Amplified region of chromosome band 11q13 in breast and squamous cell carcinomas encompasses three CpG islands telomeric of FGF3, including the expressed gene EMS1

DNA markers that map within the karyotypically defined band q13 on human chromosome 11 are amplified in a subset of mammary and squamous cell carcinomas. It is assumed that the amplified DNA includes a critical gene (or genes) whose overexpression provides a selective force in the development of the tumor. To help identify such genes, we have begun to construct a physical map of CpG islands in the region, making use of a squamous cell carcinoma cell line (UMSCC2) in which the 11q13 region is amplified 11-fold. We previously described the proximal end of this amplicon and the order of markers extending ～800 kb centromeric of the FGF3 locus (formerly INT2). We now report the use of chromosome jumping techniques to define additional CpG islands that lie distal to FGF3. These map within the amplified region in UMSCC2 cells and the most telomeric corresponds to the EMS1 gene. The data imply that the amplified DNA in UMSCC2 cells extends for over 1,500 kb and includes at least 7 potential genes. EMS1 and CCND1 (formerly PRAD1), the best candidates for the key gene on the 11q13 amplicon, are ≥800 kb apart. © 1993 Wiley-Liss, Inc.     

Homologues of insecticidal toxin complex genes in Yersinia enterocolitica biotype 1A and their contribution to virulence

Yersinia enterocolitica is an enteric pathogen that consists of six biotypes: 1A, 1B, 2, 3, 4, and 5. Strains of the latter five biotypes can carry a virulence plasmid, known as pYV, and several well-characterized chromosomally encoded virulence determinants. Y. enterocolitica strains of biotype 1A lack the virulence-associated markers of pYV-bearing strains and were once considered to be avirulent. There is growing epidemiological, clinical, and experimental evidence, however, to suggest that some biotype 1A strains are virulent and can cause gastrointestinal disease. To identify potential virulence genes of pathogenic strains of Y. enterocolitica biotype 1A, we used genomic subtractive hybridization to determine genetic differences between two biotype 1A strains: an environmental isolate, Y. enterocolitica IP2222, and a clinical isolate, Y. enterocolitica T83. Among the Y. enterocolitica T83-specific genes we identified were three, tcbA, tcaC, and tccC, that showed homology to the insecticidal toxin complex (TC) genes first discovered in Photorhabdus luminescens. The Y. enterocolitica T83 TC gene homologues were expressed by Y. enterocolitica T83 and were significantly more prevalent among clinical biotype 1A strains than other Yersinia isolates. Inactivation of the TC genes in Y. enterocolitica T83 resulted in mutants which were attenuated in the ability to colonize the gastrointestinal tracts of perorally infected mice. These results indicate that products of the TC gene complex contribute to the virulence of some strains of Y. enterocolitica biotype 1A, possibly by facilitating their persistence in vivo.     

Parkin localizes to the Lewy bodies of Parkinson disease and dementia with Lewy bodies

Mutations in alpha-synuclein (alpha S) and parkin cause heritable forms of Parkinson disease (PD). We hypothesized that neuronal parkin, a known E3 ubiquitin ligase, facilitates the formation of Lewy bodies (LBs), a pathological hallmark of PD. Here, we report that affinity-purified parkin antibodies labeled classical LBs in substantia nigra sections from four related human disorders: sporadic PD, inherited alphaS-linked PD, dementia with LBs (DLB), and LB-positive, parkin-linked PD. Anti-parkin antibodies also detected LBs in entorhinal and cingulate cortices from DLB brain and alphaS inclusions in sympathetic gangliocytes from sporadic PD. Double labeling with confocal microscopy of DLB midbrain sections revealed that approximately 90% of anti-alpha S-reactive LBs were also detected by a parkin antibody to amino acids 342 to 353. Accordingly, parkin proteins, including the 53-kd mature isoform, were present in affinity-isolated LBs from DLB cortex. Fluorescence resonance energy transfer and immunoelectron microscopy showed that alphaS and parkin co-localized within brainstem and cortical LBs. Biochemically, parkin appeared most enriched in cytosolic and postsynaptic fractions of adult rat brain, but also in purified, alpha S-rich presynaptic elements that additionally contained parkin's E2-binding partner, UbcH7. We conclude that parkin and UbcH7 are present with alphaS in subcellular compartments of normal brain and that parkin frequently co-localizes with alpha S aggregates in the characteristic LB inclusions of PD and DLB. These results suggest that functional parkin proteins may be required during LB formation.     

Urban adolescents' perceptions of their neighborhoods: An examination of spatial dependence

Spatial dependence exists when the variation between observations is dependent on spatial location. In the present study, geostatistical methods were used to examine spatial dependence in adolescents' perceptions of their neighborhoods: whether adolescents living in close proximity perceived their neighborhoods more similarly than adolescents living further apart. Participants included 343 adolescents (53% male; 91% African American) enrolled in sixth grade, residing in Baltimore City, with geocoded home addresses. These addresses were used in combination with a measure of perceived neighborhood disorder and disadvantage to compute variogram plots, a geostatistical technique used to evaluate spatial dependence. Results indicated that perceptions of youth residing in close geographic proximity were more similar than perceptions of youth residing further apart. Adjusting for census‐level characteristics, but not individual and family characteristics, resulted in decreased variation among participants but did not eliminate the spatial dependence. Findings highlight the utility of geostatistical methods for social science research. © 2004 Wiley Periodicals, Inc. J Comm Psychol 32: 277–293, 2004.     

Comparison of CHROMagar Salmonella medium and xylose-lysine-desoxycholate and Salmonella-Shigella agars for isolation of Salmonella strains from stool samples

The growth and appearance of 115 stock Salmonella isolates on a new formulation of CHROMagar Salmonella (CAS) medium were compared to those on xylose-lysine-desoxycholate agar (XLD), Salmonella-Shigella agar (SS), and Hektoen enteric agar (HEA) media. CAS medium was then compared prospectively to XLD and SS for the detection and presumptive identification of Salmonella strains in 500 consecutive clinical stool samples. All stock Salmonella isolates produced typical mauve colonies on CAS medium. Nine Salmonella strains were isolated from clinical specimens. The sensitivities for the detection of salmonellae after primary plating on CAS medium and the combination of XLD and SS after enrichment were 100%. The specificity for the detection of salmonellae after primary plating on CAS medium (83%) was significantly (P < 0.0001) higher than that after primary plating on the combination of SS and XLD media (55%) (a 28% difference in rates; 95% confidence interval, 23.0 to 34%). Twenty-nine non-Salmonella organisms produced mauve colonies on CAS medium, including 17 Candida spp. (59%) and 8 Pseudomonas spp. (28%). These were easily excluded as salmonellae by colony morphology, microscopic examination of a wet preparation, or oxidase testing. One biochemically inert Escherichia coli isolate required further identification to differentiate it from Salmonella spp. The use of plating on CAS medium demonstrated high levels of sensitivity and specificity and reduced the time to final identification of Salmonella spp., resulting in substantial cost savings. It can be recommended for use for the primary isolation of Salmonella spp. from stool specimens. Other media (e.g., XLD) are required to detect Shigella spp. concurrently.     

Electrochemically assisted co-precipitation of protein with calcium phosphate coatings on titanium alloy

A bovine serum albumin protein-containing calcium phosphate coating (BSA/brushite) was prepared by electrochemically assisted co-precipitation onto a hydroxyapatite (HA) coated Ti-6Al-4V surface. Electrochemically assisted co-precipitation of BSA/brushite coatings onto HA resulted in a 70-fold increase in BSA inclusion compared to simple adsorption, and was subsequently released by a slower mechanism (15% loss over 70 h). Thus, this electrochemically assisted co-precipitation technique provides an efficient method of protein incorporation at physiological temperature, with a potential for sustained release of therapeutic agents as may be required for metallic implant fixation.     

Degree of Immunity Induced by Killed Vaccines to Experimental Salmonellosis in Mice

Killed vaccines, deoxycholate-extracted or heated, were shown to induce an effective degree of immunity which protected against death (100%), prevented extensive multiplication, and left the mice with low residual salmonella populations in spleen and liver after intravenous (iv) or intraperitoneal (ip) challenge with virulent Salmonella typhimurium. Protection was most effective against the ip challenge route and less effective against the iv route. A study of the kinetics of the population of bacteria in the spleens and livers of immunized animals showed that after ip challenge there was an initial reduction of 99% at 6 hr after challenge, maintenance of levels of less than 10(3) bacteria per organ, and a final population of 10(2) to 10(3) per organ at 21 days. With iv challenge, after an initial reduction of 90% at 6 hr, growth ensued to levels above 10(6) bacteria per organ until 8 days, followed by a steady decline yielding residual populations of 10(3) to 10(4) in some cases. Organ hypertrophy correlated with bacterial population. Morbidity was prevented (as measured by gain in body weight) by immunization against ip challenge but not against iv challenge. Killed vaccines protected by their ability to induce an immune state which reduced the initial challenge population, prevented extensive multiplication, yet allowed "cellular immunity" to develop due to response to the living challenge infection itself. The consequence was a low-level carrier state similar to that induced by recovery from sublethal virulent infection.     

Longitudinal reliability of focus glycoprotein G-based type-specific enzyme immunoassays for detection of herpes simplex virus types 1 and 2 in women

Serologic assays that utilize herpes simplex virus (HSV) type-specific glycoproteins G-1 (HSV-1) and G-2 (HSV-2) to discriminate between antibodies against HSV-1 and HSV-2 are sensitive and specific. However, the high rates of seroreversion, defined as the change in an individual's antibody status from positive to negative over time, previously reported in longitudinal evaluations of glycoprotein G type-specific tests suggests that their use in HSV acquisitional studies would be problematic. To further explore the reliability of the glycoprotein G-based serologic tests, we evaluated HSV-1 and HSV-2 enzyme immunoassays from Focus Technologies in a longitudinal cohort of 1207 young women from Pittsburgh, Pa. On enrollment of the women in the study, HSV-1 and HSV-2 antibodies were detected in 46.6 and 24.9% of the women, respectively. Among the women with at least three visits, 3.4% (15 of 447) of those who were HSV-1 antibody positive had a subsequent negative result while fewer than 1% (2 of 227) of those who were HSV-2 antibody positive seroreverted. The median of mean positive index values for women who seroreverted to HSV-1 antibody was lower than that for women who remained seropositive (1.25 versus 7.06; P < 0.001). Similarly, the median of mean positive index values for women whose HSV-2 antibody status reverted from positive to negative was lower than that for those women who did not serorevert (1.83 versus 7.46; P = 0.02). Comparative Western blot analysis demonstrated that the lower positive index values, seen more often among the HSV seroreverters, often signified false-positive immunoassay results. Overall, the seroreversion rates were low; the use of glycoprotein G-based serologic tests for the measurement of HSV-1 and HSV-2 antibodies in incidence studies therefore appears warranted.     

Modulation of osteopontin in proteinuria-induced renal interstitial fibrosis

Proteinuria is associated with macrophage-dependent interstitial fibrosis (IF). Osteopontin (OPN), a macrophage chemoattractant, may be involved in the transition of proteinuria to IF but protective properties have also been reported. To elucidate whether OPN may be involved in the proteinuria-induced cascade of tubulointerstitial damage, renal expression of OPN was studied during the development of proteinuria-induced renal damage and during anti-proteinuric intervention with ACE inhibition (ACEi). First, the temporal relationships between proteinuria, interstitial OPN induction, and IF in adriamycin nephrosis (AN), a model of chronic proteinuria-induced renal damage, were studied. Second, the effect of anti-proteinuric treatment on OPN expression was investigated. The time course of OPN induction and markers of renal damage was studied in rats with unilateral AN at 6-week intervals until week 30. In a second study, a renal biopsy was taken 6 weeks after induction of bilateral AN; subsequently, rats were treated with ACEi until termination (week 12). In unilateral AN, proteinuria developed gradually and stabilized at week 10. In proteinuric kidneys, OPN expression was induced from week 12 onwards. Simultaneously, a progressive increase in interstitial macrophages, alpha-smooth muscle actin (alpha-SMA), collagen type III, and focal glomerulosclerosis (FGS) was observed. In bilateral AN, ACEi reduced proteinuria and OPN protein and stabilized fibrosis. In untreated animals, OPN mRNA increased, with stable OPN protein and fibrosis and increased FGS. Thus, in AN, development of proteinuria is followed by up-regulation of OPN along with markers of renal damage. The up-regulation of OPN is reversible by anti-proteinuric treatment without a corresponding reduction in fibrosis. Whereas these data are consistent with a role for OPN in the cascade of transition from proteinuria to fibrosis, intervention with ACEi showed that reduction of OPN does not attenuate established fibrosis.     

Cytadherence-deficient mutants of Mycoplasma gallisepticum generated by transposon mutagenesis

Cytadherence-related molecules of Mycoplasma gallisepticum strain R-low were identified by Tn4001 transposon mutagenesis with the hemadsorption (HA) assay as an indicator for cytadherence. Three Gm(r) HA-negative (HA(-)) colonies displaying a stable HA(-) phenotype through several successive generations in which gentamicin selection was maintained were isolated from four independent transformation experiments and characterized. Southern blot analysis showed that the transposon was inserted as a single copy within the genome of each of the HA(-) mutants, suggesting that the transposon insertion was directly responsible for their inability to attach to erythrocytes. Sequence analysis of the transposon insertion sites revealed that in two mutants, the transposon was inserted at two distinct sites within the gapA structural gene. In the third mutant, the insertion was mapped within the crmA gene, which is located immediately downstream of the gapA gene as part of the same operon. In vitro attachment experiments with the MRC-5 human lung fibroblast cell line showed that the cytadherence capabilities of the HA(-) mutants were less than 25% those of original strain R. Experimental infection of chickens, the natural host of M. gallisepticum, with each of the three mutants demonstrated significantly impaired colonization and host responses. These data demonstrate conclusively the role of both GapA and CrmA proteins in the adherence of M. gallisepticum to host cells in model systems and in vivo colonization. Furthermore, these results underscore the relevance of in vitro cytadherence model systems for studying the pathogenesis of natural infections in chickens.