Lack of direct bone effect of tofacitinib, a JAK inhibitor, in juvenile rats and evaluation of the association between offspring growth and femur length.

Bone has recently emerged as a target organ for some Janus kinase (JAK) inhibitors in adult and/or juvenile animal toxicity studies. Oral administration of tofacitinib, a JAK inhibitor, was not associated with clinical or macroscopic effects on bone growth and development in a rat juvenile animal study (JAS) with tofacitinib dosing starting on postnatal day (PND) 21. However, given that previous JAS did not include a targeted evaluation of bone, inclusive of microscopic examination, an additional rat JAS was conducted to further assess this risk. In this subsequent JAS, administration of tofacitinib from PND 7-49 or from PND 21-49 did not result in any direct effects on bone, with no histologic effects on developing bone. The only bone effect in this JAS was nonadverse shorter femur length, which was not considered to be a direct effect of tofacitinib, but rather an indicator of growth delay, as this was associated with lower body weights. There were no effects on femur length or body weight after a 2-month recovery period. To further explore the relationship between body weight and femur length, historical control data were analyzed from control rats in other JAS. This analysis clearly demonstrated that shorter femur length can occur as an indirect effect that is highly associated with lower body weight, consistent with what was observed in the JAS with tofacitinib. These analyses provide a robust and valuable data set to support the interpretation of such data in JAS, and further support the lack of direct effects of tofacitinib on bone growth and development. As with the previously conducted juvenile studies with tofacitinib, the additional JAS did not identify any special JAS-based concerns for use in pediatric patients as young as 2 years of age.

Reactions to media violence: it's in the brain of the beholder.

Media portraying violence is part of daily exposures. The extent to which violent media exposure impacts brain and behavior has been debated. Yet there is not enough experimental data to inform this debate. We hypothesize that reaction to violent media is critically dependent on personality/trait differences between viewers, where those with the propensity for physical assault will respond to the media differently than controls. The source of the variability, we further hypothesize, is reflected in autonomic response and brain functioning that differentiate those with aggression tendencies from others. To test this hypothesis we pre-selected a group of aggressive individuals and non-aggressive controls from the normal healthy population; we documented brain, blood-pressure, and behavioral responses during resting baseline and while the groups were watching media violence and emotional media that did not portray violence. Positron Emission Tomography was used with [18F]fluoro-deoxyglucose (FDG) to image brain metabolic activity, a marker of brain function, during rest and during film viewing while blood-pressure and mood ratings were intermittently collected. Results pointed to robust resting baseline differences between groups. Aggressive individuals had lower relative glucose metabolism in the medial orbitofrontal cortex correlating with poor self-control and greater glucose metabolism in other regions of the default-mode network (DMN) where precuneus correlated with negative emotionality. These brain results were similar while watching the violent media, during which aggressive viewers reported being more Inspired and Determined and less Upset and Nervous, and also showed a progressive decline in systolic blood-pressure compared to controls. Furthermore, the blood-pressure and brain activation in orbitofrontal cortex and precuneus were differentially coupled between the groups. These results demonstrate that individual differences in trait aggression strongly couple with brain, behavioral, and autonomic reactivity to media violence which should factor into debates about the impact of media violence on the public.

Enhanced striatal dopamine release during food stimulation in binge eating disorder.

Subjects with binge eating disorder (BED) regularly consume large amounts of food in short time periods. The neurobiology of BED is poorly understood. Brain dopamine, which regulates motivation for food intake, is likely to be involved. We assessed the involvement of brain dopamine in the motivation for food consumption in binge eaters. Positron emission tomography (PET) scans with [(11)C]raclopride were done in 10 obese BED and 8 obese subjects without BED. Changes in extracellular dopamine in the striatum in response to food stimulation in food-deprived subjects were evaluated after placebo and after oral methylphenidate (MPH), a drug that blocks the dopamine reuptake transporter and thus amplifies dopamine signals. Neither the neutral stimuli (with or without MPH) nor the food stimuli when given with placebo increased extracellular dopamine. The food stimuli when given with MPH significantly increased dopamine in the caudate and putamen in the binge eaters but not in the nonbinge eaters. Dopamine increases in the caudate were significantly correlated with the binge eating scores but not with BMI. These results identify dopamine neurotransmission in the caudate as being of relevance to the neurobiology of BED. The lack of correlation between BMI and dopamine changes suggests that dopamine release per se does not predict BMI within a group of obese individuals but that it predicts binge eating.

Further evidence of pleiotropy influencing speech and language: analysis of the DYX8 region.

BACKGROUND/AIMS: Genetic studies have raised the possibility of common bases for cognitive linguistic disorders such as speech sound disorder (SSD), reading disorder (RD) and language impairment (LI). Thus, some of the same genes may jointly influence cognitive components within and between these three disorders. We examined the plausibility of this theory in a sample of families ascertained on the basis of a child with SSD. METHODS: Using the method of generalized estimating equations to solve a bivariate family predictive model we obtained measures of comorbidity and familial aggregation of SSD and LI. We then used two methods of multipoint model-free linkage analysis to evaluate SSD and LI psychometric test measures over a region previously implicated in linkage studies of RD, DYX8 region, 1p34-p36. RESULTS: Bivariate phenotypic analyses show evidence of comorbidity and within family aggregation and coaggregation of SSD and LI. In addition, two regions on chromosome 1 show suggestive evidence of linkage. The first region was previously reported in dyslexia studies. Our maximum linkage signal in this region measured articulation (p = 0.0009) in SSD sibling pairs. The second region is characterized by processes involved in language production, with the maximum linkage signal measuring listening comprehension (p = 0.0019) using all sibling pairs. CONCLUSION: We conclude that the DYX8 region could bear genes controlling pleiotropic effects on SSD, LI and RD.

The Separative Bioreactor: A Continuous Separation Process for the Simultaneous Production and Direct Capture of Organic Acids.

The replacement of petrochemicals with biobased chemicals requires efficient bioprocesses, biocatalysis, and product recovery. Biocatalysis (e.g., enzyme conversion and fermentation) offers an attractive alternative to chemical processing because biocatalysis utilize renewable feedstocks under benign reaction conditions. One class of chemical products that could be produced in large volumes by biocatalysis is organic acids. However, biocatalytic reactions to produce organic acids typically result in only dilute concentrations of the product because of product inhibition and acidification that drives the reaction pH outside of the optimal range for the biocatalyst. Buffering or neutralization results in formation of the acid salt rather than the acid, which requires further processing to recover the free acid product. To address these barriers to biocatalytic organic acid production, we developed the "separative bioreactor" based on resin wafer electrodeionization, which is an electro-deionization platform that uses resin wafers fabricated from ion exchange resins. The separative bioreactor simultaneously separates the organic acid from the biocatalyst as it is produced, thus it avoids product inhibition enhancing reaction rates. In addition, the separative bioreactor recovers the product in its acid form to avoid neutralization. The instantaneous separation of acid upon formation in the separative bioreactor is one of the first truly one-step systems for producing organic acids. The separative bioreactor was demonstrated with two systems. In the first demonstration, the enzyme glucose fructose oxidoreductase (GFOR) was immobilized in the reactor and later regenerated in situ. GFOR produced gluconic acid (in its acid form) continuously for 7 days with production rates up to 1000 mg/L/hr at >99% product recovery and GFOR reactivity >30mg gluconic acid/mg GFOR/hour. In the second demonstration, the E. coli strain CSM1 produced lactic acid for up to 24 hours with a productivity of >200 mg/L/hr and almost 100% product recovery.

Kinetic and structural studies on the interaction of cholinesterases with the anti-Alzheimer drug rivastigmine.

Rivastigmine, a carbamate inhibitor of acetylcholinesterase, is already in use for treatment of Alzheimer's disease under the trade name of Exelon. Rivastigmine carbamylates Torpedo californica acetylcholinesterase very slowly (k(i) = 2.0 M(-1) min(-1)), whereas the bimolecular rate constant for inhibition of human acetylcholinesterase is >1600-fold higher (k(i) = 3300 M(-1) min(-1)). For human butyrylcholinesterase and for Drosophila melanogaster acetylcholinesterase, carbamylation is even more rapid (k(i) = 9 x 10(4) and 5 x 10(5) M(-1) min(-1), respectively). Spontaneous reactivation of all four conjugates is very slow, with <10% reactivation being observed for the Torpedo enzyme after 48 h. The crystal structure of the conjugate of rivastigmine with Torpedo acetylcholinesterase was determined to 2.2 A resolution. It revealed that the carbamyl moiety is covalently linked to the active-site serine, with the leaving group, (-)-S-3-[1-(dimethylamino)ethyl]phenol, being retained in the "anionic" site. A significant movement of the active-site histidine (H440) away from its normal hydrogen-bonded partner, E327, was observed, resulting in disruption of the catalytic triad. This movement may provide an explanation for the unusually slow kinetics of reactivation.

High-throughput assays for botulinum neurotoxin proteolytic activity: serotypes A, B, D, and F.

Botulinum neurotoxins (BoNT) are zinc metalloproteases that cleave and inactivate cellular proteins essential for neurotransmitter release. Because the paralytic effect of BoNT is a consequence of its enzymatic activity, selective inhibitors may be useful as drugs or as tools for further research. To expedite inhibitor discovery, we developed high-throughput, solid-phase protease activity assays for four of the seven BoNT serotypes: A, B, D, and F. Each assay consisted of a cleavable oligopeptide, based on the natural substrate sequence, labeled with fluorescein and covalently attached to maleimide-activated multiwell plates. Solutions of holotoxin or nontoxic catalytic domain of BoNT were incubated in substrate-coated wells, with or without test compounds, followed by transfer and assay of solubilized product in a multiwell fluorometer. Routine toxin concentrations ranged from 10 to 100 ng/ml, but concentrations as low as 2 ng/ml gave reproducible signals. The fluorescence assays were selective, gave very low background readings, and were stable upon prolonged storage. Using the nontoxic catalytic domain of BoNT A, we determined the relative inhibitory potencies of a family of structurally related pseudotripeptide compounds. Unlike previous methods, our assays did not employ antibodies or reverse-phase extraction steps, only well-to-well transfers, and were easily adapted to a high-throughput automated environment.

Mutation of the ptsG gene results in increased production of succinate in fermentation of glucose by Escherichia coli.

Escherichia coli NZN111 is blocked in the ability to grow fermentatively on glucose but gave rise spontaneously to a mutant that had this ability. The mutant carries out a balanced fermentation of glucose to give approximately 1 mol of succinate, 0. 5 mol of acetate, and 0.5 mol of ethanol per mol of glucose. The causative mutation was mapped to the ptsG gene, which encodes the membrane-bound, glucose-specific permease of the phosphotransferase system, protein EIICB(glc). Replacement of the chromosomal ptsG gene with an insertionally inactivated form also restored growth on glucose and resulted in the same distribution of fermentation products. The physiological characteristics of the spontaneous and null mutants were consistent with loss of function of the ptsG gene product; the mutants possessed greatly reduced glucose phosphotransferase activity and lacked normal glucose repression. Introduction of the null mutant into strains not blocked in the ability to ferment glucose also increased succinate production in those strains. This phenomenon was widespread, occurring in different lineages of E. coli, including E. coli B.

Active-site gorge and buried water molecules in crystal structures of acetylcholinesterase from Torpedo californica.

Buried water molecules and the water molecules in the active-site gorge are analyzed for five crystal structures of acetylcholinesterase from Torpedo californica in the resolution range 2.2-2.5 A (native enzyme, and four inhibitor complexes). A total of 45 buried hydration sites are identified, which are populated with between 36 and 41 water molecules. About half of the buried water is located in a distinct region neighboring the active-site gorge. Most of the buried water molecules are very well conserved among the five structures, and have low displacement parameters, B, of magnitudes similar to those of the main-chain atoms of the central beta-sheet structure. The active-site gorge of the native enzyme is filled with over 20 water molecules, which have poor hydrogen-bond coordination with an average of 2.9 polar contacts per water molecule. Upon ligand binding, distinct groups of these water molecules are displaced, whereas the others remain in positions similar to those that they occupy in the native enzyme. Possible roles of the buried water molecules are discussed, including their possible action as a lubricant to allow large-amplitude fluctuations of the loop structures forming the gorge wall. Such fluctuations are required to facilitate traffic of substrate, products and water molecules to and from the active-site. Because of their poor coordination, the gorge water molecules can be considered as "activated" as compared to bulk water. This should allow their easy displacement by incoming substrate. The relatively loose packing of the gorge water molecules leaves numerous small voids, and more efficient space-filling by substrates and inhibitors may be a major driving force of ligand binding.

Poly(ADP-ribose) polymerase inhibitors regulate the mechanism of sulfur mustard-initiated cell death in human lymphocytes.

Sulfur mustard (HD) produces slow-healing skin lesions that contain large, tight fluid-filled blisters. These lesions are the result of severe damage to areas of the body exposed to HD and require extensive medical care before complete recovery is achieved. Converting the mechanism of HD-initiated cell death from an inflammatory oncosis (homicide) to benign apoptosis (assisted suicide) may reduce the extent of cellular damage and the time required for healing. HD-exposed human lymphocytes lose cellular function, membrane integrity and viability, and suffer degradation of their nuclear components. The treatment of HD-exposed cells with poly(ADP-ribose) polymerase inhibitors prevents or alters the HD-initiated loss of cell viability, membrane integrity, cellular metabolic constituent (NAD) and cellular energy (ATP), while initiating alterations in nuclear constituents. It is hoped that by preventing or altering these HD-initiated changes we can limit the extent of the injury, decrease the time required for repair and reduce the loss of performance suffered by exposed individuals. The use of poly(ADP-ribose) polymerase inhibitors to assist in initiating apoptosis in affected cells should help to achieve these objectives while preventing the chance of further disease development later in the exposed individuals.

Phospholipaise A2 and arachidonic acid-mediated mechanism of neuroexocytosis: a possible target of botidinum neurotoxin A other then SNAP-25.

The vesicular neuroexocytosis process consists of two important steps: fusion of transmitter-loaded vesicles at release sites on the presynaptic nerve terminal membrane; followed by the release of transmitter molecules into the synaptic cleft. We previously reported that in nerve growth factor (NGF)-differentiated PC12 cells, arachidonic acid (AA) release is associated with acetylcholine (ACh) release, botulinum neurotoxin A (BoNT/A) inhibits both processes and AA itself or a phospholipase A(2) (PLA(2)) activator can cause ACh release in BoNT/A-poisoned cells in which SNAP-25 has supposedly been hydrolyzed. In the present study, we examined the roles of two endogenous intraterminal components in neuroexocytosis: the membrane fusogenic agent AA; and the vesicle fusion protein SNAP-25. A PLA(2) activator, mastoparan, was used to induce the release of AA and ACh from NGF-differentiated PC12 cells. Release depended upon the mastoparan concentration, as well as Ca(2+) influx via the neuronal-type voltage-sensitive Ca(2+) channels. Release of ACh followed a rise in intracellular free Ca(2+) concentration; the increased Ca(2+) activated PLA(2) and, thereby, increased the AA level. Scanning and transmission electron microscopy confirmed that mastoparan-induced ACh and AA release were not due to simple diffusion through damaged plasma membranes. Treatment of PC12 cells with appropriate antisense oligonucleotides blocked SNAP-25 expression, as judged by Western blot protein analysis with a specific monoclonal antibody. Despite apparent elimination of SNAP-25, treatment of differentiated PC12 cells with mastoparan and high (80 mM) K(+) induced ACh exocytosis. The results support the conclusion that PLA(2) and AA have important roles in neuroexocytosis that are independent of SNAP-25. Both PLA(2) and AA have been shown to be involved in actin cytoskeletal organization related to vesicle fusion and exocytosis. This mechanism may be an alternative target of BoNT/A other than SNAP-25.

Effects of dehydration, hypohydration, and hyperhydration on tolerance during uncompensable heat stress.

The present study examined the effects of dehydration from prior exercise on subsequent exercise tolerance time (TT) that involved wearing nuclear, biological, and chemical (NBC) protective clothing. It was hypothesised that TT would be reduced in the dehydrated state. Ten men undertook continuous treadmill walking at 4.8 km.h-1 at 35 degrees C and 50% relative humidity, wearing NBC clothing while euhydrated (EU) or dehydrated (D) by 2.3% of body weight. Hydration status had no impact on thermoregulatory or cardiovascular responses during exercise. Also rectal temperature at exhaustion did not differ between EU (38.52 +/- 0.39 degrees C) and D (38.43 +/- 0.45 degrees C). Exercise TT during this uncompensable heat stress was reduced significantly for D (47.7 +/- 15.3 min) compared with EU (59.0 +/- 13.6 min). It was concluded that prior exercise leading to levels of dehydration to 2.3% of body weight, together with subsequent fluid restriction during exposure to uncompensable heat stress, impaired TT while wearing the NBC protective clothing. The integration of these findings together with other comparable studies that have examined the influence of hypo- and hyperhydration on TT while wearing NBC protective clothing revealed that hydration status has less effect on TT as the severity of uncompensable heat stress increases.

A preliminary comparison of structural models for catalytic intermediates of acetylcholinesterase.

Determination of the three dimensional structure of Torpedo Californica acetylcholinesterase (TcAChE) provided an experimental tool for directly visualizing interaction of AChE with cholinesterase inhibitors of fundamental, pharmacological and toxicological interest. The structure revealed that the active site is located near the bottom of a deep and narrow gorge lined with 14 conserved aromatic amino acids. The structure of a complex of TcAChE with the powerful 'transition state analog' inhibitor, TMTFA, suggested that its orientation in the experimentally determined structure was very similar to that proposed for the natural substrate, acetylcholine, by manual docking. The array of enzyme-ligand interactions visualized in the TMFTA complex also are expected to envelope the unstable TI that forms with acetylcholine during acylation, and to sequester it from solvent. In our most recent studies, the crystal structures of several 'aged' conjugates of TcAChE obtained with OP nerve agents have been solved and compared with that of the native enzyme. The methylphosphonylated-enzyme obtained by reaction with soman provides a useful structural analog for the TI that forms during deacylation after the reaction of TcAChE with acetylcholine. By comparing these structures, we conclude that the same 'oxyanion hole' residues, as well as the aromatic side chains constituting the 'acyl pocket', participate in acylation (TMTFA-AChE) and deacylation (OP-AChE), and that AChE can accommodate both TIs at the bottom of the gorge without major conformational movements.

Protein engineering of a human enzyme that hydrolyzes V and G nerve agents: design, construction and characterization.

Because of deficiencies in the present treatments for organophosphorus anticholinesterase poisoning, we are attempting to develop a catalytic scavenger that can be administered as prophylactic protection. Currently known enzymes are inadequate for this purpose because they have weak binding and slow turnover, so we are trying to make an appropriate enzyme by protein engineering techniques. One butyrylcholinesterase mutant, G117H, has the desired type of activity but reacts much too slowly. This communication describes an attempt to determine the reason for the slow reaction so that a more efficient enzyme might be designed. The results indicate that the mutation at residue 117 has resulted in a distortion of the transition state of the reaction of organophosphorus compounds with the active site serine. This information will be used to develop other mutants that avoid transition state stabilization sites.

Crystal structures of aged phosphonylated acetylcholinesterase: nerve agent reaction products at the atomic level.

Organophosphorus acid anhydride (OP) nerve agents are potent inhibitors which rapidly phosphonylate acetylcholinesterase (AChE) and then may undergo an internal dealkylation reaction (called "aging") to produce an OP-enzyme conjugate that cannot be reactivated. To understand the basis for irreversible inhibition, we solved the structures of aged conjugates obtained by reaction of Torpedo californica AChE (TcAChE) with diisopropylphosphorofluoridate (DFP), O-isopropylmethylphosponofluoridate (sarin), or O-pinacolylmethylphosphonofluoridate (soman) by X-ray crystallography to 2.3, 2.6, or 2.2 A resolution, respectively. The highest positive difference density peak corresponded to the OP phosphorus and was located within covalent bonding distance of the active-site serine (S200) in each structure. The OP-oxygen atoms were within hydrogen-bonding distance of four potential donors from catalytic subsites of the enzyme, suggesting that electrostatic forces significantly stabilize the aged enzyme. The active sites of aged sarin- and soman-TcAChE were essentially identical and provided structural models for the negatively charged, tetrahedral intermediate that occurs during deacylation with the natural substrate, acetylcholine. Phosphorylation with DFP caused an unexpected movement in the main chain of a loop that includes residues F288 and F290 of the TcAChE acyl pocket. This is the first major conformational change reported in the active site of any AChE-ligand complex, and it offers a structural explanation for the substrate selectivity of AChE.

Cutaneous uptake of 14C-HD vapor by the hairless guinea pig.

The hairless guinea pig (HGP) is used by our laboratory to model the human cutaneous response to sulfur mustard (HD), bis(2-chloroethylsulfide), exposure. We determined the HD content in the skin of HGP after a 7-min exposure to vapors saturated with a mixture of HD and 14C-HD. Concentration/time (CT) values in the range of 2 micrograms/cm2/min were determined by counting skin 14C disintegrations per min (dpm) in animals euthanized immediately after exposure. These values are similar to human penetration rates obtained by other investigators. A rate curve monitoring the reduction in skin 14C dpm was developed for animals euthanized between 0 and 24 hr post- exposure. This curve showed the greatest change after 1 hr. The epidermal (62%) to dermal (38%) ratio of 14C at 24 hr was measured for two animals. We saw no site preference for HD penetration among the 8 sites used. The 14C content of template adhesive tape was determined to follow HD distribution. These results contribute to a better understanding of the cutaneous response to HD in the HGP model.

Alterations in human lymphocyte DNA caused by sulfur mustard can be mitigated by selective inhibitors of poly(ADP-ribose) polymerase.

Changes in genomic DNA caused by exposure to the cytotoxic alkylating agent, 2,2'-dichlorodiethyl sulfide (sulfur mustard; HD), alone or in combination with selective inhibitors of poly(ADP-ribose) polymerase (PARP), were analyzed as a function of HD concentration and post-exposure time. Preparations of human peripheral blood lymphocytes were exposed to HD (1x10(-8) M-1x10(-3) M), and incubated at 37 degrees C for 0-24 h. Total genomic DNA was extracted from these cells and compared with DNA from control cells of the same donor using agarose gel electrophoresis. The effects of HD on genomic DNA depended on the HD concentration and the length of the post-exposure time interval. DNA fragmentation was detected as early as 2 h after exposure to 3x10(-4) M HD, or at 24 h after exposure to 6x10(-6) M HD. The qualitative DNA pattern, as well as the extent of DNA fragmentation, changed with post-exposure time. Exposure to HD caused a time-dependent shift in the DNA cleavage pattern from an oligonucleosome-sized 'DNA ladder' characteristic of apoptotic cell death, to a 'broad band' pattern characteristic of necrotic cell death. DNA fragmentation was not observed if cells were killed with heat or with Lewisite. Treatment of cells with selective PARP inhibitors consistently altered the DNA fragmentation caused by HD exposure. The inhibitors arrested DNA fragmentation at the DNA ladder stage. This effect only was observed if the PARP inhibitors were applied within 8 h of HD exposure. We conclude that early inhibition of PARP activity can induce a switch in the mechanism of cell death caused by HD. Such a switch may be useful therapeutically to convert a lytic, pro-inflammatory cell death that includes the disintegration of dying cells (necrosis), into a slower, programmed cell death that includes absorption of dying cells (apoptosis).

A novel fermentation pathway in an Escherichia coli mutant producing succinic acid, acetic acid, and ethanol.

Escherichia coli strain NZN111, which is unable to grow fermentatively because of insertional inactivation of the genes encoding pyruvate: formate lyase and the fermentative lactate dehydrogenase, gave rise spontaneously to a chromosomal mutation that restored its ability to ferment glucose. The mutant strain, named AFP111, fermented glucose more slowly than did its wild-type ancestor, strain W1485, and generated a very different spectrum of products. AFP111 produced succinic acid, acetic acid, and ethanol in proportions of approx 2:1:1. Calculations of carbon and electron balances accounted fully for the observed products; 1 mol of glucose was converted to 1 mol of succinic acid and 0.5 mol each of acetic acid and ethanol. The data support the emergence in E. coli of a novel succinic acid:acetic acid:ethanol fermentation pathway.