Stage-specific localization of cytoskeletal actin mRNA in murine seminiferous tubules and intestinal epithelia as demonstrated by in-situ hybridization

Summary In-situ hybridization experiments have been performed using isoactin ( and )-specific riboprobes in various tissues of the rat and mouse. Distribution of the grains of actin mRNAs for both and types was similar throughout sections of the rat testis. Although both mRNAs were evenly distributed in the seminiferous tubule, extremely heavy labeling was observed in about 10% of the seminiferous tubules that could be identified as stage XII of spermatogenesis. At high magnification, grains of the mRNA were found in the cytoplasm of elongating spermatids and in the Sertoli cell cytoplasm at the adluminal side. Much higher density of the grains of mRNA was observed in the neck region of the spermatids at stage XII. Thus, the dense distribution of cytoskeletal actin mRNAs is stage-specific in the tubule during spermatogenesis in the rat. The high expression of both and actin mRNAs was also observed in the epithelial cells of the intestinal crypts.     

Cross-linking of plasmalemmal cholesterol in lymphocytes induces capping, membrane shedding, and endocytosis through coated pits.

By use of a nicked and biotinylated perfringolysin O (BCtheta), which binds to cholesterol specifically, we studied consequences of cross-linking cholesterol in lymphocytes. When bound with BCtheta and then with labeled avidin or streptavidin, capping occurred in most cells within 30 min at 37 degrees C. It was inhibited by cytochalasin D or NaN3, but not by nocodazole. When BCtheta-cholesterol was capped, Thy-1 and transferrin receptor, a GPI-anchored protein and a transmembrane protein, respectively, remained evenly distributed. By fluorescence and electron microscopy, a cluster of small vesicles bound with BCtheta were observed in the cap. They were then shed in the medium or internalized through coated pits. The result indicates that cross-linking of cholesterol in lymphocytes induces capping, but does not affect distribution of membrane proteins, and that the capped cholesterol molecules are either shed as vesicles or endocytosed.     

Xenobrama microlepis,a new genus and species of bramid fish,from subantarctic waters of the South Pacific

Xenobrama microlepis gen. et sp. nov. is described on the basis of 17 adult and subadult specimens collected by surface gill nets, bottom trawl and midwater trawl from the subantarctic waters of the South Pacific Ocean. This monotypic new genus is distinguished from other bramid genera by the following characters: inner lower edges of mandible touching each other (except near symphysis); gill rakers short, thick, and stout; subpectoral region very narrow; interpelvic space flat and wide; vertebrae 49–51; and scales in longitudinal series more than 83. The new taxon is widely distributed in the high seas of the South Pacific, 38–54°S, 79–176°W, but is rather rare compared toBrama spp. in catches of drift gill nets.     

La autoantigen translocates to cytoplasm after cleavage during granzyme B-mediated cytotoxicity

Our recent report demonstrated that apoptosis-specific autoantibodies against granzyme B-induced cleavage fragments of SS-B (La) were found in the sera from patients with primary Sj?gren's syndrome. The objective of this study was identified by the intracellular redistribution of La autoantigen during granzyme B-induced apoptosis. We developed green fluorescence protein (GFP)-La and GFP-LaDelta220 (generation of granzyme B-specific cleavage of La protein) fusion proteins. GFP-La protein was localized in the nucleus, whereas the GFP-LaDelta220 protein predominantly existed in the cytoplasm in transformed A293T cells. Nuclear GFP-La protein was translocated to cytoplasm after granzyme B enriched YT cells incubation. La protein in human salivary grand HSG cells is cleaved and translocated from the nucleus to the cytoplasm after YT cell co-cultivation. These results suggest that La protein is cleaved by granzyme B and N-terminal La fragment (27 kD) translocated to the cytoplasm, thus leading to a novel autoantibody production during granzyme B-mediated cytotoxicity.     

Defective peripheral nerve myelination and neuromuscular junction formation in fukutin-deficient chimeric mice

Dystroglycan is a central component of the dystrophin-glycoprotein complex that links the extracellular matrix with cytoskeleton. Recently, mutations of the genes encoding putative glycosyltransferases were identified in several forms of congenital muscular dystrophies accompanied by brain anomalies and eye abnormalities, and aberrant glycosylation of alpha-dystroglycan has been implicated in their pathogeneses. These diseases are now collectively called alpha-dystroglycanopathy. In this study, we demonstrate that peripheral nerve myelination is defective in the fukutin-deficient chimeric mice, a mouse model of Fukuyama-type congenital muscular dystrophy, which is the most common alpha-dystroglycanopathy in Japan. In the peripheral nerve of these mice, the density of myelinated nerve fibers was significantly decreased and clusters of abnormally large non-myelinated axons were ensheathed by a single Schwann cell, indicating a defect of the radial sorting mechanism. The sugar chain moiety and laminin-binding activity of alpha-dystroglycan were severely reduced, while the expression of beta1-integrin was not altered in the peripheral nerve of the chimeric mice. We also show that the clustering of acetylcholine receptor is defective and neuromuscular junctions are fragmented in appearance in these mice. Expression of agrin and laminin as well as the binding activity of alpha-dystroglycan to these ligands was severely reduced at the neuromuscular junction. These results demonstrate that fukutin plays crucial roles in the myelination of peripheral nerve and formation of neuromuscular junction. They also suggest that defective glycosylation of alpha-dystroglycan may play a role in the impairment of these processes in the deficiency of fukutin.     

Conidioma production of the white root rot fungus in axenic culture under near-ultraviolet light radiation

 Conidiomata of the white root rot fungus were produced in axenic culture under near-ultraviolet light radiation. Pieces of sterilized Japanese pear twigs were placed on 7-day-old oatmeal agar culture in plates. The plates were further incubated for 5 days and then illuminated by near-ultraviolet light. Synnemata developed on the twigs within 5 weeks in 19 of 20 isolates tested, and conidia were observed in 12 of the 19 isolates. The synnemata and conidia produced were morphologically identical to those of Dematophora necatrix. Received: October 29, 2001 / Accepted: March 11, 2002     

Evidence for the presence and structure of asparagine-linked oligosaccharide units in the core protein of proteodermatan sulphate.

Hormonally produced changes in the synthesis and secretion of the serum copper-containing protein caeruloplasmin were studied in primary cultures of rat liver parenchymal cells isolated by the collagenase-perfusion technique. A rabbit antibody directed against rat caeruloplasmin was used to immunoprecipitate labelled caeruloplasmin. Isolated liver cells synthesized and secreted caeruloplasmin over a period of 3 days. Synthesis and secretion of this protein was enhanced when cells were treated with dexamethasone. The accumulation of copper was also moderately enhanced with glucocorticoid treatment. Inclusion of adrenaline in the culture medium resulted in elevated incorporation of copper into newly synthesized caeruloplasmin as well as an increase in 64Cu-labelled caeruloplasmin in the culture medium. However, adrenaline did not seem to increase the secretion of 3H-labelled protein, despite the elevation in secreted 64Cu-caeruloplasmin. This may be due to a large increase in the intracellular pool of 64Cu caused by enhanced accumulation of this metal when adrenaline is included in the incubation medium. Enhanced copper accumulation was also seen when cells were treated with glucagon. Adrenaline-stimulated accumulation of 64Cu could be inhibited by including phenoxybenzamine, an alpha-adrenergic blocker, in the culture medium. Elevation of extracellular copper caused enhancement in the detection of labelled caeruloplasmin in the medium of cultured cells, probably owing to the ability of this metal to stabilize the protein.     

Roles of isoamylase and ADP-glucose pyrophosphorylase in starch granule synthesis in rice endosperm

Amyloplast-targeted green fluorescent protein (GFP) was used to monitor amyloplast division and starch granule synthesis in the developing endosperm of transgenic rice. Two classical starch mutants, sugary and shrunken, contain reduced activities of isoamylase1 (ISA1) and cytosolic ADP-glucose pyrophosphorylase, respectively. Dividing amyloplasts in the wild-type and shrunken endosperms contained starch granules, whereas those in sugary endosperm did not contain detectable granules, suggesting that ISA1 plays a role in granule synthesis at the initiation step. The transition from phytoglycogen to sugary-amylopectin was gradual in the boundary region between the inner and outer endosperms of sugary. These results suggest that the synthesis of sugary-amylopectin and phytoglycogen involved a stochastic process and that ISA1 activity plays a critical role in the stochastic process in starch synthesis in rice endosperm. The reduction of cytosolic ADP-glucose pyrophosphorylase activity in shrunken endosperm did not inhibit granule initiation but severely restrained the subsequent enlargement of granules. The shrunken endosperm often developed pleomorphic amyloplasts containing a large number of underdeveloped granules or a large cluster of small grains of amyloplasts, each containing a simple-type starch granule. Although constriction-type divisions of amyloplasts were much more frequent, budding-type divisions were also found in the shrunken endosperm. We show that monitoring GFP in developing amyloplasts was an effective means of evaluating the roles of enzymes involved in starch granule synthesis in the rice endosperm.     

Evaluation of three methods for effective extraction of DNA from human hair

In this paper we evaluate three different methods for extracting DNA from human hair i.e. the Chelex method, the QIAamp DNA Mini Kit method and the ISOHAIR method. Analysis of DNA prepared from dyed hairs with the ISOHAIR method suggested that the DNA extracts contained PCR inhibitors. On the other hand, few inhibition was observed when DNA from dyed hairs were extracted using the Chelex method and the QIAamp DNA Mini Kit method. In conclusion, the Chelex method is recommended for PCR experiments in view of its simplicity and cost-effectiveness. To assess the reliability of the Chelex method for the extraction of genomic DNA from both natural and dyed hair samples, minisatellite variant repeat (MVR)-polymerase chain reaction (PCR) patterns of Chelex-extracted DNA were compared using hairs (three natural black hairs and three dyed hairs) with buccal swabs from six individuals. Complete agreement was observed between hair and swab samples in each individual, proving the utility of the Chelex method.     

Fission yeast TOR signaling is essential for the down-regulation of a hyperactivated stress-response MAP kinase under salt stress

TOR (target of rapamycin) signaling regulates cell growth and division in response to environmental stimuli such as the availability of nutrients and various forms of stress. The vegetative growth of fission yeast cells, unlike other eukaryotic cells, is not inhibited by treatment with rapamycin. We found that certain mutations including pmc1Δ (Ca²⁺-ATPase), cps9-193 (small GTPase, Ryh1) and cps1-12 (1,3-β-d-glucan synthase, Bgs1) confer a rapamycin-sensitive phenotype to cells under salt stress with potassium chloride (>0.5 M). Cytometric analysis revealed that the mutant cells were unable to enter the mitotic cell cycle when treated with the drug under salt stress. Gene cloning and overexpression experiments revealed that the sensitivity to rapamycin was suppressed by the ectopic expression of tyrosine phosphatases, Pyp1 and Pyp2, which are negative regulators of Spc1/Sty1 mitogen-activated protein kinase (MAPK). The level of tyrosine phosphorylation on Spc1 was higher and sustained substantially longer in these mutants than in the wild type under salt stress. The hyperphosphorylation was significantly suppressed by overexpression of pyp1 ⁺ with concomitant resumption of the mutant cells’ growth. In fission yeast, TOR signaling has been thought to stimulate the stress-response pathway, because mutations of TORC2 components such as Tor1, Sin1 and Ste20 result in similar sensitive phenotypes to environmental stress. The present study, however, strongly suggests that TOR signaling is required for the down-regulation of a hyperactivated Spc1 for reentry into the mitotic cell cycle. This finding may shed light on our understanding of a new stress-responsive mechanism in TOR signaling in higher organisms.