p21 functions in a post-mitotic block checkpoint in the apoptotic response to vinblastine

We have shown previously that in KB-3 (HeLa) cells vinblastine causes downregulation of the CDK inhibitor p21 through a c-Jun regulated pathway. To test the hypothesis that p21 downregulation is necessary to alleviate a protective function, we transfected p21 in KB-3 cells and examined the apoptotic response to vinblastine. The results showed that cells overexpressing p21 were apoptosis-resistant, not through an ability of p21 to cause cell cycle arrest prior to mitotic arrest, but through altering the fate of mitotically arrested cells after drug treatment. Moreover, p21 null HCT116 cells were more prone to vinblastine-induced apoptosis relative to wild-type cells. The results provide support for a model whereby p21 downregulation promotes vinblastine-induced apoptosis by alleviating its protective function following mitotic arrest.     

Phenotypic and molecular methods used for identification of oral streptococci and related microorganisms

The oral cavity contains the greatest biodiversity, over 70 species being isolated from mouth mucosa, saliva, denture surfaces and/or dental-plaque. The oral streptococci, representing over 80% of the mouth micro flora, are able to synthesize glucosyl-transferases, enzymes involved in glucans production. Glucans are involved in production of an extracellular slime layer promoting adhesion and formation of a dental plaque biofilm. The 43 isolates studied obtained from partially and/or totally edentulous, were identified by VITEK system using gram-positive identification cards. Species-specific regions within the genes coding for glucosyl-transferases (gtf genes) were targeted for PCR identification of isolates. Sequencing of 16S rRNA was used as gold standard for strain confirmation. VITEK system identified a number of 11 strains as S. mitis/oralis, 12 strains as S. anginosus/gordonii, 12 strains as S. sanguinis/parasanguinis, 3 strains as S. salivarius, 3 strains as S. plurianimalium, 1 strain as S. cristatus and 1 strain as S. alactolyticus, respectively. The PCR system targeting gtf genes was able to identify S. oralis, S. salivarius and S. gordonii strains. Sequence of 16S rRNA discriminated among streptococci species and revealed 16 strains of Leuconostoc mesenteroides. Many studies are needed in order to select the most reliable phenotypic and genotypic methods in order to improve the identification algorithm for oral streptococci used by clinical laboratories. Their accurate identification is mandatory for better understanding their role in human infections.     

TGF-beta signalling pathway factors in HPV-induced cervical lesions

Human papillomaviruses (HPV) are considered the etiological agents of cervical cancer, especially high-risk genotypes. TGF-beta (transforming growth factor-beta) is well known for its anti-proliferative effects but the neoplastic cells often lose their sensitivity to TGF-beta. A characteristic alteration associated with malignant progression is the loss of responsiveness to TGF-beta1-induced cell growth inhibition. The aim of the present study was to establish the possible role of some members of TGF-beta signalling pathway during cervical cancer development and the possible relationship with HPV infection. In order to establish TGF-beta gene expression levels in cervical oncogenesis, TGF-beta1, TGF-beta1 receptors and Smad2 were investigated in precancerous and cervical cancer samples (Quantitative Real-Time PCR). The study revealed that 84.5% of patients were positive for HPV DNA. The most prevalent HPV genotypes were high-risk HPV 16 and 18 in single or co-infections. Expression of TGF-beta1 decreased as tumor cells progressed from cervical intraepithelial neoplasia to cervical carcinoma. Furthermore, we observed that cervical lesions without HPV infection expressed significantly less TGF-beta1. TGF-betaRI and Smad2 gene expression levels were found to be decreased in SCC and AC samples in contrast with CIN1 and CIN2/3 samples. Our results showed that in human cervical cancer the disruption of TGF-beta/Smad signalling pathway might contribute to the malignant progression of cervical dysplasia. These data emphasize the importance of canonical TGF-beta pathway integrity in carcinogenesis.     

Genome-Wide Mutagenesis Reveals That ORF7 Is a Novel VZV Skin-Tropic Factor

The Varicella Zoster Virus (VZV) is a ubiquitous human alpha-herpesvirus that is the causative agent of chicken pox and shingles. Although an attenuated VZV vaccine (v-Oka) has been widely used in children in the United States, chicken pox outbreaks are still seen, and the shingles vaccine only reduces the risk of shingles by 50%. Therefore, VZV still remains an important public health concern. Knowledge of VZV replication and pathogenesis remains limited due to its highly cell-associated nature in cultured cells, the difficulty of generating recombinant viruses, and VZV''s almost exclusive tropism for human cells and tissues. In order to circumvent these hurdles, we cloned the entire VZV (p-Oka) genome into a bacterial artificial chromosome that included a dual-reporter system (GFP and luciferase reporter genes). We used PCR-based mutagenesis and the homologous recombination system in the E. coli to individually delete each of the genome''s 70 unique ORFs. The collection of viral mutants obtained was systematically examined both in MeWo cells and in cultured human fetal skin organ samples. We use our genome-wide deletion library to provide novel functional annotations to 51% of the VZV proteome. We found 44 out of 70 VZV ORFs to be essential for viral replication. Among the 26 non-essential ORF deletion mutants, eight have discernable growth defects in MeWo. Interestingly, four ORFs were found to be required for viral replication in skin organ cultures, but not in MeWo cells, suggesting their potential roles as skin tropism factors. One of the genes (ORF7) has never been described as a skin tropic factor. The global profiling of the VZV genome gives further insights into the replication and pathogenesis of this virus, which can lead to improved prevention and therapy of chicken pox and shingles.     

Effect of infliximab on mRNA expression profiles in synovial tissue of rheumatoid arthritis patients

We examined the gene expression profiles in arthroscopic biopsies retrieved from 10 rheumatoid arthritis patients before and after anti-TNF treatment with infliximab to investigate whether such profiles can be used to predict responses to the therapy, and to study effects of the therapy on the profiles. Responses to treatment were assessed using European League Against Rheumatism response criteria. Three patients were found to be good responders, five patients to be moderate responders and two patients to be nonresponders. The TNF-alpha status of the biopsies from each of the patients before treatment was also investigated immunohistochemically, and it was detected in biopsies from four of the patients, including all three of the good responders. The gene expression data demonstrate that all patients had unique gene expression signatures, with low intrapatient variability between biopsies. The data also revealed significant differences between the good responding and nonresponding patients (279 differentially expressed genes were detected, with a false discovery rate < 0.025). Among the identified genes we found that MMP-3 was significantly upregulated in good responders (log2 fold change, 2.95) compared with nonresponders, providing further support for the potential of MMP-3 as a marker for good responses to therapy. An even more extensive list of 685 significantly differentially expressed genes was found between patients in whom TNF-alpha was found and nonresponders, indicating that TNF-alpha could be an important biomarker for successful infliximab treatment. Significant differences were also observed between biopsies taken before and after anti-TNF treatment, including 115 differentially expressed genes in the good responding group. Interestingly, the effect was even stronger in the group in which TNF-alpha was immunohistochemically detected before therapy. Here, 1,058 genes were differentially expressed, including many that were novel in this context (for example, CXCL3 and CXCL14). Subsequent Gene Ontology analysis revealed that several 'themes' were significantly over-represented that are known to be affected by anti-TNF treatment in inflammatory tissue; for example, immune response (GO:0006955), cell communication (GO:0007154), signal transduction (GO:0007165) and chemotaxis (GO:0006935). No genes reached statistical significance in the moderately responding or nonresponding groups. In conclusion, this pilot study suggests that further investigation is warranted on the usefulness of gene expression profiling of synovial tissue to predict and monitor the outcome of rheumatoid arthritis therapies.     

Sterol carrier protein-2 directly interacts with caveolin-1 in vitro and in vivo

HDL-mediated reverse-cholesterol transport as well as phosphoinositide signaling are mediated through plasma membrane microdomains termed caveolae/lipid rafts. However, relatively little is known regarding mechanism(s) whereby these lipids traffic to or are targeted to caveolae/lipid rafts. Since sterol carrier protein-2 (SCP-2) binds both cholesterol and phosphatidylinositol, the possibility that SCP-2 might interact with caveolin-1 and caveolae was examined. Double immunolabeling and laser scanning fluorescence microscopy showed that a small but significant portion of SCP-2 colocalized with caveolin-1 primarily at the plasma membrane of L-cells and more so within intracellular punctuate structures in hepatoma cells. In SCP-2 overexpressing L-cells, SCP-2 was detected in close proximity to caveolin, 48 +/- 4 A, as determined by fluorescence resonance energy transfer (FRET) and immunogold electron microscopy. Cell fractionation of SCP-2 overexpressing L-cells and Western blotting detected SCP-2 in purified plasma membranes, especially in caveolae/ lipid rafts as compared to the nonraft fraction. SCP-2 and caveolin-1 were coimmunoprecipitated from cell lysates by anti-caveolin-1 and anti-SCP-2. Finally, a yeast two-hybrid assay demonstrated that SCP-2 directly interacts with caveolin-1 in vivo. These interactions of SCP-2 with caveolin-1 were specific since a functionally related protein, phosphatidyinositol transfer protein (PITP), colocalized much less well with caveolin-1, was not in close proximity to caveolin-1 (i.e., >120 A), and was not coimmunoprecipitated by anti-caveolin-1 from cell lysates. In summary, it was shown for the first time that SCP-2 (but not PITP) selectively interacted with caveolin-1, both within the cytoplasm and at the plasma membrane. These data contribute significantly to our understanding of the role of SCP-2 in cholesterol and phosphatidylinositol targeted from intracellular sites of synthesis in the endoplasmic reticulum to caveolae/lipid rafts at the cell surface plasma membrane.     

Correlation of tumor oxygen dynamics with radiation response of the dunning prostate R3327-HI tumor

Our previous studies have shown that oxygen inhalation significantly reduces tumor hypoxia in the moderately well-differentiated HI subline of the Dunning prostate R3327 rat carcinoma. To test our hypothesis that modifying hypoxia could improve the radiosensitivity of these tumors, we performed experimental radiotherapy to compare the tumor response to ionizing radiation alone or in combination with oxygen inhalation. Tumor pO(2) measurements were performed on size-selected tumors several hours before radiotherapy using (19)F nuclear magnetic resonance echo planar imaging relaxometry (FREDOM) of the reporter molecule hexafluorobenzene. In common with our previous findings, the larger tumors (>3.5 cm(3)) exhibited greater hypoxia than the smaller tumors (<2 cm(3); P < 0.001), and oxygen inhalation reduced the hypoxic fraction (<10 Torr): In the larger tumors, hypoxic fraction dropped significantly from a mean baseline value of 80% to 17% (P < 0.001). The effect of oxygen administered 30 min before and during irradiation on tumor response to a single 30-Gy dose of photons was evaluated by growth delay. For the smaller tumors, no difference in growth delay was found when treatment was given with or without oxygen breathing. By contrast, breathing oxygen before and during irradiation significantly enhanced the growth delay in the larger tumors (additional 51 days). The differential behavior may be attributed to the low baseline hypoxic fraction (<10 Torr) in small tumors (20%) as a target for oxygen inhalation. There was a strong correlation between the estimated initial pO(2) value and the radiation-induced tumor growth delay (R > 0.8). Our histological studies showed a good match between the perfused vessels marked by Hoechst 33342 dye and the total vessels immunostained by anti-CD31 and indicated extensive perfusion in this tumor line. In summary, the present results suggest that the ability to detect modulation of tumor pO(2), in particular, the residual hypoxic fraction, with respect to an intervention, could have prognostic value for predicting the efficacy of radiotherapy.     

Structure of the Rab7:REP-1 complex: insights into the mechanism of Rab prenylation and choroideremia disease

Members of the RabGDI/REP family serve as multifunctional regulators of the Rab family of GTP binding proteins. Mutations in members of this family, such as REP-1, lead to abnormalities, including progressive retinal degradation (choroideremia) in humans. The crystal structures of the REP-1 protein in complex with monoprenylated or C-terminally truncated Rab7 proteins revealed that Rab7 interacts with the Rab binding platform of REP-1 via an extended interface involving the Switch 1 and 2 regions. The C terminus of the REP-1 molecule functions as a mobile lid covering a conserved hydrophobic patch on the surface of REP-1 that in the complex coordinates the C terminus of Rab proteins. Using semisynthetic fluorescent Rab27A, we demonstrate that although Rab27A can be prenylated by REP-2, this reaction can be effectively inhibited by other Rab proteins, providing a possible explanation for the accumulation of unprenylated Rab27A in choroideremia.     

Unusually common cystic fibrosis mutation in Portugal encodes a misprocessed protein

A561E, a novel cystic fibrosis (CF) associated mutation in the first nucleotide binding domain of CFTR, is the second most common CF mutation in Portugal. Properties of the A561E-CFTR protein were studied by immunoblotting, pulse-chase, immunocytochemistry, and MQAE halide-efflux assay in stably transfected BHK cells. Altogether, results presented here suggest that A561E causes protein mislocalization in the endoplasmic reticulum where the mutant protein must be trapped by the quality control mechanism. We conclude that A561E originates a protein trafficking defect, thus belonging to class II of CFTR mutations. As it is the case for F508del-CFTR (the most common CF mutant), low temperature treatment partially rescues a functional A561E-CFTR channel, suggesting that substitution of glutamic acid for alanine at position 561 does not completely abolish CFTR function. Pharmacological strategies previously reported for treatment of CF patients with the F508del mutation could thus be also effective in CF patients bearing the A561E mutation.