Recombinant production and biochemical and in silico characterization of lactate dehydrogenase from Geobacillus thermodenitrificans DSM-465

Background: Lactate dehydrogenase (LDH) is an enzyme of glycolytic pathway, ubiquitously found in living organisms. Increased glycolysis and LDH activity are associated with many pathologic conditions including inflammation and cancer, thereby making the enzyme a suitable drug target. Studies on conserved structural and functional domains of LDH from various species reveal novel inhibitory molecules. Our study describes Escherichia coli production and characterization of a moderately thermostable LDH (LDH-GT) from Geobacillus thermodenitrificans DSM-465. An in silico 3D model of recombinant enzyme and molecular docking with a set of potential inhibitors are also described. Results: The recombinant enzyme was overexpressed in E. coli and purified to electrophoretic homogeneity. The molecular weight of the enzyme determined by MALDI-TOF was 34,798.96 Da. It exhibited maximum activity at 65°C and pH 7.5 with a KM value for pyruvate as 45 µM. LDH-GT and human LDH-A have only 35.6% identity in the amino acid sequence. On the contrary, comparison by in silico structural alignment reveals that LDH-GT monomer has approximately 80% identity to that of truncated LDH-A. The amino acids "GEHGD" as well as His179 and His193 in the active site are conserved. Docking studies have shown the binding free energy changes of potential inhibitors with LDH-A and LDH-GT ranging from −407.11 to −127.31 kJ mol−1 . Conclusions: By highlighting the conserved structural and functional domains of LDH from two entirely different species, this study has graded potential inhibitory molecules on the basis of their binding affinities so that they can be applied for in vivo anticancer studies

Can thiamine pyrophosphate prevent desflurane induced hepatotoxicity in rats?

ABSTRACT PURPOSE : To investigate the effects of thiamine pyrophosphate (TPP) against desflurane induced hepatotoxicity. METHODS : Thirty experimental animals were divided into groups as healthy (HG), desflurane control (DCG) , TPP and desflurane group (TDG). 20 mg/kg TPP was injected to intraperitoneally TDG. After one hour of TPP administration, desflurane was applied for two hours. After 24 hours, liver tissues of the animals killed with decapitation were removed. The oxidant/antioxidant levels and ALT, AST and LDH activities were measured. The histopathological examinations were performed in the liver tissues for all rats. RESULTS : Notwithstanding the levels of oxidants and liver enzymes were significantly increased (p<0.0001), antioxidant levels were significantly decreased in DCG (p<0.0001). On contrary to the antioxidant parameters were increased (p<0.05) the oxidant parameters and liver enzymes were decreased in TDG (p<0.0001). Whereas multiple prominent, congestion, hemorrhage and dilatation were observed in sinusoids and lymphocyte-rich inflammation results in the centrilobular and portal areas of liver tissue in DCG, these findings were observed less frequently in TDG. CONCLUSİON : Thiamine pyrophosphate prevented liver oxidative damage induced with desflurane and may be useful in prophylaxis of desflurane induced hepatotoxicity.

Predictive Factors of Mortality in Population of Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH): Results from a Korean PNH Registry

Paroxysmal nocturnal hemoglobinuria (PNH) is a progressive, systemic, life-threatening disease, characterized by chronic uncontrolled complement activation. A retrospective analysis of 301 Korean PNH patients who had not received eculizumab was performed to systematically identify the clinical symptoms and signs predictive of mortality. PNH patients with hemolysis (lactate dehydrogenase [LDH] > or = 1.5 x the upper limit of normal [ULN]) have a 4.8-fold higher mortality rate compared with the age- and sex-matched general population (P < 0.001). In contrast, patients with LDH < 1.5 x ULN have a similar mortality rate as the general population (P = 0.824). Thromboembolism (TE) (odds ratio [OR] 7.11; 95% confidence interval [CI] (3.052-16.562), renal impairment (OR, 2.953; 95% CI, 1.116-7.818) and PNH-cytopenia (OR, 2.547; 95% CI, 1.159-5.597) are independent risk factors for mortality, with mortality rates 14-fold (P < 0.001), 8-fold (P < 0.001), and 6.2-fold (P < 0.001) greater than that of the age- and sex-matched general population, respectively. The combination of hemolysis and 1 or more of the clinical symptoms such as abdominal pain, chest pain, or dyspnea, resulted in a much greater increased mortality rate when compared with patients with just the individual symptom alone or just hemolysis. Early identification of risk factors related to mortality is crucial for the management of PNH. This trial was registered at www.clinicaltrials.gov as NCT01224483.

Adherence to standard precautions from the standpoint of the Health Belief Model: the practice of recapping needles / Adesão às precauções padrão sob o prisma do Modelo de Crenças em Saúde: a prática de reencapar agulhas

The aim of this study was to applythe Health Belief Model to explain the adherence to the recommendation not to recap needles by dentists and dental assistants of the public health system in a municipality in the State of São Paulo. A questionnaire validated and adapted for the oral health area was used, which included variables related to the frequency of recapping and health beliefs using Likert-type scales. The relationship between beliefs and adherence to the recommendation not to recap needles was obtained by regression analysis. Of all the professionals in this study (n=79), the majority (83.5%) reported recapping needles at least once in the last month. Through regression analysis, it was observed that the relationship between the beliefs described by the model and the attitude whether or not to follow the recommendation not to recap needles was explained by a lower perception of psychological barriers and a greater perception of stimuli not to recap needles. The conclusion reached is that the acceptance of recommendations to prevent working accidents with biological material was explained by some dimensions of the Health Belief Model, enabling discussion about reformulation of training offered to professionals of the public health system.

Objetivou-se neste estudo aplicar o Modelo de Crenças em Saúde a fim de explicar a adesão à recomendação de não reencapar agulhas por cirurgiões-dentistas e auxiliares de saúde bucal da rede pública de um município paulista. Utilizou-se um questionário validado e adaptado para a área de saúde bucal, que contemplava variáveis relativas à frequência do reencape e crenças em saúde, por meio de escalas tipo Likert. A relação entre as crenças e a adesão à recomendação de não reencapar agulhas foi obtida por meio da análise de regressão. Da amostra de profissionais obtida por adesão ao estudo (n = 79), a maioria (83,5%) relatou ter reencapado agulhas pelo menos alguma vez no último mês. Por meio da análise de regressão, foi observado que a relação entre as crenças descritas pelo modelo e a atitude de aderir ou não à recomendação de não reencapar agulhas foi explicada por uma menor percepção de barreiras psicológicas e por uma maior percepção de estímulos para não reencapar agulhas. Conclui-se que a aceitação das recomendações para prevenir acidentes do trabalho com material biológico foi explicado por algumas dimensões do Modelo de Crenças em Saúde, possibilitando a discussão sobre a reformulação de capacitações oferecidas para profissionais do sistema público de saúde.

The Toxicity of Nonsteroidal Anti-inflammatory Eye Drops against Human Corneal Epithelial Cells in Vitro

This study investigated the toxicity of commercial non-steroid anti-inflammatory drug (NSAID) eye solutions against corneal epithelial cells in vitro. The biologic effects of 1/100-, 1/50-, and 1/10-diluted bromfenac sodium, pranoprofen, diclofenac sodium, and the fluorometholone on corneal epithelial cells were evaluated after 1-, 4-, 12-, and 24-hr of exposure compared to corneal epithelial cell treated with balanced salt solution as control. Cellular metabolic activity, cellular damage, and morphology were assessed. Corneal epithelial cell migration was quantified by the scratch-wound assay. Compared to bromfenac and pranoprofen, the cellular metabolic activity of diclofenac and fluorometholone significantly decreased after 12-hr exposure, which was maintained for 24-hr compared to control. Especially, at 1/10-diluted eye solution for 24-hr exposure, the LDH titers of fluorometholone and diclofenac sodium markedly increased more than those of bromfenac and pranoprofen. In diclofenac sodium, the Na+ concentration was lower and amount of preservatives was higher than other NSAIDs eye solutions tested. However, the K+ and Cl- concentration, pH, and osmolarity were similar for all NSAIDs eye solutions. Bromfenac and pranoprofen significantly promoted cell migration, and restored wound gap after 48-hr exposure, compared with that of diclofenac or fluorometholone. At 1/50-diluted eye solution for 48-hr exposure, the corneal epithelial cellular morphology of diclofenac and fluorometholone induced more damage than that of bromfenac or pranoprofen. Overall, the corneal epithelial cells in bromfenac and pranoprofen NSAID eye solutions are less damaged compared to those in diclofenac, included fluorometholone as steroid eye solution.

Escala da Satisfação com a decisão em saúde: instrumento adaptado e validado para língua portuguesa / Decision making satisfaction in health scale: instrument adapted and validated to portuguese / Escala de la satisfacción con las decisiones en la salud: instrumento adaptado y validado para el idioma portugués

A tomada de decisão é uma área de investigação na saúde que tem vindo a ganhar importância quer pelos modelos de parceria de cuidados que dão protagonismo ao paciente e família, quer pela preocupação crescente com a qualidade e satisfação do cliente com os cuidados disponibilizados. Assim, propusemo-nos efetuar a adaptação transcultural e avaliar as propriedades psicométricas da versão portuguesa da "The Satisfaction with Decision Scale" de Holmes-Rovner (1996), que visa avaliar a satisfação com as decisões tomadas em saúde. A amostra foi constituída por 521 estudantes da Escola Superior de Enfermagem do Porto. Os resultados obtidos nos testes de fiabilidade revelam uma boa consistência interna para o total dos itens (Alpha Cronbach = 0,88). O estudo psicométrico permite-nos afirmar que a versão em Português da "The Satisfaction with Decision Scale", que denominamos "Escala da Satisfação com a Decisão em Saúde", é um instrumento fidedigno e válido.

Decision making is an area of health research that has gained importance both for the partnership models of care that give prominence to the patient and family, either by growing concern about quality and customer satisfaction with the care provided. So we decided to make the cultural adaptation and to evaluate the psychometric properties of the Portuguese version "The Satisfaction with Decision Scale" de Holmes-Rovner (1996), which aims to assess satisfaction with the decisions taken in health. The sample consisted of 521 nursing students the School of Nursing of Porto. The results of reliability tests show good internal consistency for the total items (Alpha Cronbach = 0.88). The psychometric study allows us to state that the Portuguese version of "The Satisfaction with Decision Scale", we call "Escala da Satisfação com a Decisão em Saúde", is an instrument comparable with the original in terms of validity and reliability.

La toma de decisiones es un área de investigación en salud que ha ganado importancia tanto por los modelos de atención dirigida al paciente y su familia, como por la creciente preocupación por la calidad y satisfacción del cliente con la atención recibida. Por esta razón decidimos hacer la adaptación transcultural y evaluar las propiedades psicométricas de la versión portuguesa "The Satisfaction with Decision Scale" de Holmes-Rovner (1996), que tiene como objetivo evaluar la satisfacción con las decisiones adoptadas en materia de salud. La muestra consta de 521 estudiantes de la Escuela de Enfermería del Porto. Los resultados de las pruebas de fiabilidad muestran una buena consistencia interna para la escala total (Alpha Cronbach = 0,88). El estudio psicométrico nos permite afirmar que la versión en portugués de "The Satisfaction with Decision Scale", que nosotros llamamos "Escala da Satisfação com a Decisão em Saúde", es válida.

Expression of Glucose Transporters 1 and 3 in Metastatic and Non-Metastatic Lower Lip Squamous Cell Carcinoma

This study aimed to evaluate the immunoexpression of glucose transporters 1 (GLUT-1) and 3 (GLUT-3) in metastatic and non-metastatic lower lip squamous cell carcinoma (LLSCC). Twenty LLSCCs with regional nodal metastasis and 20 LLSCCs without metastasis were selected. The distribution of staining and the percentage of GLUT-1 and GLUT-3 staining in each tumor core and at the deep invasive front were assessed. Most tumors (70%) exhibited peripheral staining for GLUT-1 in nests, sheets and islands of neoplastic cells, whereas predominantly central staining was observed for GLUT-3 (72.5%). A high percentage of GLUT-1-positive cells was observed at the deep invasive front and in the tumor core of metastatic and non-metastatic tumors (p>0.05). The percentage of GLUT-1-positive cells was much higher than that of GLUT-3-positive cells both in the deep invasive front (p<0.001) and in the tumor core (p<0.001) of LLSCCs. No significant differences in the percentage of GLUT-1- and GLUT-3-positive cells were observed according to nodal metastasis, clinical stage or histological grade of malignancy (p>0.05). In conclusion, the results of the present study suggest an important role of GLUT-1 in glucose uptake in LLSCCs, although this protein does not seem to be involved in the progression of these tumors. On the other hand, GLUT-3 expression may represent a secondary glucose uptake mechanism in LLSCCs.

Este estudo objetivou avaliar a imunoexpressão dos transportadores de glicose 1 (GLUT-1) e 3 (GLUT-3) em carcinomas de células escamosas de lábio inferior (CCELI) metastáticos e não-metastáticos. Vinte CCELIs com metástase nodal regional e 20 CCELI sem metástase foram selecionados. Foram analisados a distribuição da imunomarcação e o percentual de imunorreatividade para GLUT-1 e GLUT-3 no centro tumoral e no front de invasão tumoral. A maioria dos tumores (70%) revelou marcação para GLUT-1 em áreas periféricas dos ninhos, lençóis e ilhas de células neoplásicas, ao passo que GLUT-3 revelou predomínio de marcação em áreas centrais (72.5%). Um alto percentual de células positivas para GLUT-1 foi observado no front de invasão e no centro tumoral das lesões metastáticas e não-metastáticas (p>0,05). O percentual de células positivas para GLUT-1 foi superior ao percentual de células positivas para GLUT-3, tanto no front de invasão (p<0,001) quanto no centro tumoral (p<0,001) dos CCELIs. Não foram observadas diferenças significativas no percentual de células positivas para GLUT-1 e GLUT-3 em relação à mestástase nodal, ao estádio clínico ou ao grau histológico de malignidade (p>0,05). Em conclusão, os resultados do presente estudo sugerem um importante papel para GLUT-1 na absorção de glicose nos CCELIs, embora esta proteína não pareça estar envolvida na progressão destes tumores. Por outro lado, a expressão de GLUT-3 pode representar um mecanismo secundário para a absorção de glicose nos CCELIs.

Putative role of ischemic postconditioning in a rat model of limb ischemia and reperfusion: involvement of hypoxia-inducible factor-1? expression

Hypoxia-inducible factor-1α (HIF-1α) is one of the most potent angiogenic growth factors. It improves angiogenesis and tissue perfusion in ischemic skeletal muscle. In the present study, we tested the hypothesis that ischemic postconditioning is effective for salvaging ischemic skeletal muscle resulting from limb ischemia-reperfusion injury, and that the mechanism involves expression of HIF-1α. Wistar rats were randomly divided into three groups (n=36 each): sham-operated (group S), hindlimb ischemia-reperfusion (group IR), and ischemic postconditioning (group IPO). Each group was divided into subgroups (n=6) according to reperfusion time: immediate (0 h, T0), 1 h (T1), 3 h (T3), 6 h (T6), 12 h (T12), and 24 h (T24). In the IPO group, three cycles of 30-s reperfusion and 30-s femoral aortic reocclusion were carried out before reperfusion. At all reperfusion times (T0-T24), serum creatine kinase (CK) and lactate dehydrogenase (LDH) activities, as well as interleukin (IL)-6, IL-10, and tumor necrosis factor-α (TNF-α) concentrations, were measured in rats after they were killed. Histological and immunohistochemical methods were used to assess the skeletal muscle damage and HIF-1α expression in skeletal muscle ischemia. In groups IR and IPO, serum LDH and CK activities and TNF-α, IL-6, and IL-10 concentrations were all significantly increased compared to group S, and HIF-1α expression was up-regulated (P<0.05 or P<0.01). In group IPO, serum LDH and CK activities and TNF-α and IL-6 concentrations were significantly decreased, IL-10 concentration was increased, HlF-1α expression was down-regulated (P<0.05 or P<0.01), and the pathological changes were reduced compared to group IR. The present study suggests that ischemic postconditioning can reduce skeletal muscle damage caused by limb ischemia-reperfusion and that its mechanisms may be related to the involvement of HlF-1α in the limb ischemia-reperfusion injury-triggered inflammatory response.

Rutin ameliorates glycemic index, lipid profile and enzymatic activities in serum, heart and liver tissues of rats fed with a combination of hypercaloric diet and chronic ethanol consumption.

Alcoholism and obesity are strongly associated with several disorders including heart and liver diseases. This study evaluated the effects of rutin treatment in serum, heart and liver tissues of rats subjected to a combination of hypercaloric diet (HD) and chronic ethanol consumption. Rats were divided into three groups: Control: rats fed a standard diet and drinking water ad libitum; G1: rats fed the HD and receiving a solution of 10% (v/v) ethanol; and G2: rats fed the HD and ethanol solution, followed by injections of 50 mg/kg-1 rutin as treatment. After 53 days of HD and ethanol exposure, the rutin was administered every three days for nine days. At the end of the experimental period (95 days), biochemical analyses were carried out on sera, cardiac and hepatic tissues. Body weight gain and food consumption were reduced in both the G1 and G2 groups compared to control animals. Rutin effectively reduced the total lipids (TL), triglycerides (TG), total cholesterol (TC), VLDL, LDL-cholesterol and glucose levels, while it increased the HDL-cholesterol in the serum of G2 rats, compared to G1. Although rutin had no effect on total protein, albumin, uric acid and cretinine levels, it was able to restore serum activities of alkaline phosphatase (ALP), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and creatine kinase (CK) in animals fed HD and receiving ethanol. Glycogen stores were replenished in both hepatic and cardiac tissues after rutin treatment. Moreover, rutin consistently reduced hepatic levels of TG and TC and cardiac AST, ALT and CK activities. Thus, rutin treatment was effective in reducing the risk factors for cardiac and hepatic disease caused by both HD and chronic ethanol consumption.

Factors predictive of the failure of medical treatment in patients with pleural infection

BACKGROUND/AIMS: The clinical outcomes of some patients with pleural infection may be favorable with medical treatment alone, but in others, the disease progresses and requires additional surgical treatment. However, little is known about the factors affecting this difference. The aim of this study was to investigate the factors predictive of failure of medical treatment in patients with pleural infection. METHODS: A cohort of 127 consecutive patients who were admitted to the hospital with pleural infection was studied. Clinical manifestations and laboratory findings in patients in whom medical treatment succeeded or failed were reviewed. RESULTS: In univariate analysis, the significant factors associated with medical treatment outcome were age, smoking history, duration of chief complaint, serum albumin level, and pleural fluid glucose and lactate dehydrogenase levels (p < 0.05). Multivariate logistic regression analysis identified age and duration of chief complaint as independent predictive factors for failure of medical treatment, with odds ratios of 0.871 (p = 0.013) and 0.797 (p = 0.026), respectively. Receiver operating characteristic curve analysis determined cutoff values of 50.5 years for age and 4.5 days for duration of chief complaint. CONCLUSIONS: We demonstrated that a younger age < 50.5 years and shorter duration of chief complaint < 4.5 days were independent predictive factors for the failure of medical treatment in patients with pleural infection. This suggests their role as evaluative criteria in setting indications for the optimal treatment in patients with pleural infection. A larger, prospective study is required to confirm these findings.

Intravenous human umbilical cord blood improves elctrophysiological and metabolic properties in ISO induced myocardial necrosis in rats.

Rats treated with isoproterenol (ISO, 85 mg/kg, sc, twice at an interval of 24 h) showed a significant increase in heart rate, mean arterial blood pressure, pressure rate index, ST elevation on ECG, and a significant increase in the levels of cardiac marker enzymes- lactate dehydrogenase, and creatine kinase in serum and a significant reduction in superoxide dismutase, and catalase and increase in thiobarbituric acid reactive substance activity in heart tissue. Treatment with Human umbilical cord blood (hUCBC; 500 and 1000 µL, iv, via the tail vein; 2 h after the second dose of ISO) significantly restored back to normal levels and showed a lesser degree of cellular infiltration and infarct size in histopathological and planimetry studies respectively. Thus, hUCBC ameliorates cardiotoxic effects of isoproterenol and may be of value in the treatment of myocardial infarction.

Combined pleural fluid cholesterol and total protein in differentiation of exudates and transudates.

Background. The management strategy to be adopted in pleural effusion depends on whether an effusion is a transudate or exudate. Objective. To evaluate the usefulness of pleural fluid cholesterol and/or total protein measurements for differentiating between exudates and transudates, and to compare it with Light’s criteria. Methods. In this prospective study 60 patients with pleural effusion were included. Pleural fluid total protein, lactate dehydrogenase (LDH) and cholesterol as well as serum total protein and LDH levels along with other investigations were studied. Clinical classification of transudate or exudate was done on the basis of aetiology. Results. Based on clinical signs and symptoms, chest radiograph, other investigations and response to treatment, 49 of these effusions were classified as exudates and 11 as transudates. Using pleural fluid cholesterol levels at a cut-off point of greater than 60 mg/dL and/or total protein at a cut-off point of greater than 3 g/dL for distinguishing transudates and exudates, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV), were 100 percent. Using Light’s criteria for discriminating transudates and exudates, sensitivity, specificity, PPV and NPV were found to be 98%; 100%; 100% and 92%, respectively. The differences resulted from a mis-classification of one expected exudate as transudate by Light’s criteria. Conclusion. Pleural fluid cholesterol and total protein are simple, cost-effective, and useful parameters in distinguishing pleural transudates from exudates, with the advantage of requiring only two laboratory determinations and no simultaneous blood sample, compared to the use of Light’s criteria.

Interleukin-10 Down-Regulates Cathepsin B Expression in Fetal Rat Alveolar Type II Cells Exposed to Hyperoxia

PURPOSE: Hyperoxia has the chief biological effect of cell death. We have previously reported that cathepsin B (CB) is related to fetal alveolar type II cell (FATIIC) death and pretreatment of recombinant IL-10 (rIL-10) attenuates type II cell death during 65%-hyperoixa. In this study, we investigated what kinds of changes of CB expression are induced in FATIICs at different concentrations of hyperoxia (65%- and 85%-hyperoxia) and whether pretreatment with rIL-10 reduces the expression of CB in FATIICs during hyperoxia. MATERIALS AND METHODS: Isolated embryonic day 19 fetal rat alveolar type II cells were cultured and exposed to 65%- and 85%-hyperoxia for 12 h and 24 h. Cells in room air were used as controls. Cytotoxicity was assessed by lactate dehydrogenase (LDH) released into the supernatant. Expression of CB was analyzed by fluorescence-based assay upon cell lysis and western blotting, and LDH-release was re-analyzed after preincubation of cathepsin B-inhibitor (CBI). IL-10 production was analyzed by ELISA, and LDH-release was re-assessed after preincubation with rIL-10 and CB expression was re-analyzed by western blotting and real-time PCR. RESULTS: LDH-release and CB expression in FATIICs were enhanced significantly in an oxygen-concentration-dependent manner during hyperoxia, whereas caspase-3 was not activated. Preincubation of FATIICs with CBI significantly reduced LDH-release during hyperoxia. IL-10-release decreased in an oxygen-concentration-dependent fashion, and preincubation of the cells with rIL-10 significantly reduced cellular necrosis and expression of CB in FATIICs which were exposed to 65%- and 85%-hyperoxia. CONCLUSION: Our study suggests that CB is enhanced in an oxygen-concentration-dependent manner, and IL-10 has an inhibitory effect on CB expression in FATIICs during hyperoxia.

Mechanisms involved in the cytotoxic effects of berberine on human colon cancer HCT-8 cells

Berberine, a constituent of some traditional Chinese medicinal plants, has been reported to have cytotoxicity effects on different human cancer cell lines. There is no available information about the effects and mechanism of action of berberine on human colon cancer cell line HCT-8. In this paper, the cytotoxicity of berberine on HCT-8 cancer cells was investigated by MTT assay, fluorescence microscopy and flow cytometry analysis. Our data revealed that berberine could significantly inhibit the growth of HCT-8 cells in a dose- and time-dependent manner. Morphology of apoptotic cells was studied with acridine orange/ethidium bromide staining. The concentrations of lactate dehydrogenase and both acid and alkaline phosphatases were significantly increased in cell supernatants after berberine treatment, suggesting cell death. Furthermore, flow cytometry analysis showed that berberine could arrest HCT-8 cells at S phase in a time-dependent manner. To further investigate the apoptotic molecular mechanism, reverse transcription-polymerase chain reaction (RT-PCR) and western blotting methods were used. The up-regulated mRNA and/or protein expressions of Fas, FasL, TNF-a, caspase-3 and down-regulation of pro-caspase-3 suggest that the death receptor pathway may be involved in the apoptotic pathway induced by berberine. Decrease of Bcl-2 and increase of Bax in mRNA and/or protein expressions showed that the Bcl-2 family proteins were involved in berberine-induced apoptosis. We also found that berberine-induced apoptosis was associated with an up-regulated expressions of p53 and prohibitin (PHB), and decreased vimentin expression. These results suggest that berberine can suppress cell growth and reduce cell survival by arresting the cell-cycle and by inducing apoptosis of HCT-8 cells.

Simultaneous allergen inactivation and detoxification of castor bean cake by treatment with calcium compounds

Ricinus communis L. is of great economic importance due to the oil extracted from its seeds. Castor oil has been used for pharmaceutical and industrial applications, as a lubricant or coating agent, as a component of plastic products, as a fungicide or in the synthesis of biodiesel fuels. After oil extraction, a castor cake with a large amount of protein is obtained. However, this by-product cannot be used as animal feed due to the presence of toxic (ricin) and allergenic (2S albumin) proteins. Here, we propose two processes for detoxification and allergen inactivation of the castor cake. In addition, we establish a biological test to detect ricin and validate these detoxification processes. In this test, Vero cells were treated with ricin, and cell death was assessed by cell counting and measurement of lactate dehydrogenase activity. The limit of detection of the Vero cell assay was 10 ng/mL using a concentration of 1.6 x 10(5) cells/well. Solid-state fermentation (SSF) and treatment with calcium compounds were used as cake detoxification processes. For SSF, Aspergillus niger was grown using a castor cake as a substrate, and this cake was analyzed after 24, 48, 72, and 96 h of SSF. Ricin was eliminated after 24 h of SSF treatment. The cake was treated with 4 or 8% Ca(OH)2 or CaO, and both the toxicity and the allergenic properties were entirely abolished. A by-product free of toxicity and allergens was obtained.

Presence of bound substrate in lactate dehydrogenase from carp liver.

While attempting to purify UDP-galactose 4-epimerase from carp liver extract at pH 8.0, it was observed that the preparation even after dialysis could reduce NAD to NADH, interfering epimerase assay. The NAD reduction activity and the epimerase were co-eluted in a series of chromatographic steps. Mass spectrometric analysis of semi-purified fraction revealed that carp liver lactate dehydrogenase (LDH) contained bound lactate which was converted to pyruvate in the presence of NAD. The enzyme-bound lactate and the association with epimerase stabilized LDH from trypsin digestion and thermal inactivation at 45°C by factors of 2.7 and 4.2 respectively, as compared to substrate-free LDH. LDH and epimerase do not belong to any one pathway, but are the rate-limiting enzymes of two different pathways of carbohydrate metabolism. Typically, strongly associated enzymes work in combination, such as two enzymes of the same metabolic pathway. In that background, co-purification of LDH and epimerase as reloaded in this study was an unusual phenomenon.

Effect of Inula racemosa root extract on cardiac function and oxidative stress against isoproterenol-induced myocardial infarction.

The cardioprotective potential of Inula racemosa root hydroalcoholic extract against isoproterenol-induced myocardial infarction was investigated in rats. The rats treated with isoproterenol (85 mg/kg, s.c.) exhibited myocardial infarction, as evidenced by significant (P<0.05) decrease in mean arterial pressure, heart rate, contractility, relaxation along with increased left ventricular end diastolic pressure, as well as decreased endogenous myocardial enzymatic and non-enzymatic antioxidants. Isoproterenol also significantly (P<0.05) induced lipid peroxidation and increased leakage of myocyte injury marker enzymes. Pretreatment with I. racemosa extract (50, 100 or 200 mg/kg per day, p.o.) for 21 consecutive days, followed by isoproterenol injections on days 19th and 20th significantly (P<0.05) improved cardiac function by increasing the heart rate, mean arterial pressure, contractility and relaxation along with decreasing left ventricular end diastolic pressure. Pretreatment with I. racemosa also significantly (P<0.05) restored the reduced form of glutathione and endogenous antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase from the heart, which were depleted after isoproterenol administration. In addition to restoration of antioxidants, I. racemosa significantly (P<0.05) inhibited lipid peroxidation and prevented the leakage of myocytes specific marker enzymes creatine phosphokinase-MB and lactate dehydrogenase from the heart. Thus, it is concluded that I. racemosa protects heart from isoproterenol-induced myocardial injury by reducing oxidative stress and modulating hemodynamic and ventricular functions of the heart. Present study findings demonstrate the cardioprotective effect of I. racemosa and support the pharmacological relevance of its use and cardioprotection mechanism in ischemic heart disease as well as substantiate its traditional claim

Effect and mechanism of intermedin in acute rat cardiac ischemic injury / 法医学杂志

OBJECTIVE@#To investigate the effect and potential mechanism of intermedin (IMD) in acute cardiac ischemic injury and to provide a new approach for exploring mechanism of sudden cardiac death.@*METHODS@#Seventy-two healthy male rats were randomly divided into 3 groups: control, ischemic and the IMD-treated group. The activity of lactate dehydrogenase (LDH), malondialdehyde (MDA) and superoxide dismutase (SOD) in heart blood were tested by enzyme chemistry method. The mRNA changes of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMPs) in cardiac were measured by real-time PCR analysis. Myocardial cyclic adenosine monophosphate (cAMP) content was determined by enzyme linked immunosorbent assay (ELISA). Apoptosis related factors Bcl-2 and Bax were detected by immunohistochemistry.@*RESULTS@#Comparing with the control group, LDH and MDA activity of ischemic group in heart blood increased and SOD activity decreased. The concentration of cAMP increased in ventricular muscle, Bcl-2 and Bax proteins expression ratio level decreased. The intravenation of IMD decreased the level of increased activity of LDH and MDA, and lessened the level of decreased activity of SOD. The mRNA expression of CRLR and RAMPs obviously increased in ventricular muscle.@*CONCLUSION@#The protective effect of IMD against myocardial ischemic injury could be caused by decreasing the oxidative stress of ischemia and inhibiting the myocardial apoptosis.

Down-regulation of survivin suppresses uro-plasminogen activator through transcription factor JunB

Survivin, a member of the inhibitors of apoptosis protein family, is expressed during development and in various human cancers. However, the clinical relevance of survivin in cancer is still a matter of debate. Genes induced by hepatocyte growth factor (HGF) were screened using cDNA microarray technology in the stomach cancer cell lines, NUGC3 and MKN28. The levels of JunB, survivin, and uro-plasminogen activator (uPA) were up-regulated in cells treated with HGF in a dose-dependent manner. HGF-induced up regulation of JunB, survivin, and uPA was inhibited by pre-treatment with a MEK inhibitor (PD 98059). HGF-induced up-regulation of uPA was repressed by survivin knockdown. HGF enhanced the binding activity of JunB to the survivin promoter in control cells, but not in the JunB-shRNA cells. Transfection with survivin-shRNA resulted in a decrement of cell proliferation, as determined with MTT assays. In an in vitro invasion assay, significantly fewer cells transfected with survivin shRNA than control cells were able to invade across a Matrigel membrane barrier. In conclusion, survivin appeared to play an important role in the up-regulation of uPA induced by HGF via JunB and might contribute to HGF-mediated tumor invasion and metastasis, which may serve as a promising target for gastric cancer therapy.

The analysis of pericardial fluid in forensic practice / 法医学杂志

Pericardial fluid is a kind of serous fluid in pericardial cavity. Because blood undergoes postmortem changes such as autolysis and putrefaction, vitreous humor is limited,cerebrospinal fluid is easily mixed with blood, pericardial fluid, on the other hand, exists in a closed cavity and can be hardly contaminated by postmortem changes, and also is easily obtained. Pericardial fluid not only plays an important role in clinic practice, but also is widely applicable in forensic practice. This paper briefly presented the properties of pericardial fluid and its clinical significance. It reviewed biochemical changes in decedents died of heart diseases, drowning and asphyxia, and explored the significance in medico-legal investigation. Moreover, application of pericardial fluid in forensic serology, forensic toxicological analysis and other fields were also discussed. Pericardial fluid analysis may provide important information for determination of the cause of death with further investigation concerning forensic applicability of pericardial fluid.