Various Degrees Of Inhibition Research Articles

Differential Effect of 4H-Benzo[d] [1, 3]oxazines on the Proliferation of Breast Cancer Cell Lines.

A family of 4H-benzo[d][1,3]oxazines were obtained from a group of N-(2-alkynyl)aryl benzamides precursors via gold(I) catalysed chemoselective 6-exo-dig C-O cyclization. The precursors and oxazines obtained were studied in breast cancer cell lines MCF-7, CAMA-1, HCC1954 and SKBR-3 with differential biological activity showing various degrees of inhibition with a notable effect for those that had an aryl substituted at C-2 of the molecules. 4H-benzo[d][1,3]oxazines showed an IC50 rating from 0.30 to 157.4 µM in MCF-7, 0.16 to 139 in CAMA-1, 0.09 to 93.08 in SKBR-3, and 0.51 to 157.2 in HCC1954 cells. We observed that etoposide is similar to benzoxazines while taxol effect is more potent. Four cell lines responded to benzoxazines while SKBR-3 cell line responded to precursors and benzoxazines. Compounds 16, 24, 25 and 26 have the potent effect in cell proliferation inhibition in the 4 cell lines tested and correlated with oxidant activity suggesting a possible mechanism by ROS generation. These compounds represent possible drug candidates for the treatment of breast cancer. However, further trials are needed to elucidate its full effect on cellular and molecular features of cancer.

Influence of Methods of Corn Starch Modification and Used Sweetener on the Functional Properties of Blackberry Jelly-like Dessert.

A study was conducted on selected physicochemical properties of blackberry jelly-like desserts (kissel) prepared from physically modified starches (with various degrees of inhibition) and chemically modified starches (with various degrees of cross-linking). The desserts were conventionally sweetened with saccharose (S) or, as a dietary alternative, xylitol (X). The characteristics of changes in the viscosity of the kissels as a function of temperature and time were determined. It was noted that regardless of the sweetener used, the viscosity of the kissels increased with the decreasing degree of inhibition (high < medium < low). Regardless of the kind of modification of the starch used for the preparation of the kissels and of the kind of sweetener, thixotropy was observed. Desserts prepared from inhibited starch with xylitol (CL + X) were characterised by the biggest range of their hysteresis loop. Progressing retrogradation was noted with the decrease in the temperature of the experiment (+20 °C and +4 °C). After 7 days of storage, kissels sweetened with saccharose were characterised by a low transparency, which may indicate retarded retrogradation; however, on day 28, the transparency significantly increased, exceeding the values of transmittance for samples sweetened with xylitol. The tendency towards syneresis was tested at +4 °C and −22 °C. The substitution of saccharose with xylitol only caused a slight modification of viscosity. Regardless of the sweetener used and of the level of starch inhibition, lower ranges of the hysteresis loop were noted (apart from CL + X) than in the case of kissels obtained from chemically modified starches. Distinctly lower values of kissel “aging” indices were noted in the case of samples obtained from inhibited starches, and their colour did not significantly differ in relation to the dessert prepared from native starch.

Preparation and Properties of Papain Precipitated from Fresh Latex of Papaya Fruits (Carica papaya)

Papain was precipitated from cysteine hydrochloride extract of fresh latex of papaya fruits planted in AlexandriaGovernorate, Egypt. Three precipitants; namely, ethyl alcohol, acetone and sodium chloride resulted in proteolyticactivity recovery 84.8 (of three fractions), 85.2 and 76.7 %, respectively. Acetone was found to be the most promisingrapid and simple conditions for preparing acetone dry powder of partially purified crude papain. Papain propertiesindicated that the maximum activity was attained at pH 7.5–8.0, and temperature of 75 -85°C. The activity was notaffected by Na+ or Ca++ ions. Various degrees of inhibition were exerted by Ni+,+, Co++, Pb ++,and Hg++., while Cu++,caused complete inhibition .On the other hand, the proteolytic activity increased to various levels, in the presenceof Sn++, cyanide, thioglycolic acid, and cysteine hydrochloride. The catalytic activation (49%) of Sn++ was found todepend to some extent on the concentration. A mixed stored papain-oryzae protease preparation ,was found to retain71, 63, and 54 % of its proteolytic activity upon storage at 25°C for 1, 2 ,and 3 months ,respectively. The retainedactivity was more pronounced, being 92, 80,and 69 %, respectively upon storage at –5°C for the aformentioned durations,accordingly, storage at –5°C for up to 2 months, was recommended. The storage stability of papain powder andsolutions were studied. A papain-oryzae protease preparation was formulated for further use in meat tenderization.

Inhibition of Streptococcus pneumoniae hyaluronidase by honeys of Malaysian origins

Streptococcus pneumoniae, a major human pathogen causing meningitis, pneumonia and bacteraemia produce hyaluronidase as its virulence factor. This enzyme increase bacterial permeability to the host tissue by degrading hyaluronic acids that maintain the viscosity of connective tissues. Limited hyaluronidase inhibitor are available at the moment, thus lead us to identify potential hyaluronidase inhibitor from local products which is honeys. Three local honeys (Tualang, Kelulut and Gelam) have been selected and undergo hyaluronidase inhibition test through hyaluronic acids turbidity reduction assay. Honeys that show highest and lowest anti-hyaluronidase were analysed for flavonoid and phenolic content, to identify association between the contents and hyaluronidase inhibition rate. All honeys showed various degrees of inhibition against hyaluronidase of S. pneumoniae where honey with the highest anti-hyaluronidase activity is Kelulut with 18% inhibition while honey with the lowest anti-hyaluronidase activity is Gelam with 2% inhibition. Kelulut also demonstrated highest phenolic content, where significant association was found between the inhibition and the content. Therefore, this study supported the claim that higher phenolic contents indicate greater inhibitory activity on hyaluronidase enzyme. The findings suggest that local Malaysian honey have potential as hyaluronidase inhibitor which could be beneficial for future treatment against S. pneumoniae infections.

Monitoring Antifungal Agents of Artemisia annua against Fusarium oxysporum and Fusarium solani, Associated with Panax notoginseng Root-Rot Disease.

Root rot of Panax notoginseng has received great attention due to its threat on the plantation and sustainable utilization of P. notoginseng. To suppress the root-rot disease, natural ingredients are of great importance because of their environment friendly properties. In this study, we found that the methanol extract from Artemisia annua leaves has strong antifungal effects on the growth of Fusarium oxysporum and Fusarium solani resulting into root-rot disease. Essential oil (EO) thereof was found to be the most active. GC-MS analysis revealed 58 ingredients and camphor, camphene, β-caryophyllene, and germacrene D were identified as the major ingredients. Further antifungal assays showed that the main compounds exhibit various degrees of inhibition against all the fungi tested. In addition, synergistic effects between A. annua EO and chemical fungicides were examined. Finally, in vivo experiments were conducted and disclosed that P. notoginseng root rot could be largely inhibited by the petroleum ether extract from A. annua, indicating that A. annua could be a good source for controlling P. notoginseng root-rot.

High Temperature Affects Cytokine Release by Human Peripheral Blood Mononuclear Cells

Background: Fever is one of the leading signs of the inflammatory process and it is one of the mechanisms that activate the immune system to defend the organism from various pathogens. For this goal, the peripheral blood mononuclear cells (PBMC) are among the first to be mobilized by triggering their capacity for phagocytosis and inflammatory cytokine production. However, their activation in the living organism is the outcome of several factors, including fever. The aim of the present work was to examine the effect of elevated temperature only on the capacity of human PBMC to produce inflammatory cytokines Methods: Stimulated and non-stimulated PBMC from healthy donors were treated with 37°C and 40°C for 4 and 24 hours. At the end of the incubation period the level of TNFα, IL-1β, IL-6, IL-10, IL-1ra, IL-2 and IFNγ was detected using ELISA kits. Results: Non-stimulated PBMC incubated at 37°C produced similar amounts of TNFα and IL-1β at 4 and 24 hrs. The expression of IL-6, IL-10 and IL-1ra was higher after 24 hrs as compared with that produced after 4 hrs. As for LPS-stimulated cells the production of all cytokines tested was increased significantly during 24 hrs of incubation except for the level of TNFα that did not differ significantly between 4 and 24 hrs of stimulation. Non-stimulated PBMC did not secrete detectable amounts of IL-2 or IFNγ, and that produced by PMA/ionomycin stimulated cells was significantly higher after 24 hrs of incubation as compared to 4 hrs. Elevation of the incubation temperature from 37°C to 40°C caused various degrees of inhibition in the generation of both pro- and anti-inflammatory cytokines. Conclusions: Exposure of unstimulated and stimulated PBMC to 40°C induced various degrees of inhibition in the production of both pro- and anti-inflammatory cytokines compared to cells incubated at 37°C. This observation may clarify the way the immune cells react in cases with fever.

Synergy between rhinacanthins from Rhinacanthus nasutus in inhibition against mosquito cytochrome P450 enzymes.

The cytochrome P450 monooxygenases play a major role in insecticide detoxification and become a target for development of insecticide synergists. In this study, a collection of rhinacanthins (rhinacanthin-D, -E, -G, -N, -Q, and -H/I) purified from Rhinacanthus nasutus, in addition to previously purified rhinacanthin-B and -C, were isolated. These compounds displayed various degrees of inhibition against benzyloxyresorufin-O-debenzylation mediated by CYP6AA3 and CYP6P7 which were implicated in pyrethroid resistance in Anopheles minimus malaria vector. Inhibition modes and kinetics were determined for each of rhinacanthins. Cell-based inhibition assays by rhinacanthins employing 3-(4, 5-dimethylthiazol-2-y-l)-2, 5-diphenyltetrazolium bromide (MTT) cytotoxicity test were explored their synergistic effects with cypermethrin toxicity on CYP6AA3- and CYP6P7-expressing Spodoptera frugiperda (Sf9) cells. Rhinacanthin-B, -D, -E, -G, and -N exhibited mechanism-based inhibition against CYP6AA3, an indication of irreversible inhibition, while rhinacanthin-B, -D, -G, and -N were mechanism-based inhibitors of CYP6P7. There was structure-function relationship of these rhinacanthins in inhibition effects against both enzymes. In vitro enzymatic inhibition assays revealed that there were synergistic interactions among rhinacanthins, except rhinacanthin-B and -Q, in inhibition against both enzymes. These rhinacanthins exerted synergism with cypermethrin toxicity on Sf9 cells expressing each of the two P450 enzymes via P450 inhibition and in addition could interact in synergy to further increase cypermethrin toxicity. The inhibition potentials, synergy among rhinacanthins in inhibition against the P450 detoxification enzymes, and synergism with cypermethrin toxicity of the R. nasutus constituents of reported herein could be beneficial to implement effective resistance management of mosquito vector control.

English

The indigenous Ganoderma mushroom belongs to the class Basidiomycetous. It has been used in different systems of traditional medication for the treatment of diseases of human beings and animals. It contains various triterpenes, polysaccharides, alkaloids and steroids known to have broad effects pharmacologically and anti-bacterial properties. The indigenous Ganoderma mushroom has been used by locals in Namibia traditionally as a source of medicine to fight skin and wound infections, and other ailments. There is a need to validate the traditional usage of Ganoderma mushroom extracts on Escherichia coli,Alcaligenes faecalis, Proteus vulgaris, Neisseria meningitidis, Bacillus cereus andStaphylococcus aureus. This is important for the Namibian local communities, where people do not have access to modern medicines and use traditional medicines. This study analyzed the anti-bacterial effects of crude organic and aqueous extracts of the mycochemical components of the indigenous Ganoderma mushroom. Using the agar disc diffusion method, the crude extracts of the Ganoderma mushroom exhibited various degrees of inhibition against the tested organisms. The widest inhibitory zones (19.0 mm) were obtained with the crude benzene extract of G. lucidum against E. coli and N. meningitidis. The lowest zone of inhibition (6.0 mm) was demonstrated with the aqueous extract against E. coli. The study has concluded that the crude extracts of the indigenous Namibian Ganoderma mushroom possess antibacterial properties to all Gram positive and negative strains tested. Key words: Ganoderma lucidum, antibacterial properties, Escherichia coli, Alcaligenesfaecalis, Proteus vulgaris, Neisseria meningitidis, Bacillus cereus and Staphylococcusaureus.

Inhibition of Digestive α-Amylases from Chilo Suppressalis Walker (Lepidoptera: Crambidae) by a Proteinaceous Extract of Citrullus Colocynthis L. (Cucurbitaceae)

Abstract The striped rice-stem borer, Chilo suppressalis (Walker) is one of the most important pest of rice worldwide. In this study, a proteinaceous extract from Citrullus colocynthis L. shows various degrees of inhibition to digestive α-amylases of C. suppressalis. Digestive α-amylases of larvae were inhibited by different concentrations (approximately 50%) of C. colocynthis amylase inhibitor (CCAI). One of the isozymes totally disappeared and the sharpness of another decreased on native-PAGE electrophoresis. The pH dependency of inhibition revealed that the enzyme was inhibited in a wide range of pH from 7-10, and the optimal pH of the enzyme occurred in lepidopteran larvae. The highest inhibition of α-amylase by CCAI was observed at 25°C; the temperature near the optimal temperature of amylolytic activity. A time-course experiment demonstrated that enzymatic activity was the highest, 30 min after the onset of the experiment, when the highest inhibition occurred. The enzyme kinetic studies using Lineweaver-Burk analysis, revealed a mixed inhibition of CCAI on α-amylase activity. The current study is the first basic one using α-amylase inhibitor against C. suppressalis. This study opens the way for transgenic rice varieties containing inhibitors

A hyperpolarization-activated ion current of amphibian oocytes

A comparative analysis of a hyperpolarization-activated ion current present in amphibian oocytes was performed using the two-electrode voltage-clamp technique in Xenopus laevis, Xenopus tropicalis, and Ambystoma mexicanum. This current appears to be driven mainly by Cl(-) ions, is independent of Ca(2+), and is made evident by applying extremely negative voltage pulses; it shows a slow activating phase and little or no desensitization. The pharmacological profile of the current is complex. The different channel blocker used for Cl(-), K(+), Na(+) and Ca(2+) conductances, exhibited various degrees of inhibition depending of the species. The profiles illustrate the intricacy of the components that give rise to this current. During X. laevis oogenesis, the hyperpolarization-activated current is present at all stages of oocytes tested (II-VI), and the amplitude of the current increases from about 50nA in stage I to more than 1μA in stage VI; nevertheless, there was no apparent modification of the kinetics. Our results suggest that the hyperpolarization-activated current is present both in order Anura and Urodela oocytes. However, the electrophysiological and pharmacological characteristics are quite perplexing and seem to suggest a mixture of ionic conductances that includes the activation of both anionic and cationic channels, most probably transiently opened due to the extreme hyperpolarizion of the plasma membrane. As a possible mechanism for the generation of the current, a kinetic model which fits the data suggests the opening of pores in the plasma membrane whose ion selectivity is dependent on the extracellular Cl(-) concentration. The extreme voltage conditions could induce the opening of otherwise latent pores in plasma membrane proteins (i.e., carriers), resembling the ´slippage´ events already described for some carriers. These observations should be valuable for other groups trying to express cloned, voltage-dependent ion channels in oocytes of amphibian in which hyperpolarizing voltage pulses are applied to activate the channels.

Comparative evaluation of nonylphenol isomers on steroidogenesis of rat Leydig Cells

Nonylphenol (NP) has been proven to be one of the most investigated xenohormones interacting with the estrogen receptor. Technical nonylphenol (t-NP) contains at least 20 para-substituted isomers. It has been shown that NP isomers vary in their estrogenic potency. So the use of mixtures or impure substances can lead to misinterpretations and unsatisfying conclusions. In the present study, experiments were performed to examine effects of NP isomers on steroidogenesis of rat Leydig cells. Primary cultured Leydig cells were exposed to NP isomers (p33-NP, p262-NP, p353-NP, p363-NP) at the optimized inhibitory concentration 5μmol/L for 6h. NP isomers showed various degrees of inhibition of testosterone biosynthesis, with p363-NP leading to the most significant decrease and others sharing the similar efficacy. The expression of 3b-HSD, Cyp11a1, Star and the apoptosis of Leydig cells were further measured to investigate the underlying mechanisms. We demonstrated that NP isomers can affect the steroidogenesis of rat Leydig cells, at least in part, through their influence on gene expression and cell apoptosis, but varied in their individual degree. However, the final results were not completely coincident with their estrogenic potency tested in vitro, which implies that effects of NP isomers on steroidogenesis appear to be mediated through some other underlying mechanisms besides their various estrogenic potency.

Variation in antifungal activity in extracts from &lt;i&gt;Momordica&lt;/i&gt; plants

Antifungal activity in aqueous and organic extracts from Momordica charantia and Momordica balsamina were evaluated against three pathogens: Fusarium oxysporum f. sp. melonis, Macrophomina phaseolina, and Alternaria solani. Cold-water extracts of 12 Momordica accessions exhibited various degrees of inhibition of the three pathogens: A. solani was more suppressed by the different extracts than F. oxysporum or M. phaseolina. Extracts from accessions 5 and 10 from M. balsamina were the most effective at inhibiting the tested fungi. Accession 10 exhibited the highest suppression level, more than 30% higher inhibition relative to the untreated control. Heating the cold extracts resulted in loss of antifungal activity in all cases. Hot ethanol extracts of the studied accessions exhibited a somewhat different pattern: F. oxysporum and M. phaseolina were strongly inhibited relative to A. solani. Interestingly, accession 10, which excelled in its inhibitory activity in the cold-water extract, was not effective whe...

Gastroprotective effect of Dodonaea viscosa on various experimental ulcer models

Aim of the study Dodonaea viscosa Linn. (Sapindaceae) is used as a medicinal herb by the tribes of Shola forest regions of Western Ghats. It is used for headaches, backaches, stomach pain, piles and simple ulcers. The present study was performed to evaluate the gastroprotective effect and acute toxicity of this plant in various experimental models. Materials and methods Studies were performed in two different models (ethanol and indomethacin induced gastric ulcer) in wistar rats. Gastric protection was evaluated by measuring the ulcer index, gastric glutathione assay, alkaline phosphate assay and histopathological studies. Gastric secretion studies were done by pyloric ligation experiment. Results and conclusions Water and ethanol extract (500 mg/kg body weight) showed moderate activity compared to hexane extract. Hexane extract of Dodonaea viscosa dose dependently inhibited ethanol induced gastric lesions, causing 90% protection at 500 mg/kg, 81% protection at 250 mg/kg, and 70% protection at 125 mg/kg and it also dose dependently inhibited indomethacin induced gastric lesions, causing 92% protection at 500 mg/kg, 77% protection at 250 mg/kg, and 52% protection at 125 mg/kg. The various degrees of inhibition were statistically significant ( p ≤ 0.05). Further in our gastric secretion studies, we found that hexane extract of Dodonaea viscosa (500 mg/kg) decreased the amount of total acid in gastric juice. Dodonaea viscosa hexane extract thus inhibits acid secretion to prevent ulcer aggravation. Acute toxicity study with a higher dose of 1250 mg/kg did not manifest any toxicological signs in rats. We also performed preliminary phytochemical screening of the crude extract which gave positive results for the presence of flavanoids, saponins, bitter principles and phenols.

Common methodology is inadequate for studies on the microbicidal activity of neutrophils

Microbicidal activity of neutrophils is usually measured by colony-counting techniques after cell lysis in distilled water. While studying the effect of the reduced nicotinamide adenine dinucleotide phosphate-oxidase inhibitor diphenyleneiodonium (DPI) on the staphylocidal activity of neutrophils, we obtained inconsistent results: various degrees of inhibition in some experiments and no effect in others. The lysis step, i.e., dilution of neutrophils in distilled water, was the source of error. Cell-associated microorganisms were not dispersed effectively by this treatment. We overcame this problem by using water at pH 11 for cell lysis. Under these conditions, killing was inhibited completely and reproducibly by DPI. Here, we show that cell lysis in distilled water is incomplete and leads to an overestimate of microbial killing. This hinders identification of partial defects and makes complete defects appear as partial. We found that DPI-treated neutrophils and chronic granulomatous disease neutrophils were completely defective in killing of Staphylococcus aureus and Candida albicans and partially defective in killing of Escherichia coli after lysis with water pH 11, whereas after lysis in distilled water, killing of S. aureus and C. albicans was approximately 60% and approximately 70% of control killing, respectively, and killing of E. coli was normal. Likewise, killing of S. aureus by myeloperoxidase-deficient neutrophils was severely impaired after lysis in water pH 11 but appeared normal after lysis in distilled water. As most studies about neutrophil microbicidal activity have been performed using distilled water, our findings indicate that previous data about killing defects and the effects of agents that modulate microbicidal activity of neutrophils should be re-evaluated.

Antifungal activities of selected aromatic plants growing wild in Greece.

Essential oils of Origanum onites, Satureja thymbra, Salvia fruticosa (Greek sage), and Salvia pomifera subsp. calycina plants growing wild in Greece and their components carvacrol, camphor, and 1,8-cineole, were assayed for antifungal activity against 13 fungal species. Among the fungi tested were food poisoning, plant, animals and human pathogenic species. The oils presented various degrees of inhibition against all the fungi investigated. The highest and broadest activity was shown by the carvacrol content oils (O. onites and S. thymbra), while the oil of sage was the least effective. Carvacrol exhibited the highest and 1,8-cineole the lowest level of antifungal activity among the components tested.

Induction and inhibition of the in vitro N1-oxidation of 9-benzyladenine and isomeric 9-(nitrobenzyl)adenines.

The present study investigated some aspects of the enzymology of the in vitro N1-oxidation of 9-benzyladenine (BA) and isomeric 9-(nitrobenzyl)adenines (NBAs) using various potential inducers and inhibitors of cytochrome P-450 (CYP). When incubated with phenobarbital-induced rabbit hepatic microsomes, the N1-oxidation rates of BA and 9-(4-nitrobenzyl)adenine were about 6- and 2-fold higher than that of the control, respectively; while the N1-oxidation of 9-(2-nitrobenzyl)adenine and 9-(3-nitrobenzyl)adenine was not markedly affected. In contrast, beta-naphthoflavone and Arochlor 1254 showed no inductive effects towards the N1-oxidation of any of these substrates. Using 12 typical CYP inhibitors, it was found that nifedipine (CYP3A inhibitor) and haloperidol (CYP2D inhibitor) showed significant inhibition towards the N1-oxidation of BA and NBAs. Therefore, the N1-oxidation of BA and NBAs is probably catalysed by CYP3A and CYP2D subfamilies. Furthermore, when 9-(4-nitrobenzyl)adenine was incubated with compounds which possessed a certain chemical similarity to the adenine substrate, various degrees of inhibition of N1-oxidation of 9-(4-nitrobenzyl)adenine were observed. These observations allowed a preliminary indication as to the structure-metabolism relationship of 9-substituted adenine derivatives.

The effects of aromatase and 5 alpha-reductase inhibitors, antiandrogen, and sex steroids on Bidder's organs development and gonadal differentiation in Bufo bufo tadpoles.

Embryos of toads (Bufo bufo) were treated with aromatase (4-OHA) and 5 alpha-reductase (17 beta C) inhibitors, antiandrogen (CPA), estradiol-17 beta, testosterone, and 5 alpha-dihydrotestosterone in order to study the role played by sex steroids in the development and sex differentiation of gonads. Test compounds were administered to tadpoles in water and morphometric and cytometric analyses were carried out on histological sections of the cephalic Bidder's organ (a rudimentary ovary) and of the gonadal region. In Bidder's organs, the number and size of oogonia and oocytes were modified by the treatments. However, the female commitment of the Bidder's organ occurs independently from steroid treatments that lead to an acceleration or slackening of the processes of proliferation and differentiation of oogonia. 4-OHA and androgens caused various degrees of inhibition of ovarian differentiation, with gonads maintaining an undifferentiated condition. Estrogen provoked a shift of the sex ratio towards the female sex, yet slackened gonadal growth. 17 beta C accelerated ovarian differentiation in females while CPA enhanced gonadal differentiation in both sexes by promoting the germ and somatic cell proliferation. We suggest that sex hormones may have a local regulatory role in gonadal differentiation during early developmental stages. Furthermore, the strong tendency of Bidderian germ cells to develop in the oogenetic way regardless of sex genotype and steroid treatments, and the quantitative sex differences found in the control Bidder's organs and gonads, suggest that other factors (such as intracellular mechanisms) may be involved in the initial steps of the process of germ cell differentiation.

Inhibition of Listeria monocytogenes on cold-smoked salmon by nisin and carbon dioxide atmosphere

The bacteriostatic and bacteriocidal effect of nisin in combination with carbon dioxide, NaCl and low temperature on the survival of Listeria monocytogenes was investigated in in vitro model studies and in trials with cold-smoked salmon. Addition of nisin caused various degrees of inhibition and sometimes death of L. monocytogenes in model experiments performed at 10°C. The antilisterial effect of nisin was improved in the presence of 100% CO2 and increasing NaCl concentrations (0.5 to 5.0% w/v). Minimal bactericidal concentrations (MBC) of nisin varied from 30 to more than 500 IU/ml. The most pronounced effect of nisin was found when 102 cfu/ml was grown in media with 5.0% NaCl and incubated in CO2 atmosphere (MBC=30 IU/ml). The bactericidal effect of nisin was reduced in air and vacuum, and did not increase systematically with increasing NaCl concentrations. In general, nisin concentration ≤50 IU/ml resulted in the survival and growth of L. monocytogenes in all combinations with other preservatives (NaCl, CO2). Addition of nisin (500 or 1000 IU/g) to cold-smoked salmon inoculated with L. monocytogenes and stored at 5°C delayed, but did not prevent growth of L. monocytogenes in vacuum-packs. Numbers of L. monocytogenes increased to 108 cfu/g in vacuum packed cold-smoked salmon in 8 days, whereas CO2 packing of cold-smoked salmon resulted in an 8-day lag phase of L. monocytogenes, with numbers eventually reaching 106 cfu/g in 27 days. Addition of nisin to CO2 packed cold-smoked salmon resulted in a 1 to 2 log reduction of L. monocytogenes followed by a lag phase of 8 and 20 days in salmon with 500 and 1000 IU nisin/g, respectively. The levels of L. monocytogenes remained below 103 cfu/g during 27 days of storage at both concentrations of nisin.

Inhibition of Neurospora crassa Growth by a Glucan Synthase-1 Antisense Construct

We have used the filamentous fungus, Neurospora crassa, as a model system to test the concept that antisense targeting of the cell-wall assembly enzyme, (1,3)beta-glucan synthase [E.C. 2.4.1.34; UDPglucose: 1,3-beta-D-glucan 3-beta-D-glucosyltransferase], leads to a corresponding decrease in growth of the organism. Previously, our laboratory isolated a gene (glucan synthase-1, gs-1) that is required for (1,3)beta-glucan synthase activity. Wild-type cells were transformed with DNA vectors encoding various RNAs complementary to the gs-1 messenger RNA (antisense RNA) cloned downstream from an inducible promoter (quinic acid-2[qa-2p]). Stable transformants, expressing a partially inverted antisense message of gs-1 (pMYX107), exhibited dramatic reduction ingrowth compared with empty vector controls. Hyphal measurements of these transformants grown on race tubes indicated that all of the transformants showed various degrees of inhibition. Microscopic observations of transformants revealed shorter hyphal lengths when grown under conditions expressing antisense. Further characterization revealed that the specific activities of (1,3)beta-glucan synthase were decreased by as much as 63% relative to empty vector controls. Together, these observations suggest that antisense against (1,3)beta-glucan synthase led to a reduction in enzyme levels that resulted in altered cell-wall morphology and inhibition of growth. It is possible that antisense oligonucleotides against gs-1 may be useful antifungal agents.

Comparison of the antibody response to bee venom phospholipase A2 induced by natural exposure in humans or by immunization in mice.

Two human and twelve murine monoclonal antibodies directed against the main bee venom allergen phospholipase A2 (PLA) were evaluated for their fine specificity of binding to antigen and their ability to inhibit the enzymatic activity of the antigen. Antibodies were induced by natural exposure of beekeepers to bee venom or immunization of mice via different methods. Both human monoclonal antibodies (hmAbs) were previously shown to recognize the native three-dimensional conformation of PLA and are directed against discontinuous epitopes which include lysine residue at position 25 as a contact residue. In contrast, six of the murine monoclonal antibodies (mmAbs) bind to the denatured structure of the protein as determined by enzyme-linked immunosorbent assay. The epitopes recognized are located near the C-terminal end (n = 8), in the centre of the polypeptide (n = 1), near the N-terminal end (n = 1) or include the carbohydrate part (n = 2) of the PLA molecule. The capacity of the antibodies to modify the enzymatic activity was also determined. The hmAbs significantly inhibit the enzyme (70-79%), whereas the mmAbs produced various degrees of inhibition (39-100%). Since the X-ray structure of PLA is known, the epitopes can be visualized in the context of the three-dimensional structure of the antigen. A qualitative correlation was found between the location of epitopes and the inhibition pattern. Strong inhibition was seen with those antibodies that recognize epitopes that lie on the surface of the enzyme that is thought to contact the phospholipid bilayer. The results show that even though both hmAbs and most mmAbs inhibit the enzymatic activity of PLA, the antigen-binding properties of antibodies from different species raised after different routes of immunization differ significantly. Thus, detailed epitope mapping studies using murine antibodies prepared by artificial immunization may have limited value in predicting epitope patterns relevant to an antibody response to allergens in humans naturally exposed to antigen/allergen.