Effect of sonication on paraffin-embedded tissue preparation for DNA flow cytometry

Since the publication of paraffin block extraction procedures, flow cytometric analysis of DNA ploidy and S-phase of tumor specimens has been widely applied. DNA aneuploidy, DNA tetraploid (elevated G2/M), and elevated S-phase are clinically significant in some tumor systems. True DNA tetraploid cell lines will contain a large 4c population and perhaps an 8c population; samples with cell aggregates will also contain a 6c population. Microscopic examination of samples having a 6c peak revealed nuclei with adhering debris and doublets, triplets, and larger nuclear aggregates. After sonication, a uniform suspension of single nuclei without adherent debris was seen. In addition to reducing the percent of G2/M cells, sonication also reduced S-phase percent such that it was closer to the bromodeoxyuridine labeling index. The DNA ploidy classification of specimens was also compared pre- and post-sonication. Four of 96 breast cancer samples changed classification; all were specimens in which the histogram became cleaner and a small DNA aneuploid peak became apparent after sonication.     

Innate resistance to herpes simplex virus infection. Human lymphocyte and monocyte inhibition of viral replication

Peripheral blood monocytes and lymphocytes isolated from most humans are resistant to HSV infection in vitro. Viral replication is inhibited very early in the cycle, prior to the onset of alpha-protein synthesis; no viral protein or DNA synthesis is detectable even up to 1 week later. The enhanced expression of two 62-kDa and 57-kDa cellular proteins, however, is induced in the lymphocyte population within 3 to 5 h after infection. A 30-kDa protein is induced in the monocyte population immediately after infection. The induced expression of 62-kDa and 57-kDa lymphocyte proteins appears to be virus-mediated because: a) HSV and pseudorabies virus (although not vaccinia virus) induce the expression of 62-kDa and 57-kDa proteins, b) heat shock or exposure of lymphocytes to uninfected cell extracts does not induce expression of either protein, c) 62-kDa protein is not induced in lymphocytes stimulated with a mitogenic concentration of PHA. UV-inactivated HSV induces expression of 62-kDa and 57-kDa proteins in a manner similar to that observed with untreated virus. In contrast, expression of 30-kDa monocyte protein is induced nonspecifically by either uninfected cell extracts or cell extracts containing virus. Sixty-two-kilodalton and 57-kDa protein induction appears to be a marker for human lymphocytes that express profound intracellular resistance to infection with HSV. Induced expression of these proteins occurs only in lymphocytes that inhibit viral replication very early in the growth cycle, prior to the onset of alpha-protein synthesis. Expression of 62-kDa and 57-kDa proteins is not induced in lymphocytes that are permissive or partially permissive to infection with HSV.     

Morphological and Anatomical Characterization of the Coleoptile of Triticum aestivum with Regard to the Evolution of Forms with Different Ploidy Levels

Details of the morphology and anatomy of the coleoptile of wheatplants are given; these have not been described adequately previously.The investigations focussed on the hexaploid summer wheat Triticumaestivum L. cv. Hatri, and three taxa with different ploidylevels. In darkness the longitudinal growth of the coleoptile was delayedby nearly 24 h but the final length, reached after 120 h, wasdouble that of coleoptiles of plants cultivated under continuouslight. As soon as the coleoptile has grown, the primary leafpushes through a pore pre-formed during the meristematic stageand located 1–1·5 mm behind the apex. The poreis stabilized mechanically by anastomosing of the originallyfree ends of the vascular bundles, as well as by increased lignificationin this region. The species investigated differ in length, f.wt and d. wt, size of epidermal cells, and especially in thesize of guard cells of the coleoptiles. The number of parenchymalayers, however, shows no specificity. Triticum aestivum L. cv. Hatri, wheat, coleoptile, morphology, anatomy, Aegilops spp     

Molecular and genetic characterization of an ornithine decarboxylase-deficient Chinese hamster cell line.

The ornithine decarboxylase (ODC)-deficient Chinese hamster ovary (CHO) cell line C55.7 has normal amounts of ODC mRNA with very low amounts of immunologically detectable ODC protein, suggesting a structural mutation; however, 5-azacytidine treatment leads to phenotypical reversion (Steglich, C., and Scheffler, I. E. (1985) Somat. Cell Mol. Genet. 11, 11-23). We have demonstrated by chemical cleavage a single base mismatch in DNA heteroduplexes composed of wild-type and mutant cDNA strands. DNA sequencing showed that the mutant phenotype results from an aspartate-glycine substitution at amino acid 381 of the protein. When 5-azacytidine-revertant cell lines were selected for resistance to alpha-difluoromethylornithine, the resulting amplified ODC gene was structurally indistinguishable from the wild type gene. These results suggested the existence of a single active ODC locus in CHO cells. Using the methylation-sensitive restriction endonucleases AvaI and HpaII, we found evidence for two differentially methylated alleles in wild type, ODC-deficient and alpha-difluoromethylornithine-resistant cells. One of the alleles appeared completely inactivated by hypermethylation but could be reactivated by demethylation in spontaneous or 5-azacytidine-induced revertants.     

Interactions of lipid micelles with blood proteins

Interaction of lipid micelles (LM), containing cholesterol and hydroxycholesterol, with human serum lipoproteins was investigated. It was shown that cholesterol-containing LM interact with low density lipoproteins (LDL). Selectivity of LM-LDL interaction depended on the cholesterol content of micelles and almost did not depend on the composition of LM core. Up to 90% of LDL were bound with cholesterol-saturated LM. By means of gel chromatography it was shown that interaction of cholesterol- and 7-hydroxycholesterol-containing micelles with serum led to the partial fusion of LDL with LM and LDL-LM complex formation, as well as to the cholesterol and 7-hydroxycholesterol transfer from micelles to LDL. The obtained results indicate that cholesterol-containing LM can be used for the delivery of oxidized cholesterol to cells involving LDL and receptor-dependent pathway of their capture by peripheral cells.     

Sequence and relatedness in other bacteria of the Pseudomonas aeruginosa oprP gene coding for the phosphate-specific porin P

The oprP gene encoding the Pseudomonas aeruginosa phosphate-specific outer membrane porin protein OprP was sequenced. Comparison of the derived amino acid sequence with the known sequences of other bacterial porins demonstrated that OprP could be no better aligned to these porin sequences than it could to the periplasmic phosphate-binding protein PhoS of Escherichia coli. Southern hybridization and restriction mapping of the oprP gene in 37 clinical isolates and the 17 serotype strains of P. aeruginosa revealed that restriction sites in the vicinity of the oprP gene were highly conserved. Several species from the Pseudomonas fluorescens rRNA homology group contained DNA that hybridized to an oprP gene probe.