Autoradiographic imaging and quantification of the high-affinity GHB binding sites in rodent brain using 3H-HOCPCA.

GHB (γ-hydroxybutyric acid) is a compound endogenous to mammalian brain with high structural resemblance to GABA. GHB possesses nanomolar-micromolar affinity for a unique population of binding sites, but the exact nature of these remains elusive. In this study we utilized the highly selective GHB analogue, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) as a tritiated version (3H-HOCPCA) to radioactively label the specific GHB high-affinity binding site and gain further insight into the density, distribution and developmental profile of this protein. We show that, in low nanomolar concentrations, 3H-HOCPCA displays excellent signal-to-noise ratios using rodent brain autoradiography, which makes it a valuable ligand for anatomical quantification of native GHB binding site levels. Our data confirmed that 3H-HOCPCA labels only the high-affinity specific GHB binding site, found in high density in cortical and hippocampal regions. The experiments revealed markedly stronger binding at pH 6.0 (Kd 73.8 nM) compared to pH 7.4 (Kd 2312 nM), as previously reported for other GHB radioligands but similar Bmax values. Using 3H-HOCPCA we analyzed the GHB binding protein profile during mouse brain development. Due to the high sensitivity of this radioligand, we were able to detect low levels of specific binding already at E15 in mouse brain, which increased progressively until adulthood. Collectively, we show that 3H-HOCPCA is a highly sensitive radioligand, offering advantages over the commonly used radioligand 3H-NCS-382, and thus a very suitable in vitro tool for qualitative and quantitative autoradiography of the GHB high-affinity site.

The orthosteric GABAA receptor ligand Thio-4-PIOL displays distinctly different functional properties at synaptic and extrasynaptic receptors.

BACKGROUND AND PURPOSE: Explorations into the heterogeneous population of native GABA type A receptors (GABAA Rs) and the physiological functions governed by the multiple GABAA R subtypes have for decades been hampered by the lack of subtype-selective ligands. EXPERIMENTAL APPROACH: The functional properties of the orthosteric GABAA receptor ligand 5-(4-piperidyl)-3-isothiazolol (Thio-4-PIOL) have been investigated in vitro, ex vivo and in vivo. KEY RESULTS: Thio-4-PIOL displayed substantial partial agonist activity at the human extrasynaptic GABAA R subtypes expressed in Xenopus oocytes, eliciting maximal responses of up to ∼30% of that of GABA at α5 ß3 γ2S , α4 ß3 δ and α6 ß3 δ and somewhat lower efficacies at the corresponding α5 ß2 γ2S , α4 ß2 δ and α6 ß2 δ subtypes (maximal responses of 4-12%). In contrast, it was an extremely low efficacious agonist at the α1 ß3 γ2S , α1 ß2 γ2S , α2 ß2 γ2S , α2 ß3 γ2S , α3 ß2 γ2S and α3 ß3 γ2S GABAA Rs (maximal responses of 0-4%). In concordance with its agonism at extrasynaptic GABAA Rs and its de facto antagonism at the synaptic receptors, Thio-4-PIOL elicited robust tonic currents in electrophysiological recordings on slices from rat CA1 hippocampus and ventrobasal thalamus and antagonized phasic currents in hippocampal neurons. Finally, the observed effects of Thio-4-PIOL in rat tests of anxiety, locomotion, nociception and spatial memory were overall in good agreement with its in vitro and ex vivo properties. CONCLUSION AND IMPLICATIONS: The diverse signalling characteristics of Thio-4-PIOL at GABAA Rs represent one of the few examples of a functionally subtype-selective orthosteric GABAA R ligand reported to date. We propose that Thio-4-PIOL could be a useful pharmacological tool in future studies exploring the physiological roles of native synaptic and extrasynaptic GABAA Rs.

Biocorrosion and biofilm formation in a nutrient limited heating system subjected to alternating microaerophilic conditions.

Severe biofilm formation and biocorrosion have been observed in heating systems even when the water quality complied with existing standards. The coupling between water chemistry, biofilm formation, species composition, and biocorrosion in a heating system was investigated by adding low concentrations of nutrients and oxygen under continuous and alternating dosing regimes. Molecular analysis of 16S rRNA gene fragments demonstrated that the amendments did not cause changes in the overall bacterial community composition. The combined alternating dosing of nutrients and oxygen caused increased rates of pitting (bio-) corrosion. Detection of bacteria involved in sulfide production and oxidation by retrieval of the functional dsrAB and apsA genes revealed the presence of Gram-positive sulfate- and sulfite-reducers and an unknown sulfur-oxidizer. Therefore, to control biocorrosion, sources of oxygen and nutrients must be limited, since the effect of the alternating operational conditions apparently is more important than the presence of potentially corrosive biofilm bacteria.

Microbial diversity in biofilms from corroding heating systems.

Culture-independent investigations of the bacterial diversity and activity in district heating systems with and without corrosion did not make it possible to relate one group of microorganisms with the observed corrosion. Fluorescence in situ hybridization by oligonucleotide probes revealed the dominance of beta-proteobacteria, sulphate reducing prokaryotes and alpha-proteobacteria. Analysis of a clone library from one Danish heating (DH) system showed that the most sequences formed two clusters within the alpha-proteobacteria affiliated to the families Rhizobiaceae and Acetobacteraceae and two clusters within the beta-proteobacteria belonging to the family Comamonadaceae. Functional groups were determined by microautoradiography showing aerobic and anaerobic bacteria (sulphate reducing and methanogenic bacteria). The corrosion study showed that pitting corrosion rates were five to ten times higher than the general corrosion rates, suggesting the presence of biocorrosion. The results indicate that several bacterial groups could be involved in corrosion of DH system piping including sulphate reducing prokaryotes, Acidovorax (within the beta-proteobacteria), methanogenic bacteria and others.

Monitoring of microbial souring in chemically treated, produced-water biofilm systems using molecular techniques.

The identification of bacteria in oil production facilities has previously been based on culture techniques. However, cultivation of bacteria from these often-extreme environments can lead to errors in identifying the microbial community members. In this study, molecular techniques including fluorescence in situ hybridization, PCR, denaturing gradient gel electrophoresis, and sequencing were used to track changes in bacterial biofilm populations treated with nitrate, nitrite, or nitrate+molybdate as agents for the control of sulfide production. Results indicated that nitrite and nitrate+molybdate reduced sulfide production, while nitrate alone had no effect on sulfide generation. No long-term effect on sulfide production was observed. Initial sulfate-reducing bacterial numbers were not influenced by the chemical treatments, although a significant increase in sulfate-reducing bacteria was observed after termination of the treatments. Molecular analysis showed a diverse bacterial population, but no major shifts in the population due to treatment effects were observed.

MIC mitigation in a 100 MW district heating peak load unit.

During inspection of AISI316 stainless steel plate heat exchangers in a district heating peak load unit, localised corrosion attacks along with indications of microbiological activity were found on the boiler side beneath patches of sturdy black deposits. Bacteria and sulphide were detected within black deposits. Thorough investigation of the boiler system revealed several incidents of localised corrosion on low alloy steel along with deposits of organic matter and bacteria primarily in places with stagnant water or places operating at a low flow rate. A relatively large amount of bacteria was detected within the system, primarily in deposits and around corrosion sites. The observations suggested the combination of deposits and bacterial activity, being the major reason for the observed corrosion. Prior to the investigation, the boiler system had operated with cat-/anion-exchanged, de-aerated water for 3 years, during which the water fulfilled strict chemical limits set to minimise corrosion. Based on these findings, the system has been modified in order to minimise the risk of microbiologically influenced corrosion and a monitoring program for fouling and corrosion has been established.

Monitoring and characterisation of bacteria in corroding district heating systems using fluorescence in situ hybridisation and microautoradiography.

Presence of biofilm and biocorrosion has been observed in Danish district heating (DH) systems despite very good water quality that was expected to prevent significant microbial growth. The microbiological water quality was investigated in order to identify the dominating bacterial groups on surfaces with corrosion problems. Water samples from 29 DH systems were investigated for the total number of bacteria and presence of sulphate reducing bacteria (SRBs). SRBs were found to be present in more than 80% of the DH systems. The microbial population in samples from 2 DH system (biofilm from a test coupon and an in situ sample from a heat exchanger) was investigated with fluorescence in situ hybridisation, and the results showed significant differences in population composition. Betaproteobacteria was the dominant population in both samples. SRBs were present in both samples but were most numerous in the biofilm from the test coupon. Examination of functional groups based on uptake of radiolabelled acetate (microautoradiography) showed presence of both aerobic and anaerobic bacteria despite the fact that oxygen is not anticipated in DH systems.

Characterization of GABA(A) receptor ligands in the rat cortical wedge preparation: evidence for action at extrasynaptic receptors?

1. GABA(A) receptor agonists have previously been characterized at human GABA(A) receptors expressed in Xenopus oocytes. The correlation between these data and functional in vivo data of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) has shown that THIP is 100 fold more potent in clinical studies than in oocytes. 2. THIP and a series of agonists (GABA, Isoguvacine), partial agonists (Imidazole acetic acid; P4S, 4-PIOL, thio-4-PIOL) and one antagonist (SR95531) were characterized in the rat cortical wedge preparation using inhibition of spontaneous activity in Mg(++) free medium as the measurable parameter. 3. Agonists were in general 40 times more potent in the wedge preparation than at alpha(1)beta(3)gamma(2s) containing receptors expressed in Xenopus oocytes, whereas the antagonist was equipotent under these two conditions. 4. Partial agonists with responses above 6% at alpha(1)beta(3)gamma(2s) containing receptors were full agonists in the rat cortical wedge preparation, whereas partial agonists with maximum responses below 6% behaved as partial agonists in the rat cortical wedge preparation. 5. These data suggest that only a small fraction of the GABA(A) receptors in the rat cortical wedge needs to be activated by GABA(A) agonists in order to obtain a maximum response. Results therefore indicate a significant contribution of extrasynaptic receptors to pharmacological activity of exogenous applied GABA(A) agonists in this system.

GABA uptake inhibitors. Design, molecular pharmacology and therapeutic aspects.

In the mid seventies a drug design programme using the Amanita muscaria constituent muscimol (7) as a lead structure, led to the design of guvacine (23) and (R)-nipecotic acid (24) as specific GABA uptake inhibitors and the isomeric compounds isoguvacine (10) and isonipecotic acid (11) as specific GABAA receptor agonists. The availability of these compounds made it possible to study the pharmacology of the GABA uptake systems and the GABAA receptors separately. Based on extensive cellular and molecular pharmacological studies using 23, 24, and a number of mono- and bicyclic analogues, it has been demonstrated that neuronal and glial GABA transport mechanisms have dissimilar substrate specificities. With GABA transport mechanisms as pharmacological targets, strategies for pharmacological interventions with the purpose of stimulating GABA neurotransmission seem to be (1) effective blockade of neuronal as well as glial GABA uptake in order to enhance the inhibitory effects of synaptically released GABA, or (2) selective blockade of glial GABA uptake in order to increase the amount of GABA taken up into, and subsequently released from, nerve terminals. The bicyclic compound (R)-N-Me-exo-THPO (17) has recently been reported as the most selective glial GABA uptake inhibitor so far known and may be a useful tool for further elucidation of the pharmacology of GABA transporters. In recent years, a variety of lipophilic analogues of the amino acids 23 and 24 have been developed, and one of these compounds, tiagabine (49) containing (R)-nipecotic acid (24) as the GABA transport carrier-recognizing structure element, is now marketed as an antiepileptic agent.

Equilibrium binding characteristics of [3H]thiomuscimol.

The equilibrium binding characteristics of the tritiated GABAA agonist, 5-aminomethyl-3-isothiazolol (thiomuscimol) are described. Using the filtration technique to separate bound- from free-ligand, [3H]thiomuscimol was shown to bind to the GABA(A) receptor site(s) in a saturable manner with a Kd value of 28+/-6.0 nM and a Bmax value of 50+/-4.0 fmol/mg original tissue. In parallel binding experiments, the Kd and Bmax values for [3H]muscimol were determined to be 5.4+/-2.8 nM and 82+/-11 fmol/mg original tissue, respectively. In binding assays using the centrifugation technique, Kd and Bmax values for [3H]thiomuscimol were found to be 116+/-22 nM and 154 13 fmol/mg original tissue, respectively, whereas a Kd value of 16+/-1.8 nM and a Bmax value of 155+/-8.0 fmol/mg original tissue were determined for [3H]muscimol. In comparative inhibition studies using the GABA(A) antagonist SR 95531 and a series of specific GABAA agonists, the binding sites for [3H]thiomuscimol and [3H]muscimol were shown to exhibit similar pharmacological profiles. Autoradiographic studies disclosed similar regional distribution of [3H]thiomuscimol and [3H]muscimol binding sites in rat brain. Highest densities of binding sites were detected in cortex, hippocampus, and cerebellum, whereas low densities were measured in the midbrain structures of rat cortex. In conclusion, the equilibrium GABA(A) receptor binding characteristics of [3H]thiomuscimol are very similar to those of [3H]muscimol.

Bioisosterically modified dipeptide excitatory amino acid receptor antagonists containing 3-oxygenated isothiazole ring systems.

The AMPA receptor agonist Thio-AMPA, the 3-isothiazolol analogue of AMPA was converted into the selective NMDA antagonist, 2, in which a 3-isothiazolone unit is a bioisosteric analogue of the peptide bond of the NMDA antagonist, gamma-(R)-Glu-Gly. The isomeric 3-oxygenated isothiazole amino acid, 3, and the corresponding isothiazole phosphono amino acid 4 were also synthesized, and were shown to be selective AMPA receptor antagonists. Compound 1, in which the peptide bond of gamma-(R)-Glu-Gly is replaced by an ester group, was synthesized and shown to be unstable in the test buffer system.

AMPA receptor agonists: synthesis, protolytic properties, and pharmacology of 3-isothiazolol bioisosteres of glutamic acid.

A number of 3-isothiazolol bioisosteres of glutamic acid (1) and analogs of the AMPA receptor agonist, (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA, 2a), including (RS)-2-amino-3-(3-hydroxy-5-methylisothiazol-4-yl)propionic acid (thio-AMPA, 2b), were synthesized. Comparative in vitro pharmacological studies on this series of 3-isothiazolol and the corresponding 3-isoxazolol amino acids were performed using a series of receptor binding assays (IC50 values) and the electrophysiological rat cortical slice model (EC50 values). Whereas 2a (IC50 = 0.04 +/- 0.005 microM, EC50 = 3.5 +/- 0.2 microM) is markedly more potent than the tert-butyl analog ATPA (3a) (IC50 = 2.1 +/- 0.16 microM, EC50 = 34 +/- 2.4 microM) in [3H]AMPA binding and electrophysiological studies, 2b (IC50 = 1.8 +/- 0.13 microM, EC50 = 15.0 +/- 2.4 microM) was approximately equipotent with thio-ATPA (3b) (IC50 = 0.63 +/- 0.07 microM, EC50 = 14 +/- 1.3 microM). (RS)-2-Amino-3-(3-hydroxyisoxazol-5-yl)propionic acid (HIBO, 4a) was approximately equipotent with its thio analog 4b, whereas 4-Br-HIBO (5a) (IC50 = 0.65 +/- 0.12 microM, EC50 = 22 +/- 0.6 microM) turned out to be much more potent than the corresponding 3-isothiazolol 5b (IC50 = 17 +/- 2.2 microM, EC50 = 500 +/- 23 microM). 2b (ED50 = 130 mumol/kg) was more potent than 2a (220 mumol/kg) as a convulsant after subcutaneous administration in mice. The protolytic properties of 2a,b-4a,b were determined using 13C NMR spectroscopy. For each pair of compounds, the alpha-amino acid groups showed similar protolytic properties, whereas the 3-isoxazolol moieties typically showed pKa values 2 units lower than those of the 3-isothiazolols. Accordingly, calculations of ionic species distributions revealed pronounced differences between 3-isoxazolol and 3-isothiazolol amino acids. No simple correlation between activity as AMPA agonists in vitro and pKa values of these compounds was apparent. On the other hand, the relative potencies of AMPA (2a) and thio-AMPA (2b) in vitro and in vivo may reflect that these compounds predominantly penetrate the blood-brain barrier as net uncharged diprotonated ionic species.

Deflocculation of Activated Sludge by the Dissimilatory Fe(III)-Reducing Bacterium Shewanella alga BrY.

The influence of microbial Fe(III) reduction on the deflocculation of autoclaved activated sludge was investigated. Fe(III) flocculated activated sludge better than Fe(II). Decreasing concentrations of Fe(III) caused an increase in sludge bulk water turbidity, while bulk water turbidity remained relatively constant over a range of Fe(II) concentrations. Cells of the dissimilatory metal-reducing bacterium Shewanella alga BrY coupled the oxidation of H(inf2) to the reduction of Fe(III) bound in sludge flocs. Cell adhesion to the Fe(III)-sludge flocs was a prerequisite for Fe(III) reduction. The reduction of Fe(III) in sludge flocs by strain BrY caused an increase in bulk water turbidity, suggesting that the sludge was deflocculated. The results of this study support previous research suggesting that microbial Fe(III) respiration may have an impact on the floc structure and colloidal chemistry of activated sludge.

Changes in the composition of extracellular polymeric substances in activated sludge during anaerobic storage.

Changes in the chemical composition of organic compounds in total activated sludge, activated sludge extracellular polymeric substances (EPS), and sludge bulk water during anaerobic storage (12 days) were studied. The background for the study was that anaerobic storage of activated sludge, which often takes place at wastewater treatment plants before dewatering, causes a deterioration of the dewaterability. The reasons are not known at present, but may be related to changes in exopolymer composition of the flocs. The results showed that a fast decrease in total sludge protein and carbohydrate took place within 3 days of anaerobic storage as a result of degradation processes, which accounted for approximately 20% of the organic fraction. The amount of uronic acids and humic compounds remained almost constant in the sludge. The EPS were extracted from the floc matrix using a cation-exchange resin. In the EPS matrix a similar initial (2-3 days) degradation of proteins and carbohydrate took place, whereas the content of DNA and uronic acids showed minor changes. The extractability of humic compounds increased during the first 3 days of storage. No changes in extractability of the carbohydrate were observed. A fraction of the EPS protein was found to be difficult to extract but was observed to be degraded during the anaerobic storage. The EPS composition was further characterized by high-performance size-exclusion chromatography analysis obtained by on-line UV detection and post-column detection of proteins, carbohydrates, humic compounds and DNA. Four fractions of polysaccharides were found, of which only one was responsible for the decrease in the carbohydrate content observed with storage time. The fraction was presumably of low molecular mass. Humic compounds and volatile fatty acids (acetate and propionate) were released to the bulk water from the flocs during the storage. A possible mechanism to explain the reduced dewaterability developed during anaerobic storage, partly because of the observed changes in EPS, is discussed.

Neuroactive polyamine wasp toxins: nuclear magnetic resonance spectroscopic analysis of the protolytic properties of philanthotoxin-343.

Acid-base properties (pKa values and proton distribution patterns) of philanthotoxin-343(PhTX-343) were investigated by 1H and 13C NMR titration. Chemical shift data and the total ionization shifts were used to assign carbon atoms of the polyamine chain. Nonlinear analysis of the 13C NMR titration curves gave four pKa values (pK1 8.5, pK2 9.5, pK3 10.4, pK4 11.4) and the intrinsic chemical shifts of the non-, mono-, di-, tri-, and tetraprotonated forms. The changes of intrinsic chemical shifts enabled analysis of the deprotonation sequence of fully protonated PhTX-343. The results of analysis of the 13C NMR titration curves were supported by 1H NMR data obtained from two-dimensional 1H, 13C chemical shift correlation experiments. Thus, the first deprotonation mainly takes place at the inner amino group. The phenol group is deprotonated in the second and third deprotonation steps. The preferential deprotonation of the inner amino group is also apparent in the deprotonated form. The monoprotonated form carries a practically fully ionized phenol group and the proton shared between the three amino groups. This characteristic is in agreement with existing data on polyamines. At physiological pH, the tetraprotonated form of PhTX-343 predominates, but the proportion of the triprotonated form becomes significant at low ionic strength. The terminal, primary amino group, which has been shown to be essential for biological activity, remains practically fully protonated at biologically relevant pH values, and this charge is likely to participate in the receptor-binding event. Protonation of the central amino group does not appear to be necessary for biological activity.

Influence of protein conditioning films on binding of a bacterial polysaccharide adhesin from Hyphomonas MHS-3.

A putative polysaccharide adhesin which mediates non-specific attachment of Hyphomonas MHS-3 (MHS-3) to hydrophilic substrata has been isolated and partially characterized. A polysaccharide-enriched portion of the extracellular polymeric substance (EPS(P)) from MHS-3 was separated into four fractions using high performance size exclusion chromatography (HPSEC). Comparison of chromatograms of EPS(P) from MHS-3 and a reduced adhesion strain (MHS-3 rad) suggested that one EPS(P) fraction, which consisted of carbohydrate, served as an adhesin. Adsorption of this fraction to germanium (Ge) was investigated using attenuated total reflection Fourier transform infrared (ATR/FT-IR) spectrometry. Binding curves indicated that the isolated fraction had a relatively high affinity for Ge when ranked against an adhesive protein from Mytilis edulis, mussel adhesive protein (MAP) and an acidic polysaccharide (alginate from Macrocystis pyrifera). Spectral features were used to identify the fraction as a polysaccharide previously reported to adsorb preferentially out of the EPS(P) mixture. Conditioning the Ge substratum with either bovine serum albumin (BSA) or MAP decreased the adsorption of the adhesive polysaccharide significantly. Conditioning Ge with these proteins also decreased adhesion of whole cells.

Adhesion of biofilms to inert surfaces: A molecular level approach directed at the marine environment.

Protein/ligand interactions involved in mediating adhesion between microorganisms and biological surfaces have been well-characterized in some cases (e.g. pathogen/host interactions). The strategies microorganisms employ for attachment to inert surfaces have not been so clearly elucidated. An experimental approach is presented which addresses the issues from the point of view of molecular interactions occurring at the interface.

Partial GABAA receptor agonists. Synthesis and in vitro pharmacology of a series of nonannulated analogs of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol.

5-(4-Piperidyl)isoxazol-3-ol (4-PIOL, 10), a structural analog of 4-aminobutanoic acid (GABA, 1) and the GABAA agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 5), is a low-efficacy partial GABAA agonist. A number of compounds bioisosterically derived from 10, including 5-(4-piperidyl)isothiazol-3-ol (11), 3-(4-piperidyl)isoxazol-5-ol (12), 5-(1,2,3,6-tetrahydropyrid-4-yl)isoxazol-3-ol (13), and 5-(1,2,3,6-tetrahydropyrid-4-yl)isothiazol-3-ol (14), were synthesized and tested as GABAA receptor ligands. Whereas none of these compounds significantly affected GABAB receptor binding or GABA uptake, they showed affinities for GABAA receptor sites in the low-micromolar range. Using cultured cerebral cortical neurons and whole-cell patch-clamp techniques, the efficacies of these compounds relative to that of the full GABAA agonist, isoguvacine (8) (20 microM), were determined. The relative efficacy of 11, which has a higher receptor affinity (IC50 = 1.3 +/- 0.3 microM) than 10 (IC50 = 9.3 +/- 2.6 microM), was comparable with that of 10 (30-35%). The tetrahydropyridine analog of 10, compound 13, showed a markedly lower receptor affinity (IC50 = 32 +/- 10 microM) and apparently a lower relative efficacy than 10. The corresponding unsaturated analog of 11, compound 14, showed a slightly weaker receptor affinity (IC50 = 4.0 +/- 2.0 microM) but a significantly higher relative efficacy (50-55%) than 11. The 5-isoxazolol isomer of 10, compound 12, showed a reduced receptor affinity (IC50 = 26 +/- 7 microM) and a very low relative efficacy. Substitution of propanoic or propenoic acid moieties for the acidic heterocyclic units of these compounds gave the monocyclic amino acids 15-18, which have very little or no affinity for GABAA receptor sites.