Mapping the active-site tyrosine of vaccinia virus DNA topoisomerase I.

Site-directed mutagenesis of the vaccinia virus gene encoding a type I DNA topoisomerase implicates Tyr-274 as the active-site residue that forms a covalent adduct with DNA during cycles of DNA-strand breakage and reunion. Replacement of Tyr-274 by phenylalanine results in loss of the ability of the enzyme to relax negatively supercoiled DNA as well as to form the covalent DNA-protein intermediate. Substitution of phenylalanine for tyrosine at nine other sites in the protein has no apparent effect on enzyme activity. Amino acid sequence alignment reveals Tyr-274 to be homologous to Tyr-727 and Tyr-771, respectively, of the type I topoisomerases from Saccharomyces cerevisiae and Saccharomyces pombe; Tyr-727 and Tyr-771 have been shown to represent the active-site tyrosines of those enzymes. Sequence comparison of the active-site regions defines a motif Ser-Lys-Xaa-Xaa-Tyr common to the viral and cellular type I topoisomerases, including the human enzyme.     

A simple method for the isolation and culture of epithelial and stromal cells from benign and neoplastic prostates

Objectives. Current primary prostate cell culture techniques use an overnight digestion or extensive media preparation. In this report, we describe a method for the culture of benign and neoplastic cells from human prostatectomy specimens that is rapid and contains no undefined factors in the medium.Methods. Characterization of the human cultured prostate cells was performed using immunohistochemical methods and monoclonal antibodies AE1/AE3 and cytokeratin 8, as well as monoclonal antibodies against prostate-specific antigen (PSA). Polymerase chain reaction was used to measure the exclusive epithelial and stromal cell products, c-met and hepatocyte growth factor (HGF), respectively. Electron microscopy was performed to assess the cell junctions and morphologic features of epithelial cells. Optimum cell growth in different media was tested using a cell replication assay.Results. Microscopic evidence revealed that the cells demonstrate typical epithelial morphology, with polyhedral cells forming tight junctions in a continuous monolayer. Desmosomes were present in electron micrographs of epithelial cells. The cultured epithelial cells described in this report also demonstrate positive cytokeratin staining. The epithelial cells reacted positively with PSA antibody, indicating that the cells retain their secretory role in cell culture for a limited period. Epithelial cells expressed the HGF receptor, c-met; stromal cells secreted HGF. Insulin, transferrin, and selenium increased the growth of cells in the chemically defined media, compared with minimum essential media (MEM) and Ham's F12.Conclusions. In summary, essentially pure cultures of prostate stromal or epithelial cells have been established using simple isolation and culture methods. These cells will be useful for the investigation of related growth factors, such as insulin-like growth factor I and insulin-like growth factor II, and in understanding the basis for stromal-epithelial cell interactions.     

The evolution of ischemic cerebral infarction in infancy: a sonographic evaluation

Cranial sonography provides a noninvasive, portable method for imaging the infant brain. This study describes the time-dependent, sonographic findings of infantile cerebral infarction, as well as computed tomographic (CT) scan and neuropathologic confirmation. Three hundred ninety-five infants under 18 months of age were sonogrammed over a period of 18 months. Three infants were diagnosed by cranial sonography and confirmed by CT scan and/or autopsy to have acute ischemic cerebral infarcts. The cases were followed with serial cranial sonograms for up to 18 months of age. The acute sonographic findings included a hyperechoic zone around the infarcted tissue. The subacute infarct had a checkerboard pattern, while the chronic infarcts were anechoic.     

Identification of Legionella pneumophila genes required for growth within and killing of human macrophages.

Legionella pneumophila was mutagenized with Tn903dIIlacZ, and a collection of mutants was screened for defects in macrophage killing (Mak-). Of 4,564 independently derived mutants, 55 (1.2%) showed a reduced or complete lack in the ability to kill HL-60-derived human macrophages. Forty-nine of the Mak- mutants could be assigned to one of 16 DNA hybridization groups. Only one group (9 of the 10 members) could be complemented for macrophage killing by a DNA fragment containing icm and dot, two recently described L. pneumophila loci that are required for macrophage killing. Phenotypic analysis showed that none of the mutants were any more sensitive than the wild type to human serum, oxidants, iron chelators, or lipophilic reagents nor did they require additional nutrients for growth. The only obvious difference between the Mak-mutants and wild-type L. pneumophila was that almost all of the Mak- mutants were resistant to NaCl. The effects of LiCl paralleled the effects of NaCl but were less pronounced. Resistance to salt and the inability to kill human macrophages are linked since both phenotypes appeared when Tn903dIIlacZ mutations from two Mak- strains were transferred to wild-type backgrounds. However, salt sensitivity is not a requisite for killing macrophages since a group of Mak- mutants containing a plasmid that restored macrophage killing remained resistant to NaCl. Mak- mutants from groups I through IX associated with HL-60 cells similarly to wild-type L. pneumophila. However, like the intracellular-multiplication-defective (icm) mutant 25D, the Mak- mutants were unable to multiply within macrophages. Thus, the ability of L. pneumophila to kill macrophages seems to be determined by many genetic loci, almost all of which are associated with sensitivity to NaCl.     

Analphoid 3qter markers

Two cases of marker chromosomes derived from a non-centromeric location were studied to determine the characteristics of these markers with respect to the presence of functional centromeres and whether an associated phenotype could be described. The markers were characterized by fluorescence in situ hybridization and centromeric protein studies. Assessments were done to identify clinical features. Case 1 is a girl referred at age 1.5 years with swirly areas of hyperpigmentation, bilateral preauricular pits, hypotonia, developmental delay, and seizures. Case 2 is a male first evaluated as a newborn and then later during the first year of life. He had streaky hypopigmentation, right preauricular pit, accessory nipples, postaxial polydactyly, asymmetric cerebral ventricles, duplicated right kidney, a right pulmonary artery stenosis, and seizures. Mosaicism for an extra marker from the 3qter region was present in both cases. Both markers had a constriction near one end and were C-band negative. Centromeric protein studies indicated absence of CENP-B, presence of CENP-C (data for case 1 only), and presence of CENP-E. Marker chromosomes were thus identified with a chromosomal origin far from their usual centromeric region and yet appeared to have functional centromeres. These two cases did not permit a specific clinical phenotype to be ascribed to the presence of tetrasomy for 3q26.2 approximately 3q27.2-->3qter.     

Insertional mutagenesis of the vaccinia virus gene encoding a type I DNA topoisomerase: evidence that the gene is essential for virus growth

Vaccinia virus encodes a type I DNA topoisomerase whose function in virus replication is not known. To determine whether topoisomerase is required for growth of vaccinia in cell culture, we attempted to isolate null mutations in the topoisomerase gene through insertional mutagenesis. Plasmids containing mutant topoisomerase alleles were constructed by intragenic insertion of the Escherichia coli gpt gene. Recombinant viruses containing the gpt insertion were isolated by selection for growth in the presence of mycophenolic acid. Analysis of the genome structures of drug-resistant viruses revealed that in every case (n = 22) both the wild-type and the gpt-inserted allele were present in viral DNA. We interpret the retention of the wild-type allele as indicative of the essential nature of the topoisomerase gene for vaccinia virus growth.     

Ultrastructure,function and composition of smooth muscle

Filamentous myosin is present in both relaxed (myosin light chains unphosphorylated) and contracted (light chains phosphorylated) vascular smooth muscle. The organization of myosin and actin filaments and the insertion of the latter on cytoplasmic and plasma membrane bound dense bodies is consistent with a mini sarcomere-like organization and a sliding filament mechanism of contraction in smooth muscle. Mitochondria are high capacity, low affinity Ca stores in smooth muscle. They do not play a role in the regulation of cytoplasmic Ca²⁺ at physiological levels. The localization and Ca content of the junctional sarcoplasmatic reticulum (SR) is consistent with this organelle being the major intracellular source of activator Ca released by excitatory transmitters. Repeated contractions in the absence of extracellular Ca²⁺ (thought to represent recycling of intracellular activator Ca²⁺) can be demonstrated if the excitatory agent is not allowed to remain in contact with the smooth muscle throughout relaxation; the demonstration of “recycling” is facilitated if the efflux of cellular Ca²⁺ is blocked. The rise in total cytoplasmic calcium measured with electron probe analysis during a maintained (30 min) contracture in rabbit portal-anterior mesenteric vein smooth muscle (∼0.9 mmol/kg dry cytoplasm) is greater than the amount of Ca that could be bound to calmodulin.