Specific [3H]Piflutixol Binding to CHAPS-Solubilised Rat Striatal Preparations Involves Dopamine D-2 but Not D-1 Binding Sites

[3H]Piflutixol binding to rat striatal membrane preparations identifies both D-1 and D-2 sites. We used [3H]piflutixol to characterise those binding sites present in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)-solubilised rat striatal preparations. The specific binding of [3H]piflutixol, as defined using cis-flupenthixol, to CHAPS-solubilised rat striatal tissue was saturable and of high affinity. Specific [3H]piflutixol binding to the solubilised preparations was displaced stereoselectively by the isomers of butaclamol and to an equal extent by both cis-flupenthixol and (+/-)-sulpiride. A positive correlation was found between the capacity of a range of drugs to displace [3H]piflutixol binding and the displacement of [3H]spiperone to the same preparations. The Bmax of [3H]piflutixol binding was not different from that of [3H]spiperone binding to the same preparation. These studies suggest that, in contrast to specific binding of membrane preparations, the specific binding of [3H]piflutixol to CHAPS-solubilised preparations involves mainly D-2 sites. Specific [3H]piflutixol binding, in contrast to [3H]spiperone binding, showed only slow dissociation from soluble preparations. The binding of [3H]piflutixol to CHAPS-solubilised preparations was retained during passage through a gel filtration column. This prelabelling of solubilised striatal preparations using [3H]piflutixol may aid in the purification of CHAPS-solubilised rat striatal D-2 sites.     

Preferential synthesis, localisation and solubilisation of a precursor form of Bacillus subtilis levansucrase in an Escherichia coli minicell system

Abstract In the presence of phenethylalcohol, plasmidencoded levansucrase (2,6β- d -fructan: 6,β- d -fructosyltransferase, EC 2.4.1.10) was accumulated as a precursor form in the membrane fraction of Escherichia coli minicells. Immunoblotting experiments were used to evaluate the amount of the newly synthesised precursor form relative to the immunoreactivity of the purified mature form of levansucrase. Solubilisation of both forms with Triton X100 and Triton X114 was compared. Triton X114 allowed separation of the precursor form from the mature form, which were recovered in the detergent and the aqueous phases, respectively, after separation of the phases at 30°C.     

Activation of proteases in an anaerobic sulphidogenic bioreactor

Activities of proteases were stimulated by specific sulphur metabolites during the enhanced hydrolysis of complex polymeric organic carbon in an anaerobic sulphidogenic environment. While sulphate at 1000 mg l(-1) inhibited proteases by 50%, there was a 2.5-fold increase in activity of proteases by added sulphite and a 3.6-fold increase from added sulphide. Two hypothetical models are proposed. First the sulphur species, sulphite (HSO3-) and sulphide (HS-), liberated at different times during the sulphate reduction process, directly activate the proteases, which are associated with the organic particulate matter, leading to a subsequent enhancement of hydrolysis of polymeric material. Second, they indirectly activate the proteases by neutralising the cations on the floc surface disrupting the integrity of the organic particulate floc therebye releasing further entrapped enzymes from the organic particulate matter.     

[3H]norharman ([3H]β-carboline) binds reversibly and with high affinity to a specific binding site in rat liver

In addition to the known binding of norharman (NH) to monoamine oxidase (MAO) and benzodiazepine (BZ) binding sites (at M concentrations), a distinct class of high-affinity NH binding sites was discovered in rat brain (1,2). Investigations of several organs of the rat led to the discovery of high affinity binding sites in the liver, which successfully could be solubilized from P₂ membrane homogenate (0.25% w/v Triton X-100). Scatchard analysis revealed an apparent K_D value of 26±8 nM and a maximum number of binding sites of 11±3 pmol/mg protein (n=14). Association kinetics showed that equilibrium was nearly reached after two hours. Dissociaton was totally complete only after more than 16 hours. The MAO-inhibitors examined did not influence the binding characteristics. No displacement of specific binding could be found by haloperidol.     

Bioconversion of wheat stalk to hydrogen by dark fermentation: effect of different mixed microflora on hydrogen yield and cellulose solubilisation

This study determined hydrogen production, volatile fatty acids (VFAs) generation and cellulose solubilisation from anaerobic dark fermentation of wheat stalk and showed the effect of different mixed microflora. The cumulative hydrogen yields of anaerobic digested activated sludge (AS)-inoculated and anaerobic digested dairy manure (DM)-inoculated system were 23.3 and 37.0 mL/g VS at 204 h, respectively. A modified Gompertz equation was able to adequately describe the production of hydrogen from the batch fermentation by both mixed microflora. During the process, acetate and butyrate accounted for more than 76.1% of total VFAs for both fermentations. The extent of cellulose solubilisation approached 46.6% and 75.2% for AS- and DM-inoculated fermentation, respectively. The X-ray diffraction (XRD) showed that the crystallinities of both fermented stalks were partly disrupted by the mixed microflora, and DM-inoculated fermentation had more disruption than AS-inoculated one.     

Some isolates of the nematophagous fungus Pochonia chlamydosporia promote root growth and reduce flowering time of tomato

The fungal parasite of nematode eggs Pochonia chlamydosporia is also a root endophyte known to promote growth of some plants. In this study, we analysed the effect of nine P. chlamydosporia isolates from worldwide origin on tomato growth. Experiments were performed at different scales (Petri dish, growth chamber and greenhouse conditions) and developmental stages (seedlings, plantlets and plants). Seven P. chlamydosporia isolates significantly (P < 0.05) increased the number of secondary roots and six of those increased total weight of tomato seedlings. Six P. chlamydosporia isolates also increased root weight of tomato plantlets. Root colonisation varied between different isolates of this fungus. Again P. chlamydosporia significantly increased root growth of tomato plants under greenhouse conditions and reduced flowering and fruiting times (up to 5 and 12 days, respectively) versus uninoculated tomato plants. P. chlamydosporia increased mature fruit weight in tomato plants. The basis of the mechanisms for growth, flowering and yield promotion in tomato by the fungus are unknown. However, we found that P. chlamydosporia can produce Indole‐3‐acetic acid and solubilise mineral phosphate. These results suggest that plant hormones or nutrient ability could play an important role. Our results put forward the agronomic importance of P. chlamydosporia as biocontrol agent of plant parasitic nematodes with tomato growth promoting capabilities.     

Screening for PGPR to improve growth of Cistus ladanifer seedlings for reforestation of degraded mediterranean ecosystems

A screening for PGPRs was carried out in the rhizosphere of wild populations of Cistus ladanifer. Two hundred and seventy bacteria were isolated, purified and grouped by morphological criteria. Fifty percent of the isolates were selected and tested for aminocyclopropanecarboxylic acid (ACC) degradation, auxin and siderophore production and phosphate solubilisation. Fifty-eight percent of the isolates showed at least one of the evaluated activities, with phosphate solubilisation and siderophore production being the most abundant traits. After PCR-RAPDs (Randomly amplified polymorphic DNA) analysis, 11 groups appeared with 85% similiarity, revealing the low diversity in the system. One strain of each group was tested in a biological assay, and those that enhanced Cistus growth were identified by 16S rDNA sequencing.Although seven of the 11 assayed strains were phosphate solubilisers and able to produce siderophores, only one was really effective in increasing all biometric parameters in Cistus ladanifer seedlings, the lack of effect of the other six probably being due to the rich substrate used. This suggests that other mechanisms apart from nutrient mobilisation might be involved in growth promotion by this strain. However, the low diversity together with the high redundancy detected by PCR-RAPDs and the predominance of strains able to mobilise nutrients in the rhizosphere of Cistus reveals that the plant selects for bacteria that can help to supply scarce nutrients. This type of plant growth promoting rhizobacteria (PGPR) strains should be succesful in reforestation practices.     

Alcaligenes aquatilis GTE53: Phosphate solubilising and bioremediation bacterium isolated from new biotope “phosphate sludge enriched-compost”

The isolation and identification of beneficial bacteria from the active phase of composting is considered to be a key bio-quality parameter for the assessment of the process. The aim of this work was the selection and identification of beneficial bacteria from a co-composting experiment of vegetable waste (VW), olive oil mill waste (O₂MW), and phosphate sludge (PS). Phosphate-solubilizing strains were isolated from the thermophilic phase using Pikovskaya (PVK) solid medium supplemented with tricalcium phosphate Ca3(PO4) (TCP) as the sole source of phosphorus (P). Therefore, the selected isolate Alcaligenes aquatilis GTE53 was tested to tolerate abiotic stresses (different levels of temperature, variable pH, high salinity and water stress). The isolate was also assessed for indole acetic acid (IAA) and siderophores synthesis, nitrogen fixation, phenol degradation and pathogens inactivation. The quality of the co-composting process was also investigated by monitoring the physico-chemical parameters. The obtained results showed that A. aquatilis GTE53 displayed a higher solubilization index of 2.4 and was efficiently dissolved, up to 162.8 and 247.4 mg·mL⁻¹ of inorganic phosphate from PS and phosphate rock (PR), respectively. A. aquatilis GTE53 exhibited siderophores and IAA release, along with atmospheric nitrogen fixation. In addition to that, A. aquatilis GTE53 showed a high resistance to heat and tolerance to acidic and alkaline pH, high salinity and water stress. Moreover, A. aquatilis GTE53 could degrade 99.2% of phenol from a high-concentrated medium (1100 mg·L⁻¹ of phenol) and can inactivate the most abundant pathogens in industrial wastes: Escherichia coli, Streptococcus sp., Salmonella sp., and Fusarium oxysporum albedinis. Analysis of temperature, pH, electrical conductivity, carbon/nitrogen (C/N) ratio, indicated successful co-composting. An efficient transformation of P to the available form and a great abatement of polyphenols, were also recorded during the process. The findings of this study will help to advance the understanding of A. aquatilis GTE53 functions and will facilitate its application to promote beneficial microbial organisms during composting, thus obtaining a high-quality product.     

Actinobacteria may influence white truffle (Tuber magnatum Pico) nutrition,ascocarp degradation and interactions with other soil fungi

To test the hypothesis that truffle-associated bacteria may improve truffle nutrition, we isolated bacteria from white truffle ascocarps and tested Actinobacteria for their ability to solubilise phosphate and iron, nutrients that have limited availability in white truffle grounds. Two isolates with sequence similarities to Curtobacterium flaccumfaciens and Rhodococcus sp. were characterized in detail. Both solubilised Ca₃(PO₄)₂ in a way that was dependent on the nitrogen and carbon sources present. Neither strain broke down phytate, but both produced chelating compounds, performed ammonification, and broke down β-glucan. Additionally, C. flaccumfaciens decomposed chitin, pectin, lipids and proteins, while Rhodococcus sp. exhibited urease activity. Three potentially fungicolous fungi were isolated from diseased white truffle ascocarps and bioassayed against the isolated Actinobacteria. The Rhodococcus isolate inhibited Verticillium leptobactrum, neither bacterium affected Clonostachys rosea, while both isolates promoted growth of Trichoderma sp. The results suggest that Actinobacteria might be involved in improving truffle nutrition, ascocarp degradation and establishing relationships with other soil fungi.