An enzyme-linked immunosorbent assay (ELISA) for the detection of IgM antibodies against Leishmania chagasi in dogs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Chemical properties and enzyme activity in a sewage sludge-treated soil

A soil sample was taken from the top 0-20cm at Jaboticabal county, São Paulo State, Brazil, air dried, sieved to 5mm, and placed into pots (2700g per pot). Sewage sludge was air-dried, ground to 2mm, and thoroughly mixed to the top 0-10cm soil of each pot, which were irrigated with distilled water in a total volume equivalent to the last 30years average rainfall in the region. Sorghum was sowed 120days after sewage sludge incorporation and then the irrigation was made according to the plants' requirement. When the plants were about 10 cm high, they were thinned to two per pot. Soil samples (0-10, 10-20, and 20-30 cm depth) were obtained immediately after the incorporation of sewage sludge and at 30, 60, 120, and 170 days after, air dried, sieved to 2 mm and analyzed for organic matter (OM), pH (0,01 mol L-1 CaCl2), extractable P (resin), potassium (K), calcium (Ca), and magnesium (Mg), amylase and cellulase activity. Sewage sludge increased soil OM, pH, extractable phosphorus (P), K. Ca. amylase and cellulase activity, especially at the rate 16 t ha(-1). Organic matter, extractable P, K, Ca, Mg. and amylase activity were higher in the top 0-10cm, while pH was higher in the 20-30cm layer. Amylase activity was not affected by sampling depth. Organic matter, pH, extractable P. K, Ca, and Mg decreased during the experimental period. Amylase activity decreased until sorghum was sowed and increased afterwards. Cellulase activity increased until 90 days after sewage sludge application and then decreased. Sewage sludge used in the experiment should already contain some amylase activity or a substance that was a soil enzyme activator and also a substance that was an inhibitor of soil cellulase inhibitor. Sonic of the plant nutrients contained in sewage sludge, mainly P, did not migrate down the soil column. an indication that sewage sludge should be incorporated into the soil to improve nutrient bioavailability. Sorghum roots increased amylase activity but did not affect cellulase activity.

The zymogen-enteropeptidase system: A practical approach to study the regulation of enzyme activity by proteolytic cleavage

The present research describes an efficient procedure to obtain high levels of trypsinogen and chymotrypsinogen by using a simple, rapid, and easily reproducible method. The extraction process and the time-course of activation of zymogens can be carried out in a single laboratory period, without sophisticated equipment. The main objective was to prepare a laboratory class that would stimulate student interest in enzyme regulation, exploring the fact that the catalytic activity of some enzymes is regulated by different mechanisms. The regulation of proteolytic enzymes requires the synthesis of an inactive zymogen and its being irreversibly switched on by specific proteolytic cleavage.

Development of a scar marker for Pierce's Disease strains of Xylella fastidiosa

O objetivo deste trabalho foi desenvolver um oligonucleotídeo iniciador para reação em cadeia da polimerase (PCR) específico para as estirpes de Xylella fastidiosa que causam o mal de Pierce (PD) em videira (Vitis vinifera). Amplificações de DNA de 23 diferentes hospedeiros, usando o conjunto de oligonucleotídeos REP1-R (5'-IIIICGICGIATCCIGGC-3') e REP 2 (5'-ICGICTTATCI GGCCTAC-3') utilizando o programa: 94 ºC/2 min; 35 X (94 ºC/1 min, 45 ºC/1 min; 72 ºC/1 min and 30 s) 72 ºC/5 min, produziu um fragmento de 630 pb que diferenciou as estirpes de videiras dos demais. Entretanto, padrões de bandeamento REP não são considerados confiáveis para detecção devido ao par de oligonucleotídeos REP 1 e REP 2 corresponderem a seqüências repetitivas encontradas por todo o genoma bacteriano. Desse modo, o produto amplificado de 630 pb foi eluído do gel de agarose, purificado e seqüenciado. A informação da seqüência nucleotídica foi usada para identificar e sintetizar um oligonucleotídeo específico para o isolado de X. fastidiosa causadora do mal de Pierce denominado Xf-1 (5'-CGGGGGTGTAGGAGGGGTTGT-3'), que foi utilizado juntamente com o oligonucleotídeo REP-2 nas condições 94 ºC/2 min; 35 X (94 ºC/1 min, 62 ºC/1 min; 72 ºC/1 min and 30 s) 72 ºC/10 min. Os DNAs das estirpes de X. fastidiosa de outros hospedeiros [amêndoa (Prumus amygdalus), citros (Citrus spp.), café (Coffea arabica), olmo (Ulmus americana), amora (Morus rubra), carvalho (Quercus rubra), vinca (Catharantus roseus), ameixa (Prunus salicina) e ragweed (Ambrosia artemisiifolia)] e de bactérias Gram negativas e positivas foram submetidos a amplificação com o conjunto de oligonucleotídeos Xf-1/REP 2. Um fragmento, de aproximadamente 350 pb, foi amplificado apenas com o DNA de X. fastidiosa isolada de videira.

Mating system of a population of Myracrodruon urundeuva F.F. & M.F. Allemão using the fAFLP molecular marker

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Optimization of fibrolytic enzyme production by Aspergillus japonicus C03 with potential application in ruminant feed and their effects on tropical forages hydrolysis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of the spouted bed dried leaf extract of Bauhinia forficata for the treatment of experimental diabetes in rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Large-scale transcriptome analyses reveal new genetic marker candidates of head, neck, and thyroid cancer

A detailed genome mapping analysis of 213,636 expressed sequence tags (EST) derived from nontumor and tumor tissues of the oral cavity, larynx, pharynx, and thyroid was done. Transcripts matching known human genes were identified; potential new splice variants were flagged and subjected to manual curation, pointing to 788 putatively new alternative splicing isoforms, the majority (75%) being insertion events. A subset of 34 new splicing isoforms (5% of 788 events) was selected and 23 (68%) were confirmed by reverse transcription-PCR and DNA sequencing. Putative new genes were revealed, including six transcripts mapped to well-studied chromosomes such as 22, as well as transcripts that mapped to 253 intergenic regions. In addition, 2,251 noncoding intronic RNAs, eventually involved in transcriptional regulation, were found. A set of 250 candidate markers for loss of heterozygosis or gene amplification was selected by identifying transcripts that mapped to genomic regions previously known to be frequently amplified or deleted in head, neck, and thyroid tumors. Three of these markers were evaluated by quantitative reverse transcription-PCR in an independent set of individual samples. Along with detailed clinical data about tumor origin, the information reported here is now publicly available on a dedicated Web site as a resource for further biological investigation. This first in silico reconstruction of the head, neck, and thyroid transcriptomes points to a wealth of new candidate markers that can be used for future studies on the molecular basis of these tumors. Similar analysis is warranted for a number of other tumors for which large EST data sets are available.

Effects and toxicity of Eugenia punicifolia extracts in streptozotocin-diabetic rats

Extracts and decoctions of Eugenia jambolana Lam., Eugenia uniflora L., and Eugenia punicifolia (Humb., Bonpl. & Kunt) DC. are used in traditional medicine to treat diabetes mellitus. Although there have been reports that Eugenia jambolana and Eugenia uniflora have antidiabetic effects, no study has yet been made on Eugenia punicifolia . We investigated the effects of aqueous, butanol, and methanol extracts of Eugenia punicifolia leaves administered by gavage to streptozotocin-diabetic rats for 26 to 29 days. Body weight, food and fluid intake, urine volume, and urinary glucose and urea were evaluated every 7 days. At the end of the experiment, we measured serum cholesterol, high-density lipoprotein (HDL)-cholesterol, triglycerides and bilirubin, hepatic glycogen and serum marker-enzymes (alanine and aspartate aminotransferases, alkaline phosphatase, gamma-glutamyltransferase, L-lactate dehydrogenase, creatine kinase, alpha-amylase, and angiotensin I converting enzyme). We found that in rats treated with the aqueous extracts, food and liquid intake, urinary volume, and body weight were all reduced, while for rats treated with the methanol extract, not only were liquid intake, urinary volume and body weight reduced, but urinary glucose and urea also decreased. Rats treated with the butanol extract showed no significant alterations in any of the parameters measured. Chronic treatment with extracts had no effect on the marker enzymes nor on serum bilirubin levels. The results indicate that aqueous extracts of Eugenia punicifolia leaves produced an anorexic effect and that methanol extracts had a beneficial effect on the diabetic state by improving carbohydrate and protein metabolism without provoking hepatobiliary, microvascular, muscular, or pancreatic toxic effects.

Enzyme Biomarkers of Renal Tubular Injury in Arterial Surgery Patients

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Menstrual irregularity: a possible clinical marker of metabolic dysfunction in women with class III obesity

Purpose. To evaluate whether menstrual irregularity in morbidly obese women is indicative of metabolic dysfunction.Patients and Methods. Fifty-seven women with morbid obesity were evaluated. They were divided into two groups: one comprising women without menstrual dysfunctions or hirsutism (Group 1), and another obese women showing menstrual dysfunction with or without hirsutism (Group 2). The following were evaluated: age, colour, childbirth, marital status, profession, socio-economic level, education, age at menarche, body weight, height, body mass index, presence of hirsutism (Ferriman Gallwey Index), abdominal circumference, hip circumference, waist-to-hip ratio, menstrual cycle, blood pressure, presence of acanthosis nigricans, insulin resistance (IR), fasting glycaemia, total cholesterol, HDL-C, LDL-C, triglycerides, thyroid-stimulating hormone, free T4, luteinising hormone (LH), follicle-stimulating hormone, prolactin, total testosterone, dehydroepiandrosterone sulfate, insulin and the Homeostasis Model Assessment (HOMA test).Results. Clinical and epidemiological aspects did not present statistical differences. Clinical and laboratory parameters did not show statistically significant alterations; however, HOMA test values for Group 2 were significantly higher than those for Group 1.Conclusions. The presence of IR in class III obese women can cause menstrual dysfunctions such as amenorrhoea or oligomenorrhoea even in the absence of hyperandrogenism, suggesting that IR plays an important role in the ovarian mechanisms involved in the menstrual cycle control.

CDX2, a highly sensitive and specific marker of adenocarcinomas of intestinal origin - An immunohistochemical survey of 476 primary and metastatic carcinomas

CDX2 is a recently cloned homeobox gene that encodes an intestine-specific transcription factor, expressed in the nuclei of epithelial cells throughout the intestine, from duodenum to rectum. While expression of CDX2 protein in primary and metastatic colorectal carcinomas has been previously documented, neither the sensitivity nor the specificity of CDX2 expression, as determined by immunohistochemistry, for colorectal adenocarcinoma has been determined. We performed an immunohistochemical survey of 476 tumors with a monoclonal antibody, CDX2-88, including 89 tumors from the colon and duodenum and 95 tumors from other gastrointestinal sites, including the esophagus, stomach, pancreatobiliary system, gastrointestinal carcinoids, and liver. CDX2 was expressed uniformly (that is, in 76-100% of tumor cells) in all but one of the evaluated colorectal and duodenal tumors. High-level expression of CDX2 was also found, however, in mucinous ovarian carcinomas and adenocarcinomas primary to the urinary bladder of which 64% and 100% were positive, respectively. Gastric, gastroesophageal, and pancreatic adenocarcinomas and cholangiocarcinomas all showed similar, heterogeneous patterns of CDX2 expression. Most tumors in each group showed CDX2 expression by a minority of cells, whereas a substantial minority of cases in each group was completely negative and a smaller minority was uniformly positive. Gastrointestinal carcinoids gave similarly varied results, but the majority (58%) was negative. Hepatocellular carcinomas showed no expression of CDX2. Only very rare examples of carcinomas of the genitourinary and gynecologic tracts, breast, lung, and head and neck showed significant levels of CDX2 expression. In this study of primary and metastatic epithelial tumors, uniform CDX2 expression is demonstrated to be an exquisitely sensitive and highly, but incompletely, specific marker of intestinal adenocarcinomas. Compared with villin, a previously described marker of GI adenocarcinomas, CDX2 demonstrated superior sensitivity and comparable specificity. CDX2 expression can be seen, however, in selected non-GI adenocarcinomas such as mucinous ovarian carcinomas and adenocarcinomas of the urinary bladder.

Cocaine mutagenicity and hepatocarcinogenicity evaluations in rodents

The mutagenicity (clastogenicity) and the carcinogenicity (promoting potential) of cocaine were evaluated, respectively, by the mouse bone marrow micronucleus test (study I) and by the initiated rat liver bioassay (study II). In study I, two administration routes (i.p. and i.v.) and two sampling times (24 and 48 hours) after cocaine treatment were studied. Swiss male mice were treated with cocaine at doses of 0, 18, 37, and 75 mg/kg and 0, 2, 4, and 8 mg/kg by i.p. and i.v. routes, respectively. No significant differences were observed between treated and negative control groups regarding the frequencies of micronuclei and the polichromatic/normochromatic erythrocyte (PCE/NCE) ratios. In study II, the development of putative preneoplastic foci of hepatocytes expressing the enzyme glutathione S-transferase placental form (GST-P+) was utilized as the end-point marker in a 8-week rat liver bioassay. The animals were initiated for carcinogenesis by a single i.p. sub-carcinogenic dose of diethylnitrosamine (DEN). After a 6-week exposure to 5 or 10 mg/kg of cocaine i.v. twice a week there was no enhancement of GST-P+ foci development above the values of the control DEN-only treated animals. Also, cocaine did not induce any toxicity as evidenced by the absence of alterations of rat body and liver weights and of liver biochemical function and morphology. The results suggest that cocaine does not have a mutagenic effect on the mouse bone marrow cells or promoting activity on the rat hepatocarcinogenesis process. Teratogenesis Carcinog. Mutagen. 18:199-208, 1998. (C) 1998 Wiley-Liss, Inc.

Evaluation of estrogen receptor alpha and beta and progesterone receptor expression and correlation with clinicopathologic factors and proliferative marker Ki-67 in breast cancers

To elucidate the molecular profile of hormonal steroid receptor status, we analyzed ER-alpha, ER-beta, and PGR mRNA and protein expression in 80 breast carcinomas using reverse transcriptase polymerase chain reaction (RT-PCR), quantitative RT-PCR, and immunohistochemical analysis. Qualitative analysis revealed positive expression of ER-alpha, ER-beta, and PGR mRNA in 48%, 59%, and 48% of the breast carcinomas, respectively. ER-alpha, ER-beta, and PGR transcript overexpression was observed in 51%, 0%, and 12% of the cases, respectively, whereas moderate or strong protein expression was detected in 68%, 78%, and 49% of the cases, respectively. Tumor grade was negatively correlated with transcript and protein levels of ER-alpha (P = .0169 and P = .0006, respectively) and PGR (P = .0034 and P = .0005, respectively). Similarly, proliferative index Ki-67 was negatively associated with transcript and protein levels of ER-alpha (P = .0006 and P < .0001, respectively) and PGR (P = .0258 and P =. 0005, respectively). These findings suggest that ER-alpha and PGR expression are associated with well-differentiated breast tumors and less directly related to cell proliferation. A significant statistical difference was observed between lymph node status and ER-beta protein expression (P = .0208). In ER-alpha-negative tumors, we detected a correlation between ER-beta protein expression and high levels of Ki-67. These data suggest that ER-beta could be a prognostic marker in human breast cancer. (C) 2008 Elsevier B.V. All rights reserved.