Sample records for autolysis

  1. [Overexpression of FKS1 to improve yeast autolysis-stress]. (United States)

    Li, Jia; Wang, Jinjing; Li, Qi


    With the development of high gravity brewing, yeast cells are exposed to multiple brewing-associated stresses, such as increased osmotic pressure, enhanced alcohol concentration and nutritional imbalance. These will speed up yeast autolysis, which seriously influence beer flavor and quality. To increase yeast anti-autolytic ability, FKS1 overexpression strain was constructed by 18S rDNA. The concentration of β-1,3-glucan of overexpression strain was 62% higher than that of wild type strain. Meantime, FKS1 overexpression strain increased anti-stress ability at 8% ethanol, 0.4 mol/L NaCl and starvation stress. Under simulated autolysis, FKS1 showed good anti-autolytic ability by slower autolysis. These results confirms the potential of FKS1 overexpression to tackle yeast autolysis in high-gravity brewing.

  2. Autolysis and extension of isolated walls from growing cucumber hypocotyls. (United States)

    Cosgrove, D J; Durachko, D M


    Walls isolated from cucumber hypocotyls retain autolytic activities and the ability to extend when placed under the appropriate conditions. To test whether autolysis and extension are related, we treated the walls in various ways to enhance or inhibit long-term wall extension ('creep') and measured autolysis as release of various saccharides from the wall. Except for some non-specific inhibitors of enzymatic activity, we found no correlation between wall extension and wall autolysis. Most notably, autolysis and extension differed strongly in their pH dependence. We also found that exogenous cellulases and pectinases enhanced extension in native walls, but when applied to walls previously inactivated with heat or protease these enzymes caused breakage without sustained extension. In contrast, pretreatment of walls with pectinase or cellulase, followed by boiling in methanol to inactivate the enzymes, resulted in walls with much stronger expansin-mediated extension responses. Crude protein preparations from the digestive tracts of snails enhanced extension of both native and inactivated walls, and these preparations contained expansin-like proteins (assessed by Western blotting). Our results indicate that the extension of isolated cucumber walls does not depend directly on the activity of endogenous wall-bound autolytic enzymes. The results with exogenous enzymes suggest that the hydrolysis of matrix polysaccharides may not induce wall creep by itself, but may act synergistically with expansins to enhance wall extension.

  3. Cell wall degradation in the autolysis of filamentous fungi. (United States)

    Perez-Leblic, M I; Reyes, F; Martinez, M J; Lahoz, R


    A systematic study on autolysis of the cell walls of fungi has been made on Neurospora crassa, Botrytis cinerea, Polystictus versicolor, Aspergillus nidulans, Schizophyllum commune, Aspergillus niger, and Mucor mucedo. During autolysis each fungus produces the necessary lytic enzymes for its autodegradation. From autolyzed cultures of each fungus enzymatic precipitates were obtained. The degree of lysis of the cell walls, obtained from non-autolyzed mycelia, was studied by incubating these cell walls with and without a supply of their own lytic enzymes. The degree of lysis increased with the incubation time and generally was higher with a supply of lytic enzymes. Cell walls from mycelia of different ages were obtained. A higher degree of lysis was always found, in young cell walls than in older cell walls, when exogenous lytic enzymes were present. In all the fungi studied, there is lysis of the cell walls during autolysis. This is confirmed by the change of the cell wall structure as well as by the degree of lysis reached by the cell wall and the release of substances, principally glucose and N-acetylglucosamine in the medium.

  4. Autolysis of Blakeslea trispora during carotene production from cheese whey in an airlift reactor. (United States)

    Varzakakou, Maria; Roukas, Triantafyllos; Papaioannou, Emmanuel; Kotzekidou, Parthena; Liakopoulou-Kyriakides, Maria


    The phenomenon of autolysis in Blakeslea trispora during carotene production from deproteinized hydrolyzed whey in an airlift reactor was investigated. The process of cellular autolysis was studied by measuring the changes in carotene concentration, dry biomass, residual sugars, pH, intracellular protein, specific activity of the hydrolytic enzymes (proteases, chitinase), and micromorphology of the fungus using a computerized image analysis system. All these parameters were useful indicators of autolysis, but image analysis was found to be the most useful indicator of the onset and progress of autolysis in the culture. Autolysis of B. trispora began early in the growth phase, continued during the stationary phase, and increased significantly in the decline phase. The morphological differentiation of the fungus was a result of the degradation of the cell membrane by hydrolytic enzymes. The biosynthesis of carotenes was carried out in the exponential phase, where the phenomenon of autolysis was not intense.

  5. Comparative proteome and transcriptome analysis of lager brewer's yeast in the autolysis process. (United States)

    Xu, Weina; Wang, Jinjing; Li, Qi


    The autolysis of brewer's yeast during beer production has a significant effect on the quality of the final product. In this work, we performed proteome and transcriptome studies on brewer's yeast to examine changes in protein and mRNA levels in the process of autolysis. Protein and RNA samples of the strain Qing2 at two different autolysis stages were obtained for further study. In all, 49 kinds of proteins were considered to be involved in the autolysis response, eight of which were up-regulated and 41 down-regulated. Seven new kinds of proteins emerged during autolysis. Results of comparative analyses showed that important changes had taken place as an adaptive response to autolysis. Functional analysis showed that carbohydrate and energy metabolism, cellular amino acid metabolic processes, cell response to various stresses (such as oxidative stress, salt stress, and osmotic stress), translation and transcription were repressed by the down-regulation of the corresponding proteins, and starvation and DNA damage responses could be induced. The comparison of data on transcriptomes with proteomes demonstrated that most autolysis-response proteins as well as new proteins showed a general correlation between mRNA and protein levels. Thus these proteins were thought to be transcriptionally regulated. These findings provide important information about how brewer's yeast acts to cope with autolysis at molecular levels, which might enhance global understanding of the autolysis process.

  6. Acinar autolysis and mucous extravasation in human sublingual glands: a microscopic postmortem study (United States)

    AZEVEDO-ALANIS, Luciana Reis; TOLENTINO, Elen de Souza; de ASSIS, Gerson Francisco; CESTARI, Tânia Mary; LARA, Vanessa Soares; DAMANTE, José Humberto


    Although some morphological investigations on aged human sublingual glands (HSG) found eventual phenomena identified as autolysis and mucous extravasation, the exact meaning of these findings has not been elucidated. Objective The aim of this work is to investigate whether acinar autolysis and mucous extravasation are related to the aging process in human sublingual glands. We also speculate if autolytic changes may assist forensic pathologists in determining time of death. Material and Methods 186 cadavers’ glands were allocated to age groups: I (0–30 years); II (31–60), and III (61–90). Time and mode of death were also recorded. Acinar autolysis and mucous extravasation were classified as present or absent. Ultrastructural analysis was performed using transmission electron microscopy (TEM). Data were compared using Mann-Whitney U, Spearman’s correlation coefficient, Kruskal-Wallis, and Dunn tests (p<0.05). Results There was correlation between age and acinar autolysis (r=0.38; p=0.0001). However, there was no correlation between autolysis and time of death. No differences were observed between genders. TEM showed mucous and serous cells presenting nuclear and membrane alterations and mucous cells were more susceptible to autolysis. Conclusion Acinar autolysis occurred in all age groups and increased with age while mucous extravasation was rarely found. Both findings are independent. Autolysis degrees in HSG could not be used to determine time of death. PMID:26537715

  7. Acinar autolysis and mucous extravasation in human sublingual glands: a microscopic postmortem study

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    Luciana Reis AZEVEDO-ALANIS


    Full Text Available Although some morphological investigations on aged human sublingual glands (HSG found eventual phenomena identified as autolysis and mucous extravasation, the exact meaning of these findings has not been elucidated.Objective The aim of this work is to investigate whether acinar autolysis and mucous extravasation are related to the aging process in human sublingual glands. We also speculate if autolytic changes may assist forensic pathologists in determining time of death.Material and Methods 186 cadavers’ glands were allocated to age groups: I (0–30 years; II (31–60, and III (61–90. Time and mode of death were also recorded. Acinar autolysis and mucous extravasation were classified as present or absent. Ultrastructural analysis was performed using transmission electron microscopy (TEM. Data were compared using Mann-Whitney U, Spearman’s correlation coefficient, Kruskal-Wallis, and Dunn tests (p<0.05.Results There was correlation between age and acinar autolysis (r=0.38; p=0.0001. However, there was no correlation between autolysis and time of death. No differences were observed between genders. TEM showed mucous and serous cells presenting nuclear and membrane alterations and mucous cells were more susceptible to autolysis.Conclusion Acinar autolysis occurred in all age groups and increased with age while mucous extravasation was rarely found. Both findings are independent. Autolysis degrees in HSG could not be used to determine time of death.

  8. Death of Prorocentrum donghaiense May Be a Programmed Autolysis

    Institute of Scientific and Technical Information of China (English)

    YANG Guanpin; LIU Yongjian; LI Bingjun


    Prorocentrum donghaiense, one of the dinoflagellate, has continuously caused large scale red tides along the Chinese coast in recent years. These red tides have brought tremendous loss to the local economy and serious impacts to the local environ- ment. Unfortunately, little was known about the mechanism of the fast appearance and extinction of the red tide caused by this alga. In this study, the full-length cDNA of a caspase encoding gene ofP donghaiense was cloned and characterized, and the transcription of this gene during the senescence of the alga was semi-quantitatively determined. The cDNA was 520bp in length. It contained a 258bp open reading frame (ORF) which encoded a peptide of 85 amino acids. The amount of transcripts of the caspase encoding gene increased with the senescence of P. donghaiense and started to decrease gradually when the autolysis of P. donghaiense cells took place. We proposed that the death of P. donghaiense may be a caspase mediated programmed autolysis.

  9. Spontaneous large-scale autolysis in Clostridium acetobutylicum contributes to generation of more spores

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    Zhen eLiu


    Full Text Available Autolysis is a widespread phenomenon in bacteria. In batch fermentation of Clostridium acetobutylicum ATCC 824, there is a spontaneous large-scale autolysis phenomenon with significant decrease of cell density immediately after exponential phase. To unravel the role of autolysis, an autolysin-coding gene, CA_C0554, was disrupted by using ClosTron system to obtain the mutant C. acetobutylicum lyc::int(72. The lower final cell density and faster cell density decrease rate of C. acetobutylicum ATCC 824 than those of C. acetobutylicum lyc::int(72 indicates that CA_C0554 was an important but not the sole autolysin-coding gene responding for the large-scale autolysis. Similar glucose utilization and solvents production but obvious lower cell density of C. acetobutylicum ATCC 824 comparing to C. acetobutylicum lyc::int(72 suggests that lysed C. acetobutylicum ATCC 824 cells were metabolic inactive. On the contrary, the spore density of C. acetobutylicum ATCC 824 is 26.1% higher than that of C. acetobutylicum lyc::int(72 in the final culture broth of batch fermentation. We speculated that spontaneous autolysis of metabolic-inactive cells provided nutrients for the sporulating cells. The present study suggests that one important biological role of spontaneous large-scale autolysis in C. acetobutylicum ATCC 824 batch fermentation is contributing to generation of more spores during sporulation.

  10. Spontaneous large-scale autolysis in Clostridium acetobutylicum contributes to generation of more spores (United States)

    Liu, Zhen; Qiao, Kai; Tian, Lei; Zhang, Quan; Liu, Zi-Yong; Li, Fu-Li


    Autolysis is a widespread phenomenon in bacteria. In batch fermentation of Clostridium acetobutylicum ATCC 824, there is a spontaneous large-scale autolysis phenomenon with significant decrease of cell density immediately after exponential phase. To unravel the role of autolysis, an autolysin-coding gene, CA_C0554, was disrupted by using ClosTron system to obtain the mutant C. acetobutylicum lyc::int(72). The lower final cell density and faster cell density decrease rate of C. acetobutylicum ATCC 824 than those of C. acetobutylicum lyc::int(72) indicates that CA_C0554 was an important but not the sole autolysin-coding gene responding for the large-scale autolysis. Similar glucose utilization and solvents production but obvious lower cell density of C. acetobutylicum ATCC 824 comparing to C. acetobutylicum lyc::int(72) suggests that lysed C. acetobutylicum ATCC 824 cells were metabolic inactive. On the contrary, the spore density of C. acetobutylicum ATCC 824 is 26.1% higher than that of C. acetobutylicum lyc::int(72) in the final culture broth of batch fermentation. We speculated that spontaneous autolysis of metabolic-inactive cells provided nutrients for the sporulating cells. The present study suggests that one important biological role of spontaneous large-scale autolysis in C. acetobutylicum ATCC 824 batch fermentation is contributing to generation of more spores during sporulation. PMID:26441884

  11. Postmortem acinar autolysis in rat sublingual gland: a morphometric study

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    Leticia Rodrigues Nery


    Full Text Available ABSTRACT OBJECTIVE: To analyze and to quantify morphological acinar postmortem changes in rat sublingual glands (SLG. MATERIAL AND METHODSs: Fifty rats were divided into two groups of 25 animals each. Group I was used for morphological and morphometric evaluations and group II for the determination of gland density and processed gland volume. Acinar autolytic changes were studied at 0 (control group, 3, 6, 12 and 24 h postmortem periods. The morphometric analysis of the volume density (Vv and total volume (Vt of intact (ia and autolyzed (aa acini was performed under light microscopy using a Zeiss II integration grid with 100 symmetrically distributed points. RESULTS: Morphologically, temporal progressive nuclear alterations and gradual loss of the structural architecture of acinar cells were found. Regarding quantitative results, both the Vvaa and the Vvia showed statistically significant differences among all postmortem periods (p0.05, respectively. Vtaa increased from 0.18 mm³ at 0 h to 38.17 mm³ at 12 h, while Vtia showed a decrease from 33.47 mm³ to 0 mm³ between 3-24 h postmortem. Data concerning Vtaa were adjusted by two-variable linear regression, obtaining the equation: y=-3.54 + 3.38x (r²=0.90. The Vtaa growth rate calculated by this equation was 3.38 mm³/h between 0-12 h. CONCLUSION: Acinar autolysis on rat SLG demonstrated the most significant signs during the first 6 h postmortem and was widely spread through the gland at 12 h.

  12. Targeting the autolysis loop of urokinase-type plasminogen activator with conformation-specific monoclonal antibodies

    DEFF Research Database (Denmark)

    Bøtkjær, Kenneth Alrø; Fogh, Sarah; Bekes, Erin C;


    , with high levels correlating with a poor prognosis. This observation has stimulated efforts into finding new principles for intervening with uPA's activity. In the present study we characterize the so-called autolysis loop in the catalytic domain of uPA as a potential inhibitory target. This loop was found...

  13. Changes in Autolysis Solution of Brewer's Yeasts and the Evaluation of Autolysis%啤酒酵母自溶液中的物质变化及酵母自溶评价指标探索

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    许维娜; 王金晶; 陈希; 李崎


    以4株不同的啤酒酵母菌株为研究对象,比较其在模拟自溶条件下电镜细胞形态及溶液物质变化,寻找能够判定酵母自溶性能的较优评价指标.结果表明,α-氨基氮、甲醛氮、各种分子质量蛋白质和游离长链脂肪酸等指标虽然都随酵母自溶有一定的变化,但规律性不强,单一因素不适合作为判定酵母自溶的直接指标.随着酵母自溶性能和自溶程度的变化,(A260/A280)/死亡率呈现明显且一致的变化趋势,酵母自溶程度越大,该比值越小.该指标综合性好、灵敏度高、耗时少、操作简单,能够及时反映不同菌株自溶性能的差异,以及同一株菌在不同阶段的自溶情况,是较好的酵母自溶评价指标.%Four brewer's yeasts with different autolysis performance were used to study their microscopic morphology by scanning electron microscopy (SEM) and changes in solution during autolysis in order to find out the optimal evaluating indicator of yeast autolysis.Results showed that α-amino nitrogen,formaldehyde nitrogen,each molecule of protein content,and free long-chain fatty acid content had certain variations during autolysis,but there was no obvious regularity.Meantime,single factor was not suitable for the evaluation of autolysis as a direct indicator.Nevertheless,(A260/A280)/yeast mortality showed an obvious and consistent trend during autolysis.When the yeast autolysis degree increased,the value of (A260/A280)/yeast mortality gradually decreased.This indicator had the advantages of comprehensive,high sensitive,and less timeconsuming,also the method was easy to operate.It could reflect different autolysis performance of different strains,as well as differences of the same strain at different stages of autolysis.So (A260/A280)/yeast mortality was a good indicator to evaluate yeast autolysis.

  14. Partial autolysis of μ/m-calpain during post mortem aging of chicken muscle. (United States)

    Zhao, Liang; Jiang, Nanqi; Li, Miaozhen; Huang, Ming; Zhou, Guanghong


    The objective of this study was to investigate changes occurring in μ/m-calpain in post mortem chicken muscles and to determine the origin of the unknown bands found in calpain casein zymography. The unknown bands were reported with slightly greater mobility compared to conventional μ/m-calpain bands in casein zymography. Identification of these bands was accomplished using native polyacrylamide gel electrophoresis, liquid chromatography tandem mass spectrometry and with protein phosphatase treatment. Results showed that the unknown bands were corresponding to μ/m-calpain, and dephosphorylation by protein phosphatase did not change their appearance. The calpain samples were then incubated with various concentrations of Ca(2+) to determine the relationship between changes in μ/m-calpain and the appearance of the unknown bands. The products of μ/m-calpain partial autolysis were found to be consistent with the appearance of the unknown bands. Therefore, the appearance of these bands did not result from phosphorylation of μ/m-calpain as previously hypothesized, but from partial autolysis of μ/m-calpain. Also their presence suggests that μ/m-calpain undergoes partial autolysis during aging which may play certain roles in meat quality improvement.

  15. Lipids and lipoteichoic acid of autolysis-defective Streptococcus faecium strains. (United States)

    Shungu, D L; Cornett, J B; Shockman, G D


    Two of four previously isolated autolysis-defective mutants of Streptococcus faecium (Streptococcus faecalis ATCC 9790) incorporated substantially more [14C]glycerol into lipids and lipoteichoic acid than did the parent strain. Consistent with increased accumulation of lipids and lipoteichoic acid, significantly higher levels of phosphorus were found in the corresponding fractions of the two mutant strains than in the wild type. Although the autolysis-defective mutant strains contained the same assortment of lipids as the wild type, the relative amount of [14C]glycerol incorporated into diphosphatidylglycerol increased, accompanied by a decreased fraction of phosphatidylglycerol. These results suggested that increased cellular content of two types of substances, acylated lipoteichoic acid and lipids (notably diphosphatidylglycerol), which previously had been shown to be potent inhibitors of the N-acetylmuramoylhydrolase of this species, contributed to the autolysis-defective phenotype of these mutants. Consistent with this interpretation are observations that (i) cerulenin inhibition of fatty acid synthesis increased the rates of benzylpenicillin-induced cellular lysis and that (ii) Triton X-100 or Zwittergent 3-14 treatment could reveal the presence of otherwise cryptic but substantial levels of the active form of the autolysin in cells of three of four mutants and of the proteinase-activable latent form in all four mutants.

  16. 啤酒废酵母自溶条件的研究%Autolysis conditions of waste brewer' s yeast

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    杨建梅; 李红; 杜金华


    The key factors of waste brewer's yeast autolysis such as temperature, pH value, addition of NaCl and autolysis time were optimized by single factor tests and orthogonal tests in this article. The results indicated that the optimum conditions were obtained as follows: autolysis temperature 50℃,pH value 5.0, addition of NaCl 3% and autolysis time 24h. Under these autolysis conditions, the content of total sugar would reach 2.27g/L, yield of amino-nitragen was 3.98% and yield of the extract was 54.12% in the supernatant fluid.%通过单因素试验和正交试验对影响啤酒废酵母自溶的温度、pH值、NaCl添加量和自溶时间4个关键因素进行了优化,得到了啤酒废酵母自溶最佳工艺条件:自溶温度50℃,自溶pH值为5.0,NaCl添加量为3% (w/w),自溶时间24h,自溶上清液中总糖分含量达到2.27g/L,游离氨基酸态氮得率达到3.98%,抽提物得率达到54.12%.

  17. Identification of key peptidoglycan hydrolases for morphogenesis, autolysis, and peptidoglycan composition of Lactobacillus plantarum WCFS1

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    Rolain Thomas


    Full Text Available Abstract Background Lactobacillus plantarum is commonly used in industrial fermentation processes. Selected strains are also marketed as probiotics for their health beneficial effects. Although the functional role of peptidoglycan-degrading enzymes is increasingly documented to be important for a range of bacterial processes and host-microbe interactions, little is known about their functional roles in lactobacilli. This knowledge holds important potential for developing more robust strains resistant to autolysis under stress conditions as well as peptidoglycan engineering for a better understanding of the contribution of released muramyl-peptides as probiotic immunomodulators. Results Here, we explored the functional role of the predicted peptidoglycan hydrolase (PGH complement encoded in the genome of L. plantarum by systematic gene deletion. From twelve predicted PGH-encoding genes, nine could be individually inactivated and their corresponding mutant strains were characterized regarding their cell morphology, growth, and autolysis under various conditions. From this analysis, we identified two PGHs, the predicted N-acetylglucosaminidase Acm2 and NplC/P60 D,L-endopeptidase LytA, as key determinants in the morphology of L. plantarum. Acm2 was demonstrated to be required for the ultimate step of cell separation of daughter cells, whereas LytA appeared to be required for cell shape maintenance and cell-wall integrity. We also showed by autolysis experiments that both PGHs are involved in the global autolytic process with a dominant role for Acm2 in all tested conditions, identifying Acm2 as the major autolysin of L. plantarum WCFS1. In addition, Acm2 and the putative N-acetylmuramidase Lys2 were shown to play redundant roles in both cell separation and autolysis under stress conditions. Finally, the analysis of the peptidoglycan composition of Acm2- and LytA-deficient derivatives revealed their potential hydrolytic activities by the

  18. Autolysis of Aspergillus oryzae Mycelium and Effect on Volatile Flavor Compounds of Soy Sauce. (United States)

    Xu, Ning; Liu, Yaqi; Hu, Yong; Zhou, Mengzhou; Wang, Chao; Li, Dongsheng


    The autolyzed mycelia of Aspergillus oryzae are rich in proteins, nucleic acids, sugar, and other biomacromolecules, and are one of the main contributors to the flavor profile of commercially important fermented goods, including soy sauce and miso. We induced autolysis of the mycelia of A. oryzae over 1 to 10 d, and found that the maximum dissolved amounts of total protein and nucleic acid ratio accounted for 28.63% and 88.93%, respectively. The organic acid content, such as citric acid, tartaric acid, succinic acid, lactic acid, and acetic acid, initially increased and then decreased as autolysis progressed, corresponding to changes in pH levels. The main characteristic flavor compounds in soy sauce, namely, ethanol, 2-phenylethanol, and 2-methoxy-4-vinylphenol, were all detected in the autolysate. Subsequently, we tested the effect of adding mycelia of A. oryzae during the fermentation process of soy sauce for 60 d, and found that addition of 1.2‰ A. oryzae mycelia provided the richest flavor. Overall, our findings suggest that compounds found in the autolysate of A. oryzae may promote the flavor compounds of soy sauce, such as alcohols, aldehydes, phenols, and esters.

  19. msaABCR operon positively regulates biofilm development by repressing proteases and autolysis in Staphylococcus aureus. (United States)

    Sahukhal, Gyan S; Batte, Justin L; Elasri, Mohamed O


    Staphylococcus aureus is an important human pathogen that causes nosocomial and community-acquired infections. One of the most important aspects of staphylococcal infections is biofilm development within the host, which renders the bacterium resistant to the host's immune response and antimicrobial agents. Biofilm development is very complex and involves several regulators that ensure cell survival on surfaces within the extracellular polymeric matrix. Previously, we identified the msaABCR operon as an additional positive regulator of biofilm formation. In this study, we define the regulatory pathway by which msaABCR controls biofilm formation. We demonstrate that the msaABCR operon is a negative regulator of proteases. The control of protease production mediates the processing of the major autolysin, Atl, and thus regulates the rate of autolysis. In the absence of the msaABCR operon, Atl is processed by proteases at a high rate, leading to increased cell death and a defect in biofilm maturation. We conclude that the msaABCR operon plays a key role in maintaining the balance between autolysis and growth within the staphylococcal biofilm.

  20. Gene knockout and overexpression analysis revealed the role of N-acetylmuramidase in autolysis of Lactobacillus delbrueckii subsp. bulgaricus ljj-6.

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    Xiao-Yang Pang

    Full Text Available Autolysis of lactic acid bacteria (LAB plays a vital role in dairy processing. During cheese making, autolysis of LAB affects cheese flavor development through release of intracellular enzymes and restricts the proliferation of cells in yogurt fermentation and probiotics production. In order to explore the mechanism of autolysis, the gene for the autolytic enzymes of L. bulgaricus, N-acetylmuramidase (mur, was cloned and sequenced (GenBank accession number: KF157911. Mur gene overexpression and gene knockout vectors were constructed based on pMG76e and pUC19 vectors. Recombinant plasmids were transformed into L. bulgaricus ljj-6 by electroporation, then three engineered strains with pMG76e-mur vector and fifteen engineered strains with pUC19-mur::EryBII were screened. The autolysis of the mur knockout strain was significantly lower and autolysis of the mur overexpressed strain was significantly higher compared with that of the wild type strain ljj-6. This result suggested that the mur gene played an important role in autolysis of L. bulgaricus. On the other hand, autolytic activity in a low degree was still observed in the mur knockout strain, which implied that other enzymes but autolysin encoded by mur were also involved in autolysis of L. bulgaricus.

  1. Gene knockout and overexpression analysis revealed the role of N-acetylmuramidase in autolysis of Lactobacillus delbrueckii subsp. bulgaricus ljj-6. (United States)

    Pang, Xiao-Yang; Cui, Wen-Ming; Liu, Lu; Zhang, Shu-Wen; Lv, Jia-Ping


    Autolysis of lactic acid bacteria (LAB) plays a vital role in dairy processing. During cheese making, autolysis of LAB affects cheese flavor development through release of intracellular enzymes and restricts the proliferation of cells in yogurt fermentation and probiotics production. In order to explore the mechanism of autolysis, the gene for the autolytic enzymes of L. bulgaricus, N-acetylmuramidase (mur), was cloned and sequenced (GenBank accession number: KF157911). Mur gene overexpression and gene knockout vectors were constructed based on pMG76e and pUC19 vectors. Recombinant plasmids were transformed into L. bulgaricus ljj-6 by electroporation, then three engineered strains with pMG76e-mur vector and fifteen engineered strains with pUC19-mur::EryBII were screened. The autolysis of the mur knockout strain was significantly lower and autolysis of the mur overexpressed strain was significantly higher compared with that of the wild type strain ljj-6. This result suggested that the mur gene played an important role in autolysis of L. bulgaricus. On the other hand, autolytic activity in a low degree was still observed in the mur knockout strain, which implied that other enzymes but autolysin encoded by mur were also involved in autolysis of L. bulgaricus.

  2. Transcriptional and functional analysis of the effects of magnolol: inhibition of autolysis and biofilms in Staphylococcus aureus.

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    Dacheng Wang

    Full Text Available BACKGROUND: The targeting of Staphylococcus aureus biofilm structures are now gaining interest as an alternative strategy for developing new types of antimicrobial agents. Magnolol (MOL shows inhibitory activity against S. aureus biofilms and Triton X-100-induced autolysis in vitro, although there are no data regarding the molecular mechanisms of MOL action in bacteria. METHODOLOGY/PRINCIPAL FINDINGS: The molecular basis of the markedly reduced autolytic phenotype and biofilm inhibition triggered by MOL were explored using transcriptomic analysis, and the transcription of important genes were verified by real-time RT-PCR. The inhibition of autolysis by MOL was evaluated using quantitative bacteriolytic assays and zymographic analysis, and antibiofilm activity assays and confocal laser scanning microscopy were used to elucidate the inhibition of biofilm formation caused by MOL in 20 clinical isolates or standard strains. The reduction in cidA, atl, sle1, and lytN transcript levels following MOL treatment was consistent with the induced expression of their autolytic repressors lrgA, lrgB, arlR, and sarA. MOL generally inhibited or reversed the expression of most of the genes involved in biofilm production. The growth of S. aureus strain ATCC 25923 in the presence of MOL dose-dependently led to decreases in Triton X-100-induced autolysis, extracellular murein hydrolase activity, and the amount of extracellular DNA (eDNA. MOL may impede biofilm formation by reducing the expression of cidA, a murein hydrolase regulator, to inhibit autolysis and eDNA release, or MOL may directly repress biofilm formation. CONCLUSIONS/SIGNIFICANCE: MOL shows in vitro antimicrobial activity against clinical and standard S. aureus strains grown in planktonic and biofilm cultures, suggesting that the structure of MOL may potentially be used as a basis for the development of drugs targeting biofilms.

  3. Release of Mannoproteins during Saccharomyces cerevisiae Autolysis Induced by Pulsed Electric Field (United States)

    Martínez, Juan M.; Cebrián, Guillermo; Álvarez, Ignacio; Raso, Javier


    The potential of the application of pulsed electric fields (PEF) to induce accelerate autolysis of a commercial strain of Saccharomyces cerevisiae for winemaking use was evaluated. The influence of PEF treatments of different intensity (5–25 kV/cm for 30–240 μs) on cell viability, cytoplasmic membrane permeabilization and release of mannoproteins and compounds absorbing at 260 and 280 nm has been investigated. After 8 days of incubation at 25°C the Abs600 of the suspension containing the control cells was kept constant while the Abs600 of the suspension containing the cells treated by PEF decreased. The measurement of the absorbance at 260 and 280 nm revealed no release of UV absorbing material from untreated cells after 8 days of incubation but the amount of UV absorbing material released drastically increased in the samples that contained cells treated by PEF after the same storage period. After 18 days of storage the amount of mannoproteins released from the untreated cell was negligible. Conversely, mannoprotein concentration increased linearly for the samples containing cells of S. cerevisiae treated by PEF. After 18 days of incubation the concentration of mannoproteins in the supernatant increased 4.2 times for the samples containing cells treated by PEF at 15 and 25 kV/cm for 45 and 150 μs. Results obtained in this study indicates that PEF could be used in winemaking to accelerate the sur lie aging or to obtain mannoproteins from yeast cultures. PMID:27672386

  4. Release of mannoproteins during Saccharomyces cerevisiae autolysis induced by Pulsed Electric Field

    Directory of Open Access Journals (Sweden)

    Juan Manuel Martínez


    Full Text Available The potential of the application of PEF to induce accelerate autolysis of a commercial strain of Sacharomyces cerevisiae for winemaking use was evaluated. The influence of PEF treatments of different intensity (5-25 kV/cm for 30-240 µs on cell viability, cytoplasmic membrane permeabilization, release of mannoproteins and compounds absorbing at 260 and 280 nm has been investigated.After 8 days of incubation at 25 ºC the Abs600 of the suspension containing the control cells was kept constant while the Abs600 of the suspension containing the cells treated by PEF decreased. The measurement of the absorbance at 260 and 280 nm revealed no release of UV absorbing material from untreated cells after 8 days of incubation but the amount of UV absorbing material released drastically increased in the samples that contained cells treated by PEF after the same storage period. After 18 days of storage the amount of mannoproteins released from the untreated cell was negligible. Conversely, mannoprotein concentration increased linearly for the samples containing cells of S. cerevisiae treated by PEF. After 18 days of incubation the concentration of mannoproteins in the supernatant increased 4.2 times for the samples containing cells treated by PEF at 15 and 25 kV/cm for 45 and 150 µs.Results obtained in this study indicates that PEF could be used in winemaking to accelerate the sur lie aging or to obtain mannoproteins from yeast cultures to be used in winemaking.

  5. Effect of protein S-nitrosylation on autolysis and catalytic ability of μ-calpain. (United States)

    Liu, Rui; Li, Yupin; Wang, Mengqin; Zhou, Guanghong; Zhang, Wangang


    The effect of S-nitrosylation on the autolysis and catalytic ability of μ-calpain in vitro in the presence of 50μM Ca(2 +) was investigated. μ-Calpain was incubated with different concentrations of nitric oxide donor S-nitrosoglutathione (GSNO) and subsequently reacted with purified myofibrils. Results showed that the amount of 80kDa μ-calpain subunit significantly decreased as GSNO increased from 0 to 300μM, but increases of GSNO to 300, 500 and 1000μM did not result in further inhibition. The catalytic ability of nitrosylated μ-calpain to degrade titin, nebulin, troponin-T and desmin was significantly reduced when the GSNO concentration was higher than 300μM. The cysteine residues of μ-calpain at positions 49, 351, 384, and 592 in the catalytic subunit and at 142 in small subunit were S-nitrosylated, which could be responsible for decreased μ-calpain activity. Thus, S-nitrosylation can negatively regulate the activation of μ-calpain resulting in decreased proteolytic ability on myofibrils.

  6. Release of Mannoproteins during Saccharomyces cerevisiae Autolysis Induced by Pulsed Electric Field. (United States)

    Martínez, Juan M; Cebrián, Guillermo; Álvarez, Ignacio; Raso, Javier


    The potential of the application of pulsed electric fields (PEF) to induce accelerate autolysis of a commercial strain of Saccharomyces cerevisiae for winemaking use was evaluated. The influence of PEF treatments of different intensity (5-25 kV/cm for 30-240 μs) on cell viability, cytoplasmic membrane permeabilization and release of mannoproteins and compounds absorbing at 260 and 280 nm has been investigated. After 8 days of incubation at 25°C the Abs600 of the suspension containing the control cells was kept constant while the Abs600 of the suspension containing the cells treated by PEF decreased. The measurement of the absorbance at 260 and 280 nm revealed no release of UV absorbing material from untreated cells after 8 days of incubation but the amount of UV absorbing material released drastically increased in the samples that contained cells treated by PEF after the same storage period. After 18 days of storage the amount of mannoproteins released from the untreated cell was negligible. Conversely, mannoprotein concentration increased linearly for the samples containing cells of S. cerevisiae treated by PEF. After 18 days of incubation the concentration of mannoproteins in the supernatant increased 4.2 times for the samples containing cells treated by PEF at 15 and 25 kV/cm for 45 and 150 μs. Results obtained in this study indicates that PEF could be used in winemaking to accelerate the sur lie aging or to obtain mannoproteins from yeast cultures.

  7. Effects of Some Physico-chemical Treatments on Autolysis of Brewer's Yeast%理化处理对啤酒酵母自溶的影响

    Institute of Scientific and Technical Information of China (English)

    张晓鸣; 袁信华; 章克昌


    利用酵母自溶动力学方程考察了温度、破壁酶、酵母预处理、酵母初始质量分数、盐等理化因素对啤酒酵母自溶过程的影响.温度对酵母自溶速度有很大影响.通过回归分析得到不同体系中反映自溶温度和速度常数关系的Arrhenius方程及相应的自溶反应的活化能Ea;破壁预处理能显著加速酵母自溶,添加破壁复合酶或食盐均有利于加速酵母自溶反应,但两者复合添加有相互抑制作用;酵母初始质量分数对自溶影响不大.%The effects of some physico-chemical treatments, such as temperature, lytic enzyme, pre-treatment of yeast, salt and initial concentration of yeast on autolysis were studied based on the autolysis kinetic rate constant(Ka). The autolysis temperature had great effect on Ka. The Arrhenius-type equations for different systems, which show the relationship between Ka and the absolute temperature(Tk), were obtained by using a linear least squares estimation method. The corresponding molecular activation energy of yeast autolysis could be calculated. The autolysis was significantly accelerated by lytic enzyme, pre-treatment of yeast, and salt, respectively, but the initial yeast concentration had little effect on the autolysis. The analysis of autolysates at the optimal autolysis conditions indicated that free amino acids prevailed in the protein extracts.

  8. Effects of yogurt starters autolysis on quality of yogurt%发酵剂菌体自溶对酸乳品质的影响

    Institute of Scientific and Technical Information of China (English)

    孙洁; 沈瑾; 王希卓; 田亚洲; 吕加平; 刘鹭


    通过研究发酵剂自溶对菌株产酸、凝乳能力,及对酸乳黏度、持水率、风味物质、后酸化程度等理化指标的影响.结果表明:自溶度较高的乳酸菌菌株在酸乳发酵过程中产酸性能更优,凝乳时间较短;菌株的自溶度对酸乳的黏度、持水率、风味物质成分及含量影响不大,但对酸乳的后酸化程度有着显著影响,高自溶度菌株发酵而成的酸乳在模拟货架期储藏条件下(10~15℃)后酸化程度相对较低.该文为开发新型复合发酵剂提供了理论依据.%For developing new composite yogurt starters and finding new lactic acid bacteria strains for starters, the effects of yogurt starters autolysis on quality of yogurt, such as acid producing ability and curd ability of strains, yogurt viscosity, water-holding ratio, flavor compounds, acidification in shelf-life were studied. The results showed that coagulation time decreased significantly by using high autolysis rate lactic acid bacteria strains fermented yogurt, were positively related to acid producing ability of lactic acid bacteria starters. There was no relative correlation between autolysis rate of strains and water-holding ratio, viscosity and flavor compounds of yogurt. However, yogurt acidities were significant influenced by autolysis rate of lactic acid bacteria starters, especially in the simulated shelf life of 10-15°C storage conditions. High autolysis rate starters could decrease acidification level of yogurt.

  9. Staphylococcus aureus Alters Growth Activity, Autolysis, and Antibiotic Tolerance in a Human Host-Adapted Pseudomonas aeruginosa Lineage

    DEFF Research Database (Denmark)

    Frydenlund Michelsen, Charlotte; Christensen, Anne-Mette; Bojer, Martin Saxtorph


    Interactions among members of polymicrobial infections or between pathogens and the commensal flora may determine disease outcomes. Pseudomonas aeruginosa and Staphylococcus aureus are important opportunistic human pathogens and are both part of the polymicrobial infection communities in human....... aeruginosa DK2 strains outcompeted S. aureus during coculture on agar plates, we found that later P. aeruginosa DK2 strains showed a commensal-like interaction, where S. aureus was not inhibited by P. aeruginosa and the growth activity of P. aeruginosa was enhanced in the presence of S. aureus. This effect...... is mediated by one or more extracellular S. aureus proteins greater than 10 kDa, which also suppressed P. aeruginosa autolysis and prevented killing by clinically relevant antibiotics through promoting small-colony variant (SCV) formation. The commensal interaction was abolished with S. aureus strains mutated...

  10. 虾蛋白自溶与外源酶促水解作用研究%Study on the Autolysis and Enzymatic Hydrolysis of Shrimp Protein

    Institute of Scientific and Technical Information of China (English)

    叶伟娟; 吴少辉; 于新


    目的:研究虾蛋白的自溶与外源酶促水解作用,为开发新型虾味高档调味品提供实验依据.方法:以基围虾为原料,氨基态氮含量、水解度(DH)为指标,研究pH、温度、时间、料液比对基围虾自溶水解效果的影响,并在最优条件下添加不同外源酶,探讨其对虾蛋白的酶促作用.结果:通过单因素和L9(33)正交试验获得基围虾自溶的最佳工艺参数是:料液比1:8(g/mL),温度60℃,pH 6.5,时间8h.结论:在此条件下,氨基态氮含量1.7 g/L,水解度45.88%.外源木瓜蛋白酶具有较好的酶促水解作用,菠萝蛋白酶、中性蛋白酶、胰蛋白酶对虾蛋白的酶促水解作用不显著.%Objective:This paper studied on the autolysis and enzymatic hydrolysis of shrimp protein in order to provide experimental basis for a development of new and high-grade sauce. Method:In an autolysis experiment with Metapenaeus ensis as the main raw material, amino nitrogen content and DH were taken as indicators to determine the effects of pH, temperature, hydrolysis time and solid-liquid ratio on hydrolysis. And the effect of different exogenous enzymes on autolysis was investigated. Results: Using single-factor and L9(33) orthogonal experiments, the conditions for Metapenae us ensis autolysis were optimized, the best technological parameters being solid—liquid ratio l:8(g/mL), reaction temperature 60 ℃, pH 6.5 and reaction time 8 h. Conclusion:Under the optimized, amino nitrogen was 1.7 g/L, DH was 45.88%. The exogenous enzymes of papain promoted the autolysis of the shrimp protein preferably, while bromelain, neutral protease and trypsin were not significant.

  11. Optimizing conditions for autolysis of Spirulina using response surface methodology(RSM)%响应面法优化螺旋藻自溶条件的研究

    Institute of Scientific and Technical Information of China (English)

    冯强; 滕建文; 黄丽; 韦保耀


    The autolysis of Spirulina was optimised using response surface methodolygy.Twenty test were conducted following a Central Composite Designs-Uniform precision(CCDU) followed by ridge analysis using the TCA-solution peptides concentration,the autolysis tem%以新鲜螺旋藻藻泥为原料,通过响应面实验设计方法对螺旋藻自溶条件进行优化。以可溶性肽含量为响应变量,温度、pH以及Na+浓度为自变量,应用中心复合设计-一致精度(CCD Uniform precision)法设计实验,轴值取1.43,6个中心点,共20组实验。实验结果表明,螺旋藻自溶最适温度为48.6℃、pH为9.6以及Na+浓度为1.00mol/L,最适条件下螺旋藻自溶1hTCA-可溶性肽含量比未优化(室温25℃,加蒸馏水)下高6.35倍。

  12. Influences of food addictive on protein autolysis of Euphausia superba%食品添加剂对南极大磷虾蛋白自溶的影响

    Institute of Scientific and Technical Information of China (English)

    迟海; 李学英; 杭虞杰; 杨宪时; 郭全友


    以氨基态氮、可溶性蛋白质含量和可溶性固形物含量为指标,研究了食品添加剂对南极大磷虾(Euphausia superba)蛋白自溶的影响.结果显示,氯化镁和柠檬酸对南极大磷虾蛋白的自溶有一定的促进作用,两者作用显著的添加量分别为2%和0.2%,结合可溶性蛋白质和可溶性固形物指标,促进作用最显著的为添加0.2%柠檬酸;葡萄糖和氯化钙对南极大磷虾蛋白的自溶影响不大;氯化钾、氯化钠、EDTA-2Na对南极大磷虾蛋白的自溶有一定的抑制作用,氯化钠和EDTA-2Na的抑制作用相近,但优于氯化钾,三者作用显著的添加量分别为2%、4%、0.05%,考虑到可溶性蛋白质含量,抑制作用最显著为添加0.05% EDTA-2Na.%Based on the testing indexes of Amino Nitrogen Contents (ANC), Soluble Protein Concentration ( SPC) and Total Soluble Solids (TSS), influences of food addictive on protein autolysis of Euphausia superba were studied. Results showed that effects of magnesium chloride and citric acid on protein autolysis of Euphausia superba had certain simulative effect. Both optimum substitutabilities were 2% and 0. 2% , respectively. Combined soluble protein concentration and total soluble solid indexes, the optimum was adding 0.2% citric acid. Glucose and calcium chloride protein autolysis of Euphausia superba had little impact on protein autolysis; Potassium chloride, sodium chloride and EDTA-2Na had certain inhibition on protein autolysis of Euphausia superba, and inhibition of sodium chloride and EDTA-2Na was similar, both better than potassium chloride. The most suitable adding amount of for the three were 2% , 4% , and 0. 05%. Considering the soluble protein concentration, the best for autolysis was adding 0.05% EDTA-2Na.

  13. Effects of the strain background and autolysis process on the composition and biophysical properties of the cell wall from two different industrial yeasts. (United States)

    Schiavone, Marion; Sieczkowski, Nathalie; Castex, Mathieu; Dague, Etienne; Marie François, Jean


    The Saccharomyces cerevisiae cell surface is endowed with some relevant technological properties, notably antimicrobial and biosorption activities. For these purposes, yeasts are usually processed and packaged in an 'autolysed/dried' formula, which may have some impacts on cell surface properties. In this report, we showed using a combination of biochemical, biophysical and molecular methods that the composition of the cell wall of two wine yeast strains was not altered by the autolysis process. In contrast, this process altered the nanomechanical properties as shown by a 2- to 4-fold increased surface roughness and to a higher adhesion to the atomic force microscope tips of the autolysed cells as compared to live yeast cells. Besides, we found that the two strains harboured differences in biomechanical properties that could be due in part to higher levels of mannan in one of them, and to the fact that the surface of this mannan-enriched strain is decorated with highly adhesive patches forming nanodomains. The presence of these nanodomains could be correlated with the upregulation of flocculin encoding FLO11 as well as to higher expression of few other genes encoding cell wall mannoproteins in this mannan-enriched strain as compared to the other strain.

  14. Discrimination of three mutational events that result in a disruption of the R122 primary autolysis site of the human cationic trypsinogen (PRSS1 by denaturing high performance liquid chromatography

    Directory of Open Access Journals (Sweden)

    Férec Claude


    Full Text Available Abstract Background R122, the primary autolysis site of the human cationic trypsinogen (PRSS1, constitutes an important "self-destruct" or "fail-safe" defensive mechanism against premature trypsin activation within the pancreas. Disruption of this site by a missense mutation, R122H, was found to cause hereditary pancreatitis. In addition to a c.365G>A (CGC>CAC single nucleotide substitution, a c.365~366GC>AT (CGC>CAT gene conversion event in exon 3 of PRSS1 was also found to result in a R122H mutation. This imposes a serious concern on the genotyping of pancreatitis by a widely used polymerase chain reaction-restriction fragment length polymorphism assay, which could only detect the commonest c.365G>A variant. Materials and methods DNA samples containing either the known c.365G>A or c.365~366GC>AT variant in exon 3 of PRSS1 were used as positive controls to establish a denaturing high performance liquid chromatography (DHPLC assay. Results DHPLC could readily discriminate the two known different mutational events resulting in the R122H mutation. More importantly, under the same experimental conditions, it identified a further mutational event that also occurs in the R122 primary autolysis site but results in a different amino acid substitution: c.364C>T (CGC>TGC; R122C. Conclusions A rapid, simple, and low-cost assay for detecting both the known and new mutations occuring in the R122 primary autolysis site of PRSS1 was established. In addition, the newly found R122C variant represents a likely pancreatitis-predisposing mutation.

  15. Optimization of processing techniques for autolysis of yeast cell from waste wine yeast%葡萄酒泥酵母诱导自溶工艺的条件优化

    Institute of Scientific and Technical Information of China (English)

    李颍; 郭晓; 张彦芳; 马滕臻; 张莉; 祝霞; 韩舜愈; 杨学山


    The main factors that influence autolysis of yeast cell were screened by single factor and or-thogonal tests.The results showed that the optimal temperature was 47.5 ℃,the mass fraction of NaCl was 2%,pH was 4.5,react time was 33 h.Under these conditions,obtained extract's conductivity increased 2.49 ms/cm,amino nitrogen content increased 2.15 490 mg/mL after autolysis.The process was inexpen-sive and simple and could be applied to industrialized production.%以葡萄酒泥酵母为试验材料,采用单因素和正交试验设计,优化酵母细胞自溶的工艺条件.结果表明:在温度47.5℃,NaCl质量分数2%,pH 4.5,处理时间33h的最优条件下,酵母自溶前后电导率增加2.49 ms/cm,氨基氮含量增加2.15490 mg/mL,自溶效果最好.此方法成本低,易操作,适合工业化生产.

  16. Protease production during growth and autolysis of submerged Metarhizium anisopliae cultures Produção de protease durante o crescimento e análise de culturas submersas de Metarhizium anisopliae

    Directory of Open Access Journals (Sweden)

    Gilberto U.L. Braga


    Full Text Available The growth and autolysis of two strains of the entomopathogenic deuteromycete fungus Metarhizium anisopliae var. anisopliae were evaluated in medium containing casein or glucose as carbon source. Parameters such as economic coefficient and degree of autolysis were determined for each strain. Protease production was determined throughout the growth and autolysis phases of the cultures on medium under conditions of protease induction (in the presence of casein as sole source of carbon and nitrogen. The fungus was shown to utilize casein as a carbon/energy source in a more efficient manner than glucose. The autolysis shown by the strains was intense under both types of growth conditions, reaching up to 62.7% of the dry mass produced and started soon after the depletion of the exogenous carbon source. The relationship between the proteolytic activities of the two strains evaluated varied significantly (a maximum of 19.78 on the 5th day and a minimum of 2.03 on the 16th day of growth during the various growth and autolysis phases, clearly showing that the difference between the growth curves and the difference in the kinetics of enzyme production may decisively affect the process of strain selection for protease production.O crescimento e a autólise de duas linhagens do deuteromiceto entomopatogênico Metarhizium anisopliae var. anisopliae foram avaliados em meio contendo caseína ou glicose como fonte de carbono. Foram determinados parâmetros como o coeficiente econômico e o grau de autólise apresentado pelas linhagens. A produção de protease foi determinada durante todas as fases do crescimento e da autólise das culturas, em meio indutor da produção de proteases (meio contendo caseína como única fonte de carbono e de nitrogênio. Pôde-se verificar que o fungo foi capaz de utilizar a caseína como fonte de carbono/energia de maneira mais eficiente do que a glicose. A autólise apresentada pelas linhagens foi intensa em ambas as condi

  17. Effect of N-acetylmuramidase Knockout onAutolysis and Morphology of Lactobacillus bulgaricus LJJ%N-乙酰胞壁质酶缺失对保加利亚乳杆菌LJJ自溶及形态的影响

    Institute of Scientific and Technical Information of China (English)

    崔文明; 逄晓阳; 张书文; 刘鹭; 李红娟; 吕加平


    菌株的自溶度约为73%,而突变菌株的自溶度约为35%,自溶度降为原来的1/2。野生型菌株的单个菌体细胞长度约10μm,突变菌株的单个菌体细胞约30-40μm,相比野生型菌株约增长了3-4倍。N-乙酰胞壁质酶在保加利亚乳杆菌自溶与细胞分裂过程中起着重要作用。保加利亚乳杆菌中N-乙酰胞壁质酶的缺失会导致菌体自溶的降低和菌体增殖过程中的细胞分裂受阻,产生菌体长度增长约3-4倍的菌体细胞。%[Objective] Autolysis of lactic acid bacteria exists widely in the production of fermented dairy products. The rate and extent of autolysis not only play an important role in the whole fermentation process, but also have a vital effect on the quality, flavor and production cycle of the product. As main component of peptidoglycan hydrolase, N- acetylmuramidase can destroy the cell wall integrity in lactic acid bacteria autolysis process. It plays a crucial role in the autolysis of lactic acid bacteria. This paper mainly studied the influence of N-acetylmuramidase deletion on autolysis and morphology ofLactobacillus bulgaricus LJJ.[Method] The primers of upstream homologous arm m-up, downstream homologous arm m-down of N-acetylmuramidase and erythromycin resistance genes were designed based on the sequences ofL. bulgaricus genome and pMG36e vector. Then m-up, m-down and erythromycin resistance gene were amplified by PCR and verified by gene sequence analysis. Then erythromycin resistance gene and pUC19 were degested withKpnΙ,XbaΙ and connected together. The recombinant vector pUC:Emr was verified by double-enzyme cleavage method. Next, m-down and recombinant vector pUC: Emr were digested withKpnΙ,SacΙ and connected together. The recombinant vector pUC:Emr:m-down was confirmed by double-enzyme cleavage method. Finally, the m-up and recombinant plasmid pUC:Emr:m-down were digested withPstΙ,XbaΙ and connected together. The recombinant vector pUC:m-up:Emr:m-down was verified by

  18. Efecto de tres ambientes de transporte sobre el tiempo de aparición de la autólisis en muestras de alevines de trucha arcoíris (Oncorhynchus mykiss Effect of three transport conditions on the appearance time of autolysis in samples of rainbow trout fry (Oncorhynchus mykiss

    Directory of Open Access Journals (Sweden)

    AR Ortloff


    C. Every hour from 0 to 24 hours, groups of 4 fry per condition were fixed in 10% formalin and processed using the conventional hematoxylin and eosin stain technique (H&E. The appearance time of the autolytic changes in the different segments of the kidney and liver were determined in each group, classifying the changes as early autolytic, advanced autolytic and complete autolysis. It was observed that the autolytic changes appeared first in the livers of the corpses at 4 °C under dry condition while last ones to be affected were the corpses submerged in water at 4 °C (1 and 6 hours, respectively. The first autolytic changes appeared in the kidneys of the corpses at 4 °C in the dry atmosphere and the last in the corpses submerged in water at 4 °C (3 and 9 hours, respectively. It was concluded that out of the three transport conditions studied, the one preserving for longer the histological and cytological structure of the fish tissue for histological analysis was water preservation at 4 °C, demonstrating the key role of the temperature and atmospheric humidity in the progress of fish autolysis.

  19. Effect of Bovine Plasma Protein on Autolysis and Gelation of Protein Extracted from Giant Squid (Dosidicus gigas Mantle

    Directory of Open Access Journals (Sweden)

    Laura Raquel Marquez-Alvarez


    Full Text Available The effect of bovine plasma protein (BPP on the inhibition of autolytic activity and its effect on the gelling properties of a protein concentrate (PC obtained from jumbo squid (Dosidicus gigas mantle were investigated. Sols and gels were prepared from the PC by adding different amounts of BPP (0, 1, and 2%. Dynamic oscillatory measurements indicated that systems with 1% BPP had a higher elastic modulus (G′, in which hydrophobic interactions were favored. Concerning the technological and textural quality of the gels, BPP caused a greater water holding capacity (WHC, force, cohesiveness, and elasticity, probably due to improvement of the electrostatic and hydrophobic interactions during gel formation. Scanning electron microscopy (SEM allowed visualization of the formation of more rigid and ordered gels with less porosity when BPP was added. Therefore, the addition of BPP improved the gelling capacity of proteins extracted from giant squid.

  20. Can the development and autolysis of lactic acid bacteria influence the cheese volatile fraction? The case of Grana Padano. (United States)

    Lazzi, Camilla; Povolo, Milena; Locci, Francesco; Bernini, Valentina; Neviani, Erasmo; Gatti, Monica


    In this study, the relationship between the dynamics of the growth and lysis of lactic acid bacteria in Grana Padano cheese and the formation of the volatile flavor compounds during cheese ripening was investigated. The microbial dynamics of Grana Padano cheeses that were produced in two different dairies were followed during ripening. The total and cultivable lactic microflora, community composition as determined by length heterogeneity-PCR (LH-PCR), and extent of bacterial lysis using an intracellular enzymatic activity assay were compared among cheeses after 2, 6 and 13months of ripening in two dairies. The evolution of whole and lysed microbiota was different between the two dairies. In dairy 2, the number of total cells was higher than that in dairy 1 in all samples, and the number of cells that lysed during ripening was lower. In addition, at the beginning of ripening (2months), the community structure of the cheese from dairy 2 was more complex and was composed of starter lactic acid bacteria (Lactobacillus helveticus and Lactobacillus delbrueckii) and NSLAB, possibly arising from raw milk, including Lactobacillus rhamnosus/Lactobacillus casei and Pediococcus acidilactici. On the other hand, the cheese from dairy 1 that ripened for 2months was mainly composed of the SLAB L. helveticus and L. delbrueckii. An evaluation of the free-DNA fraction through LH-PCR identified those species that had a high degree of lysis. Data on the dynamics of bacterial growth and lysis were evaluated with respect to the volatile profile and the organic acid content of the two cheeses after 13months of ripening, producing very different results. Cheese from dairy 1 showed a higher content of free fatty acids, particularly those deriving from milk fat lipolysis, benzaldehyde and organic acids, such as pGlu and citric. In contrast, cheese from dairy 2 had a greater amount of ketones, alcohols, hydrocarbons, acetic acid and propionic acid. Based on these results, we can conclude that in the first cheese, the intracellular enzymes that were released from lysis were mainly involved in aroma formation, whereas in the second cheese, the greater complexity of volatile compounds may be associated with its more complex microbial composition caused from SLAB lysis and NSLAB (mainly L. rhamnosus/L. casei) growth during ripening.

  1. Study on Autolysis Conditions of Waste Brewer's Yeast%啤酒废酵母自溶条件的研究

    Institute of Scientific and Technical Information of China (English)




  2. Study on Autolysis Conditions of Waste Brewer's Yeast%啤酒废酵母自溶条件的研究

    Institute of Scientific and Technical Information of China (English)




  3. 啤酒废酵母变温自溶条件的研究%Study on autolysis conditiohs of spent brewer yeast by changing temperature

    Institute of Scientific and Technical Information of China (English)

    蒋雪薇; 罗晓明; 刘永乐



  4. Study on the Autolysis Kinetics of Brewer Yeast%啤酒酵母自溶动力学的研究

    Institute of Scientific and Technical Information of China (English)

    张晓鸣; 袁信华; 章克昌



  5. Autolysis of Brewers' Yeast and Its Influence on Beer Quality%啤酒酵母的自溶及对啤酒质量的影响

    Institute of Scientific and Technical Information of China (English)

    郝秋娟; 李崎; 顾国贤



  6. Impact of NaCl reduction in Danish semi-hard Samsoe cheeses on development and autolysis of DL-starter cultures

    DEFF Research Database (Denmark)

    Søndergaard, Lise; Ryssel, Mia; Svendsen, Carina


    of two commercial DL-starter cultures (C1 and C2) used in the production of Danish semi-hard Samsoe cheeses were affected by reduced NaCl levels. Cheeses containing reduced-salt) and 3.4% (normal-salted) (w/v) NaCl in moisture were produced and analyzed during 12weeks of ripening.......e. 22% and 21%), whereas no significant difference was found between the reduced-salt (i.e. 31% and 35%) and normal-salted cheeses. At the later stages of ripening (i.e. 7 and 11weeks) NaCl had no significant influence. For cheeses produced with C2, a significant influence of NaCl was only found...... in cheeses ripened for 7weeks, where the unsalted and reduced-salt cheeses had retained a significantly higher number of the initial LAB counts (cfu/g) (i.e. 39% and 38%), compared to the normal-salted cheeses (i.e. 21%). In the Samsoe cheeses, bacteria were organized as single cells, in groups of 2-3 cells...

  7. Detection of Lawsonia intracellularis in formalinfixed Porcine Intestinal Tissue Samples: Comparison of Immunofluorescense and In-situ Hybridization, and Evaluation of the Effectf of Controlled Autolysis

    DEFF Research Database (Denmark)

    Jensen, Tim Kåre; Boesen, H. T.; Vigre, Håkan;


    Two methods, an immunofluorescence assay (IFA; with a Lawsonia intracellularis-specific monoclonal antibody) and fluorescent in-situ hybridization (FISH; with a specific oligonucleotide probe targeting 16S ribosomal RNA of the bacterium), were compared for their ability to detect L. intracellularis...... (the cause of porcine proliferative enteritis [PE]) in formalin-fixed samples of intestinal tissue. Of 69 intestinal samples with gross lesions of PE, 63 were positive by both FISH and IFA, but six were positive only by IFA. This indicated that the sensitivity of FISH was 91% that of IFA. However, both...

  8. 啤酒废酵母复合促溶剂促溶效果的研究%Study on autolysis effect of spent brewer's yeast with complex promoter

    Institute of Scientific and Technical Information of China (English)

    蒋雪薇; 罗晓明; 刘永乐


    研究几种单一促溶剂及复合促溶剂的促溶效果,优化了复合促溶剂配比,得出其最佳值为NaCl 2%(W/W)、乙醇1%(V/V)、葡萄糖1%(W/W).以此促溶啤酒废酵母,其抽提液氨基氮含量比传统促溶剂(NaCl)提高了9.6%,收率提高了8%,而自溶时间则缩短了12h.

  9. Ethanol-Glycerin Fixation with Thymol Conservation: A Potential Alternative to Formaldehyde and Phenol Embalming (United States)

    Hammer, Niels; Loffler, Sabine; Feja, Christine; Sandrock, Mara; Schmidt, Wolfgang; Bechmann, Ingo; Steinke, Hanno


    Anatomical fixation and conservation are required to prevent specimens from undergoing autolysis and decomposition. While fixation is the primary arrest of the structures responsible for autolysis and decomposition, conservation preserves the state of fixation. Although commonly used, formaldehyde has been classified as carcinogenic to humans. For…

  10. Evolution of Bacterial Suicide (United States)

    Tchernookov, Martin; Nemenman, Ilya


    While active, controlled cellular suicide (autolysis) in bacteria is commonly observed, it has been hard to argue that autolysis can be beneficial to an individual who commits it. We propose a theoretical model that predicts that bacterial autolysis is evolutionarily advantageous to an individualand would fixate in physically structured environments for stationary phase colonies. We perform spatially resolved agent-based simulations of the model, which predict that lower mixing in the environment results in fixation of a higher autolysis rate from a single mutated cell, regardless of the colony's genetic diversity. We argue that quorum sensing will fixate as well, even if initially rare, if it is coupled to controlling the autolysis rate. The model does not predict a strong additional competitive advantage for cells where autolysis is controlled by quorum sensing systems that distinguish self from nonself. These predictions are broadly supported by recent experimental results in B. subtilisand S. pneumoniae. Research partially supported by the James S McDonnell Foundation grant No. 220020321 and by HFSP grant No. RGY0084/2011.

  11. High Technology Mass Spectrometry Laboratory (United States)


    Corbett, F.W. Benz and R.S. Doyle. Acrylonitrile induces autolysis Bacillus subtilis. FEMS Microbiology Letters 182: 255-258 (2000). 25. Nerland, D.E., J...Supplement, 391 (1999). 24. Benz, F.W., J Li, D Corbett, T.S. Chen and D E Nerland. Neither Direct Chemical Nor Cyanide Antagonism Can Fully Explain The


    Directory of Open Access Journals (Sweden)

    L. V. Antipova


    Full Text Available Functional foods represented an important element of a balanced, healthy diet. They play an important role in improving the structure of the diet of the population, are a means of prevention, correction and prevention of early transition premorbid conditions and various diseases. Meat and meat products based on it can be considered as a promising raw material for functional foods. Goat has certain dietary properties and can be used in manufacturing, but the production of high-grade products from goat meat, capable of long-term storage is not well developed. The chemical composition of the new raw meat - goat meat, on the example of the longissimus muscle of back is rich in protein, moisture, ash, and low-fat compared with other types of raw meat, which lets you create based on goat meat sector wide functional meat products. Therefore investigated the possibility of using goat meat in the production technology of functional foods. Showing the prospects for the development of goat in Russia. Investigated character of autolysis goat meat electro-physical methods and histological analysis. In the process of autolysis of goat meat are changing the electrical properties of raw meat, which are correlated with the morphological characteristics. Basic autolytic changes occurring in muscle tissue in the early stages of ripening, were reduced to a small extent the political process involving varying degrees of muscle structure in the early stages. In the later stages of maturation revealed changing widespread. Revealed that autolysis has characteristic periods and develops within 12 hours, further changes are irreversible. Shows the change in pH, carbohydrate fractions in the longissimus muscle of back goat. Results of the study of dynamics of change in pH, carbohydrate fractions in the longissimus muscle of back goat show compliance with the laws of classical autolysis noted in other sources, but differ in the time period. Defined stages of autolysis in

  13. Purification and partial characterisation of a cathepsin L-like proteinase from sea cucumber (Stichopus japonicus) and its tissue distribution in body wall. (United States)

    Zhou, Da-Yong; Chang, Xian-Na; Bao, Sha-Sha; Song, Liang; Zhu, Bei-Wei; Dong, Xiu-Ping; Zong, Yuan; Li, Dong-Mei; Zhang, Mao-Mao; Liu, Yu-Xin; Murata, Yoshiyuki


    A cathepsin L-like proteinase (CLP) with molecular weight of 30.9 kDa from the gut of sea cucumber (Stichopus japonicas, S. japonicus) was isolated and purified to homogeneity by several chromatographic procedures. The enzyme exhibited optimum activity at pH 5.0-5.5 and 50 °C, and showed thermostability up to 40 °C. The enzyme activity was completely inhibited by Zn(2+), strongly inhibited by Fe(2+) and Cu(2+), drastically reduced by cysteine proteinase inhibitors, but slightly enhanced by thiol-activating agents. The enzyme efficiently hydrolysed the specific substrate of cathepsin L, but hardly hydrolysed the specific substrates for cathepsin B, cathepsin H and cathepsin K. Immunohistochemical studies indicated that the CLP was more abundant in the epidermis rather than in the dermis of S. japonicus body wall. The distribution of CLP showed positive correlation with autolysis rate. Therefore, the relationship between CLP and autolysis deserved further study.

  14. Chitinase but N-acetyl-β-D-glucosaminidase production correlates to the biomass decline in Penicillium and Aspergillus species. (United States)

    Pusztahelyi, Tünde; Pócsi, István


    Hydrolytic enzyme production is typical of the autolysis in filamentous fungi; however, less attention has been given to the physiological role of the enzymes. Here, the aim was to investigate the possible relation of the chitinolytic enzymes to the changes in the biomass in some filamentous fungi of high importance for pharmaceutical or food industry. In Penicillium and Aspergillus filamentous fungi, which showed different characteristics in submerged cultures, the growth and biomass decline rates were calculated and correlated to the chitinase and N-acetyl-β-D-glucosaminidase enzyme productions. Correlation was found between the biomass decrease rate and the chitinase level at the stationary growth phase; while chitinase production covariates negatively with N-acetyl-β-D-glucosaminidase activities. The chitinase production and the intensive autolysis hindered the production of N-acetyl-β-D-glucosaminidase and, therefore, could hinder the cell death in the cultures.

  15. Influence of short-term fixation with mixed formalin or ethanol solution on the mechanical properties of human cortical bone


    Mick E.; Steinke H.; Wolfskämpf T.; Wieding J.; Hammer N.; Schulze M.; Souffrant R.; Bader R.


    Bone specimens obtained for biomechanical experiments are fresh-frozen for storage to slow down tissue degradation and autolysis in long-term storage. Alternatively, due to infectious risks related to the fresh tissues, fixative agents are commonly used. However, fixatives will likely change the mechanical properties of bone. Existing studies on this issue gave controversial results that are hardly comparable due to a variety of measurement approaches. For this reason, the influence of ethano...

  16. Intravascular "mulberry-like" bodies

    DEFF Research Database (Denmark)

    Sørensen, Flemming Brandt; Klebe, J G; Henriques, U V


    Intravascular "mulberry-like" bodies in a stillborn female infant with moderate maceration are reported. The histogenesis of these structures is discussed based on light-microscopic, immunohistochemical and ultrastructural findings. No demonstrable causal relation between the intravascular lesions...... and fetal death was found, the cause of death being attributed to intrauterine asphyxia. It is concluded, that intravascular "mulberry-bodies" most likely represent artifacts due to red blood cell autolysis....

  17. Utilization of baker's yeast (Saccharomyces cerevisiae for the production of yeast extract: effects of different enzymatic treatments on solid, protein and carbohydrate recovery

    Directory of Open Access Journals (Sweden)



    Full Text Available Yeast extract (YE was produced from commercial pressed baker's yeast (active and inactivated using two enzymes: papain and lyticase. The effects of enzyme concentration and hydrolysis time on the recovery of solid, protein and carbohydrate were investigated. Autolysis, as a basic method for cell lysis was also used and the results compared. The optimal extraction conditions were investigated. The optimal concentrations of papain and lyticase were found to be 2.5 % and 0.025 %, respectively.

  18. Proteins influencing foam formation in wine and beer: the role of yeast


    Blasco, Lucía; Viñas, Miquel; Villa, Tomás G


    This review focuses on the role of proteins in the production and maintenance of foam in both sparkling wines and beer. The quality of the foam in beer but especially in sparkling wines depends, among other factors, on the presence of mannoproteins released from the yeast cell walls during autolysis. These proteins are hydrophobic, highly glycosylated, and their molecular masses range from 10 to 200 kDa characteristics that allow mannoproteins to surround and thus stabilize the gas bubbles o...

  19. Expression of Metabolic and Apoptotic Genes During Treatment With Chemopreventive Agents for Breast Cancer (United States)


    Minneapolis, MN. Cancer -preventive properties of the components of cruciferous vegetables including indoles have been studied extensively. 3,3...carbinol (13C) is a product of autolysis from glucobrassicin in cruciferous vegetables . It is condensed to 3,3’-diindolylmethane (DIM) and other products in...AD Award Number: DAMD17-01-1-0332 TITLE: Expression of Metabolic and Apoptotic Genes During Treatment with Chemopreventive Agents for Breast Cancer

  20. Deletion of BCY1 from the Saccharomyces cerevisiae Genome Is Semidominant and Induces Autolytic Phenotypes Suitable for Improvement of Sparkling Wines (United States)

    Tabera, Laura; Muñoz, Rosario; Gonzalez, Ramon


    Autolysis of Saccharomyces cerevisiae is the main source of molecules that contribute to the quality of sparkling wines made by the traditional method. In this work the possibility of accelerating this slow process in order to improve the quality of sparkling wines by using genetically engineered wine yeast strains was explored. The effect of partial or total deletion of BCY1 (which encodes a regulatory subunit of cAMP-dependent protein kinase A) in haploid and diploid (heterozygous and homozygous) yeast strains was studied. We proved that heterozygous strains having partial or complete BCY1 deletions have a semidominant phenotype for several of the properties studied, including autolysis under simulated second-fermentation conditions, in contrast to previously published reports describing mutations in BCY1 as recessive. Considering the degree of autolysis, ethanol tolerance, and technical feasibility, we propose that deletion of the 3′ end of the open reading frame of a single copy of BCY1 is a way to improve the quality of sparkling wines. PMID:16597929

  1. The carbon starvation response of the ectomycorrhizal fungus Paxillus involutus. (United States)

    Ellström, Magnus; Shah, Firoz; Johansson, Tomas; Ahrén, Dag; Persson, Per; Tunlid, Anders


    The amounts of carbon allocated to the fungal partner in ectomycorrhizal associations can vary substantially depending on the plant growth and the soil nutrient conditions, and the fungus may frequently be confronted with limitations in carbon. We used chemical analysis and transcriptome profiling to examine the physiological response of the ectomycorrhizal fungus Paxillus involutus to carbon starvation during axenic cultivation. Carbon starvation induced a decrease in the biomass. Concomitantly, ammonium, cell wall material (chitin) and proteolytic enzymes were released into the medium, which suggest autolysis. Compared with the transcriptome of actively growing hyphae, about 45% of the transcripts analyzed were differentially regulated during C-starvation. Induced during starvation were transcripts encoding extracellular enzymes such as peptidases, chitinases and laccases. In parallel, transcripts of N-transporters were upregulated, which suggest that some of the released nitrogen compounds were re-assimilated by the mycelium. The observed changes suggest that the carbon starvation response in P. involutus is associated with complex cellular changes that involves autolysis, recycling of intracellular compounds by autophagy and reabsorption of the extracellular released material. The study provides molecular markers that can be used to examine the role of autolysis for the turnover and survival of the ectomycorrhizal mycelium in soils.

  2. Histopathologic evaluation of postmortem autolytic changes in bluegill (Lepomis macrohirus and crappie (Pomoxis anularis at varied time intervals and storage temperatures

    Directory of Open Access Journals (Sweden)

    Jami George


    Full Text Available Information is lacking on preserving fish carcasses to minimize postmortem autolysis artifacts when a necropsy cannot be performed immediately. The purpose of this study was to qualitatively identify and score histologic postmortem changes in two species of freshwater fish (bluegill—Lepomis macrochirus; crappie—Pomoxis annularis, at varied time intervals and storage temperatures, to assess the histologic quality of collected samples. A pooled sample of 36 mix sex individuals of healthy bluegill and crappie were euthanized, stored either at room temperature, refrigerated at 4 °C, or frozen at −20 °C, and then necropsied at 0, 4, 24, and 48 h intervals. Histologic specimens were evaluated by light microscopy. Data showed that immediate harvesting of fresh samples provides the best quality and refrigeration would be the preferred method of storage if sample collection had to be delayed for up to 24 h. When sample collection must be delayed more than 24 h, the preferred method of storage to minimize autolysis artifacts is freezing if evaluation of the gastrointestinal tract is most important, or refrigeration if gill histology is most important. The gill arch, intestinal tract, followed by the liver and kidney were the most sensitive organs to autolysis.

  3. 反刍动物瘤胃原虫自溶现象的初步研究

    Institute of Scientific and Technical Information of China (English)

    王欢莉; 王洪荣; 徐爱秋; 赵庆新; 姜小波


    瘤胃原虫自溶的初步研究,可为反刍动物瘤胃内微生物蛋白循环的研究及相关调控技术的开展提供基础资料。本试验采用氧诱导自溶的方法,在体外培养条件下研究原虫形态、自溶度、蛋白酶活性及脂肪酶活性的变化。结果表明,氧诱导处理原虫形态变化明显,表现出自溶特征。前期(0~4 h)自溶速度较快,4 h胞内蛋白释放达50%。自溶组蛋白酶活性较对照组降低9.92%(P<0.05);而脂肪酶活性较对照组提高41.01%(P<0.01)。胞外蛋白酶活性与原虫自溶度相关性最高(r=0.703)。脂肪酶活性对于原虫自溶度的直接效应最强(P=0.586)。综上所述,氧诱导处理使瘤胃原虫发生自溶,据此建立自溶模型。脂肪酶的作用是瘤胃原虫自溶的关键因素。%The purpose of this article is to find out the autolytic characteristic of rumen protozoa,and to provide some basic information for the study of microbial protein circulation and correlating regulation technique.The following parameters in vitro including conformation of protozoa,protein content,degree of autolysis,activity of protease and lipase were studied, with the method of oxygen inducement.The conformation change of protozoa conformation was evident induced by oxygen. There existed a sharp increase in the rate of autolysis during the prophase(0~4 h).The release of protein got 50%in 4 h.The protease activity outside of protozoa in oxygen-induced group was 9.92%lower than that of control group(P<0.05).The lipase activity outside of protozoa in oxygen-induced group was 41.01%higher than that of control group (P<0.01).There existed positive correlation between the protease activity outside of protozoa and autolysis degree of protozoa (r=0.703).The direct effect of the lipase activity on autolysis degree of protozoa was the strongest (P=0.586).The autolysis model of rumen protozoa was established in this study induced by oxygen

  4. SCCmec-associated psm-mec mRNA promotes Staphylococcus epidermidis biofilm formation. (United States)

    Yang, Yongchang; Zhang, Xuemei; Huang, Wenfang; Yin, Yibing


    Biofilm formation is considered the major pathogenic mechanism of Staphylococcus epidermidis-associated nosocomial infections. Reports have shown that SCCmec-associated psm-mec regulated methicillin-resistant Staphylococcus aureus virulence and biofilm formation. However, the role of psm-mec in S. epidermidis remains unclear. To this purpose, we analysed 165 clinical isolates of S. epidermidis to study the distribution, mutation and expression of psm-mec and the relationship between this gene and biofilm formation. Next, we constructed three psm-mec deletion mutants, one psm-mec transgene expression strain (p221) and two psm-mec point mutant strains (pM, pAG) to explore its effects on S. epidermidis biofilm formation. Then, the amount of biofilm formation, extracellular DNA (eDNA) and Triton X-100-induced autolysis of the constructed strains was measured. Results of psm-mec deletion and transgene expression showed that the gene regulated S. epidermidis biofilm formation. Compared with the control strains, the ability to form biofilm, Triton X-100-induced autolysis and the amount of eDNA increased in the p221 strain and the two psm-mec mutants pM and pAG expressed psm-mec mRNA without its protein, whereas no differences were observed among the three constructed strains, illustrating that psm-mec mRNA promoted S. epidermidis biofilm formation through up-regulation of bacterial autolysis and the release of eDNA. Our results reveal that acquisition of psm-mec promotes S. epidermidis biofilm formation.


    Directory of Open Access Journals (Sweden)

    L. V. Antipova


    Full Text Available The conducted researches allowed to establish that intensive disintegration of a muscular glycogen leads to sharp decrease in size рН muscular tissue in the sour party that in turn affects a chemical composition and physic-colloidal structure of proteins therefore: resistance of meat of fish to action of putrefactive microorganisms increases; solubility of muscle proteins, level of their hydration which is water connecting abilities decreases; there is a swelling of collagen of connecting fabric; activity of the cathepsin (an optimum рН 5,3 causing hydrolysis of proteins at later stages of an autolysis increases; the bicarbonate system of muscular tissue with release of carbon dioxide collapses; predecessors of taste and aroma of meat are formed; process of oxidation of lipids becomes more active. As a result of accumulation dairy, phosphoric and other acids in meat of fish concentration of hydrogen ions of that decrease рН is result increases. Sharply shown sour environment and availability of inorganic phosphorus is considered the reason of disintegration of an actin-myosin complex on actin and a myosin which begins after 8 hours of storage, i.e. there comes the period of relaxation of muscle fibers and the period of permission of an numbness, and then the last stage of maturing of meat – deep autolysis. Thus, on the basis of classical ideas of biochemical changes of meat of land animals and summarizing the obtained data on posthumous changes in muscular tissue of fishes, it is possible to draw a conclusion that they have similar nature of regularity in comparison with muscular tissue of land animals, but their main difference is higher speed of course of autolytic transformations. It in turn leads to faster change of FTS of meat of fishes who are the defining indicators when developing assortment groups of products taking into account stages of an autolysis in meat.

  6. Estrogen and pure antiestrogen fulvestrant (ICI 182 780) augment cell–matrigel adhesion of MCF-7 breast cancer cells through a novel G protein coupled estrogen receptor (GPR30)-to-calpain signaling axis

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yan; Li, Zheng; He, Yan; Shang, Dandan; Pan, Jigang; Wang, Hongmei; Chen, Huamei; Zhu, Zhuxia [Department of Physiology/Cancer Research Group, Guiyang Medical University School of Basic Medicine, 9 Beijing Road, Guiyang 550004, Guizhou (China); Wan, Lei [Department of Pharmacology, Guiyang Medical University School of Basic Medicine, 9 Beijing Road, Guiyang 550004, Guizhou (China); Wang, Xudong, E-mail: [Department of Physiology/Cancer Research Group, Guiyang Medical University School of Basic Medicine, 9 Beijing Road, Guiyang 550004, Guizhou (China)


    Fulvestrant (ICI 182 780, ICI) has been used in treating patients with hormone-sensitive breast cancer, yet initial or acquired resistance to endocrine therapies frequently arises and, in particular, cancer recurs as metastasis. We demonstrate here that both 17-beta-estradiol (E2) and ICI enhance cell adhesion to matrigel in MCF-7 breast cancer cells, with increased autolysis of calpain 1 (large subunit) and proteolysis of focal adhesion kinase (FAK), indicating calpain activation. Additionally, either E2 or ICI induced down-regulation of estrogen receptor α without affecting G protein coupled estrogen receptor 30 (GPR30) expression. Interestingly, GPR30 agonist G1 triggered calpain 1 autolysis but not calpain 2, whereas ER agonist diethylstilbestrol caused no apparent calpain autolysis. Furthermore, the actions of E2 and ICI on calpain and cell adhesion were tremendously suppressed by G15, or knockdown of GPR30. E2 and ICI also induced phosphorylation of extracellular regulated protein kinases 1 and 2 (ERK1/2), and suppression of ERK1/2 phosphorylation by U0126 profoundly impeded calpain activation triggered by estrogenic and antiestrogenic stimulations indicating implication of ERK1/2 in the GPR30-mediated action. Lastly, the E2- or ICI-induced cell adhesion was dramatically impaired by calpain-specific inhibitors, ALLN or calpeptin, suggesting requirement of calpain in the GPR30-associated action. These data show that enhanced cell adhesion by E2 and ICI occurs via a novel GPR30-ERK1/2-calpain pathway. Our results indicate that targeting the GPR30 signaling may be a potential strategy to reduce metastasis and improve the efficacy of antiestrogens in treatment of advanced breast cancer. - Highlights: • Estrogen and ICI augment adhesion to matrigel with calpain activation in MCF-7 cells. • GPR30 mediates cell–matrigel adhesion and calpain activation via ERK1/2. • Calpain is required in the cell–matrigel adhesion induced by E2 and ICI.

  7. Production of yeast extract from whey using Kluyveromyces marxianus

    Directory of Open Access Journals (Sweden)

    Revillion Jean P. de Palma


    Full Text Available The yeast Kluyveromyces marxianus CBS 6556 was grown on whey to produce nucleotide-rich yeast extracts. Thermal treatments of cells at 35 or 50ºC for 15-30h resulted in yeast extracts containing about 20 g/L protein, with only the second treatment resulting in the presence of small amounts of RNA. In contrast, autolysis in buffered solution was the unique treatment that resulted in release of high amounts of intracellular RNA, being, therefore, the better procedure to produce 5'-nucletide rich extract with K. marxianus.

  8. Comparison of different methods for delayed post-mortem diagnosis of falciparum malaria

    Directory of Open Access Journals (Sweden)

    Fleischmann Erna


    Full Text Available Abstract Background Between 10,000 and 12,000 cases of imported malaria are notified in the European Union each year. Despite an excellent health care system, fatalities do occur. In case of advanced autolysis, the post-mortem diagnostic is impaired. Quicker diagnosis could be achieved by using rapid diagnostic malaria tests. Methods In order to evaluate different methods for the post-mortem diagnosis of Plasmodium falciparum malaria in non-immunes, a study was performed on the basis of forensic autopsies of corpses examined at variable intervals after death in five cases of fatal malaria (with an interval of four hours to five days, and in 20 cases of deaths unrelated to malaria. Detection of parasite DNA by PCR and an immunochromatographic test (ICT based upon the detection of P. falciparum histidine-rich protein 2 (PfHRP2 were compared with the results of microscopic examination of smears from cadaveric blood, histopathological findings, and autopsy results. Results In all cases of fatal malaria, post-mortem findings were unsuspicious for the final diagnosis, and autoptic investigations, including histopathology, were only performed because of additional information by police officers and neighbours. Macroscopic findings during autopsy were unspecific. Histopathology confirmed sequestration of erythrocytes and pigment in macrophages in most organs in four patients (not evaluable in one patient due to autolysis. Microscopy of cadaveric blood smears revealed remnants of intraerythrocytic parasites, and was compromised or impossible due to autolysis in two cases. PCR and ICT performed with cadaveric blood were positive in all malaria patients and negative in all controls. Conclusion In non-immune fatalities with unclear anamnesis, ICT can be recommended as a sensitive and specific tool for post-mortem malaria diagnosis, which is easier and faster than microscopy, and also applicable when microscopic examination is impossible due to autolysis

  9. Ultrastructural investigation of the secondary excretory system in different stages of the procercoid of Triaenophorus nodulosus (Cestoda, Pseudophyllidea, Triaenophoridae). (United States)

    Korneva, J V; Kuperman, B I; Davydov, V G


    The formation of the definitive procercoid excretory system of the pseudophyllidean cestode Triaenophorus nodulosus has been investigated. This process can be divided into 3 main stages. In the first stage, active autophagic processes lead to the formation of intracellular excretory canals. In the second stage, the process of the autolysis is enhanced and the system of intercellular lacunae functioning as an excretory system is formed. A definitive excretory system of the procercoid forms at the next stage, by means of the migration and proliferation of undifferentiated cells replacing the lacunar system.

  10. 啤酒废酵母自溶提取物的制备及抗氧化活性研究%Preparation and Antioxidant Activity of Peptides from Waste Saccharomyces Cerevisiae

    Institute of Scientific and Technical Information of China (English)

    罗依雨; 罗维; 刘峻熙; 杨志民; 倪贺; 李海航


    以广州本地啤酒厂废酵母为材料,通过单因素实验研究其最佳自溶条件及最佳微波水解条件.比较自溶、微波水解与外加酶制剂处理法制备酵母提取物的效果,研究了该提取物的抗氧化活性.结果表明,啤酒酵母的最佳自溶条件为固液比1∶20、温度45.0℃、pH 6.0、时间48 h,在此条件下,酵母蛋白质的水解度为41.5%,提取蛋白质得率为42%.自溶法制备啤酒酵母提取物的效率与外加酶制剂处理的效果相当,而显著高于微波水解法,提取物具有较高的抗氧化活性.研究表明,自溶法是开发利用啤酒厂废弃的酵母和制备酵母提取物的较好方法.%The brewery waste yeast ( Saccharomyces cerevisiae) contains 50%proteins and has high values of appli-cation.The optimal conditions of autolysis of S.cerevisiae cells were explored by the single-factor tests, and its effi-ciency was compared with the microwave-assisted hydrolysis and exogenous enzyme hydrolysis.The results showed that the optimal autolysis conditions of the yeast were with a solid-liquid ratio of 1∶20, at pH 6.0 and 45.0 ℃for 48 h.Under these conditions, yeast proteins were hydrolyzed and extracted at the yield of 42%, the degree of hy-drolysis was 41.5%.The autolysis method showed the same efficiency as the exogenous enzyme hydrolysis, but higher than that of the microwave-assisted hydrolysis.The prepared yeast extracts by autolysis showed high antioxi-dant activity.

  11. Influence of early pH decline on calpain activity in porcine muscle

    DEFF Research Database (Denmark)

    Pomponio, Luigi; Ertbjerg, Per; Karlsson, Anders H;


    This study investigated the influence of post-mortem pH decline on calpain activity and myofibrillar degradation.From 80 pigs, 30 Longissimus dorsi (LD) muscles were selected on the basis of pH values at 3 h post-mortem and classified into groups of 10 as fast, intermediate and slow pH decline....... The rate of pH decline early post-mortem differed between the three groups, but the ultimate pH values were similar at 24 h. Calpain activity and autolysis from 1 to 72 h post-mortem were determined using casein zymography and studied in relation to myofibrillar fragmentation. Colour and drip loss were...... measured. A faster decrease in pH resulted in reduced level of l-calpain activity and increased autolysis of the enzyme, and hence an earlier loss of activity due to activation of l-calpain in muscles with a fast pH decline. Paralleling the l-calpain activation in muscles with a fast pH decline a higher...

  12. Antibacterial and antiparasitic activity of oleanolic acid and its glycosides isolated from marigold (Calendula officinalis). (United States)

    Szakiel, Anna; Ruszkowski, Dariusz; Grudniak, Anna; Kurek, Anna; Wolska, Krystyna I; Doligalska, Maria; Janiszowska, Wirginia


    The antibacterial and antiparasitic activities of free oleanolic acid and its glucosides and glucuronides isolated from marigold (Calendula officinalis) were investigated. The MIC of oleanolic acid and the effect on bacterial growth were estimated by A600 measurements. Oleanolic acid's influence on bacterial survival and the ability to induce autolysis were measured by counting the number of cfu. Cell morphology and the presence of endospores were observed under electron and light microscopy, respectively. Oleanolic acid inhibited bacterial growth and survival, influenced cell morphology and enhanced the autolysis of Gram-positive bacteria suggesting that bacterial envelopes are the target of its activity. On the other hand, glycosides of oleanolic acid inhibited the development of L3 Heligmosomoides polygyrus larvae, the infective stage of this intestinal parasitic nematode. In addition, both oleanolic acid and its glycosides reduced the rate of L3 survival during prolonged storage, but only oleanolic acid glucuronides affected nematode infectivity. The presented results suggest that oleanolic acid and its glycosides can be considered as potential therapeutic agents.

  13. Contribution of ClpP to stress tolerance and virulence properties of Streptococcus mutans. (United States)

    Hou, Xiang-Hua; Zhang, Jia-Qin; Song, Xiu-Yu; Ma, Xiao-Bo; Zhang, Shi-Yang


    Abilities to tolerate environmental stresses and to form biofilms on teeth surface are key virulence attributes of Streptococcus mutans, the primary causative agent of human dental caries. ClpP, the chief intracellular protease of S. mutans, along with ATPases degrades altered proteins that might be toxic for bacteria, and thus plays important roles in stress response. To further understand the roles of ClpP in stress response of S. mutans, a ClpP deficient strain was constructed and used for general stress tolerance, autolysis, mutacins production, and virulence assays. Here, we demonstrated that inactivation of ClpP in S. mutans resulted in a sensitive phenotype to several environmental stresses, including acid, cold, thermal, and oxidative stresses. The ClpP deficient strain displayed slow growth rates, poor growth yields, formation of long chains, increased clumping in broth, and reduced capacity to form biofilms in presence of glucose. Mutacins production and autolysis of S. mutans were also impaired by mutation of clpP. Animals study showed that clpP mutation increased virulence of S. mutans but not significant. However, enhanced abilities to survive lethal acid and to form biofilm in sucrose were observed in ClpP deficient strain. Our findings revealed a broad impact of ClpP on several virulence properties of S. mutans and highlighted the relevance of ClpP proteolysis with progression of diseases caused by S. mutans.

  14. A p53-like transcription factor similar to Ndt80 controls the response to nutrient stress in the filamentous fungus, Aspergillus nidulans [v1; ref status: indexed,

    Directory of Open Access Journals (Sweden)

    Margaret E Katz


    Full Text Available The Aspergillus nidulans xprG gene encodes a putative transcriptional activator that is a member of the Ndt80 family in the p53-like superfamily of proteins. Previous studies have shown that XprG controls the production of extracellular proteases in response to starvation. We undertook transcriptional profiling to investigate whether XprG has a wider role as a global regulator of the carbon nutrient stress response. Our microarray data showed that the expression of a large number of genes, including genes involved in secondary metabolism, development, high-affinity glucose uptake and autolysis, were altered in an xprGΔ null mutant. Many of these genes are known to be regulated in response to carbon starvation. We confirmed that sterigmatocystin and penicillin production is reduced in xprG- mutants. The loss of fungal mass and secretion of pigments that accompanies fungal autolysis in response to nutrient depletion was accelerated in an xprG1 gain-of-function mutant and decreased or absent in an xprG- mutant. The results support the hypothesis that XprG plays a major role in the response to carbon limitation and that nutrient sensing may represent one of the ancestral roles for the p53-like superfamily. Disruption of the AN6015 gene, which encodes a second Ndt80-like protein, showed that it is required for sexual reproduction in A. nidulans.

  15. Enhancing the value of nitrogen from rapeseed meal for microbial oil production. (United States)

    Uçkun Kiran, Esra; Salakkam, Apilak; Trzcinski, Antoine P; Bakir, Ufuk; Webb, Colin


    Rapeseed meal, a major byproduct of biodiesel production, has been used as a low-cost raw material for the production of a generic microbial feedstock through a consolidated bioconversion process. Various strategies were tested for the production of a novel fermentation medium, rich in free amino nitrogen (FAN): commercial enzymes (CEs) (2.7 mg g⁻¹ dry meal), liquid state fungal pre-treatment (LSF) using Aspergillus oryzae (4.6 mg g⁻¹), liquid state fungal pre-treatment followed by fungal autolysis (LSFA) (9.13 mg g⁻¹), liquid state pre-treatment using fungal enzymatic broth (EB) (2.1 mg g⁻¹), but the best strategy was a solid state fungal pre-treatment followed by fungal autolysis (34.5 mg g⁻¹). The bioavailability of the nitrogen sources in the novel medium was confirmed in fed-batch bioreactor studies, in which 82.3g dry cell L⁻¹ of the oleaginous yeast Rhodosporidium toruloides Y4 was obtained with a lipid content of 48%. The dry cell weight obtained was higher than that obtained using conventional yeast extract, due to a higher total nitrogen content in the novel biomedium. The fatty acids obtained from the microbial oil were similar to those derived from rapeseed oil.

  16. Using mixed inocula of Saccharomyces cerevisiae killer strains to improve the quality of traditional sparkling-wine. (United States)

    Velázquez, Rocío; Zamora, Emiliano; Álvarez, Manuel; Álvarez, María L; Ramírez, Manuel


    The quality of traditional sparkling-wine depends on the aging process in the presence of dead yeast cells. These cells undergo a slow autolysis process thereby releasing some compounds, mostly colloidal polymers such as polysaccharides and mannoproteins, which influence the wine's foam properties and mouthfeel. Saccharomyces cerevisiae killer yeasts were tested to increase cell death and autolysis during mixed-yeast-inoculated second fermentation and aging. These yeasts killed sensitive strains in killer plate assays done under conditions of low pH and temperature similar to those used in sparkling-wine making, although some strains showed a different killer behaviour during the second fermentation. The fast killer effect improved the foam quality and mouthfeel of the mixed-inoculated wines, while the slow killer effect gave small improvements over single-inoculated wines. The effect was faster under high-pressure than under low-pressure conditions. Wine quality improvement did not correlate with the polysaccharide, protein, mannan, or aromatic compound concentrations, suggesting that the mouthfeel and foaming quality of sparkling wine are very complex properties influenced by other wine compounds and their interactions, as well as probably by the specific chemical composition of a given wine.

  17. Antioxidant, ACE-Inhibitory and antibacterial activities of Kluyveromyces marxianus protein hydrolysates and their peptide fractions

    Directory of Open Access Journals (Sweden)

    Mahta Mirzaeia


    Full Text Available Background: There has been evidence that proteins are potentially excellent source of antioxidants, antihypertensive and antimicrobial peptides, and that enzymatic hydrolysis is an effective method to release these peptides from protein molecules. The functional properties of protein hydrolysates depends on the protein substrate, the specificity of the enzymes, the conditions used during proteolysis, degree of hydrolysis, and the nature of peptides released including molecular weight, amino acid composition, and hydrophobicity. Context and purpose of this study: The biomass of Kluyveromyces marxianus was considered as a source of ACE inhibitory, antioxidant and antimicrobial peptides. Results: Autolysis and enzymatic hydrolysis were completed respectively, after 96 h and 5 h. Overall, trypsin (18.52% DH and chymotrypsin (21.59% DH treatments were successful in releasing antioxidant and ACE inhibitory peptides. Autolysate sample (39.51% DH demonstrated poor antioxidant and ACE inhibitory activity compared to trypsin and chymotrypsin hydrolysates. The chymotrypsin 3-5 kDa (301.6±22.81 μM TE/mg protein and trypsin < 3 kDa (280.16±39.16 μM TE/mg protein permeate peptide fractions showed the highest DPPH radical scavenging activity. The trypsin <3 kDa permeate peptide fraction showed the highest ABTS radical scavenging (1691.1±48.68 μM TE/mg protein and ACE inhibitory (IC50=0.03±0.001 mg/mL activities. The fraction (MW=5-10 kD obtained after autolysis treatment showed antibacterial activity against St. aureus and Lis. monocytogenes in well diffusion screening. The minimum inhibitory concentration (MIC value was 13.3 mg/mLagainst St. aureus and Lis. monocytogenes calculated by turbidimetric assay and it showed bactericidal activity against St. aureus at 21.3 mg/mL protein concentration. Conclusions: Altogether, the results of this study reveal that K. marxianus proteins contain specific peptides in their sequences which can be released by

  18. Effect of varying the salt and fat content in Cheddar cheese on aspects of the performance of a commercial starter culture preparation during ripening. (United States)

    Yanachkina, Palina; McCarthy, Catherine; Guinee, Tim; Wilkinson, Martin


    Production of healthier reduced-fat and reduced-salt cheeses requires careful selection of starter bacteria, as any substantial alterations to cheese composition may prompt changes in the overall performance of starters during cheese ripening. Therefore, it is important to assess the effect of compositional alterations on the individual strain response during cheese ripening for each optimised cheese matrix. In the current study, the effect of varying fat and salt levels in Cheddar cheese on the performance of a commercial Lactococcus lactis culture preparation, containing one L. lactis subsp. lactis strain and one L. lactis subsp. cremoris strain was investigated. Compositional variations in fat or salt levels did not affect overall starter viability, yet reduction of fat by 50% significantly delayed non-starter lactic acid bacteria (NSLAB) populations at the initial ripening period. In comparison to starter viability, starter autolysis, as measured by release of intracellular lactate dehydrogenase (LDH) or post-proline dipeptidyl aminopeptidase (Pep X) into cheese juices, decreased significantly with lower salt addition levels in full-fat Cheddar. Conversely, reducing fat content of cheese resulted in a significantly higher release of intracellular Pep X, and to a lesser extent intracellular LDH, into juices over ripening. Flow cytometry (FCM) indicated that the permeabilised and dead cell sub-populations were generally lower in juices from cheeses with reduced salt content, however no significant differences were observed between different salt and fat treatments. Interestingly, fat reductions by 30 and 50% in cheeses with reduced or half added salt contents appeared to balance out the effect of salt, and enhanced cell permeabilisation, cell death, and also cell autolysis in these variants. Overall, this study has highlighted that alterations in both salt and fat levels in cheese influence certain aspects of starter performance during ripening, including

  19. 啤酒废酵母中β-D-葡聚糖非降解提取工艺%A Novel Method for Non-degradative Extraction of β-D-Glucans from Spent Yeast Cells

    Institute of Scientific and Technical Information of China (English)

    朱益波; 翟丽君; 朱明; 齐斌


    We here present a novel method to extract non-degraded β-D-glucans from spent yeast cells,mainly based on induced yeast autolysis,hot water extraction,cell wall disruption,defatting and protease hydrolysis.One-factor-at-a-time coupled with orthogonal array design method was applied to the process conditions of induced yeast autolysis and cell wall disruption.The extract obtained under optimized conditions had a total sugar content of 84.9% with an extraction yield of 13.7%.Its purity and yield were both higher than previously reported.Induced yeast autolysis and hot water extraction had a significant effect on β-D-glucan purity and yield.Moreover,protease treatment could further remove protein impurities and increase β-D-glucan purity.The absence of strong acid,strong alkali and oxidant throughout the process helped to protect the physiological activity of products and the environment.%采用单因素及正交试验研究新型非降解法提取废啤酒酵母β-D-葡聚糖工艺。该工艺主要包括诱导自溶、热水浸提、破壁、脱脂、蛋白酶处理等过程。结果表明,提取物中总糖质量百分含量为84.9%,得率为13.7%。与已有报道相比具备较高的纯度和得率。诱导自溶及热水浸提处理对于提取物的得率和纯度具有重要影响,蛋白酶处理对于进一步减少蛋白质含量和提高产物纯度也有显著作用。整个提取过程没有采用强酸、碱和氧化剂有助于保护产物的生理活性和环境。

  20. Exertional myopathy in a grizzly bear (Ursus arctos) captured by leghold snare. (United States)

    Cattet, Marc; Stenhouse, Gordon; Bollinger, Trent


    We diagnosed exertional myopathy (EM) in a grizzly bear (Ursus arctos) that died approximately 10 days after capture by leghold snare in west-central Alberta, Canada, in June 2003. The diagnosis was based on history, post-capture movement data, gross necropsy, histopathology, and serum enzyme levels. We were unable to determine whether EM was the primary cause of death because autolysis precluded accurate evaluation of all tissues. Nevertheless, comparison of serum aspartate aminotransferase and creatine kinase concentrations and survival between the affected bear and other grizzly bears captured by leghold snare in the same research project suggests EM also occurred in other bears, but that it is not generally a cause of mortality. We propose, however, occurrence of nonfatal EM in grizzly bears after capture by leghold snare has potential implications for use of this capture method, including negative effects on wildlife welfare and research data.

  1. Anticoagulant activity of Moon jellyfish (Aurelia aurita) tentacle extract. (United States)

    Rastogi, Akriti; Biswas, Sumit; Sarkar, Angshuman; Chakrabarty, Dibakar


    Moon jellyfish (Aurelia aurita) tentacle extract was studied for its anticoagulant activity in vitro. The Jellyfish Tentacle Extract (JFTE) showed very strong fibrinogenolytic activity by cleaving Aα and Bβ chain of fibrinogen molecule. The fibrinogenolytic activity was found to be stronger than some snake venom derived anticoagulants. JFTE also completely liquefied fibrin clots in 24 h. JFTE was found to contain both high and low molecular weight proteins/peptides. The fibrinogenolysis appears to be caused by high molecular weight fractions of the extract. It has been also noted that PMSF significantly reduced fibrinogenolytic activity and heating totally abolished it. Autolytic degradation of the high molecular weight protein was also noted. Autolysis slowed down, but did not abolish the fibrinogenolytic activity of the extract.

  2. Amino acid sequence alignment of vertebrate CAPN3/calpain-3/p94

    Directory of Open Access Journals (Sweden)

    Yasuko Ono


    Full Text Available CAPN3 is a calpain superfamily member that is predominantly expressed in skeletal muscle. So far, clear CAPN3 orthologs were found only in vertebrates. CAPN3 is a unique protease in that it undergoes extremely rapid and exhaustive autolysis and that autolyzed fragments spontaneously associate each other to reconstitute the proteolytic activity. These unique properties of CAPN3 are dependent on IS1 and IS2, two CAPN3-characterizing sequences that do not exist in other calpains or any other proteases. To understand how IS1 and IS2 are conserved among vertebrates, this data article provides amino acid sequence alignment of representative vertebrate CAPN3s. For further analysis and discussion, see Ono et al. [1

  3. Characterisation of an unusual bacterium isolated from genital ulcers. (United States)

    Ursi, J P; van Dyck, E; Ballard, R C; Jacob, W; Piot, P; Meheus, A Z


    The preliminary characterisation of an unusual gram-negative bacillus isolated from genital ulcers in Swaziland is reported. Like Haemophilus ducreyi, it is an oxidase positive, nitrate-reductase-positive gram-negative rod that forms streptobacillary chains in some circumstances; it was therefore called the "ducreyi-like bacterium" (DLB). Distinguishing features of DLB are production of alpha-haemolysis on horse-blood agar, stimulation of growth by a microaerophilic atmosphere and by a factor produced by Staphylococcus aureus, a strongly positive porphyrin test, and a remarkable ability to undergo autolysis. DLB had a guanine + cytosine value of c. 50 mole% but it cannot be classified, even at the genus level, until more taxonomic data are obtained.

  4. Production and functional evaluation of a protein concentrate from giant squid (Dosidicus gigas) by acid dissolution and isoelectric precipitation. (United States)

    Cortés-Ruiz, Juan A; Pacheco-Aguilar, Ramón; Elena Lugo-Sánchez, M; Gisela Carvallo-Ruiz, M; García-Sánchez, Guillermina


    A protein concentrate from giant squid (Dosidicus gigas) was produced under acidic conditions and its functional-technological capability evaluated in terms of its gel-forming ability, water holding capacity and colour attributes. Technological functionality of the concentrate was compared with that of squid muscle and a neutral concentrate. Protein-protein aggregates insoluble at high ionic strength (I=0.5M), were detected in the acidic concentrate as result of processing with no preclusion of its gel-forming ability during the sol-to-gel thermal transition. Even though washing under acidic condition promoted autolysis of the myosin heavy chain, the acidic concentrate displayed an outstanding ability to gel giving samples with a gel strength of 455 and 1160gcm at 75% and 90% compression respectively, and an AA folding test grade indicative of high gel strength, elasticity, and cohesiveness. The process proved to be a good alternative for obtaining a functional protein concentrate from giant squid muscle.

  5. Untargeted Metabolic Profiling of Winery-Derived Biomass Waste Degradation by Penicillium chrysogenum. (United States)

    Karpe, Avinash V; Beale, David J; Godhani, Nainesh B; Morrison, Paul D; Harding, Ian H; Palombo, Enzo A


    Winery-derived biomass waste was degraded by Penicillium chrysogenum under solid state fermentation over 8 days in a (2)H2O-supplemented medium. Multivariate statistical analysis of the gas chromatography-mass spectrometry (GC-MS) data resulted in the identification of 94 significant metabolites, within 28 different metabolic pathways. The majority of biomass sugars were utilized by day 4 to yield products such as sugars, fatty acids, isoprenoids, and amino acids. The fungus was observed to metabolize xylose to xylitol, an intermediate of ethanol production. However, enzyme inhibition and autolysis were observed from day 6, indicating 5 days as the optimal time for fermentation. P. chrysogenum displayed metabolism of pentoses (to alcohols) and degraded tannins and lignins, properties that are lacking in other biomass-degrading ascomycetes. Rapid fermentation (3-5 days) may not only increase the pentose metabolizing efficiency but also increase the yield of medicinally important metabolites, such as syringate.

  6. Structural analysis of Staphylococcus aureus serine/threonine kinase PknB.

    Directory of Open Access Journals (Sweden)

    Sonja Rakette

    Full Text Available Effective treatment of infections caused by the bacterium Staphylococcus aureus remains a worldwide challenge, in part due to the constant emergence of new strains that are resistant to antibiotics. The serine/threonine kinase PknB is of particular relevance to the life cycle of S. aureus as it is involved in the regulation of purine biosynthesis, autolysis, and other central metabolic processes of the bacterium. We have determined the crystal structure of the kinase domain of PknB in complex with a non-hydrolyzable analog of the substrate ATP at 3.0 Å resolution. Although the purified PknB kinase is active in solution, it crystallized in an inactive, autoinhibited state. Comparison with other bacterial kinases provides insights into the determinants of catalysis, interactions of PknB with ligands, and the pathway of activation.

  7. Structural analysis of Staphylococcus aureus serine/threonine kinase PknB. (United States)

    Rakette, Sonja; Donat, Stefanie; Ohlsen, Knut; Stehle, Thilo


    Effective treatment of infections caused by the bacterium Staphylococcus aureus remains a worldwide challenge, in part due to the constant emergence of new strains that are resistant to antibiotics. The serine/threonine kinase PknB is of particular relevance to the life cycle of S. aureus as it is involved in the regulation of purine biosynthesis, autolysis, and other central metabolic processes of the bacterium. We have determined the crystal structure of the kinase domain of PknB in complex with a non-hydrolyzable analog of the substrate ATP at 3.0 Å resolution. Although the purified PknB kinase is active in solution, it crystallized in an inactive, autoinhibited state. Comparison with other bacterial kinases provides insights into the determinants of catalysis, interactions of PknB with ligands, and the pathway of activation.

  8. Induction of Autophagy by Second-Fermentation Yeasts during Elaboration of Sparkling Wines (United States)

    Cebollero, Eduardo; Gonzalez, Ramon


    Autophagy is a transport system mediated by vesicles, ubiquitous in eukaryotic cells, by which bulk cytoplasm is targeted to a lysosome or vacuole for degradation. In the yeast Saccharomyces cerevisiae, autophagy is triggered by nutritional stress conditions (e.g., carbon- or nitrogen-depleted medium). In this study we showed that there is induction of autophagy in second-fermentation yeasts during sparkling wine making. Two methods were employed to detect autophagy: a biochemical approach based on depletion of the protein acetaldehyde dehydrogenase Ald6p and a morphological strategy consisting of visualization of autophagic bodies and autophagosomes, which are intermediate vesicles in the autophagic process, by transmission electron microscopy. This study provides the first demonstration of autophagy in second-fermentation yeasts under enological conditions. The correlation between autophagy and yeast autolysis during sparkling wine production is discussed, and genetic engineering of autophagy-related genes in order to accelerate the aging steps in wine making is proposed. PMID:16751523

  9. Effect of production phase on bottle-fermented sparkling wine quality. (United States)

    Kemp, Belinda; Alexandre, Hervé; Robillard, Bertrand; Marchal, Richard


    This review analyzes bottle-fermented sparkling wine research at each stage of production by evaluating existing knowledge to identify areas that require future investigation. With the growing importance of enological investigation being focused on the needs of the wine production industry, this review examines current research at each stage of bottle-fermented sparkling wine production. Production phases analyzed in this review include pressing, juice adjustments, malolactic fermentation (MLF), stabilization, clarification, tirage, lees aging, disgorging, and dosage. The aim of this review is to identify enological factors that affect bottle-fermented sparkling wine quality, predominantly aroma, flavor, and foaming quality. Future research topics identified include regional specific varieties, plant-based products from vines, grapes, and yeast that can be used in sparkling wine production, gushing at disgorging, and methods to increase the rate of yeast autolysis. An internationally accepted sensory analysis method specifically designed for sparkling wine is required.

  10. Ultrastructure of autophagy in plant cells: a review. (United States)

    van Doorn, Wouter G; Papini, Alessio


    Just as with yeasts and animal cells, plant cells show several types of autophagy. Microautophagy is the uptake of cellular constituents by the vacuolar membrane. Although microautophagy seems frequent in plants it is not yet fully proven to occur. Macroautophagy occurs farther away from the vacuole. In plants it is performed by autolysosomes, which are considerably different from the autophagosomes found in yeasts and animal cells, as in plants these organelles contain hydrolases from the onset of their formation. Another type of autophagy in plant cells (called mega-autophagy or mega-autolysis) is the massive degradation of the cell at the end of one type of programmed cell death (PCD). Furthermore, evidence has been found for autophagy during degradation of specific proteins, and during the internal degeneration of chloroplasts. This paper gives a brief overview of the present knowledge on the ultrastructure of autophagic processes in plants.

  11. A proteomic and ultrastructural characterization of Aspergillus fumigatus' conidia adaptation at different culture ages. (United States)

    Anjo, Sandra I; Figueiredo, Francisco; Fernandes, Rui; Manadas, Bruno; Oliveira, Manuela


    The airborne fungus Aspergillus fumigatus is one of the most common agents of human fungal infections with a remarkable impact on public health. However, A. fumigatus conidia atmospheric resistance and longevity mechanisms are still unknown. Therefore, in this work, the processes underlying conidial adaptation were studied by a time course evaluation of the proteomics and ultrastructural changes of A. fumigatus' conidia at three time-points selected according to relevant changes previously established in conidial survival rates. The proteomics characterization revealed that conidia change from a highly active metabolic to a dormant state, culminating in cell autolysis as revealed by the increased levels of hydrolytic enzymes. Structural characterization corroborates the proteomics data, with noticeable changes observed in mitochondria, nucleus and plasma membrane ultrastructure, accompanied by the formation of autophagic vacuoles. These changes are consistent with both apoptotic and autophagic processes and indicate that the changes in protein levels may anticipate those in cell morphology.

  12. Isolation and characterization of venom from nematocysts of jellyfish Rhopilema esculentum Kishinouye

    Institute of Scientific and Technical Information of China (English)

    FENG Jinhua; YU Huahua; LI Cuiping; XING Ronge; LIU Song; WANG Lin; CAI Shengbao; LI Pengcheng


    The present work is the first report of the biochemical characterization of the venom from nematocysts of the jellyfish Rhopilema esculentum Kishinouye. The nematocysts were isolated by autolysis and centrifugation and separated by flow cytometry. Four types of nematocysts were identified: mastigophores, euryteles, and atrichous and holotrichous isorhiza. SDS-PAGE and amino acid analyses demonstrated that most of the proteins in the nematocyst extract were between 10 kDa and 40 kDa, and that glutamic acid was the main amino acid. A hemolytic activity assay showed that the activity of the nematocyst venom (RNV) was strongest in Tris-HCl buffer (50 mmol/L, pH 7.8, 5% glycerol, 0.5 mmol/L EDTA, 0.1 mol/L NaCl). The hemolytic activity was related to protein concentration and the HU_(50) against chicken erythrocytes was 0.91 μg/mL.

  13. Partial purification of box jellyfish (Chironex fleckeri) nematocyst venom isolated at the beachside. (United States)

    Bloom, D A; Burnett, J W; Alderslade, P


    Chironex fleckeri, the northern Australian box jellyfish produces one of, if not, the most potent animal venoms. Study of the venom has been hampered by the limits of the animals' range and the venom's thermolability. Using retained lethality and native polyacrylamide gel electrophoresis (NPAGE), we show that lyophilization of autolysis isolated nematocysts is an effective method of transporting the venom. In addition, Sephadex G-200 chromatography, spin concentration, and NPAGE fail to demonstrate the presence of a 600 kDa protein to which the bulk of the lethal activity has been ascribed. Sodium dodecyl sulfate capillary electrophoresis of crude venom yields several protein bands with a molecular weight range of 30-200 kDa. Freeze-thaw studies show a loss of activity and NPAGE bands after two freeze thaw cycles.

  14. Living bacteria rheology: population growth, aggregation patterns and cooperative behaviour under different shear flows

    CERN Document Server

    Patricio, P; Portela, R; Sobral, R G; Grilo, I R; Cidade, T; Leal, C R


    The activity of growing living bacteria was investigated using real-time and in situ rheology -- in stationary and oscillatory shear. Two different strains of the human pathogen Staphylococcus aureus -- strain COL and its isogenic cell wall autolysis mutant -- were considered in this work. For low bacteria density, strain COL forms small clusters, while the mutant, presenting deficient cell separation, forms irregular larger aggregates. In the early stages of growth, when subjected to a stationary shear, the viscosity of both strains increases with the population of cells. As the bacteria reach the exponential phase of growth, the viscosity of the two strains follow different and rich behaviours, with no counterpart in the optical density or in the population's colony forming units measurements. While the viscosity of strain COL keeps increasing during the exponential phase and returns close to its initial value for the late phase of growth, where the population stabilizes, the viscosity of the mutant strain ...

  15. Evaluation of the effects of Streptococcus mutans chaperones and protein secretion machinery components on cell surface protein biogenesis, competence, and mutacin production. (United States)

    Crowley, P J; Brady, L J


    The respective contributions of components of the protein translocation/maturation machinery to cell surface biogenesis in Streptococcus mutans are not fully understood. Here we used a genetic approach to characterize the effects of deletion of genes encoding the ribosome-associated chaperone RopA (Trigger Factor), the surface-localized foldase PrsA, and the membrane-localized chaperone insertases YidC1 and YidC2, both singly and in combination, on bacterial growth, chain length, self-aggregation, cell surface hydrophobicity, autolysis, and antigenicity of surface proteins P1 (AgI/II, PAc), WapA, GbpC, and GtfD. The single and double deletion mutants, as well as additional mutant strains lacking components of the signal recognition particle pathway, were also evaluated for their effects on mutacin production and genetic competence.

  16. Combination of MALDI-TOF mass spectrometry with immobilized enzyme microreactor for peptide mapping

    Institute of Scientific and Technical Information of China (English)

    姜泓海; 邹汉法; 汪海林; 张强; 倪坚毅; 张清春; 郭忠; 陈小明


    Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been combined with immobilized enzyme microreactor for the rapid, sensitive, and accurate tryptic mapping of protein and polypeptides. The technique utilizes the trypsin microreactor by immobilized enzyme on the glycidyl methacrylate (GMA)-modified cellulose membrane. The membrane micro-reactor was used for the tryptic mapping of cytochrome C and the results were compared with those obtained by using free trypsin. A significant increase in the overall sensitivity of the process was observed using the membrane microreactor, as well as the elimination of background signals due to the autolysis of the trypsin. Further, membrane microreactor digestions were found to be rapid and convenient.

  17. Abnormal physiological properties and altered cell wall composition in Streptococcus pneumoniae grown in the presence of clavulanic acid. (United States)

    Severin, A; Severina, E; Tomasz, A


    Subinhibitory concentrations of clavulanate caused premature induction of stationary-phase autolysis, sensitization to lysozyme, and reductions in the MICs of deoxycholate and penicillin for Streptococcus pneumoniae. In the range of clavulanate concentrations producing these effects, this beta-lactam compound was selectively bound to PBP 3. Cell walls isolated from pneumococci grown in the presence of clavulanate showed increased sensitivity to the hydrolytic action of purified pneumococcal autolysin in vitro. High-performance liquid chromatography analysis of the peptidoglycan isolated from the clavulanate-grown cells showed major qualitative and quantitative changes in stem peptide composition, the most striking feature of which was the accumulation of peptide species carrying intact D-alanyl-D-alanine residues at the carboxy termini. The altered biological and biochemical properties of the clavulanate-grown pneumococci appear to be the consequences of suppressed D,D-carboxypeptidase activity. PMID:9055983

  18. Retrospective detection by negative contrast electron microscopy of faecal viral particles in free-living wild red squirrels (Sciurus vulgaris) with suspected enteropathy in Great Britain. (United States)

    Everest, D J; Stidworthy, M F; Milne, E M; Meredith, A L; Chantrey, J; Shuttleworth, C; Blackett, T; Butler, H; Wilkinson, M; Sainsbury, A W


    Transmission electron microscopy identified adenovirus particles in 10 of 70 (14.3 per cent) samples of large intestinal content collected at postmortem examination from free-living wild red squirrels (Sciurus vulgaris) across Great Britain between 2000 and 2009. Examination was limited to cases in which an enteropathy was suspected on the basis of predetermined macroscopic criteria such as semi-solid or diarrhoeic faeces, suspected enteritis or the presence of intussusception. In most cases, meaningful histological examination of enteric tissue was not possible due to pronounced autolysis. Two (2.9 per cent) of the samples were negative for adenovirus but were found to contain rotavirus particles, a novel finding in this species.

  19. Proteins influencing foam formation in wine and beer: the role of yeast. (United States)

    Blasco, Lucía; Viñas, Miquel; Villa, Tomás G


    This review focuses on the role of proteins in the production and maintenance of foam in both sparkling wines and beer. The quality of the foam in beer but especially in sparkling wines depends, among other factors, on the presence of mannoproteins released from the yeast cell walls during autolysis. These proteins are hydrophobic, highly glycosylated, and their molecular masses range from 10 to 200 kDa--characteristics that allow mannoproteins to surround and thus stabilize the gas bubbles of the foam. Both the production and stabilization of foam also depend on other proteins. In wine, these include grape-derived proteins such as vacuolar invertase; in beer, barley-derived proteins, such as LTP1, protein Z, and hordein-derived polypeptides, are even more important in this respect than mannoproteins.

  20. The morphology of Ganoderma lucidum mycelium in a repeated-batch fermentation for exopolysaccharide production

    Directory of Open Access Journals (Sweden)

    Wan Abd Al Qadr Imad Wan-Mohtar


    Full Text Available The morphology of Ganoderma lucidum BCCM 31549 mycelium in a repeated-batch fermentation (RBF was studied for exopolysaccharide (EPS production. RBF was optimised for time to replace and volume to replace. G. lucidum mycelium showed the ability to self-immobilise and exhibited high stability for repeated use in RBF with engulfed pellets. Furthermore, the ovoid and starburst-like pellet morphology was disposed to EPS production in the shake flask and bioreactor, respectively. Seven RBF could be carried out in 500 mL flasks, and five repeated batches were performed in a 2 L bioreactor. Under RBF conditions, autolysis of pellet core in the shake flask and shaving off of the outer hairy region in the bioreactor were observed at the later stages of RBF (R4 for the shake flask and R6 for the bioreactor. The proposed strategy showed that the morphology of G. lucidum mycelium can withstand extended fermentation cycles.

  1. 短蛸自溶中组织蛋白酶L样蛋白酶的活性及分布变化%Changes in Activity and Distribution of Cathepsin L-Like Proteases in Autolytic Octopus (Octopus ocellatus)

    Institute of Scientific and Technical Information of China (English)

    梁晓旺; 宗媛; 林竹一; 李冬梅; 周大勇


    The contents of water‐soluble total proteins and collagen proteins ,as well as the activities of endogenous total proteases and cathepsin L‐like proteases were determined in arm muscle of the autolytic octopus induced by ultraviolet (UV ) . Results showed that the octopus became insensitive after UV irradiation .The body of octopus became softened ,and mucoid degeneration took place in epidermis and subcutaneous tissue .The changes in external appearance indicated that autolysis took place .Along with autolysis ,the contents of water‐soluble total proteins and collagen proteins were found to be increased , indicating degradation of structural proteins .At the same time ,the activities of endogenous total proteases and cathepsin L‐like proteases tissue were increased in tissues in wnit mass .The distribution of cathepsin L‐like proteases in arm muscle of the autolytic octopus was in homogeneous . The stained spots of cathepsin L‐like proteases were dense in myocardial trabeculation ,but were relatively sparse in muscle fibers .Along with the progress of autolysis ,the stained spots of cathepsin L‐like proteases were scattered and eventually disappeared ,indicating the dispersion of cathepsin L‐like proteases in tissue .The findings indicated that the endogenous proteases , cathepsin L‐like proteases as representive , took part in the autolysis of octopus .%采用紫外线照射诱导短蛸自溶,检测腕部肌肉组织可溶性蛋白及可溶性胶原蛋白含量变化,监测内源总蛋白酶及内源组织蛋白酶L样蛋白酶活性变化,并应用免疫组织化学法揭示组织蛋白酶L样蛋白酶的组织分布变化。结果显示,短蛸经紫外线照射后,状态迟钝,机体瘫软,表皮及皮下组织发生黏液化,表明发生自溶。随着自溶时间的延长,可溶性总蛋白及可溶性胶原蛋白含量逐渐增加,表明结构蛋白发生降解;单位质量组织的内源蛋白酶总活力及组织蛋

  2. Effects of different types of soil on decomposition: an experimental study. (United States)

    Tumer, Ali Riza; Karacaoglu, Emre; Namli, Ayten; Keten, Alper; Farasat, Shima; Akcan, Ramazan; Sert, Osman; Odabaşi, Aysun Balseven


    Decomposition, a postmortem process including autolysis and putrefaction, is affected by many factors (e.g., humidity, microbial activity, soil properties). The purpose of this study was to determine the importance of soil type in decomposition process. Changes occurred in two intervals (3 and 6 months) were evaluated using a total of 32 Sus scrofa limbs by burying in four different types of soil (loamy, clayey, sandy and organic). The extremities in all soils had lost weight over time; however, mass loss was greater in loamy and organic soils in both intervals. Entomological findings were also assessed. Obtained findings of soil analysis and evaluation of decomposition were compatible. In conclusion, the present study revealed that soil properties and textures should be taken into account in evaluation of decomposition and estimating postmortem interval in crime scene investigation.

  3. Stability of Enzymes in Granular Enzyme Products for Laundry Detergents

    DEFF Research Database (Denmark)

    Biran, Suzan; Bach, Poul; Simonsen, Ole

    Enzymes have long been of interest to the detergent industry due to their ability to improve the cleaning efficiency of synthetic detergents, contribute to shortening washing times, and reduce energy and water consumption, provision of environmentally friendlier wash water effluents and fabric care...... particles. However, enzymes may loose a significant part of their activity over a time period of several weeks. Possible causes of inactivation of enzymes in a granule may be related to the release of hydrogen peroxide from the bleaching chemicals in a moisture-containing atmosphere, humidity, autolysis...... and humidity by bubbling nitrogen gas through their corresponding solutions. An enzyme column, acting as a plug-flow reactor, was exposed to known concentrations of H2O2 (g) and humidity in a thermally stabilized chamber. Samples were analyzed for adsorptive behavior and residual enzyme activity. Since...

  4. Combination of Maldi-tof mass spectrometry with immobilized enzyme microreactor for peptide mapping

    Institute of Scientific and Technical Information of China (English)


    Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been combined with immobilized enzyme microreactor for the rapid, sensitive, and accurate tryptic mapping of protein and polypeptides. The technique utilizes the trypsin microreactor by immobilized enzyme on the glycidyl methacrylate (GMA)-modified cellulose membrane. The membrane microreactor was used for the tryptic mapping of cytochrome C and the results were compared with those obtained by using free trypsin. A significant increase in the overall sensitivity of the process was observed using the membrane microreactor, as well as the elimination of background signals due to the autolysis of the trypsin. Further, membrane microreactor digestions were found to be rapid and convenient.

  5. Staphylococcus epidermidis recovered from indwelling catheters exhibit enhanced biofilm dispersal and "self-renewal" through downregulation of agr

    DEFF Research Database (Denmark)

    Dai, Lu; Yang, Liang; Parsons, Chris


    Background: In recent years, Staphylococcus epidermidis (Se) has become a major nosocomial pathogen and the most common cause of infections of implanted prostheses and other indwelling devices. This is due in part to avid biofilm formation by Se on device surfaces. However, it still remains unknown...... that how the process of Se biofilm development is associated with relapsed infection in such patients.Results: We have identified clinical Se isolates displaying enhanced biofilm dispersal and self-renewal relative to reference strain. These isolates also exhibit enhanced initial cell attachment......, extracellular DNA release, cell autolysis and thicker microcolonies during biofilm development relative to reference strain. Our genetic analyses suggest that these clinical isolates exhibit significant downregulation of RNAIII, the effector molecule of the agr quorum sensing system, and upregulation...

  6. Stimulation of Egg Production in Japanese Quails by Enriching Feed with Residual Yeast

    Directory of Open Access Journals (Sweden)

    Letitia Oprean


    Full Text Available Quail eggs are more and more approved for consumers because they bring many benefits to the human body. Therefore, quails breeding for eggs production have become a very profitable business. Residual yeast may be a nutritional supplement, especially rich in vitamins and proteins. This article studies the influence of residual beer yeast on egg laying in Japanese quails. In order to be integrated into the diet of quails the yeast has undergone a process of autolysis; its influence has been examined on separate groups. The results were reported as a percentage compared with the control group, where the feed does not contain this supplement. Due to its content rich in vitamins and proteins, the residual beer yeast used in feeding the quails bred for eggs stimulates egg laying.

  7. Mucosal delivery of anti-inflammatory IL-1Ra by sporulating recombinant bacteria

    Directory of Open Access Journals (Sweden)

    Ruggiero Paolo


    Full Text Available Abstract Background Mucosal delivery of therapeutic protein drugs or vaccines is actively investigated, in order to improve bioavailability and avoid side effects associated with systemic administration. Orally administered bacteria, engineered to produce anti-inflammatory cytokines (IL-10, IL-1Ra, have shown localised ameliorating effects in inflammatory gastro-intestinal conditions. However, the possible systemic effects of mucosally delivered recombinant bacteria have not been investigated. Results B. subtilis was engineered to produce the mature human IL-1 receptor antagonist (IL-1Ra. When recombinant B. subtilis was instilled in the distal colon of rats or rabbits, human IL-1Ra was found both in the intestinal lavage and in the serum of treated animals. The IL-1Ra protein in serum was intact and biologically active. IL-1-induced fever, neutrophilia, hypoglycemia and hypoferremia were inhibited in a dose-dependent fashion by intra-colon administration of IL-1Ra-producing B. subtilis. In the mouse, intra-peritoneal treatment with recombinant B. subtilis could inhibit endotoxin-induced shock and death. Instillation in the rabbit colon of another recombinant B. subtilis strain, which releases bioactive human recombinant IL-1β upon autolysis, could induce fever and eventually death, similarly to parenteral administration of high doses of IL-1β. Conclusions A novel system of controlled release of pharmacologically active proteins is described, which exploits bacterial autolysis in a non-permissive environment. Mucosal administration of recombinant B. subtilis causes the release of cytoplasmic recombinant proteins, which can then be found in serum and exert their biological activity in vivo systemically.

  8. Interaction of aspartic acid-104 and proline-287 with the active site of m-calpain. (United States)

    Arthur, J S; Elce, J S


    In an ongoing study of the mechanisms of calpain catalysis and Ca(2+)-induced activation, the effects of Asp-104-->Ser and Pro-287-->Ser large subunit mutations on m-calpain activity, the pH-activity profile, Ca(2+)-sensitivity, and autolysis were measured. The importance of these positions was suggested by sequence comparisons between the calpain and papain families of cysteine proteinases. Asp-104 is adjacent to the active-site Cys-105, and Pro-287 is adjacent to the active-site Asn-286 and probably to the active-site His-262; both Asp-104 and Pro-287 are absolutely conserved in the known calpains, but are replaced by highly conserved serine residues in the papains. The single mutants had approx. 10-15% of wild-type activity, due mainly to a decrease in kcat, since Km was only slightly increased. The Pro-287-->Ser mutation appeared to cause a local perturbation of the catalytic Cys-105/His-262 catalytic ion pair, reducing its efficiency without major effect on the conformation and stability of the enzyme. The Asp-104-->Ser mutation caused a marked narrowing of the pH-activity curve, a 9-fold increase in Ca2+ requirement, and an acceleration of autolysis, when compared with the wild-type enzyme. The results indicated that Asp-104 alters the nature of its interaction with the catalytic ion pair during Ca(2+)-induced conformational change in calpain. This interaction may be direct or indirect, but is important in activation of the enzyme. PMID:8912692

  9. Effect of Lactobacillus helveticus ATCC 15009 on the Ripening of Gouda Cheese%瑞士乳杆菌ATCC 15009对干酪成熟的影响

    Institute of Scientific and Technical Information of China (English)

    温阿祎; 王希璠; 郭慧媛; 冷小京


    干酪的成熟是形成干酪特有的风味、质地和组织状态以及影响加工成本的最关键工艺。添加辅助发酵剂是目前较为成熟的一种促进干酪成熟的方法。本文将不同剂量的瑞士乳杆菌ATCC15009作为辅助发酵剂添加到古达奶酪中,通过检测奶酪成熟过程中的乳酸菌自溶情况、可溶性氮含量以及感官评定等指标分析了ATCC15009对奶酪成熟的影响。结果证明瑞士乳杆菌ATCC15009具有很强的自溶能力,可以有效分解蛋白质并促进干酪的成熟,同时改善干酪的风味。%The ripening of cheese is the most important part that affect the lfavor, texture, and processing cost of cheese. The adding of adjunct starter is a mature way to promote the ripening of cheese. The experiment researched on the gouda cheese, which were added different amount of L. helveticus ATCC 15009, a kind of highly autolysis adjunct starter. The effect of L. helveticus ATCC 15009 on Gouda cheese ripening was analyzed by testing autolysis degree of lactobacillus, the changes of TCA-SN/TN, and making sensory evaluation of the cheese. The results showed that the L. helveticus ATCC 15009 was highly autolytic, which can accelerate the hydrolysis of protein, thereby signiifcantly promote the ripening and improve the lfavor of gouda cheese.

  10. 乳酸乳球菌与酵母菌混合培养时菌间的相互影响%Interaction of co-cultured yeasts and Lactococcus lactis

    Institute of Scientific and Technical Information of China (English)

    王永丽; 李晓东; 姚春燕; 郭晶; 王秋宇


    研究了干酪常用发酵剂——乳酸乳球菌与干酪中两株常见酵母菌——耶罗解脂酵母及汉逊德巴利酵母混合培养时的相互影响.在营养肉汤中对三株菌单独及混合培养,比浊法测定其生长浓度,同时在脱脂乳培养基中做相同培养,然后检测活菌数、球菌产酸及菌株自溶情况.结果表明:乳酸乳球菌、耶罗解脂酵母及汉逊德巴利酵母三株菌混合培养时其菌数及自溶度,均有显著增加,表明混合培养加快了菌株生长代谢的速率.这对研究干酪促熟及风味调整具有重要意义.%In this experiment, co-cultured Lactococcus lactis and two normal yeasts in cheese, Debaryomyces. hansenii and Yarrowia .lipolytica, to define the interaction among them. Three strains were alone and co-cultured in nutrient broth, determined their turbidity. Same cultured in the skim milk medium, and then detected the number of viable cells, acidity of medium and autolysis of the strains. The results showed that: plate count and autolysis were significantly increased (p<0.05) when co-cultured three strains, accelerated the rate of the strains of growth and metabolism. There is great significance to cheese ripening and flavor .

  11. Primary structure of brevilysin L4, an enzymatically active fragment of a disintegrin precursor from Gloydius halys brevicaudus venom. (United States)

    Deshimaru, Masanobu; Ichihara, Makoto; Hattori, Takahiro; Koba, Kumiko; Terada, Shigeyuki


    Brevilysin L4 (L4) is a non-hemorrhagic P-I class metalloprotease (MP) isolated from Gloydius halys brevicaudus venom. Its complete amino acid sequence has been determined. L4 is a single-chain polypeptide and highly homologous to those of other snake venom MPs. A zinc-binding motif, HExxHxxGxxH, is located at residues 142-152. A characteristic feature of L4 is the presence of a spacer sequence (LRTDTVS) at the C-terminal that links metalloprotease and disintegrin domains and is usually removed by post-translational proteolysis, suggesting that L4 is expressed together with a spacer region and a disintegrin domain at the C-terminal. The nucleotide sequence of a cDNA clone encoding L4 has revealed that L4 is a disintegrin precursor and produced as a P-II class MP. The disintegrin coded after L4 sequence was brevicaudin 1, a disintegrin previously isolated from the same venom. P-II class MPs have been suspected to undergo autoproteolysis to release disintegrins. Although being P-I class MP, L4 itself autocatalytically degrades with a half-life of 30min at pH 8.5 and 37 degrees C in the absence of Ca(2+). Sequence analysis of several fragment peptides produced during the autolysis of L4 indicated that more than 40 peptide bonds were split, and the cleavages of Ser(60)-Asn(61), Thr(99)-Ala(100), and Phe(103)-Asp(104) bonds may trigger the autoproteolysis. Addition of Ca(2+) completely suppressed the cleavage of these particular bonds, resulting in a marked prevention of autoproteolysis. Thus, L4 provides a good model for the investigation of autolysis of some MPs.

  12. Impact of the Staphylococcus epidermidis LytSR two-component regulatory system on murein hydrolase activity, pyruvate utilization and global transcriptional profile

    Directory of Open Access Journals (Sweden)

    Yu Fangyou


    Full Text Available Abstract Background Staphylococcus epidermidis has emerged as one of the most important nosocomial pathogens, mainly because of its ability to colonize implanted biomaterials by forming a biofilm. Extensive studies are focused on the molecular mechanisms involved in biofilm formation. The LytSR two-component regulatory system regulates autolysis and biofilm formation in Staphylococcus aureus. However, the role of LytSR played in S. epidermidis remained unknown. Results In the present study, we demonstrated that lytSR knock-out in S. epidermidis did not alter susceptibility to Triton X-100 induced autolysis. Quantitative murein hydrolase assay indicated that disruption of lytSR in S. epidermidis resulted in decreased activities of extracellular murein hydrolases, although zymogram showed no apparent differences in murein hydrolase patterns between S. epidermidis strain 1457 and its lytSR mutant. Compared to the wild-type counterpart, 1457ΔlytSR produced slightly more biofilm, with significantly decreased dead cells inside. Microarray analysis showed that lytSR mutation affected the transcription of 164 genes (123 genes were upregulated and 41 genes were downregulated. Specifically, genes encoding proteins responsible for protein synthesis, energy metabolism were downregulated, while genes involved in amino acid and nucleotide biosynthesis, amino acid transporters were upregulated. Impaired ability to utilize pyruvate and reduced activity of arginine deiminase was observed in 1457ΔlytSR, which is consistent with the microarray data. Conclusions The preliminary results suggest that in S. epidermidis LytSR two-component system regulates extracellular murein hydrolase activity, bacterial cell death and pyruvate utilization. Based on the microarray data, it appears that lytSR inactivation induces a stringent response. In addition, LytSR may indirectly enhance biofilm formation by altering the metabolic status of the bacteria.

  13. Changes in the apoplastic pH are involved in regulation of xyloglucan breakdown of azuki bean epicotyls under hypergravity conditions. (United States)

    Soga, K; Wakabayashi, K; Hoson, T; Kamisaka, S


    Hypergravity inhibited elongation growth of azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls by decreasing the mechanical extensibility of cell walls via the increase in the molecular mass of xyloglucans [Soga et al. (1999) Plant Cell Physiol. 40: 581]. Here, we report that the pH value of the apoplastic fluid in epicotyls increased from 5.8 to 6.6 by hypergravity (300 x g) treatment. When the xyloglucan-degrading enzymes extracted from cell walls of the 1 x g control epicotyls were assayed in buffer at pH 6.6 and 5.8, the activity at pH 6.6 was almost half of that at pH 5.8. In addition, when enzymically active cell wall preparations obtained from 1 x g control epicotyls were autolyzed in buffer at pH 5.8 and 6.6 and then xyloglucans were extracted from the autolyzed cell walls, the molecular mass of xyloglucans incubated at pH 5.8 decreased during the autolysis, while that at pH 6.6 did not change. Thus, the xyloglucans were not depolymerized by autolysis at the pH value (6.6) observed in the hypergravity-treated epicotyls. These findings suggest that in azuki bean epicotyls, hypergravity decreases the activities of xyloglucan-degrading enzymes by increasing the pH in the apoplastic fluid, which may be involved in the processes of the increase in the molecular mass of xyloglucans, leading to the decrease in the cell wall extensibility.

  14. Towards quantitative mRNA analysis in paraffin-embedded tissues using real-time reverse transcriptase-polymerase chain reaction: a methodological study on lymph nodes from melanoma patients. (United States)

    Abrahamsen, Helene Nortvig; Steiniche, Torben; Nexo, Ebba; Hamilton-Dutoit, Stephen J; Sorensen, Boe Sandahl


    Improved extraction techniques combined with sensitive real-time reverse transcriptase-polymerase chain reaction may allow detection of mRNA in formalin-fixed, paraffin-embedded (FFPE) materials, but the factors affecting mRNA quantification in clinical material using these methods have not been systematically analyzed. We designed analyses using real-time reverse transcriptase-polymerase chain reaction for quantification of MART-1, beta-actin, and beta(2)-microglobulin mRNAs. The analytical intra- and interassay imprecision (coefficient of variation) was in the range 10 to 20% for all three genes studied. Using these protocols, we studied the influence of tissue autolysis and length of formalin-fixation on mRNA detection in metastatic melanoma. Delay in freezing reduced detectable mRNA, although this was less than predicted and mostly occurred early in autolysis. MART-1, beta-actin, and beta(2)-microglobulin mRNAs were consistently detected in FFPE metastatic melanoma even after fixation for up to 3 weeks, although the total mRNA detected was markedly reduced in fixed compared with fresh tissues (up to 99%). Quantification of MART-1 was, however, possible if this was expressed relative to a housekeeping gene. The polymerase chain reaction product from FFPE tissues could be increased up to 100-fold amplifying short (tissue processing and in fixation length seem to be less important sources of imprecision than previously assumed. Our findings suggest that quantitative analysis of mRNA in archive and routine diagnostic tissues may be possible.

  15. Formula optimization of high autolytic complex microbial agents for yogurt%酸乳高自溶度复合发酵剂配方优化

    Institute of Scientific and Technical Information of China (English)

    孙洁; 王希卓; 刘鹭; 文一; 张书文; 吕加平


    乳酸菌的自溶特性可对发酵乳制品品质产生影响,发酵剂菌体快速自溶不仅缩短酸乳发酵周期、降低了生产成本,同时菌体自溶之后释放的胞内物质又决定着酸乳的风味和感官特性。为了复配出一种高质量的复合型酸乳发酵剂,该文对前期研究筛选出的3株高度自溶的酸乳发酵剂菌株:德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckii ssp. Bulgaricus)LD3-A3、唾液链球菌嗜热亚种(Streptococcus salivarius ssp.)GS1-A8和乳酸乳杆菌(Lactobacillus lactis)S15-A3进行复配,运用二次正交旋转组合设计复合发酵剂中各菌株比例,并讨论了3株菌株之间的共生关系和交互作用,根据所建模型优选结果,最后确定3种菌株最优组合方案为:GS1∶LD3-A3∶S15-A3为45∶1∶3。使用该复合发酵剂制作的酸乳感官性状优良,具有较好的风味和质地,在货架期贮藏条件下(10~15℃)后酸化程度小,可极好的满足生产需要。该文所得高自溶度复合发酵剂具有很好的生产实践指导和应用价值。%Cell autolysis could be induced by bacterial peptidoglycan hydrolases (autolysins) degrading the peptidoglycan structure of cell walls. Autolysis of lactobic acid bacteria shows direct influence on the characteristics of fermented milk products. Three high autolysis rate lactic acid bacteria, Lactobacillus delbrueckii ssp. Bulgaricus LD3-A3, Streptococcus salivarius ssp. and Thermophilus GS1-A8, and Lactobacillus lactis S15-A3 were tested to obtain a good yogurt starter. The three-factors quadratic regression orthogonal rotational composite experiment was designed to determine the effects of the three high autolysis strains on yogurt characteristics during fermentation, and the effects of mixed symbiosis and interactions were also analyzed. Quadratic orthogonal rotation combination design was used to compare differences among 23 yogurt samples that were

  16. Meat Spoilage Mechanisms and Preservation Techniques: A Critical Review

    Directory of Open Access Journals (Sweden)

    D. Dave


    Full Text Available Problem statement: Extremely perishable meat provides favorable growth condition for various microorganisms. Meat is also very much susceptible to spoilage due to chemical and enzymatic activities. The breakdown of fat, protein and carbohydrates of meat results in the development of off-odors, off-flavor and slim formation which make the meat objectionable for human consumption. It is, therefore, necessary to control meat spoilage in order to increase its shelf life and maintain its nutritional value, texture and flavor. Approach: A comprehensive literature review was performed on the spoliage mechanisms of meat and meat products and preservation techniques. Results: Historical data reveals that salting, drying, smoking, fermentation and canning were the traditional methods used to prevent meat spoilage and extend its shelf life. However, in order to prevent wholesomeness, appearance, composition, tenderness, flavor, juiciness and nutritive value, new methods were developed. These included: cooling, freezing and chemical preservation. Wide range of physical and chemical reactions and actions of microorganisms or enzymes are responsible for the meat spoilage. Microbial growth, oxidation and enzymatic autolysis are three basic mechanisms responsible for spoilage of meat. Microbial growth and metabolism depends on various factors including: pre-slaughter husbandry practices, age of the animal at the time of slaughtering, handling during slaughtering, evisceration and processing, temperature controls during slaughtering, processing and distribution, preservation methods, type of packaging and handling and storage by consumer. Microbial spoilage causes pH change, slime formation, structural components degradation, off odors and appearance change. Autoxidation of lipids and the production of free radicals are natural processes which affect fatty acids and lead to oxidative deterioration of meat and off-flavour development. Lipid hydrolysis can take

  17. A smallest 6 kda metalloprotease, mini-matrilysin, in living world: a revolutionary conserved zinc-dependent proteolytic domain- helix-loop-helix catalytic zinc binding domain (ZBD

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    Yu Wei-Hsuan


    Full Text Available Abstract Background The Aim of this study is to study the minimum zinc dependent metalloprotease catalytic folding motif, helix B Met loop-helix C, with proteolytic catalytic activities in metzincin super family. The metzincin super family share a catalytic domain consisting of a twisted five-stranded β sheet and three long α helices (A, B and C. The catalytic zinc is at the bottom of the cleft and is ligated by three His residues in the consensus sequence motif, HEXXHXXGXXH, which is located in helix B and part of the adjacent Met turn region. An interesting question is - what is the minimum portion of the enzyme that still possesses catalytic and inhibitor recognition?” Methods We have expressed a 60-residue truncated form of matrilysin which retains only the helix B-Met turn-helix C region and deletes helix A and the five-stranded β sheet which form the upper portion of the active cleft. This is only 1/4 of the full catalytic domain. The E. coli derived 6 kDa MMP-7 ZBD fragments were purified and refolded. The proteolytic activities were analyzed by Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay and CM-transferrin zymography analysis. SC44463, BB94 and Phosphoramidon were computationally docked into the 3day structure of the human MMP7 ZBD and TAD and thermolysin using the docking program GOLD. Results This minimal 6 kDa matrilysin has been refolded and shown to have proteolytic activity in the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay. Triton X-100 and heparin are important factors in the refolding environment for this mini-enzyme matrilysin. This minienzyme has the proteolytic activity towards peptide substrate, but the hexamer and octamer of the mini MMP-7 complex demonstrates the CM-transferrin proteolytic activities in zymographic analysis. Peptide digestion is inhibited by SC44463, specific MMP7 inhibitors, but not phosphorimadon. Interestingly, the mini MMP-7 can be processed by autolysis and producing ~ 6


    Bronfenbrenner, J; Muckenfuss, R


    We have been able to confirm the observations of Twort as well as of Gratia, that dead staphylococcus may undergo lysis if, in addition to a suitable bacteriophage, there is also present live staphylococcus. Moreover, we have endeavored to ascertain the mechanism of this phenomenon and have found that in order to elicit it it is necessary to control the numbers of live and dead bacteria in the mixture. An excess of dead bacteria interferes with lysis by adsorbing the bacteriophage before it has the opportunity to initiate necessary changes in the live bacteria, so that all lysis is prevented. The phenomenon is specific, that is, the lysis of live bacteria is accompanied by lysis of dead bacteria of the same species only. Lysis of dead bacteria occurs best with staphylococcus, an organism which easily undergoes spontaneous autolysis under appropriate conditions. In the case of B. coli or B. dysenteriae the lysis of the dead bacteria is uncertain. Dead bacteria need not be present in the mixture at the beginning of the experiment; they will be dissolved if added any time before, during, or after the completion of lysis of live bacteria. If the test is performed so that a suitable semipermeable membrane is interposed between the dead and live bacteria, the dead bacteria are not dissolved, in spite of the lysis of live bacteria on the other side of the membrane. The agent determining the lysis of dead bacteria is not diffusible, while the principle initiating the lysis of live bacteria diffuses freely and is demonstrably present on both sides of the membrane. The complete independence of the agent causing dissolution of dead bacteria from bacteriophage can also be shown by separating the two agents by means of filtration, or by adsorption on bacteria. The ferment-like substance responsible for the lysis of dead bacteria is different from the bacteriophage. It is not diffusible through collodion, it is easily adsorbed on clay filters, it is heat-labile, and is


    Bussolati, Gianni; Fulcheri, Ezio


    Collection of pathological specimens began soon after the seminal description of autopsy by Giovan Battista Morgagni in Padoa in the second half of the 18th Century. Pathologists soon realized difficulties of preserving the form and to prevent decay caused by autolysis and attack by bacteria and parasites. The ancient procedures devoted to mummification were applied to the purpose, and a number of personal experiences were reported in the first half of the 19th century, mainly in Northern Italy and France, testifying a dedicated interest of the time in those areas. A combination of chemical fixation (with corrosive sublimate/mercuric chloride and/or tannic acid) and careful drying allowed to produce dry preparations, once very numerous in the Pathological Anatomy's Museums so much popular in the 19th and early 20th Century. In fact, it was the sole way to give visual evidence of disease and pathological processes. Only a limited number of these dry preparations are still present and visible in Pathology Museums, mainly in Universities of Northern Italy, while a few examples can be traced in the other European Country.

  20. METACASPASE9 modulates autophagy to confine cell death to the target cells during Arabidopsis vascular xylem differentiation

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    Sacha Escamez


    Full Text Available We uncovered that the level of autophagy in plant cells undergoing programmed cell death determines the fate of the surrounding cells. Our approach consisted of using Arabidopsis thaliana cell cultures capable of differentiating into two different cell types: vascular tracheary elements (TEs that undergo programmed cell death (PCD and protoplast autolysis, and parenchymatic non-TEs that remain alive. The TE cell type displayed higher levels of autophagy when expression of the TE-specific METACASPASE9 (MC9 was reduced using RNAi (MC9-RNAi. Misregulation of autophagy in the MC9-RNAi TEs coincided with ectopic death of the non-TEs, implying the existence of an autophagy-dependent intercellular signalling from within the TEs towards the non-TEs. Viability of the non-TEs was restored when AUTOPHAGY2 (ATG2 was downregulated specifically in MC9-RNAi TEs, demonstrating the importance of autophagy in the spatial confinement of cell death. Our results suggest that other eukaryotic cells undergoing PCD might also need to tightly regulate their level of autophagy to avoid detrimental consequences for the surrounding cells.

  1. Population dynamics of mixed cultures of yeast and lactic acid bacteria in cider conditions

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    Leila Roseli Dierings


    Full Text Available The objective of this work was to study the malolactic bioconversion in low acidity cider, according Brazilian conditions. The apple must was inoculated with Saccharomyces cerevisiae or S. cerevisiae with Oenococcus oeni. The control contained the indigenous microorganisms. Fermentation assays were carried out with clarified apple must from the Gala variety. At the beginning of fermentation, there was a fast growth of the non-Saccharomyces yeast population. Competitive inhibition occurred in all the assays, either with inoculated or indigenous populations of the yeast. The lactic acid bacteria count was ca. 1.41·10²CFU/mL at the beginning and 10(6CFU/mL after yeast cells autolysis. The lactic bacteria O. oeni reached the highest population (10(7CFU/mL when added to the apple must after the decline of the yeast. The malic acid was totally consumed during the alcoholic fermentation period (80.0 to 95.5 % and lactic acid was still synthesized during the 35 days of malolactic fermentation. These results could be important in order to achieve a high quality brut, or sec cider obtained from the dessert apple must.

  2. Application of microbial electrolysis cells to treat spent yeast from an alcoholic fermentation. (United States)

    Sosa-Hernández, Ornella; Popat, Sudeep C; Parameswaran, Prathap; Alemán-Nava, Gibrán Sidney; Torres, César I; Buitrón, Germán; Parra-Saldívar, Roberto


    Spent yeast (SY), a major challenge for the brewing industry, was treated using a microbial electrolysis cell to recover energy. Concentrations of SY from bench alcoholic fermentation and ethanol were tested, ranging from 750 to 1500mgCOD/L and 0 to 2400mgCOD/L respectively. COD removal efficiency (RE), coulombic efficiency (CE), coulombic recovery (CR), hydrogen production and current density were evaluated. The best treatment condition was 750mgCOD/LSY+1200mgCOD/L ethanol giving higher COD RE, CE, CR (90±1%, 90±2% and 81±1% respectively), as compared with 1500mgCOD/LSY (76±2%, 63±7% and 48±4% respectively); ethanol addition was significantly favorable (p value=0.011), possibly due to electron availability and SY autolysis. 1500mgCOD/LSY+1200mgCOD/L ethanol achieved higher current density (222.0±31.3A/m(3)) and hydrogen production (2.18±0.66 [Formula: see text] ) but with lower efficiencies (87±2% COD RE, 71.0±.4% CE). Future work should focus on electron sinks, acclimation and optimizing SY breakdown.

  3. [Calpains and cardiac diseases]. (United States)

    Perrin, C; Vergely, C; Rochette, L


    Calpains are a large family of cytosolic cysteine proteases composed of at least fourteen distinct isoforms. The family can be divided into two groups on the basis of distribution: ubiquitous and tissue-specific. Our current knowledge about calpains properties apply mainly to the ubiquitous isozymes, micro- and milli-calpain (classic calpains). These forms are activated after autolysis. Translocation and subsequent interactions with phospholipids of these enzymes increase their activity. Calpains are able to cleave a subset of substrates, as enzymes, structural and signalling proteins. Cardiac pathologies, such as heart failure, atrial fibrillation or clinical states particularly ischemia reperfusion, are associated with an increase of cytosolic calcium and in this regards, calpain activation has been evoked as one of the mediators leading to myocardial damage. Calpain activities have been shown to be increased in hearts experimentally subjected to ischemia reperfusion or during hypertrophy, but also in atrial tissue harvested from patients suffering from atrial fibrillations. These activities have been related to an increase of the proteolysis of different myocardial components, particularly, troponins, which are major regulators of the contraction of cardiomyocytes. Moreover, recent works have demonstrated that calpains are involved in the development of myocardial cell death by necrosis or apoptosis.

  4. Oxidative modification of glutamine synthetase. I. Inactivation is due to loss of one histidine residue. (United States)

    Levine, R L


    Intracellular proteolytic degradation of glutamine synthetase occurs in two distinct steps in Escherichia coli (Levine, R. L., Oliver, C. N., Fulks, R. M., and Stadtman, E. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2120-2124). In the first step, a mixed function oxidation modifies the glutamine synthetase. The modified enzyme, which is catalytically inactive, becomes susceptible to proteolytic attack. In the second step, a protease specific for the modified enzyme catalyzes the actual proteolytic degradation. The oxidatively modified glutamine synthetase was studied to determine the chemical differences between it and the native enzyme. Only a single alteration was found; one of sixteen histidine residues/subunit was altered by the oxidative modification. The modification introduced a carbonyl group into the protein, permitting isolation of a stable dinitrophenylhydrazone. No other differences were detected between the native and modified proteins. Specifically, the cysteine, methionine, phenylalanine, tyrosine, and tryptophan contents were not altered. A number of other prokaryotic and eukaryotic enzymes are also susceptible to oxidative modification. This covalent modification may be important in intracellular proteolysis, in mammalian host defense systems, in prevention of autolysis, in aging processes, and in oxygen toxicity.

  5. Metamorphic changes in the stomach of the frog Rana temporaria tadpoles. (United States)

    Rovira, J; Villaro, A C; Bodegas, M E; Valverde, E; Sesma, P


    The histological transformation of amphibian stomach during metamorphosis was studied in the frog Rana temporaria. The earliest metamorphic changes occur shortly before regression of the cloacal piece of tail and appearance of forelegs. Autolysis of primary, larval epithelial cells and activity of phagocytes lead to regression of the apical mucosa, which is shed into the gastric lumen. Histogenesis takes place from the very beginning of metamorphosis in the basal region of the mucosa; undifferentiated, regenerative cells, arranged in small compact cords and surrounded by a thick basement membrane, give rise to secondary lining epithelium and glands. Lining epithelial cells differentiate into a typical mucosecretory epithelium. Oxyntic cells present in larval glands are substituted by both ion- and protein-secreting oxyntic-peptic cells. During metamorphosis, connective and muscular tissues markedly increase, a submucosa, not present in larval tadpoles, gradually develops. A muscularis mucosae is also formed and the muscular propria becomes thicker. At late metamorphosis, folded structures involving both mucosa and submucosa develop, increasing the luminal surface as in adults. Removal of the larval gastric mucosa and its replacement by a new, adult-type definitive one, together with development of peripheral connective and muscular tissues, account for metamorphosis of tadpole stomach.

  6. Mechanism of action of Melaleuca alternifolia (tea tree) oil on Staphylococcus aureus determined by time-kill, lysis, leakage, and salt tolerance assays and electron microscopy. (United States)

    Carson, Christine F; Mee, Brian J; Riley, Thomas V


    The essential oil of Melaleuca alternifolia (tea tree) has broad-spectrum antimicrobial activity. The mechanisms of action of tea tree oil and three of its components, 1,8-cineole, terpinen-4-ol, and alpha-terpineol, against Staphylococcus aureus ATCC 9144 were investigated. Treatment with these agents at their MICs and two times their MICs, particularly treatment with terpinen-4-ol and alpha-terpineol, reduced the viability of S. aureus. None of the agents caused lysis, as determined by measurement of the optical density at 620 nm, although cells became disproportionately sensitive to subsequent autolysis. Loss of 260-nm-absorbing material occurred after treatment with concentrations equivalent to the MIC, particularly after treatment with 1,8-cineole and alpha-terpineol. S. aureus organisms treated with tea tree oil or its components at the MIC or two times the MIC showed a significant loss of tolerance to NaCl. When the agents were tested at one-half the MIC, only 1,8-cineole significantly reduced the tolerance of S. aureus to NaCl. Electron microscopy of terpinen-4-ol-treated cells showed the formation of mesosomes and the loss of cytoplasmic contents. The predisposition to lysis, the loss of 260-nm-absorbing material, the loss of tolerance to NaCl, and the altered morphology seen by electron microscopy all suggest that tea tree oil and its components compromise the cytoplasmic membrane.

  7. Effect of Streptococcus pneumoniae on human respiratory epithelium in vitro. (United States)

    Steinfort, C; Wilson, R; Mitchell, T; Feldman, C; Rutman, A; Todd, H; Sykes, D; Walker, J; Saunders, K; Andrew, P W


    A total of 11 of 15 Streptococcus pneumoniae culture filtrates and all five bacterial autolysates produced by cell death in the stationary phase caused slowed ciliary beating and disruption of the surface integrity of human respiratory epithelium in organ culture. This effect was inhibited by cholesterol and was heat labile and reduced by standing at room temperature but was stable at -40 degrees C. The activity was detected at the late stationary phase of culture and was associated with the presence of hemolytic activity. Gel filtration of a concentrated culture filtrate and autolysate both yielded a single fraction of approximately 50 kilodaltons which slowed ciliary beating and were the only fractions with hemolytic activity. Rabbit antiserum to pneumolysin, a sulfhydryl-activated hemolytic cytotoxin released by S. pneumoniae during autolysis, neutralized the effect of the culture filtrate on respiratory epithelium. Both native and recombinant pneumolysin caused ciliary slowing and epithelial disruption. Electron microscopy showed a toxic effect of pneumolysin on epithelial cells: cytoplasmic blebs, mitochondrial swelling, cellular extrusion, and cell death, but no change in ciliary ultrastructure. Recombinant pneumolysin (10 micrograms/ml) caused ciliary slowing in the absence of changes in cell ultrastructure. Release of pneumolysin in the respiratory tract during infection may perturb host defenses, allowing bacterial proliferation and spread.

  8. Implementation of a method to visualize noise-induced hearing loss in mass stranded cetaceans (United States)

    Morell, Maria; Brownlow, Andrew; McGovern, Barry; Raverty, Stephen A.; Shadwick, Robert E.; André, Michel


    Assessment of the impact of noise over-exposure in stranded cetaceans is challenging, as the lesions that lead to hearing loss occur at the cellular level and inner ear cells are very sensitive to autolysis. Distinguishing ante-mortem pathology from post-mortem change has been a major constraint in diagnosing potential impact. Here, we outline a methodology applicable to the detection of noise-induced hearing loss in stranded cetaceans. Inner ears from two mass strandings of long-finned pilot whales in Scotland were processed for scanning electron microscopy observation. In one case, a juvenile animal, whose ears were fixed within 4 hours of death, revealed that many sensory cells at the apex of the cochlear spiral were missing. In this case, the absence of outer hair cells would be compatible with overexposure to underwater noise, affecting the region which transduces the lowest frequencies of the pilot whales hearing spectrum. Perfusion of cochlea with fixative greatly improved preservation and enabled diagnostic imaging of the organ of Corti, even 30 hours after death. This finding supports adopting a routine protocol to detect the pathological legacy of noise overexposure in mass stranded cetaceans as a key to understanding the complex processes and implications that lie behind such stranding events. PMID:28165504

  9. Septal membrane fusion--a pivotal event in bacterial spore formation? (United States)

    Higgins, M L; Piggot, P J


    Formation of the asymmetrically located septum divides sporulating bacilli into two distinct cells: the mother cell and the prespore. The rigidifying wall material in the septum is subsequently removed by autolysis. Examination of published electron micrographs indicates that the two septal membranes then fuse to form a single membrane. Membrane fusion would be expected to have profound consequences for subsequent development. For example, it is suggested that fusion activates processing of pro-sigma E to sigma E in the cytoplasm by exposing it to a membrane-bound processing enzyme. Asymmetry of the fused membrane could restrict processing to one face of the membrane and hence explain why sigma E is associated with transcription in the mother cell but not in the prespore. Asymmetry of the fused membrane might also provide a mechanism for restricting the activity of another factor, sigma F, to the prespore. Attachment of the flexible fused septal membrane to the condensing prespore nucleoid could help drive the engulfment of the prespore by the mother cell.

  10. Redox state and mitochondrial respiratory chain function in skeletal muscle of LGMD2A patients.

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    Mats I Nilsson

    Full Text Available Calpain-3 deficiency causes oxidative and nitrosative stress-induced damage in skeletal muscle of LGMD2A patients, but mitochondrial respiratory chain function and anti-oxidant levels have not been systematically assessed in this clinical population previously.We identified 14 patients with phenotypes consistent with LGMD2A and performed CAPN3 gene sequencing, CAPN3 expression/autolysis measurements, and in silico predictions of pathogenicity. Oxidative damage, anti-oxidant capacity, and mitochondrial enzyme activities were determined in a subset of muscle biopsies.Twenty-one disease-causing variants were detected along the entire CAPN3 gene, five of which were novel (c.338 T>C, c.500 T>C, c.1525-1 G>T, c.2115+4 T>G, c.2366 T>A. Protein- and mRNA-based tests confirmed in silico predictions and the clinical diagnosis in 75% of patients. Reductions in antioxidant defense mechanisms (SOD-1 and NRF-2, but not SOD-2, coupled with increased lipid peroxidation and protein ubiquitination, were observed in calpain-3 deficient muscle, indicating a redox imbalance primarily affecting non-mitochondrial compartments. Although ATP synthase levels were significantly lower in LGMD2A patients, citrate synthase, cytochrome c oxidase, and complex I+III activities were not different from controls.Despite significant oxidative damage and redox imbalance in cytosolic/myofibrillar compartments, mitochondrial respiratory chain function is largely maintained in skeletal muscle of LGMD2A patients.

  11. Diagnosis of Neisseria gonorrhoeae among pregnant women by culture method and PCR on cppB gene

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    Jalal Mardaneh


    Full Text Available Background: Neisseria gonorrhoeae is a human obligate pathogen and the etiological agent of gonorrhea. Health irreparable complications resulting from gonorrhea disease occur mainly in pregnant women and neonates. Aim of this study was diagnosis of Neisseria gonorrhoeae among pregnant women with using culture and molecular method by amplification of cppB gene with PCR. Material and Methods: In this cross-sectional study, two endocervical swab specimens were obtained from 1100 pregnant women who referred to Shiraz Hospitals. Culture on nonselective and selective media and nucleic acid amplification test (NAAT were performed for detection of Neisseria gonorrhoeae cppB gene. Results: All endocervical swabs cultures on selective and nonselective media were negative for Neisseria gonorrhoeae. Among examined endocervical swabs, 13samples (1.18% were positive by nucleic acid amplification of Neisseria gonorrgoeae cppB gene. Conclusion: Negative results of culture and positive results of PCR in this study indicate that however culture is gold standard method for detection of Neisseria gonorrhoeae but due to bacterial autolysis, poor sampling techniques and improper specimen storage and transport, its value decline as compared with Nucleic acid amplification test (NAAT.

  12. Dehydroabietic Acid Derivative QC4 Induces Gastric Cancer Cell Death via Oncosis and Apoptosis

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    Dongjun Luo


    Full Text Available Aim. QC4 is the derivative of rosin’s main components dehydroabietic acid (DHA. We investigated the cytotoxic effect of QC4 on gastric cancer cells and revealed the mechanisms beneath the induction of cell death. Methods. The cytotoxic effect of QC4 on gastric cancer cells was evaluated by CCK-8 assay and flow cytometry. The underlying mechanisms were tested by administration of cell death related inhibitors and detection of apoptotic and oncosis related proteins. Cytomembrane integrity and organelles damage were confirmed by lactate dehydrogenase (LDH leakage assay, mitochondrial function test, and cytosolic free Ca2+ concentration detection. Results. QC4 inhibited cell proliferation dose- and time-dependently and destroyed cell membrane integrity, activated calpain-1 autolysis, and induced apoptotic protein cleavage in gastric cancer cells. The detection of decreased ATP and mitochondrial membrane potential, ROS accumulation, and cytosolic free Ca2+ elevation confirmed organelles damage in QC4-treated gastric cancer cells. Conclusions. DHA derivative QC4 induced the damage of cytomembrane and organelles which finally lead to oncosis and apoptosis in gastric cancer cells. Therefore, as a derivative of plant derived small molecule DHA, QC4 might become a promising agent in gastric cancer therapy.

  13. Optimization of RNA Extraction from Rat Pancreatic Tissue

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    Sanaz Dastgheib


    Full Text Available Background: Optimized RNA extraction from tissues and cell lines consists of four main stages regardless of the method of extraction: 1 homogenizing, 2 effective denaturation of proteins from RNA, 3 inactivation of ribonuclease, and 4 removal of any DNA, protein, and carbohydrate contamination. Isolation of undamaged intact RNA is challenging when the related tissue contains high levels of RNase. Various technical difficulties occur during extraction of RNA from pancreatic tissue due to spontaneous autolysis. Since standard routine protocols yield unacceptable results in pancrease, we have designed a simple method for RNA extraction by comparing different protocols. Methods: We obtained 20-30 mg pancreatic tissues in less than 2 min from 30 rats. Several methods were performed to extract RNA from pancreatic tissue and evaluate its integrity. All methods were performed three times to obtain reproducible results. Results: Immersing pancreatic tissue in RNA-later for 24 h at -80ºC yielded high quality RNA by using the TriPure reagent which was comparable to the commercial RNeasy Micro Kit. The quality of RNA was evaluated by spectrophotometer, electrophoresis and RT-PCR. We separated intact 28S and 18S ribosomal RNA (rRNA when our procedure was compared with the RNeasy Micro Kit. Finally, full length of the actin gene was amplified by RT-PCR. Conclusion: We designed a simple, fast, cost-effective method for complete RNA extraction from the least amount of quantitatively intact pancreatic tissue


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    Nesterenko A. A.


    Full Text Available In spite of decrease of meat import in connection with introduction of sanctions, demand and production of specialty produce are increased. High demands are lodged to meat produce for production of summer sausages. The technological defective goods of sausages not infrequently occur under use of meat raw with non-traditional autolysis and quality. For prevention of defective goods is necessary to have an opportunity to forecast the chemical content and rheological characteristics of sausages. These indexes are important for prognosis of quality of newly worked out or modernized receipts of sausages. The quality of sausages depends on qualitative indexes of minced meat. One of the indexes of minced beef is a consistence which is assessed not only organoleptically but on rheological characteristics. For production of sausages with advanced chemical content and consistence assessed under the help of rheological characteristics is necessary to work out the method of forecasting of ready goods quality. At projecting of the receipt we were offered to use the calculation of rheological and chemical-technological characteristics. There was described the classification of minced meat as heterogeneous system consisting of dispersive medium and phase. There were considered the possibilities of calculation of rheological, chemical indexes and complex chemical characteristics of minced meat and ready sausages in the work. The application of rheological methods of forecasting allows tracing the quality of sausages on any stage of production

  15. Conjugation of D-glucosamine to bovine trypsin increases thermal stability and alters functional properties. (United States)

    Gizurarson, Jóhann Grétar Kröyer; Filippusson, Hörður


    D-Glucosamine was conjugated to bovine trypsin by carbodiimide chemistry, involving a water-soluble carbodiimide and a succinimide ester, with the latter being to increase the yield of the conjugation. Mass spectrometric data suggested that several glycoforms were formed, with around 12 D-glucosamine moieties coupled to each trypsin molecule on average. The moieties were probably coupled to eight carboxyl groups (of glutamyl and aspartyl residues) and to four tyrosyl residues on the surface of the enzyme. The glycated trypsin possessed increased thermal stability. When compared with its unmodified counterpart, T50% was increased by 7 °C, thermal inactivation of the first step was increased 34%, and long-term stability assay revealed 71-times higher residual activity at 25 °C (without stabilizing Ca(2+) ions in aqueous buffer) after 67 days. Furthermore, resistance against autolysis was increased almost two-fold. Altered functional properties of the glycated trypsin were also observed. The glycated trypsin was found to become increasingly basophilic, and was found to be slightly structurally altered. This was indicated by 1.2 times higher catalytic efficiency (k(cat)/K(m)) than unmodified trypsin against the substrate N-α-benzoyl-L-arginine-p-nitroanilide. Circular dichroism spectropolarimetry suggested a minor change in spatial arrangement of α-helix/helices, resulting in an increased affinity of the glycated trypsin for this small synthetic substrate.

  16. AKTIVITAS ANTIBAKTERI DAN ANTIOKSIDAN HIDROLISAT HASIL HIDROLISIS PROTEIN SUSU KAMBING DENGAN EKSTRAK KASAR BROMELIN [Antibacterial and Antioxidant Activity of Hydrolysate from Goat Milk Protein Hydrolized by Crude Bromelain Extract

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    Eni Kusumaningtyas


    Full Text Available Goat milk is highly nutritious foodstuffs that beneficial for improving health. The milk contains bioactive peptides which produced by hydrolysis process. The aim of this study was to evaluate antibacterial and antioxidant activities of hydrolisate produced from hydrolysis of goat milk protein by crude bromelain extract. Hydrolysis of goat milk protein was conducted using crude bromelain (0.1 U/mL at pH 6, 50°C for 60 min. Hydrolysate was fractionated by using membrane molecular weight cut off 10 kDa. hydrolysate before and after fractionation were assayed for antibacterial and antioxidant activities. Toxicity of the Hydrolysate was determined by hemolysis assay. The result showed that the hydrolysate before and after fractionation inhibited growth of E. coli, S. Typhimurium and L. monocytogenes. Hydrolysate after fractionation has higher antibacterial activity indicated that fractionation was able to improve antibacterial activities of the hydrolysate fraction. The hydrolysate showed scavenging activity to ABTS and DPPH radicals. Fraction 10 kDa not only showed absence of hemolysis but also they were able to reduce autolysis of red blood cells. The result showed that hydrolysate from goat milk hydrolyzed by bromelain were able to be antibacterial and antioxidant.

  17. Optimization of culture conditions to obtain maximal growth of penicillin-resistant Streptococcus pneumoniae

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    Rodriguez Carlos A


    Full Text Available Abstract Background Streptococcus pneumoniae, particularly penicillin-resistant strains (PRSP, constitute one of the most important causes of serious infections worldwide. It is a fastidious microorganism with exquisite nutritional and environmental requirements to grow, a characteristic that prevents the development of useful animal models to study the biology of the microorganism. This study was designed to determine optimal conditions for culture and growth of PRSP. Results We developed a simple and reproducible method for culture of diverse strains of PRSP representing several invasive serotypes of clinical and epidemiological importance in Colombia. Application of this 3-step culture protocol consistently produced more than 9 log10 CFU/ml of viable cells in the middle part of the logarithmic phase of their growth curve. Conclusion A controlled inoculum size grown in 3 successive steps in supplemented agar and broth under 5% CO2 atmosphere, with pH adjustment and specific incubation times, allowed production of great numbers of PRSP without untimely activation of autolysis mechanisms.

  18. Stress response in Pectobacterium atrosepticum SCRI1043 under starvation conditions: adaptive reactions at a low population density. (United States)

    Petrova, Olga; Gorshkov, Vladimir; Daminova, Amina; Ageeva, Marina; Moleleki, Lucy N; Gogolev, Yuri


    The adaptive reactions of plant pathogenic bacterium Pectobacterium atrosepticum SCRI1043 under starvation conditions were studied. The main emphasis was given to the peculiarities of stress responses depending on the bacterial population densities. When bacteria were subjected to starvation at high population densities (10(7)-10(9) CFU ml(-1)), their adaptive reactions conformed to the conventional conception of bacterial adaptation related to autolysis of part of the population, specific modification of cell ultrastructure, activation of expression of stress responsive genes and acquiring cross protection against other stress factors. In contrast, at low initial population densities (10(3)-10(5) CFU ml(-1)), as described in our recent work, the cell density increased due to multiple cell division despite the absence of exogenous growth substrate. Here we present data that demonstrate that such unconventional behavior is part of a stress response, which provides increased stress tolerance while retaining virulence. Cell morphology and gene expression in high- and low-cell-density starving Pba cultures were compared. Our investigation demonstrates the existence of alternative adaptive strategies enabling pathogenic bacteria to cope with a variety of stress factors, including starvation, especially necessary when residing outside of their host.

  19. Light and electron microscopic observations in connection with the developing pistil and seed-appendix (caruncle of Ricinus communis L.

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    Katherine Liszt


    Full Text Available The development of the particularly organized stigma and obturator, their fine structure, their function as well as the histological differentiation and the role of the seed-appendix i.e. the caruncle of Ricinus communis L. have been investigated in several developmental phases from the so called "gynoecial primordium" state to the ripe state, 'using the terminology introduced by S a t t 1 e r (1974. The stigma cells are characterized by dens cytoplasm, numerous vesicles mostly of ER origin, ribosomes and negatively stained mitochondria. Dilatation of ER, the appearance of electron opaque substances in it and between the plasmamembran and cell wall are frequent. The degenerating process of some stigma cells will start before the pollination because of autolysis. In the cells of the obturator and young caruncle however dictyosomes can be found more frequently than in stigma cells and the starch content of the ;plastids is remarkable. The thickening of the cell wall is connected with the function of these tissues.

  20. Development of a rapid high-efficiency scalable process for acetylated Sus scrofa cationic trypsin production from Escherichia coli inclusion bodies. (United States)

    Zhao, Mingzhi; Wu, Feilin; Xu, Ping


    Trypsin is one of the most important enzymatic tools in proteomics and biopharmaceutical studies. Here, we describe the complete recombinant expression and purification from a trypsinogen expression vector construct. The Sus scrofa cationic trypsin gene with a propeptide sequence was optimized according to Escherichia coli codon-usage bias and chemically synthesized. The gene was inserted into pET-11c plasmid to yield an expression vector. Using high-density E. coli fed-batch fermentation, trypsinogen was expressed in inclusion bodies at 1.47 g/L. The inclusion body was refolded with a high yield of 36%. The purified trypsinogen was then activated to produce trypsin. To address stability problems, the trypsin thus produced was acetylated. The final product was generated upon gel filtration. The final yield of acetylated trypsin was 182 mg/L from a 5-L fermenter. Our acetylated trypsin product demonstrated higher BAEE activity (30,100 BAEE unit/mg) than a commercial product (9500 BAEE unit/mg, Promega). It also demonstrated resistance to autolysis. This is the first report of production of acetylated recombinant trypsin that is stable and suitable for scale-up.

  1. The biomechanics of seed germination. (United States)

    Steinbrecher, Tina; Leubner-Metzger, Gerhard


    From a biomechanical perspective, the completion of seed (and fruit) germination depends on the balance of two opposing forces: the growth potential of the embryonic axis (radicle-hypocotyl growth zone) and the restraint of the seed-covering layers (endosperm, testa, and pericarp). The diverse seed tissues are composite materials which differ in their dynamic properties based on their distinct cell wall composition and water uptake capacities. The biomechanics of embryo cell growth during seed germination depend on irreversible cell wall loosening followed by water uptake due to the decreasing turgor, and this leads to embryo elongation and eventually radicle emergence. Endosperm weakening as a prerequisite for radicle emergence is a widespread phenomenon among angiosperms. Research into the biochemistry and biomechanics of endosperm weakening has demonstrated that the reduction in puncture force of a seed's micropylar endosperm is environmentally and hormonally regulated and involves tissue-specific expression of cell wall remodelling proteins such as expansins, diverse hydrolases, and the production of directly acting apoplastic reactive oxygen. The endosperm-weakening biomechanics and its underlying cell wall biochemistry differ between the micropylar (ME) and chalazal (CE) endosperm domains. In the ME, they involve cell wall loosening, cell separation, and programmed cell death to provide decreased and localized ME tissue resistance, autolysis, and finally the formation of an ME hole required for radicle emergence. Future work will further unravel the molecular mechanisms, environmental regulation, and evolution of the diverse biomechanical cell wall changes underpinning the control of germination by endosperm weakening.

  2. Food irradiation and sterilization (United States)

    Josephson, Edward S.

    Radiation sterilization of food (radappertization) requires exposing food in sealed containers to ionizing radiation at absorbed doses high enough (25-70 kGy) to kill all organisms of food spoilage and public health significance. Radappertization is analogous to thermal canning is achieving shelf stability (long term storage without refrigeration). Except for dry products in which autolysis is negligible, the radappertization process also requires that the food be heated to an internal temperature of 70-80°C (bacon to 53°C) to inactivate autolytic enzymes which catalyze spoilage during storage without refrigeration. To minimize the occurence of irradiation induced off-flavors and odors, undesirable color changes, and textural and nutritional losses from exposure to the high doses required for radappertization, the foods are vacuum sealed and irradiated frozen (-40°C to -20°C). Radappertozed foods have the characteristic of fresh foods prepared for eating. Radappertization can substitute in whole or in part for some chemical food additives such as ethylene oxide and nitrites which are either toxic, carcinogenic, mutagenic, or teratogenic. After 27 years of testing for "wholesomeness" (safety for consumption) of radappertized foods, no confirmed evidence has been obtained of any adverse effecys of radappertization on the "wholesomeness" characteristics of these foods.

  3. Analysis of axial prestretch in the abdominal aorta with reference to post mortem interval and degree of atherosclerosis. (United States)

    Horny, Lukas; Adamek, Tomas; Kulvajtova, Marketa


    It is a well-known fact that the length of an artery in situ and the length of an excised artery differs. Retraction of blood vessels is usually observed. This prestretch plays an important role in arterial physiology. We have recently determined that the decrease of axial prestretch in the human abdominal aorta is so closely correlated with age that it is suitable for forensic applications (estimation of the age at time of death for cadavers of unknown identity). Since post mortem autolysis may affect the reliability of an estimate based on axial prestretch, the present study aims to detail analysis of the effect of post mortem time. The abdominal aorta is a prominent site of atherosclerotic changes (ATH), which may potentially affect longitudinal prestretch. Thus ATH was also involved in the analysis. Axial prestretch in the human abdominal aorta, post mortem interval (PMI), and the degree of ATH were documented in 365 regular autopsies. The data was first age adjusted to remove any supposed correlation with age. After the age adjustment of the sample, the correlation analysis showed no significant PMI effects on the prestretch in non-putrefied bodies. Analysis of the prestretch variance with respect to ATH suggested that ATH is not a suitable factor to explain the prestretch variability remaining after the age adjustment. It was concluded that, although atherosclerotic plaques may certainly change the biomechanics of arteries, they do not significantly affect the longitudinal prestretch in the human abdominal aorta.

  4. Thermoacoustic imaging of prostate cancer: comparison to histology (United States)

    Patch, S. K.; Griep, S. K.; Jacobsohn, K.; See, W. A.; Hull, D.


    Ex vivo imaging of fresh prostate specimens was performed to test the hypothesis that the thermoacoustic (TA) contrast mechanism generated with very high frequency electromagnetic (EM) irradiation is sensitive to prostate cancer. Ex vivo imaging was performed immediately after radical prostatectomy, performed as part of normal care. Irradiation pulsewidth was 700 ns and duty cycle was extremely low. Typical specific absorption rate (SAR) throughout the prostate was 70-90 kW/kg during pulsing, but time-averaged SAR was below 2 W/kg. TA pressure pulses generated by rapid heating due to EM energy deposition were detected using single element transducers. 15g/L glycine powder mixed into DI water served as acoustic couplant, which was chilled to prevent autolysis. Spatial encoding was performed by scanning in tomographic "step-and-shoot" mode, with 3 mm translation between slices and 1.8-degree rotation between tomographic views. Histology slides for 3 cases scanned with 2.25 MHz transducers were marked for comparison to TA reconstructions. These three cases showed little, moderate, and severe involvement in the histology levels surrounding the verumontanum. TA signal strength decreased with percent cancerous involvement. When VHF is used for tissue heating, the TA contrast mechanism is driven by ionic content and we observed suppressed TA signal from diseased prostate tissue in the peripheral zone. For the 45 regions of interest analyzed, a reconstruction value of 0.4 mV provides 100% sensitivity but only 29% specificity.

  5. Studying the Mechanism of Phototransformation of Light Signal by Various Mammal and Bacterial Photoreceptor Pigments  Rhodopsin, Iodopsin and Bacteriorhodopsin

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    Ignat Ignatov


    Full Text Available This review article outlines the structure and function of mammal and bacterial photoreceptor pigments (rhodopsin, iodopsin, bacteriorhodopsin and their aspects of bio-nanotechnological usage. On an example of bacteriorhodopsin is described the method of its isolation from purple membranes of photo-organotrophic halobacterium Halobacterium halobium ET 1001 by cellular autolysis by distilled water, processing of bacterial biomass by ultrasound at 22 KHz, alcohol extraction of low and high-weight molecular impurities, cellular RNA, carotenoids and lipids, the solubilization with 0,5 % (w/v SDS-Na and subsequent fractionation by methanol and gel filtration chromatography on Sephadex G-200 Column balanced with 0,09 M Tris-buffer (pH = 8,35 with 0,1 % (w/v SDS-Na and 2,5 mM EDTA. Within the framework of the research the mechanism of color perception by the visual retina analyzer having the ability to analyze certain ranges of the optical spectrum as colors, was studied along with an analysis of the additive mixing of two or more colors. It was shown that at the mixing of electromagnetic waves with different wavelengths, the visual analyzer perceives them as the separate or average wave length corresponding to the mixing color.

  6. The Improved Method for Isolation of Photochrome Trans-membrane Protein Bacteriorhodopsin from Purple Membranes of Halobacterium Halobacterium Halobium ET 1001

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    Oleg Mosin


    Full Text Available It was developed the improved method for isolation of photochrome trans-membraine protein bacteriorhodopsin (output – 5 mg from 100 g of wet biomass capable to transform light energy to electrochemical energy of generated protons H+ and АТP. The protein was isolated from purple membranes of photo-organotrophic halobacterium Halobacterium halobium ET 1001 by cellular autolysis by distilled water, processing of bacterial biomass by ultrasound at 22 KHz, alcohol extraction of low and high-weight molecular impurities, cellular RNA, carotenoids and lipids, solubilization with 0.5% (w/v SDS-Na, fractionation by MeOH and column gel permeation chromatography (GPC of the final protein on Sephadex G-200 with 0.1% (w/v SDS-Na and 2.5 mM ETDA. The homogeneity of the isolated bacteriorhodopsin was proved by combination of preparative and analytical methods, including elecrtophoresis in 12.5% (w/v PAAG with 0.1% (w/v SDS-Na and regeneration of apomembranes with 13-trans-retinal.

  7. New Nano- and Biotechnological Applications of Bacterial and Animal Photoreceptor Pigments  Bacteriorhodopsin, Rhodopsin and Iodopsin

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    Ignat Ignatov


    Full Text Available This paper views predominately the structure and function of animal and bacterial photoreceptor pigments (rhodopsin, iodopsin, bacteriorhodopsin and new aspects of their nano- and biotechnological usage. On an example of bacteriorhodopsin was described the method of its isolation from purple membranes of photo-organotrophic halobacterium Halobacterium halobium by cellular autolysis by distilled water, processing of bacterial biomass by ultrasound at 22 KHz, alcohol extraction of low and high-weight molecular impurities, cellular RNA, carotenoids and lipids, the solubilization with 0,5 % (w/v SDS-Na and subsequent fractionation by methanol and gel filtration chromatography on Sephadex G-200 Column balanced with 0.09 M Tris-HCl buffer (pH = 6,76 with 0,1 % (w/v SDS-Na and 2,5 mM EDTA. Within the framework of the research the mechanism of color perception by the visual analyzer having the ability to analyze certain ranges of the optical spectrum, as colors was studied along with an analysis of the additive mixing of two colors. It was shown that at the mixing of electromagnetic waves with different wavelengths, the visual analyzer perceive them as separate or average wave length corresponding to mix color.

  8. Studying of Phototransformation of Light Signal by Photoreceptor Pigments - Rhodopsin, Iodopsin and Bacteriorhodopsin

    Directory of Open Access Journals (Sweden)

    Ignat Ignatov


    Full Text Available This review article views predominately the structure and function of animal and bacterial photoreceptor pigments (rhodopsin, iodopsin, bacteriorhodopsin and their aspects of nano- and biotechnological usage. On an example of bacteriorhodopsin is described the method of its isolation from purple membranes of photo-organotrophic halobacterium Halobacterium halobium by cellular autolysis by distilled water, processing of bacterial biomass by ultrasound at 22 KHz, alcohol extraction of low and high-weight molecular impurities, cellular RNA, carotenoids and lipids, the solubilization with 0,5 % (w/v SDS-Na and subsequent fractionation by methanol and gel filtration chromatography on Sephadex G-200 Column balanced with 0.09 M Tris-borate buffer (pH = 8,35 with 0,1 % (w/v SDS-Na and 2,5 mM EDTA. Within the framework of the research the mechanism of color perception by the visual analyzer having the ability to analyze certain ranges of the optical spectrum, as colors was studied along with an analysis of the additive mixing of two colors. It was shown that at the mixing of electromagnetic waves with different wavelengths, the visual analyzer perceive them as separate or average wave length corresponding to mix color.

  9. Introduction of Deuterated Aromatic Amino Acids  [2,3,4,5,6-2H5]phenylalanine, [3,5-2H2]tyrosine and [2,4,5,6,7-2H5]tryptophan into a Molecule of Photochrome Trans-membrane Protein Bacteriorhodopsin

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    Oleg Mosin


    Full Text Available It was carried out the introduction of functionally important deuterated aromatic amino acids  [2,3,4,5,6-2H5]phenylalanine, [3,5-2H2]tyrosine and [2,4,5,6,7-2H5]tryptophan into a molecule of photochrome trans-membrane protein bacteriorhodopsin, synthesized by a photo-organotrophic halobacterium Halobacterium halobium ET 1001. The deuterated protein (output 810 mg was isolated from purple membranes by cellular autolysis by distilled water, processing of bacterial biomass by ultrasound at 22 KHz, alcohol extraction of low and high-weight molecular impurities, cellular RNA, carotenoids and lipids, with the subsequent solubilization of final product with 0,5 % (w/v SDS-Na and fractionation by methanol, gel filtration chromatography on Sephadex G-200, reverse-phase HPLC and EI impact mass-spectrometry of methyl esters of N-Dns-[2H]derivatives of amino acids. Deuterium was detected in all residues of aromatic amino acids. However, the presence in the EI mass spectrum of the BR hydrolysate the peaks [M]+ of semi-deuterated analogues of aromatic amino acids  phenylalanine with [M]+ at m/z = 413418, tyrosine  with [M]+ at m/z = 428430 and tryptophan  with [M]+ at m/z = 453457 with different levels of contributions to the deuterium enrichment of molecules testifies about the conservation of the minor pathways of biosynthesis of aromatic amino acids de novo.

  10. Heterologous expression of a plant small heat-shock protein enhances Escherichia coli viability under heat and cold stress. (United States)

    Soto, A; Allona, I; Collada, C; Guevara, M A; Casado, R; Rodriguez-Cerezo, E; Aragoncillo, C; Gomez, L


    A small heat-shock protein (sHSP) that shows molecular chaperone activity in vitro was recently purified from mature chestnut (Castanea sativa) cotyledons. This protein, renamed here as CsHSP17. 5, belongs to cytosolic class I, as revealed by cDNA sequencing and immunoelectron microscopy. Recombinant CsHSP17.5 was overexpressed in Escherichia coli to study its possible function under stress conditions. Upon transfer from 37 degrees C to 50 degrees C, a temperature known to cause cell autolysis, those cells that accumulated CsHSP17.5 showed improved viability compared with control cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cell lysates suggested that such a protective effect in vivo is due to the ability of recombinant sHSP to maintain soluble cytosolic proteins in their native conformation, with little substrate specificity. To test the recent hypothesis that sHSPs may be involved in protection against cold stress, we also studied the viability of recombinant cells at 4 degrees C. Unlike the major heat-induced chaperone, GroEL/ES, the chestnut sHSP significantly enhanced cell survivability at this temperature. CsHSP17.5 thus represents an example of a HSP capable of protecting cells against both thermal extremes. Consistent with these findings, high-level induction of homologous transcripts was observed in vegetative tissues of chestnut plantlets exposed to either type of thermal stress but not salt stress.

  11. Identification of the major glucosinolate (4-mercaptobutyl glucosinolate) in leaves of Eruca sativa L. (salad rocket). (United States)

    Bennett, Richard N; Mellon, Fred A; Botting, Nigel P; Eagles, John; Rosa, Eduardo A S; Williamson, Gary


    The major and structurally unique glucosinolate (GLS) in leaves of Eruca sativa L. (salad rocket) was identified as 4-mercaptobutyl GLS. Both 4-methylthiobutyl GLS and 4-methylsulfinylbutyl GLS were also present, but at lower concentrations. The 4-mercaptobutyl GLS was observed to oxidise under common GLS extraction conditions, generating a disulfide GLS that may be reduced efficiently by tris(2-carboxyethyl) phosphine hydrochloride (TCEP) to reform the parent molecule. The identities of 4-mercaptobutyl GLS and of the corresponding dimeric GLS were confirmed by LC/MS, MS/MS and NMR. Myrosinase treatment of an enriched GLS fraction or of the purified dimer GLS generated a mixture of unique bi-functional disulfides, including bis-(4-isothiocyanatobutyl) disulfide (previously identified elsewhere). TCEP reduction of the purified dimer, followed by myrosinase treatment, yielded only 4-mercaptobutyl ITC. GLS-derived volatiles generated by autolysis of fresh seedlings and true leaves were 4-mercaptobutyl ITC (from the newly identified GLS), 4-methylthiobutyl ITC (from 4-methylthiobutyl GLS) and 4-methylsulfinylbutyl ITC (from 4-methylsulfinyl-butyl GLS); no unusual bi-functional disulfides were found in fresh leaf autolysate. These results led to the conclusion that, in planta, the new GLS must be present as 4-mercaptobutyl GLS and not as the disulfide found after extraction and sample concentration. This new GLS and its isothiocyanate are likely to contribute to the unique odour and flavour of E. sativa.

  12. [Changes in the lysine of spiruline algae samples after various heat treatments]. (United States)

    Adrian, J


    The spirulina algae are microorganisms which are cultivated on Mexican lakes for feeding use. After drying, they contain about 52 per cent of proteins, with 4 per cent of lysine and 1, 7 per cent of methionine. In the studied samples, pH is 6, 2; they are partially autolysed and contain 4 to 18 per cent of free lysine and methionine and 6, 5 per cent of soluble carbohydrates. During heating treatments, the spiurlina lysine reacts as the same as oilcak meal lysine; it resists rather well to autoclaving but less to roasting. The yeast lysine is more stable than the spirulina lysine. The thermic stability of spirulina lysine is caused first by the small amount of free reducing carbohydrates, and for a minor part by the natural acicity of these products. The lysine destruction is proportional to the autolysis stage of the samples, that is to say the presence of free aminoacids. All the behavior differences between the various spirulina samples disappear when are heated with xylose, which induces a strong Maillard reaction.

  13. Purification, cDNA cloning, and recombinant expression of chymotrypsin C from porcine pancreas

    Institute of Scientific and Technical Information of China (English)

    Haibo Wang; Duoduo Yuan; Rong Xu; Cheng-Wu Chi


    Chymotrypsin C is a bifunctional secretory-type serine protease in pancreas; besides proteolytical activity, it also exhibits a calcium-decreasing activity in serum, In this study, we purified activated chymotrypsin C from porcine pancreas, and identified its three active forms. Active chymotrypsin C was found to be different in the length of its 13-residue activation peptide due to carboxydipeptidase (present in the pancreas) degradation or autolysis of the activated chymotrypsin C itself, resulting in the removal of several C-terminus residues from the activation peptide. After limited chymotrypsin C cleavage with endopeptidase Lys C, several purified peptides were partially sequenced, and the entire cDNA sequence for porcine chymotrypsin C was cloned. Recombinant chymotrypsinogen C was successfully expressed in Escherichia coli cells as inclusion bodies. After refolding and activation with trypsin, the comparison of the recombinant chymotrypsin C with the natural form showed that their proteolytic and calcium-decreasing activities were at the same level. The successful expression of chymotrypsin C gene paves the way to further mutagenic structurefunction studies.

  14. A model of isoniazid treatment of tuberculosis. (United States)

    Lemmer, Yolandy; Grobler, Anne; Moody, Clint; Viljoen, Hendrik


    A mathematical model is presented of the growth and death of bacilli in a granuloma. The granuloma is treated with isoniazid (INH), a drug that inhibits the synthesis of mycolic acids (MA). Since MA is an essential component of cell walls, the organisms fail to reach maturity if deficient in MA. Cell wall turnover is a well-known feature of bacteria, at the exterior surface material sloughs off to foil attacks by hosts or other organisms, simultaneously synthesizing products for new cell wall assembly. Thus cell wall thickness is maintained in a dynamic equilibrium (Doyle et al., 1988). Presumably cell death is a result of loss in cell wall due to autolysis in combination with stinted replenishing. The mathematical model presented here uses differential equations to predict the effects of intracellular INH on cell wall thickness and cell viability. This analysis purposely distinguishes intracellular INH concentration from the concentration in the plasma. The concentration in the plasma depends only on the dosing. The intracellular INH concentration, however, depends on diffusion through the cell walls of the bacteria. This paper addresses the complex interactions between intracellular INH, cell wall thickness, and the rate of cell wall synthesis.

  15. Two-Component Signal Transduction System SaeRS Positively Regulates Staphylococcus epidermidis Glucose Metabolism

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    Qiang Lou


    Full Text Available Staphylococcus epidermidis, which is a causative pathogen of nosocomial infection, expresses its virulent traits such as biofilm and autolysis regulated by two-component signal transduction system SaeRS. In this study, we performed a proteomic analysis of differences in expression between the S. epidermidis 1457 wild-type and saeRS mutant to identify candidates regulated by saeRS using two-dimensional gel electrophoresis (2-DE combined with matrix-assisted laser desorption/lonization mass spectrometry (MALDI-TOF-MS. Of 55 identified proteins that significantly differed in expression between the two strains, 15 were upregulated and 40 were downregulated. The downregulated proteins included enzymes related to glycolysis and TCA cycle, suggesting that glucose is not properly utilized in S. epidermidis when saeRS was deleted. The study will be helpful for treatment of S. epidermidis infection from the viewpoint of metabolic modulation dependent on two-component signal transduction system SaeRS.

  16. A novel serine protease with caspase- and legumain-like activities from edible basidiomycete Flammulina velutipes. (United States)

    Iketani, Aya; Nakamura, Mayumi; Suzuki, Yuya; Awai, Koichiro; Shioi, Yuzo


    A serine protease with caspase- and legumain-like activities from basidiocarps of the edible basidiomycete Flammulina velutipes was characterized. The protease was purified to near homogeneity by three steps of chromatography using acetyl-Tyr-Val-Ala-Asp-4-methylcoumaryl-7-amide (Ac-YVAD-MCA) as a substrate. The enzyme was termed FvSerP (F. velutipes serine protease). This enzyme activity was completely inhibited by the caspase-specific inhibitor, Ac-YVAD-CHO, as well as moderately inhibited by serine protease inhibitors. Based on the N-terminal sequence, the cDNA of FvSerP was identified. The deduced protease sequence was a peptide composed of 325 amino acids with a molecular mass of 34.5 kDa. The amino acid sequence of FvSerP showed similarity to neither caspases nor to the plant subtilisin-like serine protease with caspase-like activity called saspase. FvSerP shared identity to the functionally unknown genes from class of Agaricomycetes, with similarity to the peptidase S41 domain of a serine protease. It was thus concluded that this enzyme is likely a novel serine protease with caspase- and legumain-like activities belonging to the peptidase S41 family and distributed in the class Agaricomycetes. This enzyme possibly functions in autolysis, a type of programmed cell death that occurs in the later stages of development of basidiocarps with reference to their enzymatic functions.

  17. Renal cortex copper concentration in acute copper poisoning in calves

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    Luis E. Fazzio


    Full Text Available The aim of this study was to estimate the diagnostic value of renal cortex copper (Cu concentration in clinical cases of acute copper poisoning (ACP. A total of 97 calves that died due to subcutaneous copper administration were compiled in eleven farms. At least, one necropsy was conducted on each farm and samples for complementary analysis were taken. The degree of autolysis in each necropsy was evaluated. The cases appeared on extensive grazing calf breeding and intensive feedlot farms, in calves of 60 to 200 kg body weight. Mortality varied from 0.86 to 6.96 %, on the farms studied. The first succumbed calf was found on the farms between 6 and 72 hours after the susbcutaneous Cu administration. As discrepancies regarding the reference value arose, the local value (19.9 parts per million was used, confirming the diagnosis of acute copper poisoning in 93% of the analyzed kidney samples. These results confirm the value of analysis of the cortical kidney Cu concentration for the diagnosis of acute copper poisoning.

  18. Multimodal Raman-fluorescence spectroscopy of formalin fixed samples is able to discriminate brain tumors from dysplastic tissue (United States)

    Anand, Suresh; Cicchi, Riccardo; Giordano, Flavio; Buccoliero, Anna Maria; Pavone, Francesco Saverio


    In the recent years, there has been a considerable surge in the application of spectroscopy for disease diagnosis. Raman and fluorescence spectra provide characteristic spectral profile related to biochemical and morphological changes when tissues progress from normal state towards malignancy. Spectroscopic techniques offer the advantage of being minimally invasive compared to traditional histopathology, real time and quantitative. In biomedical optical diagnostics, freshly excised specimens are preferred for making ex-vivo spectroscopic measurements. With regard to fresh tissues, if the lab is located far away from the clinic it could pose a problem as spectral measurements have to be performed immediately after dissection. Tissue samples are usually placed in a fixative agent such as 4% formaldehyde to preserve the samples before processing them for routine histopathological studies. Fixation prevents the tissues from decomposition by arresting autolysis. In the present study, we intend to investigate the possibility of using formalin fixed samples for discrimination of brain tumours from dysplastic tissue using Raman and fluorescence spectroscopy. Formalin fixed samples were washed with phosphate buffered saline for about 5 minutes in order to remove the effects of formalin during spectroscopic measurements. In case of fluorescence spectroscopy, changes in spectral profile have been observed in the region between 550-670 nm between dysplastic and tumor samples. For Raman measurements, we found significant differences in the spectral profiles between dysplasia and tumor. In conclusion, formalin fixed samples can be potentially used for the spectroscopic discrimination of tumor against dysplastic tissue in brain samples.

  19. Transfer, composition and technological characterization of the lactic acid bacterial populations of the wooden vats used to produce traditional stretched cheeses. (United States)

    Scatassa, Maria Luisa; Gaglio, Raimondo; Macaluso, Giusi; Francesca, Nicola; Randazzo, Walter; Cardamone, Cinzia; Di Grigoli, Antonino; Moschetti, Giancarlo; Settanni, Luca


    The biofilms of 12 wooden vats used for the production of the traditional stretched cheeses Caciocavallo Palermitano and PDO Vastedda della valle del Belìce were investigated. Salmonella spp. and Listeria monocytogenes were never detected. Total coliforms were at low numbers with Escherichia coli found only in three vats. Coagulase-positive staphylococci (CPS) were below the enumeration limit, whereas lactic acid bacteria (LAB) dominated the surfaces of all vats. In general, the dominance was showed by coccus LAB. Enterococci were estimated at high numbers, but usually between 1 and 2 Log cycles lower than other LAB. LAB populations were investigated at species and strain level and for their technological properties relevant in cheese production. Eighty-five strains were analysed by a polyphasic genetic approach and allotted into 16 species within the genera Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus and Streptococcus. Enterococcus faecium was found in all wooden vats and the species most frequently isolated were Enterococcus faecalis, Lactococcus lactis, Leuconostoc mesenteroides, Pediococcus acidilactici and Streptococcus thermophilus. The study of the quantitative data on acidification rate, autolysis kinetics, diacetyl production, antibacterial compound generation and proteolysis by cluster and principal component analysis led to the identification of some strains with promising dairy characteristics. Interestingly, a consistent percentage of LAB was bacteriocin-like inhibitory substances (BLIS) producer. Thus, the microbial biofilms of the wooden vats analysed in this study might contribute actively to the stability of the final cheeses.

  20. Toxin genotyping of Clostridium perfringens strains using a polymerase chain reaction protocol

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    Elisabetta Di Giannatale


    Full Text Available A polymerase chain reaction protocol consisting of a multiplex to identify the cpa, cpb1, cpetx, cpi genes and a duplex to identify the cpe and cpb2 genes encoding for a, b1, e, i, enterotoxin and b2 toxins, respectively, was applied to DNA extracted from two collections of Clostridium perfringens strains. The first collection involved 19 isolates from rabbits. The second collection of 41 isolates came from routine necropsies. The cpa gene alone, or in association with the cpb2 gene, was detected in all DNA samples examined. The cpa gene, together with cpb2 gene, were detected in seven of the rabbit C. perfringens strains (36.8% and in nine isolates from necropsies (21.9%. The cpa gene was found in 63.2% of rabbit strains and 76.9% of strains from other animal species. In rabbits, the pathological lesions associated with C. perfringens detection were predominantly forms of non-inflammatory enteropathies. In other species, C. perfringens was mainly associated with congestive-haemorrhagic enteropathy, but also with fatal traumatic lesions, degenerative diseases and organs with post-mortem autolysis. No clear correlation was observed between detection of b2 toxin gene and species-specific pathological features.

  1. Implementation of a method to visualize noise-induced hearing loss in mass stranded cetaceans (United States)

    Morell, Maria; Brownlow, Andrew; McGovern, Barry; Raverty, Stephen A.; Shadwick, Robert E.; André, Michel


    Assessment of the impact of noise over-exposure in stranded cetaceans is challenging, as the lesions that lead to hearing loss occur at the cellular level and inner ear cells are very sensitive to autolysis. Distinguishing ante-mortem pathology from post-mortem change has been a major constraint in diagnosing potential impact. Here, we outline a methodology applicable to the detection of noise-induced hearing loss in stranded cetaceans. Inner ears from two mass strandings of long-finned pilot whales in Scotland were processed for scanning electron microscopy observation. In one case, a juvenile animal, whose ears were fixed within 4 hours of death, revealed that many sensory cells at the apex of the cochlear spiral were missing. In this case, the absence of outer hair cells would be compatible with overexposure to underwater noise, affecting the region which transduces the lowest frequencies of the pilot whales hearing spectrum. Perfusion of cochlea with fixative greatly improved preservation and enabled diagnostic imaging of the organ of Corti, even 30 hours after death. This finding supports adopting a routine protocol to detect the pathological legacy of noise overexposure in mass stranded cetaceans as a key to understanding the complex processes and implications that lie behind such stranding events.

  2. Chiral MEKC-LIF of amino acids in foods: analysis of vinegars. (United States)

    Carlavilla, Davinia; Moreno-Arribas, M Victoria; Fanali, Salvatore; Cifuentes, Alejandro


    The formation of D-amino acids (D-aa's) in many fermented foods depends, among other factors, on the particular fermentation conditions, the action and autolysis of the microorganisms involved. In this sense, the analysis of chiral amino acids is an interesting analytical strategy for food scientists, since these compounds can be used as bacterial markers and can help, e.g., to detect adulterations, microbiological contaminations, etc. In this work, a fast and sensitive method based on MEKC-LIF has been developed to analyze and quantitate L-amino acid (L-aa) and D-aa in vinegars. The chiral MEKC-LIF procedure uses 100 mM sodium tetraborate, 30 mM SDS, and 20 mM beta-CD at pH 9.7 as running buffer, obtaining a good separation of the main vinegar L-/D-aa previously derivatized with fluorescein isothiocianate. Namely, L/D proline, alanine, arginine, glutamic, and aspartic acid, plus the nonchiral amino acid gamma-aminobutyric acid are separated in less than 20 min with high efficiency (up to 720,000 plates/m) and good sensitivity (LODs lower than 16.6 nM were achieved). Several D-aa's were detected and quantified in balsamic, sherry, white wine, and cider vinegars using this MEKC-LIF procedure, observing interesting differences in their L-aa and D-aa profiles and contents.

  3. Structure determination and analysis of a haemolytic gingipain adhesin domain from Porphyromonas gingivalis

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    Li, N.; Yun, P.; Nadkarni, M.A.; Ghadikolaee, N.B.; Nguyen, K.A.; Lee, M.; Hunter, N.; Collyer, C.A. (Sydney)


    Porphyromonas gingivalis is an obligately anaerobic bacterium recognized as an aetiological agent of adult periodontitis. P. gingivalis produces cysteine proteinases, the gingipains. The crystal structure of a domain within the haemagglutinin region of the lysine gingipain (Kgp) is reported here. The domain was named K2 as it is the second of three homologous structural modules in Kgp. The K2 domain structure is a 'jelly-roll' fold with two anti-parallel {beta}-sheets. This fold topology is shared with adhesive domains from functionally diverse receptors such as MAM domains, ephrin receptor ligand binding domains and a number of carbohydrate binding modules. Possible functions of K2 were investigated. K2 induced haemolysis of erythrocytes in a dose-dependent manner that was augmented by the blocking of anion transport. Further, cysteine-activated arginine gingipain RgpB, which degrades glycophorin A, sensitized erythrocytes to the haemolytic effect of K2. Cleaved K2, similar to that found in extracted Kgp, lacks the haemolytic activity indicating that autolysis of Kgp may be a staged process which is artificially enhanced by extraction of the protein. The data indicate a functional role for K2 in the integrated capacity conferred by Kgp to enable the porphyrin auxotroph P. gingivalis to capture essential haem from erythrocytes.

  4. Highly conserved salt bridge stabilizes a proteinase K subfamily enzyme, Aqualysin I, from Thermus aquaticus YT-1. (United States)

    Sakaguchi, Masayoshi; Osaku, Kanae; Maejima, Susumu; Ohno, Nao; Sugahara, Yasusato; Oyama, Fumitaka; Kawakita, Masao


    The proteinase K subfamily enzymes, thermophilic Aqualysin I (AQN) from Thermus aquaticus YT-1 and psychrophilic serine protease (VPR) from Vibrio sp. PA-44, have six and seven salt bridges, respectively. To understand the possible significance of salt bridges in the thermal stability of AQN, we prepared mutant proteins in which amino acid residues participating in salt bridges common to proteinase K subfamily members and intrinsic to AQN were replaced to disrupt the bridges one at a time. Disruption of a salt bridge common to proteinase K subfamily enzymes in the D183N mutant resulted in a significant reduction in thermal stability, and a massive change in the content of the secondary structure was observed, even at 70°C, in the circular dichroism (CD) analysis. These results indicate that the common salt bridge Asp183-Arg12 is important in maintaining the conformation of proteinase K subfamily enzymes and suggest the importance of proximity between the regions around Asp183 and the N-terminal region around Arg12. Of the three mutants that lack an AQN intrinsic salt bridge, D212N was more prone to unfolding at 80°C than the wild-type enzyme. Similarly, D17N and E237Q were less thermostable than the wild-type enzyme, although this may be partially due to increased autolysis. The AQN intrinsic salt bridges appear to confer additional thermal stability to this enzyme. These findings will further our understanding of the factors involved in stabilizing protein structure.

  5. Potential of lees from wine, beer and cider manufacturing as a source of economic nutrients: An overview. (United States)

    Pérez-Bibbins, B; Torrado-Agrasar, A; Salgado, J M; Oliveira, R Pinheiro de Souza; Domínguez, J M


    Lees are the wastes generated during the fermentation and aging processes of different industrial activities concerning alcoholic drinks such as wine, cider and beer. They must be conveniently treated to avoid uncontrolled dumping which causes environmental problems due to their high content of phenols, pesticides, heavy metals, and considerable concentrations of nitrogen, phosphate and potassium as well as high organic content. The companies involved must seek alternative environmental and economic physicochemical and biological treatments for their revalorization consisting in the recovery or transformation of the components of the lees into high value-added compounds. After describing the composition of lees and market of wine, beer and cider industries in Spain, this work aims to review the recent applications of wine, beer and cider lees reported in literature, with special attention to the use of lees as an endless sustainable source of nutrients and the production of yeast extract by autolysis or cell disruption. Lees and/or yeast extract can be used as nutritional supplements with potential exploitation in the biotechnological industry for the production of natural compounds such as xylitol, organic acids, and biosurfactants, among others.

  6. Histopathologic ear findings of syphilis: a temporal bone study. (United States)

    Hızlı, Ömer; Hızlı, Pelin; Kaya, Serdar; Monsanto, Rafael da Costa; Paparella, Michael M; Cureoglu, Sebahattin


    To the best of our knowledge, histopathologic studies of syphilitic ears have generally focused on hydropic changes; so far, no such studies have investigated peripheral vestibular otopathology using differential interference contrast microscopy, in patients with syphilis. For this study, we examined 13 human temporal bone samples from 8 patients with a history of syphilis. Using conventional light microscopy, we performed qualitative histopathologic assessment. In addition, using differential interference contrast microscopy, we performed type I and type II vestibular hair cell counts on each vestibular sense organ with minimal autolysis; in which the neuroepithelium was oriented perpendicular to the plane of section. We then compared vestibular hair cell densities (cells per 0.01 mm² surface area) in the syphilis group vs. the control group. In the syphilis group, we observed precipitate in the endolymphatic or perilymphatic spaces in 1 (7.7 %) of the samples and endolymphatic hydrops in eight (61.5 %) of the samples. Hydrops involved the cochlea (four samples) and/or saccule (four samples). In addition, the syphilis group experienced a significant loss of type II vestibular hair cells in the maculae of the utricle and saccule, and in the cristae of the lateral and posterior semicircular canals, as compared with the control group (P < 0.05).

  7. Metabolic engineering of Cyanobacteria and microalgae for enhanced production of biofuels and high-value products. (United States)

    Gomaa, M A; Al-Haj, L; Abed, R M M


    A lot of research has been performed on Cyanobacteria and microalgae with the aim to produce numerous biotechnological products. However, native strains have a few shortcomings, like limitations in cultivation, harvesting and product extraction, which prevents reaching optimal production value at lowest costs. Such limitations require the intervention of genetic engineering to produce strains with superior properties. Promising advancements in the cultivation of Cyanobacteria and microalgae have been achieved by improving photosynthetic efficiency through increasing RuBisCO activity and truncation of light-harvesting antennae. Genetic engineering has also contributed to final product extraction by inducing autolysis and product secretory systems, to enable direct product recovery without going through costly extraction steps. In this review, we summarize the different enzymes and pathways that have been targeted thus far for improving cultivation aspects, harvesting and product extraction in Cyanobacteria and microalgae. With synthetic biology advancements, genetically engineered strains can be generated to resolve demanding process issues and achieve economic practicality. This comprehensive overview of gene modifications will be useful to researchers in the field to employ on their strains to increase their yields and improve the economic feasibility of the production process.

  8. Pattern of malformations in the axial skeleton in human trisomy 18 fetuses

    Energy Technology Data Exchange (ETDEWEB)

    Kjaer, I. [Univ. of Copenhagen (Denmark); Hansen, B.F. [Hvidovre Univ. Hospital (Denmark); Keeling, J.W. [Royal Hospital for Sick Children, Edinburgh (United Kingdom)


    We examined and described the development and abnormalities of the axial skeleton in 10 human trisomy 18 fetuses. Whole-body radiographs and radiographs of midsagittal tissue blocks of the cranial base and the spine were studied. In 3 fetuses no spinal radiographs were available. Seven osseous regions or fields along the body axis were analyzed, four in the spine, and three in the cranial base and nasal bones. Malformations occurred in the occipital field in all fetuses. This was a characteristic notching, either unilateral or bilateral, of the basilar part of the occipital bone. Nasal bones were abnormal in 8 cases, either absent or hypoplastic. Malformations were found in the thoracic and/or lumbosacral field in 7 fetuses. A single abnormality was found in the cervical spine in one fetus. The pattern of axial skeletal malformation in trisomy 18 fetuses recorded in the present study has not been described previously. Axial skeletal radiography should be included in autopsies of fetuses when chromosome disorders are present or suspected. The methods applied here are unaffected by autolysis. 26 refs., 5 figs.

  9. First report of Taenia arctos (Cestoda: Taeniidae) from grizzly (Ursus arctos horribilis) and black bears (Ursus americanus) in North America. (United States)

    Catalano, Stefano; Lejeune, Manigandan; Verocai, Guilherme G; Duignan, Pádraig J


    The cestode Taenia arctos was found at necropsy in the small intestine of a grizzly (Ursus arctos horribilis) and a black bear (Ursus americanus) from Kananaskis Country in southwestern Alberta, Canada. The autolysis of the tapeworm specimens precluded detailed morphological characterization of the parasites but molecular analysis based on mitochondrial DNA cytochrome c oxidase subunit 1 gene confirmed their identity as T. arctos. This is the first report of T. arctos from definitive hosts in North America. Its detection in Canadian grizzly and black bears further supports the Holarctic distribution of this tapeworm species and its specificity for ursids as final hosts. Previously, T. arctos was unambiguously described at its adult stage in brown bears (Ursus arctos arctos) from Finland, and as larval stages in Eurasian elk (Alces alces) from Finland and moose (Alces americanus) from Alaska, USA. Given the morphological similarity between T. arctos and other Taenia species, the present study underlines the potential for misidentification of tapeworm taxa in previous parasitological reports from bears and moose across North America. The biogeographical history of both definitive and intermediate hosts in the Holarctic suggests an ancient interaction between U. arctos, Alces spp., and T. arctos, and a relatively recent host-switching event in U. americanus.

  10. Process-induced compositional changes of flaxseed. (United States)

    Wanasundara, P K; Shahidi, F


    Flaxseed has been used as an edible grain in different parts of the world since ancient times. However, use of flaxseed oil has been limited due to its high content of polyunsaturated fatty acids. Nonetheless, alpha-linolenic acid, dietary fiber and lignans of flaxseed have regained attention. New varieties of flaxseed containing low levels of alpha-linolenic acid are available for edible oil extraction. Use of whole flaxseed in foods provides a means to utilise all of its nutrients and require minimum processing steps. However, the presence of cyanogenic glucosides and diglucosides in the seeds is a concern as they may release cyanide upon hydrolysis. In addition, the polyunsaturated fatty acids may undergo thermal or autooxidation when exposed to air or high temperatures that are used in food preparation. Studies todate on oxidation products of intact flaxseed lipids have not shown any harmful effects when flaxseed is included, up to 28%, in the baked products. Furthermore, cyanide levels produced as a result of autolysis are below the harmful limits to humans. However, the meals left after oil extraction require detoxification but, by solvent extraction, to reduce the harmful effects of cyanide when used in animal rations. Flaxseed meal is a good source of proteins; these could be isolated by complexation with sodium hexametaphosphate without changing their nutritional value or composition. In addition, the effect of germination on proteins, lipids, cyanogenic glycosides, and other minor constituents of flaxseed is discussed.

  11. Characterization of the aodA, dnmA, mnSOD and pimA genes in Aspergillus nidulans. (United States)

    Leiter, Éva; Park, Hee-Soo; Kwon, Nak-Jung; Han, Kap-Hoon; Emri, Tamás; Oláh, Viktor; Mészáros, Ilona; Dienes, Beatrix; Vincze, János; Csernoch, László; Yu, Jae-Hyuk; Pócsi, István


    Mitochondria play key roles in cellular energy generation and lifespan of most eukaryotes. To understand the functions of four nuclear-encoded genes predicted to be related to the maintenance of mitochondrial morphology and function in Aspergillus nidulans, systematic characterization was carried out. The deletion and overexpression mutants of aodA, dnmA, mnSOD and pimA encoding alternative oxidase, dynamin related protein, manganese superoxide dismutase and Lon protease, respectively, were generated and examined for their growth, stress tolerances, respiration, autolysis, cell death, sterigmatocystin production, hyphal morphology and size, and mitochondrial superoxide production as well as development. Overall, genetic manipulation of these genes had less effect on cellular physiology and ageing in A. nidulans than that of their homologs in another fungus Podospora anserina with a well-characterized senescence. The observed interspecial phenotypic differences can be explained by the dissimilar intrinsic stabilities of the mitochondrial genomes in A. nidulans and P. anserina. Furthermore, the marginally altered phenotypes observed in A. nidulans mutants indicate the presence of effective compensatory mechanisms for the complex networks of mitochondrial defense and quality control. Importantly, these findings can be useful for developing novel platforms for heterologous protein production, or on new biocontrol and bioremediation technologies based on Aspergillus species.

  12. The Thanatomicrobiome: A Missing Piece of the Microbial Puzzle of Death

    Directory of Open Access Journals (Sweden)

    Gulnaz T Javan


    Full Text Available Death is a universal phenomenon; however, is there life after death? This topic has been investigated for centuries but still there are grey areas that have yet to be elucidated. Forensic microbiologists are developing new applications to investigate the dynamic and coordinated changes in microbial activity that occur when a human host dies. There is currently a paucity of explorations of the thanatomicrobiome (thanatos-, Greek for death and epinecrotic communities (microbial communities residing in and/or moving on the surface of decomposing remains. Ongoing studies can help clarify the structure and function of these postmortem microbiomes. Human microbiome studies have revealed that 75-90% of cells in the body prior to death are microbial. Upon death, putrefaction occurs and is a complicated process encompassing chemical degradation and autolysis of cells. Decomposition also involves the release of contents of the intestines due to enzymes under the effects of abiotic and biotic factors. These factors likely have predictable effects on postmortem microbial communities and can be leveraged for forensic studies. This mini review provides a critical examination of emerging research relating to thanatomicrobiome and epinecrotic communities, how each is studied, and possible strategies of stochastic processes.

  13. Molecular Events for Promotion of Vancomycin Resistance in Vancomycin Intermediate Staphylococcus aureus (United States)

    Hu, Qiwen; Peng, Huagang; Rao, Xiancai


    Vancomycin has been used as the last resort in the clinical treatment of serious Staphylococcus aureus infections. Vancomycin-intermediate S. aureus (VISA) was discovered almost two decades ago. Aside from the vancomycin-intermediate phenotype, VISA strains from the clinic or laboratory exhibited common characteristics, such as thickened cell walls, reduced autolysis, and attenuated virulence. However, the genetic mechanisms responsible for the reduced vancomycin susceptibility in VISA are varied. The comparative genomics of vancomycin-susceptible S. aureus (VSSA)/VISA pairs showed diverse genetic mutations in VISA; only a small number of these mutations have been experimentally verified. To connect the diversified genotypes and common phenotypes in VISA, we reviewed the genetic alterations in the relative determinants, including mutations in the vraTSR, graSR, walKR, stk1/stp1, rpoB, clpP, and cmk genes. Especially, we analyzed the mechanism through which diverse mutations mediate vancomycin resistance. We propose a unified model that integrates diverse gene functions and complex biochemical processes in VISA upon the action of vancomycin. PMID:27790199

  14. Another turn of the screw in shaving Gram-positive bacteria: Optimization of proteomics surface protein identification in Streptococcus pneumoniae. (United States)

    Olaya-Abril, Alfonso; Gómez-Gascón, Lidia; Jiménez-Munguía, Irene; Obando, Ignacio; Rodríguez-Ortega, Manuel J


    Bacterial surface proteins are of outmost importance as they play critical roles in the interaction between cells and their environment. In addition, they can be targets of either vaccines or antibodies. Proteomic analysis through "shaving" live cells with proteases has become a successful approach for a fast and reliable identification of surface proteins. However, this protocol has not been able to reach the goal of excluding cytoplasmic contamination, as cell lysis is an inherent process during culture and experimental manipulation. In this work, we carried out the optimization of the "shaving" strategy for the Gram-positive human pathogen Streptococcus pneumoniae, a bacterium highly susceptible to autolysis, and set up the conditions for maximizing the identification of surface proteins containing sorting or exporting signals, and for minimizing cytoplasmic contamination. We also demonstrate that cell lysis is an inherent process during culture and experimental manipulation, and that a low level of lysis is enough to contaminate a "surfome" preparation with peptides derived from cytoplasmic proteins. When the optimized conditions were applied to several clinical isolates, we found the majority of the proteins described to induce protection against pneumococcal infection. In addition, we found other proteins whose protection capacity has not been yet tested. In addition, we show the utility of this approach for providing antigens that can be used in serological tests for the diagnosis of pneumococcal disease.

  15. [Routine investigation of foetal eyes--in what way and what for?]. (United States)

    Herwig, M C; Löffler, K U


    The investigation of foetal eyes not only allows for the observation of ocular development. It is supportive and sometimes even mandatory for the diagnosis of systemic and ocular syndromes. This review gives an overview about the investigation of foetal eyes, their assignment to developmental stages, challenges related to the investigation of foetal eyes, clinically relevant syndromes, and academic questions. The morphological development of the eye has been investigated since the 19th century and will not be covered in this article. The investigation of foetal eyes that have been collected during the routine paediatric autopsy, is complicated by artifacts. Artifacts are the result of autolysis, fixation, and mechanical manipulation. They have to be distinguished from genuine findings. Besides the search for findings such as coloboma or cataract, the morphological classification of the foetal eye is of importance. The anterior-posterior diameter allows for the diagnosis of microphthalmia. The case reports comprise Goldenhar's syndrome, MIDAS syndrome and others. In conclusion, the investigation of foetal eyes is often helpful and critical for paediatric diagnostics and should be performed with great care.

  16. Metarhizium anisopliae pathogenesis of mosquito larvae: a verdict of accidental death. (United States)

    Butt, Tariq M; Greenfield, Bethany P J; Greig, Carolyn; Maffeis, Thierry G G; Taylor, James W D; Piasecka, Justyna; Dudley, Ed; Abdulla, Ahmed; Dubovskiy, Ivan M; Garrido-Jurado, Inmaculada; Quesada-Moraga, Enrique; Penny, Mark W; Eastwood, Daniel C


    Metarhizium anisopliae, a fungal pathogen of terrestrial arthropods, kills the aquatic larvae of Aedes aegypti, the vector of dengue and yellow fever. The fungus kills without adhering to the host cuticle. Ingested conidia also fail to germinate and are expelled in fecal pellets. This study investigates the mechanism by which this fungus adapted to terrestrial hosts kills aquatic mosquito larvae. Genes associated with the M. anisopliae early pathogenic response (proteinases Pr1 and Pr2, and adhesins, Mad1 and Mad2) are upregulated in the presence of larvae, but the established infection process observed in terrestrial hosts does not progress and insecticidal destruxins were not detected. Protease inhibitors reduce larval mortality indicating the importance of proteases in the host interaction. The Ae. aegypti immune response to M. anisopliae appears limited, whilst the oxidative stress response gene encoding for thiol peroxidase is upregulated. Cecropin and Hsp70 genes are downregulated as larval death occurs, and insect mortality appears to be linked to autolysis through caspase activity regulated by Hsp70 and inhibited, in infected larvae, by protease inhibitors. Evidence is presented that a traditional host-pathogen response does not occur as the species have not evolved to interact. M. anisopliae retains pre-formed pathogenic determinants which mediate host mortality, but unlike true aquatic fungal pathogens, does not recognise and colonise the larval host.

  17. Programmed cell death in cereal aleurone. (United States)

    Fath, A; Bethke, P; Lonsdale, J; Meza-Romero, R; Jones, R


    Progress in understanding programmed cell death (PCD) in the cereal aleurone is described. Cereal aleurone cells are specialized endosperm cells that function to synthesize and secrete hydrolytic enzymes that break down reserves in the starchy endosperm. Unlike the cells of the starchy endosperm, aleurone cells are viable in mature grain but undergo PCD when germination is triggered or when isolated aleurone layers or protoplasts are incubated in gibberellic acid (GA). Abscisic acid (ABA) slows down the process of aleurone cell death and isolated aleurone protoplasts can be kept alive in media containing ABA for up to 6 months. Cell death in barley aleurone occurs only after cells become highly vacuolated and is manifested in an abrupt loss of plasma membrane integrity. Aleurone cell death does not follow the apoptotic pathway found in many animal cells. The hallmarks of apoptosis, including internucleosomal DNA cleavage, plasma membrane and nuclear blebbing and formation of apoptotic bodies, are not observed in dying aleurone cells. PCD in barley aleurone cells is accompanied by the accumulation of a spectrum of nuclease and protease activities and the loss of organelles as a result of cellular autolysis.

  18. Topical Application Effect of the Isolectin Hydrogel (Cramoll 1,4 on Second-Degree Burns: Experimental Model

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    Danielle dos Santos Tavares Pereira


    Full Text Available This study aimed at evaluating the use of hydrogel isolectin in the treatment of second-degree burns. Twenty male rats were randomly divided into two groups (G1 = treatment with hydrogel containing 100 μg/mL Cramoll 1,4 and G2 = Control, hydrogel. After 7, 14, 21, 28, and 35 days, animals were euthanized. On the 7th day, G1 showed intense exudates, necrosis and edema. On the 14th day, G1 showed tissue reepithelialization and moderate autolysis. On the 21st day, G1 showed intense fibroblastic proliferation, presence of dense collagen, and moderate fibrosis. On the 28th day, G1 showed complete tissue epithelialization. On the 35th day, G1 showed modeled dense collagen. The significant wound contraction was initiated from day, 14 in the G1. There were no significant differences in biochemical and hematological parameters analyzed. These results extend the potential of therapeutic applications for Cramoll 1,4 in the treatment of thermal burns.

  19. [Urethritis syndrome and atypical germ flora of the exterior female genitalia (author's transl)]. (United States)

    Hofstetter, A; Schmiedt, E; Weissenbacher, E R; Frank, S


    A positive microbiological evidence could be obtained 54 times from the smear of the exterior genitals of 80 women suffering from complaints that were caused by urethritis, criteria of the examinations being sterile catheter specimen, negative cystoscopical findings, and missing indications to anatomical changes in the urethral region. Cytological examinations of these cases with regard to the vaginal epithelium had the following results:Group I:6 times; group II: 37 times; group IIW:8 times; group IIId: once; group IVa:twice. The cytological tests were carried out according to the method of papanicolaou as modified by Soost. Furthermore, we could state the following degrees of purity: Degree I: 8 times; degree II:16 times; degree III: 30 times. The cytological examinations of the urethral epithelium demonstrated, in 52 cases, an increased appearance of "nude" completely exposed epithelial cell nuclei--a fact corresponding to a degenerative autolysis (according to Wied). In the 26 women with missing atypical germ flora within the region of the exterior genitals, exclusively groups I (according to Papanicolaou and Soost) and degrees of purity I were stated. These persons also demonstrated remarkably grave psychical disturbances, especially in the intimate regions. In the cases of positive microbiological evidence, the following measures have proved satisfactory: Vaginal hygienization combined with a directly aimed antibacterial therapy, and the prescription of preparations containing lactic acid. A transitory discontinuation of contraceptives is being discussed. Our examination results are emphasizing the necessity of an analysis of the germ flora in cases of complaints arising from urethritis. Also psychical disturbances must be taken into consideration in cases of missing urological and gynaecological criteria of evidence.

  20. Components of yeast (Sacchromyces cervisiae) extract as defined media additives that support the growth and productivity of CHO cells. (United States)

    Spearman, Maureen; Chan, Sarah; Jung, Vince; Kowbel, Vanessa; Mendoza, Meg; Miranda, Vivian; Butler, Michael


    Yeast and plant hydrolysates are used as media supplements to support the growth and productivity of CHO cultures for biopharmaceutical production. Through fractionation of a yeast lysate and metabolic analysis of a fraction that had bioactivity equivalent to commercial yeast extract (YE), bioactive components were identified that promoted growth and productivity of two recombinant CHO cell lines (CHO-Luc and CHO-hFcEG2) equivalent to or greater than YE-supplemented media. Autolysis of the yeast lysate was not necessary for full activity, suggesting that the active components are present in untreated yeast cells. A bioactive fraction (3KF) of the yeast lysate was isolated from the permeate using a 3kDa molecular weight cut-off (MWCO) filter. Supplementation of this 3KF fraction into the base media supported growth of CHO-Luc cells over eight passages equivalent to YE-supplemented media. The 3KF fraction was fractionated further by a cation exchange spin column using a stepwise pH elution. Metabolomic analysis of a bioactive fraction isolated at high pH identified several arginine and lysine-containing peptides as well as two polyamines, spermine and spermidine, with 3.5× and 4.5× higher levels compared to a fraction showing no bioactivity. The addition of a mixture of polyamines and their precursors (putrescine, spermine, spermidine, ornithine and citrulline) as well as increasing the concentration of some of the components of the original base medium resulted in a chemically-defined (CD) formulation that produced an equivalent viable cell density (VCD) and productivity of the CHO-Luc cells as the YE-supplemented medium. The VCD of the CHO-hFcEG2 culture in the CD medium was 1.9× greater and with equivalent productivity to the YE-supplemented media.

  1. Pathways of Ca²⁺ entry and cytoskeletal damage following eccentric contractions in mouse skeletal muscle. (United States)

    Zhang, Bao-Ting; Whitehead, Nicholas P; Gervasio, Othon L; Reardon, Trent F; Vale, Molly; Fatkin, Diane; Dietrich, Alexander; Yeung, Ella W; Allen, David G


    Muscles that are stretched during contraction (eccentric contractions) show deficits in force production and a variety of structural changes, including loss of antibody staining of cytoskeletal proteins. Extracellular Ca(2+) entry and activation of calpains have been proposed as mechanisms involved in these changes. The present study used isolated mouse extensor digitorum longus (EDL) muscles subjected to 10 eccentric contractions and monitored force production, immunostaining of cytoskeletal proteins, and resting stiffness. Possible pathways for Ca(2+) entry were tested with streptomycin (200 μM), a blocker of stretch-activated channels, and with muscles from mice deficient in the transient receptor potential canonical 1 gene (TRPC1 KO), a candidate gene for stretch-activated channels. At 30 min after the eccentric contractions, the isometric force was decreased to 75 ± 3% of initial control and this force loss was reduced by streptomycin but not in the TRPC1 KO. Desmin, titin, and dystrophin all showed patchy loss of immunostaining 30 min after the eccentric contractions, which was substantially reduced by streptomycin and in the TRPC1 KO muscles. Muscles showed a reduction of resting stiffness following eccentric contractions, and this reduction was eliminated by streptomycin and absent in the TRPC1 KO muscles. Calpain activation was determined by the appearance of a lower molecular weight autolysis product and μ-calpain was activated at 30 min, whereas the muscle-specific calpain-3 was not. To test whether the loss of stiffness was caused by titin cleavage, protein gels were used but no significant titin cleavage was detected. These results suggest that Ca(2+) entry following eccentric contractions is through a stretch-activated channel that is blocked by streptomycin and encoded or modulated by TRPC1.

  2. Sensitivity and specificity of real-time reverse transcription polymerase chain reaction, histopathology, and immunohistochemical labeling for the detection of Rift Valley fever virus in naturally infected cattle and sheep. (United States)

    Odendaal, Lieza; Fosgate, Geoffrey T; Romito, Marco; Coetzer, Jacobus A W; Clift, Sarah J


    Real-time reverse transcription polymerase chain reaction (real-time RT-PCR), histopathology, and immunohistochemical labeling (IHC) were performed on liver specimens from 380 naturally infected cattle and sheep necropsied during the 2010 Rift Valley fever (RVF) epidemic in South Africa. Sensitivity (Se) and specificity (Sp) of real-time RT-PCR, histopathology, and IHC were estimated in a latent-class model using a Bayesian framework. The Se and Sp of real-time RT-PCR were estimated as 97.4% (95% confidence interval [CI] = 95.2-98.8%) and 71.7% (95% CI = 65-77.9%) respectively. The Se and Sp of histopathology were estimated as 94.6% (95% CI = 91-97.2%) and 92.3% (95% CI = 87.6-95.8%), respectively. The Se and Sp of IHC were estimated as 97.6% (95% CI = 93.9-99.8%) and 99.4% (95% CI = 96.9-100%), respectively. Decreased Sp of real-time RT-PCR was ascribed to cross-contamination of samples. Stratified analysis of the data suggested variations in test accuracy with fetuses and severely autolyzed specimens. The Sp of histopathology in fetuses (83%) was 9.3% lower than the sample population (92.3%). The Se of IHC decreased from 97.6% to 81.5% in the presence of severe autolysis. The diagnostic Se and Sp of histopathology was higher than expected, confirming the value of routine postmortem examinations and histopathology of liver specimens. Aborted fetuses, however, should be screened using a variety of tests in areas endemic for RVF, and results from severely autolyzed specimens should be interpreted with caution. The most feasible testing option for countries lacking suitably equipped laboratories seems to be routine histology in combination with IHC.

  3. 38. Morphological Changes of Testis and Sperm Subchronic Inhalation of Ethylene Oxide in Mice

    Institute of Scientific and Technical Information of China (English)


    Male mice were exposed to ethylene oxide(Et0) at concentrations of 54, 360 mg/m3 for two hours a day, on six days a week for 14 weeks. The results of sperm abnormalities test in mice showed the mutation frequencies of both low (54 mg/m3) and high (360 mg/m3) concentration groups are abvious (They are 29.40±16.24, P<0.05 and 52.91±11.97, P<0.001, respectively.). These showed that it causes evident mutagenesis to inhale ethylene oxide for a long time although in the low concentration. In light microscopic observation of the testis, the numbers of germ cells and layers of seminiferous epithelium decreased slightly in 54 mg/m3 group, but obviously in 360 mg/m3 group. Sometimes multinucleated giant cells were observed in the damaged seminiferous tubules, and more changes could be seen in the examination of seminiferous tubules under an electron microscope. The changes are: acrosomes of a lot of spermatid during spermiogenesis developmented teratoid and have not had a marked deviation of the hooked acrosome from the long axis of the nucleus, which is a charater of a sperm in mice; excessive nucleoplasm of sperm is eliminated incompletly; autolysis in a lot of spermatid was observed in development; mitochondria which located around the tails of sperm decreased and even disappeared. These abnormal ultrastructures on morphology in spermatogenesis associated the results of Sperm Abnormalities Tests, Dominat-Lethal Tests and Heritable Translocation Tests and so on, which are studied in cytogenetic, explained that Eto is a potent antispermatogenic agent and a teratogen. Indeed, the possiblility of inferti-lity and the teratogenesis in offspring among male workers potentially exposed to Eto should be studied further.

  4. Microbiological and microscopic analysis of the pulp of non-vital traumatized teeth with intact crowns

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    Kely Firmino Bruno


    Full Text Available OBJECTIVE: This study evaluated the presence of microorganisms and analyzed microscopically the pulp of 20 traumatized human teeth with intact crowns and clinical diagnosis of pulp necrosis, based on the association of at least three of the clinical criteria: crown discoloration, negative response to thermal and electric pulp vitality tests, positive response to vertical and horizontal percussion, pain on palpation or mobility. MATERIAL AND METHODS: Microbiological collection was performed from the root canals to evaluate the presence of microorganisms. The pulp samples were stained with hematoxylin and eosin (H.E. for histological evaluation of possible morphological alterations. RESULTS: Analysis of results was performed by statistical tests (linear regression test and diagnostic analysis and subjective analysis of the sections stained with H.E. and revealed that only 15% of the sample did not exhibit microbial development. The time elapsed between dental trauma and onset of endodontic intervention ranged from 15 days to 31 months; the percussion test presented high sensitivity (80% for detection of microorganisms in the root canal of traumatized teeth; 3 teeth (15% did not present pulp tissue, being characterized as complete autolysis; analysis of pulp samples was performed on the other 17 cases, among which 3 (15% exhibited partial necrosis without possibility of repair and 14 presented complete necrosis; none of the clinical criteria employed for the diagnosis of pulp necrosis in traumatized teeth was pathognomonic. CONCLUSIONS: The present results allowed the following conclusions: with regard to microbiological findings, 85% of teeth presented microorganisms in the root canal, despite the presence of an intact crown. Concerning the microscopic findings, 100% of traumatized teeth presented pulp necrosis; the pulp vitality tests based on pulp response to heat, cold and vertical percussion were the most reliable to diagnose pulp necrosis in

  5. Influence of short-term fixation with mixed formalin or ethanol solution on the mechanical properties of human cortical bone

    Directory of Open Access Journals (Sweden)

    Mick E.


    Full Text Available Bone specimens obtained for biomechanical experiments are fresh-frozen for storage to slow down tissue degradation and autolysis in long-term storage. Alternatively, due to infectious risks related to the fresh tissues, fixative agents are commonly used. However, fixatives will likely change the mechanical properties of bone. Existing studies on this issue gave controversial results that are hardly comparable due to a variety of measurement approaches. For this reason, the influence of ethanol and a formalin-based fixative agent was evaluated on the mechanical properties of human cortical bone specimens by means of four-point-bending tests. 127 prismatic specimens with rectangular cross sections (2.5 x 2.5 x 20 mm3 were obtained from different regions of two fresh human femora (medial, lateral, dorsal, ventral. Specimens were either fixed in ethanol or in a mixed formalin solution or frozen following a given scheme. After two weeks of storage the samples were re-hydrated in isotonic saline and subsequently tested mechanically. The elastic bending modulus and ultimate bending strength were computed considering the actual dimensions of each specific specimen. For statistical analysis a one-way-ANOVA and an LSD post-hoc-test were performed. For ultimate bending strength no significant differences due to formalin or ethanol fixation, as compared to unfixed-fresh bone specimens could be found. And only for few cases significant differences in elastic bending modulus were observed when the two bones were evaluated separately. Since more differences of significant level due to the anatomical region of the samples were determined, the original location seems to have more influence on the evaluated mechanical properties than the method of (chemical fixation. Consequently, ethanol and the mixed formalin solution can be recommended as a fixation agent for samples in biomechanical testing, if these samples are rinsed in isotonic saline prior to static

  6. Microbiological, physico-chemical, and sensorial modifications during the useful life of the shrimps (Penaeus brasiliensis e Penaeus Paulensis) submitted to gamma radiation.; Alteracoes microbiologicas, fisico-quimicas e sensoriais durante a vida util do camarao-rosa (Penaeus brasiliensis e Penaeus Paulensis) submetido a radiacao gama

    Energy Technology Data Exchange (ETDEWEB)

    Mayer, Mariana Del Ben


    During the last years, there has been an increase in the consumption of seafood and freshwater fish as a healthier diet option. Shrimps are the most important product in the international trade market. This kind of food easily deteriorates due to autolysis, oxidation and microbial action. This research was carried out in order to study the effectiveness of radiation in preserving shrimps. Samples of shrimps (Penaeus brasiliensis and P. paulensi) were submitted to gamma radiation doses of O kGy, 1 kGy, 2 kGy, 2.5 kGy, 3 kGy and 3.5 kGy and stored under refrigeration. The microbiological analysis was performed on days O, 2, 4, 7 10 and 14 post processing. Simultaneously, the pH and total, volatile bases (TVBN) were determined. The psychotropic population varied from <2.0 log to 8.08 log CFU/g while the mesophilic ranged from <1.0 109 to 6.03 log CFU/g; pH varied from 7.52 to 8.33 and TVBN from 28.47 to 56.00 mg N/100g, according to the radiation dose and the day of analysis. Black spots, changes in the characteristic odour and TVBN levels showed the beginning of a chemical deterioration as of the 4 th day of analysis. Doses of 3 kGy and 3.5 kGy presented the best results. Pseudomonas spp. showed the highest sensitivity to radiation. Due to the lapse of time between capturing shrimps and returning to land together with inadequate manipulation and storage in the boat, the quality of raw material is prejudiced which makes the radiation process unfeasible. (author)

  7. Left ventricular dysfunction in ischemic heart disease: fundamental importance of the fibrous matrix. (United States)

    Swan, H J


    The contractile function of the myocardium is coordinated by a fibrous matrix of exquisite organization and complexity. In the normal heart, and apparently in physiological hypertrophy, this matrix is submicroscopic. In pathological states changes are frequent, and usually progressive. Thickening of the many elements of the fine structure is due to an increased synthesis of Type I collagen, This change, which affects the myocardium in a global manner, can be observed by light microscopy using special techniques. Perivascular fibrosis, with an increase in vascular smooth muscle, is accompanied by development of fibrous septa, with a decrease in diastolic compliance. These structural changes are believed to be due to increased activation of the renin-angiotensin-aldosterone system, and to be independent of the processes of myocyte hypertrophy. Reparative or replacement fibrosis is a separate process by means of which small and large areas of necrosis heal, with the development of coarse collagen structures, which lack a specific organizational pattern. Regarding ischemic heart disease, an increase in tissue collagenase is found in experimental myocardial "stunning" and in the very early phase of acute infarction. Absence of elements of the fibrous matrix allow for myocyte slippage, and--if the affected area is large--cardiac dilatation. If, subsequently, the necrosis becomes transmural, there is further disturbance of collagen due to both mechanical strain and continued autolysis, During healing collagen synthesis increases greatly to allow for reparative scarring in the available tissue matrix. In cases of infarction with moderate or severe initial dilatation, pathological hypertrophy of the spared myocardium is progressive, accounting for late heart failure and poor survival.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. A proteomic approach to investigating gene cluster expression and secondary metabolite functionality in Aspergillus fumigatus.

    Directory of Open Access Journals (Sweden)

    Rebecca A Owens

    Full Text Available A combined proteomics and metabolomics approach was utilised to advance the identification and characterisation of secondary metabolites in Aspergillus fumigatus. Here, implementation of a shotgun proteomic strategy led to the identification of non-redundant mycelial proteins (n = 414 from A. fumigatus including proteins typically under-represented in 2-D proteome maps: proteins with multiple transmembrane regions, hydrophobic proteins and proteins with extremes of molecular mass and pI. Indirect identification of secondary metabolite cluster expression was also achieved, with proteins (n = 18 from LaeA-regulated clusters detected, including GliT encoded within the gliotoxin biosynthetic cluster. Biochemical analysis then revealed that gliotoxin significantly attenuates H2O2-induced oxidative stress in A. fumigatus (p>0.0001, confirming observations from proteomics data. A complementary 2-D/LC-MS/MS approach further elucidated significantly increased abundance (p<0.05 of proliferating cell nuclear antigen (PCNA, NADH-quinone oxidoreductase and the gliotoxin oxidoreductase GliT, along with significantly attenuated abundance (p<0.05 of a heat shock protein, an oxidative stress protein and an autolysis-associated chitinase, when gliotoxin and H2O2 were present, compared to H2O2 alone. Moreover, gliotoxin exposure significantly reduced the abundance of selected proteins (p<0.05 involved in de novo purine biosynthesis. Significantly elevated abundance (p<0.05 of a key enzyme, xanthine-guanine phosphoribosyl transferase Xpt1, utilised in purine salvage, was observed in the presence of H2O2 and gliotoxin. This work provides new insights into the A. fumigatus proteome and experimental strategies, plus mechanistic data pertaining to gliotoxin functionality in the organism.

  9. Response of methicillin-resistant Staphylococcus aureus to amicoumacin A.

    Directory of Open Access Journals (Sweden)

    Amrita Lama

    Full Text Available Amicoumacin A exhibits strong antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA, hence we sought to uncover its mechanism of action. Genome-wide transcriptome analysis of S. aureus COL in response to amicoumacin A showed alteration in transcription of genes specifying several cellular processes including cell envelope turnover, cross-membrane transport, virulence, metabolism, and general stress response. The most highly induced gene was lrgA, encoding an antiholin-like product, which is induced in cells undergoing a collapse of Δψ. Consistent with the notion that LrgA modulates murein hydrolase activity, COL grown in the presence of amicoumacin A showed reduced autolysis, which was primarily caused by lower hydrolase activity. To gain further insight into the mechanism of action of amicoumacin A, a whole genome comparison of wild-type COL and amicoumacin A-resistant mutants isolated by a serial passage method was carried out. Single point mutations generating codon substitutions were uncovered in ksgA (encoding RNA dimethyltransferase, fusA (elongation factor G, dnaG (primase, lacD (tagatose 1,6-bisphosphate aldolase, and SACOL0611 (a putative glycosyl transferase. The codon substitutions in EF-G that cause amicoumacin A resistance and fusidic acid resistance reside in separate domains and do not bring about cross resistance. Taken together, these results suggest that amicoumacin A might cause perturbation of the cell membrane and lead to energy dissipation. Decreased rates of cellular metabolism including protein synthesis and DNA replication in resistant strains might allow cells to compensate for membrane dysfunction and thus increase cell survivability.

  10. Prokaryotic caspase homologs: phylogenetic patterns and functional characteristics reveal considerable diversity.

    Directory of Open Access Journals (Sweden)

    Johannes Asplund-Samuelsson

    Full Text Available Caspases accomplish initiation and execution of apoptosis, a programmed cell death process specific to metazoans. The existence of prokaryotic caspase homologs, termed metacaspases, has been known for slightly more than a decade. Despite their potential connection to the evolution of programmed cell death in eukaryotes, the phylogenetic distribution and functions of these prokaryotic metacaspase sequences are largely uncharted, while a few experiments imply involvement in programmed cell death. Aiming at providing a more detailed picture of prokaryotic caspase homologs, we applied a computational approach based on Hidden Markov Model search profiles to identify and functionally characterize putative metacaspases in bacterial and archaeal genomes. Out of the total of 1463 analyzed genomes, merely 267 (18% were identified to contain putative metacaspases, but their taxonomic distribution included most prokaryotic phyla and a few archaea (Euryarchaeota. Metacaspases were particularly abundant in Alphaproteobacteria, Deltaproteobacteria and Cyanobacteria, which harbor many morphologically and developmentally complex organisms, and a distinct correlation was found between abundance and phenotypic complexity in Cyanobacteria. Notably, Bacillus subtilis and Escherichia coli, known to undergo genetically regulated autolysis, lacked metacaspases. Pfam domain architecture analysis combined with operon identification revealed rich and varied configurations among the metacaspase sequences. These imply roles in programmed cell death, but also e.g. in signaling, various enzymatic activities and protein modification. Together our data show a wide and scattered distribution of caspase homologs in prokaryotes with structurally and functionally diverse sub-groups, and with a potentially intriguing evolutionary role. These features will help delineate future characterizations of death pathways in prokaryotes.

  11. 丝氨酸蛋白酶抑制剂PI-9的研究进展%The present understanding of serine proteinase inhibitor-9

    Institute of Scientific and Technical Information of China (English)

    雷晓晔; 周业江


    丝氨酸蛋白酶抑制剂-9(serine proteinase inhibitor9,PI-9)是丝氨酸蛋白酶抑制剂的重要成员,也是目前发现的颗粒蛋白酶B(granzyme B,GrB)唯一的内源性丝氨酸蛋白酶抑制剂.近年研究发现,PI-9能抑制GrB所致的靶细胞DNA断裂而阻断其诱导的细胞凋亡.在生理状况下,PI-9能阻止细胞毒淋巴细胞之间的相互攻击,维持机体免疫豁免部位的功能.近年研究发现PI-9还涉及-多种病理过程,诸如调节炎性介质反应,移植免疫应答及介导肿瘤免疫耐受等多种功能.%Serine proteinase inhibitor9(P1-9),a charac-teristic member of serpins,has been identified as the only inhibitor of granzyme B(GrB).Accumulated evidence suggested that PI-9 inhibits GrB-induced apoptosis by blocking DNA fragmentation of target cell.Physiologically,PI-9 could protect cytotoxic lymphocytes from committing autolysis or fratricide,and play an important role in facilitating immunologic tolerance of immune-privileged sites.In addition,evidences in recent years suggest that PI-9 Was also involved in vailous pathologic processes,such as inflammation,trans plantation and immune tolerance of tumor.

  12. Fish Spoilage Mechanisms and Preservation Techniques: Review

    Directory of Open Access Journals (Sweden)

    Abdel E. Ghaly


    Full Text Available Problem statement: Spoilage of food products is due to chemical, enzymatic or microbial activities One-fourth of the worlds food supply and 30% of landed fish are lost through microbial activity alone. With the ever growing world population and the need to store and transport the food from one place to another where it is needed, food preservation becomes necessary in order to increase its shelf life and maintain its nutritional value, texture and flavor. The freshness and quality of fish have always gained the attention by Food Regulatory Agencies and Food Processing Industry. Proper handling, pretreatment and preservation techniques can improve the quality fish and fish products and increase their shelf life. Methodology: Historically salting, drying, smoking, fermentation and canning were the methods to prevent fish spoilage and extend its shelf life. In response to consumer demand for texture, appearance and taste, new methods were developed including: Cooling, freezing and chemical preservation. A comprehensive review of the literature on the subject of fish spoilage and modern preservation techniques was carried out. Conclusion: Fish spoilage results from three basic mechanisms: Enzymatic autolysis, oxidation, microbial growth. Low temperature storage and chemical techniques for controlling water activity, enzymatic, oxidative and microbial spoilage are the most common in the industry today. A process involving the addition of an EDTA (1 mM-TBHQ (0.02% combination and ascorbic acid and storage at refrigerated temperatures (5°C in darkness can be the most positive for controlling the spoilage of fish and fish product. The suggested process would address antimicrobial activity as well as destructive oxidation of the desired lipids and fats. However, more efforts are required to understand the role of proximate composition of fish, post harvest history, environmental conditions, initial microbial load, type and nature of bacteria and their

  13. Major role for cysteine proteases during the early phase of Acanthamoeba castellanii encystment. (United States)

    Leitsch, David; Köhsler, Martina; Marchetti-Deschmann, Martina; Deutsch, Andrea; Allmaier, Günter; Duchêne, Michael; Walochnik, Julia


    Acanthamoeba castellanii is a facultative pathogen that has a two-stage life cycle comprising the vegetatively growing trophozoite stage and the dormant cyst stage. Cysts are formed when the cell encounters unfavorable conditions, such as environmental stress or food deprivation. Due to their rigid double-layered wall, Acanthamoeba cysts are highly resistant to antiamoebic drugs. This is problematic as cysts can survive initially successful chemotherapeutic treatment and cause relapse of the disease. We studied the Acanthamoeba encystment process by using two-dimensional gel electrophoresis (2DE) and found that most changes in the protein content occur early in the process. Truncated actin isoforms were found to abound in the encysting cell, and the levels of translation elongation factor 2 (EF2) were sharply decreased, indicating that the rate of protein synthesis must be low at this stage. In the advanced stage of encystment, however, EF2 levels and the trophozoite proteome were partly restored. The protease inhibitors PMSF (phenylmethylsulfonyl fluoride) and E64d [(2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester] inhibited the onset of encystment, whereas the protein synthesis inhibitor cycloheximide was ineffective. Changes in the protein profile, similar to those of encysting cells, could be observed with trophozoite homogenates incubated at room temperature for several hours. Interestingly, these changes could be inhibited significantly by cysteine protease inhibitors but not by inhibitors against other proteases. Taken together, we conclude that the encystment process in A. castellanii is of a bipartite nature consisting of an initial phase of autolysis and protein degradation and an advanced stage of restoration accompanied by the expression of encystment-specific genes.

  14. Purification and characterization of a unique alkaline elastase from Micrococcus luteus. (United States)

    Clark, D J; Hawrylik, S J; Kavanagh, E; Opheim, D J


    Micrococcus luteus isolated from human skin secretes an alkaline protease which degrades elastin. M. luteus protease (MLP) was produced in the late logarithmic and stationary phases of growth. MLP, purified to homogeneity by a three-step process, had a molecular mass of 32,812 Da and an isoelectric point of 9.3. MLP was active and highly stable in solution for 24 h from pH 6.0 to 10.5; it had maximal activity at temperatures between 57 and 59 degrees C. The presence of calcium in the solution was essential for enzyme activity and to prevent autolysis. Optimal activity occurred between pH 9.0 and 9.5, with 60% maximal activity from pH 6.5 to 11.0. The enzyme was inhibited by the serine enzyme inhibitors phenylmethylsulfonyl fluoride and chymostatin but not by the metalloenzyme inhibitor 1,10-phenanthroline or sulfhydryl enzyme inhibitors. Casein, bovine serum albumin, ovalbumin, beta-lactoglobulin, and elastin were digested by the protease while collagen and keratin were resistant to digestion. MLP demonstrated both esterase and amidase activity on synthetic peptide substrates. MLP preferentially cleaved the Leu(15)-Tyr(16) and Phe(24)-Phe(25) bonds of the oxidized beta-chain of insulin. Longer digests of insulin and the pattern of activity against synthetic substrates suggest that MLP has a cleavage specificity for bulky, hydrophobic, or aromatic amino acids in the P(1) or P(1)' positions. Amino acid sequences from the N-terminus and internal peptides of MLP were unique.

  15. Protein-Metal Organic Framework Hybrid Composites with Intrinsic Peroxidase-like Activity as a Colorimetric Biosensing Platform. (United States)

    Yin, Yuqing; Gao, Chen Ling; Xiao, Qi; Lin, Guo; Lin, Zian; Cai, Zongwei; Yang, Huang-Hao


    Artificial enzyme mimetics have received considerable attention because natural enzymes have some significant drawbacks, including enzyme autolysis, low catalytic activity, poor recovery and low stability to environmental changes. Herein, we demonstrated a facile approach for one-pot synthesis of hemeprotein-metal organic framework hybrid composites (H-MOFs) by using bovine hemoglobin (BHb) and zeolitic imidazolate framework-8 (ZIF-8) as a model reaction system. Surprisingly, the new hybrid composites exhibits 423% increase in peroxidase-like catalytic activity compared to free BHb. Taking advantages of the unique pore structure of H-MOFs with high catalytic property, a H-MOFs-based colorimetric biosensing platform was newly constructed and applied for the fast and sensitive detection of hydrogen peroxide (H2O2) and phenol. The corresponding detection limits as low as 1.0 μM for each analyte with wide linear ranges (0-800 μM for H2O2 and 0-200 μM for phenol) were obtained by naked-eye visualization. Significantly, sensitive and selective method for visual assay of trace H2O2 in cell and phenol in sewage was achieved with this platform. The stability of H-MOFs was also examined and excellent reproducibility and recyclability without losing in its activity were observed. In addition, the general applicability of H-MOFs was also investigated by using other hemeproteins (horseradish peroxidase, and myoglobin) and the corresponding catalytic activities were 291% and 273% enhancement, respectively. This present work not only expands the application of MOFs, but also provides an alternative technique for biological and environmental sample assay.

  16. Amino acid composition and functional properties of giant red sea cucumber ( Parastichopus californicus) collagen hydrolysates (United States)

    Liu, Zunying; Su, Yicheng; Zeng, Mingyong


    Giant red sea cucumber ( Parastichopus californicus) is an under-utilized species due to its high tendency to autolysis. The aim of this study was to evaluate the functional properties of collagen hydrolysates from this species. The degree of hydrolysis (DH), amino acid composition, SDS-PAGE, emulsion activity index (EAI), emulsion stability index (ESI), foam expansion (FE), and foam stability (FS) of hydrolysates were investigated. The effects of pH on the EAI, ESI FE and FS of hydrolysates were also investigated. The results indicated that the β and α 1 chains of the collagen were effectively hydrolyzed by trypsin at 50°c with an Enzyme/Substrate (E/S) ration of 1:20 (w:w). The DH of collagen was up to 17.3% after 3 h hydrolysis with trypsin. The hydrolysates had a molecular weight distribution of 1.1-17 kDa, and were abundant in glycine (Gly), proline (Pro), glutamic acid (Glu), alanine (Ala) and hydroxyproline (Hyp) residues. The hydrolysates were fractionated into three fractions ( 10 kDa), and the fraction of 3-10 kDa exhibited a higher EAI value than the fraction of > 10 kDa ( P 10 kDa had higher FE and FS values than other fractions ( P4.0. Under pH 7.0 and pH 10.0, the 3-10 kDa fraction showed higher EAI value and the fraction of > 10 kDa showed higher FE value, respectively. They are hoped to be utilized as functional ingredients in food and nutraceutical industries.

  17. Cell wall structure and function in lactic acid bacteria. (United States)

    Chapot-Chartier, Marie-Pierre; Kulakauskas, Saulius


    The cell wall of Gram-positive bacteria is a complex assemblage of glycopolymers and proteins. It consists of a thick peptidoglycan sacculus that surrounds the cytoplasmic membrane and that is decorated with teichoic acids, polysaccharides, and proteins. It plays a major role in bacterial physiology since it maintains cell shape and integrity during growth and division; in addition, it acts as the interface between the bacterium and its environment. Lactic acid bacteria (LAB) are traditionally and widely used to ferment food, and they are also the subject of more and more research because of their potential health-related benefits. It is now recognized that understanding the composition, structure, and properties of LAB cell walls is a crucial part of developing technological and health applications using these bacteria. In this review, we examine the different components of the Gram-positive cell wall: peptidoglycan, teichoic acids, polysaccharides, and proteins. We present recent findings regarding the structure and function of these complex compounds, results that have emerged thanks to the tandem development of structural analysis and whole genome sequencing. Although general structures and biosynthesis pathways are conserved among Gram-positive bacteria, studies have revealed that LAB cell walls demonstrate unique properties; these studies have yielded some notable, fundamental, and novel findings. Given the potential of this research to contribute to future applied strategies, in our discussion of the role played by cell wall components in LAB physiology, we pay special attention to the mechanisms controlling bacterial autolysis, bacterial sensitivity to bacteriophages and the mechanisms underlying interactions between probiotic bacteria and their hosts.

  18. Improvement of experimental method for measuring Michaelis constant of invertase%蔗糖酶米氏常数测定实验方法的改进

    Institute of Scientific and Technical Information of China (English)

    吴梅芬; 王晓岗; 许新华


    介绍了蔗糖酶米氏常数测定的新方法,采用旋光仪测定蔗糖在蔗糖酶作用下分解为果糖和葡萄糖过程中溶液旋光度的变化,通过测定一系列不同初始浓度蔗糖溶液转化过程的动力学曲线,采用计算机软件处理数据,得到蔗糖酶的米氏常数值。采用成熟的物理化学实验技术,原理清晰,配合计算机数据拟合方法,实验方法简便、快速。用于该校材料、化学等专业本科物理化学实验教学,取得良好的教学效果。%A new experimental method for measuring Michaelis constant of invertase is described . The inversion of cane sucrose to glucose and fructose is catalyzed by a crude-extract from the the autolysis of a dried baker's yeast ,and the angle of rotation of polarized light passing through the solution is measured with a polarimeter .The kinetic curves of a series of solutions with varied initial concentration of cane sucrose are investigated .The data are treated with computer software and the Michaelis constant of invertase is calculated . By means of conventional experimental technology of physical chemistry and computer data fitting ,the enzyme-catalysis experiment becomes convenient and quick . The experiment is applied in the laboratory physical chemistry curriculum for the undergraduates majored in material and chemistry ,in which good effects of teaching are achieved .

  19. Restructuring upstream bioprocessing: technological and economical aspects for production of a generic microbial feedstock from wheat. (United States)

    Koutinas, A A; Wang, R; Webb, C


    Restructuring and optimization of the conventional fermentation industry for fuel and chemical production is necessary to replace petrochemical production routes. Guided by this concept, a novel biorefinery process has been developed as an alternative to conventional upstream processing routes, leading to the production of a generic fermentation feedstock from wheat. The robustness of Aspergillus awamori as enzyme producer is exploited in a continuous fungal fermentation on whole wheat flour. Vital gluten is extracted as an added-value byproduct by the conventional Martin process from a fraction of the overall wheat used. Enzymatic hydrolysis of gluten-free flour by the enzyme complex produced by A. awamori during fermentation produces a liquid stream rich in glucose (320 g/L). Autolysis of fungal cells produces a micronutrient-rich solution similar to yeast extract (1.6 g/L nitrogen, 0.5 g/L phosphorus). The case-specific combination of these two liquid streams can provide a nutrient-complete fermentation medium for a spectrum of microbial bioconversions for the production of such chemicals as organic acids, amino acids, bioethanol, glycerol, solvents, and microbial biodegradable plastics. Preliminary economic analysis has shown that the operating cost required to produce the feedstock is dependent on the plant capacity, cereal market price, presence and market value of added-value byproducts, labor costs, and mode of processing (batch or continuous). Integration of this process in an existing fermentation plant could lead to the production of a generic feedstock at an operating cost lower than the market price of glucose syrup (90% to 99% glucose) in the EU, provided that the plant capacity exceeds 410 m(3)/day. Further process improvements are also suggested.

  20. Living bacteria rheology: population growth, aggregation patterns, and collective behavior under different shear flows. (United States)

    Patrício, P; Almeida, P L; Portela, R; Sobral, R G; Grilo, I R; Cidade, T; Leal, C R


    The activity of growing living bacteria was investigated using real-time and in situ rheology-in stationary and oscillatory shear. Two different strains of the human pathogen Staphylococcus aureus-strain COL and its isogenic cell wall autolysis mutant, RUSAL9-were considered in this work. For low bacteria density, strain COL forms small clusters, while the mutant, presenting deficient cell separation, forms irregular larger aggregates. In the early stages of growth, when subjected to a stationary shear, the viscosity of the cultures of both strains increases with the population of cells. As the bacteria reach the exponential phase of growth, the viscosity of the cultures of the two strains follows different and rich behaviors, with no counterpart in the optical density or in the population's colony-forming units measurements. While the viscosity of strain COL culture keeps increasing during the exponential phase and returns close to its initial value for the late phase of growth, where the population stabilizes, the viscosity of the mutant strain culture decreases steeply, still in the exponential phase, remains constant for some time, and increases again, reaching a constant plateau at a maximum value for the late phase of growth. These complex viscoelastic behaviors, which were observed to be shear-stress-dependent, are a consequence of two coupled effects: the cell density continuous increase and its changing interacting properties. The viscous and elastic moduli of strain COL culture, obtained with oscillatory shear, exhibit power-law behaviors whose exponents are dependent on the bacteria growth stage. The viscous and elastic moduli of the mutant culture have complex behaviors, emerging from the different relaxation times that are associated with the large molecules of the medium and the self-organized structures of bacteria. Nevertheless, these behaviors reflect the bacteria growth stage.

  1. Hypergravity-induced increase in the apoplastic pH and its possible involvement in suppression of beta-glucan breakdown in maize seedlings. (United States)

    Soga, K; Wakabayashi, K; Hoson, T; Kamisaka, S


    Elongation growth of both coleoptiles and mesocotyls of maize (Zea mays L. cv. Cross Bantam T51) seedlings was inhibited under basipetal hypergravity (300 g) conditions. Hypergravity increased the pH of the apoplastic fluid of coleoptiles from 5.0 to 5.5 and mesocotyls from 5.2 to 5.7. When beta-1,3:1,4-D-glucanases (beta-glucanases) extracted from cell walls of the 1-g control coleoptiles and mesocotyls were assayed at pH 5.0 and 5.5 for coleoptiles, and at 5.2 and 5.7 for mesocotyls, respectively, the activity in the increased pH conditions was significantly lower than that in the control pH conditions. During the autolysis of the enzymically active cell wall preparations obtained from 1-g control organs, a molecular mass downshift of hemicellulosic polysaccharides occurred in cell walls. This downshift was suppressed in the increased pH conditions as compared with the control pH conditions. It was reported that hypergravity increased the molecular mass of hemicellulosic polysaccharides by decreasing the beta-glucanase activity, and thereby decreased the mechanical extensibility of cell walls in maize coleoptiles and mesocotyls. These results suggest that, in maize coleoptiles and mesocotyls, hypergravity-induced increase in the pH in the apoplastic fluid is involved in the reduction of the activity of beta-glucanases which, in turn, causes an increase in the molecular mass of hemicellulosic polysaccharides and inhibits elongation growth.

  2. A proposal to rename the hyperthermophile Pyrococcus woesei as Pyrococcus furiosus subsp. woesei

    Directory of Open Access Journals (Sweden)

    Wirojne Kanoksilapatham


    Full Text Available Pyrococcus species are hyperthermophilic members of the order Thermococcales, with optimal growth temperatures approaching 100 °C. All species grow heterotrophically and produce H2 or, in the presence of elemental sulfur (S°, H2S. Pyrococcus woesei and P. furiosus were isolated from marine sediments at the same Vulcano Island beach site and share many morphological and physiological characteristics. We report here that the rDNA operons of these strains have identical sequences, including their intergenic spacer regions and part of the 23S rRNA. Both species grow rapidly and produce H2 in the presence of 0.1% maltose and 10–100 µM sodium tungstate in S°-free medium. However,P. woesei shows more extensive autolysis than P. furiosus in the stationary phase. Pyrococcusfuriosus and P. woesei share three closely related families of insertion sequences (ISs. A Southern blot performed with IS probes showed extensive colinearity between the genomes of P. woesei and P. furiosus. Cloning and sequencing of ISs that were in different contexts in P. woesei and P. furiosus revealed that the napA gene in P. woesei is disrupted by a type III IS element, whereas in P. furiosus, this gene is intact. A type I IS element, closely linked to the napA gene, was observed in the same context in both P. furiosus and P. woesei genomes. Our results suggest that the IS elements are implicated in genomic rearrangements and reshuffling in these closely related strains. We propose to rename P. woesei a subspecies of P. furiosus based on their identical rDNA operon sequences, many common IS elements that are shared genomic markers, and the observation that all P. woesei nucleotide sequences deposited in GenBank to date are > 99% identical to P. furiosus sequences.

  3. Assembly and development of the Pseudomonas aeruginosa biofilm matrix.

    Directory of Open Access Journals (Sweden)

    Luyan Ma


    Full Text Available Virtually all cells living in multicellular structures such as tissues and organs are encased in an extracellular matrix. One of the most important features of a biofilm is the extracellular polymeric substance that functions as a matrix, holding bacterial cells together. Yet very little is known about how the matrix forms or how matrix components encase bacteria during biofilm development. Pseudomonas aeruginosa forms environmentally and clinically relevant biofilms and is a paradigm organism for the study of biofilms. The extracellular polymeric substance of P. aeruginosa biofilms is an ill-defined mix of polysaccharides, nucleic acids, and proteins. Here, we directly visualize the product of the polysaccharide synthesis locus (Psl exopolysaccharide at different stages of biofilm development. During attachment, Psl is anchored on the cell surface in a helical pattern. This promotes cell-cell interactions and assembly of a matrix, which holds bacteria in the biofilm and on the surface. Chemical dissociation of Psl from the bacterial surface disrupted the Psl matrix as well as the biofilm structure. During biofilm maturation, Psl accumulates on the periphery of 3-D-structured microcolonies, resulting in a Psl matrix-free cavity in the microcolony center. At the dispersion stage, swimming cells appear in this matrix cavity. Dead cells and extracellular DNA (eDNA are also concentrated in the Psl matrix-free area. Deletion of genes that control cell death and autolysis affects the formation of the matrix cavity and microcolony dispersion. These data provide a mechanism for how P. aeruginosa builds a matrix and subsequently a cavity to free a portion of cells for seeding dispersal. Direct visualization reveals that Psl is a key scaffolding matrix component and opens up avenues for therapeutics of biofilm-related complications.

  4. Phospholipase C from Pseudomonas aeruginosa and Bacillus cereus;characterization of catalytic activity

    Institute of Scientific and Technical Information of China (English)

    Nooran Sherif Elleboudy; Mohammad Mabrouk Aboulwafa; Nadia Abdel-Haleem Hassouna


    Objective:To study characteristics of phospholipases C (PLCs), their importance for producing microorganisms as well as the potential of their use for industrial purposes. Methods:PLC from Bacillus cereus (B. cereus) D101 was selected as an example of Gram-positive PLCs and PLC from Pseudomonas aeruginosa (P. aeruginosa) D183 of Gram-negative ones. Enzymes were partially purified by ammonium sulfate precipitation followed by membrane dialysis. Partially purified preparations were used to study effect of different factors on activities as well as in substrate specificity tests which were conducted using a turbidimetric assay method. Results: Maximum activity was at pH 7 and 8 and 40℃for P. aeruginosa PLC, and pH 8-10 and 37℃for B. cereus PLC. Both PLCs were inhibited by Pi at 5 mM or higher, whereas, PLC from B. cereus only was inhibited by EDTA. Activity of P. aeruginosa PLC was not affected by removing Zn2+ions from reaction mixture or their replacement with Ca2+, Ba2+, Mg2+or Mn2+ions. Vis-à-vis, activity of B. cereus PLC was found to be metal ion dependent. PLCs from both isolates were relatively thermostable and showed maximum affinity toward phosphatidylcholine. Sphingomyelin and phosphatidylethanolamine were not good substrates and phosphatidylinositol, phosphatidylserine, phosphatidylglycerol and cardiolipin could be considered non-substrates. Conclusions: Human body physiological conditions could favor activity of P. aeruginosa and B. cereus PLCs. These enzymes may participate in phosphate scavenging and virulence of producing isolates but not in autolysis. PLCs from both isolates are potential candidates for industrial use.

  5. Fitness of isogenic colony morphology variants of Pseudomonas aeruginosa in murine airway infection.

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    Elza Rakhimova

    Full Text Available Chronic lung infections with Pseudomonas aeruginosa are associated with the diversification of the persisting clone into niche specialists and morphotypes, a phenomenon called 'dissociative behaviour'. To explore the potential of P. aeruginosa to change its morphotype by single step loss-of-function mutagenesis, a signature-tagged mini-Tn5 plasposon library of the cystic fibrosis airway isolate TBCF10839 was screened for colony morphology variants under nine different conditions in vitro. Transposon insertion into 1% of the genome changed colony morphology into eight discernable morphotypes. Half of the 55 targets encode features of primary or secondary metabolism whereby quinolone production was frequently affected. In the other half the transposon had inserted into genes of the functional categories transport, regulation or motility/chemotaxis. To mimic dissociative behaviour of isogenic strains in lungs, pools of 25 colony morphology variants were tested for competitive fitness in an acute murine airway infection model. Six of the 55 mutants either grew better or worse in vivo than in vitro, respectively. Metabolic proficiency of the colony morphology variant was a key determinant for survival in murine airways. The most common morphotype of self-destructive autolysis did unexpectedly not impair fitness. Transposon insertions into homologous genes of strain PAO1 did not reproduce the TBCF10839 mutant morphotypes for 16 of 19 examined loci pointing to an important role of the genetic background on colony morphology. Depending on the chosen P. aeruginosa strain, functional genome scans will explore other areas of the evolutionary landscape. Based on our discordant findings of mutant phenotypes in P. aeruginosa strains PAO1, PA14 and TBCF10839, we conclude that the current focus on few reference strains may miss modes of niche adaptation and dissociative behaviour that are relevant for the microevolution of complex traits in the wild.

  6. Genomic, Transcriptomic and Metabolomic Studies of Two Well-Characterized, Laboratory-Derived Vancomycin-Intermediate Staphylococcus aureus Strains Derived from the Same Parent Strain

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    Dipti S. Hattangady


    Full Text Available Complete genome comparisons, transcriptomic and metabolomic studies were performed on two laboratory-selected, well-characterized vancomycin-intermediate Staphylococcus aureus (VISA derived from the same parent MRSA that have changes in cell wall composition and decreased autolysis. A variety of mutations were found in the VISA, with more in strain 13136p−m+V20 (vancomycin MIC = 16 µg/mL than strain 13136p−m+V5 (MIC = 8 µg/mL. Most of the mutations have not previously been associated with the VISA phenotype; some were associated with cell wall metabolism and many with stress responses, notably relating to DNA damage. The genomes and transcriptomes of the two VISA support the importance of gene expression regulation to the VISA phenotype. Similarities in overall transcriptomic and metabolomic data indicated that the VISA physiologic state includes elements of the stringent response, such as downregulation of protein and nucleotide synthesis, the pentose phosphate pathway and nutrient transport systems. Gene expression for secreted virulence determinants was generally downregulated, but was more variable for surface-associated virulence determinants, although capsule formation was clearly inhibited. The importance of activated stress response elements could be seen across all three analyses, as in the accumulation of osmoprotectant metabolites such as proline and glutamate. Concentrations of potential cell wall precursor amino acids and glucosamine were increased in the VISA strains. Polyamines were decreased in the VISA, which may facilitate the accrual of mutations. Overall, the studies confirm the wide variability in mutations and gene expression patterns that can lead to the VISA phenotype.

  7. Evolution of the lactic acid bacterial community during malt whisky fermentation: a polyphasic study. (United States)

    van Beek, Sylvie; Priest, Fergus G


    The development of the lactic acid bacterial community in a commercial malt whisky fermentation occurred in three broad phases. Initially, bacteria were inhibited by strong yeast growth. Fluorescence microscopy and environmental scanning electron microscopy revealed, in this early stage, both cocci and rods that were at least partly derived from the wort and yeast but also stemmed from the distillery plant. Denaturing gradient gel electrophoresis (DGGE) of partial 16S rRNA genes and sequence analysis revealed cocci related to Streptococcus thermophilus or Saccharococcus thermophilus, Lactobacillus brevis, and Lactobacillus fermentum. The middle phase began 35 to 40 h after yeast inoculation and was characterized by exponential growth of lactobacilli and residual yeast metabolism. Lactobacillus casei or Lactobacillus paracasei, L. fermentum, and Lactobacillus ferintoshensis were detected in samples of fermenting wort examined by DGGE during this stage. Bacterial growth was accompanied by the accumulation of acetic and lactic acids and the metabolism of residual maltooligosaccharides. By 70 h, two new PCR bands were detected on DGGE gels, and the associated bacteria were largely responsible for the final phase of the fermentation. The bacteria were phylogenetically related to Lactobacillus acidophilus and Lactobacillus delbrueckii, and strains similar to the former had previously been recovered from malt whisky fermentations in Japan. These were probably obligately homofermentative bacteria, required malt wort for growth, and could not be cultured on normal laboratory media, such as MRS. Their metabolism during the last 20 to 30 h of fermentation was associated with yeast death and autolysis and further accumulation of lactate but no additional acetate.

  8. Effects of pseudo-microgravity on symbiosis between endophyte, Neotyphodium, and its host plant, tall fescue (Festuca arundinacea) (United States)

    Tomita-Yokotani, K.; Wakabayashi, K.; Hiraishi, K.; Yoshida, S.; Hashimoto, H.; Shinozaki, S.; Yamashita, M.

    Endophyte is a group of microbes that symbiotically live in plant body Endophyte provides host plant its metabolites that protect the plant from insect pests In addition to this host plants are resistive against environmental stress In general endophyte lives in seeds to seeds of the infected plants through multiple generations The infection of fungi has never been observed and their original pathway is still unknown in nature The aim of this study is to examine whether this stable symbiosis between endophytes and its host plant would be modified under pseudo-microgravity or not We also aim to observe the infection under an exotic environment in terms of gravity We found that the internal hyphae of both the incubated plant under pseudo-microgravity and the ground control became indistinct with the number of incubation days A part of the endophyte in the seed under its autolysis was suggested because the amount of fungi in the base of the shoot that was observed with the incubated plant under the ground control was far less than that in the seed before sowing Hyphae began to grow in the germinating seed after a 3-day incubation period However a lot of aggregated fungi still existed in the 3-day incubated seed under pseudo-microgravity Moreover hyphae in the 3-day incubated seed under pseudo-microgravity were more indistinctly than that under the ground control The fungi were observed in the boundary of the seed and the shoot of the 5-day incubated seed under the ground control but not under pseudo-microgravity By this observation it was suggested that

  9. A multiplex real-time polymerase chain reaction assay with two internal controls for the detection of Brucella species in tissues, blood, and feces from marine mammals. (United States)

    Sidor, Inga F; Dunn, J Lawrence; Tsongalis, Gregory J; Carlson, Jolene; Frasca, Salvatore


    Brucellosis has emerged as a disease of concern in marine mammals in the last 2 decades. Molecular detection techniques have the potential to address limitations of other methods for detecting infection with Brucella in these species. Presented herein is a real-time polymerase chain reaction (PCR) method targeting the Brucella genus-specific bcsp31 gene. The method also includes a target to a conserved region of the eukaryotic mitochondrial 16S ribosomal RNA gene to assess suitability of extracted DNA and a plasmid-based internal control to detect failure of PCR due to inhibition. This method was optimized and validated to detect Brucella spp. in multiple sample matrices, including fresh or frozen tissue, blood, and feces. The analytical limit of detection was low, with 95% amplification at 24 fg, or an estimated 7 bacterial genomic copies. When Brucella spp. were experimentally added to tissue or fecal homogenates, the assay detected an estimated 1-5 bacteria/µl. An experiment simulating tissue autolysis showed relative persistence of bacterial DNA compared to host mitochondrial DNA. When used to screen 1,658 field-collected marine mammal tissues in comparison to microbial culture, diagnostic sensitivity and specificity were 70.4% and 98.3%, respectively. In addition to amplification in fresh and frozen tissues, Brucella spp. were detected in feces and formalin-fixed, paraffin-embedded tissues from culture-positive animals. Results indicate the utility of this real-time PCR for the detection of Brucella spp. in marine species, which may have applications in surveillance or epidemiologic investigations.

  10. Insights into the structure-function relationships of pneumococcal cell wall lysozymes, LytC and Cpl-1. (United States)

    Monterroso, Begoña; Sáiz, José Luis; García, Pedro; García, José Luis; Menéndez, Margarita


    The LytC lysozyme belongs to the autolytic system of Streptococcus pneumoniae and carries out a slow autolysis with optimum activity at 30 degrees C. Like all pneumococcal murein hydrolases, LytC is a modular enzyme. Its mature form comprises a catalytic module belonging to the GH25 family of glycosyl-hydrolases and a cell wall binding module (CBM), made of 11 sequence repeats, that is essential for activity and specifically targets choline residues present in pneumococcal lipoteichoic and teichoic acids. Here we show that the catalytic module is natively folded, and its thermal denaturation takes place at 45.4 degrees C. However, the CBM is intrinsically unstable, and the ultimate folding and stabilization of the active, monomeric form of LytC relies on choline binding. The complex formation proceeds in a rather slow way, and all sites (8.0 +/- 0.5 sites/monomer) behave as equivalent (Kd = 2.7 +/- 0.3 mm). The CBM stabilization is, nevertheless, marginal, and irreversible denaturation becomes measurable at 37 degrees C even at high choline concentration, compromising LytC activity. In contrast, the Cpl-1 lysozyme, a homologous endolysin encoded by pneumococcal Cp-1 bacteriophage, is natively folded in the absence of choline and has maximum activity at 37 degrees C. Choline binding is fast and promotes Cpl-1 dimerization. Coupling between choline binding and folding of the CBM of LytC indicates a high conformational plasticity that could correlate with the unusual alternation of short and long choline-binding repeats present in this enzyme. Moreover, it can contribute to regulate LytC activity by means of a tight, complementary binding to the pneumococcal envelope, a limited motility, and a moderate resistance to thermal denaturation that could also account for its activity versus temperature profile.

  11. Insights into the Structure-Function Relationships of Pneumococcal Cell Wall Lysozymes, LytC and Cpl-1*S⃞ (United States)

    Monterroso, Begoña; Sáiz, José Luis; García, Pedro; García, José Luis; Menéndez, Margarita


    The LytC lysozyme belongs to the autolytic system of Streptococcus pneumoniae and carries out a slow autolysis with optimum activity at 30 °C. Like all pneumococcal murein hydrolases, LytC is a modular enzyme. Its mature form comprises a catalytic module belonging to the GH25 family of glycosyl-hydrolases and a cell wall binding module (CBM), made of 11 sequence repeats, that is essential for activity and specifically targets choline residues present in pneumococcal lipoteichoic and teichoic acids. Here we show that the catalytic module is natively folded, and its thermal denaturation takes place at 45.4 °C. However, the CBM is intrinsically unstable, and the ultimate folding and stabilization of the active, monomeric form of LytC relies on choline binding. The complex formation proceeds in a rather slow way, and all sites (8.0 ± 0.5 sites/monomer) behave as equivalent (Kd = 2.7 ± 0.3 mm). The CBM stabilization is, nevertheless, marginal, and irreversible denaturation becomes measurable at 37 °C even at high choline concentration, compromising LytC activity. In contrast, the Cpl-1 lysozyme, a homologous endolysin encoded by pneumococcal Cp-1 bacteriophage, is natively folded in the absence of choline and has maximum activity at 37 °C. Choline binding is fast and promotes Cpl-1 dimerization. Coupling between choline binding and folding of the CBM of LytC indicates a high conformational plasticity that could correlate with the unusual alternation of short and long choline-binding repeats present in this enzyme. Moreover, it can contribute to regulate LytC activity by means of a tight, complementary binding to the pneumococcal envelope, a limited motility, and a moderate resistance to thermal denaturation that could also account for its activity versus temperature profile. PMID:18667432


    Directory of Open Access Journals (Sweden)

    Oladele, A.A.


    Full Text Available Background: Embalming is a process used to temporarily preserve a human cadaver to forestall decomposition and make it suitable for display at funerals; thus, are agents that prevent autolysis and putrefaction. The outbreak of maggots from a heap of inadequately embalmed bodies due to deep cuts and bodies involved in inferno, necessitated the need to re-investigate the efficacy of formalin based embalming fluid and its inability to kill maggots. Methodology: Various strength of Formaldehyde, Xylene, Kerosene, and, Lime fluid, Isopropanol, Gamalin 20, Potassium ferrocyanide, and Physiological saline as control were used in the investigation. In the present investigation, Two maggots under the same atmospheric condition were put in each of the ten selected chemical reagents/solutions, including Lime, Kerosene, and the Gamalin 20 that are naturally available were initially dispensed into ten glass universal containers. Maggot movements in each reagent solution were critically observed. Result: Maggots death occurred within the first ten minutes in test number three groups III that contains Concentrated Formalin and Xylene and Maggots died after fifteen minute of the experiment, but maggots did not died until about eight hours after the test in two of the experiment. Discussion: Results of this investigation showed clearly that Maggots were not killed as soon as expected by the embalmer when ordinary ten percent alcoholic formalin embalming fluid is used. Equal volume of concentrated formalin plus Xylene was found out to be effective at killing maggot instantly. Conclusion: It is therefore advisable to use Xylene plus Conc. Formalin when preserving cadaver infested with Maggot and this could at the same time prevent the occurrence of Maggots Infestation and better preservation of mass of burnt mutilated corpses in our Mortuary.

  13. Deletion of the glycosyltransferase bgsB of Enterococcus faecalis leads to a complete loss of glycolipids from the cell membrane and to impaired biofilm formation

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    Grohmann Elisabeth


    Full Text Available Abstract Background Deletion of the glycosyltransferase bgsA in Enterococcus faecalis leads to loss of diglucosyldiacylglycerol from the cell membrane and accumulation of its precursor monoglucosyldiacylglycerol, associated with impaired biofilm formation and reduced virulence in vivo. Here we analyzed the function of a putative glucosyltransferase EF2890 designated biofilm-associated glycolipid synthesis B (bgsB immediately downstream of bgsA. Results A deletion mutant was constructed by targeted mutagenesis in E. faecalis strain 12030. Analysis of cell membrane extracts revealed a complete loss of glycolipids from the cell membrane. Cell walls of 12030ΔbgsB contained approximately fourfold more LTA, and 1H-nuclear magnetic resonance (NMR spectroscopy suggested that the higher content of cellular LTA was due to increased length of the glycerol-phosphate polymer of LTA. 12030ΔbgsB was not altered in growth, cell morphology, or autolysis. However, attachment to Caco-2 cells was reduced to 50% of wild-type levels, and biofilm formation on polystyrene was highly impaired. Despite normal resistance to cationic antimicrobial peptides, complement and antibody-mediated opsonophagocytic killing in vitro, 12030ΔbgsB was cleared more rapidly from the bloodstream of mice than wild-type bacteria. Overall, the phenotype resembles the respective deletion mutant in the bgsA gene. Our findings suggest that loss of diglucosyldiacylglycerol or the altered structure of LTA in both mutants account for phenotypic changes observed. Conclusions In summary, BgsB is a glucosyltransferase that synthesizes monoglucosyldiacylglycerol. Its inactivation profoundly affects cell membrane composition and has secondary effects on LTA biosynthesis. Both cell-membrane amphiphiles are critical for biofilm formation and virulence of E. faecalis.

  14. Characterization of the starvation-induced chitinase CfcA and α-1,3-glucanase AgnB of Aspergillus niger. (United States)

    van Munster, Jolanda M; Dobruchowska, Justyna M; Veloo, Ruud; Dijkhuizen, Lubbert; van der Maarel, Marc J E C


    The common saprophyte Aspergillus niger may experience carbon starvation in nature as well as during industrial fermentations. Starvation survival strategies, such as conidiation or the formation of exploratory hyphae, require energy and building blocks, which may be supplied by autolysis. Glycoside hydrolases are key effectors of autolytic degradation of fungal cell walls, but knowledge on their identity and functionality is still limited. We recently identified agnB and cfcA as two genes encoding carbohydrate-active enzymes that had notably increased transcription during carbon starvation in A. niger. Here, we report the biochemical and functional characterization of these enzymes. AgnB is an α-1,3-glucanase that releases glucose from α-1,3-glucan substrates with a minimum degree of polymerization of 4. CfcA is a chitinase that releases dimers from the nonreducing end of chitin. These enzymes thus attack polymers that are found in the fungal cell wall and may have a role in autolytic fungal cell wall degradation in A. niger. Indeed, cell wall degradation during carbon starvation was reduced in the double deletion mutant ΔcfcA ΔagnB compared to the wild-type strain. Furthermore, the cell walls of the carbon-starved mycelium of the mutant contained a higher fraction of chitin or chitosan. The function of at least one of these enzymes, CfcA, therefore appears to be in the recycling of cell wall carbohydrates under carbon limiting conditions. CfcA thus may be a candidate effector for on demand cell lysis, which could be employed in industrial processes for recovery of intracellular products.

  15. 眼部热金属烧伤的临床特点分析%Clinical characteristics of ocular thermal burns by hot metal liquid

    Institute of Scientific and Technical Information of China (English)

    王玉亭; 史伟云; 韩莎莎; 李素霞


    Objective To investigate the clinical characteristics and treatment methods for ocular thermal burns by hot metal liquid .Results There were 47 patients ( 54 eyes ) with ocular thermal burns by hot metal liquid admitted to Shandong Eye Hospital between September 2004 and December 2012.Histories of these patients were analyzed retrospec-tively.The clinical characteristics of the burns , types of the metal , degree of injuries to the eye , relationship between burn-ed areas and long Pterm complications and treatment outcomes were analyzed .Results Eye metal thermal burns has the fol-lowing clinical features:(1)Burns were mainly limited to the area of hot metal direct contact: focal conjunctival ischemic necrosis were less than a quarter in 25 eyes (56.8%), and in 21 eyes, it located in lower part of the conjunctiva .(2) Se-vere tissue autolysis were found at the part of the cornea that had direct metal contact :in 20 out of 26 eyes which had the cornea autolysis, it located near the limbus, and was small, deep, and fast resolved.(3) Eyelid burns were more com-mon:in 48 eyes damaged corneal or conjunctival accompanied with eyelid injuries (88.9%), and in 29 eyes the eyelid plate progressed to local erosive necrosis and scar formation .(4) There was no significant difference in the degree of corne-al injury caused by burns with metals of different temperatures ( e.g., molten iron or molten aluminum , chi-square =0.926;P >0.05).(5) The stability of ocular surface at 6 weeks after burns was related to the injury size in conjunctiva (chipsquare=4.456, P <0.05) and cornea (chi-square=8.145, P <0.01).(6) There was a higher incidence of sym-blepharon (25 eyes, 55.6%).Regarding the therapeutic outcome: (1) 18 eyes (37.5%) received permanent tarsor-rhaphy because of damaged eyelid and consequently eyeball exposure .( 2 ) Whether corneal autolysis would occur was linked to whether early (within 3 days) amniotic membrane transplantation was performed (chi -square =5.035, P <0

  16. Study of energetic-particle-irradiation induced biological effect on Rhizopus oryzae through synchrotron-FTIR micro-spectroscopy (United States)

    Liu, Jinghua; Qi, Zeming; Huang, Qing; Wei, Xiaoli; Ke, Zhigang; Fang, Yusheng; Tian, Yangchao; Yu, Zengliang


    Energetic particles exist ubiquitously and cause varied biological effects such as DNA strand breaks, lipid peroxidation, protein modification, cell apoptosis or death. An emerging biotechnology based on ion-beam technique has been developed to serve as an effective tool for mutation breeding of crops and microbes. In order to improve the effectiveness of ion-beam biotechnology for mutation breeding, it is indispensible to gain a better understanding of the mechanism of the interactions between the energetic ions and biological systems which is still elusive. A new trend is to conduct more comprehensive research which is based on micro-scaled observation of the changes of the cellular structures and compositions under the interactions. For this purpose, advanced synchrotron FTIR (s-FTIR) microscopy was employed to monitor the cellular changes of single fungal hyphae under irradiation of α-particles from 241Am. Intracellular contents of ROS, MDA, GSSG/GSH and activities of CAT and SOD were measured via biochemical assay. Ion-irradiation on Rhizopus oryzae causes localized vacuolation, autolysis of cell wall and membrane, lipid peroxidation, DNA damage and conformational changes of proteins, which have been clearly revealed by the s-FTIR microspectroscopy. The different changes of cell viability, SOD and CAT activities can be explained by the ROS-involved chemical reactions. Evidently, the elevated level of ROS in hyphal cells upon irradiation plays the key role in the caused biological effect. This study demonstrates that s-FTIR microspectroscopy is an effective tool to study the damage of fungal hyphae caused by ionizing radiation and it facilitates the exploit of the mechanism for the interactions between the energetic ions and biological systems.

  17. Staphylococcus aureus Colonization of the Mouse Gastrointestinal Tract Is Modulated by Wall Teichoic Acid, Capsule, and Surface Proteins.

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    Yoshiki Misawa


    Full Text Available Staphylococcus aureus colonizes the nose, throat, skin, and gastrointestinal (GI tract of humans. GI carriage of S. aureus is difficult to eradicate and has been shown to facilitate the transmission of the bacterium among individuals. Although staphylococcal colonization of the GI tract is asymptomatic, it increases the likelihood of infection, particularly skin and soft tissue infections caused by USA300 isolates. We established a mouse model of persistent S. aureus GI colonization and characterized the impact of selected surface antigens on colonization. In competition experiments, an acapsular mutant colonized better than the parental strain Newman, whereas mutants defective in sortase A and clumping factor A showed impaired ability to colonize the GI tract. Mutants lacking protein A, clumping factor B, poly-N-acetyl glucosamine, or SdrCDE showed no defect in colonization. An S. aureus wall teichoic acid (WTA mutant (ΔtagO failed to colonize the mouse nose or GI tract, and the tagO and clfA mutants showed reduced adherence in vitro to intestinal epithelial cells. The tagO mutant was recovered in lower numbers than the wild type strain in the murine stomach and duodenum 1 h after inoculation. This reduced fitness correlated with the in vitro susceptibility of the tagO mutant to bile salts, proteases, and a gut-associated defensin. Newman ΔtagO showed enhanced susceptibility to autolysis, and an autolysin (atl tagO double mutant abrogated this phenotype. However, the atl tagO mutant did not survive better in the mouse GI tract than the tagO mutant. Our results indicate that the failure of the tagO mutant to colonize the GI tract correlates with its poor adherence and susceptibility to bactericidal factors within the mouse gut, but not to enhanced activity of its major autolysin.

  18. Fish protein hydrolysate production from sardine solid waste by crude pepsin enzymatic hydrolysis in a bioreactor coupled to an ultrafiltration unit

    Energy Technology Data Exchange (ETDEWEB)

    Benhabiles, M.S.; Abdi, N. [National Polytechnic school of Algiers, B.P. 182-16200, El Harrach, Algiers (Algeria); Drouiche, N., E-mail: [National Polytechnic school of Algiers, B.P. 182-16200, El Harrach, Algiers (Algeria); Silicon Technology Development Unit (UDTS) 2, Bd Frantz Fanon BP140, Alger-7 Merveilles, 16000 (Algeria); Lounici, H. [National Polytechnic school of Algiers, B.P. 182-16200, El Harrach, Algiers (Algeria); Pauss, A. [University of Technology of Compiegne, Departement Genie chimique,B.P. 20.509, 60205 Compiegne cedex (France); Goosen, M.F.A. [Alfaisal University, Riyadh (Saudi Arabia); Mameri, N. [University of Technology of Compiegne, Departement Genie chimique,B.P. 20.509, 60205 Compiegne cedex (France)


    The aims of the study were to optimize the production a fish protein hydrolysate (FPH) by enzymatic hydrolysis of sardine solid waste using crude pepsin, and to scale up the process in a bioreactor coupled to an ultrafiltration unit for product recovery. Results showed that the crude pepsin prepared by autolysis of the mucous membranes of a sheep stomach at optimal conditions (i. e. pH = 1.5-2 and incubation time of 6 h) could be satisfactory used for the enzymatic hydrolysis of fish solid waste. The optimal conditions for enzymatic reaction were: temperature 48 Degree-Sign C, and pH 1.5. The scale up of the enzymatic hydrolysis and the coupling of the reactor an ultrafiltration unit to concentrate the hydrolysate gave good results with a rejection coefficient for the protein hydrolysate product in the range of 90%. The volumetric concentration factor was 2.5, with a permeate flux of 200 L m{sup -2} bar{sup -1}. However, the results also suggest that the ultrafiltration product concentration process may be operating beyond the critical flux at which point irreversible membrane fouling occurs. - Highlights: Black-Right-Pointing-Pointer Evaluating to produce a (FPH) by enzymatic hydrolysis of sardine solid wastes was achieved. Black-Right-Pointing-Pointer Investigation of key parameters for optimal conditions for enzymatic hydrolysis have been studied. Black-Right-Pointing-Pointer Valorization of sardine waste was realized by enzymatic hydrolysis process. Black-Right-Pointing-Pointer Performances of this enzyme gave comparable results to those obtained with commercial pepsin. Black-Right-Pointing-Pointer The nutritional quality of the FPH produced appears to be satisfactory.

  19. Phenotypic Variation Is Almost Entirely Independent of the Host-Pathogen Relationship in Clinical Isolates of S. aureus.

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    Adrian D Land

    Full Text Available A key feature of Staphylococcus aureus biology is its ability to switch from an apparently benign colonizer of ~30% of the population to a cutaneous pathogen, to a deadly invasive pathogen. Little is known about the mechanisms driving this transition or the propensity of different S. aureus strains to engender different types of host-pathogen interactions. At the same time, significant weight has been given to the role of specific in vitro phenotypes in S. aureus virulence. Biofilm formation, hemolysis and pigment formation have all been associated with virulence in mice.To determine if there is a correlation between in vitro phenotype and the three types of host-pathogen relationships commonly exhibited by S. aureus in the context of its natural human host, we assayed 300 clinical isolates for phenotypes implicated in virulence including hemolysis, sensitivity to autolysis, and biofilm formation. For comparative purposes, we also assayed phenotype in 9 domesticated S. aureus strains routinely used for analysis of virulence determinants in laboratory settings.Strikingly, the clinical strains exhibited significant phenotypic uniformity in each of the assays evaluated in this study. One exception was a small, but significant, correlation between an increased propensity for biofilm formation and isolation from skin and soft tissue infections (SSTIs. In contrast, we observed a high degree of phenotypic variation between common laboratory strains that exhibit virulence in mouse models. These data suggest the existence of significant evolutionary pressure on the S. aureus genome and highlight a role for host factors as a strong determinant of the host-pathogen relationship. In addition, the high degree of variation between laboratory strains emphasizes the need for caution when applying data obtained in one lab strain to the analysis of another.

  20. Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

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    Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; Patin, Delphine; Farr, Carol L.; Grant, Joanna C.; Chiu, Hsiu-Ju; Jaroszewski, Lukasz; Knuth, Mark W.; Godzik, Adam; Lesley, Scott A.; Elsliger, Marc-André; Deacon, Ashley M.; Wilson, Ian A.



    Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.

    IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural

  1. 杂交鲍苗种“病毒性萎缩症(AbSV)”病理学研究%Studies on Pathology of “Shriveling Syndrome Associated Virus(AbSV)” in Juvenile Hybrid Abalone

    Institute of Scientific and Technical Information of China (English)



    从福州地区患病杂交鲍苗种组织中分离到一种球形病毒用以感染健康鲍,通过对感染发病的鲍苗进行光镜和电镜观察,发现:病毒主要在消化腺嗜碱性细胞的细胞核中发生;消化腺消化细胞和嗜碱性细胞细胞核异形、染色质固缩,内质网扩张,线粒体嵴断裂、消失;消化腺小叶基膜肌纤维断裂、消失;消化道肠道具微绒毛的柱状细胞和纤毛柱状细胞发生自溶现象;足部肌细胞核浓缩,染色质固缩、边集,肌纤维断裂出现空泡.据此推测,病毒以鲍消化腺嗜碱性细胞为靶细胞,造成消化腺和胃、肠道等消化系统病变,机体因长期营养不良而萎缩,最后因全身性感染出现死亡.%Pathological analysis was done on juvenile hybrid abalone (Haliotisdiscus discus Haliotis discus hannai)artificially infected by a spherical virus isolated from the diseased ones in Fuzhou.Examination of artificially infected juvenile hybrid abalone by optical and electron microscopy showed that the virion mainly developed in nucleus of basophilic cells in digestive gland,and led to nuclear heteromorphosis,chromatin condensation,endoplasmic reticulum expansion,and mitochondria cristae breakage in digestive gland cell and basophilic cell.Meanwhile,broken digestive gland lobule basal lamina muscle fibers,autolysis and pyknosis of microvillus and cilium columnar cell in digestive gland intestinal tract were observed.In foot muscle cells,muscle fibers were broken and vesicles emerged,together with chromatin condensation and margination.These results suggested that the digestive gland basophilic cells maybe the special target cell of this virus,the infection could cause lesion in digestive gland and digestive organs such as stomach,intestines.Atrophy due to long-term malnutrition and systemic infection would finally led to death.

  2. Effect of nutrient restriction and re-feeding on calpain family genes in skeletal muscle of channel catfish (Ictalurus punctatus.

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    Elena Preziosa

    Full Text Available BACKGROUND: Calpains, a superfamily of intracellular calcium-dependent cysteine proteases, are involved in the cytoskeletal remodeling and wasting of skeletal muscle. Calpains are generated as inactive proenzymes which are activated by N-terminal autolysis induced by calcium-ions. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we characterized the full-length cDNA sequences of three calpain genes, clpn1, clpn2, and clpn3 in channel catfish, and assessed the effect of nutrient restriction and subsequent re-feeding on the expression of these genes in skeletal muscle. The clpn1 cDNA sequence encodes a protein of 704 amino acids, Clpn2 of 696 amino acids, and Clpn3 of 741 amino acids. Phylogenetic analysis of deduced amino acid sequences indicate that catfish Clpn1 and Clpn2 share a sequence similarity of 61%; catfish Clpn1 and Clpn3 of 48%, and Clpn2 and Clpn3 of only 45%. The domain structure architectures of all three calpain genes in channel catfish are similar to those of other vertebrates, further supported by strong bootstrap values during phylogenetic analyses. Starvation of channel catfish (average weight, 15-20 g for 35 days influenced the expression of clpn1 (2.3-fold decrease, P<0.05, clpn2 (1.3-fold increase, P<0.05, and clpn3 (13.0-fold decrease, P<0.05, whereas the subsequent refeeding did not change the expression of these genes as measured by quantitative real-time PCR analysis. Calpain catalytic activity in channel catfish skeletal muscle showed significant differences only during the starvation period, with a 1.2- and 1.4- fold increase (P<0.01 after 17 and 35 days of starvation, respectively. CONCLUSION/SIGNIFICANCE: We have assessed that fasting and refeeding may provide a suitable experimental model to provide us insight into the role of calpains during fish muscle atrophy and how they respond to changes in nutrient supply.

  3. Recent advances in wound dressings for diabetic foot%糖尿病足创面敷料研究进展

    Institute of Scientific and Technical Information of China (English)

    朱平; 严励


    糖尿病足创而治疗是一个需要多学科参与的复杂临床课题,敷料在其中的作用不容忽视.理想的敷料应既能够保护伤口、减轻局部症状,又能够维持伤口湿润、促进创面愈合.任何一类敷料都不可能具有伞部功能,但是每一种敷料都具有各自的特点.水凝胶敷料能促进组织自溶,在治疗坏死组织较多的创面时可与外科清创结合使用.藻酸盐和泡沫敷料具有良好的吸收性,适合于渗液量大的伤口.含生长因子敷料和牛物工:程皮肤组织替代物在系统治疗3周以上仍难愈合的创面中疗效显著.感染性伤口应避免使用密闭性敷料,含抗生索的敷料可能有效.对于渗液较多的创面更换次数需要增加,避免周围组织浸渍.总之,敷料的选择应从创面的临床特点、患者自身的需求和成本效益等多方面考虑.%The managment of diabetic foot is a complicated clinical issue which involves many as-pects ,wound dressings play an important adjunctive role in the treatment of diabetic foot ulceration. Ideally, dressings should protect wound, alleviate symptoms, and maintain a moist wound environment, etc. No single dressing fulfills all the function ,however,each category of dressings has particular characteristics that aid se-lection. Hydrogels facilitate autolysis and may be beneficial in managing ulcers with necrotic tissue. Foam and alginate dressings arc highly absorbent and effective for heavily exuding wounds. The dressing containing growth factor or bioengineered tissue has been effectively and safely used to increase the incidence of com-plete wound closure. Occlusive dressings should be avoided for infected wounds, dressing impregnated with antibiotics or silver may be eligible for these wounds. In a word, decisions regarding which dressing to apply should be based on the characteristics of the ulcer, patient's preferences and cost effectiveness.

  4. Alpha-2 Heremans Schmid Glycoprotein (AHSG) Modulates Signaling Pathways in Head and Neck Squamous Cell Carcinoma Cell Line SQ20B

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    Thompson, Pamela D.; Sakwe, Amos [Department of Biochemistry and Cancer Biology, Meharry Medical College, Nashville, TN 37208 (United States); Koumangoye, Rainelli [Division of Surgical Oncology and Endocrine Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Yarbrough, Wendell G. [Division of Otolaryngology, Departments of Surgery and Pathology and Yale Cancer Center, Yale University, New Haven, CT 06520 (United States); Ochieng, Josiah [Department of Biochemistry and Cancer Biology, Meharry Medical College, Nashville, TN 37208 (United States); Marshall, Dana R., E-mail: [Department of Pathology, Anatomy and Cell Biology, Meharry Medical College, Nashville, TN 37208 (United States)


    This study was performed to identify the potential role of Alpha-2 Heremans Schmid Glycoprotein (AHSG) in Head and Neck Squamous Cell Carcinoma (HNSCC) tumorigenesis using an HNSCC cell line model. HNSCC cell lines are unique among cancer cell lines, in that they produce endogenous AHSG and do not rely, solely, on AHSG derived from serum. To produce our model, we performed a stable transfection to down-regulate AHSG in the HNSCC cell line SQ20B, resulting in three SQ20B sublines, AH50 with 50% AHSG production, AH20 with 20% AHSG production and EV which is the empty vector control expressing wild-type levels of AHSG. Utilizing these sublines, we examined the effect of AHSG depletion on cellular adhesion, proliferation, migration and invasion in a serum-free environment. We demonstrated that sublines EV and AH50 adhered to plastic and laminin significantly faster than the AH20 cell line, supporting the previously reported role of exogenous AHSG in cell adhesion. As for proliferative potential, EV had the greatest amount of proliferation with AH50 proliferation significantly diminished. AH20 cells did not proliferate at all. Depletion of AHSG also diminished cellular migration and invasion. TGF-β was examined to determine whether levels of the TGF-β binding AHSG influenced the effect of TGF-β on cell signaling and proliferation. Whereas higher levels of AHSG blunted TGF-β influenced SMAD and ERK signaling, it did not clearly affect proliferation, suggesting that AHSG influences on adhesion, proliferation, invasion and migration are primarily due to its role in adhesion and cell spreading. The previously reported role of AHSG in potentiating metastasis via protecting MMP-9 from autolysis was also supported in this cell line based model system of endogenous AHSG production in HNSCC. Together, these data show that endogenously produced AHSG in an HNSCC cell line, promotes in vitro cellular properties identified as having a role in tumorigenesis. Highlights: • Head

  5. Genetic pathway in acquisition and loss of vancomycin resistance in a methicillin resistant Staphylococcus aureus (MRSA strain of clonal type USA300.

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    Susana Gardete


    Full Text Available An isolate of the methicillin-resistant Staphylococcus aureus (MRSA clone USA300 with reduced susceptibility to vancomycin (SG-R (i.e, vancomycin-intermediate S. aureus, VISA and its susceptible "parental" strain (SG-S were recovered from a patient at the end and at the beginning of an unsuccessful vancomycin therapy. The VISA phenotype was unstable in vitro generating a susceptible revertant strain (SG-rev. The availability of these 3 isogenic strains allowed us to explore genetic correlates of antibiotic resistance as it emerged in vivo. Compared to the susceptible isolate, both the VISA and revertant strains carried the same point mutations in yycH, vraG, yvqF and lspA genes and a substantial deletion within an intergenic region. The revertant strain carried a single additional frameshift mutation in vraS which is part of two component regulatory system VraSR. VISA isolate SG-R showed complex alterations in phenotype: decreased susceptibility to other antibiotics, slow autolysis, abnormal cell division and increased thickness of cell wall. There was also altered expression of 239 genes including down-regulation of major virulence determinants. All phenotypic properties and gene expression profile returned to parental levels in the revertant strain. Introduction of wild type yvqF on a multicopy plasmid into the VISA strain caused loss of resistance along with loss of all the associated phenotypic changes. Introduction of the wild type vraSR into the revertant strain caused recovery of VISA type resistance. The yvqF/vraSR operon seems to function as an on/off switch: mutation in yvqF in strain SG-R turns on the vraSR system, which leads to increase in vancomycin resistance and down-regulation of virulence determinants. Mutation in vraS in the revertant strain turns off this regulatory system accompanied by loss of resistance and normal expression of virulence genes. Down-regulation of virulence genes may provide VISA strains with a "stealth

  6. 食用油提取番茄中番茄红素的研究%Edible Oil Extraction of Lycopene from Tomato Research

    Institute of Scientific and Technical Information of China (English)

    李莉民; 李金星


    番茄红素是一种脂溶性的天然色素,具有较强的抗氧化和防癌、抗癌作用。文章采用自溶法利用植物油脂将番茄中的番茄红索提取出来,提取效率是传统提取方法4倍。并通过实验研究了时间、料液比、浸提次数、搅拌等因素对番茄红素提取效果的影响,确定了最佳的提取工艺条件。用豆油作溶剂,在pH为8的条件下,反应温度35℃,料液比1:1,在0i断搅拌下,浚捉3h,分次进行浸提、过滤、合并浸提液,且进行2级浸捉效果最好。这种方法解决了传统有机溶剂提取番茄红素产率低,并且存在溶剂存昭的问题.简椎方便,适用于工业化生产。%Lycopene is a kind of fat-soluble natural pigment, has strong antioxidant and anti-cancer, anti-cancer effect. The paper adopts the autolysis method using plant oil to tomato lycopene extraction, the extraction efficiency was 4 times of the traditional extraction method. And through experimental study time, ratio of material to liquid, extraction times, mixing and other factors on lycopene extraction effect, the optimum extraction conditions. Using soybean oil as solvent, at pH 8 under the condition of 35 DEG C, reaction temperature, liquid ratio 1:1, constantly stirring, extracting for 3 hours, conducted leaching, filtering, and extract, and 2 stage leaching effect best. This method has solved the traditional organic solvent extraction of lycopene yield is low, and the presence of solvent retention problems, is simple and convenient, suitable for industrialized production.

  7. Application of oxygen vectors in xanthan gum production%氧载体在黄原胶发酵中的应用

    Institute of Scientific and Technical Information of China (English)

    林琳; 邓春; 龙柯; 程蓉; 张永奎


    In xanthan gum fermentation process,the level of dissolved oxygen drops with the viscosity increasing,and the synthesis of xanthan gum is inhibited.Adding oxygen vectors to the system of xanthan gum fermentation is a method to improve oxygen supply.The optimal soybean oil concentration and adding time was 6%(v/v)and 0h,and the optimal n-hexane concentration and adding time was 1%(v/v)and 12h.The addition of soybean oil and n-hexane significantly improved xanthan gum yield to 2.977% and 2.927%,respectively(increased by 49% and 47%,respectively).Although partial autolysis occurred in the soybean oil group,synthesis could still continue in most normal cells by 72h,which made it possible to extend fermentation period.Prolonging fermentation with n-hexane had little effect on the yield,however the capsule around the cell was obviously much thicker than that of control group after 72h fermentation.%黄原胶发酵过程中因发酵液粘度逐渐增加,系统供氧不足,导致黄原胶产率受到影响。为改善发酵系统供氧情况,提高黄原胶产率,本实验研究添加氧载体(大豆油、正己烷、正十二烷)对黄原胶发酵的影响。实验结果表明:发酵初期添加6%(v/v)大豆油、发酵12h添加1%(v/v)正己烷,发酵72h,可分别显著提高黄原胶产率至2.347%和2.927%,相对于空白组分别提高18%和47%;另外,实验发现:大豆油组细胞干重有明显提高,发酵72h后,仍能观察到部分形态正常的细胞,延长发酵周期至84h,黄原胶产率显著提高至2.977

  8. Estudo da produção de beta-galactosidase por fermentação de soro de queijo com Kluyveromyces marxianus Synthesis of beta-galactosidase by fermentation of cheese whey by Kluyveromyces marxianus

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    Patrícia A. Santiago


    Full Text Available A hidrólise enzimática da lactose por beta-galactosidase desempenha importante papel no processamento de produtos lácteos, como na obtenção de leite com baixo teor de lactose para consumo por indivíduos intolerantes à mesma e na prevenção da cristalização em produtos de laticínio. Neste trabalho, a enzima beta-galactosidase foi produzida pelo cultivo do microrganismo Kluyveromyces marxianus, em meio de cultura à base de soro de queijo em diferentes concentrações iniciais de lactose e extrato de levedura, de acordo com um planejamento fatorial. As fermentações foram conduzidas em incubador rotativo a 150rpm, a 30°C e pH inicial 5,5. A concentração celular inicial foi de 10(7 células/mL. Para a extração da enzima beta-galactosidase, foi realizada autólise das células utilizando como solvente o clorofórmio em tampão fosfato. No meio de cultura enriquecido com (NH42SO4, KH2PO4 e MgSO4, nas concentrações iniciais de lactose e de extrato de levedura iguais a 50g/L e 12g/L, respectivamente, obteve-se uma atividade de 28,0UGl/mL e uma concentração celular máxima de 5,3g/L.The enzymatic hydrolysis of lactose by beta-galactosidase plays an important role in the processing of milky products such as the production of lactose-hydrolyzed milk for consumption by intolerant person to lactose and the prevention of the crystallization in dairy products. In this work, the influences of nutrient concentrations in the culture medium based on cheese whey were studied with the objective of producing beta-galactosidase from Kluyveromyces marxianus. The fermentations were carried out in a shaker at 30°C and initial pH 5.5 under agitation, starting with an initial cellular concentration of 10(7 cells/mL, varying the initial concentrations of lactose and yeast extract. For extraction of the enzyme of the cells it was used autolysis with chloroform in potassium phosphate buffer. In the medium with a initial lactose concentration of 50g

  9. Cytotoxicity and metabolic stress induced by deoxynivalenol in the porcine intestinal IPEC-J2 cell line. (United States)

    Awad, W A; Aschenbach, J R; Zentek, J


    The digestive tract is a target for the Fusarium toxin deoxynivalenol (DON), a major cereal grain contaminant of animal and public health concern. Toxic effects of DON range from diarrhoea, vomiting and gastrointestinal inflammation to necrosis of several tissues. Following ingestion of contaminated food or feed, intestinal epithelial cells are exposed to a high concentration of ingested DON, potentially affecting intestinal functions. Pigs are considered to be the species most sensitive to DON toxicity. However, only few studies directly evaluated DON effects on porcine intestinal epithelial cells. Therefore, we used the porcine intestinal cell line (IPEC-J2) to assess short-term effects of DON on functional characteristics of the intestinal epithelial cells. The cytotoxic effect of DON on IPEC-J2 cells was evaluated by measuring the count of living cells and the activity of lactate dehydrogenase (LDH) released in the culture media at a DON concentration range from 0, 0.5, 2.5 and 10 μm. We demonstrated that DON at concentrations of 2.5 and 10 μm decreased significantly (p < 0.001) the cell count in a dose-dependent manner. At a concentration of 10 μm, DON caused cell damage, including rounding of cells, autolysis and cell loss from the monolayer. The mycotoxin, DON, increased LDH release into the culture medium compared with the control value. The alterations of LDH showed a good agreement with the decrease in cell count. Deoxynivalenol decreased the l-lactate concentration in the fluid supernatant of IPEC-J2 cells at 2.5 μm (p < 0.05) with a maximal effect at 10 μm of DON. To determine whether the altered lactate production may be linked to alterations of energy balance, we measured cellular ATP levels in IPEC-J2 cells. A significant decrease in ATP levels was seen at 48 h in a dose-dependent manner. It could be demonstrated that DON has a distinct cytotoxic effect on IPEC-J2 cells.

  10. PBP1a-deficiency causes major defects in cell division, growth and biofilm formation by Streptococcus mutans.

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    Zezhang T Wen

    Full Text Available Streptococcus mutans, a key etiological agent of human dental caries, lives almost exclusively on the tooth surface in plaque biofilms and is known for its ability to survive and respond to various environmental insults, including low pH, and antimicrobial agents from other microbes and oral care products. In this study, a penicillin-binding protein (PBP1a-deficient mutant, strain JB467, was generated by allelic replacement mutagenesis and analyzed for the effects of such a deficiency on S. mutans' stress tolerance response and biofilm formation. Our results so far have shown that PBP1a-deficiency in S. mutans affects growth of the deficient mutant, especially at acidic and alkaline pHs. As compared to the wild-type, UA159, the PBP1a-deficient mutant, JB467, had a reduced growth rate at pH 6.2 and did not grow at all at pH 8.2. Unlike the wild-type, the inclusion of paraquat in growth medium, especially at 2 mM or above, significantly reduced the growth rate of the mutant. Acid killing assays showed that the mutant was 15-fold more sensitive to pH 2.8 than the wild-type after 30 minutes. In a hydrogen peroxide killing assay, the mutant was 16-fold more susceptible to hydrogen peroxide (0.2%, w/v after 90 minutes than the wild-type. Relative to the wild-type, the mutant also had an aberrant autolysis rate, indicative of compromises in cell envelope integrity. As analyzed using on 96-well plate model and spectrophotometry, biofilm formation by the mutant was decreased significantly, as compared to the wild-type. Consistently, Field Emission-SEM analysis also showed that the PBP1a-deficient mutant had limited capacity to form biofilms. TEM analysis showed that PBP1a mutant existed primarily in long rod-like cells and cells with multiple septa, as compared to the coccal wild-type. The results presented here highlight the importance of pbp1a in cell morphology, stress tolerance, and biofilm formation in S. mutans.

  11. Advances in Diagnosis of Rabies

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    Shankar B.P.


    Full Text Available Rabies is a major zoonosis for which diagnostic techniques have been standardised internationally. Laboratory techniques are preferably conducted on central nervous system (CNS tissue removed from the cranium. Agent identification is preferably done using the fluorescent antibody test. A drop of purified immunoglobulin previously conjugated with fluorescein isothiocyanate is added to an acetone-fixed brain tissue smear, preferably made from several parts of the brain, including the hippocampus, cerebellum and medulla oblongata. For a large number of samples, as in an epidemiological survey, the immunoenzyme technique can provide rapid results (the rapid rabies enzyme immunodiagnosis. FAT provides a reliable diagnosis in 98-100% of cases for all genotypes if a potent conjugate is used, while RREID detects only genotype 1 virus. Infected neuronal cells have been demonstrated by histological tests and these procedures will reveal aggregates of viral material (the Negri bodies in the cytoplasm of neurones. However, the sensitivity of histological techniques is much less than that of immunological methods, especially if there has been some autolysis of the specimen. Consequently, histological techniques can no longer be recommended. As a single negative test on fresh material does not rule out the possibility of infection, inoculation tests, or other tests, should be carried out simultaneously. Newborn or 3-4-week-old mice are inoculated intracerebrally with a pool of several CNS tissues, including the brain stem, and then kept under observation for 28 days. For any mouse that dies between 5 and 28 days, the cause of death should be confirmed by FAT. Alternatively, a monolayer culture of susceptible cells is inoculated with the same material as used for mice. FAT carried out after appropriate incubation will demonstrate the presence or absence of viral antigen. Wherever possible, virus isolation in cell culture should replace mouse inoculation tests

  12. Transcriptional Analysis and Subcellular Protein Localization Reveal Specific Features of the Essential WalKR System in Staphylococcus aureus. (United States)

    Poupel, Olivier; Moyat, Mati; Groizeleau, Julie; Antunes, Luísa C S; Gribaldo, Simonetta; Msadek, Tarek; Dubrac, Sarah


    The WalKR two-component system, controlling cell wall metabolism, is highly conserved among Bacilli and essential for cell viability. In Staphylococcus aureus, walR and walK are followed by three genes of unknown function: walH, walI and walJ. Sequence analysis and transcript mapping revealed a unique genetic structure for this locus in S. aureus: the last gene of the locus, walJ, is transcribed independently, whereas transcription of the tetra-cistronic walRKHI operon occurred from two independent promoters located upstream from walR. Protein topology analysis and protein-protein interactions in E. coli as well as subcellular localization in S. aureus allowed us to show that WalH and WalI are membrane-bound proteins, which associate with WalK to form a complex at the cell division septum. While these interactions suggest that WalH and WalI play a role in activity of the WalKR regulatory pathway, deletion of walH and/or walI did not have a major effect on genes whose expression is strongly dependent on WalKR or on associated phenotypes. No effect of WalH or WalI was seen on tightly controlled WalKR regulon genes such as sle1 or saouhsc_00773, which encodes a CHAP-domain amidase. Of the genes encoding the two major S. aureus autolysins, AtlA and Sle1, only transcription of atlA was increased in the ΔwalH or ΔwalI mutants. Likewise, bacterial autolysis was not increased in the absence of WalH and/or WalI and biofilm formation was lowered rather than increased. Our results suggest that contrary to their major role as WalK inhibitors in B. subtilis, the WalH and WalI proteins have evolved a different function in S. aureus, where they are more accessory. A phylogenomic analysis shows a striking conservation of the 5 gene wal cluster along the evolutionary history of Bacilli, supporting the key importance of this signal transduction system, and indicating that the walH and walI genes were lost in the ancestor of Streptococcaceae, leading to their atypical 3 wal gene

  13. AcmB Is an S-Layer-Associated β-N-Acetylglucosaminidase and Functional Autolysin in Lactobacillus acidophilus NCFM (United States)

    Johnson, Brant R.


    ABSTRACT Autolysins, also known as peptidoglycan hydrolases, are enzymes that hydrolyze specific bonds within bacterial cell wall peptidoglycan during cell division and daughter cell separation. Within the genome of Lactobacillus acidophilus NCFM, there are 11 genes encoding proteins with peptidoglycan hydrolase catalytic domains, 9 of which are predicted to be functional. Notably, 5 of the 9 putative autolysins in L. acidophilus NCFM are S-layer-associated proteins (SLAPs) noncovalently colocalized along with the surface (S)-layer at the cell surface. One of these SLAPs, AcmB, a β-N-acetylglucosaminidase encoded by the gene lba0176 (acmB), was selected for functional analysis. In silico analysis revealed that acmB orthologs are found exclusively in S-layer- forming species of Lactobacillus. Chromosomal deletion of acmB resulted in aberrant cell division, autolysis, and autoaggregation. Complementation of acmB in the ΔacmB mutant restored the wild-type phenotype, confirming the role of this SLAP in cell division. The absence of AcmB within the exoproteome had a pleiotropic effect on the extracellular proteins covalently and noncovalently bound to the peptidoglycan, which likely led to the observed decrease in the binding capacity of the ΔacmB strain for mucin and extracellular matrices fibronectin, laminin, and collagen in vitro. These data suggest a functional association between the S-layer and the multiple autolysins noncovalently colocalized at the cell surface of L. acidophilus NCFM and other S-layer-producing Lactobacillus species. IMPORTANCE Lactobacillus acidophilus is one of the most widely used probiotic microbes incorporated in many dairy foods and dietary supplements. This organism produces a surface (S)-layer, which is a self-assembling crystalline array found as the outermost layer of the cell wall. The S-layer, along with colocalized associated proteins, is an important mediator of probiotic activity through intestinal adhesion and modulation of

  14. Dynamics of Streptococcus mutans transcriptome in response to starch and sucrose during biofilm development.

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    Marlise I Klein

    Full Text Available The combination of sucrose and starch in the presence of surface-adsorbed salivary α-amylase and bacterial glucosyltransferases increase the formation of a structurally and metabolically distinctive biofilm by Streptococcus mutans. This host-pathogen-diet interaction may modulate the formation of pathogenic biofilms related to dental caries disease. We conducted a comprehensive study to further investigate the influence of the dietary carbohydrates on S. mutans-transcriptome at distinct stages of biofilm development using whole genomic profiling with a new computational tool (MDV for data mining. S. mutans UA159 biofilms were formed on amylase-active saliva coated hydroxyapatite discs in the presence of various concentrations of sucrose alone (ranging from 0.25 to 5% w/v or in combination with starch (0.5 to 1% w/v. Overall, the presence of sucrose and starch (suc+st influenced the dynamics of S. mutans transcriptome (vs. sucrose alone, which may be associated with gradual digestion of starch by surface-adsorbed amylase. At 21 h of biofilm formation, most of the differentially expressed genes were related to sugar metabolism, such as upregulation of genes involved in maltose/maltotriose uptake and glycogen synthesis. In addition, the groEL/groES chaperones were induced in the suc+st-biofilm, indicating that presence of starch hydrolysates may cause environmental stress. In contrast, at 30 h of biofilm development, multiple genes associated with sugar uptake/transport (e.g. maltose, two-component systems, fermentation/glycolysis and iron transport were differentially expressed in suc+st-biofilms (vs. sucrose-biofilms. Interestingly, lytT (bacteria autolysis was upregulated, which was correlated with presence of extracellular DNA in the matrix of suc+st-biofilms. Specific genes related to carbohydrate uptake and glycogen metabolism were detected in suc+st-biofilms in more than one time point, indicating an association between presence of starch

  15. Sperm ultrastructure in the nudibranch genus Halgerda with reference to other Discodorididae and to Chromodorididae (Mollusca: Opisthobranchia). (United States)

    Fahey, Shireen J; Healy, John M


    nucleus of Asteronotus. Sperm features alone do not provide a means of defining the genus Halgerda or the family Discodorididae nor do they support the monophyletic status of the caryophyllidia-bearing dorids. Important sperm characters such as the acrosome, nucleus, and midpiece can often still be determined from specimens that have been initially fixed in formalin, then stored in ethanol for extended periods of time (i.e., museum material). Of all sperm features, the mitochondrial derivative of the midpiece is the most resistant to long-term fixation : the survival of acrosomal, nuclear, and axonemal components is variable, presumably a factor of prefixation autolysis, varied primary fixation times and temperatures, formalin quality, and duration of alcohol storage.

  16. Experimental intoxication of pregnant goats with Tetrapterys multiglandulosa A. Juss. (Malpighiaceae Intoxicação experimental por Tetrapterys multiglandulosa A. Juss. (Malpighiaceae em cabras gestantes

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    M.M. Melo


    Full Text Available Green leaves of Tetrapterys multiglandulosa A. Juss were fed to pregnant goats from day 35 of pregnancy. Five goats received 10g/kgBW (group I, five received 20g/kgBW (group II and five were used as control (group III, and received only hay, fresh grass and commercial ration. All animals were clinically examined daily and submitted to ultrasonography every three days. Fetal death and vulvar catarrhal discharge with subsequent abortion were observed at the end of the second month of pregnancy in group II and at the third month of pregnancy in group I. Animals from groups I and II were slaughtered after abortion and necropsied. Goats from the control group were necropsied at the same time. The main lesions in the aborted goats were focal placentitis with early involution (apoptosis and placentary coagulation necrosis, acute focal endometritis and vulvo-vaginal petechiae. All aborted fetuses were underdeveloped when compared to control fetuses, probably due to fetal malnutrition, since no congenital malformations could be noted. The majority of aborted fetuses showed some degree of autolysis, as fetal death occurred five and three days before abortion, in groups I and II, respectively. The most remarkable fetal lesions were focal or diffuse hemorrhages in the skin, meninges and visceral serosae.Com o objetivo de investigar a toxicidade da Tetrapterys multiglandulosa A. Juss. foram utilizadas 15 cabras gestantes, divididas aleatoriamente em três grupos (GI, GII e GIII com cinco animais cada. Após estabelecer o diagnóstico de gestação no tempo médio de 35 dias, eram oferecidas diariamente doses de 10 e 20g/kg PV das folhas (jovens e maduras de Tetrapterys multiglandulosa para os grupos I e II, respectivamente, junto ao capim picado, feno e concentrado. Para o grupo III, usado como controle, foram fornecidos somente capim picado, feno e concentrado. Todos os animais foram submetidos a exames clínicos diários e a ultra-sonografia a cada tr

  17. 甲醛遗体防腐剂改良研究%The study on the improved formaldehyde preservative

    Institute of Scientific and Technical Information of China (English)

    陈霜玲; 李玉光; 付慧群; 王永阔; 姜思朋; 周雪媚


    Objective To reduce the harm of formaldehyde-containing preservatives to the health of staff, a new improved preservative, EM1, was developed, which has lower concentration of formaldehyde. Methods By means of formaldehyde release test, bacterial quantitative germicidal test and fungus germicidal test, laboratory rat injection antisepsis test, and a field test on cadavers in funeral home, the disinfection and antiseptic effect of EM1 was investigated. Results EM1 showed lower formaldehyde release as 35.7%of formal antiseptic;it could effectively kill Staphylococcus aureus and Escherichia coli and other microorganisms;it could help the dead rats injected with EM1 staying for 30 days, and the morphology of which was closer tothe alive condition in comparison with that of the Dodge-treated rats; the effective embalming rate of cadaver was above 95%at room temperature. Conclusions This improved preservative EM1 has the characteristics of low toxicity, good antiseptic and fixed effect. Furthermore, the embalmed specimens shrink little, and almost show the living condition. By effectively controlling the spread of pathogenic microorganisms, the development of corruption and autolysis of remains, EM1 can do contribution to anatomy research and embalming for funeral.%目的:为减少甲醛防腐剂对人体造成的危害,研制新型改良甲醛遗体防腐剂EM1。方法通过甲醛释放量检测、细菌定量杀灭试验与真菌杀灭试验、实验室大鼠注射防腐实验、殡仪馆遗体防腐现场实验,考察遗体防腐剂的消毒、防腐效果。结果 EM1防腐剂甲醛释放量为常规防腐剂的35.7%;能有效杀灭金黄色葡萄球菌和大肠杆菌等微生物;使用EM1进行注射防腐的大鼠可保存30d,且比Dodge处理的大鼠更接近活体状态;室温下对遗体的有效防腐率达95%以上。结论 EM1防腐剂具有毒性低、防腐固定效果好、所保存的标本收缩率小、色泽接近活体状态等特点

  18. 沙蜇毒素的提取及溶血活性分析%Extraction of Stomopholus meleagris L. Agassig venom and the analysis of its hemolytic activities

    Institute of Scientific and Technical Information of China (English)

    郭容; 周玉婷; 张凤杰; 朱启忠


    Objective Formation of blood clots is the pathological basis which induces a number of seri-ous cardio-cerebrovascular diseases. Thrombolytic drugs can eliminate clots and reduce the complications. In this paper, a new material with hemolytic activities which might provide a new therapeutic approach for the thrombus treatment was extracted from biological tissue. Methods Nematocyst venom was extracted from the tentacle of Stomopholus melesgTis L.Agassig using the methods of autolysis in sterile seawater, centrifugation and mortar grinding at low temperature. The concentration-associated hemolytic curve was obtained by spectropho-tometric method, with HU50 measured. The effects of temperature, pH value were studied. The extracted crude venom was purified and separated through column chromatography with the hemolytic activity of each peak frac-tion under different pH value being measured. Results As proved by this research, venom of Stomopholus melesgris L.Agassig held relatively high hemolytic activity which was temperature sensitive and highly influenced by pH value. After separation, two different fractions were collected with one of them showed detectable hemolytic activity while the other one did not. Conclusion Results of the study indicates that nematocyst venom may have promising applications in improving the antithrombotic property of biomaterials.%目的:血栓形成是引发严重心脑血管疾病的病理基础,溶栓药物可消除血栓并减轻并发症.本研究从生物组织中提取分离的具有溶血活性的物质,有望成为有效的溶栓制剂,为血栓的治疗提供新方法.方法通过海水自溶、离心、低温研磨等方法从沙蜇触手的刺丝囊中提取沙蜇毒素,对沙蜇毒素提取液进行分离纯化,测定了毒素的溶血活性并绘制溶血曲线;深入研究了温度、介质的pH值对其溶血活性的影响.结果研究证实:沙蛰毒素具有很强的溶血活性,且对温度敏感,其活性受pH变化影

  19. Changes of Biomechanical Properties of Soft Tissues in Underwater Corpse for Postmortem Interval Estimation%水中尸体软组织生物力学性状时序性变化用于死亡时间推断

    Institute of Scientific and Technical Information of China (English)

    唐谷; 周晖; 汪家文; 钱红; 赖跃; 于晓军


    technology for estimating PMI. The specific heat capacity of the heat-eliminating medium around the corpses probably is one of the physical factors to influence algor mortis, autolysis, putrefaction and biomechanics properties.

  20. Análise imuno-histoquímica de cães naturalmente infectados pelo parvovírus canino Immunohistochemical analysis of dogs infected naturally by canine parvovirus

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    Eduardo C. Oliveira


    immunohistochemistry test was performed in tissues of small intestine, mesenteric lymph nodes, thymus, spleen, tonsils, tongue, and bone marrow in all the 96 selected cases. Parvovirus antigen was detected in 91.6% (88/96 of the dogs necropsied. The best result of the IHC test was seen in samples of small intestine which was positive in 77% (74/96 of the cases. The statistical analysis (Fisher test showed a weak association between intestinal autolysis and positive result of the IHC test. The chance of the autolysed intestine showing a positive result in the immunohistochemistry test was 0.33 less (OR=0.33, 95% CI:0.10-1.17 when compared with small intestine not autolysed.

  1. Enfermidades de cutias (Dasyprocta aguti criadas em cativeiro diagnosticadas pelo exame anatomopatológico Diseases of agouti (Dasyprocta aguti raised in captivity diagnosed by pathological examination

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    Jael S. Batista


    (3.20%, fecaloma (3.20% and esophagus obstruction (3.20%. A total of 16 (50.08% animals remained undiagnosed in which nine (28.48% showed advanced autolysis and seven (21.60% agouti had none macroscopic or microscopic lesions compatible with any disease. The present article presents reports of some diseases not yet diagnosed in agoutis and these results may produce literature review about the pathologic aspects of these diseases in this species.

  2. Influence of metal ions on pellet morphology and polygalacturonase synthesis by Aspergillus niger 3T5B8 Influência dos íons metálicos na morfologia do agregado e na síntese de poligalacturonase por Aspergillus niger 3T5B8

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    Sônia Couri


    Full Text Available The effects of cations addition on pellet morphology and polygalacturonase (PG synthesis by Aspergillus niger 3T5B8 were studied and compared with a control system. Fe(II, Cu(II, Zn(II and Mn(II were added to the fermentation medium separately, and also as combined groups of cations. The addition of Fe2+ and/or Zn2+ ions was significantly positive to the enzyme production. A positive effect in the biomass content, however, was only obtained when the same metal ions were added separately. On the other hand, Cu2+ and Mn2+ ions had almost no effect on these parameters. The morphology of the pellets was studied by image processing techniques. Small pellets with small cores were usually obtained when Fe2+ and Zn2+ ions were individually or collectively added to the medium. The pellets produced in media containing Fe2+ or Zn2+ ions were compact, while the ones produced in a medium containing both cations were considered diffuse. Autolysis of the core was observed for large control pellets, due to the deficient nutrient transfer to the interior of the pellet. The pellets obtained in a medium containing both Fe2+ and Zn2+ ions were high enzyme producers, probably due to a loose morphology, induced by the presence of combined groups of metal ions in the medium, favoring the nutrient transfer.O efeito da adição de cátions na morfologia do agregado e na síntese de poligalacturonase (PG por Aspergillus niger 3T5B8 foi estudado e comparado com um sistema controle. Fe(II, Cu(II, Zn(II and Mn(II foram adicionados ao meio de fermentação, tanto separadamente, como em grupos de cátions. A adição dos íons Fe2+ e/ou Zn2+ foi positivamente significante para a produção da enzima. Um efeito similar no teor de biomassa, contudo, só pode ser observado quando os mesmos íons foram adicionados isoladamente. Por outro lado, os íons Cu2+ e Mn2+ não afetaram significativamente estes parâmetros. Utilizando-se técnicas de processamento de imagens para o estudo

  3. Modelo experimental de tumor na cavidade oral de ratos com carcinossarcoma de Walker 256 Experimental model of Walker 256 carcinosarcoma developed in the oral cavity of rats

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    Ana Paula Negreiros Nunes Alves


    Full Text Available OBJETIVO: Estabelecer um modelo experimental de desenvolvimento tumoral na cavidade oral de ratos, permitindo, assim, o estudo da osteólise induzida pelo tumor nos ossos do complexo maxilomandibular como também nas estruturas dentais, através da caracterização histomorfológica da reabsorção óssea e dentária. MÉTODOS: Uma suspensão de células tumorais (0,1mL do Carcinossarcoma de Walker 256, na concentração de 10(6 células/mL foi implantado na cavidade alveolar de ratos previamente aberta por exodontia. Os animais foram observados durante 12 (doze dias consecutivos para determinação da curva de peso corpóreo, sendo posteriormente sacrificados e as mandíbulas removidas para exames radiográfico e histológico. RESULTADOS: No exame radiográfico foi verificada área lítica, sem evidência de reparo, na região dos alvéolos. No exame microscópico foi identificada infiltração óssea, periférica e central, de pequenas células hipercromáticas e pleomórficas, com leve infiltrado inflamatório mononuclear associado e áreas de necrose. O índice de pega foi de 100%. CONCLUSÃO: O modelo animal de invasão óssea, do tumor de Walker na cavidade oral, possibilita a avaliação in vivo de drogas antitumorais e esquemas terapêuticos no tratamento do câncer bucal.PURPOSE: To estabilish an experimental model of tumor development in the oral cavity of rats, that would enable to study the tumor-induced autolysis in the maxillomandibular bone complex as well as of the dental structures, through histomorphological characterization of bone and dental resorption. METHODS: Walker 256 carcinossarcoma cell suspension (0,1 mL containing 10(6 cell/mL was implanted in the alveoli of first and second molars. The animals were observed during twelve consecutive days and the body weigth were determined. Later, the animals were sacrificed and their mandibles removed to radiographic and hystologic analysis. RESULTS: The radiographic image

  4. NO及NOS在老年Ⅰ期压疮大鼠骨骼肌组织细胞凋亡中的作用%Effect of NO/NOS on apoptosis of skeletal muscle tissue cells in elderly rats with pressure sore in phase Ⅰ

    Institute of Scientific and Technical Information of China (English)

    王艳; 周守凤; 高金华; 陈慧敏; 郑国荣; 刁波


    Objective To observe the effect of NO and NOS during the process of cell apoptosis in elderly rats with pressure sore (phase Ⅰ),and to further investigate the mechanism of pressure sore.Methods A total of 12 rats were divided into model group (6 rats) and control group (6 rats).A pressure of 22.47kPa was pushed on model group while no pressure on control group,and two groups of rats were put to death with euthanasia after 2 h.Finally,the pathological changes of the skin and muscle were observed under light microscope,and the levels and activity of NO,NOS and SOD,and the apoptosis index (AI) were detected.Results In model group,the stratified squamous epithelium cells showed thin,and the structure became unclear,vacuolar alteration in muscle fibers,mild edema,waxy degeneration,muscle tissue atrophy and autolysis,while the color and structure of skin and muscle were normal in control group.There were significant differences between the two groups in the levels and activity of NO,NOS and SOD.NO,NOS were positively correlated with the process of cell AI.Conclusions NO and NOS play a key mediated role in the process of skin and muscle cell apoptosis in elderly rat with pressure sore (phase I).The lipid peroxidation and apoptosis induced by NO may be one of the potential mechanisms in the formation of early pressure sore.%目的 观察一氧化氮(N0)及一氧化氮合酶(NOS)在老年Ⅰ期压疮大鼠骨骼肌组织细胞凋亡中的作用,进一步探索老年压疮发生机制. 方法 将老年SD大鼠12只随机分成对照组和模型组,各6只.对照组不受压、模型组承受22.47 kPa压强2h后2组大鼠一起实施安乐死.实验终点,光镜观察皮肤肌肉组织病理学变化;检测肌组织中NO、NOS和超氧化物歧化酶(SOD)的活性和含量;检测皮肤和肌肉组织中细胞凋亡指数(AI)的变化. 结果 对照组镜下观察皮肤完整,颜色、结构正常;模型组大鼠复层鳞状上皮较薄,结构层次欠清晰.肌纤维

  5. Composição centesimal e valor protéico de levedura residual da fermentação etanólica e de seus derivados Centesimal composition and protein nutritive value of yeast from ethanol fermentation and of yeast derivatives

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    Eunice Akemi Yamada


    Full Text Available Este trabalho teve por objetivo promover a autólise e o fracionamento da levedura (Saccharomyces sp. para produção de autolisado e extrato, bem como para produção de concentrado protéico fosforilado, a partir da levedura residual das destilarias de álcool etílico. Foram estudados a composição centesimal, o perfil de aminoácidos essenciais e o valor protéico dos três derivados comparativamente à levedura íntegra não processada. Proteína e carboidrato (fibra alimentar foram os principais componentes da levedura íntegra e do autolisado. No extrato e no concentrado protéico predominaram proteína e minerais (cinzas. O autolisado e a levedura íntegra apresentaram os melhores índices de aminoácidos essenciais, seguidos pelo concentrado protéico e pelo extrato. A digestibilidade da proteína variou de 68% para a levedura íntegra a 91% para o extrato. Os índices de quociente de utilização líquida da proteína variaram de 2,1 para a levedura íntegra a 4,3 para a caseína (referência. Não houve diferença estatística no quociente de utilização líquida da proteína entre o autolisado (4,1, o extrato (3,9 e o concentrado protéico (4,2. O concentrado protéico promoveu o maior crescimento no período (21 dias, seguido do extrato e o autolisado. As células íntegras apresentaram a menor capacidade para promover crescimento em rato.The objective of this work was to promote the autolysis and the fractionation of the yeast (Saccharomyces sp. for the production of autolysate and extract, as well as phosphorylated protein concentrate, from ethanol distillery yeast. Comparative studies of centesimal composition, essential amino acid profiles and protein nutritive value were performed for the unprocessed integral cells, and for autolysate, extract and phosphorylated protein concentrate. Protein and carbohydrate (dietary fiber were the main components for the integral cells and autolysate. For the extract and the protein

  6. Estudio epidemiológico del suicidio en Sevilla en 2004 Epidemiological study of suicide in Seville in 2004

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    L. Miguel


    previous autolysis intents. Hanging was the most frequent mechanism to commit suicide in men (50.9% while in women was jumping (41,5%. In 57,1% of cases there were antecedents of mental disorder mainly depression of depressive symptoms (65,5% followed by schizophrenia (15,5%. Toxicological analyses were performed in 78,2% of cases with positive results in 69,6%. Ethanol was the toxic most detected (24,3% followed by benzodiazepines (18,3% and antidepressants (11,3%. According to the results of the study, we observe a certain stabilization of the suicide rates in Seville from the middle of the XXth century up to now.


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    Nubia Estella Cruz Casallas


    Full Text Available Abstract. One of the most consumed fish in the world is Silurid, also called "leather fish" or catfish, whose main characteristic is the absence of intramuscular bones and scales, as well as its high productivity. In recent years, the nutritional characterization of the meat of some of these species has been carried out, finding that, although the proximal composition is within the broad ranges for fish, the fat content provides a lower proportion of polyunsaturated fatty acids (PUFA particularly as regards omega-3 (w-3, furthermore the w-6/w-3 ratio is within the proscriptions of the World Health Organization (WHO for many of these species of catfish. Likewise, the contents of eicosapentaenoic acid (EPA, docosahexaenoic acid (DHA and amino acids, minerals and vitamins reveal a high variability between individuals and species associated with the type of cultivation and dietary habits and also with the age and weight at slaughter. Furthermore quality parameters have been defined in relation to susceptibility to autolysis, oxidation and hydrolysis of fats and disturbances caused by microorganisms that cause decisive changes in the physicochemical, microbiological and sensory characteristics. This review compiles current information regarding the nutritional composition of catfish meat and the quality parameters.Resumen. Una de las carnes de pescado de mayor consumo en el mundo es la de Silúridos, también denominados peces de cuero o bagres, cuya principal característica es la ausencia de espinas intramusculares y de escamas, además de su alta productividad. En los últimos años se ha logrado realizar la caracterización nutricional de la carne de algunas de estas especies, hallándose que aunque la composición proximal se encuentra dentro de los rangos generales para peces, el contenido de grasa ofrece menor proporción de ácidos grasos poliinsaturados (AGP particularmente en lo referente a la serie omega 3 (w-3, aunque la relación w-6/w-3

  8. Fermentação de trealose e glicogênio endógenos em Saccharomyces cerevisiae Fermentation of endogenous trehalose and glycogen by Saccharomyces cerevisiae

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    , depending on the yeast strain. If yeast is maintained under prolonged stressing conditions, cell autolysis occurs and nitrogen is lost to the medium, increasing from 200 to 1500mg/L. Such endogenous fermentation allows a production of 40 to 68L of ethanol per ton of dry yeast, with yeast nitrogen increasing of 25 and 27% for PE-2 and VR-1, respectively.

  9. 溴隐亭治疗垂体腺瘤后超微结构改变和相关基因表达的研究%Medical management of pituitary adenomas: a study of ultrastructural changes and receptor expressions

    Institute of Scientific and Technical Information of China (English)

    吴哲褒; 诸葛启钏; 张亚卓; 孙异临; 陈勇; 王成德; 苏志鹏; 吕庆平; 吴近森; 郑伟明


    otroph adenomas,8 sparsely granulated lactotroph adenomas showed that cytoplasm was shrunken and the karyoplasmic ratio was enlarged.Ultrastructural analysis confirmed a series of changes including nuclear chromatin aggregation and margination,marked reduction of rough endoplasmic reticulum and Golgi complexes, vascular degenerations, necrosis and autolysis of tumor cells with dark cell or naked nuclear cell Compared to prolactinomas,NFPAs had little changes in cell size and cytoplasmic apparatus but also had chromatin aggregation, cytoplasmic vacuolization and unconspicuous necrosis of tumor cell.Conclusions This study shows the differential expression of ER mRNA and D2R mRNA isoforms between prolactinomas and NFPAs.Comparinb to prolactinomas,NFPAs with medical treatment also have some ultrastructural changes according to their exression levels of D2R isoforms.Ultrastructural changes in those adenomas are closely correlated with the expression levels of D2R isoforms.

  10. Intoxicação aguda e abortos em cobaias pelas favas de Enterolobium contortisiliquum (Leg. Mimosoideae Acute poisoning and abortions in guinea pigs by the pods of Enterolobium contortisiliquum (Leg. Mimosoideae

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    Josiane Bonel-Raposo


    properties of Enterolobium contortisiliquum pods in guinea pigs. Pods of E. contortisiliquum were administered orally to 4 groups of 3 guinea pigs each. Another group of 3 guinea-pigs was used as control. Group 1 and 2 were fed with one dose of 5 and 10g of pods for kg body weight, respectively. The guinea pigs of Group 3 and 4 received 10 and 15g/kg, respectively, divided into daily doses of 5g/kg. One guinea pig from Group 2 and one from Group 4 died 12 and 18 hours after the end of the administration. Gross lesions were hemorrhages of the stomach and of the large and small gut, enlarged liver, and dilated gall bladder. Histologically, the liver had severe vacuolation and necrosis of periportal hepatocytes. In another experiment a ration containing 4% of pods of E. contortisiliquum was fed to 2 groups of 4 guinea-pigs, 35 days after mating. Four of the 8 guinea pigs aborted 6-15 days after the beginning of ingestion. The other 4 guinea pigs were not pregnant. All guinea pigs were euthanized after abortion or at the end of the experiment. Histologically all animals had mild to severe periportal hemorrhagic necrosis. All fetuses had variable degree of autolysis. In 4 fetuses studied no significant histologic lesions were observed. The acute lesions observed in guinea-pigs are similar than those observed in the spontaneous poisoning by Enterolobium spp. in cattle. Similar lesions are observed in guinea-pigs poisoned experimentally with saponins from E. gummiferum. The results of the experiments in pregnant guinea pigs suggest that E. contortisiliquum can be used to study the abortive effect of its pods or its toxic compounds.

  11. Doenças de bovinos no Sul do Brasil: 6.706 casos Diseases of cattle in southern Brazil: 6.706 cases

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    Ricardo B. Lucena


    exams 29.9% were necropsies performed at the LPV-UFSM and 79.1% were mailed-in organ fragments from necropsies performed at the field by veterinary practitioners. Autolysis and non-representative sampling o mailed in organs were the main reasons for non-conclusive diagnosis. Poisoning by Senecio spp. was the main cause of death in cattle in this study and poisonous plants together with toxi-infections accounted for 22.8% of the cases with conclusive diagnosis. Inflammatory diseases together with parasitic diseases accounted for more than 30% of cattle diseases and babesiosis and anaplasmosis were the main diseases in this category. Other categories were distributed in the following order: neoplasms and tumor-like lesions (13.87%, diseases caused by physical agents (2.7%, metabolic and nutritional diseases (2.46%, circulatory disturbances (1.4%, degenerative diseases (1.1%, developmental disorders (0.54%, iatrogenic diseases and sundry lesions. The high prevalence of tumors in cattle in this study was attributed to the chronic ingestion of Pteridium aquilinum, a common toxicosis in the region. The main diseases in cattle from the studied region are related to environmental factors associated to the predominantly husbandry practices adopted in the region.

  12. 测序级胰蛋白酶的制备工艺与质量检测%Preparation and Characterization of Sequencing Grade Modified Trypsin

    Institute of Scientific and Technical Information of China (English)

    喻峰; 卢大儒; 陈薇


    目的:制备高纯度、酶解效率高、酶切位点专一的测序级胰蛋白酶,应用于蛋白组学研究的蛋白质鉴定与分析中。方法:取实验室自制的猪胰蛋白酶粗酶,经硼氢化钠、甲醛还原甲基化修饰抑制胰蛋白酶自水解,采用高效液相色谱仪15RPC反相柱纯化,收集对应的甲基化胰蛋白酶峰组分,冷冻干燥;甲基化修饰的胰蛋白酶进一步经甲苯磺酰苯丙氨酰氯甲酮(TPCK)修饰,以抑制糜蛋白酶等非特异性酶切活性,并经反相色谱柱再纯化,获得终产物即质谱测序级胰蛋白酶;自制的测序级胰蛋白酶经SDS-PAGE、HPLC反相色谱分析、酶比活力测定,并应用于胶内蛋白质酶切质谱鉴定氨基酸序列等,检测其纯度、酶水解效率及酶切位点特异性。结果:自制甲基化TPCK修饰的测序级胰蛋白酶纯度大于95%,酶比活力为200 U/mgP(TAME)以上,质谱分析酶切特异性好;且酶的制备工艺流程稳定,可应用于测序级胰蛋白酶产品的生产与开发中。结论:制备的测序级胰蛋白酶纯度高、酶解效率优、酶切特异性强,可广泛应用于实验室中蛋白质和肽段测序鉴定、HPLC肽段谱图分析等蛋白组学研究分析中。%Objective: To prepare highly purified, effective, restriction site-specific and chemically stabilized se-quencing grade modified trypsin, and apply for protein identification and analysis in proteomics research. Meth-ods: Firstly, raw trypsin isolated from porcine pancreas was reductive methylated with sodium borohydride buffer and formaldehyde following purification by reverse phase chromatography of 15RPC column, producing a stable tryp-sin that was resistant to tryptic autolysis. Then, the chromatography peak composition of methyl-trypsin was collect-ed. Secondly, methyl-trypsin was further improved by TPCK treatment and was subjected to extensive purification by reverse phase chromatography

  13. Microglial Dystrophy in the Aged and Alzheimer's Disease Brain Is Associated with Ferritin Immunoreactivity

    Institute of Scientific and Technical Information of China (English)



    subpopulation of the larger microglial pool labeled with an antibody for HLA-DR antigens. The majority of these ferritin-positive microglia exhibited aberrant morphological(dystrophic)changes in the aged and particularly in the AD brain. No spatial correlation was found between ferritin-positive dystrophic microglia and senile plaques in AD tissues. Analysis of a secondary set of human postmortem brain tissues with a wide range of postmortem intervals(PMI,average 10.94 ± 5.69 h)showed that the occurrence of microglial dystrophy was independent of PMI and consequently not a product of tissue autolysis. Collectively,these results suggest that microglial involvement in iron storage and metabolism contributes to their degeneration,possibly through increased exposure of the cells to oxidative stress. We conclude that ferritin immunohistochemistry may be a useful method for detecting degenerating microglia in the human brain.

  14. Predictive factors of breast cancer evaluated by immunohistochemistry Fatores preditivos do câncer de mama avaliados pela imuno-histoqu��mica

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    Helenice Gobbi


    Full Text Available Hormone receptor and Her2 protein overexpression evaluated by immunohistochemistry (IHC is widely validated as a predictive factor in breast cancer. The quality of the IHC reaction is influenced by tissue fixation and processing. Over- and underfixation deeply affect IHC results. Antigen retrieval may improve IHC but it does not recover tissue from autolysis or overfixation. The choice of primary antibody for IHC as to its sensitivity and specificity in relation to therapeutic response represents an important stage. Apart from mouse monoclonal antibodies, new rabbit monoclonal antibodies are commercially available, such as clones anti-ER SP1 and B644, anti-PR SP2 and B645 and anti-Her2 SP3 and 4B5. They represent an alternative to hormone receptor and Her2 evaluation by IHC. New polymeric non-biotinylated detection systems are also available and allow accurate and strong marking with no stromal and no non-specific cytoplasmic staining due to endogenous biotin. The most recommended cut off for estrogen and progesterone receptors (ER and PR is more than 1% of positive cells with moderate or strong staining intensity (Allred's scoring system. New guidelines for Her2 evaluation by IHC show a cut off of more than 30% of positive cells with strong intensity (3+ that correlates better with gene amplification. The 2+ cases are now considered indeterminate and should be confirmed by fluorescence in situ hybridisation (FISH or chromogenic in situ hybridisation CISH. A quality control of pre-analytical, analytical and post-analytical phases of IHC is recommended in order to optimize results.A superexpressão de receptores hormonais e Her2 avaliada pela imuno-histoquímica (IHQ é amplamente validada como fator preditivo em câncer de mama. A qualidade da reação imuno-histoquímica é influenciada pela fixação do tecido e seu processamento. A fixação insuficiente ou demasiada afeta profundamente os resultados da IHQ. A reativação antigênica pode