Activity of a “thermostable exotoxin” of Bacillus thuringiensis subsp. morrisoni in the Salmonella/microsomal assay for bacterial mutagenicity

The purpose of this study was to employ the Salmonella/microsomal assay (Ames test) to investigate the mutagenic potential of a thermostable exotoxin of Bacillus thuringiensis subsp. morrisoni. Bacteria are ideal for the detection of infrequently occurring point mutations because the large number of organisms (200 to 400 million bacteria per plate) exposed to the mutagen at any one time increases the possibility of observing a random mutational event. The exotoxin used in this study was produced using the shaker flask fermentation procedure with mineral casein broth. A Petri dish method of bioassay using fresh bovine feces was used to determine the efficacy of the exotoxin against horn flies. The LD₅₀ was found to be 5.35 μl/g of feces. Five bacterial tester strains were identified and characterized for the genetic markers described by Ames et al. (B. N. Ames et al., 1975, Mutat. Res., 31, 347–364). Appropriate doses of the B. thuringiensis supernatant, solvent or positive control were added to agar plates. The supernatant was tested at five dose levels against all five strains of bacteria. Controls of bacteria only were included for spontaneous reversions. All treatments were performed in triplicate. The numbers of revertant colonies from each set of triplicate plates were averaged and the standard deviation calculated and compared to that found with the solvent control. The negative controls, positive controls, and sterility controls all fulfilled requirements for determination of a valid test. No detectable mutagenic activity was found for the thermostable exotoxin of B. thuringiensis morrisoni.     

Use of the beta-binomial distribution in dominant-lethal testing for “weak mutagenic activity”: Part 2

Experiments in Dominant-Lethal Testing have been simulated on the computer to estimate the type I error rates and the power of the Beta-Binomial test under various models. (1) The mating ratio is one; and p, the probability that an implant will die, is distributed over the couples. (2) The mating ratio is larger than one; and p is distributed over the males, the females mated to the same male being binomial observations of the value p supplied by the male. (3) The mating ratio is larger than one; and p is distributed over the females.The average rates of dead implants have been set at 0.08 and 0.10 for the control and treatment groups, respectively, and a nominal level of significance equal to 0.05 has been chosen. The type I error rate of the traditional chi-square test has also been estimated.A by-product of these simulations is the behaviour of the estimates α̌ and β̌ of the beta-distribution parameters, which discloses that, in the actual experiments with mice, p is distributed over the females.Our results lead to the recommendations that, for a given number of animals per group, a mating ratio larger than one should be adopted and that the males should be considered as the experimental units for the calculations. With 300 and 450 animals per group, average powers of 0.72 and 0.85 are reached, respectively, for the chosen increment of 2% in the rate of dead implants.Under these models, the type I error rate of the traditional chi-square test may grow to 0.30 for the nominal level of 0.05.     

Development of a miniaturized nitrate reduction test for the identification of oral bacteria

A miniaturized nitrate reduction test (MNRT) for oral bacteria was developed and its reliability compared with a conventional nitrate reduction test (CNRT). In the MNRT 100 μl aliquots of freshly grown heavy suspension of various oral bacterial species, in physiological saline, were added to equal volumes of 0.1% filter-sterilized KNO₃ solution in distilled water in wells of transparent plastic plates. Duplicate plates were incubated aerobically or anaerobically at 35°C for 12–15 h. At the end of the incubation period the test was performed by adding either a trace amount of a non-liquid reagent (mixture of l-(+)-tartaric acid, sulfanilic acid and 1-naphthylenediamine dihydrochloride, 10:1:1, wt/wt) or conventional liquid reagents A and B (sulfanilic acid and N,N-dimethyl-1-naphthylamine). In the conventional nitrate reduction test (CNRT), tubes of a basal anaerobic broth were inoculated with the same bacterial species used for MNRT, and the nitrate reduction tests performed after anaerobic incubation of the cultures for 4–6 days. Several hundred anaerobic and facultative bacterial isolates belonging to genera Veillonella, Bacteroides, Fusobacterium, Selenomonas, Actinomyces and Capnocytophaga were characterized by MNRT and CNRT. Analysis of the data showed that MNRT and CNRT systems were comparable. In the MNRT system Veillonella parvula and Selenomonas sputigena were capable of reducing nitrate only under anaerobic conditions. Actinomycetes reduced the nitrates under aerobic and anaerobic conditions, while all black-pigmented Bacteroides, Fusobacterium and Capnocytophaga species did not reduce nitrate. These findings suggest that the MNRT is reliable, rapid and may be conveniently used in clinical or research laboratories with a heavy microbiological work load.     

Antinociceptive and antidepressant-like action of endomorphin-2 analogs with proline surrogates in position 2

In our efforts to develop new candidate drugs with antinociceptive and/or antidepressant-like activity, two novel endomorphin-2 (EM-2, Tyr-Pro-Phe-Phe-NH₂) analogs, containing proline surrogates in position 2 were synthesized using commercially available racemic trans-4-phenylpyrrolidine-3-carboxylic acid (4-Ph-β-Pro). The obtained mixture of two diastereoisomeric peptides (2a and 2b) was separated by HPLC and both enantiopure analogs were used in the in vitro and in vivo studies. To assign the absolute configuration to the 4-Ph-β-Pro residues in both peptides, the stereoselective synthesis of (3R,4S)-4-phenylpyrrolidine-3-carboxylic acid was performed and this enantiomer was introduced into position 2 of EM-2 sequence. Based on the HPLC retention times we were able to assign the absolute configuration of 4-Ph-β-Pro residues in both peptide analogs. Analog 2a incorporating (3R,4S)-4-Ph-β-Pro residue produced strong analgesia in mice after intracerebroventricular (icv) administration which was antagonized by the μ-opioid receptor (MOR) antagonist, β-funaltrexamine (β-FNA). This analog also influenced an emotion-related behavior of mice, decreasing immobility time in the forced swimming and tail suspension tests, without affecting locomotor activity. The antidepressant-like effect was reversed by the δ-selective antagonist, naltrindole (NLT) and κ-selective nor-binaltorphimine (nor-BNI). Thus, the experiments with selective opioid receptor antagonists revealed that analgesic action of analog 2a was mediated through the MOR, while the δ- and κ-receptors (DOR and KOR, respectively) were engaged in the antidepressant-like activity. Analog 2b with (3S,4R)-4-Ph-β-Pro in position 2 showed no antinociceptive or antidepressant-like activity in animal studies.     

The immunological response of CBA mice to Trypanosoma musculi: mechanisms of protective immunity

CBA mice which had recovered from infection with Trypanosoma musculi were immune to challenge with all strains of the homologous species that were tested but were still full susceptible to challenge with T. cruzi, T. brucei or T. evansi. A heavy challenge inoculum of T. musculi was cleared rapidly from the blood of mice which had recently recovered from infection but, in mice which had recovered 11 months earlier, the parasitaemia changed very little for 3–4 days but then fell abruptly within a few hours. Immunization with a parasite extract in multiple emulsion conferred a strong though not complete protection against homologous challenge.Serum from mice which had recovered from infection had a marked neutralizing effect in vitro on the infectivity of the homologous parasites although the numbers of live organisms were not reduced during the period of in vitro incubation. The test did not reveal antigenic differences among three isolates of the parasite.A summary is given of the sequence of events that is thought to make up the immune response of mice to T. musculi.     

Intercalibration of ecotoxicity testing protocols with Artemia franciscana

The brine shrimp, Artemia spp., is widely used in ecotoxicology as a target biological model. Although several protocols were available in the early 1980s, only the 24-h acute mortality toxicity test was evaluated in a European intercalibration exercise during that period. Nevertheless, documentation of standard methods serving to provide specifications, guidelines or detailed characteristics of the 24-h protocol is still unavailable. This paper present the results of an intercalibration study of three toxicity-testing protocols using Artemia franciscana: (a) the 24-h static acute mortality test, (b) the 48-h static hatching test and (c) the 14-d static-renewal long-term mortality test. A first tier of experiments was conducted by a reference laboratory, which investigated the repeatability of the three methods. The feasibility and reproducibility of these protocols were then investigated by an intercomparison exercise involving 11 participants for the acute mortality test, seven for the acute hatching test and nine for the long-term mortality test. Protocols were tested on reference toxicants (copper sulphate pentahydrate and sodium dodecyl sulphate). The coefficients of variation were <20% and <50% for intra- and interlaboratory activities, respectively. These results encourage the standardization of the proposed methods and their use as regulatory procedures.     

Mutagenicity assessment of Salacia chinensis by bacterial reverse mutation assay using histidine dependent Salmonella typhimurium tester strains

Background and objectiveGenotoxicity analysis is one of the most important non-clinical environmental safety investigations required for pharmaceutical and agrochemical product registration. Any medicinal product must undergo a risk evaluation to determine its mutagenicity and carcinogenicity.Materials and methodsThe Ames test is a commonly used in vitro test for determining a test chemical's mutagenic activity. Histidine-dependent Salmonella typhimurium strains with a defective gene that causes the bacteria to synthesis the necessary amino acid histidine for life were tested for mutagenic potential. In order to reveal pro-mutagens and mutagens, the mutagenic potential of both plate integration and pre-incubation techniques was examined in the presence and absence of metabolizing system. Salacia chinensis has been widely used in ayurveda to treat various ailments. However, the information of mutagenicity of Salacia chinensis is scarce as per available literature.ResultsThe mutagenicity of a Salacia chinensis root extract was investigated utilizing the Ames assay with plate incorporation and pre-incubation protocols using the appropriate Salmonella typhimurium tester strains: TA98, TA100, TA1537, TA1535, and TA102 in the presence and absence of S9. The concentrations used were 0.3123, 0.625, 1.25, 2.5 and 5 mg/plate. The extract of Salacia chinensis root did not show any mutagenic effect in any of the Salmonella typhimurium strains at the concentrations tested in the absence or presence of metabolic activation.ConclusionThe root of Salacia chinensis was hence confirmed to be non-mutagenic and at least according to the results of this genotoxicity evaluation can be regarded as being safe for human use.     

Molecular cloning and biologically active production of IpaD N-terminal region

Shigella is known as pathogenic intestinal bacteria in high dispersion and pathogenic bacteria due to invasive plasmid antigen (Ipa). So far, a number of Ipa proteins have been studied to introduce a new candidate vaccine. Here, for the first time, we examined whether the N-terminal region of IpaD^72–162 could be a proper candidate for Shigella vaccine. Initially, the DNA sequence coding N-terminal region was isolated by PCR from Shigella dysenteriae type I and cloned into pET-28a expression vector. Then, the heterologous protein was expressed, optimized and purified by affinity Ni–NTA column. Western blot analysis using, His-tag and IpaD^72–162 polyclonal antibodies, confirmed the purity and specificity of the recombinant protein, respectively. Subsequently, the high immunogenicity of the antigen was shown by ELISA. The results of the sereny test in Guinea pigs showed that IpaD^72–162 provides a protective system against Shigella flexneri 5a and S. dysenteriae type I.     

Bovine toxoplasmosis: experimental infections.

Three calves dosed per os with 0.75 × 10⁶, 0.75 × 10⁶ and 1.5 × 10⁶Toxoplasma oocysts developed high titres of Toxoplasma antibodies as measured by the indirect fluorescent antibody test and the dye test. Toxoplasma gondii was reisolated from 2 of these animals. Two calves given 1 × 10³ tissue cysts and 2 × 10⁶ tachyzoites intramuscularly did not develop such high fitres, but T. gondii was reisolated from the calf injected with tachyzoites.Of 4 pregnant cows given 7 × 10⁴, 7 × 10⁴ and 1.6 × 10⁵ oocysts and 1.5 × 10² tissue cysts only 2 developed significant levels of Toxoplasma antibodies. There was no evidence of Toxoplasma infection in the calves born by these cows.It was concluded that cattle do not readily acquire persistent T. gondii infections, and this may be due, in part at least, to early elimination of Toxoplasma from their tissues.     

Seed viability of five wild Saudi Arabian species by germination and X-ray tests

Our objective was to evaluate the usefulness of the germination vs. the X-ray test in determining the initial viability of seeds of five wild species (Moringa peregrina, Abrus precatorius, Arthrocnemum macrostachyum, Acacia ehrenbergiana and Acacia tortilis) from Saudi Arabia. Usually several days were required to determine the viability of all five species via germination tests. However, X-ray test will give immediate results on filled/viable seeds. Seeds of all species, except Acacia ehrenbergiana and Acacia tortilis showed high viability in both germination (96–72% at 25/15 °C, 94–70% at 35/25 °C) and X-ray (100–80%) test. Furthermore, there was a general agreement between the germination (19%, 14% at 25/15 °C and 17% and 12% at 35/25 °C) and X-ray (8%, 4%) tests in which seed viability of Acacia ehrenbergiana and Acacia tortilis was very low due to insect damaged embryo as shown in X-ray analysis. Seeds of Abruspreca torius have physical dormancy, which was broken by scarification in concentrated sulfuric acid (10 min), and they exhibited high viability in both the germination (83% at 25/15 °C and 81% at 35/25 °C) and X-ray (96%) tests. Most of the nongerminated seeds of the five species except those of Acacia ehrenbergiana and Acacia tortilis, were alive as judged by the tetrazolium test (TZ). Thus, for the five species examined, the X-ray test was proved to be a good and rapid predictor of seed viability.     

Determination of seed viability of eight wild Saudi Arabian species by germination and X-ray tests

Our purpose was to evaluate the usefulness of the germination vs. the X-ray test in determining the initial viability of seeds of eight wild species (Salvia spinosa, Salvia aegyptiaca, Ochradenus baccatus, Ochradenus arabicus, Suaeda aegyptiaca, Suaeda vermiculata, Prosopisfarcta and Panicumturgidum) from Saudi Arabia. Several days were required to determine viability of all eight species via germination tests, while immediate results on filled/viable seeds were obtained with the X-ray test. Seeds of all the species, except Sa.aegyptiaca, showed high viability in both the germination (98–70% at 25/15 °C, 93–66% at 35/25 °C) and X-ray (100–75%) test. Furthermore, there was general agreement between the germination (10% at 25/15 °C and 8% at 35/25 °C) and X-ray (5%) tests that seed viability of Sa.aegyptiaca was very low, and X-ray analysis revealed that this was due to poor embryo development. Seeds of P.farcta have physical dormancy, which was broken by scarification in concentrated sulfuric acid (10 min), and they exhibited high viability in both the germination (98% at 25/15 °C and 93% at 35/25 °C) and X-ray (98%) test. Most of the nongerminated seeds of the eight species except those of Sa.aegyptiaca were alive as judged by the tetrazolium test (TZ). Thus, for the eight species examined, the X-ray test was a good and rapid predictor of seed viability.     

Development of EST-SSR for preliminary analysis of genetic diversity of Cordyceps militaris

Through previous research, different populations of Cordyceps militaris were determined to have varying contents of cordyceps polysaccharides and cordycepic acid, which is involved inantioxidant activity and immune stimulation. This study aimed to exploit expressed sequence tags-simple sequence repeats (EST-SSRs) and to analyse the population genetic differentiation of C. militaris. The SSR frequency of C. militaris in ESTs was 24.3%. Mono-repeats were the most abundant motif (83.4%), and the most frequent mono-repeat was A/T (98.8%). The percentage of polymorphic bands (PPB) of the seven populations of C. militaris ranged from 11.7% to 73.7% with a mean of 34.7%.Shannon's information index ranged from 0.0576 to 0.3021 with a mean of 0.1623. The total genetic diversity of C. militaris was 0.1907, and the genetic diversity within the population was 0.1049. The coefficient of gene differentiation was 0.4500, indicating extensive genetic differentiation of this species. The mean Nei's genetic distance among the C. militaris populations was 0.1184. The UPGMA dendrogram exhibited a low correlation between the genetic and geographic distances, which can also be confirmed by the Mantel test. The high level of diversification among populations may be due to deforestation and forest fragmentation in China.     

Trypanosoma gambiense: immunity with thymic cell transfer in mice.

This report deals with the enhanced agglutinin production and protection in thymectomized, lethally irradiated mice (TI-mice) with transferred thymic cells from mice immune to T. gambiense. Such mice, when sensitized with trypanosome antigen showed protection against experimental infection and also produced agglutinins. Thymic cells from cortisone-treated immune mice were able to induce the production of agglutinins in TI-mice subsequently injected with antigen. However, these agglutinin titers were very low. In bovine serum albumin gradient centrifugation experiments, agglutinin production could be efficiently induced by inoculation of TI-mice with a rather high density thymic cell subpopulation taken from immune mice. Fractionated by Sephadex G-200, the agglutinins displayed a division into two parts, a first and second peak. The main agglutination reaction was seen in the first or macroglobulin peak. In the fractionation of serum by DEAE-cellulose column chromatography, agglutinins were eluted in two parts, the 0.0175 M and 0.4 M effluents. The agglutination by the 0.4 M effluent was much stronger than that of the 0.0175 M effluent, in agreement with the gel filtration results. The sera containing agglutinins were able to enhance the phagocytosis of trypanosomes by cultured macrophages from the peritoneal cavity of normal and irradiated mice. Delay of parasitemia was evident in some of the TI-mice having detectable agglutinins. The delayed parasitemia resulted from antigenically altered trypanosomes which were able to withstand the lethal factors of TI-mice. Transplantation of thymic cells was considered to be responsible for agglutinins induced by the antigenic stimulation in TI-mice and for protection against experimental infection.     

Estimating the flexural rigidity of Arabidopsis inflorescence stems: Free-vibration test vs. three-point bending test

The mechanical strength of a plant stem (a load-bearing organ) helps the plant resist drooping, buckling and fracturing. We previously proposed a method for quickly evaluating the stiffness of an inflorescence stem in the model plant Arabidopsis thaliana based on measuring its natural frequency in a free-vibration test. However, the relationship between the stiffness and flexural rigidity of inflorescence stems was unclear. Here, we compared our previously described free-vibration test with the three-point bending test, the most popular method for calculating the flexural rigidity of A. thaliana stems, and examined the extent to which the results were correlated. Finally, to expand the application range, we present an example of a modified free-vibration test. Our results provide a reference for improving estimates of the flexural rigidity of A. thaliana inflorescence stems.     

Targeted disruption of the Pak5 and Pak6 genes in mice leads to deficits in learning and locomotion

PAK6 is a member of the group B family of PAK serine/threonine kinases, and is highly expressed in the brain. The group B PAKs, including PAK4, PAK5, and PAK6, were first identified as effector proteins for the Rho GTPase Cdc42. They have important roles in filopodia formation, the extension of neurons, and cell survival. Pak4 knockout mice die in utero, and the embryos have several abnormalities, including a defect in the development of motor neurons. In contrast, Pak5 knockout mice do not have any noticeable abnormalities. So far nothing is known about the biological function of Pak6. To address this, we have deleted the Pak6 gene in mice. Since Pak6 and Pak5 are both expressed in the brain, we also generated Pak5/Pak6 double knockout mice. These mice were viable and fertile, but had several locomotor and behavioral deficits. Our results indicate that Pak5 and Pak6 together are not required for viability, but are required for a normal level of locomotion and activity as well as for learning and memory. This is consistent with a role for the group B PAKs in the nervous system.     

Synthesis of benzimidazole-linked-1,3,4-oxadiazole carboxamides as GSK-3β inhibitors with in vivo antidepressant activity

Recent findings of potential implications of glycogen synthase kinase-3β (GSK-3β) dysfunction in psychiatric disorders like depression, have increased focus for development of GSK-3β inhibitors with possible anti-depressant activity. Keeping this in view, we synthesized a series of benzimidazole-linked-1,3,4-oxadiazole carboxamides and evaluated them for in vitro GSK-3β inhibition. Active compounds were investigated for in vivo antidepressant activity in Wistar rats. Docking studies of active compounds have also been performed. Among nineteen compounds synthesized, compounds 7a, 7r, 7j, and 7d exhibited significant potency against GSK-3β in sub-micromolar range with IC₅₀ values of 0.13 μM, 0.14 μM, 0.20 μM, 0.22 μM respectively and significantly reduced immobility time (antidepressant-like activity) in rats compared to control group. Docking study showed key interactions of these compounds with GSK-3β. These compounds may thus serve as valuable candidates for subsequent development of effective drugs against depression and related disorders.     

Unrecognized fine-scale recombination can mimic the effects of adaptive radiation

Gene sequences can undergo accelerated nucleotide changes and rapid diversification. The rapid sequence changes can then potentially lead to phylogenetic incongruence. Recently, Bodilis et al. (2011) observed artificial phylogenetic incongruence using the Pseudomonas surface protein gene oprF, and hypothesized that it was the result of a long-branch attraction artifact ultimately caused by adaptive radiation. In this study, an alternative hypothesis, namely fine-scale recombination, was tested on the same dataset. The results reveal that regions in oprF are of different evolutionary origins, and the mosaic gene structure resulted in confounding phylogenetic signals. These findings demonstrate that unrecognized fine-scale recombination can confound the phylogenetic interpretation and emphasize the limitation of using whole genes as the unit of phylogenetic analysis.     

Prediction of protein structural classes by recurrence quantification analysis based on chaos game representation

In this paper, we intend to predict protein structural classes (α, β, α+β, or α/β) for low-homology data sets. Two data sets were used widely, 1189 (containing 1092 proteins) and 25PDB (containing 1673 proteins) with sequence homology being 40% and 25%, respectively. We propose to decompose the chaos game representation of proteins into two kinds of time series. Then, a novel and powerful nonlinear analysis technique, recurrence quantification analysis (RQA), is applied to analyze these time series. For a given protein sequence, a total of 16 characteristic parameters can be calculated with RQA, which are treated as feature representation of protein sequences. Based on such feature representation, the structural class for each protein is predicted with Fisher's linear discriminant algorithm. The jackknife test is used to test and compare our method with other existing methods. The overall accuracies with step-by-step procedure are 65.8% and 64.2% for 1189 and 25PDB data sets, respectively. With one-against-others procedure used widely, we compare our method with five other existing methods. Especially, the overall accuracies of our method are 6.3% and 4.1% higher for the two data sets, respectively. Furthermore, only 16 parameters are used in our method, which is less than that used by other methods. This suggests that the current method may play a complementary role to the existing methods and is promising to perform the prediction of protein structural classes.     

Isolation and molecular characterization of causal agent of blue mold on Allium cepa L. and its control by Pennisetum flaccidum Griseb

Blue mold pathogen, isolated from infected Allium cepa L., was identified as a Penicillium species through morphological and molecular characterisation. Internal Transcribed spacer (ITS) region of ribosomal DNA (rDNA) was utilised for DNA sequencing. Basic Local Alignment Search Tool (BLAST) analysis has found the maximum similarity index of the fungus to be 82.39% with the Uncultured Penicillium clone (Accession: MF535522). So, the isolated Penicillium specie is the first reported specie of the genus that infects onion. A phylogenetic tree was constructed to establish a relationship of the isolated fungus with the most relevant species reported on GenBank. Extracts of Pennisetum flaccidum Griseb. were evaluated against the isolated fungus as a potential biocontrol agent. Among the five tested methanol concentrations (0.5%, 1%, 1.5%, 2% and 2.5%) of each plant part (root, inflorescence and foliage), 0.5% root extract showed maximum growth retardation, i.e. 89%. For bioassay-guided fractionation, the root extract was partitioned in n-hexane, chloroform, n-butanol and ethyl acetate. Ethyl acetate (1%) was proved to be the most potent one. Phytochemical screening has confirmed the occurrence of terpenoids, tannins, saponins and alkaloids. The applied molecular approach has deduced that the Penicillium specie collected from Pakistan might be novel. This study can be concluded that P. flaccidum contains potent phytochemicals which might be used as antifungal agent against Penicillium species.     

High congruence of intraspecific variability in floral scent and genetic patterns in Gentianella bohemica Skalický (Gentianaceae)

Gentianella bohemica Skalický (Gentianaceae) is a critically endangered species endemic to the Bohemian Massif in the border region of Germany, Czechia and Austria. It consists of a restricted number of extremely scattered populations which are known to form distinct genetic groups. The objective of this work was to test for differences in the floral scent between Gentianella bohemica and Gentianella germanica and within these two species among populations, and to test for a correlation of scent and genetic similarity among the populations of G. bohemica. Floral scent was collected from the inflorescences/plants of eight flowering populations of G. bohemica and three populations of G. germanica using dynamic headspace methods, followed by GC/MS analyses. Both species emitted several aromatic and terpenoid compounds and multivariate analyses revealed differences in scent between the two species and within species among G. bohemica populations. Volatile components overlapped as expected for closely related species but floral scent was taxon-specific. Floral scent differentiation among G. bohemica populations was in high congruence with the genetic differentiation suggesting that scent differences among populations have a genetic basis and showing that scent is a suitable chemotaxonomic marker in this species.