Effect of Bushen Huoxuetang on Expression of Bax/Bcl-2 and Cleaved Caspase-3 in Nude Mice with Bone Metastasis of Breast Cancer / 中国实验方剂学杂志

ObjectiveTo study the effect of Bushen Huoxuetang on the apoptosis and the expression of B-cell lymphoma （Bcl-2）-associated X protein （Bax）/ Bcl-2 and cleaved cysteine-containing aspartate proteolytic enzyme-3 （cleaved Caspase-3） in the nude mouse model of bone metastasis of breast cancer， and explore the mechanism of Bushen Huoxuetang in inhibiting bone destruction. MethodThirty BALB/c female nude mice were randomly assigned into blank group （n=6） and model group （n=24）. The suspension of 4T1 breast cancer cells was injected into the tibia of mouse right lower limb to establish model of bone metastasis of breast cancer. The successfully modeled nude mice were randomly assigned into model group， Bushen Huoxuetang group， zoledronic acid group， and combined drug group， with 6 mice in each group. Bushen Huoxuetang was administrated at a dose of 36.67 g·kg-1， once a day， and zoledronic acid was administrated by subcutaneous injection at a dose of 100 μg·kg-1， twice a week. The combined drug group was administrated with the same doses of Bushen Huoxuetang group by gavage and zoledronic acid by subcutaneous injection. The mice in the blank group and the model group were administrated with the same volume of distilled water by gavage for 14 days. On the next day at the end of drug administration， the mice were sacrificed by cervical dislocation. The general situation and weight changes of the mice were examined. The right lower limb was collected， and X-ray scanning and hematoxylin-eosin （HE） staining methods were used for observation of pathological changes in the bone. The terminal deoxynucleotidyl transferase dUTP nick-end labeling （TUNEL） was employed to detect the apoptosis of bone tissue in nude mice， and Western blot to determine the expression of Bax/Bcl-2 and cleaved Caspase-3 in the bone tissue. ResultCompared with the blank group， the modeling reduced the body weight （P<0.01） and increased the right lower limb weight of the nude mice （P<0.01）. Compared with the model group， Bushen Huoxuetang， zoledronic acid， and their combination increased the body weight （P<0.01） and decreased the right lower limb weight （P<0.01）. Compared with the blank group， the other groups showed obvious tumor cell atypia， deep nuclear staining， and clear bone metastasis， and the model group showed obvious osteolytic damage in right lower limb and loss of proximal tibia and knee joint. Compared with the model group， Bushen Huoxuetang， zoledronic acid， and their combination reduced the osteolytic lesions in the right lower limb and recovered part of the bone structure， demonstrating an inhibitory effect on bone destruction. The TUNEL assay showed that the model group had lower apoptosis rate of bone metastatic tumor cells than the blank group， Bushen Huoxuetang group， zoledronic acid group， and combined drug group （P<0.01）. Compared with the blank group， the modeling down-regulated the expression of Bax and cleaved Caspase-3 （P<0.01） and up-regulated the expression of Bcl-2 （P<0.01）. Compared with the model group， Bushen Huoxuetang， zoledronic acid， and their combination up-regulated the expression of Bax （P<0.01） and cleaved Caspase-3 （P<0.05， P<0.01） and down-regulated the expression of Bcl-2 （P<0.05， P<0.01）. ConclusionBushen Huoxuetang may inhibit bone destruction in the nude mouse model of bone metastasis of breast cancer by up-regulating the expression of Bax， down-regulating the expression of Bcl-2， activating cleaved Caspase-3， and further inducing apoptosis.

Composition and microbiological quality of refrigerated raw milk in two seasons of the year in Santa Helena, Western Paraná / Composição e qualidade microbiológica do leite cru refrigerado em duas estações do ano em Santa Helena, oeste do Paraná / Composición y calidad microbiológica de la leche cruda refrigerada en dos estaciones del año en Santa Helena, oeste de Paraná

This study aimed at quantifying the occurrence of proteolytic psychotropic microorganisms and total coliforms in refrigerated raw milk, in addition to assessing the quality through its physical-chemical composition. The samples were collected in 10 dairy farms in the municipality of Santa Helena ­ Western Paraná. Two collections were made, one during the spring and the other during the summer. Samples of refrigerated raw milk were carried out directly from the cooling tank, aseptically, packed in sterile bottles and transported under isothermal conditions (± 4 ºC) to the laboratory where the physical-chemical composition and microbial populations were determined. The levels of fat had great amplitude between the seasons, being higher in the summer, while there was no variation for the contents of protein, lactose, minerals, and non-fat solids. The total coliform count did not differ between seasons. The values obtained for proteolytic psychotropic counts were higher in the summer. A significant positive correlation was found between the total coliform counts, the proteolytic psychotropic counts (r=0.73), the levels of protein (r=0.45), non-fat solids (r=0.45), and minerals (r=0.46). Also, the proteolytic psychotropic counts showed a positive correlation with the cryoscopic index. The milk components met the requirements of NI76. The quality of refrigerated raw milk in the municipality of Santa Helena, Western Paraná was not satisfactory for total coliforms, due to its high incidence, indicating the need for good practices in milking management. Proteolytic psychotropic bacteria presented low proliferation, thus not affecting milk quality.(AU)

O presente estudo teve como objetivo quantificar a ocorrência de microrganismos psicrotróficos proteolíticos e de coliformes totais em leite cru refrigerado além de avaliar a qualidade por meio da sua composição físico-química. As amostras foram coletadas em 10 propriedades leiteiras no município de Santa Helena ­ Oeste do Paraná. Foram realizadas duas coletas, uma durante a primavera e outra no verão. As amostragens do leite cru refrigerado foram realizadas diretamente no tanque de resfriamento, de forma asséptica, acondicionado em frascos esterilizados e transportado sob condições isotérmicas (± 4ºC) ao laboratório onde foram determinadas a composição físico-química e as populações microbianas. Os teores de gordura tiveram grande amplitude entre as estações do ano, sendo superior no verão, enquanto que não houve variação para os teores de proteína, lactose, minerais e sólidos desengordurados. A contagem de coliformes totais não diferiu entre as estações. Os valores obtidos para contagens de psicrotróficos proteolíticos foram superiores no verão. Foi constatada correlação significativa positiva entre as contagens de coliformes totais com as contagens de psicrotróficos proteolíticos (r=0,73) e os teores de proteína (r=0,45), sólidos desengordurados (r=0,45) e minerais (r=0,46). Além disto, as contagens de psicrotróficos proteolíticos apresentaram correlação positiva com o índice crioscópico. Os componentes do leite atenderam as exigências da IN76. A qualidade do leite cru refrigerado no município de Santa Helena, Oeste do Paraná não foi satisfatória em relação aos coliformes totais, devido sua alta incidência, indicando a necessidade de boas práticas no manejo de ordenha. As bactérias psicrotróficas proteolíticas tiveram baixa proliferação, não afetando a qualidade do leite.(AU)

El presente estudio tuvo como objetivo cuantificar la ocurrencia de microorganismos psicrotróficos proteolíticos y de coliformes totales en leche cruda refrigerada, además de evaluar la calidad a través de su composición fisicoquímica. Las muestras fueron recolectadas en 10 granjas lecheras del municipio de Santa Helena ­ Oeste de Paraná. Se realizaron dos colecciones, una en primavera y otra en verano. Las muestras de leche cruda enfriada se realizaron directamente en el tanque de enfriamiento, de forma aséptica, se empacaron en botellas estériles y se transportaron en condiciones isotérmicas (± 4ºC) al laboratorio donde se determinó la composición fisicoquímica y las poblaciones microbianas. Los niveles de grasa tuvieron gran amplitud entre las estaciones, siendo más altos en verano, mientras que no hubo variación para los contenidos de proteínas, lactosa, minerales y sólidos desgrasados. El recuento total de coliformes no difirió entre temporadas. Los valores obtenidos para los recuentos de psicotrópicos proteolíticos fueron mayores en verano. Se encontró una correlación positiva significativa entre los recuentos de coliformes totales y los recuentos de psicrótroficos proteolíticos (r=0.73) y los niveles de proteínas (r=0.45), sólidos desgrasados (r=0.45) y minerales (r=0.46). Además, los recuentos de psicrótroficos proteolíticos mostraron una correlación positiva con el índice crioscópico. Los componentes de la leche cumplieron con los requisitos de IN76. La calidad de la leche cruda refrigerada en el municipio de Santa Helena, Oeste de Paraná no fue satisfactoria en relación a los coliformes totales, debido a su alta incidencia, lo que indica la necesidad de buenas prácticas en el manejo del ordeño. Las bacterias psicrotróficas proteolíticas tuvieron baja proliferación, no afectando la calidad de la leche.(AU)

Asociación de polimorfismos en los genes CAPN y CAST con propiedades fisicoquímicas de la carne bovina: una revisión / Association of polymorphisms in CAPN and CAST genes with physicochemical properties of beef: a review / Associação de polimorfismos nos genes CAPN e CAST com propriedades físico-químicas da carne bovina: uma revisão

Resumen Las propiedades fisicoquímicas como terneza, color, jugosidad, sabor, marmoleo, pH y capacidad de retención de agua, son consideradas como los atributos que tienen mayor importancia en la calidad de la carne. El manejo post mortem influye en el funcionamiento del sistema proteolítico de las enzimas µ-calpaínas y calpastatinas, que están codificadas por los genes CAPN1 y CAST, respectivamente. Esta revisión muestra las asociaciones de los polimorfismos CAPN1 y CAST con el fin de explicar las propiedades fisicoquímicas antes mencionadas, que determinan las características relacionadas con la calidad de la carne. Existen polimorfismos de CAPN1 y CAST asociados a propiedades fisicoquímicas particulares de la carne, pero no todos los polimorfismos se asocian con las propiedades mencionadas. La actividad del sistema proteolítico de las enzimas varía según el manejo peri y post mortem. La presencia o ausencia de un polimorfismo varía entre razas. Por consiguiente, las asociaciones de polimorfismos de nucleótido simple con las propiedades fisicoquímicas de la carne mencionadas en esta revisión podrían ayudar a mejorar los indicadores de calidad de la carne bovina.

Abstract Physicochemical properties such as tenderness, color, juiciness, flavor, marbling, pH and water retention capacity, are considered the most important attributes in the meat quality. Posmortem management influences the functioning of the proteolytic system of the enzymes calpains and calpastatins, which are encoded by CAPN1 and CAST genes, respectively. This review displays the associations of CAPN1 and CAST polymorphisms in order to explain the aforementioned physicochemical properties, which determine the characteristics relate to the beef quality. There are CAPN1 and CAST polymorphisms associated with particular physicochemical properties of meat, but not all polymorphisms are associated with the mentioned properties. The activity of enzymes proteolytic system varies according to peri and posmortem management. The presence or absence of a polymorphism varies between races. Therefore, the associations of single nucleotide polymorphisms with the physicochemical properties of meat mentioned in this review could help to improve the quality indicators of beef.

Resumo Propriedades físico-químicas como maciez, cor, suculência, sabor, marmorização, pH e capacidade de retenção da água, são consideradas os atributos mais importantes na qualidade da carne. O manejo pós-morte influencia o funcionamento do sistema proteolítico das enzimas calpaína e calpastatina, que são codificadas pelos genes CAPN1 e CAST, respectivamente. Esta revisão mostra as associações dos polimorfismos CAPN1 e CAST para explicar as propriedades físico-químicas citadas, que determinam as características relacionadas à qualidade da carne. Existem polimorfismos CAPN1 e CAST associados a propriedades físico-químicas específicas da carne, mas nem todos os polimorfismos estão associados às propriedades mencionadas. A atividade das enzimas do sistema proteolítico varia de acordo com o manejo peri e pos-mortem. A presença ou ausência de um polimorfismo varia entre as raças. Portanto, as associações de polimorfismos de nucleotídeo único com as propriedades físico-químicas da carne mencionadas nesta revisão poderiam ajudar a melhorar os indicadores de qualidade da carne bovina.

Jinxiangdan Inhibits NLRP3/IL-1β/Caspase-1 Pathway to Alleviate Myocardial Ischemia-reperfusion Injury in Rats / 中国实验方剂学杂志

Objective:To observe the effect of Jinxiangdan （JXD） on NOD-like receptor pyrin domain-containing-3 （NLRP3）/cysteine-dependent aspartate-directed protease-1 （Caspase-1）/interleukin-1<italic>β</italic> （IL-1<italic>β</italic>） signaling pathway in myocardium of rats with myocardial ischemia-reperfusion injury （MIRI） and explore the protective effect and mechanism of JXD against MIRI. Method:Fifty male SD rats were randomly divided into the sham operation group， model group， high- and low-dose JXD groups， and positive drug （Di'ao Xinxuekang） group， with 10 rats in each group. Seven days before modeling， rats in the JXD groups were separately treated with intragastric administration of 0.72 and 0.18 g·kg^-1 JXD tablets， the ones in the sham operation group and model group with the same volume of normal saline， and those in the positive drug group with 1.29 g·kg^-1 Di'ao Xinxuekang. Twelve hours after the last intragastric administration， the anterior descending branch of the left coronary artery was ligated for 30 min and then re-perfused for 60 min for inducing MIRI. ST segment elevation was detected by electrocardiogram（ECG） for model evaluation. The contents of creatine kinase （CK） and lactate dehydrogenase （LDH） in cardiac tissue were measured by colorimetry. Hematoxylin-eosin （HE） staining was conducted for observing myocardial histopathological changes， followed by the detection of cardiomyocyte apoptosis by DNA in situ nick end-labeling （TUNEL） assay. The protein and mRNA expression levels of NLRP3， Caspase-1， and IL-1<italic>β</italic> were detected by Western blot and real-time polymerase chain reaction （Real-time PCR）， respectively. Result:Compared with sham operation group， the model group exhibited obviously elevated ST segment （<italic>P</italic><0.01）， enhanced CK and LDH activities in the myocardium （<italic>P</italic><0.01）， increased apoptotic cardiomyocytes （<italic>P</italic><0.01）， and up-regulated NLRP3， Caspase-1， and IL-1<italic>β</italic> protein and mRNA expression （<italic>P</italic><0.01）. Compared with model group， JXD at both the high and low doses and Di'ao Xinxuekang significantly lowered the ST segment （<italic>P</italic><0.05，<italic>P</italic><0.01）， diminished the CK and LDH activities in myocardial tissue （<italic>P</italic><0.05，<italic>P</italic><0.01）， improved the apoptosis of cardiomyocytes （<italic>P</italic><0.05，<italic>P</italic><0.01）， and down-regulated the mRNA and protein expression levels of NLRP3， Caspase-1， and IL-1<italic>β</italic> in myocardial tissue （<italic>P</italic><0.05，<italic>P</italic><0.01）. The ST segment of ECG in the low-dose JXD group was increased as compared with that in the Di'ao Xinxuekang group （<italic>P</italic><0.05）， while the ST segment in the high-dose JXD group was obviously elevated （<italic>P</italic><0.05）. Besides， the green fluorescence intensities in the low- and high-dose JXD groups and the Di'ao Xinxuekang group remarkably declined （<italic>P</italic><0.05，<italic>P</italic><0.01）. The mRNA and protein expression levels of NLRP3， Caspase-1， and IL-1<italic>β</italic> in the high-dose JXD group were down-regulated （<italic>P</italic><0.05）. Conclusion:JXD alleviates MIRI possibly by lowering NLRP3 and IL-1<italic>β</italic> expression and inhibiting cardiomyocyte apoptosis.

POST1/C12ORF49 regulates the SREBP pathway by promoting site-1 protease maturation

Sterol-regulatory element binding proteins (SREBPs) are the key transcriptional regulators of lipid metabolism. The activation of SREBP requires translocation of the SREBP precursor from the endoplasmic reticulum to the Golgi, where it is sequentially cleaved by site-1 protease (S1P) and site-2 protease and releases a nuclear form to modulate gene expression. To search for new genes regulating cholesterol metabolism, we perform a genome-wide CRISPR/Cas9 knockout screen and find that partner of site-1 protease (POST1), encoded by C12ORF49, is critically involved in the SREBP signaling. Ablation of POST1 decreases the generation of nuclear SREBP and reduces the expression of SREBP target genes. POST1 binds S1P, which is synthesized as an inactive protease (form A) and becomes fully mature via a two-step autocatalytic process involving forms B'/B and C'/C. POST1 promotes the generation of the functional S1P-C'/C from S1P-B'/B (canonical cleavage) and, notably, from S1P-A directly (non-canonical cleavage) as well. This POST1-mediated S1P activation is also essential for the cleavages of other S1P substrates including ATF6, CREB3 family members and the α/β-subunit precursor of N-acetylglucosamine-1-phosphotransferase. Together, we demonstrate that POST1 is a cofactor controlling S1P maturation and plays important roles in lipid homeostasis, unfolded protein response, lipoprotein metabolism and lysosome biogenesis.

Milk-clotting proteases from Pleurotus albidus: an innovative alternative for the production of Minas frescal cheese

Pleurotus albidus, a naturally growing species in the Amazon region, has been considered a promising source of milk-clotting proteases. The production of such enzymes using lignocellulosic residues is a sustainable alternative to replace mammalian rennet. The application of P. albidus milk-clotting proteases in cheese making has not yet been reported in the scientific literature. The aim of this study was to characterize the milk-clotting proteases of P. albidus and use these enzymes in the production of Minas frescal cheese. For the production of coagulating proteases, the mushroom was grown in açaí seeds supplemented with rice bran (10%, w/w). The parameters affecting the production of coagulant, such as inoculum size, fermentation time, initial pH of cultivation medium and age of the inoculum were evaluated. The coagulant extract obtained under optimal production conditions was evaluated for optimal pH and temperature, pH and temperature stability, effect of ions and inhibitors. Significant production of coagulating proteases was obtained under the following conditions: inoculum size (2.5%), fermentation time (10 days), initial pH of the cultivation medium (6), and inoculum age (10 days). The coagulant exhibited significant catalytic activity in pH 5.0 at 55°C, with stability at 45°C and was completely inhibited by iodoacetic acid. The milk-clotting proteases of P. albidus were efficient for making Minas frescal cheese that presented 55.0% of moisture, 20.0% of lipids and 17.20% of protein. Pleurotus albidus is a potential source of milk-clotting proteases that can be applied in dairy industry for production of fresh Minas frescal cheese.

Clinical Efficacy of Modified Qingjin Huatan Tang on Bronchiectasis with Syndrome of Phlegm-heat Accumulating Lung at Acute Exacerbation / 中国实验方剂学杂志

<b>Objective::To observe the clinical efficacy of modified Qingjin Huatan Tang on bronchiectasis with syndrome of phlegm-heat accumulating lung at acute exacerbation and its inhibitory effect on pro-inflammatory factors and proteolytic activity. <b>Method::One hundred and thirty patients were randomly divided into control group and observation group by random number table. Patients in control group got tazobactam sodium and piperacillin sodium for injection, 3.375 g/time, 1 time/6 hours, and the types of antibiotics were regulated according to the bacterial culture results. And patients in control group also got Ambroxol Hydrochloride injection, 30 mg/time, 2 time/days, and postural drainage. In addition to the therapy of control group, patients in observation group were also given modified Qingjin Huatan Tang, 1 dose/day. Before and after treatment, symptoms and signs were scored. And levels of white blood cell count (WBC), neutrophile granulocyte (GRAN), C-reactive protein (CRP), procalcitonin (PCT) were detected. And scores of forced expiratory volume in one second (FEV₁), forced vital capacity (FVC), peak expiratory flow rate (PEFR) and BODE were graded. And levels of tumor necrosis factor-<italic>α</italic> (TNF-<italic>α</italic>), interleukin-4 (IL-4), IL-6 and IL-8 in sputum, peripheral neutrophil elastase (NE) and cathepsin G were detected. <b>Result::By rank sum test, the clinical efficacy in observation group was better than that in control group (Z=2.086, <italic>P</italic><0.05), while scores of symptoms and signs in observation group were lower than those in control group (<italic>P</italic><0.01). WBC, GRAN, CRP, PCT, airflow limitation (O), dyspnea (D), motor ability (E) score, BODE index, TNF-<italic>α</italic>, IL-4, IL-6, IL-8, plasma NE and cathepsin G were all lower than those in control group (<italic>P</italic><0.01). And levels of FEV₁, FVC, PEF and FEV₁/FVC were higher than those in control group (<italic>P</italic><0.01). <b>Conclusion::In addition to routine anti-infection and expectoration western medicine therapy, modified Qingjin Huatan Tang can be added to control symptoms and signs, alleviate the degree of illness, improve pulmonary function and the quality of life of patients, and inhibit expression of airway pro-inflammatory factor and proteolysis, with a better clinical efficacy than pure western medicine.

Evaluation of biogenic amines producing potential of bacteria associated with proteinous food

@#Aim: The aim of this study was to assay for the biogenic amine-producing capacity of bacteria isolated from proteinous food. Methodology and results: Previously characterized bacterial isolates (Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae) obtained from proteinous food samples (smoked fish and yoghurt) were subjected to proteolytic analysis using nutrient agar supplemented with 0.2 g/mL casein and decarboxylase activity using nutrient broth supplemented with 0.004 g/mL amino acids (histidine, tyrosine, asparagines, leucine and lysine). Isolates that expressed proteolytic and decarboxylase activities were screened for biogenic amine producing capacity using decarboxylase broth which was supplemented with an amino acid (tyrosine). Biogenic amines obtained in this research were classified into primary amine and secondary amine based on their qualitative characteristics. Confirmatory and quantitative analysis of biogenic amines produced was done using high-performance liquid chromatography. The confirmatory screening revealed the presence of methylamine, ethylamine, putrescine, cadaverine, histamine, spermidine, phernylethylamine, spermine, agmatine, tyramine, dopamine, tryptamine, norepinephrine and serotonin respectively. Total biogenic amines produced by S. aureus was 70.12 mg/kg, K. pneumoniae (62.58 mg/kg) and E. coli (56.57 mg/kg) respectively. Conclusion, significance and impact of study: Enzymatic decarboxylation of free amino acids and other metabolic processes by the test organisms (S. aureus, E. coli and K. pneumoniae) leads to production of biogenic amines which can be used as a quality indicator in food in terms of degree of spoilage, use of non-hygienic raw material and poor manufacturing environment. Thus, effect of biogenic amines obtained in this research would be determined by individual toxicological threshold which can be extremely variable from few mg/kg in sensitive person to several hundred mg/kg in healthy person. The concentrations of each biogenic amine quantified are within the limit but their toxic effects depend on the type of amine, the presence of modulating compounds and the efficiency of an individual’s detoxification mechanism.

Influence of time and storage temperature on raw milk deteriorating microbiota / Influência do tempo e temperatura de estocagem sobre a microbiota deteriorante do leite cru

The quality of raw milk depends on initial microbial contamination and conditions of storage until industry processing. Considering the influence of time and storage temperature on raw milk microbiota, the objective of this work was to quantify and monitor the multiplication of these groups under different conditions. For this purpose, 41 samples of raw milk were collected immediately after milking, stored in the following storage conditions: 25 °C/2 h; 35 °C/2 h; 7 °C/24 h; 7 °C/48 h and 7 °C/60 h and analyses of aerobic mesophilic, psychrotrophic and proteolytic psychrotrophic microorganisms. The milk samples analyzed in the study had an initial mean count of mesophilic aerobes of 5.38 Log CFU/mL at Time Zero. The milk stored at 25 °C/2 h and 35 °C/2 h kept the mesophilic aerobic counts within the limits established by the legislation (5.48 Log CFU/mL), with an increase in counts of psychrotrophic and proteolytic microorganisms. When stored at 7 °C/24 h and 7 °C/48 h, the count of mesophiles exceeded the established parameters. A significant increase in the count of proteolytic psychrotrophs and psychrotrophs was also observed during storage at 7 °C from 24 h. The results of this study indicate that the temperature of 7 °C is not suitable for the milk conservation, since it was not able to control the microbial multiplication. Thus, the results contribute to the change in milk storage temperature proposed by the new Brazilian legislation.(AU)

A qualidade do leite cru depende da contaminação microbiana inicial e das condições de armazenamento até o processamento na indústria. Considerando a influência do tempo e da temperatura de armazenamento na microbiota do leite cru, o objetivo deste trabalho foi quantificar e monitorar a multiplicação desses grupos de microrganismos sob diferentes condições. Para tanto, foram coletadas 41 amostras de leite cru imediatamente após a ordenha, armazenadas nas seguintes condições de armazenamento: 25 °C/2 h; 35 °C/2 h; 7 °C/24 h; 7 °C/48 h e 7 °C/60 h para análise de microrganismos psicrotróficos, aeróbios mesófilos, psicrotróficos e proteolíticos. As amostras de leite analisadas no estudo apresentaram uma contagem média inicial de aeróbios mesófilos de 5.38 Log UFC/mL no Tempo Zero. O leite armazenado a 25 °C/2 h e 35 °C/2 h manteve as contagens aeróbias mesófilas dentro dos limites estabelecidos pela legislação (5,48 Log UFC/mL), com aumento nas contagens de microrganismos psicrotróficos e proteolíticos. Quando armazenado a 7 °C/24 h e 7 °C/48 h a contagem de mesófilos excedeu os parâmetros estabelecidos. Um aumento significativo na contagem de psicrotróficos e psicrotróficos proteolíticos também foi observado durante o armazenamento a 7 °C a partir das 24 h. Os resultados deste estudo indicam que a temperatura de 7 °C não é adequada para a conservação do leite, uma vez que não foi capaz de controlar a multiplicação microbiana. Assim, os resultados contribuem para a mudança na temperatura de armazenamento de leite proposta pela nova legislação brasileira.(AU)

Effect of ions and inhibitors on the catalytic activity and structural stability of S. aureus enolase

Constantly rising energy demands, finite fossil fuel reserves and deteriorating environmental conditions have invokedworldwide interest to explore the sustainable sources of renewable biofuels. Locally adapted photosynthetic oleaginousmicroalgae with rapid growth on variable temperatures could be an ideal way for bioremediating the wastewater (WW) whileproducing the feedstock for biodiesel. To test this notion, an unknown strain was isolated from a sewage fed lake (Neela-Hauz).It was discerned as Chlorella sorokiniana-I using the 16S rDNA and 18S rDNA barcodes. The culture conditions such as pH,illumination, different temperature ranges and growth medium were cohesively optimized prior to the assessment of C.sorokiniana-I’s efficacy to remediate the WW and biodiesel production. The strain has thrived well up to 40C when continuously grown for 15 days. The highest lipid accumulation and biomass productivity were recorded in 100% WW. Fatty acidmethyl ester (FAME) content was observed to be more than twice in WW (47%), compared to control synthetic media, TAP(20%) and BG11 (10%), which indicate the importance of this new isolate for producing economically viable biodiesel.Moreover, it is highly efficient in removing the total nitrogen (77%), total phosphorous (81%), iron (67%) and calcium (42%)from the WW. The quality of WW was considerably improved by reducing the overall chemical oxygen demand (48%),biological oxygen demand (47%) and alkalinity (15%). Thus, C. sorokiniana-I could be an ideal alga for the tropical countriesin the remediation of WW while producing feedstock for biodiesel in a cost-effective manner.

Combating protein energy wasting in end stage kidney disease: role of exogenous proteolytic enzyme as an adjuvant to dietary protein

Background: Despite interventions, the prevalence of protein energy wasting in patients on dialysis continues to be unacceptably high. The objective of the study is to evaluate the efficacy of exogenous proteolytic enzyme Aminace 70000 (Hemoglobin Tyrosine Unit), as an adjuvant to dietary protein in improving the nutritional status.Methods: This is a retrospective, real world, single centre, observational study, aimed at assessing the changes in key nutritional indices, over 6 months in patients with chronic kidney disease (CKD) initiated on continuous ambulatory peritoneal dialysis (CAPD). The intervention included addition of egg protein and use of an exogenous proteolytic enzyme. Three cohorts were identified. Cohort 1, had access to a nephrologist and CAPD counsellor; Cohort 2, in addition had access to a dietician who emphasized the need for increase in dietary protein in form of 4-6 eggs a day; and cohort 3, were in addition given an exogenous proteolytic enzyme with the major protein meal.Results: The absolute fall in serum albumin at 6 months for the cohort 1, 2 and 3 is 0.48, 0.29 and 0.09 gm/dl respectively. Not only was the fall in serum albumin significantly less with the use of exogenous proteolytic enzyme, a higher proportion of patients were able to maintain or improve their serum albumin. The fall in midarm circumference was maximum in cohort 1 (2.08 cm) and least in cohort 3 (0.45 cm). This positive trend however, did not achieve statistical significance.Conclusions: Use of exogenous proteolytic enzyme, when combined with egg protein, improves key nutritional indices in patients of CKD on CAPD.

Proteolytic Enzymes Screening for Enzymolysis Preparation Technology of Active Parts of Periplaneta americana against Liver Fibrosis / 中国药房

OBJECTIVE： To optimize the proteolytic enzymes for enzymolysis technology of degreasing ointment from Periplaneta americana， and to improve the extraction rate and activity of anti-liver fibrosis active part from P. americana. METHODS： Using degreasing ointment of P. americana as control， ninhydrin method and folin-ciocalteu method were used to investigate the hydrolysis degree of trypsin （TR）， pepsin （PE）， alkaline protease （AL）， papain （PA） and neutral protease （NE） to the degreasing ointment. Macroporous resin isolation and purification method was used to investigate the yield of elution part from hydrolyzate， with 50％， 60％， 70％， 95％ ethanol as eluting solvents. Inhibition test in vitro of rat hepatic stellate cells HSC-T6 was performed， and anti-liver fibrosis activity of elution part from hydrolyzate was investigated. RESULTS： The hydrolysis degree of PA and NE were 14.15％ and 15.70％， showing strong enzymatic hydrolysis ability. The yield of 95％ ethanol elution part from PA， NE and AL hydrolyzate were （0.73±0.04）％，（0.65±0.01）％ and（0.64±0.05）％， improving 30.36％， 16.07％， 14.29％ compared with degreasing ointment without enzyme. Results of inhibition test in vitro showed that inhibitory rate of 50％， 60％， 70％ ethanol elution parts isolated and  purified from hydrolyzate had a low inhibition rate or a growth-promoting effect on HSC-T6 cells. Inhibition rates of 95％ ethanol elution parts to HSC-T6 cells were all more than 20％. IC50 of 95％ ethanol elution part isolated and purified from PA and NE hydrolyzate for 24-72 h were 94.5-112.3 and 117.1-120.0 μg/mL， which were lower than that （116.1-123.0 μg/mL） of degreasing ointment without enzyme. CONCLUSIONS： PA is the best hydrolyzate for enzymolysis technology of active parts against liver fibrosis in degreasing ointment from P. americana， followed by NE and AL； PE and TR， which have poor effect， are not suitable for the enzymatic hydrolysis technology.

Antimicrobial activities of Plantactinospora sp. KBS50, a rare marine-derived actinomycete from the coastal area of Sarawak

Aims@#An actinomycete strain, designated KBS50, was isolated from a beach sediment sample collected from the Santubong area in Sarawak, Malaysia. This study reports on the identification, characterization and evaluation of the antimicrobial potential of this rare actinomycete. @*Methodology and results@#KBS50 was identified as a potentially new species of Plantactinospora genus using the 16S rRNA gene sequence analysis. The rare actinomycete showed distinct morphological and physiological characteristics from other species of Plantactinospora. KBS50 exhibited strong antagonistic activities against Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) and fungi (Aspergillus niger, Ganoderma boninense, and Rhizoctonia solani). The actinomycete also tested positive for proteolytic activity. Meanwhile, secondary screening of the cell-free culture broths and the ethyl acetate crude extracts detected antimicrobial activity against the Gram-positive bacteria only. The minimum inhibitory concentration of the crude extract against B. subtilis and S. aureus was 5.21±1.30 μg/mL and 15.63±0.00 μg/mL, respectively. @*Conclusion, significance and impact of study@#The results presented in this paper provided an insight into the capability of Plantactinospora sp. KBS50 as a potential source of bioactive secondary metabolites compounds. This study also showed that the marine-associated environment such as the coastal area in Sarawak can be a valuable source of unique actinomycetes that can be exploited for natural product discovery.

Proteolytic and lipolytic potential of Pseudomonas spp. from goat and bovine raw milk / Potencial proteolítico e lipolítico de espécies de Pseudomonas do leite cru caprino e bovino

Pseudomonas, the main genus of gram-negative microorganisms isolated from milk, is psychrotrophic, biofilm-forming, and thermo-resistant deteriorating enzyme producers. The aim of this study was to quantify Pseudomonas spp. in goat's and cow's milk produced in the Paraná state, Brazil, to evaluate the deteriorating activity of the isolates at mesophilic and psychrotrophic conditions and to identify, at the species level, the isolates with alkaline metalloprotease (aprX gene) production potential. Microbiological, biochemical and molecular methods were used for isolating, confirming and identifying of isolates. The mean counts were 1.6 (±6.3)x104 and 0.89(±3)x102 CFU/mL for goat and bovine milk samples, respectively, immediately after milking. Of the Pseudomonas colonies isolated from goat milk (n=60), 91.7% showed proteolytic potential when incubated at 35°C/48 h and 80% at 7°C/10 days, and lipolytic potential was observed in 95% of the isolates incubated in mesophilic and 78.3% at refrigeration conditions. From the isolates of bovine milk (n=20), 35% showed proteolytic activity only when incubated at 35°C/48 h, and lipolytic potential was observed in 25% of the isolates incubated at 7°C/10d and 35°C/48h. It was observed that 83.3% and 25% of the isolates genetically confirmed as Pseudomonas spp. of goat and bovine milk showed the potential for alkaline metalloprotease production, with the species P. azotoformans, P. koreensis, P. gessardii, P. monteilii and P. lurida being the most frequent in goat milk and P. aeruginosa the only species identified in cow milk.(AU)

Pseudomonas é o principal gênero de micro-organismos Gram negativos isolados do leite, são psicrotróficos, formadores de biofilmes e produtores de enzimas deteriorantes termodúricas. O objetivo do presente trabalho foi quantificar Pseudomonas spp. no leite de cabras e vacas produzido no estado do Paraná, Brasil, avaliar a atividade deteriorante em temperatura mesofílica e psicrotrófica e identificar, em nível de espécie, os isolados com potencial de produção de metaloprotease alcalina (geneaprX). Foram utilizados métodos microbiológicos, bioquímicos e moleculares para isolamento, confirmação e identificação dos isolados. As contagens médias foram de 1,6 (±6,3) x 104 e 0,9 (±3) x 102 UFC/mL para as amostras de leite caprino e bovino, respectivamente. Dos isolados de Pseudomonas do leite de cabra (n=60), 91,7% demonstraram potencial proteolítico quando incubadas a 35°C/48h e 80% a 7°C/10dias e lipolíticos em 95% dos isolados incubados em mesofilia e em 78,3% dos incubados em temperatura de refrigeração. Dos isolados do leite bovino (n=20), foi verificada atividade proteolítica de 35% apenas quando incubadas a 35°C/48h e lipolítica em 25% dos isolados incubados a 7°C/10d e 35°C/48h. Foi observado que 83,3% e 25% dos isolados confirmados geneticamente como Pseudomonas spp. do leite caprino e bovino, respectivamente, apresentaram o potencial de produção de metaloprotease alcalina, sendo as espécies P. azotoformans, P. koreensis, P. gessardii, P. monteilii e P. lurida as mais frequentes no leite de cabras e P. aeruginosa a única identificada do leite de vacas.(AU)

Operatoria dental y endodoncia 5: protección de la interfase resina-dentina mediante inhibidores de las enzimas colagenolíticas / Operative dentistry and endodontics 5: protection of the resin-dentin interface by colagenolytic enzyme inhibitors

La adhesión entre las resinas hidrófilas y la dentina se encuentra sometida a una degradación permanente cuya intensidad aumenta en función del tiempo transcurrido. Esto es producto de la actividad de las metaloproteinasas, de las catepsinas y otras enzimas colagenolíticas, responsables de la destrucción paulatina de las fibras colágenas de la capa híbrida. La mayoría de las estrategias para inhibir estas enzimas han sido ensayos de laboratorio, mientras que las investigaciones clínicas son escasas. En este trabajo se analiza la literatura relacionada con las estrategias sugeridas para prevenir la degradación del colágeno de la capa híbrida en la interfaz resina-dentina (AU)

The bonds between hidrophylic resins and dentin are subjected to a time-dependent collagenolytic degradation. Endogenous enzymes such as matrix metalloproteinases, catepsins and other enzymes are responsible for the hybrid layer destruction. The majority of strategies developed to inhibit these enzymes are laboratory evaluations while clinical studies are scarce. This review examines the literature related to the strategies suggested to prevent collagen degradation of the hybrid layer in resin-dentin interfaces (AU)

Proteolytic activity of excretory/secretory products of Cochliomyia hominivorax larvae (Diptera: Calliphoridae) / Atividade proteolítica de produtos de excreção/secreção de Cochliomyia hominivorax (Diptera: Calliphoridae)

The protein profiles and proteolytic activity of the excretory secretory products (E/SP) of the first (L1), second (L2) and third (L3) larval stages of Cochliomyia hominivorax were studied in the laboratory. Analysis on the E/SP protein profile was carried out using polyacrylamide gel containing sodium dodecyl sulfate (SDS-PAGE). The E/SP of each larval stage (L1, L2 and L3) treated with protease inhibitors, containing 30µg, 40µg and 50µg of protein, was applied to the 10% polyacrylamide gel. The proteolytic activity of the crude E/SP was analyzed in gels copolymerized with gelatin and by colorimetric assays using azocasein as a substrate, with the characterization of the proteases using synthetic inhibitors. Different protein profiles were observed for the larval instars, with L1 presenting the most complex profile. Nevertheless, various protein bands were observed that were common to all the larval instars. The E/SP of all the instars showed proteolytic activity on gelatin, evidenced by proteolysis zones, predominantly with apparently higher molecular masses in L1, while for L2 and L3 the proteolysis zones could also be observed in regions with lower masses. Tests with protease inhibitors using gelatin as substrate showed that the E/SP of larvae were mainly composed of serine proteases. Additionally, inhibition was observed in L2 E/SP treated previously with EDTA, an inhibitor of metalloproteases. The assays with azocasein revealed a gradual increase of proteolytic activity on this substrate with larval development progress, with the strongest inhibitions being observed after treatments with 3,4-dichloroisocoumarin (DCI) for E/SP of L1, L2 and L3. These results suggest that C. hominivorax larvae produce different proteases, a fact that can be related to the parasite's vital processes for survival, such as penetration into the host's tissues and nutrition during the larval stage.(AU)

Os perfis protéicos e a atividade proteolítica dos produtos de excreção/secreção (PE/S) das larvas de primeiro (L1), segundo (L2) e terceiro (L3) estágios de Cochliomyia hominivorax foram estudados em laboratório. Os perfis protéicos foram obtidos por eletroforese em géis de poliacrilamida (SDS-PAGE). Os PE/S de cada fase larval (L1, L2 e L3), tratados com inibidores de proteases, contendo 30µg, 40µg e 50µg de proteína, foram aplicados em géis de poliacrilamida a 10%. A atividade proteolítica dos PE/S na sua forma nativa, foi analisada em géis co-polimerizados com gelatina e por testes colorimétricos usando a azocaseína como substrato, com a caracterização das proteases feita por meio de inibidores sintéticos. Diferentes perfis protéicos foram observados para os instares larvais, com L1 apresentando o perfil mais complexo. Apesar disso, foram observadas várias bandas protéicas comuns a todos os estágios larvais. Os PE/S de todos os instares mostraram atividade proteolítica sobre a gelatina, evidenciada por zonas de proteólise, com predominância de massas moleculares aparentes mais altas em L1, enquanto que para L2 e L3 as zonas de proteólise puderam ser observadas também em regiões de menores massas. Os testes com inibidores de proteases usando a gelatina como substrato mostraram que os PE/S de L1, L2 e L3 eram compostos principalmente de serina-proteases. Adicionalmente, inibição foi observada nos PE/S de L2 tratada previamente com EDTA, um inibidor de metalo-proteases. Os ensaios com a zocaseína revelaram um aumento gradual da atividade proteolítica sobre este substrato com o progresso do desenvolvimento larval, com a mais forte inibição sendo observada após o tratamento com 3,4 dicloroisocumarina (DCI) para os PE/S de L1, L2 e L3. Estes resultados sugerem que as larvas de C. hominivorax produzem diferentes proteases, fato que pode estar relacionado a processos vitais para a sobrevivência do parasita, tais como a penetração nos tecidos dos hospedeiros e nutrição durante os estágios larvais.(AU)

A review on the Scorpaena plumieri fish venom and its bioactive compounds

The most poisonous fish species found along the Brazilian coast is the spotted scorpionfish Scorpaena plumieri. Though hardly ever life-threatening to humans, envenomation by S. plumieri can be quite hazardous, provoking extreme pain and imposing significant socioeconomic costs, as the victims may require days to weeks to recover from their injuries. In this review we will walk the reader through the biological features that distinguish this species as well as the current epidemiological knowledge related to the envenomation and its consequences. But above all, we will discuss the challenges involved in the biochemical characterization of the S. plumieri venom and its compounds, focusing then on the successful isolation and pharmacological analysis of some of the bioactive molecules responsible for the effects observed upon envenomation as well as on experimental models. Despite the achievement of considerable progress, much remains to be done, particularly in relation to the non-proteinaceous components of the venom. Therefore, further studies are necessary in order to provide a more complete picture of the venoms chemical composition and physiological effects. Given that fish venoms remain considerably less studied when compared to terrestrial venoms, the exploration of their full potential opens a myriad of possibilities for the development of new drug leads and tools for elucidating the complex physiological processes.

A review on the Scorpaena plumieri fish venom and its bioactive compounds

The most poisonous fish species found along the Brazilian coast is the spotted scorpionfish Scorpaena plumieri. Though hardly ever life-threatening to humans, envenomation by S. plumieri can be quite hazardous, provoking extreme pain and imposing significant socioeconomic costs, as the victims may require days to weeks to recover from their injuries. In this review we will walk the reader through the biological features that distinguish this species as well as the current epidemiological knowledge related to the envenomation and its consequences. But above all, we will discuss the challenges involved in the biochemical characterization of the S. plumieri venom and its compounds, focusing then on the successful isolation and pharmacological analysis of some of the bioactive molecules responsible for the effects observed upon envenomation as well as on experimental models. Despite the achievement of considerable progress, much remains to be done, particularly in relation to the non-proteinaceous components of the venom. Therefore, further studies are necessary in order to provide a more complete picture of the venom's chemical composition and physiological effects. Given that fish venoms remain considerably less studied when compared to terrestrial venoms, the exploration of their full potential opens a myriad of possibilities for the development of new drug leads and tools for elucidating the complex physiological processes.(AU)

Proteolytic and Milk Clotting Activity of Fractionated Protein of Lagenaria siceraria.

The study was undertaken to evaluate milk clotting and proteolytic activity of protein fractions of seeds of Lagenaria siceraria. Protein fractions were isolated by the method of differential solubility, and their protein contents were estimated using Bio-Rad protein assay reagent and bovine serum albumin. The effects of pH and temperature on the milk-clotting and proteolytic activity were also evaluated. The isolated protein fractions showed highest milk clotting activity over a broad temperature range of 30-80 ºC and pH range of 3-9. Based upon the observations, milk clotting enzymes present in different fraction of proteins of Lagenaria siceraria are promising candidates for application in industrial scale for production of cheese and might be a potential substitute for commercial animal rennet.

Evaluación de la capacidad neutralizante de extractos de plantas de uso popular en Guatemala como antídotos para el envenenamiento por la mordedura de Bothrops asper / Neutralizing activities of ethanolic extracts of six plants traditionally used in Guatemala as antidotes for the envenomation caused by the snake Bothrops asper

Se determinó la capacidad de los extractos de seis plantas de uso etnomédico (Acacia hindsii, Aristolochia maxima, Cissampelos pareira, Hamelia patens, Piper peltatum y Sansevieria hyacinthoides) para neutralizar los efectos proteolítico y fosfolipasa A2 (PLA2) del veneno de Bothrops asper, la principal especie causante de envenenamiento en el país. Estos efectos, indicadores de la capacidad miotóxica, hemorrágica e inflamatoria del veneno, se evaluaron en ensayos controlados in vitro. Las plantas fueroncolectadas, secadas y extraídas por percolación con etanol. Los resultados demuestran que ninguno de los extractos posee actividad PLA2 o proteo-lítica intrínseca a las dosis estudiadas. Se determinó que tres de los extractos neutralizaron pobremente (< 50%) los efectos estudiados: S. hyacinthoides neutralizó 13.90 ± 6.41% del efecto PLA2 y P. peltatum y C. pareira el 32.98 ± 5.51% y 24.52 ± 7.45%, respectivamente, del efecto proteolítico. Por ello, ningún extracto se evaluó en pruebas de neutralización de la letalidad en ratones. Se concluye que no es recomendable el uso aislado de estas plantas en el tratamiento del envenenamiento por mordedura de B. asper, aunque posiblemente las que demostraron alguna actividad puedan resultar potenciadas al usarse en combinación con otras plantas, como se hace en las recetas tradicionales. Dada la complejidad de los componentes del veneno y sus efectos fisiopatológicos, falta investigar la capacidad de las plantas estudiadas para neutralizar las coagulopatías, edema y miotoxicidad producidas durante el envenenamiento.

Many plants are reported to be used in Guatemalan traditional medicine as antidotes against various effects of the snakebite; however, very few attempts have been made to evaluate their neutralizing capacity in controlled experiments. Six plants (Acacia hindsii, Cissampelos pareira; Hamelia patens, Piper peltatum, Sansevieria hyacinthoides and Aristolochia maxima) were evaluated in vitro for their ability to neutralize phospholipase A2 (PLA2) and proteolytic effects of the venom of Bothrops asper, the snake responsible for approximately half of the snakebite envenomations in Central America. These effects are indicatives of the ability of B. asper venom to produce myotoxicity, hemorrhage and inflammation. Plants were collected, dried and extracted by maceration with ethanol. After pre-incubation of several amounts of each extract with a challenge dose of venom, S. hyacinthoides demonstrated a low neutralizing capacity (< DE 50) of the PLA2 effect (13.90 ± 6.41%); C. pareira (32.98 ± 5.51%) and P. peltatum (24.52 ± 7.45%) neutralized less than 50% of the proteolytic effect. The results suggest that neither of the tested plants should be used individually to treat the main effects of B. asperenvenomation. However, the three low-active extracts might be potentiated when used in mixtures composed of several plants, as prepared by traditional healers. Given the complexity of the venom components and the multiple pathologic effects produced by B. asper envenomation, more tests are required to fully investigate the ability of this plants to neutralize the coagulant, fibrin(ogen)olytic, edematizing and myotoxic effects of the venom.