Impact of Hydrocodone Rescheduling on Dental Prescribing of Opioids.

INTRODUCTION: In the United States, dentists frequently prescribe hydrocodone. In October 2014, the US Drug Enforcement Administration rescheduled hydrocodone from controlled substance schedule III to II, introducing more restricted prescribing and dispensing regulations, which may have changed dental prescribing of opioids. OBJECTIVE: The study aim was to evaluate the impact of the hydrocodone rescheduling on dental prescribing of opioids in the United States. METHODS: This was a cross-sectional study of opioids prescribed by dentists between October 2012 and October 2016, using the IQVIA Longitudinal Prescription Dataset. Monthly dentist-based opioid prescribing rate (opioid prescription [Rx]/1,000 dentists) and monthly average opioid dosages per prescription (mean morphine milligram equivalent per day [MME/d]) were measured in the 24 mo before and after hydrocodone rescheduling in October 2014 (index or interruption). An interrupted time-series analysis was conducted using segmented ordinary least square regression models, with Newey-West standard errors to handle autocorrelation. RESULTS: Dentists prescribed 50,412,942 opioid prescriptions across the 49 mo. Hydrocodone was the most commonly prescribed opioid pre- and postindex (74.9% and 63.8%, respectively), followed by codeine (13.8% and 21.6%), oxycodone (8.1% and 9.5%), and tramadol (2.9% and 4.8%). At index, hydrocodone prescribing immediately decreased by -834.8 Rx/1,000 dentists (95% confidence interval [CI], -1,040.2 to -629.4), with increased prescribing of codeine (421.9; 95% CI, 369.7-474.0), oxycodone (85.3; 95% CI, 45.4-125.2), and tramadol (111.8; 95% CI, 101.4-122.3). The mean MME increased at index for all opioids except for hydrocodone, and dosages subsequently decreased during the postindex period. CONCLUSION: Following the rescheduling, dentist prescribing of hydrocodone declined while prescribing of nonhydrocodone opioids increased. Understanding the impact of this regulation informs strategies to ensure appropriate prescribing of opioids for dental pain. KNOWLEDGE TRANSFER STATEMENT: The study findings can be used by policy makers to make informed decisions in developing future risk mitigation strategies aimed to regulate opioid prescribing behaviors. Furthermore, dentist-specific resources and guidelines are needed subsequent to these policies in order to meet the dental population needs.

Risk factors for community-associated multidrug-resistant Pseudomonas aeruginosa in veterans with spinal cord injury and disorder: a retrospective cohort study.

STUDY DESIGN: Retrospective cohort studyObjectives:To identify independent risk factors associated with community-associated multidrug-resistant Psedomonas aeruginosa (MDRPA) in a population of veterans with spinal cord injury and disorders (SCI/D). SETTING: A total of 127 Veterans Affairs healthcare facilities. METHODS: Laboratory results from 1 January 2012 to 31 December 2013 were collected, and MDRPA cultures were compared with non-MDRPA cultures. RESULTS: One thousand four hundred forty-one cultures were collected from Veterans with SCI/D, including 227 cultures with MDRPA isolates. Characteristics associated with an increased odds of MDRPA include age 50-64 (adjusted odds ratio (aOR)=1.80, 95% confidence interval (CI)=1.13-2.87), MDRPA culture in the past 365 days (aOR=9.12, 95% CI=5.88-14.15) and carbapenem exposure in the past 90 days (aOR=2.56, 95% CI=1.35-4.87). In contrast, paraplegia was associated with a 53% decreased odds of MDRPA compared with those with tetraplegia (aOR=0.47, 95% CI=0.32-0.69). CONCLUSIONS: Risk factors for community-associated MDRPA include prior history of MDRPA and exposure to carbapenems. Awareness of these factors is important for targeted prevention and treatment of MDRPA in patients with SCI/D.

Probiotics and prevention of Clostridium difficile infection.

The role of probiotics as adjunctive measures in the prevention of Clostridium difficile infection (CDI) has been controversial. However, a growing body of evidence has suggested that they have a role in primary prevention of CDI. Elements of this controversy are reviewed and the proposed mechanisms of action, the value and cost effectiveness of probiotics are addressed with a focus on three agents, Saccharomyces boulardii, Lactobacillus rhamnosus GG and the combination of Lactobacillus acidophilus CL1285, Lactobacillus casei LBC80R, Lactobacillus rhamnosus CLR2 (Bio-K+).

Infections in the spinal cord-injured population: a systematic review.

STUDY DESIGN: Spinal cord injury (SCI) patients are an increasing population due to recent military conflicts. SCI patients are at an increased risk of infection, but the epidemiology management and prevention strategies for these infections are unclear. OBJECTIVE: To review the incidence, microbiology and management of pneumonia, skin and soft tissue infections (SSTI), urinary tract infections (UTI) and bloodstream infections in the SCI population via literature review. METHODS: With the assistance of an experienced medical librarian, we developed a search strategy for the Ovid MEDLINE database and then adapted it for the Ovid Embase, Scopus and Web of Science databases. The databases were searched from their inception to April 2014 with no restrictions on language or time period. Data were extracted using a standardized form. All studies were reviewed by two independent investigators. RESULTS: Forty-one studies reporting on the described infections were identified. UTIs were the most commonly identified infections, but studies failed to identify consistently effective preventive strategies. SSTIs were also common, and the best preventive strategies focused on decubitus ulcer prevention and skin decolonization protocols. Pneumonia management and course were not significantly different from the general population. Bloodstream infections were associated with delays in recognition, and were most often secondary to UTI, pneumonia or SSTI. CONCLUSION: There is a paucity of literature on consistently effective infection prevention strategies in SCI patients. Identification and implementation of evidence-based interventions that optimize prevention and management of infections in this patient population are needed.

Bacterial susceptibility patterns in patients with spinal cord injury and disorder (SCI/D): an opportunity for customized stewardship tools.

STUDY DESIGN: Retrospective observational study of bacterial susceptibilities in Veterans with SCI/D as compared to a general patient population. OBJECTIVES: The purpose of this project was to evaluate the prevalence and susceptibility of bacteria isolated from spinal cord injury and disorder (SCI/D) patients as compared with a general patient population and determine whether a SCI/D-specific antibiogram, a report of bacterial susceptibilities used to guide empiric antibiotic selection, would be a useful stewardship tool. SETTING: Veterans Affairs Medical Center located in Cook county, IL, USA. METHODS: Microbiology reports from 1 October 2012 to 30 September 2013 were compiled into a SCI/D-specific antibiogram and compared to a non-SCI/D antibiogram. RESULTS: Persons with positive cultures and SCI/D were younger and had a higher Charlson Index as compared to non-SCI/D patients (P<0.0001 for both). Five thousand one hundred and thirty-one unique isolate cultures were evaluated (SCI/D=23.0%). Frequencies of pathogens isolated in SCI/D and non-SCI/D differed. Methicillin-resistant Staphylococcus aureus occurred more frequently in SCI/D (27.8% vs 55.4%; P<0.0001). Gram-negatives had generally lower susceptibilities in SCI/D and a higher frequency of organisms producing extended-spectrum Beta-lactamases (17.6% vs 5.0%; P<0.0001), carbapenem-resistant Enterobacteriaceae (2.4% vs 0.5%; P<0.0001), carbapenem resistance (7.6% vs 2.4%; P<0.0001) and isolates resistant to ⩾3 antibiotic classes (60.7% vs 28.0%; P=0.0001). CONCLUSION: Different pathogens with poorer susceptibilities are isolated in SCI/D. Thus an SCI/D-specific antibiogram reflective of resistance patterns in these patients may increase the appropriateness of empiric antibiotic selection. The frequency of multi-drug resistant organisms in cultures obtained from patients with SCI/D is worrisome.

Implementing a declination form programme to improve influenza vaccine uptake by staff in Department of Veterans Affairs spinal cord injury centres: a pilot study.

BACKGROUND: Individuals with spinal cord injuries and disorders are at high risk for respiratory and influenza-related complications after developing influenza. These individuals often have frequent contact with the healthcare system. Vaccination rates in healthcare workers at Department of Veterans Affairs (VA) spinal cord injury (SCI) centres have been approximately 50% for several years. Efforts are needed to increase vaccination uptake among SCI HCWs. Declination form programmes (DFPs) in combination with other strategies have resulted in significant increases in influenza vaccination uptake in HCWs. AIM: Use of external and internal facilitation including local teams and consensus processes to pilot a DFP in two VA SCI centres and evaluate factors influencing implementation. METHODS: Implementation meetings and a consensus-building process with leadership and implementation team members were conducted, along with semi-structured post-implementation interviews with members of each implementation team (N = 7). FINDINGS: The DFP was well accepted and easy to use. Leadership was a key facilitator for DFP implementation. Barriers included difficulty communicating with HCWs working during early/late shifts. Participation was 100% at Site 1 and 48% at Site 2. CONCLUSION: Use of local teams and consensus to identify strategies to implement a DFP is feasible and effective for achieving moderate-to-high levels of participation in the programme.

Hypertension and antihypertensive treatment in veterans with spinal cord injury and disorders.

OBJECTIVES: Hypertension (HTN) is an important risk factor for cardiovascular disease, a major cause of morbidity and mortality among people with spinal cord injury and disorders (SCI/D). Our study examined prevalence, associated factors, and pharmacological treatment of HTN in Veterans with SCI/D compared with a matched control group. METHODS: A retrospective review was conducted of Veterans with traumatic SCI/D (TSCI/D; n=6672), non-traumatic SCI/D (NTSCI/D; n=3566) and a matched, non-injured cohort. RESULTS: Over half of patients with TSCI/D (56.6%) had HTN, compared with 68.4% of matched controls (P<0.001). Paraplegic and tetraplegic Veterans with TSCI/D had significantly lower odds of having a HTN diagnosis compared with control (odds ratios (OR)=0.84 (0.77-0.91); OR=0.38 (0.35-0.42)). About 71.8% of patients with NTSCI/D had HTN compared with 72.3% of matched controls (P>0.05). Paraplegic and tetraplegic Veterans with NTSCI/D did not have significantly different odds of a HTN diagnosis compared with control (OR=0.92 (0.79-1.05); OR=0.85 (0.71-1.01)). Adjusted analysis indicates that Veterans with tetraplegia and HTN were less likely to receive antihypertensive therapy (TSCI/D, OR=0.62 (0.53-0.71); NTSCI/D, OR=0.81 (0.66-0.99)). CONCLUSION: HTN appears to be more prevalent in SCI/D Veterans than previously reported. TSCI/D Veterans have a significantly lower prevalence of HTN whereas NTSCI/D Veterans have a comparable prevalence of HTN to those without SCI/D. The level of injury (tetraplegia vs paraplegia) has a large impact on the prevalence of HTN in the traumatic cohort. Subsequent antihypertensive therapy is used less in both TSCI/D and NTSCI/D Veterans with tetraplegia and more in TSCI/D Veterans with paraplegia.

Bloodstream infections and setting of onset in persons with spinal cord injury and disorder.

STUDY DESIGN: A retrospective cohort study. OBJECTIVE: Health-care-associated (HCA) bloodstream infection (BSI) has been shown to be a distinct epidemiologic category in the general adult population, but few studies have examined specific patient populations. The objective of this study was to assess characteristics associated with BSI that occurred in the hospital (hospital-acquired, HA BSI), from health-care contact outside the hospital (HCA BSI) or in the community (community-acquired, CA BSI) in veterans with spinal cord injury and disorder (SCI&D). SETTING: Two United States Department of Veterans Affairs hospitals. METHODS: All patients with SCI&D with a positive blood culture admitted to study hospitals over a 7-year period (1 October 1997 to 30 September 2004). Demographics, medical characteristics and causative organisms were collected. RESULTS: Four hundred and thirteen episodes of BSI occurred in 226 patients, with a rate of 7.2 BSI episodes per 100 admissions: 267 (64.7%) were HA BSI, 110 (26.6%) were HCA BSI and 36 (8.7%) were CA BSI. Antibiotic resistance was more common in those with HA BSI (65.5%) compared with that in those with HCA (49.1%, P=0.001) and CA BSI (22.2%, P<0.0001). Methicillin resistance in Staphylococcus aureus was highly prevalent; HA BSI (84.5%), HCA BSI (60.6%) and CA BSI (33.3%). CONCLUSION: HCA BSI comprises one-quarter of all BSIs in hospitalized patients with SCI&D. Although those with HCA and CA BSI share similarities, several differences in medical characteristics and causal microorganism are noted. Treatment and management strategies for HCA and CA infections need to vary.

Management of community-acquired pneumonia in persons with spinal cord injury.

STUDY DESIGN: Retrospective case series. OBJECTIVES: Respiratory disorders are the leading cause of death in persons with spinal cord injury (SCI), but the epidemiology and medical management of pneumonia in persons with chronic SCI is not well characterized. We describe the clinical presentation of persons with SCI with community-acquired pneumonia (CAP), characterize its management and compare practice to recommendations for CAP in the general population. SETTING: Three United States Veterans Affairs Medical Centers with specialized SCI services. METHODS: Chart abstraction was performed for all persons with chronic SCI seen at participating centers for treatment of CAP during a 2-year period. Collected data included presenting signs and symptoms, laboratory and imaging results, initial antibiotic therapy, secretion mobilization techniques, in-patient vs outpatient management, length of stay, and mortality. RESULTS: In all, 41 persons with SCI received treatment for CAP during the study period. A total of 32 (78.0%) patients were admitted for treatment; two (4.8%) required intubation and mechanical ventilation. Initial antibiotic coverage met guideline recommendations for only half of inpatients and infrequently provided adequate antipseudomonal coverage. Microbiologic testing was performed on 26 cases (63.4%) and demonstrated a specific pathogen in only five cases (12.2% of total). Three cases (7.3%) died during treatment for CAP, and 16 (42.1%) of 38 CAP survivors died within a median follow-up of 3 years. CONCLUSION: The majority of chronic SCI patients who present to specialized SCI centers with CAP are admitted for treatment. Short-term mortality is comparable to CAP in the general population.

Role of citrate synthase in aldosterone-mediated sodium reabsorption.

Aldosterone and other mineralocorticoids increase citrate synthase activity in the kidney and enhance renal sodium reabsorption, but it is unclear whether the increased citrate synthase activity is involved in renal sodium transport. We used the Wistar-Furth rat, an inbred strain found to be deficient in renal citrate synthase activity, as an experimental model to investigate this issue. We confirmed that renal citrate synthase activity from adrenalectomized Wistar-Furth rats was decreased compared with that from control Wistar rats (by 28%). Similarly, urinary citrate excretion was 23% lower in Wistar-Furth rats. Subnormal citrate formation in Wistar-Furth rats could not be accounted for by differences in systemic pH or circulating potassium levels. Because renal citrate synthase activity was reduced in Wistar-Furth rats, we hypothesized that renal sodium excretory responses to mineralocorticoids would be reduced as well. Four-hour sodium excretion after intraperitoneal injection of 5 microg of aldosterone was reduced by 56% in adrenalectomized Wistar rats and by 52% in adrenalectomized Wistar-Furth rats (both P<0.01 compared with vehicle injection). Similarly, the pattern of urinary sodium excretion in response to subcutaneous injections of deoxycorticosterone acetate over a 2-week period was similar in adrenalectomized Wistar and Wistar-Furth rats. In summary, acute and chronic antinatriuretic responses to mineralocorticoids are maintained in Wistar-Furth rats at the level of Wistar rats, despite the marked reduction in citrate synthase activity. These findings are not consistent with an important role for citrate synthase activity in mineralocorticoid-mediated renal sodium transport.

Active site mutants of pig citrate synthase: effects of mutations on the enzyme catalytic and structural properties.

We examined the catalytic efficiency of 18 pig citrate synthase mutants. The residues mutated were selected according to two criteria: the conservation of that residue in all known citrate synthase sequences, and the importance of that residue in substrate-amino acid interactions suggested by the extensive crystal structure information on the enzyme and its complexes. Several changes were made at certain residues to probe the effects of size, hydrogen bonding, and charge on the kinetics of the enzyme. The mutations, as expected, affected the kcats and Kms for OAA and acetyl-CoA to varying degrees. The catalytic efficiency of each of the mutants was determined by the kcat/Km for the individual substrates, OAA and acetyl-CoA. All mutations affected kcat. There was only one mutant, Asp327 Asn, in which the Kms primarily were affected. Most mutations affected both kcat and Km and included the following: His274Gly, His274Arg, Asp375Gly, Asp375Asn, Asp375Glu, Asp375Gln, His320Gly, His320Gln, His320Asn, His320Arg, Arg401His, Gly275Val, and Gly275Ala. The mutations, Arg401Gly, Arg401Lys, His235Gln, and Asn242Glu, had smaller effects on kcat and Km. The CS mutant Arg401Lys exhibited a modestly improved kcat/Km for both substrates compared to the nonmutant enzyme. X-ray crystallographic studies at 2.7 A resolution of one of the mutants, His274Gly, have been undertaken. The mutant enzyme crystallizes in an "open" conformation essentially isomorphous to wild type. The refined model has good geometry and a crystallographic R factor of 0.187 for 11 441 reflections observed between 6.0 and 2.7 A resolution. The refined model revealed a localized relaxation of the structure to relieve strain imposed by a high-energy main and side chain conformation of His274 in the nonmutant, but otherwise the mutation does not result in major structural alterations. Preliminary electrostatic calculations provide support for the concept that the transition state in the rate-limiting step of the citrate synthase catalyzed reaction may be an "enolized" version of acetyl-CoA that is neither neutral nor fully negatively charged and that a possible role for the catalytically essential His274 is to stabilize this by charge delocalization mediated by a hydrogen bond. These results provide the basis for further studies of the effects of these changes on the several reactive intermediates, activated substrates, and transition states which may occur along the reaction coordinate for this type of Claisen enzyme.

Regulation of eicosanoid synthesis in fibroblasts from inflamed gallbladders.

Gallbladder cell cultures obtained from rabbits subjected to sham or 72 h of bile duct ligation (72 h BDL, cholecystitis model) were incubated with calcium ionophore (A23187), dibutyryl cAMP (cAMP), and phorbol 12,13-diacetate (phorbol) to determine the intracellular signal transduction mechanisms responsible for increased inflamed gallbladder eicosanoid synthesis. Incubation of sham and 72 h BDL cell cultures with A23187 or phorbol significantly increased, whereas cAMP decreased, release of 6-keto-PGF1 alpha, PGE2, thromboxane B2 (measured by enzyme immunoassay) in a dose-related manner. Seventy-two-hour BDL cell cultures contained a specific 2-fold increased level of prostacyclin synthase compared to sham cell cultures which was not altered by preincubation with A23187, phorbol or cAMP. These findings suggest that increased PGI2 release in the sham and inflamed cell cultures following A23187 and phorbol stimulation was mediated in part via the inositol triphosphate pathway and protein kinase C activation and was not associated with altered cyclooxygenase or prostacyclin synthase content.

Metabolic engineering of a non-allosteric citrate synthase in an Escherichia coli citrate synthase mutant.

This study examined the organization of the Krebs tricarboxylic acid (TCA) cycle by metabolic engineering and high-resolution 13C NMR. The oxidation of [1,2,3-13C]propionate to glutamate via the TCA cycle was measured in wild-type (WT) and a citrate synthase mutant (CS-) strain of Escherichia coli transformed with allosteric E. coli citrate synthase (ECCS) or non-allosteric pig citrate synthase (PCS). The 13C fractional enrichment in glutamate C-2, C-3, and C-4 in ECCS and PCS were similar; although quantitative differences in total citrate synthase activity and total C-4 labeling of glutamate were observed in ECCS and PCS. Allosteric ECCS cells contained 10-fold less total enzyme activity than PCS but only 50% less total labeling in glutamate C-4 and equivalent doubling times. The observed spectra were mathematically fitted using an iterative procedure (TCACALC) and yielded an acetate/succinyl-CoA flux ratio of 10 for both ECCS and PCS, a result that is in agreement with the isotopomer analyses of the 13C spectra of cells presented with [3-13C]propionate or [2-13C]propionate. The results are consistent with the presence of an allosteric citrate synthase in ECCS and a non-allosteric citrate synthase in PCS. The former maintains TCA cycle flux via alternative propionate pathways activated by positive allosteric mechanisms and the latter via elevated enzyme levels.

Catalytic strategy of citrate synthase: subunit interactions revealed as a consequence of a single amino acid change in the oxaloacetate binding site.

The active site of pig heart citrate synthase contains a histidine residue (H320) which interacts with the carbonyl oxygen of oxaloacetate and is implicated in substrate activation through carbonyl bond polarization, a major catalytic strategy of the enzyme. We report here the effects on the catalytic mechanism of changing this important residue to glycine. H320G shows modest impairment in substrate Michaelis constants [(7-16)-fold] and a large decrease in catalysis (600-fold). For the native enzyme, the chemical intermediate, citryl-CoA, is both hydrolyzed and converted back to reactants, oxaloacetate and acetyl-CoA. In the mutant, citryl-CoA is only hydrolyzed, indicating a major defect in the condensation reaction. As monitored by the carbonyl carbon's chemical shift, the extent of oxaloacetate carbonyl polarization is decreased in all binary and ternary complexes. As indicated by the lack of rapid H320G--oxaloacetate catalysis of the exchange of the methyl protons of acetyl-CoA or the pro-S-methylene proton of propionyl-CoA, the activation of acetyl-CoA is also faulty. Reflecting this defect in acetyl-CoA activation, the carboxyl chemical shift of H320G-bound carboxymethyl-CoA (a transition-state analog of the neutral enol intermediate) fails to decrease on formation of the H3020G-oxaloacetate-carboxymethyl-CoA ternary complex. Progress curves and steady-state data with H320G using citryl-CoA as substrate show unusual properties: substrate inhibition and accelerating progress curves. Either one of two models with subunit cooperativity [Monod, J., Wyman, J., & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88; Koshland, D. E., Jr., Nemethy, G., & Filmer, D. (1966) Biochemistry 5, 365] quantitatively accounts for both the initial velocity data and the individual progress curves. The concentrations of all enzyme forms and complexes are assumed to rapidly reach their equilibrium values compared to the rate of substrate turnover. The native enzyme also behaves according to models for subunit cooperativity with citryl-CoA as substrate. However, the rates of formation/dissociation and reaction of complexes are kinetically significant. Comparisons of the values of kinetic constants between the native and mutants enzymes lead us to conclude that the mutant less readily undergoes a conformation change required for efficient activation of substrates.

Increased splanchnic prostacyclin synthase and cyclooxygenase content and activity during ischemia is due to new protein synthesis.

BACKGROUND: This study examines the hypothesis that the exaggerated splanchnic release of prostacyclin is due to new synthesis of both cyclooxygenase and prostacyclin synthase (PS) in the ileum muscularis/serosa. METHODS: Sprague-Dawley rats were anesthetized and subjected to acute hemorrhage to 30 mm Hg for 30 minutes (shock) or sham shock. The superior mesenteric artery (SMA) was cannulated and removed with its end-organ intestine and perfused in vitro with Krebs-Henseleit buffer with and without cycloheximide (50 micrograms/ml) or indomethacin (20 micrograms/ml). Venous effluent was analyzed for eicosanoids by radioimmunoassay. The SMA, aorta and ileal mucosa, and muscularis/serosa were analyzed for PS and cyclooxygenase content by immunoblot analysis. RESULTS: The sham splanchnic bed released threefold more 6-keto-PGF1 alpha than prostaglandin E2 and thromboxane. Acute ischemia increased splanchnic release of 6-keto-PGF1 alpha threefold compared with sham, which was abolished by cycloheximide or indomethacin treatment. Acute ischemia increased content of PS and cyclooxygenase in the ileal muscularis/serosa twofold and PS in the aorta and SMA by 50%. CONCLUSIONS: Acute ischemia increased release of 6-keto-PGF1 alpha, which was dependent on new protein synthesis. The immunoblot data suggest that the location of the increased enzymes responsible for increased 6-keto-PGF1 alpha release is the ileal muscularis/serosa and in the aorta and SMA.

Acute cholecystitis potentiates bradykinin stimulated fibroblast prostanoid release in the rabbit.

Gallbladder explants from control rabbits and rabbits subjected to bile duct ligation (BDL) for 24 and 72 h (cholecystitis model) were placed in cell culture to determine the source for increased gallbladder prostanoid synthesis during cholecystitis. Cultures from control and 24 h BDL gallbladders grew spindle shaped fibroblasts which did not exhibit increased prostanoid synthesis. 72 h BDL gallbladder cell cultures grew large polygonal shaped cells which appeared to be 'stimulated fibroblasts' by light and electron microscopy and were associated with increased basal and bradykinin stimulated 6-keto-PGF1 alpha release and increased content of prostacyclin synthase when measured by enzyme immunoassay and protein immunoblot analysis respectively. Use of bradykinin antagonists showed that the bradykinin BK2 subtype receptor was the most prominent in the 72 h BDL cell cultures. The 'stimulated fibroblasts' were the source of bradykinin stimulated gallbladder 6-keto-PGF1 alpha synthesis in the inflamed rabbit gallbladder which was mediated by the bradykinin B2 subtype receptor.

[13C]propionate oxidation in wild-type and citrate synthase mutant Escherichia coli: evidence for multiple pathways of propionate utilization.

The metabolism of propionate was examined in wild-type Escherichia coli and cells lacking citrate synthase by high-resolution 13C n.m.r. Spectra of cell extracts from wild-type E. coli show that glutamate becomes highly enriched in 13C when 13C-enriched propionate is the sole carbon source. No glutamate labelling was detected when the tricarboxylic acid cycle was blocked either by deletion of citrate synthase or by inhibition of succinate dehydrogenase by malonate. The 13C fractional enrichment in glutamate C-2, C-3 and C-4 in wild-type cells was quantitatively and qualitatively different when [2-13C]propionate as opposed to [3-13C]propionate was supplied. Approximately equal labelling occurred in the C-2, C-3 and C-4 positions of glutamate when [3-13C]propionate was available, and multiplets due to carbon-carbon spin-spin coupling were observed. However, in cells supplied with [2-13C]propionate, very little 13C appeared in the glutamate C-4 position, and the remaining glutamate resonances all appeared as singlets. The unequal and non-identical labelling of glutamate in cells supplied with [2-13C]- as opposed to [3-13C]propionate is consistent with the utilization of propionate by E. coli via two pathways, oxidation of propionate to pyruvate and carboxylation of propionate to succinate. These intermediates are further metabolized to glutamate by the action of the tricarboxylic acid cycle. The existence of an organized tricarboxylic acid cycle is discussed as a consequence of the ability to block utilization of propionate in tricarboxylic acid-cycle-defective E. coli.

Increased gall-bladder prostanoid synthesis after bile-duct ligation in the rabbit is secondary to new enzyme formation.

Ligation of the common bile duct (BDL) in the male rabbit resulted in increased gall-bladder microsomal total cyclo-oxygenase activity with prostaglandin E2 (PGE2) and 6-oxoprostaglandin F1 alpha [6-oxo-PGF1 alpha, stable metabolite of prostaglandin I2 (PGI2; prostacyclin)] as the major prostanoids synthesized after 24 and 72 h. Kinetic analysis of gallbladder microsomal membrane fractions incubated with increasing levels of [14C]arachidonic acid indicated that BDL for 24 and 72 h did not change substrate affinity (apparent Km) but markedly increased the rate of conversion (apparent Vmax.) suggesting the presence of more total enzyme responsible for synthesis of 6-oxo-PGF1 alpha and PGE2. BDL for 24 and 72 h significantly increased gall-bladder tissue slice basal release of 6-oxo-PGF1 alpha, but not PGE2, when compared with the controls. Gall-bladder slice release of PGE2 was 3-fold less than 6-oxo-PGF1 alpha in the control gall-bladder slices. Immunoblot analysis of 72 h BDL gall-bladder microsomal membrane fractions showed a slight increase in cyclo-oxygenase content and a 5-fold increase in the content of prostacyclin synthase as compared with the control. These data suggest that the BDL-stimulated total gall-bladder cyclo-oxygenase activity was the result of an increase in the level of specific prostaglandin-synthetic enzymes, in particular prostacyclin synthase, and not from a change in enzyme affinity.

Catalytic strategy of citrate synthase: effects of amino acid changes in the acetyl-CoA binding site on transition-state analog inhibitor complexes.

Acetyl-CoA enol has been proposed as an intermediate in the citrate synthase (CS) reaction with Asp375 acting as a base, removing a proton from the methyl carbon of acetyl-CoA, and His274 acting as an acid, donating a proton to the carbonyl [Karpusas, M., Branchaud, B., & Remington, S.J. (1990) Biochemistry 29, 2213]. CS-oxaloacetate (OAA) complexes with the transition-state analog inhibitor, carboxymethyl-CoA (CMCoA), mimic those with acetyl-CoA enol. Asp375 and His274 interact intimately with the carboxyl of the bound inhibitor. While enzymes in which these residues have been changed to other amino acids have very low catalytic activity, we find that they retain their ability to form complexes with substrates and the transition-state analog inhibitor. In comparison with the value of the chemical shift of the protonated CMCoA carboxyl in acidic aqueous solutions or its value in the wild-type ternary complex, the values in the Asp375 mutants are unusually low. Model studies suggest that these low values result from complete absence of one hydrogen bond partner for the Gly mutant and distortions in the active site hydrogen bond systems for the Glu mutant. The high affinity of Asp375Gly-OAA for CMCoA suggests that the unfavorable proton uptake required to stabilize the CMCoA-OAA ternary complex of the wild-type enzyme [Kurz, L.C., Shah, S., Crane, B.R., Donald, L.J., Duckworth, H.W., & Drysdale, G.R. (1992) Biochemistry (preceding paper in this issue)] is not required by this mutant; the needed proton is most likely provided by His274. This supports the proposed role of His274 as a general acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of the citrate synthase (CS) gene to the p12-p13 bands of chromosome 5 in pigs by in situ hybridization.

Citrate synthase (CS) is a key enzyme of the Krebs tricarboxylic acid cycle. A 1.4 kb porcine CS cDNA probe was used to chromosomally localize the CS gene in pigs by in situ hybridization. Two in situ hybridization experiments were conducted. Although the first experiment indicated a distinct signal on the 5p12-p13 bands, a secondary signal was observed on the 13q24-q32 bands. Hence, a second in situ hybridization experiment was conducted at higher stringency. The results demonstrated a consistent signal on the 5p12-p13 bands, and the signal on chromosome 13 was scattered with no prominent secondary peak. The CS gene was therefore assigned to the p12-p13 bands of chromosome 5 in pigs.