Regulation of Serotonin2A Receptor Expression by an Antisense Oligodeoxynucleotide

Abstract: The regulation of 5-HT_2A receptor expression by an antisense oligodeoxynucleotide, complementary to the coding region of rat 5-HT_2A receptor mRNA, was examined in a cortically derived cell line and in rat brain. Treatment of A₁A₁ variant cells, which express the 5-HT_2A receptor coupled to the stimulation of phosphatidylinositol (PI) hydrolysis, with antisense oligodeoxynucleotide decreased the maximal stimulation of PI hydrolysis by the partial agonist quipazine and the number of 5-HT_2A receptor sites as measured by the binding of 2-[¹²⁵I]-iodolysergic acid diethylamide. Treatment of cells with random, sense, or mismatch oligodeoxynucleotide did not alter the stimulation of PI hydrolysis by quipazine or 5-HT_2A receptor number. Intracerebroventricular infusion of antisense, but not mismatch, oligodeoxynucleotide for 8 days resulted in a significant increase in cortical 5-HT_2A receptor density and an increase in headshake behavior induced by the 5-HT₂ receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane. The density of cortical 5-HT_2A receptors was not altered by administration of antisense oligodeoxynucleotide for 1, 2, or 4 days. We hypothesize that in brain this antisense oligodeoxynucleotide relieved some form of translational suppression, resulting in an increase in 5-HT_2A receptor expression.     

Neuronal co-processing of course deviation and head movements in locusts

Summary Descending deviation detector neurons (DDNs) of Locusta migratoria are characterized physiologically by their responses to light on/off stimuli, simulated course deviation (rotation of an artificial horizon), passive rotation of the head, frontal wind, and flight activity. The investigation emphasises on the co-processing of exteroceptive input signalling course deviation (mainly movement of the retinal image, but also wind), and proprioceptive input signalling head movement and position. Stimuli were presented in combinations as expected during natural behavior. Eight DDNs are described for the first time, and 3 previously described DDNs are characterized further. Responses to horizon rotation and imposed head movements are assigned to one of 4 response types: (1) the horizon-only type codes retinal slip and/or the position of the horizon in the visual field but ignores cervical proprioception; (2) the head-only type ignores visually simulated course deviation but codes for movement or position of the head; (3) in the compensating type, head rolling causes visual input and cervical proprioceptive input of opposite signs, so that head movements themselves are ignored, whereas course deviations are recognized; (4) in the amplifying type, head rolling causes visual input and cervical proprioceptive input of the same sign, i.e. one input amplifies the other. This classification does not take the various responses to wind into account. In several DDNs, responses to phasic and tonic stimuli of the same modality, and/or responses to deviations about different axes could be assigned to different response types. Activity in DDNs has been shown previously to result in steering responses of wings, legs, abdomen and/or the head. It is proposed that different kinds of flight steering (e.g. corrective course control, intentional steering, orientation towards or away from a target) may be controlled by selective enhancement or suppression of responses or motor effects of DDN-subpopulations.Abbreviations AP action potential - DDN descending deviation detector neuron - DNI, DNC, DNM descending deviation detector neurons receiving major input from the ipsilateral, contralateral, and median ocellus respectively - PDDSMD protocerebral, descending direction-selective motion-detecting neuron - PI(2)5 descending deviation detector neuron with the cell body in the pars intercerebralis medialis - TCG tritocerebral commissure giant neuron     

Optimization of batch fermentor sterilization

This article presents a calculation procedure useful for the optimization and scale up of batch sterilization cycles in large-scale fermentors. This technique determines the sterilization temperature and hold-time necessary to minimize nutrient damage in a specific fermentor. The method can also be used for "scaledown" experiments to eliminate sterilization conditions as a scale up parameter. A method for the systematic evaluation of different sterilization conditions on product yield is also presented. This procedure is useful in determining if scale up of sterilization conditions is important for a given process. The validity of the techniques presented are supported by data showing significant yield improvements in a 1.2 x 10(5) L antibiotic fermentation.     

Ethanol consumption and serotonin-1A (5-HT1A) receptor function in heterozygous BDNF (+/-) mice

Heterozygous brain-derived neurotrophic factor (BDNF) (+/-) mice display abnormalities in central serotonergic neurotransmission, develop decrements in serotonergic innervation of the forebrain, and exhibit enhanced intermale aggressiveness. As disturbances of serotonin neurotransmission are implicated in alcohol abuse and aggression, we have examined in BDNF (+/-) mice alcohol drinking behavior, as well as central 5-hydroxytryptamine (5-HT)1A receptor function at the level of 5-HT1A receptor-G protein interaction. BDNF (+/-) mice displayed increased ethanol intake in a two-bottle choice procedure. There was no difference in the preference ratio for non-alcoholic tastants (i.e. quinine or saccharin) between genotypes. In the brains of alcohol-naive mice, we measured [35S]GTP gamma S binding stimulated by the 5-HT1A receptor agonist (+/-)-8-hydroxy-2-dipropyl-aminotetralin hydrobromide (8-OH-DPAT; 1 microM). In BDNF (+/-) versus wild-type (WT) mice, 5-HT1A receptor-stimulated [35S]GTP gamma S binding was significantly attenuated in the median raphe nucleus. There was a decrease in (+/-)8-OH-DPAT-stimulated [35S]GTP gamma S binding in the dorsal raphe, which did not reach statistical significance. In the hippocampus, 5-HT1A receptor-stimulated [35S]GTP gamma S binding was significantly attenuated in BDNF (+/-) mice. 5-HT1A receptor-stimulated [35S]GTP gamma S binding was attenuated in the anterior cingulate cortex and lateral septum, although these reductions did not reach statistical significance. 5-HT1A receptor number was not different between genotypes in any area of brain examined, suggesting that 5-HT1A receptor function, specifically the capacity of the 5-HT1A receptor to activate G proteins, is attenuated in BDNF (+/-) mice.     

Novel bacterial metabolite merochlorin A demonstrates in vitro activity against multi-drug resistant methicillin-resistant Staphylococcus aureus

Background

We evaluated the in vitro activity of a merochlorin A, a novel compound with a unique carbon skeleton, against a spectrum of clinically relevant bacterial pathogens and against previously characterized clinical and laboratory Staphylococcus aureus isolates with resistance to numerous antibiotics.

Methods

Merochlorin A was isolated and purified from a marine-derived actinomycete strain CNH189. Susceptibility testing for merochlorin A was performed against previously characterized human pathogens using broth microdilution and agar dilution methods. Cytotoxicity was assayed in tissue culture assays at 24 and 72 hours against human HeLa and mouse sarcoma L929 cell lines.

Results

The structure of as new antibiotic, merochlorin A, was assigned by comprehensive spectroscopic analysis. Merochlorin A demonstrated in vitro activity against Gram-positive bacteria, including Clostridium dificile, but not against Gram negative bacteria. In S. aureus, susceptibility was not affected by ribosomal mutations conferring linezolid resistance, mutations in dlt or mprF conferring resistance to daptomycin, accessory gene regulator knockout mutations, or the development of the vancomycin-intermediate resistant phenotype. Merochlorin A demonstrated rapid bactericidal activity against MRSA. Activity was lost in the presence of 20% serum.

Conclusions

The unique meroterpenoid, merochlorin A demonstrated excellent in vitro activity against S. aureus and C. dificile and did not show cross-resistance to contemporary antibiotics against Gram positive organisms. The activity was, however, markedly reduced in 20% human serum. Future directions for this compound may include evaluation for topical use, coating biomedical devices, or the pursuit of chemically modified derivatives of this compound that retain activity in the presence of serum.     

Preservation of Metabolic Flexibility in Skeletal Muscle by a Combined Use of n-3 PUFA and Rosiglitazone in Dietary Obese Mice

Insulin resistance, the key defect in type 2 diabetes (T2D), is associated with a low capacity to adapt fuel oxidation to fuel availability, i.e., metabolic inflexibility. This, in turn, contributes to a further damage of insulin signaling. Effectiveness of T2D treatment depends in large part on the improvement of insulin sensitivity and metabolic adaptability of the muscle, the main site of whole-body glucose utilization. We have shown previously in mice fed an obesogenic high-fat diet that a combined use of n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) and thiazolidinediones (TZDs), anti-diabetic drugs, preserved metabolic health and synergistically improved muscle insulin sensitivity. We investigated here whether n-3 LC-PUFA could elicit additive beneficial effects on metabolic flexibility when combined with a TZD drug rosiglitazone. Adult male C57BL/6N mice were fed an obesogenic corn oil-based high-fat diet (cHF) for 8 weeks, or randomly assigned to various interventions: cHF with n-3 LC-PUFA concentrate replacing 15% of dietary lipids (cHF+F), cHF with 10 mg rosiglitazone/kg diet (cHF+ROSI), cHF+F+ROSI, or chow-fed. Indirect calorimetry demonstrated superior preservation of metabolic flexibility to carbohydrates in response to the combined intervention. Metabolomic and gene expression analyses in the muscle suggested distinct and complementary effects of the interventions, with n-3 LC-PUFA supporting complete oxidation of fatty acids in mitochondria and the combination with n-3 LC-PUFA and rosiglitazone augmenting insulin sensitivity by the modulation of branched-chain amino acid metabolism. These beneficial metabolic effects were associated with the activation of the switch between glycolytic and oxidative muscle fibers, especially in the cHF+F+ROSI mice. Our results further support the idea that the combined use of n-3 LC-PUFA and TZDs could improve the efficacy of the therapy of obese and diabetic patients.     

Pyrrolidine bis-cyclic guanidines with antimicrobial activity against drug-resistant Gram-positive pathogens identified from a mixture-based combinatorial library

The rapid rise in antibiotic-resistant Gram-positive bacterial infections prompted us to explore the development of novel strategies for synthesis of large chemical libraries amenable to high-throughput screening for antimicrobial activities. Here we report the solid-phase synthesis of a 738,192 member pyrrolidine bis-cyclic guanidine chemical library with 26 different amino acids at three positions of diversity and 42 carboxylic acids at the fourth position. This synthetic combinatorial library was developed for positional scanning and screened for bacteriostatic and bactericidal activities against the important human pathogen methicillin-resistant Staphylococcus aureus (MRSA). The eight compound mixtures exhibiting bactericidal activity (10 microg/mL) against MRSA were used to direct the synthesis of 36 individual compounds that were then screened for activity against MRSA, vancomycin-resistant Enterococcus faecalis (VRE), and two Gram-negative bacterial species. At least 20 individual compounds were bactericidal for MRSA at 2.5 microg/mL, with a subset of these compounds showing bactericidal activities (10 microg/mL) against the other species tested. This approach demonstrates the capability to synthesize and screen a complex library to yield promising antimicrobials that address a critical need for novel infectious disease therapeutics.     

Carbohydrate Catabolism in Phaeobacter inhibens DSM 17395, a Member of the Marine Roseobacter Clade

Since genome analysis did not allow unambiguous reconstruction of transport, catabolism, and substrate-specific regulation for several important carbohydrates in Phaeobacter inhibens DSM 17395, proteomic and metabolomic analyses of N-acetylglucosamine-, mannitol-, sucrose-, glucose-, and xylose-grown cells were carried out to close this knowledge gap. These carbohydrates can pass through the outer membrane via porins identified in the outer membrane fraction. For transport across the cytoplasmic membrane, carbohydrate-specific ABC transport systems were identified. Their coding genes mostly colocalize with the respective “catabolic” and “regulatory” genes. The degradation of N-acetylglucosamine proceeds via N-acetylglucosamine-6-phosphate and glucosamine-6-phosphate directly to fructose-6-phosphate; two of the three enzymes involved were newly predicted and identified. Mannitol is catabolized via fructose, sucrose via fructose and glucose, glucose via glucose-6-phosphate, and xylose via xylulose-5-phosphate. Of the 30 proteins predicted to be involved in uptake, regulation, and degradation, 28 were identified by proteomics and 19 were assigned to their respective functions for the first time. The peripheral degradation pathways feed into the Entner-Doudoroff (ED) pathway, which is connected to the lower branch of the Embden-Meyerhof-Parnas (EMP) pathway. The enzyme constituents of these pathways displayed higher abundances in P. inhibens DSM 17395 cells grown with any of the five carbohydrates tested than in succinate-grown cells. Conversely, gluconeogenesis is turned on during succinate utilization. While tricarboxylic acid (TCA) cycle proteins remained mainly unchanged, the abundance profiles of their metabolites reflected the differing growth rates achieved with the different substrates tested. Homologs of the 74 genes involved in the reconstructed catabolic pathways and central metabolism are present in various Roseobacter clade members.