Freeze-fracture studies of muscle plasma membranes in myopathic patients with hypo-and hyperthyroidism

To examine the influence of thyroid hormone on the skeletal muscle plasma membrane, we analyzed the changes in ultrastructural architecture and membrane area complexed with digitonin of muscle plasma membrane in myopathic patients with hypo-and hyperthyroidism by the conventional freeze-fracture (F-F) technique and F-F cytochemistry using the sterol-specific ligand digitonin. The densities of flask-shaped invaginations, which are mainly thought to correspond to caveolae, intramembranous particles, and orthogonal arrays, and the changes of digitoninreacted membrane areas in the muscle plasma membranes in three patients with hypothyroid myopathy and one patient with both myasthenia gravis and hyperthyroidism were compared with those in age-matched controls. In the conventional F-F study, the muscle plasma membrane of hypothyroid patients showed increased invagination density, whereas that of the hyperthyroid patient was normal ultrastructurally. In the F-F cytochemistry study, however, the ratio of digitonin-reacted membrane areas versus fractured membrane areas was not different between hypothyroid patients and controls, whereas that of the hyperthyroid patient was lowered in comparison with that of control. These results suggest that thyroid hormone may alter the biochemical properties and ultrastructural architecture of muscle plasma membrane.This study was presented at the 29th annual meeting of the Clinical Electron Microscopy Society of Japan, Sapporo, October 2–3, 1997     

An immunohistochemical study of bone morphogenetic protein in pleomorphic adenoma of the salivary gland

Bone morphogenetic protein (BMP) is a potent induction factor for new bone formation including heterotopic chondro-ossification in soft tissues. The immunohistochemical reaction for BMP was studied in 23 cases of pleomorphic adenoma of salivary gland by using a monoclonal antibody produced by hybridoma technique. Positive BMP immunoreactivity was seen in 87% of tumours. Immunohistochemical expression of BMP was observed in modified myoepithelial cells (88% cases), luminal tumour cells of tubulo-ductal structures (78% cases) and chondroid cells in hyaline tissue (22% cases). The authors concluded that the simultaneous presence of glycosaminoglycans as matrix substance and S-100 protein for calcium signalling are associated with BMP-mediated cellular activity of modified myoepithelial cells in the formation of chondroid structures in pleomorphic adenomas of the salivary glands.     

Induction of interferon-inducible protein-10 and monokine induced by interferon-gamma from human endothelial cells infected with Influenza A virus

Primary human umbilical vein endothelial cells (HUVECs) were infected with Influenza virus A/Aichi/2/68 (H3N2) in order to determine the role of endothelial cells in mediating inflammation induced upon virus infection. Structural proteins of the virus and mRNA of the M2 protein were detected in the infected cells, indicating that virus infection had occurred in HUVECs. The Influenza A virus-infected HUVECs showed elevated levels of gene expression of interferon (IFN)-inducible protein (IP)-10 and monokine induced by IFN-gamma (Mig), while heat-, formalin- and diethyl ether-inactivated viruses did not enhance the IP-10 and Mig gene expression. The results thus indicate that infection of live Influenza A virus is responsible for elevation of IP-10 and Mig gene expression. The elevation of IP-10 and Mig gene expression in infected HUVECs was not accompanied by the elevation of IFN-gamma gene expression, indicating that the elevation of IP-10 and Mig gene expression was independent of the IFN-gamma pathway.     

Precursor genes of future pandemic influenza viruses are perpetuated in ducks nesting in Siberia

Summary.  Influenza A viruses of different subtypes were isolated from fecal samples of ducks in their nesting areas in Siberia in summer from 1996 to 1998. Phylogenetic analysis of the NP genes of the isolates in Siberia and those in Hokkaido, Japan on their flyway of migration from Siberia to the south in autumn revealed that they belong to the Eurasian lineage of avian influenza viruses. It is noted that the genes of the isolates in Siberia are closely related to those of H5N1 influenza virus strains isolated from chickens and humans in Hong Kong in 1997 as well as to those of isolates from domestic birds in southern China. The results indicate that influenza viruses perpetuated in ducks nesting in Siberia should have contributed genes in the emergence of the H5N1 virusin Hong Kong. Vaccine prepared from avirulent A/duck/Hokkaido/4/96 (H5N3) influenza virus was potent enough to protect mice from challenge with lethal dose of the pathogenic H5N1 virus [19]. Intensive surveillance study of aquatic birds especially in Siberia is, therefore, stressed to provide information on the future pandemic influenza virus strains and for vaccine preparation. Received August 24, 1999/Accepted January 7, 2000     

The T-cell receptor alpha, beta and gamma polymorphism in Japanese

Polymorphism in the genes encoding the alpha (alpha), beta (beta) and gamma (gamma) chains of the human T-cell receptors was analyzed both in population and family studies. Against twelve unrelated Japanese, several out of the 15 restriction endonucleases tested, revealed restriction fragment length polymorphism. The segregation of the polymorphic fragments were confirmed among 15 members of three families. In most of the cases paternal and/or maternal haplotypes could be assigned. By testing the polymorphic enzymes among the random healthy Japanese, the frequency of each polymorphic fragment was then determined. Although the polymorphism found in this study was similar to that reported in Caucasians, some differences were observed. Such differences are discussed. The restriction fragment length polymorphism in both population and family studies, derived from alpha, beta and gamma chains of the T-cell receptor found in this report, might be useful markers for genetic analysis of the T-cell function in relation to immunological disorders.     

Distribution of 5-hydroxytryptamine (5HT) in tissue of a mutant mouse deficient in mast cell (W/W v ). Demonstration of the contribution of mast cells to the 5HT content in various organs

The content of 5-hydroxytryptamine (5HT) in various tissues of mutant mouse (W/W ^v) deficient in mast cells and of control mouse (+/+) was determined by high performance liquid chromatography. The depletion of mast cells in the mutant mouse (W/W ^v) was expected to cause a decrease in the 5HT content. In the control mice, 5HT was most densely accumulated in the lung (9.66±5.23 g/g). Large intestine (6.40±2.61 g/g) and stomach (6.10±2.14 g/g) followed the lung in the rating of the 5HT content. The 5HT content ofW/W ^v mice was only 23.4% and 4.1% that of the control in the stomach (p<0.01) and the skin (p<0.01), respectively. The results were consistent with the expectation. In other organs (small intestine, caecum, large intestine, brain, lung, blood and salivary gland), the difference between theW/W ^v and normal mice was not statistically significant. The difference in the 5HT content of the stomach between the two genotypes was 4.67 g/g and was much larger than the 5HT content (0.49g/g) of normal mouse skin. With regard to the relatively small number of mast cells present in the stomach, the great difference in the 5HT content in the stomach between the two genotypes cannot be explained by the loss of mast cells. Hence, besides mast cells other cells may contribute to the high 5HT content of the stomach.This work was supported in part by grants from Takeda Science Foundation (1982), from the Ministry of Education, Science and Culture of Japan (Grant-in-Aid for Special Project Research. 1981–83) and from National Center of Nervous, Mental and Muscular Disorders (NCNMMD) of the Ministry of Health and Welfare, Japan (1981–83).To whom correspondence should be addressed     

Immunohistochemical,ultrastructural and biochemical studies of an amylase-producing breast carcinoma

Summary We describe a breast cancer with ectopic production of amylase, found in the patient's serum, urine and in the tumour. Clinically, serum amylase levels reflected both the progression of the disease and regression induced by various therapies. Using agarose gel electrophoresis and a wheat protein inhibitor assay, the predominant serum amylase appeared to be identical to pancreatic-type isoenzyme. However, the action mode analysis using a new fluorogenic substrate revealed that the serum contained non-salivary, non-pancreatic amylase. The tumour had microscopic features of invasive ductal carcinoma with some argyrophilic differentiation. The component cells stained positively for amylase, and ultrastructurally numerous secretory granules were seen.     

Loss of Hrs in the Central Nervous System Causes Accumulation of Ubiquitinated Proteins and Neurodegeneration

The endosomal sorting complex required for transport (ESCRT) proteins form multimolecular complexes that control multivesicular body formation, endosomal sorting, and transport ubiquitinated membrane proteins (including cell-surface receptors) to the endosomes for degradation. There is accumulating evidence that endosomal dysfunction is linked to neural cell degeneration in vitro, but little is known about the relationship between neural disorders and ESCRT proteins in vivo. Here we specifically deleted the hrs gene, ESCRT-0, in the neurons of mice by crossing loxP-flanked hrs mice with transgenic mice expressing the synapsin-I Cre protein (SynI-cre). Histological analyses revealed that both apoptosis and a loss of hippocampal CA3 pyramidal neurons occurred in the hrs^flox/flox;SynI-cre mice. Notably, the hrs^flox/flox;SynI-cre mice accumulated ubiquitinated proteins, such as glutamate receptors and an autophagy-regulating protein, p62. These molecules are particularly prominent in the hippocampal CA3 neurons and cerebral cortex with advancing age. Accordingly, we found that both locomotor activity and learning ability were severely reduced in the hrs^flox/flox;SynI-cre mice. These data suggest that Hrs plays an important role in neural cell survival in vivo and provide an animal model for neurodegenerative diseases that are known to be commonly affected by the generation of proteinaceous aggregates.