Fenobam promoted the neuroprotective effect of PEP-1-FK506BP following oxidative stress by increasing its transduction efficiency

We examined the ways in which fenobam could promote not only the transduction of PEP-1-FK506BP into cells and tissues but also the neuroprotective effect of PEP-1-FK506BP against ischemic damage. Fenobam strongly enhanced the protective effect of PEP-1-FK506BP against H₂O₂-induced toxicity and DNA fragmentation in C6 cells. In addition, combinational treatment of fenobam with PEP-1-FK506BP significantly inhibited the activation of Akt and MAPK induced by H₂O₂, compared to treatment with PEP-1-FK506BP alone. Interestingly, our results showed that fenobam significantly increased the transduction of PEP-1-FK506BP into both C6 cells and the hippocampus of gerbil brains. Subsequently, a transient ischemic gerbil model study demonstrated that fenobam pretreatment led to the increased neuroprotection of PEP-1-FK506BP in the CA1 region of the hippocampus. Therefore, these results suggest that fenobam can be a useful agent to enhance the transduction of therapeutic PEP-1-fusion proteins into cells and tissues, thereby promoting their neuroprotective effects. [BMB Reports 2013; 46(11): 561-566]     

The mGluR5 Antagonist Fenobam Induces Analgesic Conditioned Place Preference in Mice with Spared Nerve Injury

Antagonists of metabotropic glutamate receptors (mGluRs) have the potential to act as analgesic drugs that may help alleviate chronic pain. This study was done to look at the possible rewarding properties of the mGluR5 antagonist, fenobam, in a cognitive assay. Analgesic conditioned place preference (aCPP) was used to examine the effects of fenobam (30 mg/kg) and the prototypical mGluR5 antagonist, MPEP, and these effects were compared to those of a drug with known analgesic properties, morphine (10 mg/kg). In each experiment, one group of mice received spared nerve injury (SNI) surgery to model chronic pain; the other group received a control sham surgery. Both fenobam and MPEP induced preference in the SNI mice, such that SNI mice spent significantly more time in the mGluR5 antagonist-paired chamber compared to a vehicle-paired chamber. No such preference developed for sham mice. Morphine induced preference in male and female mice in both the SNI and sham groups. The results showed that fenobam and MPEP likely reduced on-going distress in the SNI mice, causing them to prefer the chamber paired with the drug compared to the vehicle-paired chamber. Since sham animals did not prefer the drug-paired chamber, these data demonstrate that mGluR5 antagonism is non-rewarding in the absence of pain-like injury.     

Phenyl ureas of creatinine as mGluR5 antagonists. A structure-activity relationship study of fenobam analogues

Fenobam (1) was developed by McNeil Laboratories as an anxiolytic agent with an unknown molecular target in the late 1970s. In a recent publication, it was revealed that fenobam is a non-competitive mGluR5 antagonist. Herein, we present the structure-activity relationship of fenobam and its analogues and similarities between the SAR of mGluR5 antagonism and the SAR of CNS properties originally reported by McNeil are discussed.     

Rational design, synthesis, and structure-activity relationship of benzoxazolones: new potent mglu5 receptor antagonists based on the fenobam structure

A novel class of potent and stable mGlu5 receptor antagonists was developed by combining information from a high-throughput screening campaign with the structure of the known anxiolytic fenobam. Representative compounds from this class show favorable pharmacokinetic properties and are active in an in vivo model of anxiety.     

Comparison of the binding pockets of two chemically unrelated allosteric antagonists of the mGlu5 receptor and identification of crucial residues involved in the inverse agonism of MPEP

Fenobam [N-(3-chlorophenyl)-N'-(4,5-dihydro-1-methyl-4-oxo-1H-imidazole-2-yl)urea], a clinically validated non-benzodiazepine anxiolytic, has been shown to be a potent and non-competitive metabotropic glutamate (mGlu)-5 receptor antagonist. In the present study, we have used the site-directed mutagenesis coupled with three-dimensional receptor-based pharmacophore modelling to elucidate the interacting mode of fenobam within the seven-transmembrane domain (7TMD) of mGlu5 receptor and its comparison with that of 2-methyl-6-(phenylethynyl)pyridine (MPEP), the prototype antagonist. The common residues involved in the recognition of MPEP and fenobam include Pro654(3.36), Tyr658(3.40), Thr780(6.44), Trp784(6.48), Phe787(6.51), Tyr791(6.55) and Ala809(7.47). The differentiating residues between both modulators' interacting modes are Arg647(3.29), Ser657(3.39) and Leu743(5.47). Our data suggest that these chemically unrelated mGlu5 antagonists act similarly, probing a functionally unique region of the 7TMD. Using [3H]inositol phosphates accumulation assay, we have also identified the critical residues involved in the inverse agonist effect of MPEP. The mutation W784(6.48)A completely blocked the inverse agonist activity of MPEP; two mutations F787(6.51)A and Y791(6.55)A, caused a drastic decrease in the MPEP inverse agonism. Furthermore, these three mutations led to an increased efficacy of quisqualate without having any effect on its potency. The fact that the residues Trp784(6.48) and Phe787(6.51) are essential equally in antagonism and inverse agonism effects emphasizes again the key role of these residues and the involvement of a common transmembrane network in receptor inactivation by MPEP.     

Isoxazolo[3,4-d]pyridazinones positively modulate the metabotropic glutamate subtypes 2 and 4

Isoxazolo[3,4-d] pyridazinones ([3,4-d]s) are selective positive modulators of the metabotropic glutamate receptors (mGluRs) subtypes 2 and 4, with no functional cross reactivity at mGluR_1a, mGLuR₅ or mGluR₈. Modest binding for two of the [3,4-d]s is observed at the allosteric fenobam mGluR₅ site, but not sufficient to translate into a functional effect. The structure activity relationship (SAR) for mGluR₂ and mGluR₄ are distinct: the compounds which select for mGluR₂ all contain fluorine on the N-6 aryl group. Furthermore, the [3,4-d]s in this study showed no significant binding at inhibitory GABA_A, nor excitatory NMDA receptors, and previously we had disclosed that they lack significant activity at the System Xc-Antiporter. A homology model based on Conn’s mGluR₁ crystal structure was examined, and suggested explanations for a preference for allosteric over orthosteric binding, subtype selectivity, and suggested avenues for optimization of efficacy as a reasonable working hypothesis.     

Satellite glial cell P2Y12 receptor in the trigeminal ganglion is involved in lingual neuropathic pain mechanisms in rats

Background

Interstitial cystitis/painful bladder syndrome (IC/PBS), is a severely debilitating chronic condition that is frequently unresponsive to conventional pain medications. The etiology is unknown, however evidence suggests that nervous system sensitization contributes to enhanced pain in IC/PBS. In particular, central nervous system plasticity of glutamatergic signaling involving NMDA and metabotropic glutamate receptors (mGluRs) has been implicated in a variety of chronic pain conditions. Here, we test the hypothesis that mGluR5 mediates both non-inflammatory and inflammatory bladder pain or nociception in a mouse model by monitoring the visceromotor response (VMR) during graded bladder distention.

Results

Using a combination of genetic and pharmacologic approaches, we provide evidence indicating that mGluR5 is necessary for the full expression of VMR in response to bladder distention in the absence of inflammation. Furthermore, we observed that mice infected with a uropathogenic strain of Escherichia coli (UPEC) develop inflammatory hyperalgesia to bladder distention, and that the selective mGluR5 antagonist fenobam [N-(3-chlorophenyl)-N'-(4,5-dihydro-1-methyl-4-oxo-1H-imidazole-2-yl) urea], reduces the VMR to bladder distention in UPEC-infected mice.

Conclusions

Taken together, these data suggest that mGluR5 modulates both inflammatory and non-inflammatory bladder nociception, and highlight the therapeutic potential for mGluR5 antagonists in the alleviation of bladder pain.     

Pcp4l1 contains an auto-inhibitory element that prevents its IQ motif from binding to calmodulin

Purkinje cell protein 4-like 1 (Pcp4l1) is a small neuronal IQ motif protein closely related to the calmodulin-binding protein Pcp4/PEP-19. PEP-19 interacts with calmodulin via its IQ motif to inhibit calmodulin-dependent enzymes and we hypothesized Pcp4l1 would have similar properties. Surprisingly, full-length Pcp4l1 does not interact with calmodulin in yeast two-hybrid or pulldown experiments yet a synthetic peptide constituting only the IQ motif of Pcp4l1 binds calmodulin and inhibits calmodulin-dependent kinase II. A nine-residue glutamic acid-rich sequence in Pcp4l1 confers these unexpected properties. This element lies outside the IQ motif and its deletion or exchange with the homologous region of PEP-19 restores calmodulin binding. Conversion of a single isoleucine (Ile36) within this motif to phenylalanine, the residue present in PEP-19, imparts calmodulin binding onto Pcp4l1. Moreover, only aromatic amino acid substitutions at position 36 in Pcp4l1 allow binding. Thus, despite their sequence similarities PEP-19 and Pcp4l1 have distinct properties with the latter harboring an element that can functionally suppress an IQ motif. We speculate Pcp4l1 may be a latent calmodulin inhibitor regulated by post-translational modification and/or co-factor interactions.     

Simultaneous determination of various aromatic amines and metabolites of aromatic nitro compounds in urine for low level exposure using gas chromatography-mass spectrometry

A newly developed method permits the simultaneous quantitative determination of various aromatic amines (or metabolites of aromatic nitro compounds, respectively) in human urine in one analytical run. Applying this method it is possible to determine aniline, toluidines, 4-isopropylaniline, o-anisidine, 3- and 4-chloroaniline, 4-bromoaniline, aminonitrotoluenes, aminodinitrotoluenes, 3,5- and 3,4-dichloroaniline, alpha- and beta-naphtylamine and 4-aminodiphenyl. After separation from the urinary matrix by a simple liquid-liquid extraction at pH 6.2-6.4 the analytes are converted into their pentafluoropropionic acid amides. Separation and quantitative analysis is carried out by capillary gas chromatography and mass-selective detection in the single ion monitoring mode. The limits of detection were within the range from 0.05 microg/l (4-aminobiphenyl, o-anisidine, 3,5-dichloroaniline) to 2 microg/l urine (4-amino-2,6-dinitrotoluene). The relative standard deviation of the within-series imprecision (determined at spiked concentrations of 2.0 microg/l and 10 microg/l) was between 2.9 and 13.6% depending on analyte and concentration. The relative recovery rates were in the range of 70-121%. The analytes that do not contain a nitro function showed better performance regarding the analytical reliability criteria. In order to determine the suitability of this new method for biological monitoring we analysed 20 12-h urine samples of persons without known exposure to aromatic amines, nitroaromatics or precursors in a pilot study. In these samples various aromatic amines could be clearly identified. The general population renally excretes aniline (median: 3.5 microg/l; 95th percentile: 7.9 microg/l), o- (0.12 microg/l; 2.7 microg/l), m- (0.17 microg/l; 2.2 microg/l) and p-toluidine (0.11 microg/l; 0.43 microg/l), and o-anisidine (0.22 microg/l; 0.68 microg/l). Additionally, we found that the persons investigated also excrete 3- (<0.05 microg/l; 0.55 microg/l) and 4-chloroaniline (0.11 microg/l; 0.57 microg/l) as well as 3,5-dichloroaniline (0.18 microg/l; 1.5 microg/l). 3,4-Dichloroaniline was found in some specimens (20%) in concentrations near the limit of detection (<0.05 microg/l; 0.12 microg/l). We did not detect alpha- or beta-naphtylamine, 4-aminobiphenyl or metabolites of explosives in the samples.     

A human SP-C promoter fragment targets α1-proteinase inhibitor gene expression to lung alveolar type II cells in transgenic mice

A 1.277 kb promoter fragment of the gene encoding one of the lung surfactant proteins, SP-C, was cloned from a human genomic library and characterized using the human 1-proteinase inhibitor (1PI) gene as reporter. Messenger RNA for human 1PI isolated from a single transgenic mouse line was detected solely in lung tissue. Using immunogold electron microscopy, accumulation of human 1PI was shown unambiguously to occur only in type II pulmonary cells and, in discrete amounts, in the alveolar lining fluid. The protein was secreted and glycosylated showing a molecular weight close to that of plasma-derived human 1PI.     

Do Casuarina cunninghamiana seedlings dependent on symbiotic N2 fixation have higher phosphorus requirements than those supplied with adequate fertilizer nitrogen?

P requirements of Casuarina cunninghamiana seedlings inoculated with Frankia and reliant on symbiotic N2 fixation were compared with those of uninoculated seedlings grown with 4 levels of fertilizer N in a solution culture system.Growth responses to increasing P supply depended on the N treatment that had been imposed. At the two lowest levels of N supply (0 t M and 100 t M) growth was relatively poor and there was no response to increasing P supply above 10 t M and 50 t M P respectively. In contrast, inoculated seedlings and those with higher levels of combined N (500 t M and 1000 t M) had significantly greater shoot dry weights (except at 0.1 t M P), and they responded to increasing P supply to between 50 and 100 t M P. At each level of P supply, the dry weights of seedlings in these 3 N treatments were similar. Nitrogen concentrations in shoots of seedlings provided with fertilizer N decreased with increasing P supply to 50 t M and then remained unchanged. In contrast, N concentrations in shoots of inoculated seedlings increased with increasing P supply to 25 t M P. At the 2 highest levels of P supply, N concentrations in shoots of inoculated plants were significantly higher than those in seedlings provided with fertilizer N. In all N treatments, P concentrations in shoots increased with increasing P supply; concentrations were similar in inoculated seedlings and those with the 2 highest levels of fertilizer N across all levels of P supply.Alleviation of P deficiency in inoculated seedlings increased nodule number, nodule dry weight, N2 fixation g-1 nodule dry weight (nodule 'efficiency'), P concentration in nodules, proportion of total seedling biomass allocated to nodules and average nodule size. However, all these parameters reached their maximum values at levels of P supply at, or below, those required for maximum host-plant growth (50 to 100 t M P).The results indicate that the P requirement for host plant growth per se is similar to, or higher than, that required for symbiotic N2 fixation processes.     

Hspa4l-deficient mice display increased incidence of male infertility and hydronephrosis development

The Hspa4l gene, also known as Apg1 or Osp94, belongs to the HSP110 heat shock gene family, which includes three genes encoding highly conserved proteins. This study shows that Hspa4l is expressed ubiquitously and predominantly in the testis. The protein is highly expressed in spermatogenic cells, from late pachytene spermatocytes to postmeiotic spermatids. In the kidney, the protein is restricted to cortical segments of distal tubules. To study the physiological role of this gene in vivo, we generated mice deficient in Hspa4l by gene targeting. Hspa4l-deficient mice were born at expected ratios and appeared healthy. However, approximately 42% of Hspa4l(-/-) male mice suffered from fertility defects. Whereas the seminiferous tubules of Hspa4l(-/-) testes contained all stages of germ cells, the number of mature sperm in the epididymis and sperm motility were drastically reduced. The reduction of the sperm count was due to the elimination of a significant number of developing germ cells via apoptosis. No defects in fertility were observed in female mutants. In addition, 12% of null mutant mice developed hydronephrosis. Concentrations of plasma and urine electrolytes in Hspa4l(-/-) mice were similar to wild-type values, suggesting that the renal function was not impaired. However, Hspa4l(-/-) animals were preferentially susceptible to osmotic stress. These results provide evidence that Hspa4l is required for normal spermatogenesis and suggest that Hspa4l plays a role in osmotolerance.     

Dissociation of CO from carboxyhemoglobin.

The reaction between carboxyhemoglobin and reduced microperoxidase (MP): Hb4(CO)4 + 4MP=Hb4 + 4MPCO, recently reported by us, has been further studied. By generating species Hb4(CO), Hb4(CO)2, and Hb(CO)3 in the stopped flow cuvette by the reaction of dithionite with the species of the general formula Hb4(O2)x(CO)y(x + y=4) in the presence of microperoxidase it has been possible to determine the stepwise CO dissociation rate constants l4, l3, l2, and l1. The overall CO dissociation rate constant l, which is the same in this system as l4, is not affected by 2,3-diphosphoglyceric acid. The activation energy of the reaction is 21,400 cal in 15-25 degrees range. The ratio deltal/deltapH is approximately 3 in 6.5 to 7.5 pH range. The kinetic data indicate that, compared to HbO2, the contribution to the cooperativity of the dissociation rate constants of carboxyhemoglobin is greatly reduced. The ligand-dependent differences in the reactions of Hb with CO, O2, and NO suggest that in the combination reactions the ligand plays an active role in the rate-limiting step.     

Carotenoid pigments of Sorangium compositum (Myxobacterales) including two new carotenoid glucoside esters and two new carotenoid rhamnosides

Summary The carotenoid pigments of the myxobacterium Sorangium compositum were analyzed by chromatographical and chemical techniques and by visible, infra red, and mass spectroscopy. Besides -carotene, neurosporene, torulene, lycopene, and 1,2-dihydro-1-hydroxy--carotene, four new carotenoid glycosides were found. These pigments were identified as 1,2-dihydro-1-hydroxy-torulene glucoside ester (I), 1,2-dihydro-3,1-dihydroxy-torulene glucoside ester (III), 1,2-dihydro-1-hydroxy-torulene rhamnoside (II), and 1,2-dihydro-3,1-dihydroxytorulene rhamnoside (IV).Fifth communication on the carotenoids of myxobacteria. Fourth communication see Arch. Mikrobiol. 76, 364–380 (1971).     

Simultaneous and comparative assessment of parent and mutant strain of Rhizobium meliloti for nutrient limitation and enhanced polyhydroxyalkanoate (PHA) production using optimization studies

Nutrient limitation conditions, optimization and comparison of polyhydroxyalkanoate (PHA) yields and biomass production by parent and mutant strains of Rhizobium meliloti were investigated. Complex interactions among concentrations of sucrose (5–55 g/l), urea (0.05–0.65 g/l) inoculum (10–250 ml/l) and K₂HPO₄ (0.5–2 g/l), were studied using central composite rotatable design (CCRD) experiments. Phosphate-limiting medium (0.33 g K₂HPO₄/l) in the presence of excess carbon (sucrose 42.5 g/l) results in more production of PHA (2.2 g/l) in the parent strain. In comparison, the mutant strain required moderate levels of sucrose (30 g/l), along with excess of phosphate (1 g/l) for high PHA content of cell biomass (80%) and PHA yield (3.3 g/l). Optimised PHA production (biomass 4.8 g/l and PHA 3.09 g/l) by the parent strain occurred at: sucrose 51.58 g/l, urea 0.65 g/l, K₂HPO₄ 0.48 g/l and inoculum 10 ml/l. In the mutant strain, higher yields of biomass (9.05 g/l) and PHA (5.66 g/l) were obtained in Optimised medium containing: sucrose 55 g/l, urea 0.65 g/l, K₂HPO₄ 1.0 g/l and inoculum 150.58 ml/l.