Immunostimulating activity of Uncaria tomentosa in RAW 264.7 macrophages.

Uncaria tomentosa is a plant native to the Amazon that has immunomodulatory and antitumor properties due to the alkaloids found in the plant, being able to modify the immune response by potentiating or suspending the action of cytokines secreted by macrophages that induce the immune response, either by the classical route (M1) or through the alternative route (M2). Macrophages activated by M1 convert L-arginine into L-citrulline and nitric oxide (NO), whereas macrophages activated by the M2 pathway use the enzymatic activity of arginase to convert the same substrate into L-ornithine and urea. The aim of this work was to evaluate the immunostimulating activity of the crude hydroalcoholic extract from the bark of the U. tomentosa stem in RAW 264.7 macrophages. Concentrations of 0.2, 0.1 and 0.05 mg/mL of U. tomentosa extract associated with LPS, INF-γ and IL-4 inducers were tested by determining NO production and arginase enzyme activity. Nitric oxide production was enhanced by the extract when associated with LPS and LPS + INF-γ inducers. In the activity of the arginase enzyme, the extract decreased the stimulation of IL-4 on the enzyme, mainly at 0.2 mg/mL concentration. Therefore, it is concluded that the crude hydroalcoholic extract of the stem bark of U. tomentosa in RAW 264.7 cells, at a concentration of 0.2 mg/mL, showed considerable pro-inflammatory activity.

Increased expression of interleukin-6 gene in gastritis and gastric cancer.

Helicobacter pylori (H. pylori) induces an intense inflammatory response, mediated by proinflammatory cytokines, including interleukin (IL)-6 and its membrane receptor (IL-6R), which activates important signaling pathways in the development of gastric disease and cancer. We investigated the gene and protein expression of IL-6 and IL-6R and the influence of polymorphisms rs1800795, rs1800796, and rs1800797 on its gene expression together with H. pylori infection. Furthermore, an in-silico analysis was performed to support our results. Gastric biopsies were obtained from patients with gastric symptoms and patients with gastric cancer (GC) and were divided into groups (Control, Gastritis, and Cancer). H. pylori was detected by PCR. Real-time-qPCR was employed to determine gene expression, and western blot assay was used to analyze protein expression levels. PCR-RFLP was used to characterize IL-6 polymorphisms. Bioinformatics analyses were performed using the Gene Expression Omnibus (GEO) database and GEO2R to screen out differentially expressed genes (DEGs). H. pylori was detected in 43.3% of the samples. Statistically significant differences were found for IL-6 (P=0.0001) and IL-6R (P=0.0005) genes among the three groups, regardless of the presence of H. pylori. Among patients with H. pylori infection, the IL-6 and IL-6R gene and protein expressions were significantly increased, highlighting IL-6 gene overexpression in patients with GC. No statistically significant differences were found for the rs1800795, rs1800796, and rs1800797 polymorphisms compared to IL-6 gene expression. The results indicated that the IL-6 polymorphisms do not influence its expression, but IL-6 and IL-6R expression seems to be altered by the presence of H. pylori.

Learning complex texture discrimination.

Higher-order spatial correlations contribute strongly to visual structure and salience, and are common in the natural environment. One method for studying this structure has been through the use of highly controlled texture patterns whose obvious structure is defined entirely by third- and higher-order correlations. Here we examine the effects that longer-term training has on discrimination of 17 such texture types. Training took place in 14 sessions over 42 days. Discrimination performance increased at different rates for different textures. The time required to complete a visit reduced by 25.4% (p=0.0004). Factor analysis was applied to data from the learning and experienced phases of the experiment. This indicated that the gain in speed was accompanied by an increase in the number of mechanisms contributing to discrimination. Learning was not affected by sleep quality but was affected by extreme tiredness (p<0.01). The improved discrimination and speed were retained for 2.5 months. Overall, the effects were consistent with perceptual learning. The observed learning is likely related to the adaptation of innate mechanisms that underlie our ability to identify nonredundant, visually salient structure in natural images. It may involve cortical V2 and appears to involve increased strength, speed, and breadth of connections within our internal representation of this complex perceptual space.

Increased expression of interleukin-6 gene in gastritis and gastric cancer

Helicobacter pylori (H. pylori) induces an intense inflammatory response, mediated by proinflammatory cytokines, including interleukin (IL)-6 and its membrane receptor (IL-6R), which activates important signaling pathways in the development of gastric disease and cancer. We investigated the gene and protein expression of IL-6 and IL-6R and the influence of polymorphisms rs1800795, rs1800796, and rs1800797 on its gene expression together with H. pylori infection. Furthermore, an in-silico analysis was performed to support our results. Gastric biopsies were obtained from patients with gastric symptoms and patients with gastric cancer (GC) and were divided into groups (Control, Gastritis, and Cancer). H. pylori was detected by PCR. Real-time-qPCR was employed to determine gene expression, and western blot assay was used to analyze protein expression levels. PCR-RFLP was used to characterize IL-6 polymorphisms. Bioinformatics analyses were performed using the Gene Expression Omnibus (GEO) database and GEO2R to screen out differentially expressed genes (DEGs). H. pylori was detected in 43.3% of the samples. Statistically significant differences were found for IL-6 (P=0.0001) and IL-6R (P=0.0005) genes among the three groups, regardless of the presence of H. pylori. Among patients with H. pylori infection, the IL-6 and IL-6R gene and protein expressions were significantly increased, highlighting IL-6 gene overexpression in patients with GC. No statistically significant differences were found for the rs1800795, rs1800796, and rs1800797 polymorphisms compared to IL-6 gene expression. The results indicated that the IL-6 polymorphisms do not influence its expression, but IL-6 and IL-6R expression seems to be altered by the presence of H. pylori.

Multidrug-resistant Escherichia coli isolated from a dog with a history of urolithiasis: case report / Escherichia coli multirresistente isolada de cão com histórico de urolitíase: relato de caso

Bacterial resistance is a reality in both human and veterinary health, it limits the therapeutic arsenal and raises the costs of the patient's treatment. A dog with signs of cystitis received treatment with 5mg/kg enrofloxacin at three consecutive times, with low effectiveness. The presence of urethral uroliths was identified and urohydropulsion was done. The animal presented a new obstruction, for which a cystotomy was performed, but continued with signs of infection. Uroculture and antimicrobial susceptibility test were then performed. Escherichia coli was identified, which was resistant to 13 antibiotics, being sensitive only to piperacillin-tazobactam and amikacin. In the screening test for ß-lactamase, the production of ESßL was detected. The qPCR indicated the presence of the bla CTXm, bla DHA, bla OXA, bla IMP, bla TEM, bla GIM, bla SIM, bla SPM and bla SME genes, which may lead to a phenotypic resistance profile for ampicillin, amoxicillin-clavulanate, aztreonam, cefepime cefoxitin, cefuroxime, ceftazidime, ceftriaxone, imipenem, and piperacillin-tazobactam. This case reaffirms the value that laboratory analysis adds to the diagnosis and treatment of cystitis and urolithiasis, which can define the direction of evolution of the prognosis and the speed at which the patient's health will be restored.(AU)

A resistência bacteriana aos antibióticos é uma realidade, tanto na saúde humana quanto veterinária, limita o arsenal terapêutico e eleva os custos relacionados ao tratamento do paciente. Um cão, com sinais de cistite, recebeu tratamento com enrofloxacina, na dose de 5mg/kg, em três momentos seguidos, com baixa efetividade. Identificou-se presença de urólitos uretrais e foi feita uro-hidropropulsão. O animal apresentou nova obstrução, para a qual foi realizada uma cistotomia, mas continuou com sinais de infecção. Realizou-se, então, urocultura e teste de antibiograma. Foi identificada Escherichia coli, que se mostrou resistente a 13 antibióticos, sendo sensível somente à piperacilina-tazobactam e amicacina. No teste de triagem para ß-lactamase, detectou-se a produção de ESßL. A qPCR indicou presença dos genes blaCTXm, blaDHA, blaOXA, blaIMP, blaTEM, blaGIM, blaSIM, blaSPM e blaSME, que podem conduzir um perfil fenotípico de resistência para ampicilina, amoxicilina-ácido clavulânico, aztreonam, cefepima, cefoxitina, cefuroxima, ceftazidima, ceftriaxona, imipenem, piperacilina-tazobactam. Este caso reafirma o valor que a análise laboratorial agrega ao diagnóstico e tratamento da cistite e da urolitíase, podendo definir o sentido de evolução do prognóstico e a velocidade em que a saúde do paciente será restabelecia.(AU)

Atividade biológica in vitro de própolis e óleos essenciais sobre o fungo Colletotrichum musae isolado de bananeira (Musa spp. ) / In vitro biological activity of propolis and essential oils on the fungus Colletotrichum musae isolated from banana Musa spp

RESUMO: No Brasil existem várias doenças fúngicas que acometem a bananeira. Destas, pode-se citar a antracnose, responsável por grandes prejuízos à cultura, cujo agente causal é o fungo Colletotrichum musae. A principal forma de controle dessa enfermidade é através da aplicação de fungicidas a base de tiabendazol ou tiofanato metílico. Esse manejo, embora eficiente, favorece o desenvolvimento de resistência do patógeno, causa danos ao ambiente e ao produtor, deixando ainda resíduos nos frutos. Esses fatores têm favorecido a busca por substâncias alternativas com capacidade de controlar o fungo e que não sejam nocivas ao ambiente e, principalmente, que sejam seguras ao consumidor final. Dentre as opções, surge o interesse pelo uso de certos óleos essenciais e da própolis, ambos conhecidos por possuírem propriedades fungicidas. O presente trabalho foi desenvolvido com o objetivo de determinar o potencial fungitóxico "in vitro" da própolis e dos óleos essenciais de palmarosa (Cymbopogon martinii), de teatree (Melaleuca alternifolia), de cravo (Eugenia caryophyllata), e de eucalipto (Corymbia citriodora), sobre Colletotrichum musae. O desenvolvimento experimental consistiu em adicionar inóculos fúngicos de 5 mm, obtidos a partir de colônias puras, ao meio de cultura BDA (batata-dextrose-ágar) acrescido das referidas substâncias em diferentes concentrações (0, 25, 50, 75, 100 e 125 µL/L). Paralelo aos tratamentos realizou-se teste com o fungicida padrão para comparações das médias. A eficiência das substâncias sobre o fungo foi determinada através das avaliações do crescimento micelial das colônias (média de duas medidas diametralmente opostas). Os valores de crescimento micelial obtidos foram utilizados também para o cálculo do índice de velocidade de crescimento micelial. O delineamento experimental utilizado foi inteiramente casualizado em esquema fatorial 5 x 6 + 1, (cinco substâncias em seis concentrações + fungicida), com cinco repetições. Os óleos de tea tree, cravo e palmarosa foram eficientes no controle do fungo Colletotrichum musae não diferindo do fungicida a partir da dose de 50 µL/L em todas as avaliações, apresentando potencial para controle em cultivos orgânicos ou em sistemas de manejo integrado.

ABSTRACT: In Brazil there are several fungi that cause diseases on banana plants. These include the "anthracnose", which is responsible for major crop losses and whose causative agent is the fungus Colletotrichum musae. The main way to control this disease is through the application of fungicides based on thiabendazole and thiophanate-methyl. Although this management is effective, it favors the development of pathogen resistance, which causes damage to the environment and producer and also leaves residues in fruits. These factors have encouraged the search for alternative substances to control the fungus and that are not harmful to the environment and particularly to the final consumer. Among the options, there is interest in the use of essential oils and propolis, both known to have antifungal activity. The present work was developed with the objective of determining the potential of propolis and essential oils of palmarosa (Cymbopogon martinii), tea tree (Melaleuca alternifolia), clove (Eugenia caryophyllata) and eucalyptus (Corymbia citriodora) in the in vitro control of the fungus Colletotrichum musae. The experimental development consisted in adding 5 mm fungal inoculants, obtained from pure colonies, in PDA culture (potato-dextrose-agar) plus the aforementioned substances in different concentrations (0, 25, 50, 75, 100 and 125 µL/L). At the same time as these treatments, we carried out a test with the fungicide to compare the averages. The efficiency of the substances on the fungus was determined through evaluations of the mycelial growth of the colonies (average of two diametrically opposed measures). The values of mycelial growth obtained were also used for the calculation of the speed index of the mycelial growth. The experimental design was completely randomized in 5 x 6 + 1 (5 substances in 6 concentrations + fungicide) factorial design, with 5 repetitions. The tea tree, clove and palmarosa oils were efficient in the control of the fungus Colletotrichum musae, which can be used as a control option in organic crops or in integrated management systems.

Purification and characterization of the bacteriocin produced by Lactobacillus sakei MBSa1 isolated from Brazilian salami.

AIMS: The study aimed at determining the biochemical characteristics of the bacteriocin produced by Lactobacillus sakei MBSa1, isolated from salami, correlating the results with the genetic features of the producer strain. METHODS AND RESULTS: Identification of strain MBSa1 was performed by 16S rDNA sequencing. The bacteriocin was tested for spectrum of activity, heat and pH stability, mechanism of action, molecular mass and amino acid sequence when purified by cation-exchange and reversed-phase HPLC. Genomic DNA was tested for bacteriocin genes commonly present in Lact. sakei. Bacteriocin MBSa1 was heat-stable, unaffected by pH 2·0 to 6·0 and active against all tested Listeria monocytogenes strains. Maximal production of bacteriocin MBSa1 (1600 AU ml(-1)) in MRS broth occurred after 20 h at 25°C. The molecular mass of produced bacteriocin was 4303·3 Da, and the molecule contained the SIIGGMISGWAASGLAG sequence, also present in sakacin A. The strain contained the sakacin A and curvacin A genes but was negative for other tested sakacin genes (sakacins T-α, T-ß, X, P, G and Q). CONCLUSIONS: In the studied conditions, Lact. sakei MBSa1 produced sakacin A, a class II bacteriocin, with anti-Listeria activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The study covers the purification and characterization of the bacteriocin produced by a lactic acid bacteria isolated from salami (Lact. sakei MBSa1), linking genetic and expression information. Its heat-resistance, pH stability in acid conditions (pH 2·0-6·0) and activity against L. monocytogenes food isolates bring up a potential technological application to improve food safety.

Mathematical characterization of three-dimensional gene expression patterns.

MOTIVATION: The importance of a systematic methodology for the mathematical characterization of three-dimensional gene expression patterns in embryonic development. METHODS: By combining lacunarity and multiscale fractal dimension analyses with computer-based methods of three-dimensional reconstruction, it becomes possible to extract new information from in situ hybridization studies. Lacunarity and fractality are appropriate measures for the cloud-like gene activation signals in embryonic tissues. The newly introduced multiscale method provides a natural extension of the fractal dimension concept, being capable of characterizing the fractality of geometrical patterns in terms of spatial scale. This tool can be systematically applied to three-dimensional patterns of gene expression. RESULTS: Applications are illustrated using the three-dimensional expression patterns of the myogenic marker gene Myf5 in a series of differentiating somites of a mouse embryo.

The effect of a T cell-specific NF-kappa B inhibitor on in vitro cytokine production and collagen-induced arthritis.

NF-kappa B plays a key role in the production of cytokines in inflammatory diseases. The effects of a novel T cell-specific NF-kappa B inhibitor, SP100030, were evaluated in cultured Jurkat cells and in murine collagen-induced arthritis (CIA). Chemical libraries were screened for NF-kappa B-inhibitory activity. SP100030, a compound identified in this process, inhibited NF-kappa B activation in PMA/PHA-activated Jurkat cells by EMSA at a concentration of 1 microM. Jurkat cells and the monocytic cell line THP-1 were transfected with an NF-kappa B promotor/luciferase construct and activated. SP100030 inhibited luciferase production in the Jurkat cells (IC50 = 30 nM). ELISA and RT-PCR confirmed that IL-2, IL-8, and TNF-alpha production by activated Jurkat and other T cell lines were inhibited by SP100030. However, cytokine expression was not blocked by the compound in THP-1 cells, fibroblasts, endothelial cells, or epithelial cells. Subsequently, DBA/1J mice were immunized with type II collagen. Treatment with SP100030 (10 mg/kg/day i.p. beginning on day 21) significantly decreased arthritis severity from onset of clinical signs to the end of the study on day 34 (arthritis score, 5.6 +/- 1.7 for SP100030 and 9.8 +/- 1.5 for control; p < 0.001). Histologic evaluation demonstrated a trend toward improvement in SP100030-treated animals. EMSA of arthritic mouse ankles in CIA showed that synovial NF-kappa B binding was suppressed in the SP100030-treated mice. SP100030 inhibits NF-kappa B activation in T cells, resulting in reduced NF-kappa B-regulated gene expression and decreased CIA. Its selectivity for T cells could provide potent immunosuppression with less toxicity than other NF-kappa B inhibitors.

The mitogen-activated protein kinase p38-2 is necessary for the inhibition of N-type calcium current by bradykinin.

Calcium influx via voltage-dependent calcium channels (ICa,V) links depolarization of excitable cells to critical cellular processes, such as secretion, contraction, and gene transcription. Fast regulation of ICa,V (<1 sec) by G-protein-coupled receptors is a relatively well-defined mechanism, whereas slow (30-60 sec) actions of transmitters and hormones on the same current remain poorly understood. In NG108-15 cells, the kinetically slow inhibition of N-type ICa,V by bradykinin (BK) requires the sequential activation of two G-proteins, heterotrimeric G13 and monomeric Rac1/Cdc42. We have now defined a role in this pathway for the relatively fast-acting p38 mitogen-activated protein kinase (MAPK). The slow inhibition of ICa,V by BK was suppressed specifically by SB203580, a compound that inhibits the p38 family of MAPKs. BK potently and selectively activated a newly discovered p38 family member, p38-2. These data provide the first evidence that a MAPK is involved in the regulation of ICa,V by a receptor-mediated process.

Transcription factor Sp1 mediates cell-specific trans-activation of the human cytomegalovirus DNA polymerase gene promoter by immediate-early protein IE86 in glioblastoma U373MG cells.

Human cytomegalovirus (HCMV) gene expression is highly cell and tissue specific. Cell factor-mediated regulatory interactions are involved in regulating the restricted expression of the HCMV major immediate-early (IE) gene (J. F. Baskar, P. P. Smith, G. Nilaver, R. A. Jupp, S. Hoffmann, N. J. Peffer, D. J. Tenney, A. M. Colberg-Poley, P. Ghazal, and J. A. Nelson, 70:3207-3213, 1996). To gain an understanding of HCMV early gene activation, we studied the effect of each of the three major IE proteins, IE72, IE86, and IE55, on the HCMV DNA polymerase gene (pol; UL54) promoter. In transient-expression assays, the IE86 protein alone was able to transactivate the pol promoter, but IE72 and IE55 were not, in permissive U373MG cells. However, we were unable to detect IE86-mediated transactivation in nonpermissive HeLa or C33-A cells. Using electrophoretic mobility shift assays (EMSAs), we found that expression of the IE86 protein in U373MG cells resulted in specific binding of a DNA complex to an inverted-repeat element, IR1, of the pol promoter. Antibody supershifting and EMSA-Western blotting experiments further showed that IE86 and the cellular transcription factor Sp1 were components of the IR1 DNA-binding complex. Furthermore, we found that binding of DNA by Sp1 was dramatically increased in the presence of IE86. Interestingly, this IE86-induced DNA-binding activity of Sp1 was inhibited by a repressor activity presented in HeLa cells. In summary, our study suggests that a viral regulatory protein can modulate the DNA binding activity of a cellular transcription factor, resulting in cell-specific transactivation of viral genes.

HSP70 binding sites in the tumor suppressor protein p53.

Mutations within conserved regions of the tumor suppressor protein, p53, result in oncogenic forms of the protein with altered tertiary structures. In most cases, the mutant p53 proteins are selectively recognized and bound by members of the HSP70 family of molecular chaperones, but the binding site(s) in p53 for these chaperones have not been clearly defined. We have screened a library of overlapping biotinylated peptides, spanning the entire human p53 sequence, for binding to the HSP70 proteins, Hsc70 and DnaK. We show that most of the high affinity binding sites for these proteins map to secondary structure elements, particularly beta-strands, in the hydrophobic core of the central DNA binding domain, where the majority of oncogenic p53 mutations are found. Although peptides corresponding to the C-terminal region of p53 also contain potential binding sites, p53 proteins with C-terminal deletions are capable of binding to Hsc70, indicating that this region is not required for complex formation. We propose that mutations in the p53 protein alter the tertiary structure of the central DNA binding domain, thus exposing high affinity HSP70 binding sites that are cryptic in the wild-type molecule.

p38-2, a novel mitogen-activated protein kinase with distinct properties.

Mitogen-activated protein (MAP) kinases are involved in many cellular processes. Here we describe the cloning and characterization of a new MAP kinase, p38-2. p38-2 belongs to the p38 subfamily of MAP kinases and shares with it the TGY phosphorylation motif. The complete p38-2 cDNA was isolated by polymerase chain reaction. It encodes a 364-amino acid protein with 73% identity to p38. Two shorter isoforms missing the phosphorylation motif were identified. Analysis of various tissues demonstrated that p38-2 is differently expressed from p38. Highest expression levels were found in heart and skeletal muscle. Like p38, p38-2 is activated by stress-inducing signals and proinflammatory cytokines. The preferred upstream kinase is MEK6. Although p38-2 and p38 phosphorylate the same substrates, the site specificity of phosphorylation can differ as shown by two-dimensional phosphopeptide analysis of Sap-1a. Additionally, kinetic studies showed that p38-2 appears to be about 180 times more active than p38 on certain substrates such as ATF2. Both kinases are inhibited by a class of pyridinyl imidazoles. p38-2 phosphorylation of ATF2 and Sap-1a but not Elk1 results in increased transcriptional activity of these factors. A sequential kinetic mechanism of p38-2 is suggested by steady state kinetic analysis. In conclusion, p38-2 may be an important component of the stress response required for the homeostasis of a cell.

Enhanced transcriptional activation by E2 proteins from the oncogenic human papillomaviruses.

A systematic comparison of transcriptional activation by papillomavirus E2 proteins revealed that the E2 proteins from high-risk human papillomaviruses (human papillomavirus type 16 [HPV-16] and HPV-18) are much more active than are the E2 proteins from low-risk HPVs (HPV-6b and HPV-11). Despite the tropism of HPVs for particular epithelial cell types, this difference in transcriptional activation was observed in a number of different epithelial and nonepithelial cells. The enhanced activities of the E2 proteins from high-risk HPVs did not result from higher steady-state levels of protein in vivo, and in vitro DNA-binding assays revealed similar binding properties for these two classes of E2 proteins. These results demonstrate that the E2 proteins from high-risk HPVs have an intrinsically enhanced potential to activate transcription from promoters with E2-responsive elements. We found that there are also substantial differences between the activation properties of the bovine papillomavirus type 1 E2 protein and those of either of the two classes of HPV E2 proteins, especially with regard to requirements for particular configurations of E2 binding sites in the target promoter. Our results indicate that there are at least three distinct functional classes of E2 proteins and that these classes of E2 proteins may perform different roles during the respective viral life cycles.

Cloning and characterization of MEK6, a novel member of the mitogen-activated protein kinase kinase cascade.

Mitogen-activated protein kinases are members of a conserved cascade of kinases involved in many signal transduction pathways. They stimulate phosphorylation of transcription factors in response to extracellular signals such as growth factors, cytokines, ultraviolet light, and stress-inducing agents. A novel mitogen-activated protein kinase kinase, MEK6, was cloned and characterized. The complete MEK6 cDNA was isolated by polymerase chain reaction. It encodes a 334-amino acid protein with 82% identity to MKK3. MEK6 is highly expressed in skeletal muscle like many other members of this family, but in contrast to MKK3 its expression in leukocytes is very low. MEK6 is a member of the p38 kinase cascade and efficiently phosphorylates p38 but not c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) family members in direct kinase assays. Coupled kinase assays demonstrated that MEK6 induces phosphorylation of ATF2 by p38 but does not phosphorylate ATF2 directly. MEK6 is strongly activated by UV, anisomycin, and osmotic shock but not by phorbol esters, nerve growth factor, and epidermal growth factor. This separates MEK6 from the ERK subgroup of protein kinases. MEK6 is only a poor substrate for MEKK, a mitogen-activated protein kinase kinase kinase that efficiently phosphorylates the related family member JNKK.

The oncogenic role of human papillomavirus proteins.

Each human papillomavirus (HPV) type is genotypically distinct and infects epithelial cells at unique anatomic sites. Among the HPV types described, a subgroup is associated with genital disease and a subset of these is found in 90% of genital cancers. Although in benign infections the viral genome is present as an episome, in cancers it is integrated. The integration event invariably results in the expression of two viral proteins, E6 and E7. These two proteins are capable of transforming cells individually and cooperate to immortalize primary human epithelial cells. Molecular analysis has revealed that the E6 protein encoded by the HPV "high risk" types prevalent in cancers forms a tripartite complex with the p53 tumor suppressor protein and a cellular protein termed E6-AP, resulting in the degradation of p53. The E7 protein encoded by "high-risk" HPV types shows high-affinity association with the retinoblastoma tumor suppressor, pRb. The E7 protein associates also with other cellular factors known to play a role in cell cycle regulation. This review discusses the evidence, molecular and biological, in vitro and in vivo, supporting a direct role for the "high-risk" HPV type encoded E6 and E7 proteins in cervical carcinogenesis.

Distinct regions of p53 have a differential role in transcriptional activation and repression functions.

The wild type p53 tumor suppressor protein transactivates genes carrying p53 responsive elements and represses several TATA containing promoters. We report in vivo gene regulation assays where deletion of the N-terminal 75 residues (delta N75) results in loss of transactivation of p53CON and repression of an HPV 6 reporter. In contrast, removal of the C-terminal 75 (delta C75) amino acids resulted in a truncated protein capable of trans-activating p53CON but not able to repress the HPV 6 reporter. In vitro protein association assays revealed that the delta N75 protein, but not the delta C75 truncated protein, could oligomerize with the wild type p53 protein. Co-transfection assays with wild type p53 showed that the delta N75 mutant protein has a dominant negative effect on trans-activation function. However, it does not affect the ability of wild type p53 to repress transcription from the HPV 6 receptor. The delta C75 protein had no effect on the ability of the wild type p53 to activate p53CON or repress the HPV 6 reporter. These results suggest that distinct regions of p53 have a differential role in transcriptional activation and repression functions.