DNA topoisomerase inhibitors: biflavonoids from Ouratea species

Topoisomerase inhibitors are agents with anticancer activity. 7"-O-Methyl-agathisflavone (I) and amentoflavone (II) are biflavonoids and were isolated from the Brazilian plants Ouratea hexasperma and O. semiserrata, respectively. These biflavonoids and the acetyl derivative of II (IIa) are inhibitors of human DNA topoisomerases I at 200 µM, as demonstrated by the relaxation assay of supercoiled DNA, and only agathisflavone (I) at 200 µM also inhibited DNA topoisomerases II-alpha, as observed by decatenation and relaxation assays. The biflavonoids showed concentration-dependent growth inhibitory activities on Ehrlich carcinoma cells in 45-h culture, assayed by a tetrazolium method, with IC50 = 24 ± 1.4 µM for I, 26 ± 1.1 µM for II and 10 ± 0.7 µM for IIa. These biflavonoids were assayed against human K562 leukemia cells in 45-h culture, but only I showed 42% growth inhibitory activity at 90 µM. Our results suggest that biflavonoids are targets for DNA topoisomerases and their cytotoxicity is dependent on tumor cell type.

Trypanosoma cruzi: inhibition of alpha-hydroxyacid dehydrogenase isozyme II by N-allyl and N-propyl oxamates and their effects on intact epimastigotes

N-allyl (NAOx) and N-propyl (NPOx) oxamates were designed as inhibitors of alpha-hydroxyacid dehydrogenase (HADH) isozyme II from Trypanosoma cruzi. The kinetic studies showed that NAOx and NPOx were competitive inhibitors of HADH-isozyme II (Ki = 72 µM, IC50 = 0.33 mM and 70 µM, IC50 = 0.32 mM, respectively). The attachment of the allylic and propylic chains to nitrogen of the competitive inhibitor oxamate (Ki = 0.91 mM, IC50 = 4.25 mM), increased 12.6 and 13-folds respectively, the affinity for T. cruzi HADH-isozyme II. NAOx and NPOx were selective inhibitors of HADH-isozyme II, because other T. cruzi dehydrogenases were not inhibited by these substances. Since HADH-isozyme II participates in the energy metabolism of T. cruzi, a trypanocidal effect can be expected with these inhibitors. However, we were not able to detect any trypanocidal activity with these oxamates. When the corresponding ethyl esters of N-allyl (Et-NAOx) and N-propyl (Et-NPOx) oxamates were tested as a possible trypanocidal prodrugs, in comparison with nifurtimox and benznidazole, the expected trypanocidal effects were obtained.

alpha-Tocopherol enhances tumour growth inhibition by cis-dichlorodiammine platinum (II)

Present studies indicate that alpha-tocopherol enhances the efficacy of cisplatin as demonstrated by inoculation of Dalton's lymphoma cells incubated with either cisplatin (5 or 10 µg/ml) alone or cisplatin + alpha-tocopherol (25 or 50 µg/ml) into C3H/He mice. Tumour cells (3 x 10(6) cells/mouse) incubated with cisplatin grow slowly in syngeneic mice as indicated by the late appearance of tumour. However, mice failed to develop tumour when inoculated with tumour cells incubated with cisplatin + alpha-tocopherol. When the animals were challenged with tumour cells (3 x 10(6) cells/mouse) on the 15th day after the initial inoculation, 30-50% survived more than 60 days, with 10% tumour-free survivors being observed in some groups. Antitumour activity was higher in mice receiving lymphoma cells (3 x 10(6) cells/mouse) preincubated with cisplatin + alpha-tocopherol compared to cisplatin alone. Tumour-bearing mice receiving cisplatin in combination with different concentrations of alpha-tocopherol exhibited significantly higher (P<0.001) intratumour platinum content (123-306%) but without any change in the kidney platinum content as compared to those receiving cisplatin (5 or 10 µg/ml) alone. Enhancement of cisplatin-induced tumour growth inhibition is probably due to the modulation of tumour cell membrane permeability by alpha-tocopherol. alpha-Tocopherol might increase the influx of cisplatin into tumour cells, causing the DNA repair machinery to be less efficient due to increased efficiency of adduct formation in the DNA molecule. This effect of alpha-tocopherol can render cisplatin more effective as an antitumour agent.

Proteomic data show an increase in autoantibodies and alpha-fetoprotein and a decrease in apolipoprotein A-II with time in sera from senescence-accelerated mice

We evaluated changes in levels by comparing serum proteins in senescence-accelerated mouse-prone 8 (SAMP8) mice at 2, 6, 12, and 15 months of age (SAMP8-2 m, -6 m, -12 m, -15 m) to age-matched SAM-resistant 1 (SAMR1) mice. Mice were sacrificed, and blood was analyzed by 2-dimensional electrophoresis combined with mass spectrometry. Five protein spots were present in all SAMP8 serum samples, but only appeared in SAMR1 samples at 15 months of age except for spot 3, which also showed a slight expression in SAMR1-12 m sera. Two proteins decreased in the sera from SAMP8-2 m, -6 m, and -12 m mice, and divided into 2 spots each in SAMP8-15 m sera. Thus, the total number of altered spots in SAMP8 sera was 7; of these, 4 were identified as Ig kappa chain V region (M-T413), chain A of an activity suppressing Fab fragment to cytochrome P450 aromatase (32C2_A), alpha-fetoprotein, and apolipoprotein A-II. M-T413 is a monoclonal CD4 antibody, which inhibits T cell proliferation. We found that M-T413 RNA level was significantly enhanced in splenocytes from SAMP8-2 m mice. This agreed with serum M-T413 protein alterations and a strikingly lower blood CD4+ T cell count in SAMP8 mice when compared to the age-matched SAMR1 mice, with the latter negatively correlating with serum M-T413 protein volume. Age-related changes in serum proteins favored an increase in autoantibodies and alpha-fetoprotein and a decrease of apolipoprotein A-II, which occurred in SAMP8 mice at 2 months of age and onwards. These proteins may serve as candidate biomarkers for early aging.

Long term follow-up and patterns of response of ALT in patients with chronic hepatitis NANB/C treated with recombinant interferon-alpha

The response to interferon treatment in chronic hepatitis NANB/C has usually been classified as complete, partial or absent, according to the behavior of serum alanine aminotransferase (ALT). However, a more detailed observation of the enzymatic activity has shown that the patterns may be more complex. The aim of this study was to describe the long term follow-up and patterns of ALT response in patients with chronic hepatitis NANB/C treated with recombinant interferon-alpha. A follow-up of 6 months or more after interferon-a was achieved in 44 patients. We have classified the serum ALT responses into six patterns and the observed frequencies were as follows: I. Long term response = 9 (20.5%); II. Normalization followed by persistent relapse after IFN = 7 (15.9%); III. Normalization with transient relapse = 5 (11.9%); IV. Temporary normalization and relapse during IFN = 4 (9.1%); V. Partial response (more than 50% of ALT decrease) = 7 (15.9%); VI. No response = 12 (27.3%). In conclusion, ALT patterns vary widely during and after IFN treatment and can be classified in at least 6 types.

Cryoglobulinemia in chronic hepatitis C: clinical aspects and response to treatment with interferon alpha and ribavirin

INTRODUCTION: The main extra-hepatic manifestation of hepatitis C is mixed cryoglobulinemia (MC). The aim of this study was to evaluate its prevalence among patients with chronic hepatitis C (CHC), to correlate its presence to host and virological variables and to the response to combined therapy with interferon-alpha and ribavirin. CASUISTIC AND METHODS: 202 CHC naive patients (136 with chronic hepatitis and 66 with cirrhosis) were consecutively evaluated for the presence of cryoglobulins. Cryoprecipitates were characterized by immunoelectrophoresis and classified according to the Brouet's criteria. RESULTS: The prevalence of MC was 27% (54/202), and 24% of them (13/54) showed major clinical manifestation of the disease. Even though type III MC was more frequent (78%), symptomatic MC was more common in type II MC. The presence of cirrhosis (RR = 2.073; IC95% = 1.029 - 4.179; p = 0.041), and age of the patients (RR = 1.035; IC95% = 1.008 - 1.062; p = 0.01) were independently associated with the presence of cryoglobulins. No relationship was found with viral load and genotype. 102 patients were treated with interferon alpha and ribavirin. Among these, 31 had MC. Sustained virological response (around 30%) was similar in patients with and without MC (p = 0.971). CONCLUSION: MC represents a prevalent complication in patients with CHC, specially older and cirrhotic patients. Only 24% of these patients show clinical manifestation of the disease, specially those with type II MC. The presence of MC did not affect the response to therapy.

S100, CD68, and MHC class II molecule expression in cervical high- and low-grade HPV-induced lesions

INTRODUCTION: Some human papillomavirus (HPV) types are involved in malignant processes in the cervical epithelium, with 99% of cases attributed to oncogenic HPV infection. This study aimed to detect S100, CD68, and major histocompatibility complex class II (MHC-II) molecules in cervical uterine epithelial samples in patients with high- and low-grade lesions induced by HPV. METHODS: Fifty-eight samples from patients who were confirmed positive or negative for high-risk oncogenic HPV DNA, had histopathological diagnosis of cervical intraepithelial neoplasia (CIN) of grades I, II, or III, or were negative for intraepithelial lesion or malignancy were subjected to immunohistochemistry reaction to S100 protein, CD68, and MHC-II (HLA-DR alpha chain). RESULTS: The presence of MHC-II predominated in samples exhibiting histopathological alterations (p < 0.05). S100 detection was more numerous in carcinoma samples (CIN III) (75%). Presence of this protein correlated significantly (p < 0.05) with histopathological findings and viral load. CONCLUSIONS: A small expression of CD68 was observed, which may be explained by the observation in our study having been made on random microscopic fields and not on specific areas. The findings, such as the presence of S100 protein and MHC-II expression in samples with histological alterations, could suggest that the immune system fails to control HPV replication at the early stages of infection. Further studies with larger prospective data are necessary to confirm this result.

Preoperative serum levels of ca 72-4, cea, ca 19-9, and Alpha-fetoprotein in patients with gastric cancer

INTRODUCTION: The clinical importance of preoperative serum levels of CA 72-4, carcinoembryonic antigen (CEA), CA 19-9, and alpha-fetoprotein (AFP) was prospectively evaluated in 44 patients with gastric cancer. METHOD: The serum tumor marker levels were determined by commercial radioimmunoassay kits. Positivity for CA 72-4 (>4 U/mL), CEA (>5 ng/mL), CA 19-9 (>37 U/mL), and AFP (>10 ng/mL) were correlated according to the stage, histology, and lymph node metastasis. RESULTS AND DISCUSSION: CA 72-4 showed a higher positivity rate for gastric cancer (47.7%) than CEA (25%), CA 19-9 (25%), and AFP (0%). The combination of CA 72-4 with CEA and CA 19-9 increased the sensitivity to 61.4%. The positivity rates of CA 72-4 in patients at stages I and II (initial disease) and in patients at stages III and IV (advanced disease) were 9% and 60.6%, respectively (P < 0.005). No correlation was found between CEA and CA 19-9 levels and the stage of gastric cancer. There was a tendency of positivity for CA 72-4 to suggest lymph node involvement, but it was not significant (P = 0.075). Serum levels of tumor markers did not show a correlation with the histological types of gastric cancer. CONCLUSION: Preoperative serum levels of CA 72-4 provided a predictive value in indicating advanced gastric cancer.

Reações de inserção intramolecular de diazo compostos polifuncionais catalisadas por ródio(II): síntese de oxetan-3-ona-2-carboxilato e outros heterociclos funcionalizados

gamma-Hydroxy-alpha-diazo-beta-ketoesters are key intermediates in the chemistry of penicilin-based antibiotics and natural products. The method developed here for the synthesis of ethyl 2-diazo-4-hydroxy-3-oxo-butanoate 17 (in two steps from the diazo mercurial 2) compares very favorably with those reported in the literature for similar compounds. The Rh2(OAc)4-mediated intramolecular OH-insertion reaction of the diazo hydroxy ester 17 was investigated, furnishing the oxetan-3-one-2-carboxilate 18 in good yield. When the diazo ester lacks a free hydroxyl group as in the case of the phenoxy diazo ester 11 an intramolecular CH-insertion takes place, affording the 2H-chromene 20 in almost quantitative yield. The behavior of other functionalized diazo esters towards Rh2(OAc)4 was also investigated.

Aplicação de metodologias FTIR de transmissão e fotoacústica à caracterização de materiais altamente energéticos: parte II

Materials obtained during the synthesis of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) were characterized by Fourier transform infrared (FTIR) transmission spectroscopy and/or Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS). By these techniques the spectrometric alterations that occurred during the process were observed. The characterized species during the synthesis of HMX were alpha-HMX, beta-HMX, hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and HMX/RDX mixtures. The FTIR-PAS was verified to be a promising technique of great usefulness of the characterization of highly energetic materials because it is fast, simple and requires no sample preparation unlike Fourier transform infrared transmission technique (KBr pellet). The FTIR-PAS analysis showed that with small sample quantity is possible to distinguish between thealpha-HMX and beta-HMX and to detect even in a qualitative way different HMX / RDX ratios.

alpha-Globin genes: thalassemic and structural alterations in a Brazilian population

Seven unrelated patients with hemoglobin (Hb) H disease and 27 individuals with alpha-chain structural alterations were studied to identify the alpha-globin gene mutations present in the population of Southeast Brazil. The -alpha3.7, --MED and -(alpha)20.5 deletions were investigated by PCR, whereas non-deletional alpha-thalassemia (alphaHphalpha, alphaNcoIalpha, aaNcoI, alphaIcalpha and alphaTSaudialpha) was screened with restriction enzymes and by nested PCR. Structural alterations were identified by direct DNA sequencing. Of the seven patients with Hb H disease, all of Italian descent, two had the -(alpha)20.5/-alpha3.7 genotype, one had the --MED/-alpha3.7 genotype, one had the --MED/alphaHphalpha genotype and three showed interaction of the -alpha3.7 deletion with an unusual, unidentified form of non-deletional alpha-thalassemia [-alpha3.7/(aa)T]. Among the 27 patients with structural alterations, 15 (of Italian descent) had Hb Hasharon (alpha47Asp->His) associated with the -alpha3.7 deletion, 4 (of Italian descent) were heterozygous for Hb J-Rovigo (alpha53Ala->Asp), 4 (3 Blacks and 1 Caucasian) were heterozygous for Hb Stanleyville-II (alpha78Asn->Lys) associated with the alpha+-thalassemia, 1 (Black) was heterozygous for Hb G-Pest (alpha74Asp->Asn), 1 (Caucasian) was heterozygous for Hb Kurosaki (alpha7Lys->Glu), 1 (Caucasian) was heterozygous for Hb Westmead (alpha122His->Gln), and 1 (Caucasian) was the carrier of a novel silent variant (Hb Campinas, alpha26Ala->Val). Most of the mutations found reflected the Mediterranean and African origins of the population. Hbs G-Pest and Kurosaki, very rare, and Hb Westmead, common in southern China, were initially described in individuals of ethnic origin differing from those of the carriers reported in the present study and are the first cases to be reported in the Brazilian population.

Incorporation of conjugated linoleic and alpha linolenic fatty acids into Pimedolus maculatus fillets

The objective of this study was to investigate the fatty acid composition of Pimedolus maculatus fillets of fish fed with different diets. The fish were submitted to an adaptation period (over 30 days) and fed only a control diet (diet I). After this period, the fish were divided into two groups: one group received a diet enriched with flaxseed oil (diet II), and the other received a diet enriched with conjugated linoleic acid (CLA) (diet III). Subsequent collections were undertaken after 25 and 50 days. Compared to the control samples, fish fed diet II showed an increase in concentrations of total fatty acid omega-3 (n-3) in the fillets, with an improvement ratio of n-6/n-3. The incorporation of CLA into fish fed diet III was successful. Supplementing the diet of Pimedolus maculatus with CLA and flaxseed oil led to improvements in nutritional quality and fatty acid profile of the fish fillets.

Hidropsia endolinfática experimental sob ação de inibidor da óxido nítrico sintase tipo II: avaliação com emissões otoacústicas e eletrococleografia

No modelo experimental de hidropsia endolinfática há redução na amplitude das emissões otoacústicas produtos de distorção (EOAPD) e elevação nos limiares eletrofisiológicos na eletrococleografia. Estudos mostraram que há expressão da óxido nítrico sintase tipo II (ONS II) na cóclea com hidropsia, sugerindo a participação do óxido nítrico (ON) na patogênese desta doença. O objetivo deste trabalho foi avaliar a ação de um inibidor da ONS II nas EOAPD e eletrococleografia em cobaias com hidropisia endolinfática experimental. MATERIAL E MÉTODOS: Foram estudadas 16 cobaias nas quais se induziu hidropsia endolinfática experimental por obliteração do ducto e saco endolinfático na orelha direita durante 16 semanas, divididas em dois grupos: oito cobaias recebendo um inibidor da ONS II, a aminoguanidina, por via oral e um grupo de oito cobaias como controle. Comparamos as amplitudes das EOAPD nas médias geométricas de freqüências de 1062, 2187, 4375 e 7000Hz, os limiares eletrofisiológicos nas freqüências de 1000, 2000, 4000 e 6000Hz e a relação entre os potenciais de somação e de ação (PS/PA) entre os grupos. RESULTADOS: Não houve diferença significante nas EOAPD e na relação PS/PA entre os grupos. O grupo que recebeu a aminoguanidina apresentou menor elevação nos limiares eletrofisiológicos nas freqüências de 2000 (p<0,05) e 6000 Hz (p<0,05) na 12ª semana e nas freqüências de 1000 (p<0,05), 2000 (p<0,001), 4000 (p<0,001) e 6000Hz (p<0,001) na 16ª semana. CONCLUSÕES: O inibidor da ONS II reduziu a elevação dos limiares eletrofisiológicos na eletrococleografia na hidropsia endolinfática experimental.

Processo de adaptação de próteses auditivas em usuários atendidos em uma instituição pública federal: parte II: resultados dos questionários de auto-avaliação

Uma reabilitação eficiente deve reduzir os efeitos da deficiência sobre as habilidades auditivas e comunicativas do indivíduo e aumentar o bem-estar psicossocial. OBJETIVOS:Verificar a viabilidade do uso de questionários de auto-avaliação e comparar os resultados da protetização em usuários de uma instituição pública federal, com e sem queixas relacionadas às características da amplificação. MATERIAL E MÉTODOS:25 indivíduos, de 13 a 77 anos de idade, usuários de próteses auditivas. Foram aplicados os questionários de auto-avaliação HHIE-S/HHIA (Hearing Handicap Inventory for the Elderly Screening Version ou for Adult) e APHAB (Abbreviated Profile of Hearing Aid Benefit), nos indivíduos sem (Grupo 1) e com queixas relacionados às características da amplificação (Grupo 2). RESULTADOS: Diferenças significantes não foram encontradas entre os grupos nos protocolos HHIE-S/HHIA e APHAB, exceto na subescala facilidade de comunicação do APHAB, onde o Grupo 1 obteve melhor benefício. Também evidenciou-se redução significativa da incapacidade auditiva com o uso das próteses em situações favoráveis de comunicação, ambientes reverberantes e na presença de ruído ambiental para ambos os grupos. CONCLUSÃO: Os questionários revelaram ser excelentes preditores das dificuldades enfrentadas pelos usuários, e diferenças significantes foram encontradas em situações favoráveis de comunicação, onde o grupo sem queixas obteve melhor benefício.