Antibacterial and proteolytic activity of rabbit phagocytes and the effect of temperature and endotoxin on this activity

Proteolytic activity at pH 2 (caused by cathepsin E) and at pH 3.5 (caused by cathepsin D and E) was found in extracts of rabbit polymorphonuclear leucocytes, macrophages and lymphocytes, while antibacterial activity was detected only in polymorphonuclear leucocyte extracts, against microorganisms of theEnterobacteriaceae family. Comparison of the character of antibacterial and proteolytic activity showed that the two systems were quite different. If polymorphonuclear leucocyte extract was heated at 56° C for 30 minutes, the effect on antibacterial activity was non-significant, while proteolytic activity at pH 2 and 3.5 fell by about 60%. The addition of a heat-inactivatedEscherichia coli suspension or of purifiedSalmonella paratyphi B bacterial lipopolysaccahride to leucocyte extract completely destroyed antibacterial activity, while proteolytic activity was unaffected.     

Antibacterial and cytotoxic activities of extracts from (Tunisian)Rhamnus alaternus (Rhamnaceae)

The petroleum ether, chloroformic, ethyl acetate, methanolic, Total Oligomers Flavonoids (TOF) enriched extracts, water extract as well as its fractions A₁, A₂, A₃ obtained from aerial parts ofRhamnus alaternus, a Tunisian-Mediterranean medicinal species, were investigated for the contents of phenolic compounds, cytotoxic activity against the K562 human chronic myelogenous leukaemia cell line and L1210 leukaemia murine cells and for antibacterial activity against Gram positive and Gram negative bacterial reference strains. A pronounced cytotoxic effect on both the cell lines was shown in the TOF, ethyl acetate, methanolic, aqueous extracts and A₂ fraction, with respectively IC₅₀ values 75, 232, 298, 606 and 571 μg/ml on K562 cells and 198, 176, 767, 560 and 614 μg/ml on L1210 cell line. Significant activity against bacterial reference strains:Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Salmonella enteritidis andSalmonella typhimurium was shown with ethyl acetate, TOF extracts and A2 fraction. The antimicrobial and cytotoxic activities showed byR. alatemus depended on the chemical composition of the tested extracts.     

Cleavage of the protein III and major iron-regulated protein of Neisseria gonorrhoeae by lysosomal cathepsin G

Incubation of either 125I-labelled or unlabelled Neisseria gonorrhoeae with enzymically active preparations of human polymorphonuclear leucocyte lysosomal cathepsin G revealed that surface-exposed outer-membrane proteins were susceptible to proteolytic modification. Electroimmunoblotting experiments confirmed that outer-membrane protein III (PIII) and the major iron-regulated protein (MIRP), two conserved gonococcal proteins, were cleaved by cathepsin G. A direct relationship was observed between susceptibility to the antibacterial properties of cathepsin G and cleavage of PIII among isogenic strains differing in their level of resistance to the bactericidal activity of cathepsin G. Although the antibacterial property of cathepsin G is known to be independent of serine-esterase activity, the data suggest that gonococcal outer-membrane proteins are involved in the binding of cathepsin G, and that variation in the level of resistance reflects the degree to which target outer-membrane proteins such as PIII are exposed.     

The recombinant prepro region of TvCP4 is an inhibitor of cathepsin L-like cysteine proteinases of Trichomonas vaginalis that inhibits trichomonal haemolysis

Trichomonas vaginalis expresses multiple proteinases, mainly of the cysteine type (CPs). A cathepsin L-like 34 kDa CP, designated TvCP4, is synthesized as a 305-amino-acid precursor protein. TvCP4 contains the prepro fragment and the catalytic triad that is typical of the papain-like CP family of clan CA. The aim of this work was to determine the function of the recombinant TvCP4 prepro region (ppTvCP4r) as a specific inhibitor of CPs. We cloned, expressed, and purified the recombinant TvCP4 prepro region. The conformation of the purified and refolded ppTvCP4r polypeptide was verified by circular dichroism spectroscopy and fluorescence emission spectra. The inhibitory effect of ppTvCP4r was tested on protease-resistant extracts from T. vaginalis using fluorogenic substrates for cathepsin L and legumain CPs. In 1-D zymograms, the inhibitory effect of ppTvCP4r on trichomonad CP proteolytic activity was observed in the ∼97, 65, 39, and 30 kDa regions. By using 2-D zymograms and mass spectrometry, several of the CPs inhibited by ppTvCP4r were identified. A clear reduction in the proteolytic activity of several cathepsin L-like protein spots (TvCP2, TvCP4, TvCP4-like, and TvCP39) was observed compared with the control zymogram. Moreover, pretreatment of live parasites with ppTvCP4r inhibited trichomonal haemolysis in a concentration dependent manner. These results confirm that the recombinant ppTvCP4 is a specific inhibitor of the proteolytic activity of cathepsin L-like T. vaginalis CPs that is useful for inhibiting virulence properties depending on clan CA papain-like CPs.     

Analysis of anti-bacterial and anti oxidative activity of Azadirachta indica bark using various solvents extracts

Herbal medications have been used for relief of symptoms of disease. Regardless of the great advances observed in current medicine in recent decades, plants still make a significant contribution to health care. An alarming increase in bacterial strains resistant to a number of antimicrobial agents demands that a renewed effort be made to seek antibacterial agents effective against pathogenic bacteria resistant to or less sensitive to current antibiotics. Anti-bacterial activity of Azadirachta indica stem bark was tested against pathogenic Salmonella paratyphi and Salmonella typhi using various solvent extracts. The in vitro anti-bacterial activity was performed by agar well diffusion method and the results were expressed as the average diameter of zone of inhibition of bacterial growth around the well. The ethanol and methanol extracts showed better anti-bacterial activity with zone of inhibition (20–25 mm) when compared with other tested extracts and standard antibiotic Erythromycin (15 mcg) with zone of inhibition (13–14 mm). Using Fisher’s exact test of significance difference was found between two Salmonella strains sensitivity patterns against tested extracts (P ⩽ 0.035). Extracts of A. indica stem bark also exhibited significant antioxidant activity, thus establishing the extracts as an antioxidant. The results obtained in this study give some scientific support to the A. indica stem bark for further investigation of compounds and in future could be used as drug.     

Separation of the human leucocyte enzymes alanine aminopeptidase, cathepsin G, collagenase, elastase and myeloperoxidase

A simple and rapid procedure is described for the separation of the human leucocyte enzymes alanine aminopeptidase, cathepsin G, collagenase, elastase and myeloperoxidase. The enzymes are prepared from leucocytes, obtained from buffy coat, by repeated extraction with buffer A(1 M salt concentration). The pooled extracts are successively subjected to batch adsorption on concanavalin A-Sepharose, gel filtration on Sephacryl S-300, affinity chromatography on collagen-Sepharose 4-B, batch adsorption on CM-Sephadex C-50 and adsorption chromatography on hydroxyapatite. The yields of the isolated enzymes of a typical preparation are 47% alanine aminopeptidase, 9% cathepsin G, 90% latent and active collagenase, 23% elastase and approximately 100% myeloperoxidase with respect to the pooled extracts. The cathepsin G, collagenase and elastase preparations are essentially free from other proteolytic enzymes and may be used without further purifications.     

Intracellular Proteolytic Activity in Mycelia of Arthrobotrys oligospora Bearing Mycoparasitic or Nematode Trapping Structures

Persson, Y., and Friman, E. 1993. Intracellular proteolytic activity in mycelia of Arthrobotrys oligospora bearing mycoparasitic or nematode trapping structures. Experimental Mycology 17, 182-190. The fungus Arthrobotrys oligospora parasitizes other fungi with the aid of coils and captures and digests nematodes by means of adhesive traps. We have compared proteolytic activities of mycelial extracts from coils and traps with those of vegetative hyphae. A. oligospora produced a number of proteases active at both alkaline and acidic pH. Coil extract had significantly higher proteolytic activity than extracts of vegetative hyphae. Several coil culture-specific bands were found after substrate gel electrophoresis. Pepstatin-sensitive proteolytic activity at acidic pH was higher in coil extract than in normal mycelial extracts, although the total proteolytic activity was the same. No proteolytic activity was connected solely to mycelial extracts with traps and no enhancement of proteolytic activity was observed during infection of nematodes.     

Phytochemical, antimicrobial, antioxidant and antigenotoxic potentials of Cyperus rotundus extracts

The aqueous, ethyl acetate, methanolic and Total Oligomer Flavonoids (TOF) enriched extracts, obtained from the aerial parts of Cyperus rotundus, were investigated for their contents in phenolic compounds. Antioxidative activity using the NBT/riboflavin assay system, antimicrobial activity against Gram positive and Gram negative bacterial reference strains as well as antigenotoxic activity tested with the SOS chromotest assay were also studied. Significant antibacterial activity against reference strains; Staphylococcus aureus, Enterococcus faecalis, Salmonella enteritidis and Salmonella typhimurium, was detected in the presence of ethyl acetate and TOF enriched extracts. In addition to their antimicrobial activity, the same extracts showed a significant ability to inhibit nitroblue tetrazolium reduction by the superoxide radical in a non enzymatic O₂^.− generating system, and were also able to reduce significantly the genotoxicity induced by nifuroxazide and Aflatoxin B1. The antioxidant, antimicrobial and antigenotoxic activities exhibited by C. rotundus depend on the chemical composition of the tested extracts.     

Neutral proteinase activity ofCandida albicans

Neutral proteinase activity (pH optimum 6–7.5) was demonstrated in cell extracts of the yeast form ofCandida albicans H-317. Proteolytic activity which required activation by treatment with sodium dodecyl sulfate was measured by a colorimetric assay for Azocoll hydrolysis. The activity was inhibited by antipain, chymostatin, phenylmethylsulfonyl fluoride, andp-chloromercuribenzoate, but was not affected by ethylenediaminetetraacetate, leupeptin, or pepstatin A. These results give the first evidence of a neutral proteinase inC. albicans.     

Sulfur metabolism in Thiorhodaceae. V. Enzymes of sulfur metabolism inThiocapsa floridana andChromatium species

In extracts ofThiocapsa floridana strain 6311 andChromatium strains 1611, 2811 and 6412 a high specific activity of adenosine 5-phosphosulfate reductase was found. In contrast, little activity of this enzyme was found inChromatium strain D.Adenosine 5-diphosphate sulfurylase is present in extracts fromThiocapsa floridana strain 6311. The enzyme appears to be involved in the oxidation of reduced sulfur compounds to sulfate.Sulfite oxidase could not be demonstrated in any of the strains examined.     

UV-induced mutability in repair-deficientrad6-1 strains ofSaccharomyces cerevisiae is caused by a suppressor gene

TheRAD6 gene is a multifunctional gene required for DNA repair, induced mutagenesis and sporulation. The survival and revertibility of two loci in fourrad6-1 mutant strains of different origin after UV irradiation were followed. As expected, all therad6-1 strains tested were more sensitive to UV radiation in comparison withRAD6 strains. The reversion frequency per survivor intrpl-289 andarg4–17 alleles was significantly higher in all fourrad6-1 mutant strains than in wild-type strains after equal doses of UV radiation. On the basis of genetic analysis we suggest that the phenomenon of increased frequency of induced mutagenesis is caused by a suppressor gene.     

Bioactivity in free-living and symbiotic cyanobacteria of the genus Nostoc

Fifty cyanobacterial strains from different habitats(symbioses, soil, fresh and marine waters) belongingto the genus Nostoc were cultured and tested forbioactivity. Thirty-seven strains were isolated in ourlaboratory, the remaining were supplied by officialculture collections. All the organisms were grownunder controlled laboratory conditions. The biomasseswere lyophilised and extracted with ethanol:water toobtain a hydrophilic extract and then withdichloromethane:isopropanol to obtain a lipophilicextract. Both crude extracts were tested forantifungal (against Penicillium expansum andRhizoctonia solani) and antibacterial activity(against Agrobacterium vitis, Escherichiacoli and Staphylococcus epidermidis), and fortoxicity against Artemia salina nauplii.Twenty-four strains showed activity against at leastone of the target organisms. Bioactivity was equallydistributed between lipophilic and hydrophilicextracts, and was mostly directed against fungi (15strains) and Artemia nauplii (12 strains);antibacterial activity was less frequent (8 strains).The presence of bioactivity was independent of thestrain origin.     

Antibacterial metabolites synthesized by psychrotrophic bacteria isolated from cold-freshwater environments

The ability of three psychrotrophic Gram-negative bacilli isolated from Chilean Patagonian cold freshwater rivers to produce bioactive metabolites was evaluated. The strains were isolated from cold waters rivers and identified by their biochemical properties and 16S rRNA gene analysis. The metabolites fractions showing antibacterial activity were obtained by solvent extraction and partially characterized by gas–mass chromatography (GC-MS). Antibacterial activity of the fractions was evaluated by an agar-well diffusion test upon 14 bacterial strains, both Gram positive and Gram negative. Thermal and proteolytic resistances of the antibacterial metabolites fractions were also evaluated. Molecular analysis allows the identification of the three Patagonian strains as Pseudomonas sp. RG-6 (Pseudomonas brenneri 99.6 % identity), Pseudomonas sp. RG-8 (Pseudomonas trivialis 99.6 % identity) and Yersinia sp. RP-3 (Yersinia aldovae 99.5 % identity). These extracts were able to inhibit both Gram-positive and Gram-negative bacteria but not Listeria monocytogenes. The antibacterial activity of the filtrated supernatants was lost at temperatures ≥60 °C, and was not affected by proteinase K treatment. The chemical structure of the active molecule remains to be elucidated, although the GC-MS analysis of the filtrates suggests that compounds like sesquiterpenes derivatives from β-maaliene or δ-selinene could be responsible of this antibacterial activity. Pristine cold freshwater streams showed to be interesting sources of metabolites-producing microorganisms with antibacterial activity.     

On the mode of action of 5-diazouracil on bacterial cell division

Cell division by strains ofEscherichia coli andSalmonella typhimurium is inhibited by 5-diazouracil (5-DU). Division recovers in the presence of the inhibitor after a period which is temperature-dependent. Recovery is probably due to breakdown of 5-DU and the rate of this breakdown is apparently increased at alkaline pH. Growth with 5-DU caused only a slight reduction in the rate of murein synthesis and no alteration in the properties or composition of membranes ofS. typhimurium. The agent caused chaining inStreptococcus fecalis and inhibition of the penicillin-induced lysis ofS. typhimurium. These effects may have been due to direct inhibition of lysin activity but an indirect effect seems more likely. The most marked effect of 5-DU onS. typhimurium was to cause a transient inhibition of DNA synthesis. Since 5-DU did not stop uncoupled cell division (i.e. division occurring independently of DNA replication) and sincelon^? strains were more sensitive to 5-DU thanlon⁺ strains, it was concluded that 5-DU acts on cell division via an inhibitory effect on DNA replication.     

Some properties and protective activity of specific DLE against Salmonella cholerae suis infection

From a rabbit lymphoid tissue, twice immunized with a Salmonella ch. suis vaccine, was obtained a dialysable leucocyte extract (DLE) (m.w. 10 000Da; protein content 1.14 mg/ml; content of ribose 2.7 mg/ml; A₂₆₀/A₂₈₀ ratio 2.17 and pH 6.8). By gel filtration on Sephadex G-25, six peaks were obtained and activity was found in peak IV. The activity of the extract was determined by a dermo-application test (DAT) on 10 cows. The protective effect was tested by a challenge with Salmonella ch. suis and Salmonella dublin pathogen strains on white mice intraperitoneally treated with DLE. The DAT proved to be positive in 8 of the 10 cows. When applied on white mice, it induced a high specific protective effect against Salmonella ch. suis (70%), but not against Salmonella dublin infection.     

Production and characterization of antibacterial compounds produced by Pediococcus damnosus and Pediococcus pentosaceus

The broad-spectrum antibacterial activity exhibited by three Pediococcus strains isolated from beer was preliminarily characterized. Factors affecting the production rate of bacterial inhibitors were screened and the effects of simultaneous cultivation of Lactococcus and Pediococcus on the production of inhibitory substances were studied. The antibacterial activity against a range of Gram-negative test organisms was not affected by catalase or proteolytic enzymes and was extremely thermotolerant. Production of the inhibitors was maximal between pH 6 and pH 7. A growth medium containing unhopped end-fermented wort was beneficial for the production of inhibitors, particularly by the Pediococcus damnosus strain, and anaerobic growth conditions were preferable. The antagonistic activity against the Gram-negative test organism Salmonella infantis could be demonstrated after an incubation period of only 2 d if the Pediococcus and Lactococcus strains were incubated simultaneously as a mixed population.     

Elucidation of cathepsin B-like activity associated with extracts of human myelin basic protein

Myelin basic protein (MBP) extracted from human delipidated white matter was found to be degraded at pH 3.0 by endogenous proteolytic activities of extracts. Electrophoretic peptide patterns were consistent with limited proteolysis of MBP. Based on pH, activation by EDTA and DTE, and inhibition by p-CMPS, E-64 and, in particular, by leupeptin, the protease involved was tentatively identified as cathepsin B or a cathepsin B-like enzyme. As pepstatin failed to inhibit acid proteolysis of MBP cathepsin D was ruled out.     

Insecticidal effect of Canavalia ensiformis major urease on nymphs of the milkweed bug Oncopeltus fasciatus and characterization of digestive peptidases

Jackbean (Canavalia ensiformis) ureases are entomotoxic upon the release of internal peptides by insect’s digestive enzymes. Here we studied the digestive peptidases of Oncopeltus fasciatus (milkweed bug) and its susceptibility to jackbean urease (JBU). O. fasciatus nymphs fed urease showed a mortality rate higher than 80% after two weeks. Homogenates of midguts dissected from fourth instars were used to perform proteolytic activity assays. The homogenates hydrolyzed JBU in vitro, yielding a fragment similar in size to known entomotoxic peptides. The major proteolytic activity at pH 4.0 upon protein substrates was blocked by specific inhibitors of aspartic and cysteine peptidases, but not significantly affected by inhibitors of metallopeptidases or serine peptidases. The optimal activity upon N-Cbz-Phe-Arg-MCA was at pH 5.0, with complete blockage by E-64 in all pH tested. Optimal activity upon Abz-AIAFFSRQ-EDDnp (a substrate for aspartic peptidases) was detected at pH 5.0, with partial inhibition by Pepstatin A in the pH range 2-8. Fluorogenic substrates corresponding to the N- and C-terminal regions flanking a known entomotoxic peptide within urease sequence were also tested. While the midgut homogenate did not hydrolyze the N-terminal peptide, it cleaved the C-terminal peptide maximally at pH 4.0-5.0, and this activity was inhibited by E-64 (10 ??M). The midgut homogenate was submitted to ion-exchange chromatography followed by gel filtration. A 22 kDa active fraction was obtained, resolved in SDS-PAGE (12%), the corresponding band was in-gel digested by trypsin, the peptides were analyzed by mass spectrometry, retrieving a cathepsin L protein. The purified cathepsin L was shown to have at least two possible cleavage sites within the urease sequence, and might be able to release a known insecticidal peptide in a single or cascade event. The results suggest that susceptibility of O. fasciatus nymphs to jackbean urease is, like in other insect models, due mostly to limited proteolysis of ingested protein and subsequent release of entomotoxic peptide(s) by cathepsin-like digestive enzymes.     

Survey of Phytochemical Composition and Biological Effects of Three Extracts from a Wild Plant (Cotoneaster nummularia Fisch. et Mey.): A Potential Source for Functional Food Ingredients and Drug Formulations

This study was focused on the analysis of the phenolic content, antioxidant, antibacterial, anti-cholinesterase, anti-tyrosinase, anti-amylase and anti-glucosidase activity of three solvent extracts from Cotoneaster nummularia. Moreover, water extract was tested in terms of mutagenic/anti-mutagenic effects. The antioxidant activities of these extracts were evaluated by DPPH, ABTS, O₂, metal chelating, phosphomolybdenum, β-carotene/linoleic acid, ferric and cupric reducing power assays. Enzyme inhibitory activities were also examined with colorimetric methods. Generally, methanol and water extracts exhibited excellent biological activities. These extracts were rich in phenolic and flavonoid content. Furthermore, Cotoneaster extracts indicated appreciable antibacterial properties against human pathogen strains. HPLC analysis showed that ferulic acid, chlorogenic acid, (-) – epicatechin and (+)-catechin were the major phenolics in extracts tested. These data offer that these extracts from C. nummularia may be considered as a potential source of biological agents for developing functional foods or drug formulations.     

Antimicrobial compounds produced by probiotic Lactobacillus brevis isolated from dairy products

A total of 38 lactic acid bacteria, belonging to Lactobacillus, isolated from 24 samples of traditional Egyptian dairy products, were screened for antimicrobial activity against different Gram-positive and Gram-negative bacteria. A strain of Lactobacillus brevis showed the best inhibitory activity when tested by well diffusion assay. The antibacterial activity was pronounced between early logarithmic and early stationary phases. The strain produced a heat-stable antimicrobial compound showing no reduction in activity after heat treatment from 60 to 100°C for 15 and 30 min. Since it was inactivated by proteolytic enzymes, it is considered to be proteinaceous in nature and, therefore, referred to as a bacteriocin-like substance. This compound was also active over a wide pH range (pH 2–6). The antimicrobial compound was partially purified by 40% ammonium sulfate precipitation. Lactobacillus brevis was tested for its in vitro antibiotics susceptibility, tolerance to bile salts, resistance to low pH values, acidifying activity, proteolytic activity, and haemolytic activity. The results showed the potential of L. brevis strain as a probiotic culture, and hence it can be utilized in the manufacturing of pharmaceuticals and dietary supplements.