Localization of anti-clathrin antibody in the sarcomere and sensitivity of myofibril structure to chloroquine suggest a role for clathrin in myofibril assembly

Immunofluorescence microscopy has been used to demonstrate that X22, a monoclonal antibody specific for clathrin heavy chain, localizes in repetitive bands that appear soon after the fusion of skeletal myoblasts into multinucleate fibers. This organization has been found in cultures containing myotubes that develop in vitro from explants of newborn rat hindlimb cells and in myotubes derived from the L8E63 myogenic line. Bands were also prominent in skinned fibers prepared from adult rat soleus muscle and in cardiac myocytes grown in vitro from 4-day heart ventricles. Immunofluorescence banding was localized in the sarcomere as a doublet, with one element on either side of the Z line. Evidence that supports the conclusion that the reaction with X22 antibody is specific and indicative of the localization of clathrin in the sarcomere includes: (1) Identical titration of X22 antibody reactivity with the determinant in coated vesicles and in the sarcomere. (2) Conditions (eg., pH and Tris) that disrupt clathrin baskets or prevent its assembly likewise disrupt the localization of X22 in bands. (3) Chloroquine inhibits both the normal trafficking of clathrin in the cell and X22 banding in the sarcomere. (4) Immunoblot analysis of myotube lysates reveals a single band with an electrophoretic mobility identical to the 180,000-Da clathrin heavy chain. (5) The assembly of clathrin into sarcomeric bands occurs early in the development of the myofibrillar apparatus. Quantitation of the appearance of X22 banding in primary cultures of myotubes indicates that it precedes that of other myofibrillar proteins and that assembly takes place in the following order: X22, titin, myosin heavy chain, actin, and desmin. The assembly of myosin, titin, and actin into sarcomeric bands, as well as X22, is inhibited by chloroquine. Upon prolonged exposure to chloroquine previously assembled proteins are drastically reduced or no longer evident in the sarcomere. On the basis of these results and considering the role of clathrin in intracellular transport and its capacity to interact with actin and alpha-actinin, we suggest that clathrin may have diverse roles in the assembly, integrity, and functioning of the sarcomere and its integration with the sarcolemma. The early organization of X22 into bands further suggests that clathrin may also function early in the assembly of the contractile system.     

The pattern of X-chromosome inactivation in the embryonic and extra-embryonic tissues of post-implantation digynic triploid LT/Sv strain mouse embryos

Spontaneously cycling LT/Sv strain female mice were mated to hemizygous Rb(X.2)2Ad males in order to facilitate the distinction of the paternal X chromosome, and the pregnant females were autopsied at about midday on the tenth day of gestation. Out of a total of 222 analysable embryos recovered, 165 (74.3%) were diploid and 57 (25.7%) were triploid. Of the triploids, 26 had an XXY and 31 an XXX sex chromosome constitution. Both embryonic and extra-embryonic tissue samples from the triploids were analysed cytogenetically by G-banding and by the Kanda technique to investigate their X-inactivation pattern. The yolk sac samples were separated enzymatically into their endodermally-derived and mesodermally-derived components, and these were similarly analysed, as were similar samples from a selection of control XmXp diploid embryos. In the case of the XmXmY digynic triploid embryos, a single darkly-staining Xm chromosome was observed in 485 (82.9%) out of 585, 304 (73.3%) out of 415, and 165 (44.7%) out of 369 metaphases from the embryonic, yolk sac mesodermally-derived and yolk sac endodermally-derived tissues, respectively. The absence of a darkly staining X-chromosome in the other metaphase spreads could either indicate that both X-chromosomes present were active, or that the Kanda technique had failed to differentially stain the inactive X-chromosome(s) present. In the case of the XmXmXp digynic triploid embryos, virtually all of the tissues analysed comprised two distinct cell lineages, namely those with two darkly-staining X-chromosomes, and those with a single darkly staining X-chromosome.(ABSTRACT TRUNCATED AT 250 WORDS)     

The stress response in Chinese hamster ovary cells. Regulation of ERp72 and protein disulfide isomerase expression and secretion

Expression of the glucose-regulated proteins (GRPs), GRP78 and GRP94, is induced by a variety of stress conditions including treatment of cells with tunicamycin or the calcium ionophore A23187. The stimulus for induction of these resident endoplasmic reticulum (ER) proteins appears to be accumulation of misfolded or underglycosylated protein within the ER. We have studied the induction of mRNAs encoding two other resident ER proteins, ERp72 and protein disulfide isomerase (PDI), during the stress response in Chinese hamster ovary cells. ERp72 shares amino acid sequence homology with PDI within the presumed catalytic active sites. ERp72 mRNA and, to a lesser degree, PDI mRNA were induced by treatment of Chinese hamster ovary cells with tunicamycin or A23187. These results identify ERp72 as a member of the GRP family. Stable high level overproduction of ERp72 or PDI from recombinant expression vectors did not alter the constitutive or induced expression of other GRPs. High level overexpression resulted in secretion of the overproduced protein specifically but not other resident ER proteins. This suggests that the ER retention mechanism is mediated by more specific interactions than just KDEL sequence recognition.     

Exfoliative colonic cytology. A simplified method of collection and initial results

Exfoliative colonic cytology for the diagnosis of colorectal cancer has been largely abandoned due to (1) the widespread use of colonoscopy, (2) the cumbersome methods of cell collection and (3) the occasional difficulty of interpreting the cytologic findings in the presence of inflammatory bowel disease or adenomas. This paper describes a newly formulated bowel preparation for routine colonoscopy, based on imbibing 2 L to 4 L of a balanced electrolyte solution, in which the recovered precolonoscopic effluent (using a convenient disposable collecting kit) yielded cells for cytologic evaluation from 70% of a group of 80 patients at high risk for large bowel neoplasia. Cytology demonstrated neoplastic cells in most cases of endoscopically proven cancer. These results suggest that colonic exfoliative cytology may be useful as a supplemental test to routine colonoscopy. This could be enhanced by further methodologic modifications to the collecting and cytologic methods; large long-term studies are needed to evaluate the potential usefulness of colonic exfoliative cytology.     

Splicing defect at the ornithine aminotransferase (OAT) locus in gyrate atrophy.

Gyrate atrophy (GA), a recessive eye disease involving progressive vision loss due to chorioretinal degeneration, is associated with the deficiency of the mitochondrial enzyme ornithine aminotransferase (OAT), with consequent hyperornithinemia. We and others have reported a number of missense mutations at the OAT locus which result in GA. Here we report a GA patient of Danish/Swedish ancestry in whom one OAT allele produces an mRNA that is missing a single 96-bp exon relative to the normal mRNA. Polymerase-chain-reaction amplification and sequencing revealed a 9-bp deletion covering the splice acceptor region of exon 5, resulting in the absence of exon 5 sequences from the mRNA with no disruption to the reading frame. This mutation, which was not present in 15 other independent GA patients, adds to the array of allelic heterogeneity observed in GA and represents the first example of a splicing mutation associated with this disorder.     

Survival of Paracoccidioides brasiliensis yeast cells under microaerophilic conditions

The ability of P. brasiliensis yeast cells to withstand microaerophilic conditions was investigated in a liquid medium distributed in tall columns in screw-capped tubes. Young cells of three isolates were inoculated on top of the medium, and the tubes were incubated aerobically and anaerobically at 36 degrees C for 28 days. The viability of cells that had sedimented to the bottoms of the tubes was studied by fluorescent microscopy and by their capacity to resume growth when transferred to fresh medium under continuous agitation. The proportion of viable cells in the sediments diminished with time of incubation. However, after 28 days, 27% of the cells were still viable and fully capable of active growth when placed under adequate aeration. On the other hand, drastic reduction of oxygen access elicited an accelerated death rate, with no survival after 7 days of incubation.     

A rapid and specific method for the immunological identification of mycelial form cultures ofParacoccidioides brasiliensis

We developed two simple methods for extracting specific, cell-free, soluble antigens of the mold form ofParacoccidioides brasiliensis. Detection of these antigens by a microimmunodiffusion test permits the rapid and accurate identification of cultures ofP. brasiliensis. ThirtyP. brasiliensis isolates treated by these techniques produced specific exoantigens detectable by the immunodiffusion test. None of the other 78 fungal pathogens or saprophytes tested produced identical exoantigens. Personnel in any diagnostic laboratory who want a rapid and specific method for identifying or confirming suspected isolates ofP. brasiliensis can use the simple procedures described.