Changes in antibacterial and proteolytic activity of polymorphonuclear leucocytes following the whole body x-irradiation of rabbits

In whole body x-irradiated rabbits with 150 r, 500 r and 1000 r an antibacterial and proteolytic activity in extracts of polymorphonuclear leucocytes obtained from peritoneal exudate was tested immediately and 1, 3, 6, 10 and 21 days following irradiation. The changes in antibacterial activity tested inEscherichia coli, Bethesda andSalmonella adelaide strains depended on the intensity of radiation and time interval between radiation and collection of leucocytes. With increasing radiation the antibacterial activity inBethesda andSalmonella adelaide strains was decreased. In rabbits irradiated 150 r and 500 r a decrease or even a disappearance of bactericidal activity on the sixth day in all strains tested was found, whereas after an irradiation with 1000 r the antibacterial activity was found to be low immediately after irradiation and this decrease could be detected up to the tenth day. UsingEscherichia coli strains the antibacterial activity of leucocyte extracts did not show such a regular dependence on the intensity of radiation and time as with other strains used. The proteolytic activity of leucocyte extracts tested at pH 3.5 (cathepsin D and E) and pH 2.0 (cathepsin E) within 3 days after irradiation was higher with increasing radiation. At other time intervals the activity did not show regular changes. The dynamics of changes in proteolytic activity at both pH was different and also a relation between proteolytic and antibacterial activity was not found.     

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.     

Digestive proteolytic activity in the pistachio green stink bug,Brachynema germari Kolenati (Hemiptera: Pentatomidae)

Proteolytic activity in the digestive system of the pistachio green stink bug, Brachynema germari, was investigated. The maximum total proteolytic activity in the midgut extract was observed at pH 5, suggesting the presence of cysteine proteases. Hydrolyzing the specific substrates for cysteine proteases revealed the presence of cathepsin B and cathepsin L activities in the midgut extract. The presence of cysteine proteases was confirmed by their noticeable inhibition and activation due to specific inhibitors and activators, respectively. The significant inhibition of chymotryptic activity by the inhibitors showed the presence of chymotrypsin in the midgut. No considerable tryptic activity was observed in the midgut extract. There was no detectable total proteolytic activity in the salivary gland extract. Tryptic activity of the salivary gland extract was also inhibited by the specific inhibitors. The substrates for cysteine proteases were also slightly hydrolyzed by the salivary gland extract. Zymogram analysis showed at least one distinct band due to cysteine protease activity in the midgut extract, and the cysteine protease inhibitor caused almost complete disappearance of the band. Cathepsin B and L activities were mainly detected in midgut divisions m₁ and m₃, respectively, and maximum chymotrypsin and trypsin activities were observed in m₃. In general, the results revealed the significant presence of cathepsin B, cathepsin L, and chymotrypsin proteases in the midgut extract. The major proteolytic activity in the salivary glands seems to be conducted by trypsin-like proteases.     

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.     

Progesterone-dependent cathepsin L proteolytic activity in cat uterine flushings

Sequence analysis of a cDNA clone for the progesterone-dependent protein (PDP) of the cat uterus revealed that PDP may be cathepsin L. This study was undertaken to directly measure the cathepsin L activity in uterine flushings from pregnant and ovariectomized steroid-treated animals in order to confirm that PDP is cathepsin L. Optimum activity toward the substrate Z-Phe-Arg-NMec was observed at a pH of 5-6. Z-Phe-Phe-CHN2, a specific inhibitor of cathepsin L, significantly inhibited the proteolytic activity present in uterine flushings. Immunoabsorption of PDP from uterine flushings obtained from progesterone (P)-treated cats reduced cathepsin L proteolytic activity to levels observed in ovariectomized and estradiol (E2)-treated animals. In E2-primed and E2 + P-treated animals, proteolytic activity in uterine flushings was detectable after 7 days and peaked after 11-13 days of E2 + P treatment. This proteolytic activity was also dramatically increased before implantation (10-12 days after coitus) in pregnant cats. Thus, our data indicate that changes in cathepsin L activity in uterine flushings are correlated with changes in PDP, the uterine protein synthesized and released from the epithelial cells of the deep uterine glands. PDP, via its cathepsin L proteolytic activity, may play a role in the implantation process.     

In vitro evaluation of Datura innoxia (thorn-apple) for potential antibacterial activity

Various parts of Datura innoxia were examined for potential antibacterial activity by preparing their crude aqueous and organic extracts against Gram-negative bacteria (Escherichia coli and Salmonella typhi) and Gram-positive bacteria (Bacillus cereus, Bacillus subtilis and Staphylococcus aureus). The results of agar well diffusion assay indicated that the pattern of inhibition depends largely upon the plant part, solvent used for extraction and the organism tested. Extracts prepared from leaves were shown to have better efficacy than stem and root extracts. Organic extracts provided potent antibacterial activity as compared to aqueous extracts. Among all the extracts, methanolic extract was found most active against almost all the bacterial species tested. Gram-positive bacteria were found most sensitive as compared to Gram-negative bacteria. Staphylococcus aureus was signifi cantly inhibited by almost all the extracts even at very low MIC followed by other Gram-positives. For Escherichia coli (a Gram-negative bacterium), the end point was not reached for ethyl acetate extract while it was very high for other extracts. The study promises an interesting future for designing a potentially active antibacterial agent from Datura innoxia.     

Proteolytic activity in relationship to senescence and cotyledonary development in Pisum sativum L.

Changes in the weight and in the chlorophyll, free amino-acid and protein content of developing and senescing, vegetative and reproductive organs of Pisum sativum L. (cv. Burpeeana) were measured, and the proteolytic activity in extracts from the senescing leaf and the subtended pod was followed in relation to these changes. Protein content decreased in the ageing leaf and pod while it increased in the developing cotyledon. The proteolytic activity of the leaf did not increase as the leaf protein content decreased. In contrast, proteolytic activity in the subtended pod increased while the protein level decreased. The proteolytic activity in the extracts from the ageing organs was greater than the rates of protein loss. The proteolytic activity of leaf and pod extracts was greater on protein prepared from the respective organ than on non-physiological substrates. Proteolysis was increased by 2-mercaptoethanol and ethylenediaminetetraacetate but was not influenced by addition of ATP to the reaction mixture. The pH optimum was at 5.0. Free amino acids did not accumulate in the senescing leaf or pod when protein was degraded in each organ. It is suggested that these amino acids were quickly metabolized in situ or translocated to sink areas in the plant, especially to the developing seeds.     

The isolation and properties of a non-pepsin proteinase from human gastric mucosa.

1. A non-pepsin proteinase, proteinase 2, was successfully isolated free from pepsinogen (by repetitive chromatography on DEAE- and CM-celluloses) from the gastric mucosa of a patient with a duodenal ulcer and the uninvaded mucosa of a patient with a gastric adenocarcinoma. 2. Proteinases 1a and 1b, found in gastric adenocarcinoma, were not found in the gastic mucosa of these patients. 3. Proteinase 2 was shown to have an asymmetrical broad pH-activity curve with a maximum over the pH range 3.0-3.7. 4. Proteolytic activity of proteinase 2 was inhibited by pepstatin; the concentration of pepstatin giving 50% inhibition is of the order of 3nm. 5. Inhibition of proteolytic activity by carbenoxolone and related triterpenoids indicated that at pH 4.0 proteinase 2 possesses structural characteristics relating it to the pepsins and at pH 7.4 to the pepsinogens. 6. The sites of cleavage of the B-chain of oxidized insulin for proteinase 2 at pH 1.7 and pH 3.5 were shown to be similar to those previously established for human pepsin 3 and for the cathepsin E of rabbit bone marrow. 7. The non-pepsin proteinase 2 (cathepsin) of human gastric mucosa has properties more similar to cathepsin E than to the cathepsins D.     

Entamoeba histolytica and E. invadens: Sulfhydryl-Dependent Proteolytic Activity1

Sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (2-ME) activated proteolytic enzymes present in extracts of Entamoeba histolytica and E. invadens; SDS (0.5%) and 2-ME (1.4 and 715 mM) doubled the enzymatic activity when assayed on a stained insoluble substrate. Urea (4 M) did not reduce this activity, suggesting that amebic proteases are stable in the above denaturant conditions. Specific reagents for sulfhydryl (-SH) groups completely inhibited proteolytic activity regardless of pH. Inhibition with alkylating agents, such as N-ethylmaleimide and iodoacetamide, was reversed with 715 mM 2-ME as was also observed with papain. We conclude from these results that the main proteolytic enzymes contained in extracts of E. histolytica and E. invadens are dependent on free thiol groups.     

Inhibition of cysteine and aspartyl proteinases in the alfalfa weevil midgut with biochemical and plant-derived proteinase inhibitors

Proteolytic activities in alfalfa weevil (Hypera postica) larval midguts have been characterized. Effects of pH, thiol activators, low-molecular weight inhibitors, and proteinase inhibitors (PIs) on general substrate hydrolysis by midgut extracts were determined. Hemoglobinolytic activity was highest in the acidic to mildly acidic pH range, but was maximal at pH 3.5. Addition of thiol-activators dithiothreitol (DTT), 2-mercaptoethanol (2-ME), or L-cysteine had little effect on hemoglobin hydrolysis at pH 3.5, but enhanced azocaseinolytic activity two to three-fold at pH 5.0. The broad cysteine PI E-64 reduced azocaseinolytic activity by 64% or 42% at pH 5 in the presence or absence of 5 mM L-cysteine, respectively. Inhibition by diazomethyl ketones, Z-Phe-Phe-CHN(2) and Z-Phe-Ala-CHN(2), suggest that cathepsins L and B are present and comprise approximately 70% and 30% of the cysteine proteolytic activity, respectively. An aspartyl proteinase component was identified using pepstatin A, which inhibited 32% (pH 3.5, hemoglobin) and 50% (pH 5, azocasein) of total proteolytic activity. This activity was completely inhibited by an aspartyl proteinase inhibitor from potato (API), and is consistent with the action of a cathepsin D-like enzyme. Hence, genes encoding PIs with specificity toward cathepsins L, B and D could potentially be effective for control of alfalfa weevil using transgenic plants.     

Effect of culture conditions on mycelial growth, antibacterial activity, and metabolite profiles of the marine-derived fungus Arthrinium c.f. saccharicola

The effects of culture conditions and competitive cultivation with bacteria on mycelial growth, metabolite profile, and antibacterial activity of the marine-derived fungus Arthrinium c.f. saccharicola were investigated. The fungus grew faster at 30°C, at pH 6.5 and in freshwater medium, while exhibited higher antibacterial activity at 25°C, at pH 4.5, 5.5, and 7.5, and in 34 ppt seawater medium. The fungus grew faster in a high-nitrogen medium that contained 0.5% peptone and/or 0.5% yeast extract, while exhibiting higher bioactivity in a high-carbon medium that contained 2% glucose. The fungal growth was inhibited when it was co-cultured with six bacterial species, particularly the bacterium Pseudoalteromonas piscida. The addition of a cell free culture broth of this bacterium significantly increased the bioactivity of the fungus. Metabolite profiles of the fungus revealed by gas chromatography (GC)-mass spectrometry showed clear difference among different treatments, and the change of relative area of three peaks in GC profile followed a similar trend with the bioactivity variation of fungal extracts. Our results showed clear differences in the optimal conditions for achieving maximal mycelial growth and bioactivity of the fungus, which is important for the further study on the mass cultivation and bioactive compounds isolation from this fungus.     

苹果渣乙醇提取物抗菌活性及防腐作用研究

为提高苹果渣资源利用率,探究苹果渣乙醇提取物的抗菌活性和防腐性能,该文采用微波辅助提取法制取苹果渣乙醇提取物,用抑菌圈实验测定其抗菌活性,并研究了其防腐作用.结果表明:(1)苹果渣乙醇提取物对酵母菌抑制作用不明显(抑菌圈直径<1 mm),对金黄色葡萄球菌和大肠杆菌的抑菌作用较明显(抑菌圈直径为6～9 mm),最佳抑菌浓...     

A homologue of cathepsin L identified in conditioned medium from Sf9 insect cells

Gelatin zymography revealed the presence of proteolytic activity in conditioned medium (CM) from a serum-free, non-infected Spodoptera frugiperda, Sf9 insect cell culture. Two peptidase bands at about 49 and 39 kDa were detected and found to be proform and active form of the same enzyme. The 49-kDa form was visible on zymogram gels in samples of CM taken on days 4 and 5 of an Sf9 culture, while the 39-kDa form was seen on days 6 and 7. On basis of the inhibitor profile and substrate range, the enzyme was identified as an Sf9 homologue of cathepsin L, a papain-like cysteine peptidase. After lowering the pH of Sf9 CM to 3.5, an additional peptidase band at 22 kDa appeared. This peptidase showed the same inhibitor profile, substrate range and optimum pH (5.0) as the 39-kDa form, indicating that Sf9 cathepsin L has two active forms, at 39 and 22 kDa. Addition of the cysteine peptidase inhibitor E-64c to an Sf9 culture inhibited all proteolytic activities of Sf9 cathepsin L but did not influence the proliferation of Sf9 cells.     

Reducing power and radical scavenging activity of four Parmeliaceae species

The methanol extracts of four Parmeliaceae lichens (Hypogymnia physodes, Evernia prunastri, Flavoparmelia caperata and Parmelia sulcata) were screened for antioxidant properties and total phenol content. The H. physodes extract was the most effective at reducing iron(III) and scavenging 1,1-diphenyl-2-picrylhydrazyl radicals, while the P. sulcata extract was the most effective in reducing molybdenum(VI) in an acidic medium. The E. prunastri and H. physodes extracts contained more Folin-Ciocalteu reagent reactive substances than the F. caperata and P. sulcata extracts. Significant activity of the H. physodes extract in DPPH and reducing Fe(III) assays suggest that this lichen can be considered as a potential source of antioxidants.     

Phytochemical Profiling and Pharmacological Evaluation of Leaf Extracts of Ruellia tuberosa L.: An In Vitro and In Silico Approach

The present study was designed to appraise the photoprotective, antioxidant, and antibacterial bioactivities of Ruellia tuberosa leaves extracts (RtPE, RtChl, RtEA, RtAc, RtMe, and RtHMe). The results showed that, RtHMe extracts of R. tuberosa was rich in total phenolic content, i. e., 1.60 mgGAE/g dry extract, while highest total flavonoid content was found in RtAc extract, i. e., 0.40 mgQE/g. RtMe showed effective antioxidant activity (%RSA: 58.16) at the concentration of 120 μL. RtMe, RtEA and RtHMe exhibited effective in vitro antibacterial activity against Gram-negative bacteria (E. coli). In silico docking studies revealed that paucifloside (−11.743 kcal/mol), indole-3-carboxaldehyde (−7.519 kcal/mol), nuomioside (−7.275 kcal/mol), isocassifolioside (−6.992 kcal/mol) showed best docking score against PDB ID 2EX8 [penicillin binding protein 4 (dacB) from Escherichia coli, complexed with penicillin-G], PDB ID 6CQA (E. coli dihydrofolate reductase protein complexed with inhibitor AMPQD), PDB ID 2Y2I [Penicillin-binding protein 1B in complex with an alkyl boronate (ZA3)] and PDB ID 2OLV (from S. aureus), respectively. Docked phytochemicals also showed good drug likeness properties.     

Chemical composition and antibacterial activity of Cordia verbenacea extracts obtained by different methods

The present study describes the chemical composition and the antibacterial activity of extracts from Cordia verbenacea DC (Borraginaceae), a traditional medicinal plant that grows widely along the southeastern coast of Brazil. The extracts were obtained using different extraction techniques: high-pressure operations and low-pressure methods. The high-pressure technique was applied to obtain C. verbenacea extracts using pure CO₂ and CO₂ with co-solvent at pressures up to 30 MPa and temperatures of 30, 40 and 50 °C. Organic solvents such as n-hexane, ethyl acetate, ethanol, acetone and dichloromethane were used to obtain extracts by low-pressure processes. The antibacterial activity of the extracts was also subjected to screening against four strains of bacteria using the agar dilution method. The extraction yields were up to 5.0% w/w and up to 8.6% w/w for supercritical fluid extraction with pure CO₂ and with ethyl acetate as co-solvent, respectively, while the low-pressure extraction indicates yields up to 24.0% w/w in the soxhlet extraction using water and aqueous mixture with 50% ethanol as solvents. The inhibitory activity of the extracts in Gram-positive bacteria was significantly higher than in Gram-negative. The quantification and the identification of the extracts recovered were accomplished using GC/MS analysis. The most important components identified in the extract were artemetin, β-sitosterol, α-humulene and β-caryophyllene, among others.     

Comparative activity against pathogenic bacteria of the root,stem, and leaf of <Emphasis Type="Italic">Raphanus sativus</Emphasis> grown in India

Aqueous, methanol, ethyl acetate, and chloroform extracts of the root, stem, and leaf of Raphanus sativus were studied for antibacterial activity against food-borne and resistant pathogens. All extracts except the aqueous extracts had significant broad-spectrum inhibitory activity. The ethyl acetate extract of the root had the potent antibacterial activity, with a minimum inhibitory concentration (MIC) of 0.016–0.064 mg/ml and a minimum bactericidal concentration (MBC) of 0.016–0.512 mg/ml against health-damaging bacteria. This was followed by the ethyl acetate extracts of the leaf and stem with MICs of 0.064–0.256 and 0.128–0.256 mg/ml, respectively and MBCs of 0.128–2.05 and 0.256–2.05 mg/ml, respectively. The ethyl acetate extracts of the different parts of R. sativus retained their antibacterial activity after heat treatment at 100°C for 30 min, and their antibacterial activity was enhanced when pH was maintained in the acidic range. Hence this study, for the first time, demonstrated that the root, stem, and leaf of R. sativus had significant bactericidal effects against human pathogenic bacteria, justifying their traditional use as anti-infective agents in herbal medicines.     

Preservation of the proteolytic activity of a bovine spleen lysosomal-enriched extract using various freezing conditions

The preservation of the proteolytic activity of a bovine spleen lysosomal-enriched (BSLE) extract was investigated. The BSLE extract (pH = 5.8), was subjected to storage under different conditions: refrigeration at 0 degrees C for 60 days; freezing at -20 degrees C -either directly or previously frozen in liquid nitrogen-, -80 degrees C and in liquid nitrogen; freeze-drying and stored at 0 degrees C; and freezing at -20 degrees C or in liquid nitrogen in the presence of glycerol and sorbitol as cryoprotectants. Freezing at low temperatures (-80 degrees C and in liquid nitrogen) was most effective for preserving about 100% of the initial activity of all cathepsins (B, B+L and D), as well as the activity of the extract on myofibrils, for two years. Freezing at -20 degrees C, on the contrary, led to significant (P < 0.01) losses of activity. Freeze-drying was able to preserve cathepsin activity, while it failed to maintain activity on myofibrils. Both cryoprotectants sorbitol and glycerol significantly (P < 0.01) enhanced enzyme preservation, particularly cathepsin D and the activity on myofibrils, even at a freezing temperature of -20 degrees C.     

Antioxidant and antimicrobial activity of some lichen species

The aim of the research is to explore the overall in vitro antioxidant activity, total phenol content, reduction power and antimicrobial activity of the methanol extracts of the lichens Cetraria pinastri, Cladonia digitata, Cladonia fimbriata, Fulgensia fulgens, Ochrolechia parella and Parmelia crinita. The methanol extract of the Cetraria pinastri showed a strong antioxidant activity, whereas the extracts of the species Fulgenesi fulgens, Cladonia fimbriata and Parmelia crinita showed the moderate one and the extract of the species Ochrolechia parella and Cladonia digitata the weak one. The methanol extract of the lichen Cetraria pinastri had the biggest total phenol content (32.9 mg/g of the dry extract). A certain correlation was established between the antioxidant activity and the total phenol content for the researched lichen extracts. The work also explores the antimicrobial activity of the methanol extracts of the mentioned species of lichens against six bacterial and eleven fungi species by the disc-diffusion method and by establishing the minimum inhibitory concentration (MIC). The methanol extracts of the lichens Cetraria pinastri and Parmelia crinita showed the strongest both antibacterial and antifungal activity against most of the tested microorganisms. These researches suggest that the lichens Cetraria prunastri can be used as new sources of the natural antioxidants and the substances with antimicrobial features.     

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.