Isolation and characterization of a Mr = 110,000 glycoprotein localized to the hepatocyte bile canaliculus

A Mr = 110,000 glycoprotein, GP 110, was partially purified using wheat germ agglutinin-Sepharose affinity chromatography from a bile canalicular-enriched membrane fraction denoted N2u of rat liver. This fraction was subjected to preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the Mr = 110,000 polypeptide was excised and used as an immunogen in rabbits. The antisera were found to specifically recognize a Mr = 110,000 polypeptide, named GP 110, in the N2u membrane fraction. In isolated hepatocytes, GP 110 was readily accessible to cell surface iodination catalyzed by lactoperoxidase at 4 degrees C and was judged by immunoprecipitation studies to contain about 2% of total radioactivity incorporated into externally oriented proteins of the cell. Immunoprecipitated GP 110 was shown by two-dimensional polyacrylamide gel electrophoresis to migrate with an approximate pI of 4.9. Indirect immunofluorescence on frozen liver sections demonstrated that GP 110 was primarily localized in the bile canaliculus. In corroborative studies employing subcellular fractionation, it was found that GP 110 was enriched nearly 19-fold in P2, a plasma membrane fraction primarily derived from the sinusoidal domain, and 44-fold in N2u. In contrast, only low levels of GP 110 were present in endoplasmic reticulum, mitochondrial, cytosolic, and nuclear-enriched fractions of liver. The physiological function of GP 110 is as yet unknown; antisera to it did not immunoprecipitate other known bile canalicular proteins of similar molecular weights. GP 110 was found to be extensively glycosylated relative to other known membrane proteins; approximately 33% of the apparent molecular weight appear to be carbohydrate. In agreement, limited removal of N-linked carbohydrate chains indicated that there are approximately eight chains/GP 110 polypeptide. Neuraminidase treatment of GP 110 resulted in a desialylated Mr = 85,000 polypeptide suggesting that the majority of carbohydrate chains on GP 110 are of the complex type.     

Dinucleoside tetraphosphate variations in cultured tumor cells during their cell cycle and growth

Asynchronous and synchronized cultures of A549 and HTC cells were used to detect possible, cell cycle or cell density specific variations in the intracellular pools of dinucleoside tetraphosphates (Ap4X). No important variations of the nucleotide pools were observed during cell growth. When HTC cells were released from mitotic arrest, a decrease by a factor of N3 Ap4X and ATP levels was observed when the cells entered the G1 phase. This decrease is essentially due to cell doubling. When A549 cells were released from an arrest at the G1/S boundary, the nucleotide pool size increased slightly during the G2 phase just before mitosis. This result is in agreement with both earlier data from our laboratory and the observed decrease in Ap4X pool after release from mitotic-arrested HTC cells. These results suggest that the Ap4X and ATP pools are only subjected to very small variations during the cell cycle, essentially in the G2 phase and after mitosis.     

Down syndrome with duplication of a region of chromosome 21 containing the CuZn superoxide dismutase gene without detectable karyotypic abnormality

Summary We report the case of an 18-month-old boy with many typical Down syndrome features but a normal cytogenetic analysis. High-resolution banding techniques on lymphocytes and fibroblasts of the propositus and his parents did not show any detectable abnormality including that of trisomy 21 mosaicism. However, CuZn superoxide dismutase (CuZn SOD) in the patient's red cells was increased as in trisomy 21. DNA analysis (Southern blots) using a human CuZn SOD probe showed that the genotype of the propositus contained three CuZn SOD genes. In situ hybridization on metaphase chromosomes with the same probe confirmed the gene location in a segment enclosing the distal part of 21q21 and 21q22.1. There was no significant labeling on other chromosomes of the patient. These results indicate that the Down syndrome phenotype of this patient is due to microduplication of a chromosome 21 fragment containing the CuZn SOD gene.     

De novo t(2;13)(p24.3;q14.2) and retinoblastoma

Summary The two probes H3-8 and H2-42, known to be located in 13q14, were mapped by in situ hybridization to either side of the 13 breakpoint of an apparently balanced de novo t(2;13)(p24.3;q14.2) detected in a patient with retinoblastoma as the only phenotypic manifestation.     

The effect of alkaline borohydride treatment onN-linked carbohydrates of glycoproteins

The effects of treatments of the glycoprotein ribonuclease-B, the proteins ribonuclease-A and myoglobin, and the glyco-amino acid GlcNAc(1-N) Asn with alkalil alkaline sodium borohydride, and aqueous sodium borohydride were systematically studied as a function of the concentration of the reagents, the temperature, and the length of the treatment. High-field¹H-NMR spectroscopy, chromatographic methods and amino-acid analysis were used to characterize products of the treatments of the various compounds. Our results indicate that mild alkaline borohydride treatment, as well as aqueous borohydride treatment alone, is capable of extensively degrading polypeptides and of partially releasing theN-linked glycans from ribonuclease-B. Initially, glycopeptides are produced, the peptide portion of which consists of several amino acids, which are further hydrolyzed to yield a mixture of glyco-asparagines and oligosaccharide-alditols in the ratio of 4:1. Strong alkaline borohydride treatment of ribonuclease-B is capable of completely releasing theN-linked carbohydrates as oligosaccharide-alditols.Abbreviation RNase ribonuclease     

Binding of a radiolabeled sea anemone cytolysin to erythrocyte membranes

Stichodactyla helianthus cytolysin III, a 17 kDa basic polypeptide isolated from a Caribbean sea anemone, is one of the most potent hemolysins yet found in a living organism. This toxin has been reported to form new ion channels in artificial lipid bilayer membranes. The ability of this toxin to attack cell membranes is greatly enhanced by the presence of sphingomyelin. In order to investigate the mechanism by which the cytolysin causes cell lysis, we have prepared a highly active [3H]cytolysin derivative by reductive methylation with sodium cyanoborohydride and [3H]formaldehyde. A dimethylated toxin derivative was used to investigate the basis for the differential lytic activity of this polypeptide upon erythrocytes from six mammalian species. Using both direct [3H]toxin binding and indirect (Thron method) binding techniques, we found that the interspecies differences are due to variable membrane susceptibilities toward the bound toxin, rather than to differences in membrane affinity for the toxin. Similarly, we showed the enhanced lytic activity of the toxin for rat erythrocytes at elevated pH to be caused by enhanced activity of the bound toxin.     

Sodium lactate delays toxin production by Clostridium botulinum in cook-in-bag turkey products.

Comminuted raw turkey, containing 1.4% sodium chloride, 0.3% sodium phosphate, and 0 (control), 2.0, 2.5, 3.0, or 3.5% sodium lactate, was inoculated with a 10-strain mixture of proteolytic type A and B Clostridium botulinum spores. The inoculated turkey was vacuum packaged and cooked by immersion in heated water to an internal temperature of 71.1 degrees C. Samples were incubated at 27 degrees C for up to 10 days. Five samples per treatment were examined for botulinal toxin at specific intervals. Sodium lactate exhibited an antibotulinal effect which was concentration dependent. Processed turkey containing 0, 2.0, 2.5, 3.0, or 3.5% sodium lactate was toxic after 3, 4 to 5, 4 to 6, 7 or 7 to 8 days, respectively. Subsequent studies with a broth medium revealed that lactate, not the sodium ion, was the principal factor in delaying botulinal-toxin formation.     

Fate of Listeria monocytogenes in processed meat products during refrigerated storage.

The fate of Listeria monocytogenes during refrigerated storage was determined on several processed meat products, including ham, bologna, wieners, sliced chicken, sliced turkey, fermented semidried sausage, bratwurst, and cooked roast beef. The meats were surface inoculated with a five-strain mixture of less than or equal to 200 or ca. 10(5) L. monocytogenes cells per package, vacuum packaged, and stored at 4.4 degrees C. Survival or growth of listeriae was determined for up to 12 weeks of storage or until the product was spoiled. The organism survived but did not grow on summer sausage, grew only slightly on cooked roast beef, grew well on some wiener products but not on others, grew well (10(3) to 10(5) CFU/g increase within 4 weeks) on ham, bologna, and bratwurst, and grew exceptionally well (10(3) to 10(5) CFU/g increase within 4 weeks) on sliced chicken and turkey. The rate of growth depended largely upon the type of product and the pH of the product. Growth was most prolific on processed poultry products. The organism generally grew well on meats near or above pH 6 and poorly or not at all on products near or below pH 5. These results indicate the importance of preventing postprocessing contamination of L. monocytogenes in a variety of ready-to-eat meat products.     

Immunological relationship among hydrogenases.

We examined the immunological cross-reactions of 11 different hydrogenase antigens with 9 different hydrogenase antibodies. Included were antibodies and antigens of both subunits of the hydrogenases of Bradyrhizobium japonicum and Thiocapsa roseopersicina. The results showed a strong relationship among the Ni-Fe dimeric hydrogenases. The two subunits of Ni-Fe dimeric hydrogenases appeared immunologically distinct: specific interactions occurred only when antibodies to the 60- and 30-kilodalton subunits reacted with the 60- and 30-kilodalton-subunit antigens. The interspecies cross-reactions suggested that at least one conserved protein region exists among the large subunits of these enzymes, whereas the small subunits are less conserved. Antibodies to the Fe-only bidirectional hydrogenase of Clostridium pasteurianum reacted with the Desulfovibrio vulgaris bidirectional hydrogenase. Surprisingly, antibodies to the clostridial uptake hydrogenase did not react with any of the Fe-only bidirectional hydrogenases but did react with several of the Ni-Fe dimeric hydrogenases. The two hydrogenases from C. pasteurianum were found to be quite different immunologically. The possible relationship of these findings to the structure and catalytic functions of hydrogenase are discussed.