Reduction in pain: Is it worth the gain? The effect of opioids on the GI tract.

The use of opioid medications for acute and chronic pain has increased significantly in the past 20 years in the United States. Given the high density of opioid receptors in the gastrointestinal tract, side effects are common in these patients including constipation, dysphagia, bloating, nausea, and vomiting. These side effects, which are experienced by most patients who take opioids, can lead to significant impairment in quality of life. Unlike other side effects from opioids, gastrointestinal side effects do not diminish with continued use, often leading patients to reduce or discontinue their opioid treatment to relieve these side effects. Therefore, physicians must be aware and anticipate potential side effects in patients receiving opioids to ensure appropriate pain management.

Emergency department utilisation for inflammatory bowel disease in the United States from 2006 to 2014.

BACKGROUND: Despite advances in treatment, patients with inflammatory bowel disease (IBD) frequently require emergency department (ED) visits and hospitalisations. AIMS: To analyse trends in ED visits and subsequent hospitalisations for IBD in the United States (US). METHODS: Data were analysed from the Nationwide Emergency Department Sample (NEDS) years 2006-2014. The NEDS is the largest all-payer ED database in the US, weighted to represent 135 million visits/year. IBD was identified using ICD-9 codes for Crohn's disease (CD) or ulcerative colitis (UC). Surgeries were identified using procedure codes. RESULTS: The frequency of IBD-ED visits increased 51.8%, from 90 846 visits in 2006 to 137 946 in 2014, which was statistically significant in linear regression. For comparison, all-case ED use between 2006 and 2014 increased 14.8%. In-patient hospitalisations from the ED decreased 12.1% for IBD (from 64.7% rate of hospitalisation from the ED in 2006 to 52.6% in 2014), with a UC:CD ratio of 1.2:1 in 2006 and 1.3:1 in 2014. Chi-square analysis revealed that this was a significant decrease. Surgery rates also showed a statistically significant decrease. The mean ED charge per patient rose 102.5% and the aggregate national cost of IBD-ED visits increased 207.5%. CD accounted for over twice as many visits as UC in both years. UC, age, male gender, highest income quartile, private insurance, Medicaid/Medicare, and tobacco use were associated with in-patient admissions. CONCLUSIONS: The number of ED visits due to IBD and associated charges have continued to rise, while the rates of in-patient hospitalisations referred from the ED and surgeries have decreased.

Severity of ineffective esophageal motility is associated with utilization of skeletal muscle relaxant medications.

BACKGROUND: Ineffective esophageal motility (IEM) is the most common finding on high-resolution esophageal manometry (HREM). The underlying mechanisms for IEM remain to be fully elucidated. The aim of this study was to determine if utilization of skeletal muscle relaxants is associated with IEM, and with more severe subtypes of the disorder. METHODS: Patients with diagnosis of IEM were gender and age matched to patients with normal HREM. Demographic information, symptoms, endoscopic findings, medication usage and medical comorbidities were recorded. Patients with a diagnosis of IEM were divided into subgroups based on mean distal contractile integral (DCI) and percentage of ineffective swallows, and assessed for clinically significant differences among patients with varying severity of underlying IEM. KEY RESULTS: A total of 118 patients were included in each group. There were no significant clinical differences between the group of patients with IEM and the group of patients with normal manometry. Within the group of IEM patients, those with mean DCI < 250 mm Hg/s/cm were more likely to be prescribed skeletal muscle relaxants (27.8% vs 11.0%, P = .044), and those using skeletal muscle relaxants had a larger mean percentage of ineffective swallows (81.1% vs 71.5%, P = .029). There were no significant differences across mean DCI subgroups in usage of any other medication, or in any of the demographic variables or disease comorbidities examined in this study. CONCLUSIONS & INFERENCES: Use of skeletal muscle relaxants is associated with more severe IEM, which may suggest a causal association between this class of medications and weaker esophageal peristalsis.

Lofty missions, down-to-earth plans.

Most nonprofits make program decisions based on a mission rather than a strategy. They rally under the banner of a particular cause, be it "fight homelessness" or "end hunger." And since their causes are so worthwhile, they support any programs that are related--even tangentially--to their core missions. It's hard to fault people for trying to improve the state of the world, but that approach to making decisions is misguided. Acting without a clear long-term strategy can stretch an agency's core capabilities and push it in unintended directions. The fundamental problem is that many nonprofits don't have a strategy; instead, they have a mission and a portfolio of programs. But they hardly make deliberate decisions about which programs to run, which to drop, and which to turn down for funding. What most nonprofits call "strategy" is really just an intensive exercise in resource allocation and program management. This article outlines for nonprofits a four-step process for developing strategy. The first step is to create a broad, inspiring mission statement. The second step is to translate that core mission into a smaller, quantifiable operational mission. For instance, an agency whose core mission is to fight homelessness must decide if its focus is rural or urban and if it should concentrate on low-income housing loans or on establishing more shelters. The third step is to create a strategy platform; that is, the nonprofit decides how it will achieve its operational mission. Decisions about funding and about client, program, and organizational development are all made here. Once that platform is established, the nonprofit is ready to move to step four--making reasoned, strategic decisions about which programs to run and how to run them. The agency that follows these steps will improve its focus and its effectiveness at fulfilling its mission.

Mapping the functional topology of the animal fatty acid synthase by mutant complementation in vitro.

An in vitro mutant complementation approach has been used to map the functional topology of the animal fatty acid synthase. A series of knockout mutants was engineered, each mutant compromised in one of the seven functional domains, and heterodimers generated by hybridizing all possible combinations of the mutated subunits were isolated and characterized. Heterodimers comprised of a subunit containing either a beta-ketoacyl synthase or malonyl/acetyltransferase mutant, paired with a subunit containing mutations in any one of the other five domains, are active in fatty acid synthesis. Heterodimers in which both subunits carry a knockout mutation in either the dehydrase, enoyl reductase, keto reductase, or acyl carrier protein are inactive. Heterodimers comprised of a subunit containing a thioesterase mutation paired with a subunit containing a mutation in either the dehydrase, enoyl reductase, beta-ketoacyl reductase, or acyl carrier protein domains exhibit very low fatty acid synthetic ability. The results are consistent with a model for the fatty acid synthase in which the substrate loading and condensation reactions are catalyzed by cooperation of an acyl carrier protein domain of one subunit with the malonyl/acetyltransferase or beta-ketoacyl synthase domains, respectively, of either subunit. The beta-carbon-processing reactions, responsible for the complete reduction of the beta-ketoacyl moiety following each condensation step, are catalyzed by cooperation of an acyl carrier protein domain with the beta-ketoacyl reductase, dehydrase, and enoyl reductase domains associated exclusively with the same subunit. The chain-terminating reaction is carried out most efficiently by cooperation of an acyl carrier protein domain with the thioesterase domain of the same subunit. These results are discussed in the context of a revised model for the fatty acid synthase.

The human thioesterase II protein binds to a site on HIV-1 Nef critical for CD4 down-regulation.

A HIV-1 Nef affinity column was used to purify a 35-kDa Nef-interacting protein from T-cell lysates. The 35-kDa protein was identified by peptide microsequence analysis as the human thioesterase II (hTE) enzyme, an enzyme previously identified in a yeast two-hybrid screen as a potential Nef-interacting protein. Immunofluorescence studies showed that hTE localizes to peroxisomes and that coexpression of Nef and hTE leads to relocalization of Nef to peroxisomes. Interaction of Nef and hTE was abolished by point mutations in Nef at residues Asp(108), Leu(112), Phe(121), Pro(122), and Asp(123). All of these mutations also abrogated the ability of Nef to down-regulate CD4 from the surface of HIV-infected cells. Based on the x-ray and NMR structures of Nef, these residues define a surface on Nef critical for CD4 down-regulation. A subset of these mutations also affected the ability of Nef to down-regulate major histocompatibility complex class I. These results, taken together with previous studies, identify a region on Nef critical for most of its known functions. However, not all Nef alleles bind to hTE with high affinity, so the role of hTE during HIV infection remains uncertain.

Dibromopropanone cross-linking of the phosphopantetheine and active-site cysteine thiols of the animal fatty acid synthase can occur both inter- and intrasubunit. Reevaluation of the side-by-side, antiparallel subunit model.

The objective of this study was to test a new model for the homodimeric animal FAS which implies that the condensation reaction can be catalyzed by the amino-terminal beta-ketoacyl synthase domain in cooperation with the penultimate carboxyl-terminal acyl carrier protein domain of either subunit. Treatment of animal fatty acid synthase dimers with dibromopropanone generates three new molecular species with decreased electrophoretic mobilities; none of these species are formed by fatty acid synthase mutant dimers lacking either the active-site cysteine of the beta-ketoacyl synthase domain (C161A) or the phosphopantetheine thiol of the acyl carrier protein domain (S2151A). A double affinity-labeling strategy was used to isolate dimers that carried one or both mutations on one or both subunits; the heterodimers were treated with dibromopropanone and analyzed by a combination of sodium dodecyl sulfate/polyacrylamide gel electrophoresis, Western blotting, gel filtration, and matrix-assisted laser desorption mass spectrometry. Thus the two slowest moving of these species, which accounted for 45 and 15% of the total, were identified as doubly and singly cross-linked dimers, respectively, whereas the fastest moving species, which accounted for 35% of the total, was identified as originating from internally cross-linked subunits. These results show that the two polypeptides of the fatty acid synthase are oriented such that head-to-tail contacts are formed both between and within subunits, and provide the first structural evidence in support of the new model.

Fatty acid synthase dimers containing catalytically active beta-ketoacyl synthase or malonyl/acetyltransferase domains in only one subunit can support fatty acid synthesis at the acyl carrier protein domains of both subunits.

A double-tagging, dual affinity chromatographic procedure, which permits isolation of dimers independently mutated in each subunit, has been exploited to probe the functional topology of the animal fatty acid synthase. Dimers were engineered in which the chain-terminating thioesterase reaction was compromised by mutation of the (active-site) serine residue in both subunits; these dimers assembled two long-chain fatty acyl moieties, which remained covalently linked to the 4'-phosphopantetheine residues of the two acyl carrier protein domains. Significantly, dimers that contained an additional mutation that compromised the activity of either the beta-ketoacyl synthase or malonyl/acetyltransferase activity in only one subunit also assembled two long-chain acyl moieties. In contrast, in a control experiment, introduction of an additional mutation that compromised the function of the acyl carrier protein domain in only one subunit resulted in the assembly of only one long-chain acyl moiety per dimer. Because the beta-ketoacyl synthase and malonyl/acetyltransferase domains are located near the amino terminus of the polypeptide and the acyl carrier protein domain near the carboxyl terminus, these results support a modified model for the animal fatty acid synthase in which head-to-tail functional contacts are possible both within as well as between subunits.

Differential affinity labeling of the two subunits of the homodimeric animal fatty acid synthase allows isolation of heterodimers consisting of subunits that have been independently modified.

To explore the domain interactions that are required for catalytic activity of the multifunctional, homodimeric fatty acid synthase (FAS), we have formulated a strategy that allows isolation of modified dimers containing independently mutated subunits. Either a hexahistidine or a FLAG octapeptide tag was incorporated into the FAS at either the amino terminus, within an internal noncatalytic domain, or at the carboxyl terminus. The presence of the tags had no effect on the activity of the wild-type FAS. His-tagged dimers were mixed with FLAG-tagged dimers, and the subunits were randomized to produce a mixture of His-tagged homodimers, FLAG-tagged homodimers, and doubly tagged heterodimers. The doubly tagged heterodimers could be purified to homogeneity by chromatography on an anti-FLAG immunoaffinity column followed by a metal ion chelating column. This procedure for isolation of FAS heterodimers was utilized to determine whether the two centers for fatty acid synthesis in the FAS dimer can function independently of each other. Doubly tagged heterodimers, consisting of one wild-type subunit and one subunit in which the thioesterase activity had been eliminated, either by mutation or by treatment with phenylmethanesulfonyl fluoride, have 50% of the wild-type thioesterase activity and, in the presence of substrates, accumulate a long chain fatty acyl moiety on the modified subunit, thus blocking further substrate turnover at this center. Nevertheless, the ability of the heterodimer to synthesize fatty acids is also 50% of the wild-type FAS, demonstrating that an individual center for fatty acid synthesis has the same activity when paired with either a functional or nonfunctional catalytic center.

Alteration of the substrate specificity of the malonyl-CoA/acetyl-CoA:acyl carrier protein S-acyltransferase domain of the multifunctional fatty acid synthase by mutation of a single arginine residue.

The structural basis for the dual specificity of the malonyl-CoA/acetyl-CoA:acyl carrier protein S-acyltransferase associated with the multifunctional animal fatty acid synthase has been investigated by mutagenesis. Arginine 606, which is positionally conserved in the transacylase domains of all multifunctional fatty acid and polyketide synthases, was replaced by alanine or lysine in the context of the isolated transacylase domain, and the mutant proteins were expressed in Escherichia coli. Malonyl transacylase activity of the Arg-606 --> Ala and Arg-606 --> Lys mutant enzymes was reduced by 100- and 10-fold, respectively. In contrast, acetyl transacylase activity was increased 6.6-fold in the Arg-606 --> Ala mutant and 1.7-fold in the Arg-606 --> Lys mutant. Kinetic studies revealed that selectivity of the enzyme for acetyl-CoA was increased >16,000-fold by the Ala mutation and 16-fold by the Lys mutation. Activity toward medium chain length acyl thioesters was also increased >3 orders of magnitude by mutation of Arg-606, so that the Ala-606 enzyme is an effective medium chain length fatty acyl transacylase. These results indicate that Arg-606 plays an important role in the binding of malonyl moieties to the transacylase domain but is not required for binding of acetyl moieties; these results are also consistent with a mechanism whereby interaction between the positively charged guanidinium group of Arg-606 and the free carboxylate anion of the malonyl moiety serves to position this substrate in the active site of the enzyme.

Regulatory elements in the first intron of the rat fatty acid synthase gene.

Sequence elements have been identified within the 1.2 kb-long first intron of the fatty acid synthase (FAS) gene that mediate both positive and negative effects on transcription. The negative regulatory element, when positioned downstream of either the FAS or simian virus 40 promoter, down-regulates the expression of a coupled reporter gene in an orientation-dependent manner. Sequences mediating this effect have been mapped, by deletion mutagenesis, to two regions approximately within nucleotides +405 to +768 and +924 to +1083. Both regions contain sequence elements that are strongly protected from DNase I digestion by nuclear extracts prepared from liver, but not by those prepared from spleen. The results of run-on assays performed with nuclei derived from tissues that express FAS at either high or low levels indicate that the different rates of transcription of the endogenous FAS gene result from differences in the extent of initiation, so it is unlikely that the negative effect is caused by transcriptional pausing in the first intron. The positive element maps to nt +292 to +297 and corresponds to an authentic binding site for upstream stimulatory factor (USF). This USF-binding element can up-regulate transcription from a heterologous promoter in a position- and orientation-independent manner. However, in the context of the entire FAS first intron, the effect of the USF-binding site is masked unless the effect of the negative elements is ablated by mutagenesis. These results suggest that the dominant negative element of the first intron may play a role in determining the tissue-specific expression of the FAS gene.

Characterization of the malonyl-/acetyltransacylase domain of the multifunctional animal fatty acid synthase by expression in Escherichia coli and refolding in vitro.

cDNAs of various lengths encoding the second domain of the multifunctional fatty acid synthase (FAS) have been expressed in Escherichia coli and the recombinant proteins refolded in vitro to catalytically active monomeric malonyl-/acetyltransacylases. FAS residues 428-487, previously thought to represent the amino terminus of the malonyl-/acetyltransacylase, can be omitted from the recombinant enzyme with no loss in catalytic activity. This shortened transacylase, consisting of FAS residues 488-809, can be repeatedly denatured and renatured in vitro with reproducibly high recovery and no loss in specific activity. When expressed as a soluble enzyme in Spodoptera frugiperda cells, this transacylase has the same specific activity as the enzyme that has been refolded in vitro. The refolded transacylase consisting of FAS residues 488-809, but not the longer enzyme consisting of residues 428-815, can be crystallized readily. These results suggest that FAS residues 428-487, previously thought to represent the amino terminus of the malonyl-/acetyltransacylase, are not required for catalysis of the transacylase reaction. This region of the FAS is less well conserved than the transacylase catalytic domain and may constitute an extended structural linker that facilitates the functional interaction between the transacylase and acyl carrier protein domains.

Expression in Escherichia coli and refolding of the malonyl-/acetyltransferase domain of the multifunctional animal fatty acid synthase.

A cDNA encoding residues 429-815 of the multifunctional rat fatty acid synthase has been expressed in Escherichia coli and the recombinant protein refolded in vitro as a catalytically active malonyl-/acetyltransferase. Kinetic properties of the refolded recombinant enzyme were indistinguishable from those of a transferase preparation derived from the natural fatty acid synthase by limited proteolysis, indicating that the transferase domain is capable of folding correctly as an independent protein. Replacement of the active site Ser-581 (full-length fatty acid synthase numbering) with alanine completely eliminated catalytic activity, whereas replacement with cysteine resulted in retention of about 1% activity. The wild type transferase was extremely susceptible to inhibition by diethyl pyrocarbonate, and protection against inhibition was afforded by both malonyl- and acetyl-CoA. Replacement of the highly conserved residue His-683 with Ala reduced activity by 99.95%, and the residual activity was relatively unaffected by diethyl pyrocarbonate. The rate of acylation of the active site serine residue was also reduced by several orders of magnitude in the His-683 --> Ala mutant. These results indicate that His-683 plays an essential role in catalysis, likely by accepting a proton from the active site serine, thus increasing its nucleophilicity.

Transcriptional regulation of the rat fatty acid synthase gene: identification and functional analysis of positive and negative effectors of basal transcription.

The gene for fatty acid synthase (FAS), which contains both GC-rich sequences and a TATA box in its promoter region, is expressed in a tissue-specific manner in response to developmental, nutritional and hormonal signals. Here we report the identification of sequence elements in the 5'-flanking region responsible for modulation of basal promoter activity. Transient transfection of H4IIE hepatoma cells and 3T3-30A5 preadipocytes with plasmids containing the chloroamphenicol acetyltransferase gene driven by FAS promoter sequences of different lengths revealed that two regions between nucleotides -249 and -30 contain elements capable of enhancing transcription. One of these positive regulatory elements was localized to nucleotides -241/-236 using DNase I footprinting, electrophoretic mobility-shift assays and mutagenesis. The sequence element is a typical GC box and the nuclear protein binding to this region appears immunochemically indistinguishable from Sp1. The second positive regulatory element, an inverted CCAAT box, was localized to nucleotides -98/-92 by electrophoretic mobility-shift assays and mutagenesis. A putative negative regulatory element, initially identified by reporter gene transfection experiments, was localized between nucleotides -319 and -301 by DNase I footprinting, electrophoretic mobility-shift assays and deletion mutagenesis; this region consists of 78% G residues. In conclusion, initiation of FAS transcription from a single start site is enhanced by the presence of an adjacent TATA motif, an inverted CCAAT box and an upstream binding site for the transcription factor Sp1; further modulation of transcription is achieved through complex interactions between these promoter elements and an upstream negative regulatory element.

Identification of an inverted CCAAT box motif in the fatty-acid synthase gene as an essential element for modification of transcriptional regulation by cAMP.

The antagonistic effect of cAMP on the insulin-induced expression of fatty acid synthase (FAS) in liver could be mimicked in vitro using H4IIE hepatoma cells, both by measuring the response of the endogenous FAS gene and by assaying expression of transfected reporter genes containing promoter elements of the FAS gene. 5'-Deletion analysis and replacement mutagenesis revealed that an essential element required for cAMP antagonism of the insulin effect is an inverted CCAAT box located between nucleotides -99 and -92. DNase I foot-printing and gel shift analysis revealed that this region can bind a protein present in nuclei of liver and spleen, organs that express high and undetectable levels of FAS, respectively. This protein is not a CCAAT/enhancerbinding protein, C/EBP. Thus, the FAS gene appears unusual in that the sequence element required for transcriptional regulation by cAMP is neither a cAMP response element (CRE) nor a binding site for AP-1, AP-2, or C/EBP. These results suggest that essential to the regulation of FAS transcription by cAMP is the interaction of an inverted CCAAT box motif with a constitutively produced trans-acting factor that either itself undergoes modification in response to cAMP or associated with a protein that is produced or modified by cAMP exposure.

Isolation of a functional transferase component from the rat fatty acid synthase by limited trypsinization of the subunit monomer. Formation of a stable functional complex between transferase and acyl carrier protein domains.

Limited trypsinization of rat fatty acid synthase monomers results in cleavage at sites protected in the native dimer. A 47,000-Da polypeptide containing the transferase component was isolated from the digest and its location in the multifunctional polypeptide established. Both acetyl and malonyl moieties are transferred stoichiometrically from CoA ester to this polypeptide and each can replace the other, confirming that a single common site is utilized in the loading of these substrates onto the fatty acid synthase. Transferase activity of the 47,000-Da polypeptide decreases with increasing acyl donor chain length (malonyl = acetyl greater than butyryl greater than hexanoyl greater than octanoyl). Activity is inhibited by certain thiol-directed reagents, and protection is afforded by substrate suggesting the presence of a sensitive cysteine residue near the substrate binding site. The transferase was also able to utilize as acyl acceptor the Escherichia coli acyl carrier protein and the acyl carrier protein domain of the multifunctional fatty acid synthase. When the fatty acid synthase monomer was trypsinized under milder conditions, the 47,000-Da transferase domain could be isolated in association with the 8,000-Da acyl carrier protein domain. The transferase was capable of translocating substrate moieties from CoA ester donors to the associated acyl carrier protein. The results provide the first direct evidence that, in the head-to-tail oriented fatty acid synthase homodimer, functional communication between the transferase domain located near the end of one polypeptide and the acyl carrier protein domain located at the opposite end of the other polypeptide is facilitated by a stable physical interaction between these domains.

Structural organization of the multifunctional animal fatty-acid synthase.

The amino acid sequence of the multifunctional fatty-acid synthase has been examined to investigate the exact location of the seven functional domains. Good agreement in predicting the location of interdomain boundaries was obtained using three independent methods. First, the sites of limited proteolytic attack that give rise to relatively stable, large polypeptide fragments were identified; cryptic sites for protease attack at the subunit interface were unmasked by first dissociating the dimer into its component subunits. Second, polypeptide regions exhibiting higher-than-average rates of non-conservative mutation were identified. Third, the sizes of putative functional domains were compared with those of related monofunctional proteins that exhibit similar primary or secondary structure. Residues 1-406 were assigned to the oxoacyl synthase, residues 430-802 to the malonyl/acetyl transferase, residues 1630-1850 to the enoyl reductase, residues 1870-2100 to the oxyreductase, residues 2114-2190 to the acyl-carrier protein and residues 2200-2505 to the thioesterase. The 47-kDa transferase and 8-kDa acyl-carrier-protein domains, which are situated at opposite ends of the multifunctional subunit, were nevertheless isolated from tryptic digests as a non-covalently associated complex. Furthermore, a centrally located domain encompassing residues 1160-1545 was isolated as a nicked dimer. These findings, indicating that interactions between the head-to-tail juxtaposed subunits occur in both the polar and equatorial regions, are consistent with previously derived electron-micrograph images that show subunit contacts in these areas. The data permit refinement of the model for the fatty-acid synthase dimer and suggest that the malonyl/acetyl transferase and oxoacyl synthase of one subunit cooperate with the reductases, acyl carrier protein and thioesterase of the companion subunit in the formation of a center for fatty-acid synthesis.

Purification and biochemical characterization of hepatocyte nuclear factor 2 involved in liver-specific transcription of the human alpha 1-antitrypsin gene.

The upstream region of the human alpha 1-antitrypsin gene binds liver-specific proteins. Two of these proteins, hepatocyte nuclear factors 1 and 2, are essential for liver-specific transcription of this gene. We report for the first time the purification of hepatocyte nuclear factor 2 from rat liver nuclei. This protein, purified to apparent homogeneity by DNA sequence-specific affinity chromatography, appeared on sodium dodecyl sulfate-polyacrylamide gels as a single polypeptide band of molecular mass of 68 kDa. The polypeptide after extraction from the gel matrix and subsequent renaturation bound specifically to the recognition sequences (-88 to -125) and protected the same region of the promoter against DNase I digestion.