Incorporation of l-lysine and 2,2′-dipyridyl in the growth media promotes desferrioxamine E production by an actinobacterium

The study describes the use of the chelating agent 2,2′-dipyridyl in conjunction with lysine to increase the production of the siderophore desferrioxamine E by a previously described actinobacterium 23F. Desferrioxamine E is a type of siderophore known to be produced by Streptomycete species. Lysine is a precursor of the siderophore and its presence in the culture medium is known to promote desferrioxamine E synthesis. The further addition of 2,2′-dipyridyl was found to enhance production of the siderophore in the presence of lysine (5 g l^?1) nearly twofold when incorporated at a concentration of 200 μM. Increasing the concentration of the chelating agent above 200 μM resulted in a decrease in siderophore production. The role of the chelating agent was thought to be in creating iron-limiting conditions in the culture medium and so promoting the induction of the desferrioxamine E biosynthetic pathway. This medium is likely to be a useful tool in the screening for producers of desferrioxamine E.     

Light-dependent quenching of chlorophyll fluorescence in pea chloroplasts induced by adenosine 5′-triphosphate

Addition of ATP to chloroplasts causes a reversible 25–30% decrease in chlorophyll fluorescence. This quenching is light-dependent, uncoupler insensitive but inhibited by DCMU and electron acceptors and has a half-time of 3 minutes. Electron donors to Photosystem I can not overcome the inhibitory effect of DCMU, suggesting that light activation depends on the reduced state of plastoquinone. Fluorescence emission spectra recorded at ?196°C indicate that ATP treatment increases the amount of excitation energy transferred to Photosystem I. Examination of fluorescence induction curves indicate that ATP treatment decreases both the initial (F_o) and variable (F_v) fluorescence such that the ratio of F_v to the maximum (F_m) yield is unchanged. The initial sigmoidal phase of induction is slowed down by ATP treatment and is quenched 3-fold more than the exponential slow phase, the rate of which is unchanged. A plot of F_v against area above the induction curve was identical plus or minus ATP. Thus ATP treatment can alter quantal distribution between Photosystems II and I without altering Photosystem II-Photosystem II interaction. The effect of ATP strongly resembles in its properties the phosphorylation of the light-harvesting complex by a light activated, ATP-dependent protein kinase found in chloroplast membranes and could be the basis of physiological mechanisms which contribute to slow fluorescence quenching in vivo and regulate excitation energy distribution between Photosystem I and II. It is suggested that the sensor for this regulation is the redox state of plastoquinone.     

Effect of adenosine 3′,5′ monophosphate on the mutation frequency induced by N-methyl-N′-nitro-N-nitrosoguanidine

The effect of increased cellular concentrations of adenosine 3′,5′ monophosphate (cAMP) upon mutation frequency induced by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was studied in V79 Chinese hamster lung cells. Incubation with either forskolin, which increased the accumulation of cAMP, or 8BrcAMP, an analogue of cAMP, resulted in an increase in the mutation frequency which was concentration-dependent, regardless of whether these agents were added before or after mutagen treatment. Increased cAMP concentrations were shown in these cells to inhibit growth; however, this does not seem to be the mechanism responsible for the increase in mutation frequency as low serum concentrations which also retard growth reduced the mutation frequency observed with MNNG.     

Influence of mass transfer limitations on the enzymatic synthesis of β-lactam antibiotics catalyzed by penicillin G acylase immobilized on glioxil-agarose

Mass transfer effects were investigated for the synthesis of ampicillin and amoxicillin, at pH 6.5 and 25 degrees C, catalyzed by penicillin G acylase immobilized on agarose. The influence of external mass transfer was analysed using different stirring rates, ranging form 200 to 800 rpm. Above 400 rpm, the film resistance may be neglected. Intra-particle diffusion limitation was investigated using biocatalysts prepared with different enzyme loads and agarose with different mean pore diameters. When agarose with 6, 8 and 10% of crosslinking were used, for the same enzyme load, substrates and products concentration profiles presented no expressive differences, suggesting pore diameter is not important parameter. An increase on enzyme load showed that when more than 90 IU of enzyme activity were used per mL of support, the system was influenced by intra-particle mass transfer. A reactive-diffusive model was used to estimate effective diffusivities of substrates and products.     

Synthesis of 2′-deoxythymidine by an enzymatic transdeoyrinosylation

Summary 2-Deoxythymidine was synthesized by an enzymatic transdeoxyribosylation of thymine using either (i) dGuo, dCyd or dAdo, or (ii) the mixture of the same 2-deoxynucleosides resulting from enzymatic hydrolysis of DNA as donors of 2-deoxyribofuranose moiety.     

Influence of aluminium on the immune system – an experimental study on volunteers

The purpose of this study was to examine whether oral exposure to aluminum (Al) can affect the human immune system. Eighteen healthy volunteers (mean age 42, 28–57 yr) were divided into a test group (9 females, 4 males) and a referent group (3 females, 2 males). Over 6 weeks, the test subjects ingested 10 ml of antacid (aluminum hydroxide, 59 mg Al/ml) three times daily. Aluminum was analyzed in urine before and during the exposure period (ICP-MS). Blood samples were used for analysis of lymphocyte subpopulations, mitogen-induced lymphocyte proliferation and in vitro production and circulating plasma concentrations of immunoglobulin (Ig) A, IgG, IgM, interleukin (IL) -2 and IL-4. Urinary Al concentration in the test subjects was approximately 10- to 20-fold higher than in the referent group during exposure. This indicates that ingestion of an Al-containing antacid is associated with an Al absorption far above that originating from food and drinking water. In both referents and test subjects the lymphocyte subpopulations, lymphocyte proliferation and the in vitro Ig and IL production showed similar, time-dependent changes before as well as during the exposure period. No major differences were seen between the referent and test groups regarding the immune parameters, except for a slightly smaller CD8+CD45R0+ population (primed cytotoxic T-cells), in the exposed individuals as compared to the referents. The results also show that subjects on antacid therapy may constitute a suitable population for studying biological effects of high-dose oral exposure to Al.     

The common origins of the pigments of life—early steps of chlorophyll biosynthesis

The complex pathway of tetrapyrrole biosynthesis can be dissected into five sections: the pathways that produce 5-aminolevulinate (the C-4 and the C-5 pathways), the steps that transform ALA to uroporphyrinogen III, which are ubiquitous in the biosynthesis of all tetrapyrroles, and the three branches producing specialized end products. These end products include corrins and siroheme, chlorophylls and hemes and linear tetrapyrroles. These branches have been subjects of recent reviews. This review concentrates on the early steps leading up to uroporphyrinogen III formation which have been investigated intensively in recent years in animals, in plants, and in a wide range of bacteria.Abbreviations ALA 5-aminolevulinic acid - ALAS 5-aminolevulinic acid synthase - GR glutamyl-tRNA reductase - GSA glutamate-1-semialdehyde - GSAT glutamate-1-semialdehyde aminotransferase - HMB hydroxymethylbilane - PBG porphobilinogen - PBGD porphobilinogen deaminase - PBGS porphobilinogen synthase - URO uroporphyrin - URO'gen uroporphyrinogen - US uroporphyrinogen III synthase     

Glycine origin of the methyl substituent on C7′-N of octodiose for the biosynthesis of apramycin

Apramycin is unique in the aminoglycoside family due to its octodiose moiety. However, either the biosynthesis process or the precursors involved are largely unknown. Addition of glycine, as well as serine or threonine, to the Streptomyces tenebrabrius UD2 fermentation medium substantially increases the production of apramycin with little effect on the growth of mycelia, indicat-ing that glycine and/or serine might be involved in the biosynthesis of apramycin. The ¹³C-NMR analysis of [2-¹³C] glycine-fed (25% enrichment) apramycin showed that glycine specifically and efficiently incorporated into the only N-CH₃ substituent of apramycin on the C7′ of the octodiose moiety. We noticed that the in vivo concentration of S-adenosyl methionine increased in parallel with the addition of glycine, while the addition of methione in the fermentation medium significantly decreased the productivity of apramycin. Therefore, the methyl donor function of glycine is proposed to be involved in the methionine cycle but methionine itself was proposed to inhibit the methylation and methyl transfer processes as previously reported for the case of rapamycin. The ¹⁵N NMR spectra of [2-¹³C,¹⁵N]serine labeled apramycin indicated that serine may also act as a limiting precursor contributing to the ―NH₂ substituents of apramycin.     

Glycine origin of the methyl substituent on C7′-N of octodiose for the biosynthesis of apramycin

Apramycin was formerly designated factor 2 of the nebramycin complex, a mixture of aminoglyco-side-type antibiotics, produced by Streptomyces tene-brabrius[1,2]. It possesses a unique C-8 aminosugar, abicyclic aminooctodiosyl moiety named octodiose with a…     

Some observations on the minor products of the reaction of 2,2′-dilithiobiphenyl with mercuric chloride

Reaction of 2,2′-dilithiobiphenyl (formed from 2,2′-diiodobiphenyl and lithium in diethyl ether) with mercuric chloride gives the ortho-biphenylenemercury trimer (I) with 2,2′-bis(iodomercury)biphenyl (II) as an isolatable intermediate. The mass spectrum of impure 2,2′-bis(iodomercury)biphenyl at high sensitivity shows ion clusters which are interpreted as the ions of a polyphenyl iodomercury complex [Hg₃(C₆H₄)₄I₂] (III) which is identified as a further intermediate in the production of ortho-biphenylenemercury trimer and several iodomercury cations of general formula [Hg_xI_y]⁺, where x, y = 1, 2, 3. A fragmentation scheme is presented to account for these unusual iodomercury cations. Reaction mechanisms are presented to account for the production of II and III.     

The effect of intravital chelation with dimercaprol,calcium disodium edetate, 1-10-phenantroline and 2,2′-dipyridyl on the sulfide silver stainability of the rat brain

Summary Dimercaprol, calcium disodium edetate, 1-10-phenantroline and 2,2-dipyridyl were injected intraperitoneally in adult albino rats in doses ranging from 10 to 1500 mg per kg body weight. Ten animals were injected intracerebrally. At various survival times (2 minutes to several hours) their effects on the staining pattern of heavy metals as revealed by the sulfide silver method of Timm were determined. EDTA was virtually ineffective while dimercaprol, phenantroline and dipyridyl reduced the staining in many regions of the brain. A noteworthy effect of phenantroline was that it reduced the staining of a cortical area, corresponding remarkably well in extent, shape and location to the neocortical somatosensory area.     

Influence of Fluorination of the Sugar Moiety on the Anti-HIV-1 Activity of 2′,3′-Dideoxynucleosides

Abstract

The advent of AIDS has prompted the search for effective anti-HIV-1 agents, and, in view of the efficacy of azidothymidine in the treatment of AIDS, 2′,3′-dideoxynucleosides and analogues thereof have been considered as the most obvious candidates for AIDS chemotherapy. Various substituents have been introduced at the 3′-position, but only the 3′-azido and 3′-fluoro derivatives were found active against HIV-1. Introduction of a fluorine in organic compounds frequently causes a dramatic change in their biological activity. The stability of the carbon-fluorine bond and the strong electronegative character of fluorine, altering the electronic properties of the substituted molecule, led us to synthesize dideoxynucleosides with a fluorine substituent at different positions. The synthe-     

Effect of Diquat (1,1′-Ethylene-2,2′-Dipyridylium Dibromide) on the Photosynthetic Growth of Rhodospirillum rubrum

Diquat (2 x 10(-4)m) inhibited both aerobic and anaerobic growth of Rhodospirillum rubrum. With photosynthetic cultures, diquat affected the synthesis of bacteriochlorophyll more readily than cell mass (turbidity). Diquat retarded the synthesis of bacteriochlorophyll and some protein more readily than that of other cellular constituents such as ribonucleic acid, deoxyribonucleic acid, and cell mass. With cells deficient in phosphate, diquat inhibited the uptake-conversion of inorganic phosphate completely only when 3-(3,4-dichlorophenyl)-1,1'-dimethyl urea and ascorbate were also present.     

Oxidation of Morphine to 2,2′-Bimorphine by Cylindrocarpon didymum

The oxidation of morphine by whole-cell suspensions and cell extracts of Cylindrocarpon didymum gave rise to the formation of 2,2′-bimorphine. The identity of 2,2′-bimorphine was confirmed by mass spectrometry and ¹H nuclear magnetic resonance spectroscopy. C. didymum also displayed activity with the morphine analogs hydromorphone, 6-acetylmorphine, and dihydromorphine, but not codeine or diamorphine, suggesting that a phenolic group at C-3 is essential for activity.The morphine alkaloids are the major alkaloid components of opium, the dried latex material from cut seed capsules of the opium poppy, Papaver somniferum. Of all the alkaloids, the morphine alkaloid group has been studied in most detail, mainly due to the significant therapeutic properties these compounds possess. The morphine alkaloids are narcotic analgesics and are widely used by clinicians for the control of chronic pain. The use of microbial enzymes to provide biological routes for the synthesis of semisynthetic drugs that are difficult to synthesize chemically and as a means of producing new morphine alkaloid derivatives has been the subject of a significant amount of research; this topic has recently been reviewed (3). In recent years, there has been an increasing demand for new morphine alkaloid intermediates for the synthesis of novel semisynthetic drugs, and as part of a study to produce such compounds, we have been exploring fungal transformations of morphine. In this paper, we describe the conversion of morphine to pseudomorphine (2,2′-bimorphine) by Cylindrocarpon didymum 311186.

Biotransformation of morphine.

C. didymum 311186 was obtained from the International Mycological Institute (Egham, Surrey, United Kingdom). Mycelia were grown in media at pH 7.0 containing (grams per liter) yeast extract (10.0), KH₂PO₄ (10.0), (NH₄)₂SO₄ (5.0), and MgSO₄ (0.5). Trace elements were as described by Rosenberger and Elsden (9). Cultures were incubated at 30°C for 48 h with rotary shaking at 180 rpm. Washed mycelia (typically 0.5 g [wet weight]) were resuspended in 40 ml of medium containing 10 mM morphine (Macfarlan Smith Ltd., Edinburgh, United Kingdom) in 250-ml Erlenmeyer flasks. Samples (0.2 ml) were removed at regular intervals and diluted fivefold in 50 mM phosphoric acid (pH 3.5), to dissolve any insoluble metabolites. Mycelia were removed by centrifugation at 14,000 × g with an MSE Microcentaur microcentrifuge (Patterson Scientific Ltd., Dunstable, United Kingdom). The samples were analyzed by high-performance liquid chromatography (HPLC) with a Waters component system (Millipore Waters UK Ltd., Watford, United Kingdom). The HPLC system consisted of a 600E system controller connected to either a 484 absorbance detector or a model 994 programmable photodiode array detector set to 230 nm, 0 to 1 V full-scale detection. Injections of 50 μl were performed with a WISP 712 autoinjector and data processed with Millennium 2010 software. Separation of samples was achieved with a C₁₈ Spherisorb column (4.6 by 250 mm, 5-μm particle size; Anachem Ltd., Luton, United Kingdom), protected by a guard column of the same packing material with an isocratic solvent system containing 40 mM phosphoric acid buffer (pH 2.5) and acetonitrile in a ratio of 92:8 plus 2 mM pentanesulfonic acid, delivered at a flow rate of 1 ml/min. Analysis of the whole-cell incubation mixture by HPLC showed that morphine was completely removed from the medium after a period of 70 h. No other soluble metabolites were identified by HPLC; however, a white precipitate was found to accumulate in the incubation mixture. Microscopic analysis showed that the precipitate had formed regular cubic crystals. The crystalline material was found to dissolve under mildly acidic conditions, and HPLC analysis after such treatment revealed the stoichiometric conversion of morphine to an unknown compound (Fig. ​(Fig.1)1) that had a retention time that coincided with that of authentic 2,2′-bimorphine, kindly provided by M. McPherson (Macfarlan Smith Ltd.). The compound was analyzed by thin-layer chromatography (TLC) with polyester-backed plates precoated with Polygram Sil G/UV₂₅₄ (Machery-Nagel, Duren, Germany) and developed in ammonia-n-butanol (20/80 [vol/vol]). TLC analysis revealed the appearance of two compounds that were detectable under UV light at 254 nm and with Ludy Tenger reagent (7). Compound 1 had an R_f value of 0.42 corresponding to that of authentic morphine, while compound 2 had an R_f value of 0.25 which coincided with that of authentic 2,2′-bimorphine. 2,2′-Bimorphine shows greatly enhanced fluorescence characteristics, compared to those of morphine, due to extended conjugation (6). Compound 2 fluoresced with a characteristic blue color when the TLC plate was illuminated at 366 nm. Fluorescent excitation and emission spectra of compound 2 dissolved in 50 mM potassium phosphate buffer (pH 7.4) in 1-cm-path-length cuvettes were recorded with a Perkin-Elmer LS 50 B luminescence spectrometer (Perkin-Elmer Ltd., Beaconsfield, United Kingdom). Two principal excitation maxima were found at 280 and 320 nm, with a single emission maximum at 430 nm, typical of authentic 2,2′-bimorphine. Open in a separate windowFIG. 1Accumulation of 2,2′-bimorphine in whole-cell incubations of C. didymum. Whole-cell incubations contained 40 ml of minimal medium, 10 mM morphine, and 0.5 g (wet weight) of mycelia in 250-ml Erlenmeyer flasks. Morphine (•) and 2,2′-bimorphine (○) concentrations were determined by HPLC. The data are means of three replicate incubations.

Identification of 2,2′-bimorphine.

¹H nuclear magnetic resonance spectroscopy of the product was performed at 400 MHz with a Bruker AM-400 spectrometer with tetramethylsilane as an internal standard and D-6 dimethyl sulfoxide as the solvent. The ¹H nuclear magnetic resonance spectrum gave the following signals, which were indistinguishable from those of an authentic sample of 2,2′-bimorphine (5) (for the proton assignments, see Fig. ​Fig.2,2, which gives the 2,2′-bimorphine numbering system): δ H 6.31 (2H, s, 1-H and 1′-H); 5.58 (2H, dd, J = 9.6 and 2.5, 7-H and 7′-H); 5.26 (2H, d, J = 9.6, 8-H and 8′-H); 4.70 (2H, d, J = 5.7, 5-H and 5′-H); 4.10 (2H, dd, J = 5.7 and 2.5, 6-H and 6′-H); 3.29 (2H, dd, J = 6.2 and 2.6, 9-H and 9′-H); 2.91 (2H, d, J = 18.6, 10β-H and 10β′-H); 2.57 (2H, d, J = 2.6, 14-H and 14′-H); 2.50 (2H, dd, J = 12.5 and 3.5, 16β-H and 16β′-H); 2.32 (6H, s, NMe and NMe′); 2.28 (2H, d, J = 12.5, α16-H and α16′-H); 2.23 (2H, dd, J = 18.6 and 6.2, α10-H and α10′-H); 1.99 (2H, dd, J = 11.4 and 3.5, α15-H and α15′-H); 1.68 (2H, d, J = 11.4, β15-H and β15′-H). Open in a separate windowFIG. 2Morphine analogs.The ¹H spectrum agreed with that expected for a symmetrical dimer, and only one aromatic proton signal was observed, instead of the characteristic AB pair of the morphine spectra, suggesting a symmetrical substitution on the aromatic ring. Laser desorption time-of-flight mass spectrometry was performed with a Kompact Maldi III mass spectrometer, and the mass spectrum showed a molecular ion, m/z 569.4, for C₃₄H₃₆N₂O₆.

Transformations of morphine analogs by C. didymum.

Whole-cell incubations of C. didymum were challenged with a range of morphine analogs including hydromorphone, 6-acetylmorphine, dihydromorphine, codeine, and diamorphine (see Fig. ​Fig.22 for structures). The incubations contained in 250-ml Erlenmeyer flasks approximately 0.61 g (wet weight) of mycelia and morphine analogs at 5 or 10 mM in a total volume of 40 ml of minimal medium. The flasks were incubated at 30°C with shaking, and samples were removed at intervals for HPLC analysis. Figure ​Figure33 shows that C. didymum was capable of activity with morphine, hydromorphone, 6-acetylmorphine, and dihydromorphine, and precipitates were observed to accumulate. Structural information on these products was not obtained. All of these compounds possess a free phenolic group at C-3 as a common structural feature which is likely to be an essential requirement for activity. This is consistent with the chemical oxidation of morphine to 2,2′-bimorphine, which requires the formation of a phenoxy radical intermediate (1). Open in a separate windowFIG. 3Transformations of morphine analogs by C. didymum. Whole-cell incubations contained 40 ml of minimal medium, 10 mM substrate (5 mM dihydromorphine), and 0.61 g (wet weight) of mycelia in 250-ml Erlenmeyer flasks. Morphine (•), codeine (▵), diamorphine (▴), hydromorphone (○), dihydromorphine (□), and 6-acetylmorphine (■) concentrations were determined by HPLC.

Enzyme activity in cell extracts.

The whole-cell transformation of morphine to 2,2′-bimorphine prompted investigation of subcellular enzyme activity. Cell extract was prepared by the method of Rahim and Sih (8) with the following modifications. Frozen mycelia containing 10 to 14 g (wet weight) of biomass were placed in an ice-cold mortar with an equal weight of acid-washed white quartz sand (50/70 mesh; Sigma Chemical Company, Poole, United Kingdom) and an equal volume of ice-cold potassium phosphate buffer (pH 7.4). The mixture was ground with a pestle for approximately 20 min until it formed a thin paste. The paste was diluted with an equivalent volume of ice-cold buffer, and the sand and cell debris were removed by centrifugation at 20,000 × g for 15 min at 4°C in a Sorvall RC5C centrifuge fitted with an SS34 rotor. Protein was measured by the method of Bradford (2) with the Pierce protein assay reagent according to the manufacturer’s protocol. Typically, protein recoveries of approximately 7 mg of protein/g (wet weight) of cells were obtained. The fluorescent nature of 2,2′-bimorphine enabled the development of a convenient and sensitive enzyme assay. In reaction mixtures which contained potassium phosphate buffer (pH 7.4), morphine (5 mM), and cell extract, activity could be measured spectrofluorimetrically by measuring fluorescence of 2,2′-bimorphine at 440 nm when excited at 330 nm. Cell extract from mycelia harvested after 80 h of incubation with morphine had a specific activity of 0.36 U/mg of protein. One unit of activity was defined as the amount of enzyme required to produce 1 μmol of 2,2′-bimorphine from 2 μmol of morphine per min. No activity was observed in control reaction mixtures where the cell extract was replaced with boiled cell extract. Activity was inhibited completely when 0.1 mM azide was added to the reaction mixtures. Interestingly, no activity was observed in cell extract from mycelia that had not been incubated with morphine, which suggests that the activity is inducible. The development of a rapid and sensitive assay should facilitate the purification and characterization of the 2,2′-bimorphine-producing enzyme. 2,2′-Bimorphine has been shown to be a spontaneous reaction product of morphine in aqueous solutions, though the reaction was extremely slow (4). Furthermore, morphine can be oxidized to 2,2′-bimorphine with alkaline ferricyanide, a reaction which is known to proceed via a mesomeric aryloxy free radical, leading to the formation of the dimer (1). However, to the best of our knowledge, this is the first report of the microbial oxidation of morphine to 2,2′-bimorphine.     

Influence of manufacturing processes on in vitro properties of the probiotic strain Lactobacillus rhamnosus Lcr35®

Probiotics are administered as complex manufactured products and yet most studies on probiotic bacterial strains have been performed with native culture strains. Little is known about the influence of industrial processes on the properties of the microorganisms. In this study, we comparatively assessed the characteristics of the probiotic bacterial strain Lactobacillus rhamnosus (Lcr35(?)) together with four of its commercial formulations, including three intestinal formulas (BACILOR with Lcr Restituo(?) packet and capsule and FLOREA Lcr Lenio(?)) and one vaginal formula (GYNOPHILUS Lcr Regenerans(?)). Lcr35(?) grown from the intestinal formulas displayed increased resistance to acidic pH and bile stress, especially FLOREA (Lcr Lenio(?)), which showed a 4.5log higher number of viable bacteria compared to the results obtained with the control native Lcr35(?) strain. Adhesion to intestinal cells was significantly higher with Lcr Restituo(?) packet and Lcr Restituo(?) capsule vs Lcr35(?). Bacteria from the vaginal formulation GYNOPHILUS had increased ability to metabolize glycogen thereby increasing lactic acid production. In vitro growth inhibition of the pathogen Candida albicans was significantly higher with bacteria from the vaginal formulation (4.5 log difference) and in the presence of vaginal epithelial cells than with the native strain. Our results show that the manufacturing process influences strain properties and should therefore be adapted according to the strain and the therapeutic indication.     

β-Endorphinergic system involvement in the inhibitory action of clonidine on induced sodium appetite

In the present study, we investigated the degree to which β-endorphin plays a role in the alpha 2-adrenergic/imidazoline receptor agonist attenuation of salt appetite. In order to evaluate whether the inhibitory action of clonidine (an α2-adrenergic/imidazoline receptor agonist) on induced sodium intake is mediated by the β-endorphinergic system, we used a β-endorphin deficient mouse line. β-endorphin knockout (βend(-/-)), heterozygous (βend(+/-)) and wild-type (βend(+/+)) mice were submitted to acute sodium depletion by a combined treatment of furosemide and low sodium diet and, 20h later, were administered with clonidine (0.5mg/kg). An hour later, the animals were subjected to a two-bottle choice test (water/2% NaCl). The results indicate that clonidine administration during the first stage of the test exerts an equivalent inhibition on sodium intake regardless of the genotype; however, in the final stage of the test, a reversal of the inhibitory response on induced sodium appetite becomes evident in the mice lacking β-endorphin. Moreover no differences in dipsogenic response were observed between the genotypes. Considering these results and the fact that plasma half-life of clonidine at the dose administered is approximately 3h, it is possible to speculate that the inhibitory effect of clonidine on sodium appetite may be independent of β-endorphin modulation during the first stage; however, the long-lasting inhibitory effect of clonidine may be mediated by the β-endorphinergic system. This evidence supports the existence of adrenergic and β-endorphinergic system interaction in the osmoregulatory response to achieve sodium balance.     

Effect of 2, 2′-dipyridyl on the fermentation pattern of a silage simulation medium contaminated with fecal matter

Summary The fermentation pattern of a silage simulation medium contaminated with fecal matter, was followed during 7 days. Iron complexation due to 2,2-dipyridyl had no negative effect on the growth and the performance of lactic acid bacteria, but Enterobacteriaceae were effectively repressed.