Biochemical characterization of isocitrate dehydrogenase from Methylococcus capsulatus reveals a unique NAD+-dependent homotetrameric enzyme

The gene encoding isocitrate dehydrogenase (IDH) of Methylococcus capsulatus (McIDH) was cloned and overexpressed in Escherichia coli. The purified enzyme was NAD⁺-dependent with a thermal optimum for activity at 55–60°C and an apparent midpoint melting temperature (T _m) of 70°C. Analytical ultracentrifugation (AUC) revealed a homotetrameric state, and McIDH thus represents the first homotetrameric NAD⁺-dependent IDH that has been characterized. Based on a structural alignment of McIDH and homotetrameric homoisocitrate dehydrogenase (HDH) from Thermus thermophilus (TtHDH), we identified the clasp-like domain of McIDH as a likely site for tetramerization. McIDH showed moreover, higher sequence identity (48%) to TtHDH than to previously characterized IDHs. Putative NAD⁺-IDHs with high sequence identity (48–57%) to McIDH were however identified in a variety of bacteria showing that NAD⁺-dependent IDHs are indeed widespread within the domain, Bacteria. Phylogenetic analysis including these new sequences revealed a close relationship with eukaryal allosterically regulated NAD⁺-IDH and the subfamily III of IDH was redefined to include bacterial NAD⁺- and NADP⁺-dependent IDHs. This apparent relationship suggests that the mitochondrial genes encoding NAD⁺-IDH are derived from the McIDH-like IDHs.     

Left ventricular volume measurement in mice by conductance catheter: evaluation and optimization of calibration

The conductance catheter (CC) allows thorough evaluation of cardiac function because it simultaneously provides measurements of pressure and volume. Calibration of the volume signal remains challenging. With different calibration techniques, in vivo left ventricular volumes (V(CC)) were measured in mice (n = 52) with a Millar CC (SPR-839) and compared with MRI-derived volumes (V(MRI)). Significant correlations between V(CC) and V(MRI) [end-diastolic volume (EDV): R(2) = 0.85, P < 0.01; end-systolic volume (ESV): R(2) = 0.88, P < 0.01] were found when injection of hypertonic saline in the pulmonary artery was used to calibrate for parallel conductance and volume conversion was done by individual cylinder calibration. However, a significant underestimation was observed [EDV = -17.3 microl (-22.7 to -11.9 microl); ESV = -8.8 microl (-12.5 to -5.1 microl)]. Intravenous injection of the hypertonic saline bolus was inferior to injection into the pulmonary artery as a calibration method. Calibration with an independent measurement of stroke volume decreased the agreement with V(MRI). Correction for an increase in blood conductivity during the in vivo experiments improved estimation of EDV. The dual-frequency method for estimation of parallel conductance failed to produce V(CC) that correlated with V(MRI). We conclude that selection of the calibration procedure for the CC has significant implications for the accuracy and precision of volume estimation and pressure-volume loop-derived variables like myocardial contractility. Although V(CC) may be underestimated compared with MRI, optimized calibration techniques enable reliable volume estimation with the CC in mice.     

Small molecule ago-allosteric modulators of the human glucagon-like peptide-1 (hGLP-1) receptor

Following our previous publication describing the biological profiles, we herein describe the structure-activity relationships of a core set of quinoxalines as the hGLP-1 receptor agonists. The most potent and efficacious compounds are 6,7-dichloroquinoxalines bearing an alkyl sulfonyl group at the C-2 position and a secondary alkyl amino group at the C-3 position. These findings serve as a valuable starting point for the discovery of more drug-like small molecule agonists for the hGLP-1 receptor.     

Androgen dependent regulation of protein kinase A subunits in prostate cancer cells

Neuroendocrine (NE) cells may play a role in prostate cancer progression. Both androgen deprivation and cAMP are well known inducers of NE differentiation (NED) in the prostate. Gene-expression profiling of LNCaP cells, incubated in androgen stripped medium, showed that the Cbeta isoform of PKA is up-regulated during NE differentiation. Furthermore, by using semi-quantitative RT-PCR and immunoblotting analysis, we observed that the Cbeta splice variants are differentially regulated during this process. Whereas the Cbeta2 splice variant is down-regulated in growth arrested LNCaP cells, the Cbeta1, Cbeta3 and Cbeta4 variants, as well as the RIIbeta subunit of PKA, are induced in NE-like LNCaP cells. The opposite effect of Cbeta expression could be mimicked by androgen stimulation, implying the Cbeta gene of PKA as a putative new target gene for the androgen receptor in prostate cancer. Moreover, to investigate expression of PKA subunits during prostate cancer progression, we did immunoblotting of several prostatic cell lines and normal and tumor tissue from prostate cancer patients. Interestingly, multiple Cbeta subunits were also observed in human prostate specimens, and the Cbeta2 variant was up-regulated in tumor cells. In conclusion, it seems that the Cbeta isoforms play different roles in proliferation and differentiation and could therefore be potential markers for prostate cancer progression.     

A Statistical Approach for Estimation of Process Flow Data from Production of Chemicals of Fossil Origin (6 pp)

Goal, Scope and Background The life cycles of many products including textiles contain chemicals for which process flow data are not known or are too time consuming to collect. Although each chemical may not contribute significantly to the LCA results of the product, which might justify excluding them, but together their contribution could be significant. Similarly, rough estimates of the process flows for the production of a single chemical may be very uncertain and considered meaningless, while the estimates of the cumulative data of process flows for several chemicals may be less uncertain and be a meaningful contribution to the quality of the LCA results. There are methods for estimation of process flows for different types of products, with varying demands regarding input data and time and with varying accuracy of the results. This work contributes to the available methods, focusing on simple estimations for production of chemical substances. The goal was to create a fast method for estimation of emissions, resource and energy flows (process flows) for the production of chemicals, based on easily available data on the properties of the chemicals. The process flows investigated were limited to those normally associated with process industries and contributing most to depletion of resources, to global warming, acidification, eutrophication and photochemical ozone production, i.e. use of energy, crude oil, coal, natural gas, uranium in ore and emissions of CO2, SOx, NOx, NMVOC, methane, BOD, COD and total N. Toxic substances were excluded, since toxic emissions are substance specific and cannot be included in a generalization. Method Available data for the process flows for the production of chemicals of mainly fossil origin were correlated to properties of chemicals such as amount of carbon in the molecule, heat of formation and average number of chemical reaction steps in the production. The production procedures were found in readily available literature. Up to about six reaction steps were evaluated in the correlation study. The variations in the process flows among the chemicals studied were calculated. Results and Discussion There were weak correlations between average number of chemical reaction steps in the production and energy use, COD measured in water emissions, and SOx and NOx emissions to air. For the remaining properties of chemicals and process flows, there were only weak correlations for share of double bonding in the molecule if only molecules containing double bondings were included. Conclusions The precision in estimation of the process flows increases non-significantly when adding information on the number of reaction steps or share of double bonding for chemicals containing double bonding is added. Recommendations and Outlook It seems reasonable to start with a simple grouping method to estimate the process flows for the production of a chemical of fossil origin. Further investigations might investigate whether there is a correlation between process flows and the costs of chemicals, and further study the correlations between process flows and share of double bonding for chemicals containing double bondings.     

Clinical trials in systemic sclerosis: lessons learned and outcomes

The pathogenesis of systemic sclerosis (SSc) is complex and largely unclear. The clinical heterogeneity of the disease and its progression over a number of years makes the choice of endpoints in the design of clinical trials difficult. The overwhelming need in this disease is to diagnose it early and identify those patients who will benefit most from early, aggressive treatment that potentially can alter the clinical disease course. To achieve this, innumerable challenges must be overcome. This article reviews data from recent clinical trials and the lessons derived from retrospective observational studies, databases, and patient registries. Taken together, these observations will help to improve our understanding of the diverse clinical course of SSc and permit refinement of existing outcome measures for the design of future clinical trials, in which the likelihood of observing a positive treatment effect with the drugs at our disposal will be maximized.     

Gabapentin and postoperative pain: a qualitative and quantitative systematic review, with focus on procedure

Background

Gabapentin is an antiepileptic drug used in a variety of chronic pain conditions. Increasing numbers of randomized trials indicate that gabapentin is effective as a postoperative analgesic. This procedure-specific systematic review aims to analyse the 24-hour postoperative effect of gabapentin on acute pain in adults.

Methods

Medline, The Cochrane Library and Google Scholar were searched for double-blind randomized placebo controlled trials of gabapentin for postoperative pain relief compared with placebo, in adults undergoing a surgical procedure. Qualitative analysis of postoperative effectiveness was evaluated by assessment of significant difference (P < 0.05) in pain relief using consumption of supplemental analgesic and pain scores between study groups. Quantitative analyses of combined data from similar procedures, were performed by calculating the weighted mean difference (WMD) of 24-hour cumulated opioid requirements, and the WMD for visual analogue scale (VAS) pain, (early (6 h) and late (24 h) postoperatively), between study groups. Side-effects (nausea, vomiting, dizziness and sedation) were extracted for calculation of their relative risk (RR).

Results

Twenty-three trials with 1529 patients were included. In 12 of 16 studies with data on postoperative opioid requirement, the reported 24-hour opioid consumption was significantly reduced with gabapentin. Quantitative analysis of five trials in abdominal hysterectomy showed a significant reduction in morphine consumption (WMD – 13 mg, 95% confidence interval (CI) -19 to -8 mg), and in early pain scores at rest (WMD – 11 mm on the VAS, 95% CI -12 to -2 mm) and during activity (WMD -8 mm on the VAS; 95% CI -13 to -3 mm), favouring gabapentin. In spinal surgery, (4 trials), analyses demonstrated a significant reduction in morphine consumption (WMD of – 31 mg (95%CI – 53 to -10 mg) and pain scores, early (WMD – 17 mm on the VAS; 95 % CI -31 to -3 mm) and late (WMD -12 mm on the VAS; 95% CI -23 to -1 mm) also favouring gabapentin treatment. Nausea was improved with gabapentin in abdominal hysterectomy (RR 0.7; 95 % CI 0.5 to 0.9). Other side-effects were unaffected.

Conclusion

Perioperative use of gabapentin has a significant 24-hour opioid sparing effect and improves pain score for both abdominal hysterectomy and spinal surgery. Nausea may be reduced in abdominal hysterectomy.     

Uncoupling Lipid Metabolism from Inflammation through Fatty Acid Binding Protein-Dependent Expression of UCP2

Chronic inflammation in obese adipose tissue is linked to endoplasmic reticulum (ER) stress and systemic insulin resistance. Targeted deletion of the murine fatty acid binding protein (FABP4/aP2) uncouples obesity from inflammation although the mechanism underlying this finding has remained enigmatic. Here, we show that inhibition or deletion of FABP4/aP2 in macrophages results in increased intracellular free fatty acids (FFAs) and elevated expression of uncoupling protein 2 (UCP2) without concomitant increases in UCP1 or UCP3. Silencing of UCP2 mRNA in FABP4/aP2-deficient macrophages negated the protective effect of FABP loss and increased ER stress in response to palmitate or lipopolysaccharide (LPS). Pharmacologic inhibition of FABP4/aP2 with the FABP inhibitor HTS01037 also upregulated UCP2 and reduced expression of BiP, CHOP, and XBP-1s. Expression of native FABP4/aP2 (but not the non-fatty acid binding mutant R126Q) into FABP4/aP2 null cells reduced UCP2 expression, suggesting that the FABP-FFA equilibrium controls UCP2 expression. FABP4/aP2-deficient macrophages are resistant to LPS-induced mitochondrial dysfunction and exhibit decreased mitochondrial protein carbonylation and UCP2-dependent reduction in intracellular reactive oxygen species. These data demonstrate that FABP4/aP2 directly regulates intracellular FFA levels and indirectly controls macrophage inflammation and ER stress by regulating the expression of UCP2.