Chemotactic cytokines: the role of leukocytic pyrogen and epidermal cell thymocyte-activating factor in neutrophil chemotaxis

Macrophage-derived leukocytic pyrogen (LP) is thought to be similar if not identical to interleukin 1 (IL 1). In addition to macrophages, keratinocytes produce a factor that has similar biologic and biochemical characteristics to IL 1, called epidermal cell thymocyte-activating factor (ETAF). Because many diseases affecting the skin are characterized by infiltration of polymorphonuclear leukocytes (PMN) and some of these disorders are associated with fever, we investigated whether ETAF like LP had pyrogenic activity, and whether ETAF or LP were chemoattractants. ETAF or LP purified by column chromatography and isoelectric focusing were found to have chemotactic activity for PMN. The fractions containing maximal chemotactic activity corresponded to maximal fever-inducing activity and maximum thymocyte-activating activity. Furthermore, the chemotactic activity of ETAF and LP could be blocked by an antibody directed against LP. The results of this study indicate that these mediators, which arise from distinct cell populations, are closely related and may play a vital role in skin as well as distant inflammatory and immunologic events.     

The defensive role of latex in plants: detrimental effects on insects

The defensive role of the latex of Calotropis procera has recently been reported. In this study, latex proteins involved in detrimental effects on insects were evaluated on another important crop pest. The latex was fractionated to obtain its major protein fraction, which was then used to evaluate its insecticidal properties against Callosobruchus maculatus (Coleoptera: Bruchidae) in artificial bioassays. Laticifer proteins (LP) were investigated to characterize their action in such an activity. LP was highly insecticidal at doses as low as 0.1% (W/W). This effect was slightly augmented in F₁ generation reared in artificial seeds containing LP at similar proportions of F₀, but was fully reversed when F₁ developed in LP-free seeds. The insecticidal proteins were not retained in a chitin column, and did not lose their insecticidal activity, even after heat treatment or pronase digestion. However, these samples inhibited papain (EC 3.4.22.2) activity and gut proteases of C. maculatus larvae, and a reverse zymogram showed the presence of protein bands resistant to papain digestion. These activities were not observed in unheated LP as they were probably masked by abundant endogenous cysteine protease (EC 3.4.22.16) activity present in unheated LP. LP was resistant to proteolysis when assayed with C. maculatus gut extract. However, gut proteins of C. maculatus were digested when incubated with LP. These observations and the deleterious effects of LP upon C. maculatus, reinforce the hypothesis that laticifer fluids are involved in plant defense against insects and indicate C. procera latex to be a source of promising insecticidal proteins. The inhibitor of proteolysis present in the latex seems to be resistant to heat and proteolysis and is certainly involved in the detrimental effects observed.     

Characterization of lactosporin, a novel antimicrobial protein produced by Bacillus coagulans ATCC 7050

Aims:  To characterize the antimicrobial protein produced by Bacillus coagulans used in the probiotic dietary supplement (Lactospore^® Probiotic, Sabinsa Corp., Piscataway, NJ, USA).
Methods and Results:  Bacillus coagulans ATCC 7050 was grown at 37°C for 18 h. The cell free supernatant was concentrated 10-fold (lactosporin preparation, LP). The antimicrobial activity of LP was confirmed against Micrococcus luteus ATCC 10420 in a well diffusion assay. The proteinaceous nature of LP was determined following exposure to different enzymes. The activity of LP was pH-dependent but stable to heat. The isoelectric point of LP was determined to be 3·5–4·0. PCR analyses showed no similarity between lactosporin and known antimicrobial proteins produced by the Bacillus spp.
Conclusions:  Lactosporin is a novel antimicrobial protein. Initial characterization indicates that it may fall outside of the conventional classification of class I and II bacteriocins. Loss of activity after exposure to a number of proteolytic enzymes and lipase suggest that lactosporin may posses a lipid moiety which contributes to its inhibitory activity.
Significance and Impact of the Study:  The unique characteristics of lactosporin, including its antimicrobial activity against pathogenic micro-organisms, indicate that it may have potential for application in foods and personal care products.     

The lysine-peptoid hybrid LP5 maintain activity under physiological conditions and affects virulence gene expression in Staphylococcus aureus

The antimicrobial peptide, LP5, is a lysine-peptoid hybrid, with antimicrobial activity against clinically relevant bacteria. Here, we investigated how various environmental conditions affect the antimicrobial activity of LP5 against Staphylococcus aureus (S. aureus). We found that LP5 maintained activity under host physiological conditions of NaCl, MgCl₂ and pH. However, when exposed to serum, LP5 lost activity. Furthermore, when increasing NaCl concentration and lowering pH, the peptide showed reduces activity. When investigating the tolerance mechanisms of S. aureus toward antimicrobial peptides, we found that LP5 was protease resistant. However, the dltA and vraF genes, involved in reducing the net anionic charge of the bacterial cell envelope and sensing of antimicrobial peptides, respectively, played a role in the tolerance of S. aureus against LP5. In addition, the exposure of S. aureus to sub-inhibitory concentrations of LP5 affected the expression of the major virulence factors of S. aureus, revealing a potential as anti-virulence compound. Thus, these results show how environmental factors affect the peptide efficiency and further add to the knowledge on how the peptide affects S. aureus, which is crucial information for designing new peptides for optimizing antimicrobial therapy.     

A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90

P2RX7 is an ATP-gated ion channel, which can also exhibit an open state with a considerably wider permeation. However, the functional significance of the movement of molecules through the large pore (LP) and the intracellular signaling events involved are not known. Here, analyzing the consequences of P2RX7 activation in primary myoblasts and myotubes from the Dmd^mdx mouse model of Duchenne muscular dystrophy, we found ATP-induced P2RX7-dependent autophagic flux, leading to CASP3-CASP7-independent cell death. P2RX7-evoked autophagy was triggered by LP formation but not Ca²⁺ influx or MAPK1-MAPK3 phosphorylation, 2 canonical P2RX7-evoked signals. Phosphoproteomics, protein expression inference and signaling pathway prediction analysis of P2RX7 signaling mediators pointed to HSPA2 and HSP90 proteins. Indeed, specific HSP90 inhibitors prevented LP formation, LC3-II accumulation, and cell death in myoblasts and myotubes but not in macrophages. Pharmacological blockade or genetic ablation of p2rx7 also proved protective against ATP-induced death of muscle cells, as did inhibition of autophagy with 3-MA. The functional significance of the P2RX7 LP is one of the great unknowns of purinergic signaling. Our data demonstrate a novel outcome—autophagy—and show that molecules entering through the LP can be targeted to phagophores. Moreover, we show that in muscles but not in macrophages, autophagy is needed for the formation of this LP. Given that P2RX7-dependent LP and HSP90 are critically interacting in the ATP-evoked autophagic death of dystrophic muscles, treatments targeting this axis could be of therapeutic benefit in this debilitating and incurable form of muscular dystrophy.     

Ability of human leukocytic pyrogen to enhance phytohemagglutinin induced murine thymocyte proliferation

Human leukocytic pyrogen, a monokine produced by stimulated human mononuclear phagocytes, will enhance the murine thymocyte proliferation response to phytohemagglutinin (lymphocyte activating factor (LAF) activity). During all steps of purification of human LP, pyrogenicity and LAF activity are coincidental suggesting a single identity for the two monokines. The LAF assay for human LP is highly sensitive and can detect human LP at a concentration of 10^?12M. Further experiments suggests that human LP and LAF activities could be destroyed by heating to 70 °C. Furthermore, while in vivo pyrogenicity of human LP can be blocked by ibuprofen, the in vitro LAF activity of the same molecule is unaffected by ibuprofen. Immune rabbit serum directed against human LP could also block in vitro LAF activity either by preincubation with LP or by blocking during culture.     

A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90

P2RX7 is an ATP-gated ion channel, which can also exhibit an open state with a considerably wider permeation. However, the functional significance of the movement of molecules through the large pore (LP) and the intracellular signaling events involved are not known. Here, analyzing the consequences of P2RX7 activation in primary myoblasts and myotubes from the Dmd^mdx mouse model of Duchenne muscular dystrophy, we found ATP-induced P2RX7-dependent autophagic flux, leading to CASP3-CASP7-independent cell death. P2RX7-evoked autophagy was triggered by LP formation but not Ca²⁺ influx or MAPK1-MAPK3 phosphorylation, 2 canonical P2RX7-evoked signals. Phosphoproteomics, protein expression inference and signaling pathway prediction analysis of P2RX7 signaling mediators pointed to HSPA2 and HSP90 proteins. Indeed, specific HSP90 inhibitors prevented LP formation, LC3-II accumulation, and cell death in myoblasts and myotubes but not in macrophages. Pharmacological blockade or genetic ablation of p2rx7 also proved protective against ATP-induced death of muscle cells, as did inhibition of autophagy with 3-MA. The functional significance of the P2RX7 LP is one of the great unknowns of purinergic signaling. Our data demonstrate a novel outcome—autophagy—and show that molecules entering through the LP can be targeted to phagophores. Moreover, we show that in muscles but not in macrophages, autophagy is needed for the formation of this LP. Given that P2RX7-dependent LP and HSP90 are critically interacting in the ATP-evoked autophagic death of dystrophic muscles, treatments targeting this axis could be of therapeutic benefit in this debilitating and incurable form of muscular dystrophy.     

枯草芽孢杆菌HS-A38产抗菌脂肽Bacillomycin D的组分分析及鉴定

对一株枯草芽孢杆菌HS-A38产生的脂肽类物质进行分离鉴定及抑菌活性研究。通过酸沉淀分离和有机溶剂抽提的方法,从枯草芽孢杆菌HS-A38发酵液中得到脂肽粗提物LP,产率为1.956 g/L。利用薄层色谱和茚三酮染色法确定该脂肽粗提物中存在四个组分,分别为LP1、LP2、LP3和LP4;抑菌活性检测显示,组分LP3对两株海洋致病菌副溶血性弧菌和铜绿假单胞杆菌的活性较高。组分LP3经硅胶柱层析纯化分离后,应用反相高效液相色谱(RP-HPLC)和基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)对该组分做进一步纯化和鉴定。分析表明,LP3样品在保留时间20 min~28 min产生单峰团LP3-1,其纯度为85.24%;经MALDI-TOF-MS分析和数据比对,组分LP3-1中的主要成分为杆菌霉素Bacillomycin D。     

Toxicity of <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> LP1-G Against Susceptible and Resistant <Emphasis Type="Italic">Culex quinquefasciatus</Emphasis> and the Cloning of the Mosquitocidal Toxin Gene

Bacillus sphaericus LP1-G, belonging to flagellar serotype H3, has been found to have moderate toxicity against two resistant Culex quinquefasciatus colonies (RLCq1 and RLCq2) and the susceptible contrast (SLCq). With an aim of screening mosquitocidal acting factor, a partial genome library was prepared from a partial HindIII digest of the total DNA from Bacillus sphaericus LP1-G. Two thousand twenty Escherichia coli clones were screened for toxicity against susceptible SLCq, and a toxic clone, designated E-UL68, was chosen for further study. The recombinant E-UL68 performed toxicity against both susceptible and two resistant colonies, having the same level of toxicity as that of wide-type strain LP1-G. Sequence analysis revealed that the inserted fragment was composed of 3876 nucleotides and contained a complete gene, whose sequence was identical to that of the mtx gene from B. sphaericus SSII-1. Because the binary toxin produced during sporulation of strain LP1-G has no activity against the target mosquitoes, this indicates that the Mtx toxin or other active factors might perhaps be responsible for the toxicity of LP1-G against different colonies of mosquito larvae.Received: 7 October 2002 / Accepted: 4 November 2002     

Toxicity of Bacillus sphaericus LP1-G against susceptible and resistant Culex quinquefasciatus and the cloning of the mosquitocidal toxin gene

Bacillus sphaericus LP1-G, belonging to flagellar serotype H3, has been found to have moderate toxicity against two resistant Culex quinquefasciatus colonies (RLCq1 and RLCq2) and the susceptible contrast (SLCq). With an aim of screening mosquitocidal acting factor, a partial genome library was prepared from a partial HindIII digest of the total DNA from Bacillus sphaericus LP1-G. Two thousand twenty Escherichia coli clones were screened for toxicity against susceptible SLCq, and a toxic clone, designated E-UL68, was chosen for further study. The recombinant E-UL68 performed toxicity against both susceptible and two resistant colonies, having the same level of toxicity as that of wide-type strain LP1-G. Sequence analysis revealed that the inserted fragment was composed of 3876 nucleotides and contained a complete gene, whose sequence was identical to that of the mtx gene from B. sphaericus SSII-1. Because the binary toxin produced during sporulation of strain LP1-G has no activity against the target mosquitoes, this indicates that the Mtx toxin or other active factors might perhaps be responsible for the toxicity of LP1-G against different colonies of mosquito larvae.     

Penetration of lipoproteins through a capillary wall and their effects on the cardiomyocyte ultrastructure during in vitro heart perfusion

The colloidal gold-labeled lipoprotein (LP) distribution in the myocardium after the 30 min perfusion of isolated working rat heart was studied by electron microscopy. LP of physiological concentrations are shown to be able to interact with the capillary wall and they can be incorporated by endotheliocytes. The velocity and transportation mechanisms of different classes of LP differ. Very low density LP are the most intensively taken up, low density LP enter into interstitium more rapidly than others, high density LP do not leave the capillary wall at all. The passages of labeled LP through endotheliocytes by the receptor-mediated as well as the non-receptor manners are revealed. The transportation through the widened intercellular junctions may be supposed for low density LP. For the fist time LP addendum into the perfusion medium was shown to provoke the activation of interstitial macrophages. During the perfusion duration they take inside and accumulate labeled very low density LP and low density LP in their lysosomes. The internalization may be performed by the specific endocytosis or by the simple phagocytosis. The qualitative and morphometrical analyses show that LP preserve the capillary and cardiomyocyte ultrastructure from perfusion injuries. One may suppose that there are interrelations between the capillary endothelium, the interstitial macrophages and the parenchymal cells in myocardium for realization of plasma LP effects.     

Restriction of stress-induced activation of lipid peroxidation by small doses of thyroid hormones]

The experiments on 57 female rats demonstrated that small doses of thyroid hormones (thyroidin) significantly (55-118%) restrict stress induced increase in the concentration of initial and terminal products of lipid peroxidation (LP) in the myocardium and in the blood plasma. After hormone injection stress decreases the activity of key antioxidant enzyme, superoxide dismutase of erythrocytes (SOD), to a lesser degree and increases the rate of malonyldialdehyde (MDA) production induced by Fe2+ in homogenates of the myocardium to the same degree as well in comparison with rats that had not been injected thyroidin. In normal rates thyroidin does not influence the concentration of products of LP, increases the activity of SOD and decreases increment of MDA induced by Fe2+ in homogenates of the myocardium. Thus, small doses of thyroid hormones restrict significantly stress induced activity of LP membranes, increasing the power of antioxidant systems both in the myocardium and in the organism.     

In vitro inhibition of microbial flora of fish by nisin and lactoperoxidase system

AIMS: To test the antimicrobial effects of nisin and lactoperoxidase system (LP system) against sardines flora. This study is part of a programme designed to investigate the preservability of fish using these inhibitors as potential biopreservatives. METHODS AND RESULTS: Antimicrobial effects of nisin and LP system alone or in combination were tested by the agar diffusion method against bacterial strains isolated from sardines (Sardina pilchardus). Nisin inhibited only Gram-positive bacteria, whereas LP system inhibited all strains studied. The combination of nisin (100 IU ml-1) and LP system (10 level) was significantly more effective than LP system or nisin alone against all strains, excepting Aeromonas salmonicida subsp. salmonicida and Vibrio alginolyticus. CONCLUSION: These results clearly demonstrated the efficiency of LP System-nisin combination for inhibiting spoilage flora of fish. SIGNIFICANCE AND IMPACT OF THE STUDY: Because LP system has a broad activity spectrum, it may be an interesting additional hurdle to improve the safety of food preservation by nisin. Combination of nisin and LP system could be of great interest as biopreservatives for fish and fish products.     

Cytolytic mechanisms of activated macrophages. Tumor necrosis factor and L-arginine-dependent mechanisms act synergistically as the major cytolytic mechanisms of activated macrophages

We examined the cytolytic mechanisms of activated macrophages by using proteose peptone- or thioglycollate broth-induced mouse peritoneal macrophages or mouse macrophage hybridomas as effector cells, L.P3 cells, a clone of L929 cells, and P815 cells as target cells, and IFN-gamma and LPS as activators. It was determined that TNF is the main cytolytic molecule against L.P3 cells from the following results: 1) activated macrophages can produce TNF; 2) TNF shows cytotoxic activity against L.P3 cells; 3) the addition of anti-TNF antibody inhibited most of the cytolytic activity of activated macrophages against L.P3 cells. On the other hand, it was concluded that the main cytolytic mechanism against P815 cells is the production of NO2-/NO3- from L-arginine, from the following results: 1) activated macrophages can produce NO2-; 2) NaNO2 shows high cytotoxic activity against P815 cells; 3) the depletion of L-arginine from the medium inhibited most of the cytolytic activity of activated macrophages against P815 cells and NO2- production by activated macrophages. In this study, however, cytostatic effects of L-arginine-dependent effector mechanism were not studied. Thus, these results show that activated macrophages can express at least two cytolytic mechanisms independently, namely, the one that appears to be mediated by the L-arginine-dependent effector mechanism and the second that appears to be mediated directly by TNF. Furthermore, it was demonstrated that TNF and L-arginine-dependent NO2- production act synergistically as killing mechanisms of activated macrophages. These mechanisms can explain the cytolytic activity of activated macrophages against a variety of target cells.     

Changes in the phagocytic activity of macrophages under the action of different doses of antibiotics]

Penicillin, streptomycin and gentamicin, when introduced into mice in a single injection in doses of 0.5-500,000 U/kg, produced different changes in the phagocytic activity of mouse peritoneal macrophages. Bactericidal doses of these antibiotics either suppressed the activity of macrophages or left it unchanged. Their sub-bactericidal doses enhanced the phagocytic activity of macrophages. Combined use of penicillin and streptomycin produced a synergic effect.     

Antioxidant effect of a novel class of telluroacetilene compounds: Studies in vitro and in vivo

AimsThe effect of telluroacetylenes a–d on pharmacological assays was investigated in vitro. A second objective of this study was to investigate the antioxidant action of compound b against the oxidative damage induced by sodium nitroprusside (SNP) in mouse brain.Main methodsIn in vitro experiments, lipid peroxidation (LP) and protein carbonyl (PC) levels and δ-aminolevulinate dehydratase (δ-ALA-D) activity were carried out in rat brain homogenate. The thiol peroxidase-like activity and DPPH radical scavenging of telluroacetylenes a–d were investigated. In in vivo experiments, mice received SNP (0.335 µmol per site) intra cerebroventricular (i.c.v.) thirty minutes after oral administration of telluroacetylene b (10 mg/kg). After 1 h, animals were euthanized. The levels of LP and δ-ALA-D, catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST) activities were carried out in mouse brain homogenate.Key findingsTelluroacetylenes a–d, at low μM range, reduced LP and PC levels in rat brain homogenate. Telluroacetylenes a–d showed effect of scavenging DPPH radicals. δ-ALA-D activity was inhibited by telloruacetylenes a–d, at high μM range, in rat brain homogenate. Brains of mice treated with SNP showed an increase in LP and the reduction in δ-ALA-D, GR and GST activities. Telluroacetylene b protected against the oxidative stress caused by SNP in brain of rats.SignificanceThe results support an antioxidant effect of telluroacetylenes a–d in vitro. Telluroacetylene b protected against oxidative damage caused by SNP in mouse brain, suggesting an antioxidant effect of this compound.     

Activity of nucleolar organizers in central macrophage culture of bone marrow erythroblastic islands

The erythroblastic islands of the bone marrow are morphofunctional units of erythropoiesis. In this work the functional state of erythroblastic islands' cells of the bone marrow, for the first time, was defined by the estimation of the activity of the nucleolar organizers of central macrophages in the erythroblastic islands, cultivated during 24 and 48 hours with the presence of various doses of erythropoietin. The findings indicated that the increase in doses of erythropoietin was accompanied by the corresponding increase of the activity of nucleolar organizers in central macrophages of erythroblastic islands. The nucleolar organizers of central macrophages in cultures of erythroblastic islands responded to very small doses of erythropoietin by their activation.     

Curcumin inhibits 19-kDa lipoprotein of Mycobacterium tuberculosis induced macrophage apoptosis via regulation of the JNK pathway

Recently, synthetic curcumin analogs are reported as potential active compounds against Mycobacterium tuberculosis (MTB). During the process of MTB infection, macrophages show increased apoptosis. The candidate virulence factors such as 19-kDa lipoprotein secreted by the MTB (P19) strongly influences macrophages by activation of Toll-like receptor 2 (TLR2) and mitogen-activated protein kinases (MAPKs). It has been reported that curcumin could affect the apoptosis of tumor cells via regulation of MAPKs. However, its effect on the P19-induced apoptosis of macrophages is unclear. This study investigates the effect of curcumin on the MAPKs signaling and apoptosis in human macrophages. The results showed that curcumin and P19 induced macrophage apoptosis in a time- and dose-dependent manner Low doses of curcumin (10 and 20 μM) protected macrophages from P19 induced apoptosis, accompanied by decreased production of cytokines and reduced activation of the c-Jun amino-terminal kinase (JNK) and p38 MAPK. The protective effect of curcumin on P19 induced apoptosis of macrophages were enhanced by treatment with the JNK-specific inhibitors, whereas SB203580, the inhibitor of p38 MAPK had no effect. Curcumin had no effect on the activity of extracellular signal-regulated protein kinase (ERK). Taken together, our data show that the JNK pathway, but not the p38 or ERK pathway, plays an important role in the protective effect of curcumin against P19 induced macrophage apoptosis, and regulation of the JNK pathway may partially elucidate the anti-tuberculosis activity of curcumin.     

Peptides with indirect in vivo activity against an intracellular pathogen: selective lysis of infected macrophages.

A collection of natural peptides, simplified analogs of natural peptides, de novo amphipathic peptides and de novo amphipathic peptides composed of 50-80% alpha,alpha-dialkylated glycines (alpha,alpha-Dags) were synthesized on solid-phase resin as the C-terminus amides using N-alpha-fluorenylmethyloxycarbonyl protection. The synthesis of the peptides rich in alpha,alpha-Dags used acid fluoride coupling methods. The peptides show antimicrobial activity against Escherichia coli and Staphylococcus aureus but no direct antimicrobial activity against Brucella abortus at 100 microm in vitro. However, in vivo treatment with several of these peptides results in significant reductions of B. abortus in chronically infected immune BALB/c mice relative to infected control animals. The chronically infected mice were susceptible to peptide toxicity at much lower peptide doses than control animals. The highest nonlethal dose for infected mice was only 25 microg for melittin, whereas 500 microg doses were nonlethal for many of the other peptides. Several of the alpha,alpha-Dag-rich peptides selectively destroy B. abortus-infected murine macrophages in vitro. Thus, these peptides apparently reduce the bacterial load in vivo by destroying a portion of the infected macrophages and exposing the sequestered bacteria to the immune response in the mice.