Study of the dynamic behavior of the cruzain enzyme in free and complexed forms with competitive and noncovalent benzimidazole inhibitors.

There are only two drugs for the treatment of Chagas disease, namely, nifurtimox and benznidazole, that can cause several adverse effects. Despite the effectiveness of these drugs in the disease's acute phase, they are not recognized as curative in the chronic phase, establishing the need for more effective treatment in all stages of the disease. Cruzain is an enzyme that plays a vital role in the life cycle of the etiologic agent, the protozoan Trypanosoma cruzi, being relevant as a therapeutic target in the planning of new drugs. Using molecular docking and dynamics simulations, we have investigated the structural and dynamic factors that can be involved in the enzyme inhibition process at the atomic-molecular level by benzimidazole compounds that are potent cruzain inhibitors with in vitro trypanocidal activity. The study suggests that these inhibitors bind cruzain through steric and hydrogen bonding interactions without altering its secondary structure content and protein compaction. Besides, we observed that these inhibitors decrease the correlation of movements between Cα-atoms of cruzain, increasing the number of atomic communities, mainly in the α-helix that presents the catalytic Cys25 residue. As expected, we also observed a correlation between the inhibitory activity of each inhibitor and their respective binding-free energies, reinforcing that the affinity of the complexes seems to be a relevant factor for enzymatic inhibition. Hence, the results presented in this work contribute to a better understanding of the cruzain enzyme inhibition mechanism through competitive and non-covalent inhibitors.Communicated by Ramaswamy H. Sarma.

An exploration of the effects of prefabricated and customized insoles on lower limb kinetics and kinematics during walking, stepping up and down tasks: A time series analysis.

BACKGROUND: Prefabricated and customized insoles are used in clinical practice to reduce foot pronation. Although data exist on the effects at key points within the stance phase, exploring the impact of different insoles using time series analysis may reveal more detail about their efficacy. RESEARCH QUESTION: What are the effects revealed by a time series analysis of arch-supported prefabricated insoles (PREFABRICATED) versus arch-supported prefabricated insoles customized with a 6º medial wedge (CUSTOMIZED) on the lower limb biomechanics during walking, stepping up and down tasks in individuals with pronated feet? METHODS: Nineteen individuals with excessive foot pronation performed walking, stepping up and down tasks using three insoles: CONTROL (flat insole), CUSTOMIZED, and PREFABRICATED. Angles and moments of ankle and knee coronal and hip transverse planes were compared between conditions using statistical parametric mapping (SPM). RESULTS: For walking, CUSTOMIZED reduced ankle eversion moment compared to CONTROL during midstance and PREFABRICATED during propulsion. CUSTOMIZED decreased KAM during midstance and propulsion compared to PREFABRICATED. Compared to CONTROL, CUSTOMIZED and PREFABRICATED reduced hip internal rotation during propulsion and loading response, respectively. CUSTOMIZED decreased eversion movement during midstance and propulsion for the stepping up task. PREFABRICATED reduced eversion movement during midstance in comparison to CONTROL. For the stepping down task, CUSTOMIZED increased eversion movement during propulsion compared to PREFABRICATED. CUSTOMIZED reduced hip internal rotation angle for stepping up task during propulsion, decreased medial rotation movement during midstance compared to CONTROL, and reduced medial rotation during midstance compared to PREFABRICATED. CUSTOMIZED increased KAM for stepping up and down tasks during propulsion. SIGNIFICANCE: These findings suggest that both CUSTOMIZED and PREFABRICATED reduce foot pronation. However, non-local effects, such as changes in KAM and hip internal rotation, were seen only in the CUSTOMIZED. Therefore, CUSTOMIZED may be preferable if the objective is to modify the knee and hip mechanics.

Cytomegalovirus as a possibly overlooked agent of hypertensive anterior uveitis and endotheliitis in immunocompetent patients in Brazil.

Cytomegalovirus (CMV) is a member of the Herpesviridae family, including viruses that are well-known agents of keratitis, anterior uveitis, scleritis and retinitis. CMV is usually associated with ocular diseases in immunosuppressed individuals, with a notable exception of hypertensive anterior uveitis with distinctive clinical features in immunocompetent patients. This syndrome was characterized in the last two decades in Europe and Southeast Asia, and then documented in the rest of world. Definitive diagnosis in these cases is usually made by Polymerase Chain Reaction (PCR) of the anterior chamber fluid. We report three immunocompetent Brazilian adults with history of multiple glaucomatocyclitic crises and presenting with chronic hypertensive anterior uveitis invariably with mild anterior chamber inflammation and characteristic scarce nummular keratic precipitates. CMV DNA was successfully amplified and detected in the aqueous humor of all patients. Corneal endothelial counts were significantly reduced in the involved eyes, with one patient developing bullous keratopathy. All patients were then treated with topical ganciclovir gel and corticosteroids, with subsequent control of the intraocular inflammation. CMV may represent an overlooked / underestimated etiology of hypertensive anterior uveitis that may progressively lead to endothelial dysfunction, culminating in bullous keratopathy. Management of patients is challenging, with the potential use of topical antivirals to decrease the number of relapses, and corticosteroids to control anterior uveitis / endotheliitis and to protect the corneal endothelium.

Importância do diagnóstico diferencial de olho vermelho não traumático na atenção primária: uma revisão da literatura / Importance of differential diagnosis of non-traumatic red eye in primary care: a literature review

O olho vermelho é uma queixa oftalmológica frequente na atenção primária à saúde. Neste contexto, o objetivo desta revisão é descrever as principais patologias relacionadas a esse sinal na prática clínica, a fim de auxiliar os profissionais de saúde no diagnóstico, na condução dos casos, e na tomada de decisões quanto à necessidade de encaminhamento ao especialista. A maioria dos casos são benignos, porém, alguns apresentam risco de complicações e perda visual.

The red eye is a frequent eye complaint in primary health care. In this context, the objective of this review is to describe the main pathologies related to this sign in clinical practice, in order to assist health professionals in the diagnosis, in the management of cases, and in making decisions regarding the need for referral to the specialist. Most cases are benign, however, some are at risk of complications and visual loss.

The foliar application of a mixture of semisynthetic chitosan derivatives induces tolerance to water deficit in maize, improving the antioxidant system and increasing photosynthesis and grain yield.

Research has shown that chitosan induces plant stress tolerance and protection, but few studies have explored chemical modifications of chitosan and their effects on plants under water stress. Chitosan and its derivatives were applied (isolated or in mixture) to maize hybrids sensitive to water deficit under greenhouse conditions through foliar spraying at the pre-flowering stage. After the application, water deficit was induced for 15 days. Analyses of leaves and biochemical gas exchange in the ear leaf were performed on the first and fifteenth days of the stress period. Production attributes were also analysed at the end of the experiment. In general, the application of the two chitosan derivatives or their mixture potentiated the activities of the antioxidant enzymes superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase and guaiacol peroxidase at the beginning of the stress period, in addition to reducing lipid peroxidation (malonaldehyde content) and increasing gas exchange and proline contents at the end of the stress period. The derivatives also increased the content of phenolic compounds and the activity of enzymes involved in their production (phenylalanine ammonia lyase and tyrosine ammonia lyase). Dehydroascorbate reductase and compounds such as total soluble sugars, total amino acids, starch, grain yield and harvest index increased for both the derivatives and chitosan. However, the mixture of derivatives was the treatment that led to the higher increase in grain yield and harvest index compared to the other treatments. The application of semisynthetic molecules derived from chitosan yielded greater leaf gas exchange and a higher incidence of the biochemical conditions that relieve plant stress.

Synthesis, Biological Evaluation, and Molecular Modeling Studies of New Thiadiazole Derivatives as Potent P2X7 Receptor Inhibitors.

Twenty new 2-(1H-pyrazol-1-yl)-1,3,4-thiadiazole analogs were synthetized to develop P2X7 receptor (P2X7R) inhibitors. P2X7R inhibition in vitro was evaluated in mouse peritoneal macrophages, HEK-293 cells transfected with hP2X7R (dye uptake assay), and THP-1 cells (IL-1ß release assay). The 1-(5-phenyl-1,3,4-thiadiazol-2-yl)-1H-pyrazol-5-amine derivatives 9b, 9c, and 9f, and 2-(3,5-dimethyl-1H-pyrazol-1-yl)-5-(4-fluorophenyl)-1,3,4-thiadiazole (11c) showed inhibitory effects with IC50 values ranging from 16 to 122 nM for reduced P2X7R-mediated dye uptake and 20 to 300 nM for IL-1ß release. In addition, the in vitro ADMET profile of the four most potent derivatives was determined to be in acceptable ranges concerning metabolic stability and cytotoxicity. Molecular docking and molecular dynamics simulation studies of the molecular complexes human P2X7R/9f and murine P2X7R/9f indicated the putative intermolecular interactions. Compound 9f showed affinity mainly for the Arg268, Lys377, and Asn266 residues. These results suggest that 2-(1H-pyrazol-1-yl)-1,3,4-thiadiazole analogs may be promising novel P2X7R inhibitors with therapeutic potential.

Biosorption of crystal violet dye using inactive biomass of the fungus Diaporthe schini.

An inactive biomass of a new fungus recently discovered, Diaporthe schini, was evaluated for the biosorption of crystal violet (CV) in simulated textile effluents. The characterization assays were performed using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and N2 adsorption/desorption isotherms. The influences of pH and biosorbent dosage on the biosorption capacity were evaluated. Kinetics, equilibrium and thermodynamic studies were also carried out. Characterization techniques showed an amorphous biosorbent, with a rough surface containing irregular particles and surface area of 6.5 m2 g-1. The most adequate values of pH and biosorbent dosage were 7.5 and 0.4 g L-1, respectively. The Elovich kinetic model and the Sips equilibrium model were suitable to fit the experimental data. The biosorption capacity increased with temperature, reaching a maximum biosorption capacity of 642.3 mg g-1 at 328 K. The biosorption was a spontaneous and endothermic process. Diaporthe schini inactive biomass was an interesting biosorbent to treat colored effluents, presenting efficiency of 87% in the decolorization of a simulated dye house effluent.

Antifungal and antibacterial activity of extracts produced from Diaporthe schini.

The aim of this work was the production of bioactive metabolites by submerged fermentation from the fungus Diaporthe schini, followed by their extraction, separation and characterization. Different solvents (methanol, dichloromethane and hexane) were used for the extraction of metabolites from the fermentation broth and the extracts obtained were evaluated by in vitro antibacterial and antifungal activity. The separation and characterization of the extract from the hexane extraction was performed by column chromatography and GC-MS, respectively. The extracts had a great inhibitory action on the Gram-positive bacteria Staphylococcus epidermidis and Staphylococcus aureus, on the Gram-negative bacteria Enterobacter aerogenes and Klebsiella pneumoniae and on the fungus Candida krusei. The main metabolites produced were: 13-docosenamide, (Z)-; 2-hexadecene, 3,7,11,15-tetramethyl; 9-octadecenamide and 11-octadecenoic acid. Studies related to the antibacterial and antifungal activities of metabolites extracted from microorganisms are found in the literature. However, works about the identification of metabolites produced by submerged fermentation from Diaporthe schini were not found until the present moment. This work is an initial study where the conditions of the process can be optimized by looking for the production of a specific compound and can be a promising source for obtaining new drugs.

Avaliação do isolamento social em pacientes com odor em feridas neoplásicas: revisão integrativa / Evaluation of social isolation among patients with odor in neoplastic wounds: integration review

Objetivo: identificar as evidências científicas sobre o isolamento social em pacientes com odor fétido em feridas neoplásicas. Método: revisão integrativa, realizada nas bases de dados LILACS, MEDLINE e COCHRANE com a questão >. Foi utilizada a estratégia PICO com horizonte temporal de 2002 a 15 de novembro de 2014, nos idiomas português, inglês e espanhol. Após a seleção dos registros científicos, realizou-se a leitura e análise na íntegra dos textos. As informações foram tabuladas em programa Microsoft Excel® e processadas a partir de estratégia de medida de tendência central com a média aritmética descritiva simples, frequência absoluta e relativa. Resultados: identificou-se 352 artigos científicos em sua totalidade. Após aplicação dos critérios de inclusão e exclusão, foram selecionadas 23 evidências científicas. Conclusão: o odor é o principal sintoma que interfere nos aspectos psicossociais do paciente, responsável por causar constrangimento, distúrbio da imagem corporal, dificuldade de interação com a rede social, fatores que favorecem o isolamento social.

Realização de ressonância magnética em portadores de dispositivos cardíacos e vasculares implantáveis: um guia para o clínico / Magnetic resonance imaging in patients with implantable cardiac and vascular devices: a guide for the clinician

A ressonância nuclear magnética é atualmente um dos métodos não invasivos de imagem mais utilizados na prática clínica, sendo considerada o exame de escolha em diversas patologias. Sua realização em portadores de dispositivos cardíacos e vasculares implantáveis, sejam eletrônicos ou metálicos, torna-se cada vez mais frequente. Estima-se que até 75% dos pacientes com este equipamentos terão indicação de realização deste exame para avaliação de outras comorbidades...

Evidências sobre o isolamento social em pacientes com exsudato em feridas neoplásicas: revisão integrativa / Evidence about the social isolation in patients with exudate in neoplastic wounds: integrative review

Objetiva-se identificar as evidências científicas sobre o isolamento social em pacientes com feridas neoplásicas exsudativas. Revisão integrativa realizada nas bases de dados da Biblioteca Virtual de Saúde (BVS) e PubMed: Literatura Latino Americana e do Caribe em Ciências da Saúde (LILACS), Medical Literature Analysis and Retrieval Sistem on-line (MEDLINE) e Cochrane Database of Systematic Reviews (COCHRANE) com a questão norteadora: quais são as evidências científicas sobre o isolamento social em pacientes com feridas neoplásicas exsudativas? Utilizou-se estratégia PICO com recorte temporal de 2002 a 2014, nos idiomas português, inglês e espanhol. As informações foram tabuladas em programa Microsoft Excel® e processadas a partir da média aritmética descritiva simples, frequência absoluta e relativa. Identificaram-se 288 artigos em sua totalidade. Após aplicação dos critérios de elegibilidade, foram selecionadas 23 evidências científicas avaliadas a partir da categoria: os principais aspectos psicossociais prejudicados pelo exsudato em pacientes com feridas neoplásicas. Concluí-se que o exsudato é um dos sintomas que interfere negativamente nos aspectos psicossociais do paciente com feridas neoplásicas, causando alteração da imagem corporal, perda dos limites físicos, ansiedade e depressão, fatores que favorecem diretamente o isolamento social

The aim is to identify the scientific evidence on social isolation in patients with exudative neoplastic wounds. An integrative literature review conducted in the Virtual Health Library databases (VHS) and PubMed: Literature Latin American and Caribbean Health Sciences (LILACS), Medical Literature Analysis and Retrieval System online (MEDLINE) and Cochrane Database of Systematic Reviews (COCHRANE), with the guiding question: what are the scientific evidences about the social isolation in patients with exudative neoplastic wounds? We used a PICO strategy with a temporal cut from 2002 to 2014, in Portuguese, English and Spanish languages. The information was tabulated in Microsoft Excel® program and processed from simple descriptive arithmetic mean, absolute and relative frequency. It was identified 288 articles in their entirety. After applying the eligibility criteria, was selected 23 scientific evidence evaluated from the category: the main psychosocial aspects impaired by exudate in patients with neoplastic wounds. It concludes that exudate is one of the symptoms that negatively interferes the psychosocial aspects of the patient with neoplastic wounds, causing alterations in body image, loss of physical limits, anxiety and depression, factors that directly favor social isolation. Keywords: Palliative Care; Oncology Nursing; Wounds and Injuries; Exudates and Transudates; Social Isolation

Evaluation of the Propidium Monoazide-quantitative Polymerase Chain Reaction Method for the Detection of Viable Enterococcus faecalis.

INTRODUCTION: One limitation of DNA-based molecular assays is their inability to distinguish between live and dead cells. A sample treatment with propidium monoazide (PMA) before DNA amplification has been proposed to overcome this problem. The aim of this in vitro study was to test different concentrations of PMA coupled with quantitative polymerase chain reaction (qPCR) for the detection of viable Enterococcus faecalis. METHODS: Viable or heat-killed suspensions of E. faecalis (106 colony-forming units/mL) were treated with PMA at 10, 50, and 100 µg/mL before DNA extraction. qPCR was performed using primers complementary for E. faecalis 16S ribosomal RNA sequence. PMA was also tested on bacteria suspensions containing different proportions of viable and dead cells. Bacterial suspensions without PMA treatment were used as positive controls. RESULTS: The treatment of heat-killed suspensions with PMA at different concentrations significantly reduced the DNA amplification when compared with the group without treatment (P < .0001), indicating that DNA from dead cells was not used as templates. The greatest reduction in qPCR amplification of dead cell DNA was found when 100 µg/mL PMA was used (P < .005). In mixtures containing live/dead cells, PMA allowed selective detection of viable cells. CONCLUSIONS: PMA was effective in inhibiting qPCR amplification from the DNA of dead cells, enabling in vitro detection and quantification of viable cells of E. faecalis.

Effects of exogenous proline and a natural ventilation system on the in vitro growth of orchids / Efeitos da prolina exógena e sistema de ventilação natural no crescimento in vitro de orquídeas

Micropropagation is an alternative to produce orchid plants in large scale. However, this process presents losses during acclimatization. Exogenous proline use in vitro plant tissue culture can reduce the stress of the plant acclimatization phase. We aimed to verify the growth of orchids in different micropropagation systems with the addition of proline in the culture medium. Cattleya walkeriana plants were obtained from the germination of seeds in culture medium. Seeds were germinated in MS medium, added 20 g. L-1 of sucrose, solidified with 6 g. L-1 of agar and pH adjusted for 5,8. The cultures were incubated in a growth room with temperature of 24 ± 2 0C, under photoperiod of 16 h. After 5 months, 1-cm long seedlings were placed in a culture vessel according to the treatments, which were composed of two micropropagation systems (conventional and natural ventilation) and three proline concentrations (0, 1, and 2 g·L-1). The experiment was carried out in an entirely randomized design consisting of a 2 × 3 factorial, for a total of 6 treatments, each with 5 replicates. The natural ventilation system with the use of proline (1 g·L-1) promoted higher dry mass accumulation and better control of water loss by plants.

A micropropagação é uma alternativa para a produção de plantas de orquídeas em larga escala. Entretanto, este processo apresenta perdas durante a fase de aclimatização. O emprego de prolina exógena na cultura de tecidos vegetais é uma alternativa para reduzir o estresse das plantas na fase de aclimatização. O objetivo da presente pesquisa foi verificar o crescimento de orquídeas em diferentes sistemas de micropropagação com prolina adicionada no meio de cultura. Plantas de Cattleya walkeriana foram obtidas a partir da germinação de sementes em meio de cultura. Sementes foram germinadas em meio MS, adicionado de 20 g. L-1 de sacarose, solidificado com 6 g. L-1 de ágar e pH ajustado para 5,8. Após 5 meses, plântulas com 1 cm de comprimento foram inoculadas nos frascos de cultivo de acordo com os tratamentos, os quais foram compostos por dois sistemas de micropropagação (convencional e ventilação natural) em combinação com prolina (0, 1 e 2 g L-1). O experimento foi conduzido em esquema inteiramente casualizado, constando de um fatorial 2x3, totalizando 6 tratamentos com 5 repetições. O sistema de ventilação natural com o uso de prolina (1 g L-1) promoveu o maior acúmulo de massa seca e melhor controle da perda de água das plantas.

Cattleya walkeriana growth in different micropropagation systems / Crescimento de Cattleya walkeriana em diferentes sistemas de micropropagação

The aim of the present research was to verify the in vitro growth of orchids in different systems of micropropagation, being cultivated in a bioreactor, with natural ventilation and conventional systems. Cattleya walkeriana plants were obtained from the germination of seeds in culture medium. After 8 months, seedlings with 1 cm of length were placed in a culture vessel according to the treatments, which counted with two micropropagation systems (conventional and natural ventilation) in three media of culture (liquid, solid with 5 or 6g L-1 of agar). Two additional treatments in bioreactor of temporary and continuous immersion were performed. The design was entirely randomized (ERD), consisting of a 2x3 factorial with two additional treatments, totaling 8 treatments with three repetitions. The temporary immersion bioreactor promoted a bigger growth of the aerial part and of the root system, bigger accumulation of dry mass and better control of water loss by the plants. The temporary immersion bioreactor is the best micropropagation system for the C. walkeriana growth in vitro.

O objetivo do presente trabalho foi verificar o crescimento in vitro de orquídeas em diferentes sistemas de micropropagação, sendo cultivado em biorreator, sistema de ventilação natural e convencional. Plantas de Cattleya walkeriana foram obtidas a partir da germinação de sementes em meio de cultura. Após o a germinação, as plantas foram uniformizadas com aproximadamente 1,0cm de comprimento e inoculadas nos diferentes tratamentos. Os tratamentos contaram dois sistema de micropropagação (convencional e ventilação natural) e três meios de cultura (líquido, sólido com 5 e 6g L-1 de ágar). Foram realizados dois tratamentos adicionais em biorreator de imersão temporária e contínua. O delineamento foi o inteiramente casualizado, consistindo de um fatorial 2x3 com dois tratamentos adicionais, totalizando oito tratamentos com três repetições. O biorreator de imersão temporária promoveu o maior crescimento da parte aérea e do sistema radicular, maior acúmulo de massa seca e melhor controle da perda de água das plantas. O biorreator de imersão temporária é o melhor sistema de micropropagação para o crescimento in vitro de C. walkeriana.

Vector competence of the tick Ixodes ricinus for transmission of Bartonella birtlesii.

Bartonella spp. are facultative intracellular vector-borne bacteria associated with several emerging diseases in humans and animals all over the world. The potential for involvement of ticks in transmission of Bartonella spp. has been heartily debated for many years. However, most of the data supporting bartonellae transmission by ticks come from molecular and serological epidemiological surveys in humans and animals providing only indirect evidences without a direct proof of tick vector competence for transmission of bartonellae. We used a murine model to assess the vector competence of Ixodes ricinus for Bartonella birtlesii. Larval and nymphal I. ricinus were fed on a B. birtlesii-infected mouse. The nymphs successfully transmitted B. birtlesii to naïve mice as bacteria were recovered from both the mouse blood and liver at seven and 16 days after tick bites. The female adults successfully emitted the bacteria into uninfected blood after three or more days of tick attachment, when fed via membrane feeding system. Histochemical staining showed the presence of bacteria in salivary glands and muscle tissues of partially engorged adult ticks, which had molted from the infected nymphs. These results confirm the vector competence of I. ricinus for B. birtlesii and represent the first in vivo demonstration of a Bartonella sp. transmission by ticks. Consequently, bartonelloses should be now included in the differential diagnosis for patients exposed to tick bites.

Questing ticks in suburban forest are infected by at least six tick-borne pathogens.

The role of Ixodes ricinus ticks in the transmission of pathogens of public health importance such as Borrelia burgdorferi s.l. is widely recognized and is suspected in several emerging vector-borne pathogens in Europe. Here, we assess prevalence rates of several endemic and emerging zoonotic pathogens in tick populations in an area of high human population density in France, to contribute to a risk assessment for potential transmission to humans. Pathogen prevalence rates were evaluated by polymerase chain reaction detection and sequencing in questing ticks, individually for adults and in pools of 10 for nymphs. In addition to finding micro-organisms corresponding to symbionts, we found high prevalence rates of B. burgdorferi s.l. (32% of adult females and 10% of nymphs) and low to moderate ones of Anaplasma phagocytophilum (~1%), spotted fever group Rickettsia spp. (~6%), Babesia sp. EU1 (~1%), Bartonella birtlesii (0.1%), and Francisella tularensis (!1%). Our findings extend the knowledge of the geographical distribution of these endemic and emergent pathogens and support the conclusion that ticks are important vectors of pathogenic micro-organisms in suburban forests. Moreover, tick coinfection with multiple pathogens was found to occur frequently, which poses a serious challenge for diagnosis and appropriate treatment. The incrimination of these pathogens in potentially severe pathologies requires widespread surveillance to assess the risk of infection, thereby facilitating diagnosis and treatment, as well as raising local awareness of tick-borne diseases.

The limitations of the G1-S checkpoint.

It has been proposed that the G(1)-S checkpoint is the critical regulator of genomic stability, preventing the cell cycle progression of cells with a single DNA double-strand break. Using fluorescence-activated cell sorting analysis of asynchronous cells and microscopic analysis of asynchronous and synchronized cells, we show that full blockage of S-phase entry is only observed >4 hours after irradiation. The process is ataxia-telangiectasia mutated (ATM) dependent and Chk1/2 independent and can be activated throughout G(1) phase. By monitoring S-phase entry of irradiated synchronized cells, we show that the duration of arrest is dose dependent, with S-phase entry recommencing after arrest with kinetics similar to that observed in unirradiated cells. Thus, G(1)-S checkpoint arrest is not always permanent. Following exposure to higher doses (> or =2 Gy), G(1)-S arrest is inefficiently maintained, allowing progression of G(1)-phase cells into G(2) with elevated gammaH2AX foci and chromosome breaks. At early times after irradiation (< or =4 h), G(1)-S checkpoint arrest is not established but cells enter S phase at a reduced rate. This early slowing in S-phase entry is ATM and Chk2 dependent and detectable after 100 mGy, showing a novel and sensitive damage response. However, the time needed to establish G(1)-S checkpoint arrest provides a window when cells can progress to G(2) and form chromosome breaks. Our findings detail the efficacy of the G(1)-S checkpoint and define two significant limitations: At early times after IR, the activated checkpoint fails to efficiently prevent S-phase entry, and at later times, the checkpoint is inefficiently maintained.

A role for PKCzeta in potentiation of the topoisomerase II activity and etoposide cytotoxicity by wortmannin.

Enhanced cytotoxicity of etoposide by wortmannin, an inhibitor of enzymes holding a phosphatidylinositol 3-kinase domain, was investigated in eight cell lines proficient or deficient for DNA double-strand break repair. Wortmannin stimulated the decatenating activity of topoisomerase II, promoted etoposide-induced accumulation of DNA double-strand breaks, shifted the specificity for cell killing by etoposide from the S to G1 phase of the cell cycle, and potentiated the cytotoxicity of etoposide through two mechanisms. (a) Sensitization to high, micromolar amounts of etoposide required integrity of the nonhomologous end-joining repair pathway. (b) Wortmannin dramatically increased the susceptibility to low, submicromolar amounts of etoposide in a large fraction of the cell population irrespective of the status of ATM, Ku86, and DNA-PKCS. It is shown that this process correlates depression of phosphatidylinositol 3-kinase-dependent phosphorylation of the atypical, zeta isoform of protein kinase C (PKCzeta). Stable expression of a dominant-negative, kinase-dead mutant of PKCzeta in a tumor cell line reproduced the hypersensitivity pattern induced by wortmannin. The results are consistent with up-regulation of the topoisomerase II activity in relation to inactivation of PKCzeta and indicate that PKCzeta may be a useful target to improve the efficiency of topoisomerase II poisons at low concentration.