β-Glucosidase activity in fetal bovine serum renders the plant glucoside,hypoxoside, cytotoxic toward B16-F10-BL-6 mouse melanoma cells

Summary By usingp-nitrophenyl-β-d-glucopyranoside as substrate, β-glucosidase activity was observed in fetal bovine serum (FBS). This activity could be inhibited by heat inactivation of the serum. Gel chromatography of FBS indicated the presence of β-glucosidase activity with an apparent molecular mass of 29 kDa. In McCoy’s 5A medium supplemented with non-heat inactivated FBS, the diglucoside hypoxoside ([E]-1,5-bis[4′β-d-glucopyranosyloxy-3′-hydroxyphenyl]pent-4-en-1-yne) showed cytotoxicity toward B16-F10-BL-6 mouse melanoma cells. In incubations where the media were supplemented with FBS previously heat inactivated at 56° C for 1 h or more, no cytotoxicity was observed in the presence of hypoxoside. The aglucone of hypoxoside, rooperol ([E]-1,5-bis[3′,4′-dihydroxyphenyl]pent-4-en-1-yne), showed cytotoxicity regardless of whether the serum was heat inactivated or not. The kinetics of the heat inactivation of the β-glucosidase activity in FBS coincided with the loss of apparent cytotoxicity of hypoxoside. High performance liquid chromatography analysis showed that rooperol could be generated by incubation of hypoxoside in non-heat inactivated FBS, but that this ability was lost in serum that was heat inactivated for 1 h or longer. Newborn bovine serum did not contain any β-glucosidase activity whereas it was found in three different commercial sources of FBS. This observation is of practical importance because conventional heat inactivation of FBS at 56° C for 30 min was not sufficient to inactivate the β-glucosidase activity completely.     

Immunization with heat‐inactivated Staphylococcus aureus induced an antibody response mediated by IgG1 and IgG2 in patients with recurrent tonsillitis

Currently Staphylococcus aureus is the predominant pathogen isolated from the respiratory tract of patients with recurrent tonsillitis. Because of an increase in multi‐drug resistant strains of S. aureus, there is a pressing need for effective treatments and preventive approaches to reduce the risk of invasive and life‐threatening infections. A preventive vaccine against S. aureus would have a tremendous clinical impact. However, multiple clinical trials have failed to identify an agent that can induce protective responses. Most trials have been based on subunit vaccines using one or a few purified antigens, which may not be enough to confer protection. Here, the impact of a whole‐cell vaccine comprised of heat‐inactivated S. aureus was investigated in patients with RT. The vaccine was well tolerated and had no significant local or systemic reactions. Immunization with heat‐inactivated S. aureus elicited a significant antibody response characterized by production of IgG1 and IgG2 antibodies and, to a lesser extent, of IgA antibodies. Notably, this response was associated with an important decrease in the incidence of tonsillitis and bacterial colonization of the oropharyngeal mucosa. Our results show that whole‐cell inactivated S. aureus is safe and capable of evoking specific antibody responses in patients with recurrent tonsillitis.     

Effect of immunization of channel catfish with inactivated trophonts on serum and cutaneous antibody titers and survival against Ichthyophthirius multifiliis

Two trials were conducted to determine the effect of immunization of channel catfish with inactivated trophonts on serum and cutaneous antibody titers and survival against Ichthyophthirius multifiliis Fouquet (Ich). In trial I, catfish were immunized intraperitoneally (IP) with: 1) 1% formalin-inactivated trophonts, 2) 3% formalin-inactivated trophonts and 3) freeze-thawed trophonts. Positive and negative control catfish were immunized with live theronts and 5% bovine serum albumin (BSA), respectively. At day 14, 28 and 50 post-immunizations, no statistical difference was noted in serum or cutaneous anti-Ich antibody titers to formalin-inactivated trophonts or freeze-thawed trophonts. The survival of catfish challenged with live theronts ranged from 33.3% to 43.3% for the formalin-inactivated or freeze-thawed trophonts at 50 d post-immunization. The survival of catfish immunized with live theront and BSA was 93.3 and 0%, respectively. In trial II, catfish were IP immunized with sonicated trophonts at doses of 1) 5 trophonts or 10.2 microg protein g(-1) fish, 2) 10 trophonts or 20.4 microg protein g(-1) fish, 3) 20 trophonts or 40.8 microg protein g(-1) fish, and 4) 5% BSA as the control. Fish immunized with 10 or 20 trophonts g(-1) fish showed highest serum (1/210 to 1/480) and cutaneous antibody titers (1/48 to 1/52), respectively, at 22 d post-immunization and survival (63.3-60.0%). The fish immunized with 5 trophonts g(-1) fish had titers of 1/52 and 1/12 for serum and cutaneous antibody and survival of 23.3%. BSA immunized catfish had background titers and a survival of 6.7%. There was a significant correlation between doses of sonicated trophonts used to immunize and catfish survival (correlation coefficient = 0.859, p < 0.01). These results indicate that doses of sonicated trophonts, but not formalin-inactivated or freeze-thawed trophonts provided both serum and cutaneous antibody responses and survival to live trophont challenge.     

Cytoplasmic Particles in the Killers of Euplotes minuta and their Relationship to the Killer Substance*

SYNOPSIS. The killer strains of Euplotes minuta contain cytoplasmic epsilon particles absent in sensitive strains. The toxic principle of killers is associated with large particles which sediment readily in the centrifuge and may be identical with epsilon. Killing particles are inactivated by heat, certain proteolytic enzymes, and lysis with the French press.     

Comparison of the Heavy Metal Biosorption Capacity of Active,Heat‐Inactivated and NaOH‐Treated Phanerochaete chrysosporium Biosorbents

Three different kinds of Phanerochaete chrysosporium (NaOH‐treated, heat‐inactivated and active) biosorbent were used for the removal of Cd(II) and Hg(II) ions from aquatic systems. The biosorption of Cd(II) and Hg(II) ions on three different forms of Phanerochaete chrysosporium was studied in aqueous solutions in the concentration range of 50–700 mg/L. Maximum biosorption capacities of NaOH‐treated, heat‐inactivated and active Phanerochaete chrysosporium biomass were found to be 148.37 mg/g, 78.68 mg/g and 68.56 mg/g for Cd(II) as well as 224.67 mg/g, 122.37 mg/g and 88.26 mg/g for Hg(II), respectively. For Cd(II) and Hg(II) ions, the order of affinity of the biosorbents was arranged as NaOH‐treated > heat‐inactivated > active. The order of the amount of metal ions adsorbed was established as Hg(II) > Cd(II) on a weight basis, and as Cd(II) > Hg(II) on a molar basis. Biosorption equilibriums were established in about 60 min. The effect of the pH was also investigated, and maximum rates of biosorption of metal ions on the three different forms of Phanerochaete chrysosporium were observed at pH 6.0. The reusability experiments and synthetic wastewater studies were carried out with the most effective form, i.e., the NaOH‐treated Phanerochaete chrysosporium biomass. It was observed that the biosorbent could be regenerated using 10 mM HCl solution, with a recovery of up to 98%, and it could be reused in five biosorption‐desorption cycles without any considerable loss in biosorption capacity. The alkali‐treated Phanerochaete chrysosporium removed 73% of Cd(II) and 81% of Hg(II) ions from synthetic wastewater.     

Proteomics of RAW 264.7 macrophages upon interaction with heat‐inactivated Candida albicans cells unravel an anti‐inflammatory response

Murine macrophages (RAW 264.7) were allowed to interact with heat‐inactivated cells of Candida albicans SC5314 during 45 min. The proteomic response of the macrophages was then analyzed using 2‐D gel electrophoresis. Many proteins having differential expression with respect to control macrophages were identified, and their functions were related to important processes, such as cytoskeletal organization, signal transduction, metabolism, protein biosynthesis, stress response and protein fate. Several of these proteins have been described as being involved in the process of inflammation, such as Erp29, Hspa9a, AnxaI, Ran GTPase, P4hb, Clic1 and Psma1. The analysis of the consequences of their variation unravels an overall anti‐inflammatory response of macrophages during the interaction with heat‐inactivated cells. This result was corroborated by the measurement of TNF‐α and of ERK1/2 phosphorylation levels. This anti‐inflammatory effect was contrary to the one observed with live C. albicans cells, which induced higher TNF‐α secretion and higher ERK1/2 phosphorylation levels with respect to control macrophages.     

灭活原生质体作黑木耳融合育种遗传标记研究

本文采用0.83％碘乙酰胺(CIA)化学灭活剂和热处理灭活方法,对黑木耳单株原生质体灭活,观察了化学灭活与不同温度热灭活的灭活效果。并通过对灭活原生质体与未经灭活处理的原生质体融合后再生产物分析、证明,不论化学灭活还是热灭活处理,只要筛选好灭活条件,作为黑木耳种内原生质体融合遗传标记是可行的。     

Lactic acid concentration as an indicator of acceptability in refrigerated or freeze-thawed ground beef

Lactic acid concentrations increased in refrigerated and freeze-thawed anaerobically stored ground beef. Bacterial counts were higher in refrigerated samples, but the ratios of gram-positive bacteria in refrigerated and freeze-thawed samples were the same. No differences in appearance or odor between refrigerated and freeze-thawed samples were noted after 2 days of aerobic storage. Initial lactic acid concentration can be used to predict the shelf life of frozen beef.     

A PRELIMINARY STUDY ON ISOLATION OF ECLOSION HORMONE FROM ANTHERAEA PERNYI*

Abstract Eclosion hormone (EH) was isolated from 1 500 pharate adult heads of oak silkmoth, An-theraea pernyi. The crude EH which weight 420 mg was obtained by 8 steps. The A. pernyi bioassay was adopted to test biological activity of crude EH, and total EH activity was about 750 EH units. The specific activity was approximately 560 μg/unit. The crude EH was inactivated by proteolytic enzymes. It was stable in heat and had non species-specific.     

Lactic acid concentration as an indicator of acceptability in refrigerated or freeze-thawed ground beef.

Lactic acid concentrations increased in refrigerated and freeze-thawed anaerobically stored ground beef. Bacterial counts were higher in refrigerated samples, but the ratios of gram-positive bacteria in refrigerated and freeze-thawed samples were the same. No differences in appearance or odor between refrigerated and freeze-thawed samples were noted after 2 days of aerobic storage. Initial lactic acid concentration can be used to predict the shelf life of frozen beef.     

Chlamydophila pneumoniae downregulates MHC‐class II expression by two cell type‐specific mechanisms

Chlamydophila pneumoniae was shown to prevent IFNγ‐inducible upregulation of MHC‐class II molecules by secreting chlamydial protease‐like activity factor (CPAF) into the cytosol of those host cells which support the complete bacterial replication cycle. CPAF acts by degrading upstream stimulatory factor 1 (USF‐1). However, in cells like bone marrow‐derived macrophages (BMM), which restrict chlamydial replication, we show that CPAF expression is barely detectable and the expression of USF‐1 is induced upon infection with C. pneumoniae. Nevertheless, the infection still reduced base line and prevented IFNγ‐inducible MHC‐class II expression. Similar results were obtained with heat‐inactivated C. pneumoniae. In contrast, reduction of MHC‐class II molecules was not observed in MyD88‐deficient BMM. Reduction of IFNγ‐inducible MHC‐class II expression by C. pneumoniae in BMM was mediated in part by the MAP‐kinase p38. Infection of murine embryonic fibroblasts (MEF) with C. pneumoniae, which allow chlamydial replication, induced the expression of CPAF and decreased USF‐1 and MHC‐class II expression. Treatment of these cells with heat‐inactivated C. pneumoniae reduced USF‐1 and MHC‐class II expression to a much lower extent. In summary, C. pneumoniae downregulates MHC‐class II expression by two cell type‐specific mechanisms which are either CPAF‐independent and MyD88‐dependent like in BMM or CPAF‐dependent like in MEFs.     

The Extracellular Protein of Luteococcus japonicus subsp. casei Reactivates Cells Inactivated by UV Irradiation or Heat Shock

The culture liquid of Luteococcus japonicus subsp. casei was found to be able to reactivate cells of this bacterium inactivated by UV irradiation or heat shock. The antistress activity of the culture liquid was due to the presence of an extracellular exometabolite of a protein nature with a molecular mass of more than 10 kDa. When the bacterium was grown in a nutrient broth or glucose-containing mineral medium, the antistress protein was secreted by cells in the logarithmic growth phase. The reactivating effect of the antistress protein was inversely proportional to the survival rate of stressed cells.     

Enhancement of immunoregulatory effects of Lactobacillus gasseri TMC0356 by heat treatment and culture medium

Aims: The aim of this study was to investigate the influence of heat treatment and culture media on the immunoregulatory effects of a probiotic strain, Lactobacillus gasseri TMC0356 (TMC0356). Methods and Results: TMC0356 cultured in deMan–Rogosa–Sharpe and same food grade (FG) media were inactivated with the heat treatment at 70 and 90°C. Viable and heat‐killed TMC0356 were tested for their ability to induce interleukin (IL)‐12 production in the murine macrophage cell line J774.1. These TMC0356 were examined for their resistance to N‐acetylmuramidase. Their morphology was observed by scanning electron microscopy. The heat‐killed TMC0356 significantly induced IL‐12 production in J774.1 cells and exhibited enhanced resistance to N‐acetylmuramidase compared with viable TMC0356. Morphological changes were observed in TMC0356 when cultured in FG medium. Cell morphology and induction of IL‐12 production in J774.1 cells were also associated. Conclusions: These results suggest that heat treatment and culture medium composition modified the immunoregulatory effects of TMC0356 to induce IL‐12 production in macrophages. Significance and Impact of the Study: These results demonstrate that probiotic immunoregulatory effects may be modified by the processing technology of cell preparation.     

Transfer of a benomyl resistance marker by heat-inactivated Trichoderma reesei protoplasts

Summary Protoplasts from a benomyl resistant Trichoderma reesei mutant were heat inactivated at 60°C for 8 min and fused with viable protoplasts from an osmosensitive, non-sporulating T. reesei strain. Fusants recovered on 50 g/ml benomyl containing potato dextrose agar plates grew and sporulated well. Cellulolytic enzyme activities produced in liquid culture by selected fusants were higher than those produced by parental strains.     

Pyridine nucleotides and H2 as electron donors to the respiratory and photosynthetic electron-transfer chains and to nitrogenase in Anabaena heterocysts

The pathways through which NADPH, NADH and H₂ provide electrons to nitrogenase were examined in anaerobically isolated heterocysts. Electron donation in freeze-thawed heterocysts and in heterocyst fractions was studied by measuring O₂ uptake, acetylene reduction and reduction of horse heart cytochrome c. In freeze-thawed heterocysts and membrane fractions, NADH and H₂ supported cyanide-sensitive, respiratory O₂ uptake and light-enhanced, cyanide-insensitive uptake of O₂ resulting from electron donation to O₂ at the reducing side of Photosystem I. Membrane fractions also catalyzed NADH-dependent reduction of cytochrome c. In freeze-thawed heterocysts and soluble fractions from heterocysts, NADPH donated electrons in dark reactions to O₂ or cytochrome c through a pathway involving ferredoxin:NADP reductase; these reactions were only slightly influenced by cyanide or illumination. In freeze-thawed heterocysts provided with an ATP-generating system, NADH or H₂ supported slow acetylene reduction in the dark through uncoupler-sensitive reverse electron flow. Upon illumination, enhanced rates of acetylene reduction requiring the participation of Photosystem I were observed with NADH and H₂ as electron donors. Rapid NADPH-dependent acetylene reduction occurred in the dark and this activity was not influenced by illumination or uncoupler. A scheme summarizing electron-transfer pathways between soluble and membrane components is presented.     

Characteristics of a highly thermostable neutral protease produced fromBacillus stearothermophilus

A highly thermostable neutral protease was found in culture filtrates ofBacillus stearothermophilus. The optimum reaction pH and temperature of this protease were 6.0 and 60°C, respectively, and 90% activity remained even after heat treatment at 90°C for 30 min. The protease was markedly inactivated by diisopropyl fluorophosphate, but EDTA and iodoacetic acid hardly affected it. The neutral protease therefore could be defined as a highly thermostable, neutral(-serine) protease.     

2308、M28、S1330、16M四株布鲁氏菌灭活参数研究

【目的】比较不同灭活方法对布鲁氏菌灭活的效果,确定灭活参数,为制备布鲁氏菌灭活抗原提供参考。【方法】将4株布鲁氏菌参考强毒株2308(牛种)、M28(羊种)、S1330(猪种)以及16M(羊种)分别经大豆酶消化蛋白胨(TSA)培养基培养繁殖后,用生理盐水制成(4-8)×1010 CFU/m L的菌悬液,分成等份于80 oC灭活不同时间,另将同样浓度的菌悬液分别用不同浓度甲醛于37 oC灭活不同时间,通过灭活检验,确定灭活效果。取经甲醛和热灭活的16M抗原,分别以1×1010 CFU/只剂量皮下注射1.5-2.0 kg家兔2只,免疫6周内,每周采血用虎红平板凝集试验(RBT)和试管凝集试验(SAT)测定抗体效价。【结果】80 oC、5 min可灭活2308、S1330和16M三种菌株,80 oC、10 min可灭活全部4种菌株。0.2%甲醛灭活7 d,4种试验菌株均不能被彻底灭活;0.4%甲醛在12 h内只能灭活16M,72 h可灭活M28;0.4%甲醛灭活2308和S1330两次试验结果差异较大。0.6%甲醛可在72 h内灭活4种试验菌。不同方法灭活的16M抗原免疫家兔后,其血清抗体虎红平板凝集和试管凝集效价消长趋势基本一致,甲醛灭活的抗原免疫原性略高于热灭活抗原。【结论】80 oC热灭活和0.6%甲醛灭活均可用于对布鲁氏菌的灭活,且不影响布鲁氏菌的免疫原性。     

A mutant in a major heat shock protein of Escherichia coli continues to show inducible thermotolerance

Summary Escherichia coli cells carrying the dnaK756 mutation, were inactivated at 52°C faster than control cells. This suggests that the intact dnaK gene product plays a role in protecting the cell from lethal damage at 52°C. The effect of the dnaK mutation on induced thermotolerance was examined. Prior heat shock at 42°C greatly lowered the subsequent inactivation rate in both mutant and control cells. This result suggests that, although produced in large amounts in response to thermal stress, mutation in the DnaK protein has little or no effect on induced thermotolerance.     

Quantification of the influence of trimethylamine-N-oxide (TMAO) on the heat resistance of Escherichia coli K12 at lethal temperatures

Aim: To quantify the influence of trimethylamine‐N‐oxide (TMAO) on the heat resistance of Escherichia coli K12 MG1655 cells at static temperatures. Methods and Results: Stationary‐phase E. coli cells were inactivated at 52, 54 and 58°C. The heat resistance is described as reduction in the inactivation rate, k_max, and/or an increase in the time for one decimal reduction, D, and/or an increase in the time for the fourth decimal reduction, t_4D. Conclusions: Resistance of E. coli changed – increased – at all temperatures under study. Generally, the addition of TMAO to the growth medium protected E. coli cells, leading to an increase in their heat resistance, i.e. reduced k_max and increased D and t_4D values are obtained. Significance and Impact of the Study: Additional knowledge on the reaction of E. coli to heat in the presence of the organic osmolyte TMAO at lethal temperatures is provided. This work contributes to an improved understanding of the level of the resistance of bacteria to heat in the presence of osmolytes.     

Structural alterations of the mitotic apparatus induced by the heat shock response in Drosophila cells

The general architecture of the mitotic apparatus was studied at the ultrastructural level in Drosophila cultured cells. Its two main characteristics are a very polarized spindle and a strong compartmentalization, ensured by large remnants of the nuclear envelope. Such compartmentalization has previously been reported for the rapid syncytial divisions of the early embryo; a similar finding in these cells with a long cycle strongly suggests that this organization constitutes a general mechanism for mitosis in Drosophila. We followed the modifications of these structures after a heat shock of 20, 50 or 120 min at 37°C. Contrary to interphase cells, mitotic cells appear very sensitive to hyperthermia. This stress treatment induced a disruption of the mitotic spindle, a reappearance and an extension of the Golgi apparatus, an inactivation of microtubule nucleation and a disorganization of the centrosome. This organelle seems the first to be affected by the heat shock response. The centrosome is not only inactivated, but also is structurally affected. During the recovery phase after heat stress, the mitotic cells presented a remarkable ring-shaped accumulation of electrondense material around the centrioles. We conclude that in Drosophila cells the mitotic phase, and more specifically the centrosome, are targets of the stress response.