Autotransfusion of unwashed mediastinal shed blood fails to decrease banked blood requirements in patients undergoing aortocoronary bypass surgery.

Infusion of unwashed mediastinal shed blood (MSB) is one technique advocated for decreasing use of donor blood in cardiac surgery patients. A commercially available system was prospectively evaluated in 96 consecutive patients. The control group was comprised of 78 consecutive patients. All underwent elective aortocoronary bypass surgery. Student's t-test, chi-square analysis, multivariate analysis, and Fisher's exact test were used where appropriate. There was no decrease in the amount of banked blood required or percentage of patients who received transfusions in the MSB autotransfusion group.

Role of N-linked oligosaccharides in processing and intracellular transport of E2 glycoprotein of rubella virus.

The role of N-linked glycosylation in processing and intracellular transport of rubella virus glycoprotein E2 has been studied by expressing glycosylation mutants of E2 in COS cells. A panel of E2 glycosylation mutants were generated by oligonucleotide-directed mutagenesis. Each of the three potential N-linked glycosylation sites was eliminated separately as well as in combination with the other two sites. Expression of the E2 mutant proteins in COS cells indicated that in rubella virus M33 strain, all three sites are used for the addition of N-linked oligosaccharides. Removal of any of the glycosylation sites resulted in slower glycan processing, lower stability, and aberrant disulfide bonding of the mutant proteins, with the severity of defect depending on the number of deleted carbohydrate sites. The mutant proteins were transported to the endoplasmic reticulum and Golgi complex but were not detected on the cell surface. However, the secretion of the anchor-free form of E2 into the medium was not completely blocked by the removal of any one of its glycosylation sites. This effect was dependent on the position of the deleted glycosylation site.

Involvement of the X chromosome in non-Hodgkin lymphoma.

Gain of an X chromosome is observed as a secondary, acquired karyotypic alteration in a significant proportion of malignant lymphomas. To determine the potential involvement of X-linked genes in neoplastic development, we have analyzed the inactivation status of the supernumerary X chromosome in lymphomas in both male and female patients. In males, neither methylation of FMR1 nor expression of XIST was detected, demonstrating that the duplicated chromosome was not subject to inactivation. In females, both expressed polymorphisms and polymorphisms associated with methylation differences between the active and inactive X chromosome were analyzed to determine whether the duplicated chromosome was active or inactive. To facilitate this analysis, allele-specific PCR primers were designed for detection of previously described polymorphisms in the IDSX and G6PD genes. The female lymphomas were shown to be clonal in origin, and duplication of either the active (5 cases) or inactive (4 cases) X chromosome was observed. Correlations between clinical status and the inactivation status of the X chromosome involved in the duplication were not observed in our relatively small sample, although 4/4 informative cases with a t(14;18) showed duplication of the active X chromosome. In the course of these studies, we detected hypermethylation of the androgen receptor (AR) locus in an extremely high proportion of both male (7/9) and female (9/10) samples. These results are discussed with respect to whether sex chromosome aneuploidies in tumors are involved in, or simply the result of, the neoplastic process. Genes Chromosomes Cancer 28:246-257, 2000.

Mutational analysis of the arginine residues in the E2-E1 junction region on the proteolytic processing of the polyprotein precursor of rubella virus.

Endoproteolytic cleavage of precursors is a key step in biosynthesis of functional proteins. The structural proteins of rubella virus are initially translated as a precursor polyprotein in the order NH2-C-E2-E1-COOH and are cleaved by host signal peptidase to yield three structural proteins. Between regions corresponding to E2 and E1 in the precursor is a region of seven amino acid residues (R-R-A-C-R-R-R) that contains a motif for stop-transfer or a possible target for trypsin-like protease cleavage. Using site-directed mutagenesis, these arginine residues, as well as the signal peptide cleavage site at the N-terminus of E1, have been mutated individually or in combination. Results from in vitro transcription/translation analysis indicated that the mutated E2E1 precursor polyproteins were translocated into the microsome and glycosylated. Expression of mutated precursor polyproteins in COS cells revealed that the cleavage of E2E1 polyprotein precursor was impaired when the signal peptide cleavage site alone or both arginine clusters were altered, whereas partial cleavage was observed in the mutants in which either one of the two arginine clusters was modified. Our data suggest that although the arginine clusters do not function as a basic protease cleavage site, they contribute to maintain the proper configuration of that region for access of cellular signal peptidase.