Randomized clinical trial of perioperative nerve block and continuous local anaesthetic infiltration via wound catheter versus epidural analgesia in open liver resection (LIVER 2 trial).

BACKGROUND: Analgesia after liver surgery remains controversial. A previous randomized trial of continuous wound infiltration (CWI) versus thoracic epidural analgesia (TEA) after liver surgery (LIVER trial) showed a faster recovery time in the wound infiltration group but better early postoperative pain scores in the TEA group. High-level evidence is, however, limited and opinion remains divided. The aim was to determine whether there is a difference in functional recovery time between patients having CWI plus abdominal nerve blocks versus TEA after liver resection. METHODS: A randomized unblinded clinical trial of patients undergoing open liver resection was commenced in December 2012, with follow-up to August 2014. Patients were randomized to receive either wound catheter and nerve block (CWI group) or TEA for 48 h after surgery. The primary outcome measure was functional recovery time. Secondary outcomes were pain scores, complication rates, inflammatory response and central venous pressure (CVP) during transection. RESULTS: Of 50 patients randomized initially to each group, 44 received TEA and 49 CWI. Median (i.q.r.) recovery time was 6·5 (5-9·75) and 5·75 (4-7) days in the TEA and CWI groups respectively (P = 0·036). Pain scores were not significantly different between the two groups, and there were no differences in morbidity, inflammatory response or CVP during transection. CONCLUSION: Wound infiltration is associated with a reduced time to recovery after open liver resection compared with epidural analgesia. TEA does not offer an advantage over CWI in terms of attenuation of the inflammatory response or pain scores. REGISTRATION NUMBER: NCT01747122 ( http://www.clinicaltrials.gov).

Interstitial cells of Cajal generate a rhythmic pacemaker current.

Networks of interstitial cells of Cajal embedded in the musculature of the gastrointestinal tract are involved in the generation of electrical pacemaker activity for gastrointestinal motility. This pacemaker activity manifests itself as rhythmic slow waves in membrane potential, and controls the frequency and propagation characteristics of gut contractile activity. Mice that lack a functional Kit receptor fail to develop the network of interstitial cells of Cajal associated with Auerbach's plexus in the mouse small intestine and do not generate slow wave activity. These cells could provide an essential component of slow wave activity (for example, a biochemical trigger that would be transferred to smooth muscle cells), or provide an actual pacemaker current that could initiate slow waves. Here we provide direct evidence that a single cell, identified as an interstitial cell of Cajal by light microscopy, electron microscopy and expression of Kit mRNA, generates spontaneous contractions and a rhythmic inward current that is insensitive to L-type calcium channel blockers. Identification of the pacemaker of gut motility will aid in the elucidation of the pathophysiology of intestinal motor disorders, and provide a target cell for pharmacological treatment.

The effect of preoperative stoma training for patients undergoing colorectal surgery in an enhanced recovery programme.

INTRODUCTION: Stoma formation following colorectal resection is often anticipated prior to surgery. Becoming independent with stoma handling can sometimes delay discharge beyond achievement of discharge criteria. The aim of this study was to assess the impact of preoperative stoma training on length of stay. METHODS: Patients undergoing colorectal resection within an enhanced recovery after surgery (ERAS) programme were prospectively entered into a database. Retrospective analysis was performed of those who received a stoma as part of their operation. Patients who underwent preoperative stoma training were compared with those who had conventional postoperative training. The primary outcome measure was length of hospital stay. Secondary outcome measures included overall morbidity, stoma related morbidity, ERAS milestone achievement and readmission rates. RESULTS: The median length of stay was improved in the patients receiving preoperative stoma training (8 days [interquartile range: 6-10] vs 9 days [interquartile range: 7-19.5], p=0.025). No statistically significant difference was observed in overall morbidity rates, stoma specific morbidity, ERAS milestones or readmission rates. CONCLUSIONS: Preoperative stoma training can reduce length of stay and could be employed routinely for patients who are planned to have colorectal surgery. Such training can be incorporated within ERAS pathways.

Point mutations and overexpression of Ron induce transformation, tumor formation, and metastasis.

The receptor tyrosine kinase Ron is a member of the receptor family that includes the proto-oncogene Met and the avian oncogene Sea. The interaction of Ron with its ligand, known as hepatocyte growth factor-like protein (HGFL) or macrophage stimulating protein (MSP), induces crucial cellular responses including invasive growth, proliferation, cell scattering, and branching morphogenesis. Based on the homology and functional similarities between Met and Ron it was hypothesized that Ron may be important in tumor formation and metastasis. To test this hypothesis, wild-type mouse Ron and three mutant forms of Ron containing mutations similar to those found in the Met gene in human hereditary papillary renal carcinoma (HPRC), were expressed in NIH3T3 cells. A transformed phenotype was produced in cell lines expressing either wild-type Ron or the mutated Ron proteins. Further, these cell lines displayed oncogenic potential by exhibiting increased proliferation and constitutive phosphorylation of Ron. These cell lines were also tested for the ability to form solid tumors. Cells expressing wild-type Ron and the three proteins with single amino acid substitutions were highly tumorigenic in vivo. In a model of experimental metastasis, two of the cell lines with altered Ron protein formed highly aggressive tumors in the lungs. These results suggest that Ron may be an aggressive oncogene when either overexpressed or when activated by mutation.

Danazol, a new steroidal inducer of delta-aminolevulinic acid synthetase.

Danazol (a derivative of 17 alpha-ethinyl testosterone) has recently been reported to cause clinical exacerbations of acute intermittent porphyria. In the present study, we demonstrate that danazol is an inducer of hepatic delta-aminolevulinic acid synthetase and that it increases hepatocyte porphyrin production using a chick embryo model. The findings provide an explanation for the clinical observations. Although danazol is a porphyrinogenic agent in the chick embryo and humans it has been reported to be ineffective as a porphyrinogen in rats. The present findings, therefore, also confirm the superiority of the chick embryo as a model system for the screening of drugs and chemicals which may have the potential to cause clinical exacerbations of porphyria.

Characterization of the lung Krüppel-like transcription factor gene and upstream regulatory elements.

We previously described the isolation and characterization of the cDNA for lung Krüppel-like factor (LKLF), a zinc finger transcription factor that is predominately expressed in the lung of adult mice. In this study, we report the complete structure and nucleotide sequence of the mouse LKLF gene, which is comprised of three exons and two small introns. Moreover, the identification of critical sequence elements required for expression is described using reporter constructs with the LKLF promoter transfected into LA-4 lung cells. Results from these constructs reveal an important region for transcriptional activity that lies between the -490/-72bp upstream sequence. This region contains two canonical Sp1 binding sites that affect expression levels in a non tissue-specific manner. In addition, using a base-pair mutagenesis strategy, a region from -157/-72bp was found to be necessary for upregulating expression. In transfection assays, mutations of the -138/-111bp region resulted in approximately 70-80% loss of promoter activity. This cis-element does not appear to correspond to any known transcription factor consensus sequence. Moreover, mutations within this cis-region disrupt the binding of a protein complex from nuclear extracts of various tissues.

A single nucleotide is a sufficient 5' untranslated region for translation in an eukaryotic in vitro system.

The 5' untranslated region of an RNA molecule is thought to play an important role in the regulation of translation. Following a recent report that a single nucleotide is sufficient to act in this role in the unicellular organism Giardia, we show that this is also the case for a mammalian in vitro system. These results also demonstrate that an RNA can initiate translation from a start codon where an ideal translational consensus sequence is impossible.

Non-histone protein 1 (NHP1) is a member of the Ku protein family which is upregulated in differentiating mouse myoblasts and human promyelocytes.

We have previously purified and characterized a ubiquitous non-histone protein (NHP1) which has a high affinity (Kd 10(-11) M) for different avian vitellogenin gene sequences containing CpGs (Hughes et al. (1989) Biochemistry 28, 9137-9142; Hughes and Jost (1989) Nucleic Acids Res. 17, 8511-8520). Here we show by microsequencing that the peptides derived from the purified p75 and p85 subunits of NHP1 from HeLa cells have between 64 and 100% identity with the human Ku autoantigen. During the differentiation of human HL-60 promyelocytes there is an increase in the amount of p85 subunit protein whereas the level of the p75 subunit is unchanged. In differentiating mouse G8 myoblasts there is, however, an upregulation of both the p75 and p85 subunits and of the p85 mRNA. An inhibition of mouse myoblast differentiation by either cAMP, 3-aminobenzamide or sodium butyrate abolishes the upregulation of the p85 subunit. In G8 myoblasts chemical, or physical stress by UV light or X-rays does not upregulate the level of the p85 subunit. The possible involvement of NHP1 in the active demethylation of bifilarly methylated DNA will be discussed.

The 3' untranslated region of bovine preprolactin contains a transferable non-poly(A) mRNA sequence that prolongs translation.

Preprolactin transcripts, synthesized in vitro, were actively translated for a prolonged period when injected into Xenopus oocytes. As a result, prolactin continued to be secreted into the media for up to 6 days after injection of the transcript. To investigate the role of the preprolactin 3' untranslated sequence in stabilizing transcripts, it was fused to coding regions derived from signal recognition particle receptor alpha-subunit or preproinsulin receptor. The translational half-life of the chimeric RNA was increased for both coding regions, suggesting that a sequence within the preprolactin 3' untranslated region that prolongs translation is transferable. Deletion mutagenesis of this untranslated region demonstrated that a sequence of 98 nucleotides immediately following the prolactin stop codon was sufficient to prolong translation of RNAs injected into Xenopus oocytes.

Central and peripheral receptors in guinea-pigs exposed to simulated high altitude.

Guinea-pigs, chronically exposed to simulated high altitude of 6000 m, showed alterations in some, but not all, neurotransmitter-receptor systems. Thus, peripheral (cardiac) beta-adrenergic receptors were decreased in numbers (Bmax) with an increased affinity although no alterations in endogenous cardiac norepinephrine content were found. In contrast, H1-histamine receptors in the guinea-pig ventricle and brain showed no changes in Bmax or affinity. Dopamine receptors of the caudate nucleus showed no differences between control and altitude-exposed guinea-pigs, although previous reports demonstrated a significant increase (19%) in the content of endogenous dopamine of the caudate. Thus, chronic exposure to simulated high altitude altered beta-adrenergic receptors so that they were more sensitive to small ligand concentrations and less sensitive to large ligand concentrations.

Chronic administration of naltrexone alters central catecholamine levels but not the development of hypertension in spontaneously hypertensive rats.

The effect of chronic administration of the opiate antagonist naltrexone on the genesis and development of hypertension and correlated changes in central norepinephrine levels was evaluated in spontaneously hypertensive rats and Wistar-Kyoto controls. Capsules of poly-epsilon-caprolactone containing naltrexone in ethyl oleate or ethyl oleate alone were implanted in 4 week old SHR and WKY rats. Naltrexone failed to alter the genesis or magnitude of hypertension developed by the SHR and did not alter heart rates. Blood pressure and heart rate of WKY rats were also unaffected. A significant decrease in midbrain and hippocampal NE levels was observed in SHRs but not in WKYs following naltrexone.

ATP-citrate lyase as a target for hypolipidemic intervention. Sulfoximine and 3-hydroxy-beta-lactam containing analogues of citric acid as potential tight-binding inhibitors.

Citric acid analogues (+/-)-12a,b and (+/-)-17a,b, where one of the primary carboxylates has been replaced by a sulfoximinoyl and a 3-(3-hydroxy-beta-lactamyl) moiety, respectively, have been synthesized and evaluated as inhibitors of ATP-citrate lyase. The design of these inhibitors was based on methionine sulfoximine and tabtoxinine beta-lactam, potent, tight-binding inhibitors of glutamine synthetase. Both ATP-citrate lyase and glutamine synthetase employ phosphate-carboxylate anhydrides as a method for carboxylate activation during catalysis. Only one diastereomer, (+/-)-12a, displayed weak, reversible inhibition, while the remaining citrate analogues (+/-)-12b and (+/-)-17a,b were inactive against the lyase. No time-dependent inactivation of the enzyme was observed.

Alteration of dopamine metabolism in different brain regions of the rabbit by estradiol and tamoxifen.

Tamoxifen citrate, a mixed estrogen agonist-antagonist, and estradiol 17-beta administered separately for 14 days significantly reduced dopamine and dihydroxyphenylacetic acid in the cortex and hypothalamus regions of the brain in immature female rabbits. In addition to these areas, estradiol also reduced dopamine and dihydroxyphenylacetic acid in the striatum but tamoxifen treatment significantly reduced only dihydroxyphenylacetic acid concentration in the striatum. When estradiol and tamoxifen were injected together, dopamine and dihydroxyphenylacetic acid concentrations were reduced only in the cortex. Specific binding of [3H]spiperone to dopamine receptors was significantly increased by both estradiol and tamoxifen in the hypothalamus but only tamoxifen increased dopamine binding in the striatum. A low dose of tamoxifen, either alone or in combination with estradiol, increased uterine weight, but a higher dose of tamoxifen was neither an estrogen agonist nor antagonist. These studies indicate that estradiol and tamoxifen alter dopamine metabolism in the various regions of brain differentially. The estrogen agonist activity of tamoxifen does not correspond to antidopaminergic action of estradiol in the striatum.

Drug interactions represented by high resolution computer graphics.

The amount of backward walking induced in mice by co-administration of clenbuterol, a beta-adrenoceptor agonist and putative antidepressant, and the benzodiazepine chlordiazepoxide was plotted as a three-dimensional surface, using modified Uniras software. The surface was compared with the theoretical surface to be expected if the interactions between the two drugs were merely additive. The difference between the theoretical and the obtained surfaces was plotted and a functional representation of statistical variability was 'draped' over the difference model. The computer methods can be applied to small amounts of irregularly distributed data, and results are apparent from simple inspection, e.g. the highest peak of backward walking is also the most reliable statistically. The technique is useful for both laboratory and clinical studies.

A question of the specificity of rabbit atrial chronotropic histamine receptors and agents which affect their activity.

The histamine chronotropic response of rabbit atria appears to be controlled by both H1 and H2 receptors and can be blocked in part by either metiamide (an H2 antagonist) or diphenhydramine (an H1 antagonist), while both 2- and 4-methylhistamine (H1 and H2 agonists, respectively) stimulated the chronotropic response. At low agonist concentrations, the simultaneous presence of both H1 and H2 blockers results in considerably less inhibition than could be expected from calculations of individual inhibition data, suggesting that some sites behave as if they have both H1 and H2 properties. Additional compounds were tested for specific action on H1 and H2 receptors: 2-(2-pyridyl) ethylamine, reported to be an H1 agonist, appears to stimulate rabbit atria by releasing norepinephrine and guinea pig atria by releasing both norepinephrine and histamine; while dimaprit, reported to be an H2 agonist, may stimulate histamine receptors directly but has a nonspecific depressant action on rabbit atria which interferes with its use as an agonist in this species.

Age-dependent presence of antibodies in rat dams, capable of conferring protection against group B Streptococcus infection in neonates.

A rat model was used to investigate maternal age-dependent resistance on group B Streptococcus (GBS)-induced mortality of the offspring. Offspring from young (first time) or older (repeat litters) dams were challenged with GBS. There was an approximate log difference in the dose of GBS required to cause identical levels of mortality in the two groups. The sera of the dams from both groups were analysed by whole-cell ELISA, and it was demonstrated that sera from the older dams possessed circulating IgG cross-reactive to GBS. Since IgG is transplacentally transferred, we conclude that this is the method of observed protection.

Chronic vitamin D deficiency in the weanling rat alters catecholamine metabolism in the cortex.

The effect of a vitamin D deficient (--D), vitamin D replete (150 I.U. D3 twice weekly) and normal rat diets for 4 weeks in weanling male rats on the steady-state concentration in several brain sites of dopamine (DA), norepinephrine (NE), and dihydroxyphenylacetic acid (DOPAC) was investigated. The areas of the central nervous system assayed were the brainstem, cerebellum, cerebral cortex, hypothalamus-median eminence, and striatum. The results indicate that DA content of cortex and hypothalamus significantly increased in the --D group compared to the normal diet or D replete groups. The concentrations of DOPAC and NE in the cortex of both --D and D replete rats increased significantly compared to normal diet group. Plasma calcium level was significantly lower in --D group compared to the normal diet or vitamin D replete groups.

Antagonistic activity of tiotidine and ranitidine on guinea-pig and rabbit atria.

Isolated, spontaneously beating atrial pairs from rabbits and guinea-pigs were used to determine and to compare the activity of ranitidine and tiotidine as antagonists of histamine stimulated chronotropic activity. Ranitidine produced a classical competitive, reversible antagonism of histamine effects with a pA2 in rabbit atria of 8.2. In contrast, tiotidine produced both a dextral shift of the log dose-response curve, as well as a previously unreported suppression in the maximal response produced by histamine. In accordance with receptor theory, this type of activity represents a dualistic antagonism of histamine chronotropic responses.

Mapping and interpreting complex interactions between psychoactive drugs by simple computer-generated models.

Drug combinations often yield results not readily predictable from those of their separate constituents. Computer programs have been developed to generate models of dual drug effects to analyse the results of experiments involving drug combinations. Single and combined dose-response results of rat locomotor activity are expressed as three-dimensional models. A manually plotted isobol diagram, showing eqi-active dose combinations of amylobarbitone and dexamphetamine, is compared with isobols constructed by the computer. A simple dose model theory is proposed which assumes that the two drugs have merely additive effects; differences between expectations from this theoretical model and the experimental results actually obtained were found by the computer. This computer approach allows detailed analysis of results. Three-dimensional dose response surfaces can provide a simple visual guide to dose combinations whose effects deviate significantly from those expected if the constituents were simply additive. Areas of special interest can thus be highlighted.

Alterations in CNS amine levels by acclimatization to hypobaric hypoxia.

Rabbits were acclimatized to simulated high altitude (SHA) (hypobaric hypoxia) at 6000 M (350 torr) on alternate days for 70 days. The norepinephrine levels of the midbrain were lower in the acclimatized animal compared to the controls (p less than 0.06) and 3,4 dihydroxyphenylacetic acid (DOPAC) was significantly higher (p less than 0.04) in the striatum of control than in the test animals. The mean dopamine (DA) levels in the striatum of the test animals were higher than the controls. The ratio of DOPAC/DA was 2.0 for the controls and 0.4 for the SHA brains which suggests reduced dopamine turnover in the striatum of the SHA rabbits. Rats acclimatized in the same manner did not show any difference in the NE or DA levels between the control and SHA animals, possibly the result of species differences.