor-beta1 (TGF-beta1), and a peptide derived from the latency-associated peptide, Leu-Ser-Lys-Leu (LSKL), inhibits the activation of TGF-beta1. In this study, the effects of LSKL on the hepatocyte damage and fibrogenesis in dimethylnitrosamine (DMN)-induced rat liver fibrosis were examined. METHODS Animals were given an intraperitoneal (i.p.) injection of DMN or saline three times per week for 4 weeks, and treated with LSKL, a control peptide, or saline i.p. daily. RESULTS Liver atrophy caused by DMN-injection was significantly inhibited in the DMN+LSKL group. The degrees of necrosis/degeneration and fibrosis scores were significantly lower in the DMN+LSKL group than in the control groups. The hydroxyproline content was significantly higher in the control groups than in the DMN+LSKL group. The amount of active TGF-beta1 was less in the DMN+LSKL group than in the control groups, and the active/total TGF-beta1 ratio in the DMN+LSKL group was suppressed in the control groups. Phosphorylation of Smad 2 in the liver was significantly decreased in the DMN+LSKL group. CONCLUSIONS The LSKL peptide prevented the progression of hepatic damage and fibrosis through the inhibition of TGF-beta1 activation and its signal transduction in vivo.

Clinical phenotype and endocrinological investigations in a patient with a mutation in the MCT8 thyroid hormone transporter

Thyroid hormones are known to be essential for growth, development, and metabolism. Recently, the monocarboxylate transporter 8 (MCT8) was identified as a thyroid hormone transporter, and MCT8 mutations have been associated with Allan-Herndon-Dudley syndrome, an X linked condition characterized by severe mental retardation, dysarthria, athetoid movements, muscle hypoplasia, and spastic paraplegia. Here we describe in detail the clinical and biochemical features and the response to thyroid hormone (L-thyroxine (LT4)) administration in a boy with an MCT8 mutation (c.1649delA) that truncates the protein in the twelfth transmembrane domain. It is of note that brain magnetic resonance imaging (MRI) revealed delayed myelination from infancy. Endocrine functions other than thyroid hormone regulation and metabolism were intact, resulting in normal hypothalamic/pituitary function tests. While LT4 administration suppressed thyrotropin (TSH) secretion, no significant changes in thyroid hormone values or clinical symptoms were observed. Conclusion: the characteristic thyroid hormone function tests and brain MRI findings may allow screening of high-risk populations for a better understanding of MCT8 pathophysiology.

A Large Outbreak of Foodborne Infection Attributed to Providencia alcalifaciens

The enteropathogenicity of Providencia alcalifaciens, a member of the family Enterobacteriaceae, has not yet been well established. In November 1996, a large outbreak of foodborne infection occurred in Fukui, Japan. In this study, the etiology of the outbreak was investigated. No other recognized enteropathogens were detected in patient fecal samples, but P. alcalifaciens was detected in 7 of 18 samples. The isolates were found to be clonal by pulsed-field gel electrophoresis. The patients who presented with gastroenteritis had elevated levels of specific antibody against the isolated P. alcalifaciens. The isolates showed invasion of Caco-2 cells and fluid accumulation in rabbit ileal loops. This study strongly suggests that the outbreak was caused by P. alcalifaciens. This is the first report of a large outbreak of foodborne infection attributed to the organism and provides definitive evidence that P. alcalifaciens is a causative agent of gastroenteritis.

Chronic hepatitis in an infant, in association with human herpesvirus-6 infection

A 20-month-old boy was investigated for persistent liver dysfunction. Liver histologic findings showed chronic hepatitis. The presence of human herpesvirus-6 DNA in liver tissue was demonstrated both by in situ hybridization and by polymerase chain reaction. Human herpesvirus-6 may be a causative agent of chronic hepatitis in this case.

Rab11a is required for apical protein localisation in the intestine.

ABSTRACT The small GTPase Rab11 plays an important role in the recycling of proteins to the plasma membrane as well as in polarised transport in epithelial cells and neurons. We generated conditional knockout mice deficient in Rab11a. Rab11a-deficient mice are embryonic lethal, and brain-specific Rab11a knockout mice show no overt abnormalities in brain architecture. In contrast, intestine-specific Rab11a knockout mice begin dying approximately 1 week after birth. Apical proteins in the intestines of knockout mice accumulate in the cytoplasm and mislocalise to the basolateral plasma membrane, whereas the localisation of basolateral proteins is unaffected. Shorter microvilli and microvillus inclusion bodies are also observed in the knockout mice. Elevation of a serum starvation marker was also observed, likely caused by the mislocalisation of apical proteins and reduced nutrient uptake. In addition, Rab8a is mislocalised in Rab11a knockout mice. Conversely, Rab11a is mislocalised in Rab8a knockout mice and in a microvillus atrophy patient, which has a mutation in the myosin Vb gene. Our data show an essential role for Rab11a in the localisation of apical proteins in the intestine and demonstrate functional relationships between Rab11a, Rab8a and myosin Vb in vivo.

Severe mandibuloacral dysplasia caused by novel compound heterozygous ZMPSTE24 mutations in two Japanese siblings

Mandibuloacral dysplasia (MAD) is a rare autosomal recessive progeroid syndrome, characterized by mandibular hypoplasia, acroosteolysis affecting distal phalanges and clavicles, delayed closure of the cranial sutures, atrophic skin, and lipodystrophy. Recently, mutations in lamin A/C (LMNA) and zinc metalloprotease (ZMPSTE24), involved in post‐translational processing of prelamin A to mature lamin A, have been identified in MAD kindreds. We now report novel compound heterozygous mutations in exon 1 (c.121C>T; p.Q41X) and exon 6 (c.743C>T; p.P248L) in ZMPSTE24 in two Japanese sisters, 7‐ and 3‐year old, with severe MAD and characteristic facies and atrophic skin. The older sister had lipodystrophy affecting the chest and thighs but sparing abdomen. Their parents and a brother, who were healthy, had heterozygous mutations. The missense mutation, P248L, was not found in 100 normal subjects of Japanese origin. The mutant Q41X was inactive in a yeast halo assay; however, the mutant P248L retained near normal ZMPSTE24 activity. Immunoblots demonstrated accumulation of prelamin A in the patients’ cell lysates from lymphoblasts. The lymphoblasts from the patients also revealed less intense staining for lamin A/C on immunofluorescence. We conclude that ZMPSTE24 deficiency results in accumulation of farnesylated prelamin A, which may be responsible for cellular toxicity and the MAD phenotype.

Frequent Detection of the Human Herpesvirus 6-Specific Genomes in the Livers of Children with Various Liver Diseases

ABSTRACT This study was performed to investigate the frequency of human herpesvirus 6 (HHV-6) infection of the liver in children with a variety of liver diseases and to evaluate the role of HHV-6 infection in pediatric patients with prolonged non-B non-C hepatitis. Detection of the HHV-6 genomes in liver, in peripheral blood mononuclear cells (PBMC), and in plasma was performed by PCR or by in situ hybridization. Liver biopsy materials from 48 patients, in whom HHV-6 infection was serologically confirmed, were available for PCR analysis. Sequences of the HHV-6B genome were detectable in the livers of 36 of 48 patients (75%). The presence of the genome was not associated with serum transaminase activities. The genome was detectable in PBMC of 22 of 31 (71%) patients tested. In these 31 patients HHV-6 was detected in both the livers and PBMC of 20, was detected in PBMC but not in the livers of 2, was detected in the livers but not in PBMC of 3, and was detected in neither of samples of 6. In situ hybridization of the livers of six patients showed the presence of the HHV-6B genome in the nuclei of hepatocytes. The anti-HHV-6 immunoglobulin M antibody was detectable in 2 of 9 of the non-B non-C hepatitis patients, whereas none of the 22 patients with etiology-defined liver diseases tested positive. Cell-free viral DNA was not detectable in either group of patients. Our results showed that HHV-6B is frequently present in the livers of children with a variety of liver diseases but do not support the assumption that HHV-6B infection of the liver is associated with prolonged non-B non-C hepatitis.

Histological findings in the livers of patients with neonatal intrahepatic cholestasis caused by citrin deficiency

Aim:  To characterize the histological features of the livers of patients with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), we studied specimens from 30 patients diagnosed with NICCD by genetically analyzing the SLC25A13 gene.

Hypomelanosis of Ito associated with hemimegalencephaly: A clinicopathological study

A girl with hypomelanosis of Ito was studied both clinically and at postmortem examination. She manifested severe epilepsy early after birth. Magnetic resonance imaging demonstrated left-sided hemimegalencephaly. The seizures were secondarily generalized or unilateral initially, followed by infantile spasms with asymmetrical hypsarrhythmia at 1.5 months of age. Frequent complex partial seizures, refractory to anti-epileptic drug treatments appeared at 4 months of age. She died of pneumonia at the age of 14 months. Postmortem examination revealed marked asymmetry of the cerebrum and gyral abnormalities in the left cerebral hemisphere. Histopathologically, severe disorganization of the neuronal cytoarchitecture was evident. Absence of delineation between cortical gray and white matter was evident, as was increase and hypertrophy of the neurons and glial cells. We believe that the association of skin and brain lesions was not one of chance; that is, they may share a common pathogenetic mechanism.