Molecular cloning and nucleotide sequence of a pectin lyase gene from Pseudomonas marginalis N6301.

A pectin lyase (PNL;EC4.2.2.10) gene of Pseudomonas marginalis N6301 was cloned and expressed in Escherichia coli. We purified PNL from P. marginalis N6301 and determined N-terminal 33 amino acids sequence. From this sequence, we synthesized two oligonucleotide probes. From the analysis of Southern hybridization, 2. 1kb EcoRI-SmaI fragment from the chromosomal DNA of P. marginalis was found to hybridize with oligonucleotide probes. Then, we cloned the fragment into pUC119 vector and transformed into E. coli DH5 alpha. A plasmid thus obtained was designated as pPNL6301. E. coli DH5 alpha harboring pPNL6301 expressed PNL activity. The nucleotide sequence of pn1 gene in the plasmid pPNL6301 encoding PNL from P. marginalis N6301 was determined. The structural gene of pn1 consisted of 936 base pairs. An open reading frame that encodes a 34,103 dalton polypeptide composed of 312 amino acids was assigned. The molecular weight of the polypeptide predicted from the amino acid composition was close to that of PNL of P. marginalis N6301 determined. The nucleotide sequence of the 5'-flanking region of pn1 gene showed the presence of the consensus sequence of LexA binding site, Pribnow box and ribosome binding site as found in Escherichia coli. The amino acid sequence homology of PNLs and nucleotide sequence homology of pn1 gene between P. marginalis N6301 and E. carotovora Er were 60.8% and 57.2%, respectively.     

Foraging behaviour influences the outcome of predator–predator interactions

Abstract.  1. Interactions among predators may influence the total efficiency of a predator complex. The effect of intra- and interspecific interactions of the generalist predators Orthotylus marginalis (Heteroptera: Miridae) and Anthocoris nemorum (Heteroptera: Anthocoridae) was investigated in a laboratory experiment. Outcomes of the interactions were determined by comparing predation rates on eggs and larvae of the blue willow beetle Phratora vulgatissima of single individuals with those of two individuals of the same or different species.
2. A non-additive, antagonistic effect on predation rates due to intraspecific interactions was found between individuals of A. nemorum . No such effect was found in O. marginalis . These results are as expected as a consequence of differences in behaviour of the two predator species: A. nemorum is a much more active and mobile predator than O. marginalis .
3. Contrary to expectation, interspecific interactions between A. nemorum and O. marginalis did not affect the total predation rate.
4. An observation from the field corroborated the results obtained in the laboratory study; there was no negative relationship between the densities of the two predator species, indicating that the two species do not interact negatively in the field at their natural densities.
5. It is concluded that the additive effect of multiple predator species is of potential value in biological control.     

Effects of calcium hopantenate on the release of thyrotropin-releasing hormone from the rat adrenal gland in vitro

The effects of calcium hopantenate (HOPA), a GABA agonist, on the release of thyrotropin-releasing hormone (TRH) from the rat adrenal gland were studied in vitro. The adrenal glands were incubated in medium 199 with 1.0 mg/ml of bacitracin (pH 7.4) (medium) for 20 min. The amount of TRH release into the medium was measured by radioimmunoassay. The TRH release from the rat adrenal gland was inhibited significantly in a dose-related manner with the addition of HOPA to the medium. HOPA's effects on TRH release from the adrenal gland were blocked with the addition of bicuculline, a GABA receptor inhibitor. The elution profile of methanol-extracted rat adrenal gland TRH was identical to that for synthetic TRH. The findings suggest that HOPA inhibits TRH release from the rat adrenal gland, and that its effects are mediated via the GABA receptor.     

Modelling the growth,survival and death of Listeria monocytogenes

In this paper, the predictive microbiology approach has been generalized to the study of growth, survival and death of Listeria monocytogenes. As this micro-organism is involved in food poisoning, its growth, survival and death were studied as functions of low temperatures, NaCl and phenol compounds, in a synthetic medium, by a factorially designed experiment. A significant inactivation of L. monocytogenes was obtained with 20 ppm of phenol and 4% (w/v) NaCl at temperatures from 4 to 12 degrees C. An empirical model is proposed to describe, in a single step, the biomass profile vs studied factors. Thereby, the influence of temperature, NaCl and phenol concentration on L. monocytogenes biomass quantity (0.5-8 log cfu ml(-1)) are presented as a function of storage duration. The comparisons of the proposed model with existing models (Gompertz for growth, vitalistic for survival and death) were performed. The use of a single equation allows the prediction of contamination levels in all experimental conditions without knowledge a priori. The model offers considerable prospects for its use in food microbiology.     

Effects of dopamine on the release of thyrotropin-releasing hormone from the rat retina in vitro.

The effects of dopamine on the release of thyrotropin-releasing hormone (TRH) from the rat retina in vitro were studied. The rat retina was incubated in the medium 199 (pH 7.4) with 1.0 mg/ml of bacitracin and 100 micrograms/ml of ascorbic acid. The amount of TRH release into the medium was measured by radioimmunoassay. The TRH release from the rat retina was inhibited significantly in a dose-related manner with the addition of dopamine, but not with pimozide. The inhibitory effects of dopamine on TRH release from the rat retina were blocked with an addition of pimozide to the medium. The elution profile of methanol-extracted rat retina on sephadex G-10 was identical to that of synthetic TRH. From these findings it is concluded that the dopaminergic system inhibits TRH release from the rat retina in vitro.     

Effects of acetylcholine on thyrotropin-releasing hormone release from the rat caecum in vitro

Effects of acetylcholine on the release of thyrotropin-releasing hormone (TRH) from the rat caecum in vitro were studied. The rat caecum was incubated in medium 199 with 1.0 mg/ml of bacitracin and 100 micrograms/ml of ascorbic acid (pH 7.4) (medium). The amount of TRH release into the medium was measured by radioimmunoassay. The immunoreactive TRH (ir-TRH) release from the rat caecum was enhanced significantly in a dose-related manner with the addition of acetylcholine, but not changed with atropine. The stimulatory effect of acetylcholine on ir-TRH release from the rat caecum was blocked with an addition of atropine. Elution profile of acid-methanol-extracted rat caecum on Sephadex G-10 was identical to that of synthetic TRH. The findings suggest that the cholinergic system stimulates TRH release from the rat caecum in vitro.     

Optimisation of synthetic medium composition for levorin biosynthesis by Streptomyces levoris 99/23 and investigation of its accumulation dynamics using mathematical modelling methods

The composition of a synthetic culture medium for levorin biosynthesis by Streptomyces levoris 99/23 was optimised using mathematical modelling methods. The optimal concentrations of the medium components were established by means of an optimum composition design at three factor variation levels. An adequate regression model was obtained. Levorin biosynthesis by Streptomyces levoris 99/23 in the optimised synthetic medium was over 38% higher than in the initial medium. The antibiotic biosynthesis dynamics in the optimised culture medium was studied by means of a non-linear differential equation system. The resultant model was valid.     

Effects of opioid peptides on thyrotropin-releasing hormone release from the rat caecum in vitro

Effects of opioid peptides (beta-endorphin, dynorphin (1-13). alpha-neoendorphin, beta-neoendorphin, leucine-enkephalin, methionine-enkephalin) on the release of thyrotropin-releasing hormone (TRH) from the rat caecum were studied in vitro. The rat caecum was incubated in medium 199 with 1.0 mg/ml of bacitracin (pH 7.4) (medium). The amount of TRH release from the rat caecum into the medium was measured by radioimmunoassay. The immunoreactive TRH (ir-TRH) release from the rat caecum was inhibited significantly in a dose-related manner with the addition of opioid peptides. The inhibitory effects of opioid peptides on ir-TRH release from the rat caecum were blocked with an addition of naloxone. The elution profile of acid-methanol-extracts of rat caecum on Sephadex G-10 was identical to that of synthetic TRH. The findings suggest that opioid peptides inhibit TRH release from the rat caecum in vitro.     

Dopamine inhibits thyrotropin-releasing hormone release from rat adrenal gland in vitro

The effects of dopamine on the release of thyrotropin-releasing hormone (TRH) from the rat adrenal gland were studied in vitro. The rat adrenal glands were incubated in medium 199 with 1.0 mg/ml of bacitracin and 100 micrograms/ml of ascorbic acid (pH 7.4) (medium) for 20 min. The amount of TRH release into the medium was measured by radioimmunoassay. The immunoreactive TRH (ir-TRH) release from the rat adrenal gland was inhibited significantly in a dose-related manner with the addition of dopamine and enhanced with the addition of pimozide or domperidone to the medium. Dopamine's effects on ir-TRH release from the adrenal gland were blocked with the addition of pimozide or domperidone. The elution profile of methanol-extracted rat adrenal gland was identical to that of synthetic TRH. The findings suggest that the dopaminergic system inhibits TRH release from the rat adrenal gland.     

Toxic exoproducts of citrobacter freundii

A strain of Citrobacter freundii isolated from the feces of a patient with diarrhoea was examined for growth kinetics and toxic exoproduct formation using the complete (BHI) and synthetic culture media. It was found that the test organism in synthetic medium grew distinctly slower than in BHI. Fractionations on Sephadex G-100 column yielded 3 fractions from the complete medium culture filtrate and 2 fractions from the culture filtrate obtained from synthetic medium. The first culture filtrate fractions (F1) were represented by components of the molecular weight over 100,000, the respective second fractions (F2) from complete and synthetic medium were of the molecular weights of about 40,000 and 10,000. In the early skin test on rabbits the toxicity of culture filtrates and their fractions manifested itself by an increased permeability of blood vessels, in the late skin test by a hemorrhagic reaction associated with dilatation of blood vessels and induration of the skin tissue. In a test on mouse foot pad all separated filtrate fractions gave a positive edematous reaction. In cultured Vero cells samples of synthetic medium fractions gave a distinct cytotoxic reaction. Immunochemically, the presence of LPS in culture filtrates as well as some variations in the antigenicity of components from the complete and synthetic medium fractions were found. Apart from LPS some additional high-molecular-weight components were also present in the toxic complex of both first filtrate fractions (F1). Much more attention should be given to analysis of these first fraction complexes as well as to toxinogenicity of second fractions (F2) using some additional tests.     

Co-immobilized aerobic/anaerobic mixed cultures in stirred tank and gaslift loop reactors

Co-immobilized Aspergillus awamori and Zymomonas mobilis cultures were investigated in a stirred tank reactor on synthetic medium with starch as substrate at various dissolved oxygen concentrations. In a gaslift loop reactor, freely suspended and immobilized A. awamori were cultivated on synthetic medium and soluble potato starch. In the same reactor, the growth and ethanol production of freely suspended and immobilized Z. mobilis cultures were studied on synthetic medium and glucose. Co-immobilized A. awamori and Z. mobilis were cultivated in batch and continuous operations in the gaslift loop reactor on synthetic medium with starch substrate at different dissolved oxygen concentrations. The interrelations between the different process variables are discussed.     

The role of glucose and glycosylation in the regulation of lipoprotein lipase synthesis and secretion in rat adipocytes

Several studies have suggested that insulin and glucose increase adipose tissue lipoprotein lipase (LPL). To study the mechanism of the glucose-induced stimulation of LPL, the effects of glucose and glycosylation were examined in primary rat adipocyte cultures. In cells cultured in the presence of 1 mg/ml glucose, a 55-kDa LPL protein was synthesized and secreted into the medium, whereas cells cultured in glucose-free medium synthesized a 49-kDa form of LPL which was not secreted. The treatment of the mature 55-kDa form of LPL with peptide:N-glycosidase-F resulted in the formation of a 49-kDa form of LPL. When cells were cultured in the presence of tunicamycin, a 49-kDa form of LPL was synthesized by the cells but was not secreted. In addition, LPL activity was reduced by 90% when glycosylation was blocked by either tunicamycin or glucose deprivation. LPL synthetic rate was examined in cells cultured in a spectrum of glucose concentrations. LPL synthetic rate increased directly with medium glucose concentration and was decreased 80% in the absence of glucose compared to the synthetic rate in the presence of 1 mg/ml glucose. In addition, LPL synthetic rate in the presence of insulin was approximately 200% of the synthetic rate in untreated control cells at all glucose concentrations and even in the absence of glucose. In spite of the effect of glucose on LPL synthetic rate, glucose had no effect on the level of LPL mRNA. In contrast, the mRNA for the 78-kDa glucose-regulated protein (GRP78) was increased in adipocytes cultured in glucose-free medium. In summary, glucose was essential for glycosylation of LPL, and glycosylation was essential for LPL catalytic activity and secretion. In addition, glucose stimulated LPL synthetic rate and potentiated the stimulatory effects of insulin, but had no specific effect on LPL mRNA. Whereas insulin stimulates LPL by increasing the level of LPL mRNA, glucose stimulates LPL translation and post-translational processing.     

The discovery of adamantyl-derived, inhaled, long acting beta(2)-adrenoreceptor agonists

The design and profile of a series of adamantyl-containing long acting beta(2)-adrenoreceptor agonists are described. An optimal pharmacokinetic profile of low oral bioavailability was combined with a strong pharmacology profile when assessed using a guinea pig trachea tissue model. A focus was then placed on developing a robust synthetic route to ensure rapid delivery of material for clinical trials.     

Kinetic analysis of red pigment and citrinin production by Monascus ruber as a function of organic acid accumulation

In submerged cultures performed in synthetic medium containing glucose and glutamate, the filamentous fungus Monascus ruber produced a red pigment and a mycotoxin, citrinin. In oxygen-limiting conditions, the production of these two metabolites was growth-associated, as was the production of primary metabolites. In oxygen-excess conditions, the profile of citrinin production was typical of a secondary metabolite, since it was produced mostly during the stationary phase. In contrast, the production of the pigment decreased rapidly throughout the culture, showing a profile characteristic of an inhibitory mechanism. The organic acids produced during the culture, L-malate and succinate, were shown to be slightly inhibitory against pigment production, while citrinin production was unaffected. However, this inhibition could not account for the observed profile of pigment production in batch cultures. Other dicarboxylic acids such as fumarate or tartrate showed a similar effect to that provoked by malate and succinate as regards pigment production. It was concluded that the decrease in red pigment production during the culture was due to the inhibitory effect of an unknown product whose accumulation was favored in aerobic conditions.     

Influence of culture medium of the fatty-acid profile in enteric bacteria.

Enteric bacteria having a high content of cyclopropane fatty acids steeply increase their synthesis when grown on insufficiently propitious culture media (meat-peptone agar or modified Drobot'ko synthetic medium) as compared with bacteria grown under more favourable conditions (meat-peptone broth). Simultaneously, a decrease in monounsaturated fatty acids and increase in palmitic acid are observed. One of the main factors underlying the change in the proportion of fatty acids in bacteria grown on synthetic medium is an increase in medium pH in the process of their growth. Enteric bacteria containing minute amounts/or not containing cyclopropane fatty acids at all (under the experimental conditions used) change their fatty-acid profile little if the culture medium is changed. When grown under insufficiently favourable conditions, these bacteria mainly display an enhanced content of palmitic acid and a lowered content of octadacenoic acid as compared with bacteria grown under more favourable conditions. Of the culture media used, meat-peptone broth, which affords the most favourable conditions for eneteric bacteria growth, is the most suitable medium for obtaining data of taxonomic value.     

Rhythmical sporogenesis in Streptomyces colonies. A one-dimensional model

A one-dimensional model imitating the growth and zone formation of a colony of the radiant fungus streptomycete on a synthetic agar nutrient medium with pyruvate, glucose, or succinate as a limiting source of energy and carbon was considered. The key parameters were determined whose values depend on the composition of nutrient medium and environmental conditions.     

An effective medium for the selective growth of yeast or mycelial forms of Candida albicans: Biochemical aspects of the two forms

A new synthetic medium, based on a modification of a commercially available tissue culture medium, allows Candida albicans to be grown in the yeast or mycelial form. Salient features of the system are described and comparisons with previous physiological investigations are discussed. A concise biochemical profile of these two forms of C. albicans is also presented. The results indicate vast metabolic differences between the two forms.     

Preparation,structure, drug release and bioinspired mineralization of chitosan-based nanocomplexes for bone tissue engineering

Chitosan-based nanocomplexes with various forms were prepared by ionically crosslinking with tripolyphosphate (TPP) in different acidic media under mild conditions. It was found that the self-assembly and ionic interactions of chitosan and TPP were greatly affected by reaction media, and chitosan-based nanofibers could be obtained in adipic acid medium while nanoparticles were formed in acetic acid medium. Using bovine serum albumin (BSA) as a macromolecular model-drug, in vitro drug release studies indicated that chitosan-based nanofibers and nanoparticles exhibited a similar prolonged release profile. In addition, the bioinspired mineralization of both chitosan-based nanofibers and nanoparticles was carried out by soaking them in synthetic body fluids (SBF). Transmission electron microscopy (TEM) and X-ray Diffraction (XRD) results indicated that chitosan-based nanofibers have better inductivity for nano-hydroxyapatite formation than chitosan-based nanoparticles. The results suggested that biomimetic chitosan-based nanofibers with controlled release capacity of bioactive factors may be of use in bone tissue engineering for enhancing the bioactivity and bone inductivity.     

Preliminary description of biocidal (syringomycin) activity in fluorescent plant pathogenic Pseudomonas species

Strains representing the fluorescent plant pathogenic Pseudomonas spp., Ps. agarici , Ps. asplenii , Ps. avellanae , Ps. beteli , Ps. caricapapayae , Ps. cichorii , Ps. corrugata , Ps. ficuserectae , Ps. flectens , Ps. fuscovaginae , Ps. marginalis , Ps. meliae , Ps. savastanoi , Ps. syringae , Ps. tolaasii and Ps. viridiflava were tested for biocidal activity using Aspergillus niger as assay organism. Inhibitory behaviour was found in strains of Ps. asplenii , Ps. blatchfordae , Ps. cichorii , Ps. corrugata , Ps. fuscovaginae , Ps. marginalis , Ps. marginalis pv. pastinacea , Ps. syringae pv. syringae , Ps. syringae pv. aptata , Ps. syringae pv. atrofaciens , Ps. syringae pv. lapsa , Ps. tolaasii , and strains of a Pseudomonas sp. pathogenic to Actinidia , in the Ps. savastanoi genomic sp. Antifungal activity could be identified with the production of members of the syringomycin family of toxins by strains in Ps. syringae , Ps. asplenii and Ps. fuscovaginae . These toxin reactions support suggestions made elsewhere of the synonymy of the latter two species. In a preliminary characterization using tests for stability to heat, protease, acid and alkaline treatments, unknown toxins consistent with syringomycin-like toxins the strains from Actinidia speciesColour RGB 0,0,128. The toxins from Ps. cichorii and from Ps. corrugata differed in their reactions from all other agents. Pseudomonas tolaasii produces the antifungal compound tolaasin. The white line reaction with ' Ps. reactans ', a test for tolaasin production by strains of Ps. tolaasii , was confirmed as specific for this compound. Some of these low molecular weight toxins may be produced by some of these plant pathogenic strains.     

Effects of histamine and related compounds on the release of immunoreactive thyrotropin-releasing hormone from the rat stomach in vitro

Effects of histamine and related compounds on the release of immunoreactive thyrotropin-releasing hormone (ir-TRH) from the rat stomach in vitro were studied. The rat stomach was incubated in medium 199 with 1.0 mg/ml of bacitracin and 100 micrograms/ml of ascorbic acid (pH 7.4) for 20 min. The amount of TRH release into the medium was measured by radioimmunoassay. The ir-TRH release from the rat stomach was enhanced significantly in a dose-related manner with the addition of histamine and inhibited with the addition of famotidine, but not with mepyramine. The stimulatory effect of histamine on ir-TRH release from the stomach was partially blocked with the addition of famotidine, but not with mepyramine. The elution profile of acid-methanol-extracted rat stomach on Sephadex G-10 was identical to that of synthetic TRH. These findings suggest that histamine stimulated ir-TRH release from the rat stomach in vitro, and that histamine's effects may be mediated via a H2-receptor.