System Xc- Antiporter Inhibitors: Azo-Linked Amino-Naphthyl-Sulfonate Analogues of Sulfasalazine.

The cystine/glutamate antiporter system Xc- (SXc-) mediates the exchange of intracellular L-glutamate (L-Glu) with extracellular L-cystine (L-Cys2). Both the import of L-Cys2 and the export of L-Glu take on added significance in CNS cells, especially astrocytes. When the relative activity of SXc- overwhelms the regulatory capacity of the EAATs, the efflux of L-Glu through the antiporter can be significant enough to trigger excitotoxic pathology, as is thought to occur in glioblastoma. This has prompted considerable interest in the pharmacological specificity of SXc- and the development of inhibitors. The present study explores a series of analogues that are structurally related to sulfasalazine, a widely employed inhibitor of SXc-. We identify a number of novel aryl-substituted amino-naphthylsulfonate analogues that inhibit SXc- more potently than sulfasalazine. Interestingly, the inhibitors switch from a competitive to noncompetitive mechanism with increased length and lipophilic substitutions, a structure-activity relationship that was previously observed with aryl-substituted isoxazole. These results suggest that the two classes of inhibitors may interact with some of the same domains on the antiporter protein and that the substrate and inhibitor binding sites may be in close proximity to one another. Molecular modeling is used to explore this possibility.

Quantifying the dark data in museum fossil collections as palaeontology undergoes a second digital revolution.

Large-scale analysis of the fossil record requires aggregation of palaeontological data from individual fossil localities. Prior to computers, these synoptic datasets were compiled by hand, a laborious undertaking that took years of effort and forced palaeontologists to make difficult choices about what types of data to tabulate. The advent of desktop computers ushered in palaeontology's first digital revolution-online literature-based databases, such as the Paleobiology Database (PBDB). However, the published literature represents only a small proportion of the palaeontological data housed in museum collections. Although this issue has long been appreciated, the magnitude, and thus potential significance, of these so-called 'dark data' has been difficult to determine. Here, in the early phases of a second digital revolution in palaeontology--the digitization of museum collections-we provide an estimate of the magnitude of palaeontology's dark data. Digitization of our nine institutions' holdings of Cenozoic marine invertebrate collections from California, Oregon and Washington in the USA reveals that they represent 23 times the number of unique localities than are currently available in the PBDB. These data, and the vast quantity of similarly untapped dark data in other museum collections, will, when digitally mobilized, enhance palaeontologists' ability to make inferences about the patterns and processes of past evolutionary and ecological changes.

Correction to: Process-induced degradation of bioresorbable PDLGA in bone tissue scaffold production.

The original version of this article unfortunately contained a mistake. E. Themistou was missing from the author group and so is now included with this erratum.

Process-induced degradation of bioresorbable PDLGA in bone tissue scaffold production.

Process-induced degradation of clinically relevant resorbable polymers was investigated for two thermal techniques, filament extrusion followed by fused deposition modelling (FDM). The aim was to develop a clear understanding of the relationship between temperature, processing time and resultant process-induced degradation. This acts to address the current knowledge gap in studies involving thermal processing of resorbable polymers. Poly(DL-lactide-co-glycolide) (PDLGA) was chosen for its clinically relevant resorption properties. Furthermore, a comparative study of controlled thermal exposure was conducted through compression moulding PDLGA at a selected range of temperatures (150-225 °C) and times (0.5-20 min). Differential scanning calorimetry (DSC) and gel permeation chromatography (GPC) were used to characterise thermally induced degradation behaviour. DSC proved insensitive to degradation effects, whereas GPC demonstrated distinct reductions in molecular weight allowing for the quantification of degradation. A near-exponential pattern of degradation was identified. Through the application of statistical chain scission equations, a predictive plot of theoretical degradation was created. Thermal degradation was found to have a significant effect on the molecular weight with a reduction of up to 96% experienced in the controlled processing study. The proposed empirical model may assist prediction of changes in molecular weight, however, accuracy limitations are highlighted for twin-screw extrusion, accredited to high-shear mixing. The results from this study highlight the process sensitivity of PDLGA and proposes a methodology for quantification and prediction, which contributes to efforts in understanding the influence of manufacture on performance of degradable medical implants.

The importance of glucocorticoids in alcohol dependence and neurotoxicity.

Alterations in hypothalamo-pituitary adrenal (HPA) function have been described in alcoholics and in rodents after chronic alcohol consumption but the role of glucocorticoids in alcohol consumption, and the mechanisms involved, has received little attention until recently. Both alcohol consumption and withdrawal from chronic alcohol intake raise circulating glucocorticoid levels, and prolonged high concentrations of glucocorticoids are known to have detrimental effects on neuronal function and cognition. This minireview covers the ways in which glucocorticoids may be involved in drinking behavior, from social drinking to dependence, and the negative consequences of alcohol consumption seen during withdrawal which may have a detrimental effect on treatment outcome. Research shows prolonged increases in brain glucocorticoid concentrations and decreased brain glucocorticoid receptor availability (consistent with increased levels of endogenous ligand) after withdrawal from chronic alcohol treatment. Evidence suggests that increased glucocorticoid levels in the brain after chronic alcohol treatment are associated with the cognitive deficits seen during abstinence which impact on treatment efficacy and quality of life. Studies on organotypic cultures also demonstrate the importance of glucocorticoids in the neuropathological consequences of alcohol dependence.

Selective increases in regional brain glucocorticoid: a novel effect of chronic alcohol.

The hypothalamo-pituitary-adrenal axis shows functional changes in alcoholics, with raised glucocorticoid release during alcohol intake and during the initial phase of alcohol withdrawal. Raised glucocorticoid concentrations are known to cause neuronal damage after withdrawal from chronic alcohol consumption and in other conditions. The hypothesis for these studies was that chronic alcohol treatment would have differential effects on corticosterone concentrations in plasma and in brain regions. Effects of chronic alcohol and withdrawal on regional brain corticosterone concentrations were examined using a range of standard chronic alcohol treatments in two strains of mice and in rats. Corticosterone was measured by radioimmunoassay and the identity of the corticosterone extracted from brain was verified by high performance liquid chromatography and mass spectrometry. Withdrawal from long term (3 weeks to 8 months) alcohol consumption induced prolonged increases in glucocorticoid concentrations in specific regions of rodent brain, while plasma concentrations remained unchanged. This effect was seen after alcohol administration via drinking fluid or by liquid diet, in both mice and rats and in both genders. Shorter alcohol treatments did not show the selective effect on brain glucocorticoid levels. During the alcohol consumption the regional brain corticosterone concentrations paralleled the plasma concentrations. Type II glucocorticoid receptor availability in prefrontal cortex was decreased after withdrawal from chronic alcohol consumption and nuclear localization of glucocorticoid receptors was increased, a pattern that would be predicted from enhanced glucocorticoid type II receptor activation. This novel observation of prolonged selective increases in brain glucocorticoid activity could explain important consequences of long term alcohol consumption, including memory loss, dependence and lack of hypothalamo-pituitary responsiveness. Local changes in brain glucocorticoid levels may also need to be considered in the genesis of other mental disorders and could form a potential new therapeutic target.

Nimodipine prior to alcohol withdrawal prevents memory deficits during the abstinence phase.

Effects of the dihydropyridine, nimodipine, an antagonist at L-type calcium channels, on the memory loss in rats caused by long term alcohol consumption were examined. Either a single dose of nimodipine or 2 weeks of repeated administration was given prior to withdrawal from 8 months of alcohol consumption. Memory was measured by the object recognition test and the T maze. Both nimodipine treatments prevented the memory deficits when these were measured between 1 and 2 months after alcohol withdrawal. At the end of the memory testing, 2 months after cessation of chronic alcohol consumption, glucocorticoid concentrations were increased in specific regions of rat brain without changes in plasma concentrations. Both nimodipine treatment schedules substantially reduced these rises in brain glucocorticoid. The data indicate that blockade of L-type calcium channels prior to alcohol withdrawal protects against the memory deficits caused by prolonged alcohol intake. This shows that specific drug treatments, such as nimodipine, given over the acute withdrawal phase, can prevented the neuronal changes responsible for subsequent adverse effects of long term consumption of alcohol. The results also suggest the possibility that regional brain glucocorticoid increases may be involved in the adverse effects of long term alcohol intake on memory. Such local changes in brain glucocorticoid levels would have major effects on neuronal function. The studies indicate that L-type calcium channels and brain glucocorticoid levels could form new targets for the treatment of cognitive deficits in alcoholics.

Sidney George Shaw, DPhil (1948-2017).

On March 4, 2017 at the age of 68, Sidney George Shaw (Sid) unexpectedly died from complications following surgery, only four years after retiring from the University of Bern. Trained in biochemistry at Oxford University, Sid had quickly moved into molecular pharmacology and became a key investigator in the field of enzyme biochemistry, vasoactive peptide research, and receptor signaling. Sid spent half his life in Switzerland, after moving to the University of Bern in 1984. This article, written by his friends and colleagues who knew him and worked with him during different stages of his career, summarizes his life, his passions, and his achievements in biomedical research. It also includes personal memories relating to a dear friend and outstanding scientist whose intellectual curiosity, humility, and honesty will remain an example to us all.

Protein kinase C delta inhibits Caco-2 cell proliferation by selective changes in cell cycle and cell death regulators.

PKC-delta is a serine/threonine kinase that mediates diverse signal transduction pathways. We previously demonstrated that overexpression of PKC-delta slowed the G1 progression of Caco-2 colon cancer cells, accelerated apoptosis, and induced cellular differentiation. In this study, we further characterized the PKC-delta dependent signaling pathways involved in these tumor suppressor actions in Caco-2 cells overexpressing PKC-delta using a Zn2+ inducible expression vector. Consistent with a G1 arrest, increased expression of PKC-delta caused rapid and significant downregulation of cyclin D1 and cyclin E proteins (50% decreases, P<0.05), while mRNA levels remained unchanged. The PKC agonist, phorbol 12-myristate 13-acetate (TPA, 100 nM, 4 h), induced two-fold higher protein and mRNA levels of p21(Waf1), a cyclin-dependent kinase (cdk) inhibitor in PKC-delta transfectants compared with empty vector (EV) transfected cells, whereas the PKC-delta specific inhibitor rottlerin (3 microM) or knockdown of this isoenzyme with specific siRNA oligonucleotides blocked p21(Waf1) expression. Concomitantly, compared to EV control cells, PKC-delta upregulation decreased cyclin D1 and cyclin E proteins co-immunoprecipitating with cdk6 and cdk2, respectively. In addition, overexpression of PKC-delta increased binding of cdk inhibitor p27(Kip1) to cdk4. These alterations in cyclin-cdks and their inhibitors are predicted to decrease G1 cyclin kinase activity. As an independent confirmation of the direct role PKC-delta plays in cell growth and cell cycle regulation, we knocked down PKC-delta using specific siRNA oligonucleotides. PKC-delta specific siRNA oligonucleotides, but not irrelevant control oligonucleotides, inhibited PKC-delta protein by more than 80% in Caco-2 cells. Moreover, PKC-delta knockdown enhanced cell proliferation ( approximately 1.4-2-fold, P<0.05) and concomitantly increased cyclin D1 and cyclin E expression ( approximately 1.7-fold, P<0.05). This was a specific effect, as nontargeted PKC-zeta was not changed by PKC-delta siRNA oligonucleotides. Consistent with accelerated apoptosis in PKC-delta transfectants, compared to EV cells, PKC-delta upregulation increased proapoptotic regulator Bax two-fold at mRNA and protein levels, while antiapoptotic Bcl-2 protein was decreased by 50% at a post-transcriptional level. PKC-delta specific siRNA oligonucleotides inhibited Bax protein expression by more than 50%, indicating that PKC-delta regulates apoptosis through Bax. Taken together, these results elucidate two critical mechanisms regulated by PKC-delta that inhibit cell cycle progression and enhance apoptosis in colon cancer cells. We postulate these antiproliferative pathways mediate an important tumor suppressor function for PKC-delta in colonic carcinogenesis.

Mechanisms of proteinuria in diabetic nephropathy. II. A study of the size-selective glomerular filtration barrier.

We evaluated the size-selective properties of the glomerular barrier in 30 patients in whom diabetic nephropathy was associated with urinary IgG losses. Neutral dextrans of graded size were used to characterize glomerular membrane-pore structure. A fractional IgG clearance (relative to freely permeable inulin) smaller or greater than 0.001 was used to distinguish patients with minor (group 1, N = 14) and major (group 2, N = 16) urinary IgG leakage, respectively. Fractional clearances of dextrans (theta D) of smaller size (radii 20-40 A) were similar, but those of larger dextrans (radii 42-60 A) were elevated in group 2 relative to group 1 patients. When plotted on log-normal probability coordinates, the correlation between theta D and radius in healthy subjects is linear, suggesting that glomerular pores form one population with a normal distribution. In diabetic nephropathy with urinary IgG leakage, however, theta D for large molecules was elevated and departed from linearity, suggesting a bimodal pore size distribution within the glomerular membrane. A pore model of solute transport revealed (1) the upper pore mode was highly permeable to large dextrans equivalent in size to IgG and (2) the fraction of glomerular filtrate permeating the large pores was greater in group 2 than in group 1 patients with diabetic nephropathy, 6% versus 3%, respectively. We conclude that urinary IgG leakage in diabetic nephropathy is determined by the development of a subpopulation of enlarged pores. The magnitude of urinary IgG losses appears to be a function of the membrane area-fraction occupied by the enlarged pores.

How has molecular pharmacology contributed to our understanding of the mechanism(s) of general anesthesia?

This review discusses the mechanism(s) of general anesthesia from a pharmacological viewpoint; in particular, the ability of drugs to produce many different effects is emphasised. The problems of experimental measurement of general anesthesia are discussed, and the possibilities for antagonism and potentiation of anesthesia considered. Physicochemical studies on anesthesia are described, as are the advancement of ideas beyond consideration of lipids and proteins as separate sites of action. The importance of studies on different areas of the brain is highlighted, and the review finishes with a survey of the effects of general anesthetics on synaptic transmission which emphasises the problems of extrapolation from in vitro to in vivo.

The contribution of electrophysiology to knowledge of the acute and chronic effects of ethanol.

This review describes the effects of ethanol on the components of neuronal transmission and the relationship of such effects to the behavioural actions of ethanol. The concentrations of ethanol with acute actions on voltage-sensitive ion channels are first described, then the actions of ethanol on ligand-gated ion channels, including those controlled by cholinergic receptors, 5-hydroxytryptamine receptors, the various excitatory amino acid receptors, and gamma-aminobutyric acid receptors. Acute effects of ethanol are then described on brain areas thought to be involved in arousal and attention, the reinforcing effects of ethanol, the production of euphoria, the actions of ethanol on motor control, and the amnesic effects of ethanol; the acute effects of ethanol demonstrated by EEG studies are also discussed. Chronic effects of alcohol on neuronal transmission are described in the context of the various components of the ethanol withdrawal syndrome, withdrawal hyperexcitability, dysphoria and anhedonia, withdrawal anxiety, craving, and relapse drinking. Electrophysiological studies on the genetic influences on the effects of ethanol are discussed, particularly the acute actions of ethanol and electrophysiological differences reported in individuals predisposed to alcoholism. The conclusion notes the concentration of studies on the classical transmitters, with relative neglect of the effects of ethanol on peptides and on neuronal interactions between brain areas and integrated patterns of neuronal activity.

The role of neuronal calcium channels in dependence on ethanol and other sedatives/hypnotics.

This review discusses the importance of neuronal calcium currents in dependence on ethanol, barbiturates, benzodiazepines and opiates. The main sections describe the actions of ethanol on control of intracellular calcium and on calcium and calcium-dependent conductance mechanisms. In particular, the effects of both acute and chronic ethanol treatment on dihydropyridine-sensitive, voltage-dependent, calcium channels are described. The later sections cover the effects of barbiturates, benzodiazepines and opiates on these systems. The conclusions suggest that dihydropyridine calcium channel antagonists may offer a new therapeutic approach to the treatment of ethanol and opiate dependence.

Thiamine deficiency in the pathogenesis of chronic ethanol-associated cerebellar damage in vitro.

Nutritional deficiencies associated with long-term ethanol consumption may cause neuronal damage in ethanol-dependent individuals. Thiamine deficiency, in particular, is thought to contribute to ethanol-associated cerebellar degeneration, although damage may occur in adequately nourished alcoholics. Thus, the present study examined the effects of thiamine depletion and ethanol exposure on cytotoxicity in rat cerebellum. Organotypic cerebellar slice cultures were treated starting at 25 days in vitro with 100 mM ethanol for 11 days or 10 days followed by a 24-h withdrawal period. This exposure paradigm has previously been shown in hippocampal slice cultures to result in spontaneous cytotoxicity upon ethanol withdrawal. Additional cerebellar cultures were exposed to the thiamine depleting agent pyrithiamine (10-500 microM) for 10 or 11 days, some in the presence of ethanol exposure or withdrawal. Other cultures were co-exposed to thiamine (1-100 microM), 500 microM pyrithiamine, and ethanol for 10 or 11 days. The results demonstrated that neither 11-day ethanol treatment nor withdrawal from 10-day exposure significantly increased cerebellar cytotoxicity, as measured by propidium iodide fluorescence. The 11-day treatment with 100 or 500 microM pyrithiamine significantly increased propidium iodide fluorescence approximately 21% above levels observed in control tissue. Cultures treated with both ethanol (11 days or 10 days plus withdrawal) and 500 microM pyrithiamine displayed a marked increase in cytotoxicity approximately 60-90% above levels observed in control cultures. Pyrithiamine and ethanol-induced cytotoxicity was prevented in cultures co-exposed to thiamine (10-100 microM) for the duration of pyrithiamine treatment. Findings from this report suggest that the cerebellum may be more sensitive to the toxic effects of thiamine deficiency, as compared with alcohol withdrawal, associated with alcohol dependence.

Social defeat increases alcohol preference of C57BL/10 strain mice; effect prevented by a CCKB antagonist.

RATIONALE: In humans, social stress over long and short term can increase alcohol consumption, but the mechanisms involved are not understood. OBJECTIVES: This study was conducted to examine the effects of social defeat, using the resident/intruder paradigm, on the alcohol preference of "low alcohol drinking" individuals in a colony of C57BL/10 strain mice and the effects of two anxiolytic drugs. METHODS: Alcohol preference, in a two-bottle choice (8% v/v alcohol or water), was measured, in separate experiments, after either a single experience of social defeat by a resident male mouse, five consecutive daily defeat experiences or one experience per week for 4 weeks. Comparison was made with effects of repeated social defeat on the preference for dilute sucrose. In addition, the actions of the CCKB receptor antagonist, CAM1028, and of diazepam were examined on the effects of repeated defeat experiences. RESULTS: Five consecutive daily defeat experiences had a slow onset effect in increasing alcohol preference and consumption, compared with five daily exposures to a novel environment. A single defeat, or one defeat per week, did not significantly alter alcohol preference or intake. There were no effects of five daily defeat experiences on sucrose preference or consumption. The effect of repeated defeats on alcohol preference was significantly decreased by administration of the CCKB receptor antagonist, CAM1028, prior to each experience, but not by corresponding administration of diazepam. CONCLUSION: The results show that social stress increases alcohol intake in low alcohol preference C57BL/10 mice and suggest that CCK transmission may be involved in this effect.

Inhibition of nitric oxide synthase activity diminishes the acute effects of relaxin on growth, but not softening, of the cervix in the rat.

Relaxin promotes growth and softening of the cervix during pregnancy in the rat. This study examined the hypothesis that nitric oxide (NO) mediates the effects of relaxin on the rat cervix. To test that hypothesis, N omega-nitro-L-arginine methyl ester (L-NAME) was used to inhibit NO synthase, the enzyme that converts arginine to NO and L-citrulline. Nonpregnant rats were ovariectomized when they were 78 days old (day 1 of treatment). At ovariectomy each animal was fitted with silicon tubing implants containing progesterone (P) and estrogen (E) in doses that provide blood levels similar to those during late pregnancy. Rats were assigned to three treatment groups. The control group OPE (n = 6 rats) received 0.5 ml L-NAME vehicle (PBS) sc at 6-h intervals from 0600 h on day 7 through 1200 h on day 8 and 0.5 ml relaxin vehicle (PBS) sc at 0600 and 1200 h on day 8. Group OPER (n = 6 rats) was treated in the same way as group OPE, except that 20 microg porcine relaxin were administered. Group OPERI (n = 7 rats) was treated in the same way as group OPER, except that L-NAME was administered at a dose of 100 mg/kg x 6 h. Between 1400-1500 h on day 8, the cervices were removed and weighed. Cervical wet weight and extensibility were markedly greater (P < 0.01) in relaxin-treated group OPER rats than in group OPE controls. Treatment with L-NAME diminished relaxin's effects on cervical wet weight, but not cervical extensibility. In conclusion, this study provides evidence that NO contributes to the acute effects of relaxin on the growth, but not the softening, of the rat cervix.

Upward subluxation of the axis in ankylosing spondylitis. A clinical pathologic report.

Upward subluxation of the axis associated with cord compression and death was noted in a patient with a long history of idiopathic ankylosing spondylitis. Upward subluxation of the axis has been recognized in up to 8 per cent of patients with rheumatoid arthritis but it is an exceedingly rare complication of ankylosing spondylitis. In this patient psoriasis and then psoriatic dactylitis developed 26 years after the onset of his ankylosing spondylitis. It is tempting to speculate that the unusual destruction of the joints around the atlas might be due to an added effect of psoriasis on idiopathic ankylosing spondylitis.

Prolonged effects of chronic ethanol treatment on responses to repeated nicotine administration: interactions with environmental cues.

The most common combination of dependence on two drugs occurs with alcohol and nicotine, but little is known of the way in which these drugs interact in the brain. This study investigated the effects in mice of prolonged consumption of alcohol, by liquid diet, on the actions of nicotine on locomotor activity, and the influence of environmental cues on these effects. Administration of nicotine after chronic alcohol intake did not show any significant changes on first administration, but after 28 days of daily nicotine injections, nicotine produced significant locomotor stimulation and increased rearing activity in the mice which had previously received the alcohol diet, compared with the activity of animals that received the control diet. However, significantly increased locomotor activity was also seen, after the repeated nicotine administration, immediately prior to the nicotine injection, only in the mice that had previously consumed alcohol. Examination of the influence of the environment in which the activity was tested demonstrated that the effect on activity prior to injections was seen only if measurement was made in a test environment that was familiar to the animals. The results demonstrate an interaction between chronic alcohol consumption and the effects of environment on the actions of nicotine, that may have relevance to the consumption of these drugs in humans.

The benzodiazepine antagonist, Ro 15-1788, prevents the effects of flurazepam on the high pressure neurological syndrome.

The benzodiazepine antagonist, Ro 15-1788, was used to investigate the anticonvulsant effect of flurazepam on the high pressure neurological syndrome (HPNS). Flurazepam raised the threshold pressures for the onset of tremor and of clonic convulsions caused by high pressure helium. Administration of the benzodiazepine antagonist Ro 15-1788 completely prevented the changes in threshold pressures produced by flurazepam. Alone, Ro 15-1788 did not affect the onset pressures. The concentration of Ro 15-1788 which blocked the actions of flurazepam on the pressure signs was the same as that required to prevent its effects on convulsions due to infusion of bicuculline. It is concluded that the effect of flurazepam on thresholds for the high pressure neurological syndrome is due to action at benzodiazepine receptors and not to a nonspecific effect.

The novel anticonvulsant, gabapentin, protects against both convulsant and anxiogenic aspects of the ethanol withdrawal syndrome.

The effects of the anticonvulsant, gabapentin, were investigated, in mice, on the withdrawal convulsive behaviour and anxiety-related behaviour that are produced by cessation of prolonged intake of ethanol. When given at 50 or 100 mg/kg, this compound decreased the rise in handling-induced hyperexcitability which occurs during the withdrawal period; the effects were most pronounced for the first 4 hr after administration. Gabapentin also decreased the convulsive response to an audiogenic stimulus during the withdrawal period. The elevated plus-maze, with both traditional and ethological indices of activity was used as a test of anxiety-related behaviour after cessation of chronic ethanol treatment. Gabapentin, at 50 and 100 mg/kg, was found to decrease some, although not all, of the signs of withdrawal-induced anxiety. At doses up to and including 200 mg/kg, gabapentin had no effect on motor co-ordination or spontaneous locomotor activity in control animals. The results demonstrated that gabapentin has a selective action in decreasing both convulsive and anxiety-related aspects of withdrawal behaviour after chronic ethanol treatment. It is possible that further studies with this compound may shed further light on the mechanisms involved in the withdrawal syndrome.