A RAC-GEF network critical for early intestinal tumourigenesis.

RAC1 activity is critical for intestinal homeostasis, and is required for hyperproliferation driven by loss of the tumour suppressor gene Apc in the murine intestine. To avoid the impact of direct targeting upon homeostasis, we reasoned that indirect targeting of RAC1 via RAC-GEFs might be effective. Transcriptional profiling of Apc deficient intestinal tissue identified Vav3 and Tiam1 as key targets. Deletion of these indicated that while TIAM1 deficiency could suppress Apc-driven hyperproliferation, it had no impact upon tumourigenesis, while VAV3 deficiency had no effect. Intriguingly, deletion of either gene resulted in upregulation of Vav2, with subsequent targeting of all three (Vav2-/- Vav3-/- Tiam1-/-), profoundly suppressing hyperproliferation, tumourigenesis and RAC1 activity, without impacting normal homeostasis. Critically, the observed RAC-GEF dependency was negated by oncogenic KRAS mutation. Together, these data demonstrate that while targeting RAC-GEF molecules may have therapeutic impact at early stages, this benefit may be lost in late stage disease.

The psychological and physiological health effects of fatigue.

Background: The issue of employee fatigue is becoming increasingly prominent, particularly in safety-critical industries. Aims: To produce an in-depth review collating the known psychological and physiological health and work effects of fatigue to guide mitigation strategies in safety-critical industries. Methods: Literature searches were conducted via scientific databases using appropriate filters and keywords. The available results were collated into a review and commentary. Results: Decreased sleep duration and chronodisruption have been shown to cause both significant morbidity and mortality. There is a large body of evidence showing strong associations between fatigue, reduced cognition and occupational accidents, as well as increased metabolic and reproductive health sequelae, some forms of cancer and mortality. Additional evidence links fatigue with mental, gastrointestinal, neurological and chronic pain sequelae. Conclusions: Fatigue risk mitigation strategies should be implemented, not only to reduce these short- and long-term health risks in employees of safety-critical industries, but also to create more efficient, productive and effective workplace personnel with longer and more fulfilling careers. This requires improved acute fatigue mitigation, as well as the prevention of cumulative fatigue build-up and the formation of acute-on-chronic fatigue. The health recommendations for fatigue mitigation outlined in this paper are pertinent to all professions where employees have high rates of both acute and chronic fatigue.

Wnt ligands influence tumour initiation by controlling the number of intestinal stem cells.

Many epithelial stem cell populations follow a pattern of stochastic stem cell divisions called 'neutral drift'. It is hypothesised that neutral competition between stem cells protects against the acquisition of deleterious mutations. Here we use a Porcupine inhibitor to reduce Wnt secretion at a dose where intestinal homoeostasis is maintained despite a reduction of Lgr5+ stem cells. Functionally, there is a marked acceleration in monoclonal conversion, so that crypts become rapidly derived from a single stem cell. Stem cells located further from the base are lost and the pool of competing stem cells is reduced. We tested whether this loss of stem cell competition would modify tumorigenesis. Reduction of Wnt ligand secretion accelerates fixation of Apc-deficient cells within the crypt leading to accelerated tumorigenesis. Therefore, ligand-based Wnt signalling influences the number of stem cells, fixation speed of Apc mutations and the speed and likelihood of adenoma formation.

First trial and StartReact effects induced by balance perturbations to upright stance.

Postural responses (PR) to a balance perturbation differ between the first and subsequent perturbations. One explanation for this first trial effect is that perturbations act as startling stimuli that initiate a generalized startle response (GSR) as well as the PR. Startling stimuli, such as startling acoustic stimuli (SAS), are known to elicit GSRs, as well as a StartReact effect, in which prepared movements are initiated earlier by a startling stimulus. In this study, a StartReact effect paradigm was used to determine if balance perturbations can also act as startle stimuli. Subjects completed two blocks of simple reaction time trials involving wrist extension to a visual imperative stimulus (IS). Each block included 15 CONTROL trials that involved a warning cue and subsequent IS, followed by 10 repeated TEST trials, where either a SAS (TESTSAS) or a toes-up support-surface rotation (TESTPERT) was presented coincident with the IS. StartReact effects were observed during the first trial in both TESTSAS and TESTPERT conditions as evidenced by significantly earlier wrist movement and muscle onsets compared with CONTROL. Likewise, StartReact effects were observed in all repeated TESTSAS and TESTPERT trials. In contrast, GSRs in sternocleidomastoid and PRs were large in the first trial, but significantly attenuated over repeated presentation of the TESTPERT trials. Results suggest that balance perturbations can act as startling stimuli. Thus first trial effects are likely PRs which are superimposed with a GSR that is initially large, but habituates over time with repeated exposure to the startling influence of the balance perturbation.

Startle induces early initiation of classically conditioned postural responses.

Startling acoustic stimuli (SAS) induce the early release of prepared motor responses. The current study used SAS, in conjunction with a classical conditioning paradigm, to examine advanced motor preparation of conditioned postural responses (PRs). After generalized startle responses were induced, standing posture was perturbed in 2 blocks of 15 Conditioning trials, where in each trial the onset of a nonstartling auditory cue [i.e., a conditioned stimulus (CS)] preceded a leftward support-surface translation. Upon completion of each block, a single trial was conducted. After block 1, a CS-Only trial was used to induce conditioned PRs in the absence of balance perturbations. After block 2, a post-Conditioning Startle trial that involved a CS subsequently followed by a SAS was used to examine motor preparation of conditioned PRs. PRs were quantified in terms of center of pressure displacements, ankle and hip kinematics, as well as surface electromyography of proximal and distal bilateral muscle pairs. Results indicated that repeated experience with cued balance perturbations led to PR conditioning and, more importantly, motor preparation of PRs. Conditioning was evidenced in biomechanical and electromyographic responses observed in CS-Only trials, as well as the progressive changes to evoked response parameters during repeated Conditioning trials. SAS presented in post-Conditioning Startle trials evoked early onsets of biomechanical and electromyographic responses, while preserving relative response parameters that were each distinct from generalized startle responses. These results provide important insight into both the consequences of using cues in dynamic postural control studies and the neural mechanisms governing PRs.

Postural responses explored through classical conditioning.

The purpose of the study was to determine whether the central nervous system (CNS) requires the sensory feedback generated by balance perturbations in order to trigger postural responses (PRs). In Experiment 1, twenty-one participants experienced toes-up support-surface tilts in two blocks. Control blocks involved unexpected balance perturbations whereas an auditory tone cued the onset of balance perturbations in Conditioning blocks. A single Cue-Only trial followed each block (Cue-Only(Control) and Cue-Only(Conditioning) trials) in the absence of balance perturbations. Cue-Only(Conditioning) trials were used to determine whether postural perturbations were required in order to trigger PRs. Counter-balancing the order of Control and Conditioning blocks allowed Cue-Only(Control) trials to examine both the audio-spinal/acoustic startle effects of the auditory cue and the carryover effects of the initial conditioning procedure. In Experiment 2, six participants first experienced five consecutive Tone-Only trials that were followed by twenty-five conditioning trials. After conditioning, five Tone-Only trials were again presented consecutively to first elicit and then extinguish the conditioned PRs. Surface electromyography (EMG) recorded muscle activity in soleus (SOL), tibialis anterior (TA) and rectus femoris (RF). EMG onset latencies and amplitudes were calculated together with the onset latency, peak and time-to-peak of shank angular accelerations. Results indicated that an auditory cue could be conditioned to initiate PRs in multiple muscles without balance-relevant sensory triggers generated by balance perturbations. Postural synergies involving excitation of TA and RF and inhibition of SOL were observed following the Cue-Only(Conditioning) trials that resulted in shank angular accelerations in the direction required to counter the expected toes-up tilt. Postural synergies were triggered in response to the auditory cue even 15 min post-conditioning. Furthermore, conditioned PRs were quickly extinguished as participants became unresponsive by the third trial in extinction. In conclusion, our results reveal that the CNS does not require sensory feedback from postural perturbations in order to trigger PRs.

The identification of alpha-ketoamides as potent inhibitors of hepatitis C virus NS3-4A proteinase.

Peptides based upon the non-prime side residues of the NS4A-4B cleavage site of hepatitis C virus (HCV) NS3-4A proteinase containing an alpha-ketoamide moiety in place of the scissile amide bond are potent inhibitors of this enzyme.

The design and synthesis of potent inhibitors of hepatitis C virus NS3-4A proteinase.

Hepatitis C virus (HCV) is the cause of the majority of transfusion-associated hepatitis and a significant proportion of community-acquired hepatitis worldwide. Infection by HCV frequently leads to persistent infections that result in a range of clinical conditions including an asymptomatic carrier state, severe chronic active hepatitis, cirrhosis and, in some cases, hepatocellular carcinoma. The HCV genome consists of a single-stranded, positive sense RNA containing an open reading frame of approximately 9060 nucleotides. This is translated into a single polyprotein of approximately 3020 amino acids (C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B), which in turn is processed by a series of host and viral proteinases into at least 10 cleavage products. The N-terminal portion of the NS3 protein encodes a serine proteinase that is responsible for the cleavage at the NS3-4A, NS4A-4B, NS4B-5A and NS5A-5B junctions. The 54 amino acid NS4A protein is a cofactor that binds to the NS3 protein and enhances its proteolytic activity. This report describes the expression of a recombinant NS3-4A proteinase fusion protein in Escherichia coli and the in vitro characterization of the enzyme activity using synthetic peptide substrates. It then demonstrates how these results were employed to guide the design of potent inhibitors of this enzyme.

Serotonin-3 receptor and ethanol-stimulated somatodendritic dopamine release.

The effects of local application of the 5-HT3 receptor agonist, 1-(m-chlorophenyl)-biguanide (CPBG), and i.p. administration of ethanol on the extracellular levels of dopamine (DA) in the ventral tegmental area (VTA) were studied using in vivo microdialysis. Adult female Wistar rats were implanted with microdialysis probes in the VTA at least 24 h before each experiment. Stable extracellular levels of DA (101 +/- 9 fmol/20 min) were established before initiating the experiments. Application of 10-250 microM CPBG through the microdialysis probe dose-dependently enhanced the extracellular concentrations of DA but did not alter the levels of either 3,4-dihydroxyphenylacetic acid or homovanillic acid in the dialysate. The effects of CPBG were reversible and dependent upon Ca2+. Co-perfusion with the 5-HT3 receptor antagonist, 3-tropanyl-indole-3-carboxylate (ICS 205-930), inhibited the effects of CPBG on enhancing extracellular DA levels. The i.p. administration of 2 g/kg ethanol significantly (p < 0.005) enhanced the levels of DA to 150% of baseline values; this ethanol-induced increase was prevented by local perfusion with 100 microM ICS 205-930. These results suggest that 5-HT3 receptors in the VTA are involved in regulating the somatodendritic release of DA and in mediating the stimulatory effects of ethanol on this neuronal system.

Epidemic of women battering. Recognition and intervention by health care professionals.

Domestic violence impacts every member of society and takes an emotional toll on the victim, her family (especially children), friends, and law enforcement and health professionals called on to assist. It affects the economic system in medical expenses, social services to protect the victim and her children, time lost from work by the victim, and expense to jail and prosecute the abuser.

Serotonin-3 receptor and ethanol-stimulated dopamine release in the nucleus accumbens.

The present study was undertaken to examine the involvement of activation of 5-HT3 receptors in the rat nucleus accumbens (Acb) on the effects of ethanol-induced increases of dopamine (DA) using the selective agonist 1-(m-chlorophenyl)-biguanide (CPBG). Perfusion of CPBG through the microdialysis probe concentration-dependently (3.3-100 microM) enhanced the extracellular levels of DA in the Acb. Extracellular DA concentrations increased as high as 1000% of baseline. The CPBG-induced increases in DA levels were Ca++ dependent and inhibited by local perfusion with the 5-HT3 antagonist ICS 205-930 (100 microM). In addition, CPBG at high concentrations caused significant decreases in the extracellular levels of DA metabolites. Intraperitoneal (IP) injection of 1 g/kg ethanol produced no changes in extracellular DA levels in the Acb; coadministration of 1 g/kg ethanol (IP) and 5 microM CPBG (local) produced increases equal to 5 microM CPBG alone. Administration of 2 g/kg ethanol (IP) alone enhanced extracellular DA levels by approximately 60% above baseline, whereas local perfusion of 5 microM CPBG alone produced an increase of approximately 100% above baseline. The coadministration of 2 g/kg ethanol (IP) and 5 microM CPBG (local) enhanced DA levels by approximately 170% above baseline; this apparent additive enhancement was almost completely prevented when 100 microM ICS 205-930 was locally coperfused. Local administration of 3.3-100 microM CPBG did not alter the extracellular levels of serotonin or 5-hydroxyindoleacetic acid. The results support an involvement of 5-HT3 receptors in regulating DA release in the Acb, and also in mediating ethanol-induced DA release.(ABSTRACT TRUNCATED AT 250 WORDS)

Cross-tolerance between ethanol and neurotensin in mice selectively bred for ethanol sensitivity.

Neurotensin (NT), a tridecapeptide that satisfies criteria as a neurotransmitter, mimics many actions of ethanol, and evidence indicates that some of the acute effects of ethanol are mediated in part by NT. Recent studies have shown that chronic ethanol treatment produced a downregulation of NT receptors in mesolimbic brain regions of long sleep (LS) mice and that reduced NT binding capacity was associated with acquisition and decay of tolerance to ethanol-induced locomotor inhibition and hypothermia in these mice. The present study was undertaken to determine whether cross-tolerance develops between NT and ethanol and whether chronic NT infusion produces NT receptor downregulation. Animals chronically treated with ethanol were tolerant to NT-mediated locomotor inhibition at a dose of 1.8 pmol NT, ICV, and were tolerant to NT-induced hypothermia at 1.8 and 6.0 pmol NT. Following repeated injections or continuous infusion of NT ICV, LS mice showed tolerance to both NT- and ethanol-induced hypothermia and locomotor inhibition. Indeed, ethanol doses that are hypnotic in control mice (2.8 g/kg) were not effective in abolishing locomotor activity following chronic NT administration. Results with chronic saline infusion ICV indicate that stress alters sensitivity to ethanol-induced hypothermia. Chronic infusion of NT ICV produced a region-specific downregulation of high-affinity NT receptors in the striatum. The results demonstrate that cross-tolerance develops between NT and ethanol, and further support a role for neurotensinergic systems in the actions of ethanol.

The 5-HT3 receptor agonist 1-(m-chlorophenyl)-biguanide interacts with the dopamine transporter in rat brain synaptosomes.

The ability of the 5-HT3 receptor agonist 1-(m-chlorophenyl)-biguanide to bind to the dopamine transporter and inhibit [3H]dopamine uptake was investigated in rat brain synaptosomes from the nucleus accumbens and caudate putamen. Competitive displacement experiments showed that 1-(m-chlorophenyl)-biguanide inhibited the binding of [3H]GBR-12935 in a biphasic manner (IC50 values of 0.4 and 2.0 microM [high affinity] and 34.8 and 52.7 microM [low affinity] for caudate putamen and nucleus accumbens, respectively), and the high affinity binding site differed between brain regions. Serotonin was ineffective at competing for [3H]GBR-12935 binding, while the selective 5-HT3 receptor antagonist ICS 205-930 exhibited an IC50 > 100 microM. The maximum density of [3H]GBR-12935 binding sites was more than two-fold greater in the caudate putamen than in the nucleus accumbens (6.9 vs. 2.7 pmol/mg protein), and KD values were similar (4.7 and 4.2 nM). 1-(m-chlorophenyl)-biguanide was able to inhibit [3H]dopamine uptake into synaptosomes of both brain regions, however it was significantly more potent in the caudate putamen (IC50: 5.1 vs. 6.5 microM). The results demonstrate that some of the reported dopamine releasing effects of 1-(m-chlorophenyl)-biguanide may be due in part to activity at the dopamine transporter, and further suggest a possible difference in dopamine uptake parameters between the caudate putamen and nucleus accumbens.

The human mu opioid receptor: modulation of functional desensitization by calcium/calmodulin-dependent protein kinase and protein kinase C.

Opioids are some of the most efficacious analgesics used in humans. Prolonged administration of opioids, however, often causes the development of drug tolerance, thus limiting their effectiveness. To explore the molecular basis of those mechanisms that may contribute to opioid tolerance, we have isolated a cDNA for the human mu opioid receptor, the target of such opioid narcotics as morphine, codeine, methadone, and fentanyl. The receptor encoded by this cDNA is 400 amino acids long with 94% sequence similarity to the rat mu opioid receptor. Transient expression of this cDNA in COS-7 cells produced high-affinity binding sites to mu-selective agonists and antagonists. This receptor displays functional coupling to a recently cloned G-protein-activated K+ channel. When both proteins were expressed in Xenopus oocytes, functional desensitization developed upon repeated stimulation of the mu opioid receptor, as observed by a reduction in K+ current induced by the second mu receptor activation relative to that induced by the first. The extent of desensitization was potentiated by both the multifunctional calcium/calmodulin-dependent protein kinase and protein kinase C. These results demonstrate that kinase modulation is a molecular mechanism by which the desensitization of mu receptor signaling may be regulated at the cellular level, suggesting that this cellular mechanism may contribute to opioid tolerance in humans.

Changes in mouse brain neurotensin receptor density following chronic infusion of neurotensin.

Many centrally acting drugs affect neurotensin (NT) systems by increasing levels of the peptide in specific brain regions. If these changes represent increases in extracellular NT levels, then changes in NT receptors would be expected. The focus of this study was to examine the effects of continuous exposure of NT receptors to agonist. Continuous infusion of NT (0.6 or 6 nmol/h) into the lateral ventricle via an osmotic minipump for 3 days caused a significant increase (over saline infusion) in total and low-affinity NT receptor density in the cerebellum of LS mice. High-affinity NT receptor density was increased in the frontal cortex. Seven days of NT infusion (6 nmol/h) caused no changes in NT receptor density.

LE-ACS4, a fruit ripening and wound-induced 1-aminocyclopropane-1-carboxylate synthase gene of tomato (Lycopersicon esculentum). Expression in Escherichia coli, structural characterization, expression characteristics, and phylogenetic analysis.

ACC (1-aminocyclopropane-1-carboxylic acid) synthase is the key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene and is encoded by a highly divergent multigene family in tomato (Rottmann, W. H., Peter, G. F., Oeller, P. W., Keller, J. A., Shen, N. F., Nagy, B. P., Taylor, L. P., Campbell, A. D., and Theologis, A. (1991) J. Mol. Biol. 222, 937-961). Two members of the family, LE-ACS2 and LE-ACS4, are induced during fruit ripening and upon treatment of mature green fruits with exogenous ethylene (C2H4) in a dose-dependent manner. Both genes are superinduced by wounding of pericarp tissue during various stages of ripening. The wound-induced accumulation of LE-ACS2 mRNA is more rapid and greater than that of LE-ACS4. Both mRNAs accumulate in the absence of protein synthesis, suggesting that their induction is a primary response to the inducer. The LE-ACS4 gene was isolated and structurally characterized. The function of the LE-ACS4 protein (53,509 Da, pI 5.4) was verified by expression experiments in Escherichia coli. The promoters of LE-ACS2 and LE-ACS4 contain potential cis-acting regulatory elements responsible for induction by ethylene, wounding, and anaerobiosis. In addition, elements for binding the transcriptional factors EmBP1, GBF-1, and OCSBF-1 are also present. Phylogenetic analysis of 20 ACC synthases from dicots and monocots indicate that the LE-ACS2 and LE-ACS4 proteins belong to an unique sublineage that includes an additional member of the tobacco family, NT-ACS1. The divergence of this sublineage is a relatively recent event in the evolution of ACC synthase protein.

Pharmacogenetics of cocaine: II. Mesocorticolimbic and striatal dopamine and cocaine receptors in C57BL and DBA mice.

Studies were conducted to determine whether genetic differences in behavioural effects of cocaine in C57BL/6 and DBA/2 mice might be mediated by strain differences in dopamine and serotonin transporters and dopamine D1 and D2 receptors in specific brain regions. Binding characteristics of [3H]CFT, a cocaine analogue, in the presence of either GBR12909, a dopamine uptake blocker or fluoxetine, a serotonin uptake blocker and binding of [3H]-paroxetine, a specific serotonin uptake receptor antagonist, were evaluated. We observed regional differences in [3H]CFT binding parameters in the presence of GBR12909 or fluoxetine, but no strain differences by brain region were observed. There were no differences in [3H]paroxetine binding characteristics between corresponding brain regions from C57BL and DBA/2 mice. The D1 antagonist, [3H]SCH23390 and the D2 ligands [3H]sulpiride or [125I]epidepride were used to determine dopamine receptor characteristics. Regional differences were found in [3H]SCH23390 and [3H]sulpiride, with higher affinities and lower densities in frontal cortex compared to striatum; with no differences in [3H]SCH23390 binding in corresponding tissues from C57BL and DBA/2 brains. There were strain-related differences in [3H]sulpiride and in [3H]epidepride binding in striatal membranes with higher densities in C57BL than in DBA/2. Our findings suggest striatal D2 receptor differences are possibly involved in genetic differences in cocaine-related behaviours.