Fusion and Matrix Protein Gene Sequence Analysis of Paramyxoviruses of Type 1(PMV-1) Isolated from Pigeons in Slovenia

Paramyxoviruses of type 1 (PMV-l) isolated from pigeons were genetically analyzed. A part of the fusion and the matrix protein genes were amplified and sequenced, Typical amino acid sequences associated with virulence were determined at the fusion protein cleavage site in all PMV-1 isolates. All Slovene pigeon PMV-1 strains share high amino acid sequence similarity with other pigeon strains. In the phylogenetic tree, they are clustered together with pigeon PMV-1 isolates with moderate pathogenicity. Phylogenetic analysis obtained from the fusion and the matrix protein gene alignments showed the same branching order. Viruses circulating among pigeons were found to form quite unique lineage of virulent NDV strains.     

Induction of protein oxidation by cobalt and chromium ions in human U937 macrophages

Metal particles and ions from hip prostheses have the potential to induce the production of reactive oxygen species (ROS), making them prime suspects for disturbing the cellular balance of oxidants/antioxidants (redox state of the cell). To better understand the cellular effect of metal ions from metal-on-metal prostheses, the aim of this study was to examine the effect of cobalt (Co2+) and chromium (Cr3+) ions on protein oxidation in human U937 macrophages. Protein oxidation was measured by Western blot using antibodies directed against dinitrophenylhydrazine (DNP)-derivatized protein carbonyls, the most commonly measured products of protein oxidation in biological samples. Three DNP-derived proteins were detected. The first has a molecular weight of 16 kDa and is expressed at a very low level. The second has a molecular weight of 48 kDa and its level is not regulated by metal ions. The third is a 69 kDa protein and its level is regulated by Co2+ and Cr3+ ions. Therefore, the last band served as a marker of protein oxidation in the present study. Results showed that Co2+ and Cr3+ ions induced a time- and dose-dependent protein oxidation reaching 6.5 and 2.9 times the control after 72 h, respectively, which were inhibited by the antioxidant glutathione monoethyl-ester. Finally, results showed that the oxidized proteins are mainly found in the cytoplasmic fraction of the cells and are absent from the nucleus. In conclusion, our results suggest that metal ions from metal-on-metal prostheses have the potential to modify the redox state of cells both locally (periprosthetic environment) or systematically (circulating cells). The long term effect of these ions on protein oxidation in vivo remains to be investigated.     

Effect of cobalt and chromium ions on bcl-2, bax, caspase-3, and caspase-8 expression in human U937 macrophages

The bcl-2 and caspase families of proteins play a central role in the modulation of apoptosis. The purpose of this study was to analyze the effect of Co(2+) and Cr(3+) ions on the expression of bcl-2, bax, caspase-3 and caspase-8 to better understand the mechanisms leading to ion-induced apoptosis in macrophages. U937 human macrophages were exposed to Co(2+) and Cr(3+) ions. The expression of proteins was measured by Western blot while caspase activities were measured by colorimetric assay. Results show that Co(2+) ions inhibited bcl-2 expression with significant effect (p<0.05) after 16 h and a maximal 52% inhibitory effect after 24 h. Co(2+) stimulated bax expression with a significant stimulation (p<0.05) after 8 h and a maximal 1.75-fold increase after 16 h. Co(2+) also stimulated the expression of the active fragment of caspase-3 as well as caspase-3 activity maximal increase after 24 h. Co(2+) ions had no effect on caspase-8 expression or activity.Cr(3+) ions inhibited bcl-2 expression with significant effect (p<0.05) after 16 h and a maximal 43% inhibitory effect after 24 h. Cr(3+) stimulated bax expression with significant stimulation (p<0.01) after 8h and a maximal 2.25-fold increase after 24 h. Cr(3+) ions also stimulated the expression of the active fragments of caspase-3 and -8, as well as the activities of both proteases. The effect of Cr(3+) ions on the expression of both caspase active fragments was maximal after 16 h incubation. In conclusion, our results suggest that the modulation of the expression of proteins from the bcl-2 and the caspase families of proteins are implicated in the induction of macrophage apoptosis by Co(2+) and Cr(3+) ions.     

Coamplification of 12p11 and 12q13 approximately q22 in multiple ring chromosomes in a spindle cell sarcoma resolved by novel multicolor fluorescence in situ hybridization analysis

An 80-year-old male presented with a lobulated mass in the lower abdominal wall. A diagnosis of an intermediate grade myofibroblastic spindle cell sarcoma was made. Cytogenetic analysis demonstrated a complex karyotype with a der(6), a small marker and five, different in size, ring chromosomes. Fluorescence in situ hybridization (FISH), multiplex FISH, and multicolor banding analysis was used to further delineate this complex karyotype. The der(6) was shown to be a der(18)t(6;18;9;12;18), the marker chromosome was identified as del(17), and the ring chromosomes as r(9) and r(12;18)x4. Amplification of 18 and coamplification of 12p and 12q was detected in the ring and marker chromosomes. No intercellular heterogeneity was observed although a few micronuclei containing chromosome 18 and anaphase bridges, containing chromosome 12 material, the result of bridge-fusion-bridge (BFB) cycles, were observed. Our findings combined with results from others indicate that amplification of chromosomes 12 and 18 as well as BFB phenomena characterize this type of sarcoma.     

Immune Responses to Bile-Tolerant Helicobacter Species in Patients with Chronic Liver Diseases, a Randomized Population Group, and Healthy Blood Donors

Bile-tolerant Helicobacter species such as Helicobacter pullorum, Helicobacter bilis, and Helicobacter hepaticus are associated with hepatic disorders in animals and may be involved in the pathogenesis of chronic liver diseases (CLD) in humans. Antibody responses to cell surface proteins of H. pullorum, H. bilis, and H. hepaticus in serum samples from patients with CLD, a randomized population group, and healthy blood donors were evaluated by using enzyme linked immunosorbent assay (ELISA). The results were compared with the antibody responses to Helicobacter pylori. For analysis of a possible cross-reactivity between bile-tolerant Helicobacter species and H. pylori, sera from a subpopulation of each group were absorbed with a whole-cell extract of H. pylori and retested by ELISA. Results before absorption showed that the mean value of the ELISA units for H. pullorum was significantly higher in patients with CLD than in healthy blood donors (P = 0.01). Antibody reactivity to cell surface protein of H. hepaticus was also significantly higher in the CLD patients than in the healthy blood donors and the population group (P = 0.005 and P = 0.002, respectively). Following the absorption, antibody responses to H. pullorum decreased significantly in all three groups (P = 0.0001 for CLD patients, P = 0.0005 for the population group, and P < 0.0001 for the blood donors), indicating that cross-reactivity between H. pylori and other Helicobacter spp. occurs. The antibody responses to H. hepaticus and H. bilis in CLD patients remained high following absorption experiments compared to ELISA results before absorption. The significance of this finding requires further investigations.     

Apoptosis of myocytes and proliferation markers as prognostic factors in end-stage dilated cardiomyopathy.

INTRODUCTION: The aim of the study was to evaluate the role of apoptosis, proliferation markers, volume density of interstitium, and myofibril volume fraction for the prognosis in patients with end-stage dilated cardiomyopathy (DCM). METHODS: Endomyocardial biopsy was performed during open-heart surgery in 56 patients with end-stage DCM. Patients were divided into two groups, one group with shorter survival (24+/-9 months, mean+/-S.D.) and another group with survival of more than 7 years after operation. The TUNEL method was used for the detection of apoptosis, and immunohistochemical methods were used for the evaluation of inhibitor of apoptosis (bcl-2) and proliferation markers (PCNA and Ki-67). RESULTS: The increased percentage of apoptotic myocytes, decreased expression of bcl-2, and decreased expression of PCNA and Ki-67 antigen was found in the group with early mortality compared to that with longer survival. Myofibril volume fraction was lower and volume density of interstitium was higher in the group with early mortality compared to that with longer survival. CONCLUSION: Apoptosis, bcl-2 expression, and proliferation activity of myocytes, myofibril volume fraction, and volume density of interstitial tissue might be useful in predicting the prognosis (progressive vs. nonprogressive form) of patients with heart failure due to DCM.     

Update on Pneumocystis carinii f. sp. hominis Typing Based on Nucleotide Sequence Variations in Internal Transcribed Spacer Regions of rRNA Genes

Pneumocystis carinii f. sp. hominis isolates from 207 clinical specimens from nine countries were typed based on nucleotide sequence variations in the internal transcribed spacer regions I and II (ITS1 and ITS2, respectively) of rRNA genes. The number of ITS1 nucleotides has been revised from the previously reported 157 bp to 161 bp. Likewise, the number of ITS2 nucleotides has been changed from 177 to 192 bp. The number of ITS1 sequence types has increased from 2 to 15, and that of ITS2 has increased from 3 to 14. The 15 ITS1 sequence types are designated types A through O, and the 14 ITS2 types are named types a through n. A total of 59 types of P. carinii f. sp. hominis were found in this study.     

Loss of cell polarity causes severe brain dysplasia in Lgl1 knockout mice

Disruption of cell polarity is seen in many cancers; however, it is generally considered a late event in tumor progression. Lethal giant larvae (Lgl) has been implicated in maintenance of cell polarity in Drosophila and cultured mammalian cells. We now show that loss of Lgl1 in mice results in formation of neuroepithelial rosette-like structures, similar to the neuroblastic rosettes in human primitive neuroectodermal tumors. The newborn Lgl1(-/-) pups develop severe hydrocephalus and die neonatally. A large proportion of Lgl1(-/-) neural progenitor cells fail to exit the cell cycle and differentiate, and, instead, continue to proliferate and die by apoptosis. Dividing Lgl1(-/-) cells are unable to asymmetrically localize the Notch inhibitor Numb, and the resulting failure of asymmetric cell divisions may be responsible for the hyperproliferation and the lack of differentiation. These results reveal a critical role for mammalian Lgl1 in regulating of proliferation, differentiation, and tissue organization and demonstrate a potential causative role of disruption of cell polarity in neoplastic transformation of neuroepithelial cells.     

Relating cistrons and functions in bacteriophage PM2

An approach to correlate functions with cistrons in bacteriophage PM2 is presented. Two of the temperature-sensitive mutants obtained differed from the wild type in heat sensitivity and four differed in host range. Comparison of the electrophoretic patterns of DNA from cells infected at the restrictive temperature with those obtained in wildtype infection revealed that viral DNA bands were absent with three additional mutants. Complementation analysis assigned the four host range mutants to cistron I and the three mutants defective in DNA synthesis to cistron IV. Recombination frequencies were used to locate cistrons III and IV on the partial genetic map of PM2.     

Analysis of the allelic diversity of the mycobacterial interspersed repetitive units in Mycobacterium tuberculosis strains of the Beijing family: practical implications and evolutionary considerations

A study set comprised 44 Mycobacterium tuberculosis strains of the Beijing family selected for their representativeness among those previously characterized by IS6110-RFLP and spoligotyping (Northwest Russia, 1997 to 2003). In the present study, these strains were subjected to mycobacterial interspersed repetitive units (MIRU) typing to assess a discriminatory power of the 12-MIRU-loci scheme (P. Supply et al., J. Clin. Microbiol. 39:3563-3571, 2001). The 44 Russian Beijing strains were subdivided into 12 MIRU types with identical profiles: 10 unique strains and two major types shared by 10 and 24 strains. Thus, basically, two distinct sublineages appear to shape the evolution of the Beijing strains in Russia. Most of the MIRU loci were found to be (almost) monomorphic in the Russian Beijing strains; the Hunter-Gaston discriminatory index (HGDI) for all 12 loci taken together was 0.65, whereas MIRU26 (the most variable in our study) showed a moderate level of discrimination (0.49). The results were compared against all available published MIRU profiles of Beijing strains from Russia (3 strains) and other geographic areas (51 strains in total), including South Africa (38 strains), East Asia (7 strains), and the United States (4 strains). A UPGMA (unweighted pair-group method with arithmetic averages)-based tree was constructed. Interestingly, no MIRU types were shared by Russian and South African strains (the two largest samples in this analysis), whereas both major Russian types included also isolates from other locations (United States and/or East Asia). This implies the evolution of the Beijing genotype to be generally strictly clonal, although a possibility of a convergent evolution of the MIRU loci cannot be excluded. We propose a dissemination of the prevailing local Beijing clones to have started earlier in South Africa rather than in Russia since more monomorphic loci were identified in Russian samples than in South African samples (mean HGDI scores, 0.08 versus 0.17). To conclude, we suggest to use a limited number of MIRUs for preliminary subdivision of Beijing strains in Russian (loci 26 + 31), South African (10 + 26 + 39), and global settings (10 + 26 + 39).