Prevalência da baixa densidade mineral óssea em mulheres na pós-menopausa tratadas de câncer de mama / Prevalence of low bone mineral density in postmenopausal breast cancer survivors

OBJETIVO: Avaliar a prevalência da baixa densidade mineral óssea (DMO) em mulheres na pós-menopausa tratadas de câncer de mama. MÉTODOS: Estudo de corte transversal que incluiu 115 mulheres tratadas de câncer de mama atendidas em Hospital Universitário do Sudeste do Brasil. Foram incluídas mulheres com amenorreia há 12 meses ou mais e 45 anos ou mais de idade, tratadas de câncer de mama e livres de doença há pelo menos 5 anos. A DMO foi mensurada pelos raios-X de dupla energia em coluna lombar (L1 a L4) e colo de fêmur. Considerou-se baixa DMO quando valores de T-score de coluna total e/ou colo de fêmur <-1,0 Score de Delphi (DP) (osteopenia e osteoporose). Por meio de entrevista, foram avaliados fatores de risco para baixa DMO. Na análise estatística, empregaram-se os testes do χ2 ou Exato de Fisher. RESULTADOS: A média de idade das pacientes foi 61,6±10,1 anos e o tempo de menopausa, 14,2±5,6 anos, com tempo médio de seguimento de 10,1±3,9 anos. Considerando coluna e colo de fêmur, 60% das mulheres tratadas de câncer de mama apresentavam baixa DMO. Avaliando os fatores de risco para baixa DMO, foi encontrada diferença significativa na distribuição percentual quanto à idade (maior porcentagem de mulheres com mais de 50 anos e baixa DMO), história pessoal de fratura prévia (11,6% com baixa DMO e nenhuma com DMO normal) e índice de massa corpórea. Maior frequência de obesidade foi observada entre mulheres com DMO normal (63%) quando comparadas àquelas com baixa DMO (26,1%; p<0,05). CONCLUSÃO: Mulheres na pós-menopausa tratadas de câncer de mama apresentaram elevada prevalência de baixa DMO (osteopenia e/ou osteoporose). .

PURPOSE: To evaluate the prevalence of low bone mineral density (BMD) in postmenopausal breast cancer survivors. METHODS: In this cross-sectional study, 115 breast cancer survivors, seeking healthcare at a University Hospital in Brazil, were evaluated. Eligibility criteria included women with amenorrhea ≥12 months and age ≥45 years, treated for breast cancer and metastasis-free for at least five years. BMD was measured by DEXA at the lumbar spine (L1-L4) and femoral neck. Low BMD was considered when total-spine and/or femoral-neck T-score values were <-1.0 Delphi Score (DP) (osteopenia and osteoporosis). The risk factors for low BMD were assessed by interview. Data were analyzed statistically by the χ2 test and Fisher's exact test. RESULTS: The mean age of breast cancer survivors was 61.6±10.1 years and time since menopause was 14.2±5.6 years, with a mean follow-up of 10.1±3.9 years. Considering spine and femoral neck, 60% of breast cancer survivors had low BMD. By evaluating the risk factors for low BMD, a significant difference was found in the percent distribution for age (higher % of women >50 years with low BMD), personal history of previous fracture (11.6% with low BMD versus 0% with normal BMD) and BMI. A higher frequency of obesity was observed among women with normal BMD (63%) compared to those with low BMD (26.1%) (p<0.05). CONCLUSION: Postmenopausal breast cancer survivors had a high prevalence of osteopenia and osteoporosis. .

Determinants of emergency contraception non-use among women in unplanned or ambivalent pregnancies / Determinantes del no uso de la anticoncepción de emergencia entre mujeres con embarazo no planeado u ambivalente / Determinantes do não uso da anticoncepção de emergência entre mulheres com gravidez não planejada ou ambivalente

Objective To analyze the determinants of emergency contraception non-use among women in unplanned and ambivalent pregnancies. Method Cross-sectional study with a probabilistic sample of 366 pregnant women from 12 primary health care units in the city of São Paulo, Brazil. A multinomial logistic regression was performed, comparing three groups: women who used emergency contraception to prevent ongoing pregnancies (reference); women who made no use of emergency contraception, but used other contraceptive methods; and women who made no use of any contraceptive methods at all. Results Cohabitation with a partner was the common determinant of emergency contraception non-use. No pregnancy risk awareness, ambivalent pregnancies and no previous use of emergency contraception also contributed to emergency contraception non-use. Conclusion Apart from what is pointed out in the literature, knowledge of emergency contraception and the fertile period were not associated to its use. .

Objetivo Analizar los determinantes del no uso de la anticoncepción de emergencia entre las mujeres con embarazo no planeado o ambivalente. Método Estudio transversal en una muestra probabilística de 366 mujeres embarazadas de 12 Unidades Básicas de Salud de São Paulo. Mediante regresión logística multinomial, se comparó tres grupos de mujeres: aquellas que usaron la anticoncepción de emergencia para prevenir el embarazo en curso (referencia), aquellas que usaron algún método anticonceptivo, pero no la anticoncepción de emergência; y aquellas que no usaron ningún método. Resultados Los hallazgos mostraron que vivir com la pareja fue el determinante común del no uso de la anticoncepción de emergencia. No tener conciencia del riesgo de embarazo, estar en un embarazo ambivalente y nunca tener utilizado la anticoncepción de emergencia también fueron associados con su no uso para prevenir el embarazo en curso. Conclusión Contrariamente a lo que reporta la literatura, el conocimiento de la anticoncepción de emergencia y el período fértil no mostró asociación con el no uso. .

Objetivo Analisar os determinantes do não uso da anticoncepção de emergência entre mulheres com gravidez não planejada ou ambivalente. Método Estudo transversal com amostra probabilística de 366 gestantes de 12 Unidades Básicas de Saúde da cidade de São Paulo. Por meio de regressão logística multinomial, compararam-se três grupos de mulheres: as que usaram anticoncepção de emergência para prevenir a gravidez em curso (referência); as que usaram algum método contraceptivo, mas não anticoncepção de emergência; e as que não usaram nenhum método. Resultados Os achados mostraram que morar com o parceiro foi o determinante comum do não uso da anticoncepção de emergência. Não ter consciência do risco de engravidar, estar em uma gravidez ambivalente e nunca ter usado anticoncepção de emergência também foram associados ao seu não uso para prevenir a gravidez em curso. Conclusão Diferentemente do que relata a literatura, o conhecimento sobre anticoncepção de emergência e sobre o período fértil não mostrou qualquer associação ao não uso. .

COMP-Angiopoietin-1 Promotes Cavernous Angiogenesis in a Type 2 Diabetic Rat Model

Cartilage oligomeric matrix protein-angiopoietin-1 (COMP-Ang1) is an angiogenic factor for vascular angiogenesis. The aim was to investigate the effect of an intracavernosal injection of COMP-Ang1 on cavernosal angiogenesis in a diabetic rat model. Male Otsuka Long-Evans Tokushima Fatty (OLETF) rats made up the experimental group (1 yr old) and Long-Evans Tokushima Otsuka (LETO) rats made up the control group. The experimental group was divided into vehicle only, 10 microg COMP-Ang1, and 20 microg COMP-Ang1. COMP-Ang1 was injected into the corpus cavernosum of the penis. After 4 weeks, the penile tissues of the rats were obtained for immunohistochemistry and Western blot analysis. The immunoreactivity of PECAM-1 and VEGF was increased in the COMP-Ang1 group compared with the vehicle only group. Moreover, the expression of PECAM-1 and VEGF was notably augmented in the 20 microg Comp Ang-1 group. In the immunoblotting study, the expression of PECAM-1 and VEGF protein was significantly less in the OLEFT rats than in the control LETO rats. However, this expression was restored to control level after intracavernosal injection of COMP-Ang1. These results show that an intracavernosal injection of COMP-Ang1 enhances cavernous angiogenesis by structurally reinforcing the cavernosal endothelium.

Construction of three different recombinant scorpion fusion proteins with bifunctional activity.

This is the first report of three different fusion proteins with an antitumor-analgesic peptide obtained from Chinese scorpion Buthus martensii Karsch (BmKAGAP). The fusion proteins were constructed in the form of chimeric toxins, aiming to obtain bifunctional analgesic and antitumor activity. The fusion proteins consisted of luteinizing hormone-releasing hormone (LHRH), three different types of flexible linkers (L1, Ser-Ser-His-His-His-His-His-His-Ser-Ser-Gly-Leu-Val-Pro-Arg-Gly-Ser-His-Met; L2, Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser; L3, Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Ser-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser), and BmKAGAP. The genes coding three fusion proteins were cloned and expressed in E. coli in soluble form. Following two successive column chromatographic separations, purified fusion proteins were obtained. These fusion proteins exhibited analgesic activity in mice and were cytotoxic to a hepatocellular carcinoma cell line Hep3B.

Preparation and identification of scFv and bsFv against transferrin receptor / 华中科技大学学报（医学）（英德文版）

To obtain single chain variable fragment (scFv) and bivalent single chain variable fragment (bsFv) against transferrin receptor, up-stream and down-stream primers were designed according to the complementary sequences of FR1 region of variable heavy (VH) and FR4 of variable light (VL), respectively, which contained inter-linker G4S and the restriction endonuclease SfiI, AscI and NotI. Two pieces of scFv fragments were first amplified through PCR and then inserted into plasmid pAB1, which could express scFv protein once induced by IPTG in the host bacteria. To express scFv and bsFv, E. coli TG1 was cultured in LB broth and was induced by IPTG. The restriction enzyme digestion map and DNA sequencing demonstrated that scFv and bsFv genes were successfully inserted into the expression plasmid. SDS-PAGE and Western blotting revealed the protein band at 35kD and 60kD, which were consistent with the molecular weight of scFv and bsFv respectively. Flow cytometry showed that scFv and bsFv harbored the specific binding activity with TfR expressed in various tumor cells, and the avidity of bsFv was higher than that of the parent scFv.

Inhibition of LPS-induced cyclooxygenase 2 and nitric oxide production by transduced PEP-1-PTEN fusion protein in Raw 264.7 macrophage cells

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a tumor suppressor. Although it is well known to have various physiological roles in cancer, its inhibitory effect on inflammation remains poorly understood. In the present study, a human PTEN gene was fused with PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame PEP-1-PTEN fusion protein. The expressed and purified PEP-1-PTEN fusion protein were transduced efficiently into macrophage Raw 264.7 cells in a time- and dose- dependent manner when added exogenously in culture media. Once inside the cells, the transduced PEP-1-PTEN protein was stable for 24 h. Transduced PEP-1-PTEN fusion protein inhibited the LPS-induced cyclooxygenase 2 (COX-2) and iNOS expression levels in a dose-dependent manner. Furthermore, transduced PEP-1-PTEN fusion protein inhibited the activation of NF-kappa B induced by LPS. These results suggest that the PEP-1-PTEN fusion protein can be used in protein therapy for inflammatory disorders.

Human Monoclonal Antibody Inhibiting Reverse Transcriptase Activity of Hepatitis B Virus Polymerase Protein / 대한소화기학회지

BACKGROUND/AIMS: To develop a novel treatment method for hepatitis B virus (HBV) infection, we aimed to make a human monoclonal antibody inhibiting reverse transcriptase (RT) activity of P protein which was important in HBV replication by using phage display technique. Therefore, we analysed the usability of human monoclonal antibody as a protein based gene therapy. METHODS: Reverse transcriptase/polymerase (RT/POL) functional motif of P protein of HBV was cloned in pMAL-c vector and expressed as maltose binding fusion protein form. The RT/POL recombinant protein (pMRT/POL) was purified by amylose resin column. Using human single chain Fv phage antibody library with 1.1x10(10) size, human antibody against pMRT/POL was selected with BIAcore panning. Selected antibody fragments were analyzed for the activity of RT inhibition. Finally, they were analyzed for the affinity with BIAcore and the complementarity determining regions with nucleotide sequencing. RESULTS: pMRT/POL recombinant protein expressed in E. coli showed RT activity, 1microgram of recombinant protein had an activity equivalent to 5 unit of MMLV RT. By BIAcore panning, we could select 3 clones; POL-A5, POL-B8 and POL-B12. Each clone's RT inhibiting activity were 52-82%, affinity against antigen were 8.15x10(-8) M to 1.75x10(-6) M. CONCLUSIONS: Human monoclonal antibodies produced in this study showed low affinity, but efficiently inhibited the activity of RT in vitro. If POL-A5, POL-B8, and POL-B12 can be converted to intracellular antibody form, it can be used for protein-based gene therapy by inhibiting the replication through the neutralization of polymerase protein of HBV.

Proteins in a DNA world: expression systems for their study / Proteínas en el mundo del DNA: Sistemas de expresión para su estudio

Every day, new proteins are discovered and the need to understand its function arises. Human proteins have a special interest, because to know its role in the cell may lead to the design of a cure for a disease. In order to obtain such information, we need enough protein with a high degree of purity, and in the case of the human proteins, it is almost impossible to achieve this by working on human tissues. For that reason, the use of expression systems is needed. Bacteria, yeast, animals and plants have been genetically modified to produce proteins from different species. Even "cell-free" systems have been developed for that purpose. Here, we briefly review the options with their advantages and drawback, and the purification systems and analysis that can be done to gain understanding on the function and structure of the protein of interest.

Cada día, nuevas proteínas son descubiertas y surge la necesidad de caracterizarlas, siendo las de origen humano las que presentan un mayor interés. Conocer su función nos ayudará a entender padecimientos y diseñar una posible cura. Sin embargo, obtener suficiente cantidad de proteínas humanas en cantidad para llevar a cabo los análisis pertinentes, presenta una gran dificultad. Por tal razón, es necesario el uso de sistemas de expresión de proteínas heterólogas. Bacterias, levaduras, animales y plantas han sido modificados genéticamente para expresar proteínas de otras especies, e incluso sistemas in vitro han sido desarrollados para producir proteínas. En este artículo se revisan brevemente las opciones con sus ventajas y desventajas, así como las estrategias de purificación y los análisis que se pueden llevar a cabo para avanzar en el conocimiento de la función y estructura de la proteína de interés.

Enhanced immune response with foot and mouth disease virus VP1 and interleukin-1 fusion genes

The capsid of the foot and mouth disease (FMD) virus carries the epitopes that are critical for inducing the immune response. In an attempt to enhance the specific immune response, plasmid DNA was constructed to express VP1/interleukin-1alpha (IL-1alpha) and precursor capsid (P1) in combination with 2A (P1-2A)/IL-1alpha under the control of the human cytomegalovirus (HCMV) immediateearly promoter and intron. After DNA transfection into MA104 (monkey kidney) cells, Western blotting and an immunofluorescence assay were used to confirm the expression of VP1 or P1-2A and IL-1alpha. Mice were inoculated with the encoding plasmids via the intradermal route, and the IgG1 and IgG2a levels were used to determine the immune responses. These results show that although the immunized groups did not carry a high level of neutralizing antibodies, the plasmids encoding the VP1/ IL-1alpha, and P1-2A /IL-1alpha fused genes were effective in inducing an enhanced immune response.

Anticuerpos monoclonales (AcMo) y recombinantes en medicina transfusional: desarrollos y desafíos / Monoclonal antibodies and recombinant in transfusion medicine: developments and challenges

Desde el advenimiento de los AcMo logrados a través de tecnologías de biología celular, el campo del inmunodiagnóstico, se ha visto altamente beneficiado al cambiar los reactivos policlonales, obtenidos de animales o humanos sensibilizados, por reactivos monoclonales, altamente específicos y muy potentes producidos en laboratorios. En un comienzo se utilizaron técnicas de fusión celular (las cuales se continúan utilizando para cierto tipo de desarrollos tecnológicos). Posteriormente, la biología molecular y la biología celular se unieron para producir anticuerpos quiméricos y humanizados, y la tecnología recombinante generó la producción de los repertorios o muestrarios de fagos (phage display). Estos anticuerpos han sido utilizados para múltiples propósitos: investigación básica y aplicada, para producción de reactivos y en el campo terapéutico, por lo que su impacto en el campo de la medicina tranfusional ha sido innegable. Todos estos avances nos permitirán en el futuro producir en forma rápida y barata, anticuerpos genéticamente diseñados según el objetivo que se persiga.

Overexpression for commercial production of recombinant human insulin as A-chain and B-chain fusion protein in Escherichia coli through genetically engineered plasmids.

The construction of expression vectors encoding either the human insulin A- or B-chains fused to a synthetic peptide and the temperature-induced expression of the recombinant genes in Escherichia coli are reported. Using this two-chain approach we also describe the separate isolation of the insulin A- and B-chains from inclusion bodies and their subsequent assembly into native human insulin. The expression yields about 600 mg of the insulin B-chain per litre of culture. Under similar conditions the expression yield of the insulin A-chain corresponds to approximately 500 mg per litre of culture. This is the highest yield from shake flask experiments.

Distribution of Adenoviral Vector in Brain after Intravenous Administration

The delivery of transgenes to the central nervous system (CNS) can be a valuable tool to treat CNS diseases. Various systems for the delivery to the CNS have been developed; vascular delivery of viral vectors being most recent. Here, we investigated gene transfer to the CNS by intravenous injection of recombinant adenoviral vectors, containing green fluorescence protein (GFP) as a reporter gene. Expression of GFP was first observed 6 days after the gene transfer, peaked at 14 days, and almost diminished after 28 days. The observed expression of GFP in the CNS was highly localized to hippocampal CA regions of cerebral neocortex, inferior colliculus of midbrain, and granular cell and Purkinje cell layers of cerebellum. It is concluded that intravenous delivery of adenoviral vectors can be used for gene delivery to the CNS, and hence the technique could be beneficial to gene therapy.

Prokaryotic expression and characterization of human AP DNA endonuclease

The expression of major human apurinic/apyrimidinic DNA endonuclease (APEX) from its cDNA in E. coli (DH5 alpha) was attempted in order to obtain a biologically active recombinant APEX. E. coli cells were transformed by a prokaryotic translation vector (pGEX-4T-3) harboring APEX cDNA. GST-APEX fusion protein with a molecular weight of 6.3 KDa was induced by IPTG (1.0 mM) treatment. Western blot immunodetection identified the induced protein as the GST-APEX fusion protein. The survival rate of E. coli cells (DH5 alpha) transformed with pGEX-4T-3-APEX increased when the cells were treated with N-diethyl-N-nitrosamine (DENA) or 3'-methyl-4-monomethylaminoazobenzene (3'-MeMAB), indicating that APEX expression had a protective effect on the cytotoxicity of these carcinogens. The fusion protein extracted from E. coli cells and purified by GSH-agarose gel affinity chromatography exhibited APEX activity. Treatment of thrombin to the GST-APEX fusion protein and affinity purification followed by Sephacryl S-100 gel filtration resulted in APEX peptide with MW 36 KDa, which exhibited AP DNA repair activity (8,7000 EU/mg protein). N-ethylmaleimide (0.1 mM) or AMP (0.98 mM) inhibited APEX activity by 50% and kinetic analysis indicated that the recombinant APEX (rAPEX) had a Km value of 0.022 microM (AP sites for AP DNA) and the Ki value was 0.48 mM for AMP. These results indicated that E. coli cells expressing biologically active GST-APEX were resistant to the cell damage caused by chemical carcinogens and that rAPEX purified from E. coli cells transformed with APEX cDNA-inserted translation vector was similar to native APEX in some properties.

Expression of the Japanese encephalitis virus NS3 and NS2b proteins as glutathione S-transferase fusions.

Flaviviruses generate their structural and nonstructural proteins by proteolytic processing of a single large polyprotein precursor. These proteolytic events are brought about both by host cell signalase and a virally encoded protease. The virally encoded proteolytic activity has been shown to reside within the nonstructural protein 3 (NS3) and requires the product of the nonstructural 2b (NS2b) gene. In order to obtain sufficient quantities of pure NS2b and NS3 proteins for kinetic analysis, we have expressed both these proteins in recombinant systems as fusions to glutathione S-transferase (GST). The fusion constructs were driven by the strong bacteriophage T7 promoter. Transfection of these constructs into the African green monkey kidney cell line CV-1 previously infected with a recombinant vaccinia virus expressing the T7 RNA polymerase resulted in synthesis of the fusion proteins. Both the fusion proteins could be purified to homogeneity in a single step using a glutathione agarose affinity matrix.

Gene cloning of the human cytomegalovirus (HCMV) antigen reactive with the serum from a HCMV-infected patient

The human cytomegalovirus(HCMV) gene encoding the protein reactive with the sera of HCMV-infected patient was cloned and characterized. A reactive phage clone was screened from a lambda gt11 expression library of cDNA of HCMV AD169 strain using HCMV-infected patient sera. The recombinant protein was expressed as 138 kDa-fusion protein with beta-galactosidase, which was reactive with IgM or IgG HCMV antibody-positive sera, but not with anti-HCMV antibody-negative sera. A homology search of the DNA sequence of the cloned gene with HCMV AD169 sequences revealed that it was composed of 709 base pairs spanning between 0.174 and 0.177 map units of the UL32 region of the HCMV AD169 strain genome. This position corresponded to a part of the gene encoding 150 kDa phosphoprotein-(pp150), a major tegument protein, which was reported as an immunogenic protein which evoked strong and longstanding antibody response and had no sequence homology with the proteins of other herpesviruses. These results suggested that pp150 was an immunogenic protein in natural HCMV infection and therefore this clone was regarded as a useful candidate for developing an antigen for the serodiagnosis of HCMV.

Patterns of transient expression of the arenavirus nucleocapsid protein gene in transfected cells

Los genes clonados de las proteínas de nucleocápside, N, de los arenavirus Junín y LCM (choriomeningitis linfocitaria) se insertaron en el vector de expresión pKG4 regulado por el promotor tardío del virus SV40. Cuando estas construcciones se utilizaron para transfectar las líneas celulares BHK-21 (fibroblastos de hamster lactante) y CV-1 (fibroblastos de riñón de mono verde africano) se observó la expresión transiente de un polipéptido de tamaño e inmunoreactividad indistinguible de la proteína N sintetizada durante una infección viral. El análisis por inmunofluorescencia reveló un patrón de distribución intracelular semejante al observado en células infectadas. Este patrón presentó variaciones desde una tinción citoplásmica difusa hasta gránulos citoplásmicos dispersos o concentrados en la zona perinuclear. La asociación de la proteína N con gránulos basófilos es semejante a la descripta en el efecto citópático causado por los arenavirus en las células infectadas, y podría relacionarse con las características fisicoquímicas de la proteina N, que contiene numerosas secuencias de aminoácidos básicos capaces de interactuar con ácidos ribonucleicos celulares