The Modified β-Ketoadipate Pathway in Rhodococcus rhodochrous N75: Enzymology of 3-Methylmuconolactone Metabolism

Rhodococcus rhodochrous N75 is able to metabolize 4-methylcatechol via a modified β-ketoadipate pathway. This organism has been shown to activate 3-methylmuconolactone by the addition of coenzyme A (CoA) prior to hydrolysis of the butenolide ring. A lactone-CoA synthetase is induced by growth of R. rhodochrous N75 on p-toluate as a sole source of carbon. The enzyme has been purified 221-fold by ammonium sulfate fractionation, hydrophobic chromatography, gel filtration, and anion-exchange chromatography. The enzyme, termed 3-methylmuconolactone-CoA synthetase, has a pH optimum of 8.0, a native M_r of 128,000, and a subunit M_r of 62,000, suggesting that the enzyme is homodimeric. The enzyme is very specific for its 3-methylmuconolactone substrate and displays little or no activity with other monoene and diene lactone analogues. Equimolar amounts of these lactone analogues brought about less than 30% (most brought about less than 15%) inhibition of the CoA synthetase reaction with its natural substrate.     

Transbilayer movement of Glc-P-dolichol and its function as a glucosyl donor: protein-mediated transport of a water-soluble analog into sealed ER vesicles from pig brain

The results described in the accompanying article support the model in which glucosylphosphoryldolichol (Glc-P-Dol) is synthesized on the cytoplasmic face of the ER, and functions as a glucosyl donor for three Glc-P-Dol:Glc0-2Man9-GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) in the lumenal compartment. In this study, the enzymatic synthesis and structural characterization by NMR and electrospray-ionization tandem mass spectrometry of a series of water-soluble beta-Glc-P-Dol analogs containing 2-4 isoprene units with either the cis - or trans - stereoconfiguration in the beta-position are described. The water- soluble analogs were (1) used to examine the stereospecificity of the Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) and (2) tested as potential substrates for a membrane protein(s) mediating the transbilayer movement of Glc-P-Dol in sealed ER vesicles from rat liver and pig brain. The Glc-P-Dol-mediated GlcTases in pig brain microsomes utilized [3H]Glc-labeled Glc-P-Dol10, Glc-P-(omega, c )Dol15, Glc-P(omega, t,t )Dol20, and Glc-P-(omega, t,c )Dol20as glucosyl donors with [3H]Glc3Man9GlcNAc2-P-P-Dol the major product labeled in vitro. A preference was exhibited for C15-20 substrates containing an internal cis -isoprene unit in the beta-position. In addition, the water-soluble analog, Glc-P-Dol10, was shown to enter the lumenal compartment of sealed microsomal vesicles from rat liver and pig brain via a protein-mediated transport system enriched in the ER. The properties of the ER transport system have been characterized. Glc- P-Dol10was not transported into or adsorbed by synthetic PC-liposomes or bovine erythrocytes. The results of these studies indicate that (1) the internal cis -isoprene units are important for the utilization of Glc-P-Dol as a glucosyl donor and (2) the transport of the water- soluble analog may provide an experimental approach to assay the hypothetical "flippase" proposed to mediate the transbilayer movement of Glc-P-Dol from the cytoplasmic face of the ER to the lumenal monolayer.      

Biological Reduction of Trichloroethene Supported by Fe(0)

An anaerobic culture reductively transformed trichloroethene (TCE) in an aqueous medium containing elemental iron as the sole electron source. The TCE disappearance rate was enhanced and the product distribution was markedly altered when the culture was present. In abiotic samples containing Fe(0) but no culture, 11 µmol TCE (equivalent to an aqueous concentration of 260 µM) disappeared over a period of 39 days, with ethene and ethane as the major reduction products. Small amounts of cis-dichloroethene (cis-DCE), 1,1-DCE, and vinyl chloride (VC) also were detected. When the culture was incubated with TCE and Fe(0), the same amount of TCE was transformed in less than 2 weeks. The major products after 39 days were VC, ethene, and ethane. VC accounted for 65% of the initial TCE and appeared to be reduced further to ethene at slow rates. The significant VC production in the culture-amended samples indicates that most TCE was transformed microbially rather than chemically. The data indicate that abiotic and biological reduction of chlorinated ethenes can be coupled to enhance treatment efficiency. The results also suggest that microbial dechlorination within and downgradient from iron walls is potentially important for evaluating the long-term performance of permeable iron barriers.     

Enhancement ofcis,cis-muconate productivity by overexpression of catechol 1,2-dioxygenase inPseudomonas putida BCM114

For enhancement ofcis,cis-muconate productivity from benzoate, catechol 1,2-dioxygenase (C12O) which catalyzes the rate-limiting step (catechol conversion tocis,cis-muconate) was cloned and expressed in recombinantPseudomonas putida BCM114. At higher benzoate concentrations (more than 15 mM),cis,cis-muconate productivity gradually decreased and unconverted catechol was accumulated up to 10 mM in the case of wildtypeP. putida BM014, whereascis,cis-muconate productivity continuously increased and catechol was completely transformed tocis,cis-muconate forP. putida BCM114. Specific C12O activity ofP. putida BCM114 was about three times higher than that ofP. putida BM014, and productivity was enhanced more than two times.     

QuantiFERON-TB Gold In-Tube Assay for Screening Arthritis Patients for Latent Tuberculosis Infection before Starting Anti-Tumor Necrosis Factor Treatment

Background

Patients undergoing anti-tumor necrosis factor (TNF) treatment are at an increased risk of reactivating a latent tuberculosis infection (LTBI). This study evaluated the effectiveness of the QuantiFERON-TB Gold In-Tube (QFT) assay for diagnosing LTBI in arthritis patients undergoing anti-TNF treatment.

Methods

We enrolled 342 consecutive patients from August 2007 to October 2013: 176 (51.5%) patients with ankylosing spondylitis and 166 (48.5%) with rheumatoid arthritis. Screening tests included tuberculin skin test (TST) and QFT assay. Positive QFT results, regardless of TST results, were considered an indicator for LTBI treatment.

Results

Bacillus Calmette-Guérin scars were found in 236 (69.0%) patients. Of 342 patients, TST and QFT were positive in 122 (35.7%) and 103 (30.1%) patients, respectively, and discordant in 101 (29.5%) patients. During a median follow-up duration of 41.7 months, five patients (1.5%) developed TB in a median of 20.8 months after initiation of anti-TNF treatment (428/100,000 person-years). TB did not occur in 62 TST+/QFT+ patients who received LTBI treatment. Of 41 TST−/QFT+ patients who received LTBI treatment, one (2.4%) developed TB 20.5 months after starting anti-TNF treatment (705/100,000 person-years). Of 60 TST+/QFT− patients who did not receive LTBI treatment, two (3.3%) developed TB 20.8 and 22.0 months after starting anti-TNF treatment (871/100,000 person-years). Of 179 TST−/QFT− patients, two (1.1%) developed TB 7.2 and 22.7 months, respectively, after initiating anti-TNF treatment (341/100,000 person-years). TB incidence rate during the follow-up period did not differ among TST−/QFT+, TST+/QFT−, and TST−/QFT− patients (P = 0.661).

Conclusion

QFT might be used instead of TST for diagnosing LTBI in patients before starting anti-TNF therapy in countries, such as Korea, where the TB prevalence is intermediate and the BCG vaccination is mandatory at birth. In the absence of a true gold standard test for LTBI, however, there is still a risk of TB development during anti-TNF treatment.     

Voxel Based Morphometry Alterations in Mal de Debarquement Syndrome

Background

Mal de debarquement syndrome (MdDS) is a disorder of chronic self-motion perception that occurs though entrainment to rhythmic background motion, such as from sea voyage, and involves the perception of low-frequency rocking that can last for months or years. The neural basis of this persistent sensory perception abnormality is not well understood.

Methods

We investigated grey matter volume differences underlying persistent MdDS by performing voxel-based morphometry on whole brain and pre-specified ROIs in 28 individuals with MdDS and comparing them to 18 age, sex, and handedness matched controls.

Results

MdDS participants exhibited greater grey matter volume in the left inferior parietal lobule, right inferior occipital gyrus (area V3v), right temporal pole, bilateral cerebellar hemispheric lobules VIII/IX and left lobule VIIa/VIIb. Grey matter volumes were lower in bilateral inferior frontal, orbitofrontal, pregenual anterior cingulate cortex (pgACC) and left superior medial gyri (t = 3.0, p<0.005_uncorr). In ROI analyses, there were no volume differences in the middle occipital gyrus (region of V5/MT) or parietal operculum 2 (region of the parietoinsular vestibular cortex). Illness duration was positively related to grey matter volume in bilateral inferior frontal gyrus/anterior insula (IFG/AI), right posterior insula, superior parietal lobule, left middle occipital gyrus (V5/MT), bilateral postcentral gyrus, anterior cerebellum, and left cerebellar hemisphere and vermian lobule IX. In contrast, illness duration was negatively related to volume in pgACC, posterior middle cingulate gyrus (MCC), left middle frontal gyrus (dorsolateral prefrontal cortex-DLPFC), and right cerebellar hemispheric lobule VIIIb (t = 3.0, p<0.005_uncorr). The most significant differences were decreased volume in the pgACC and increased volume in the left IFG/AI with longer illness duration (qFDR_corr <0.05). Concurrent medication use did not correlate with these findings or have a relationship with duration of illness. MdDS participants showed positive correlations between grey matter volume in pgACC and bilateral cerebellar lobules VIII/IX, which was not seen in controls.

Conclusions

Individuals with MdDS show brain volume differences from healthy controls as well as duration of illness dependent volume changes in (a) visual-vestibular processing areas (IPL, SPL, V3, V5/MT), (b) default mode network structures (cerebellar IX, IPL, ACC), (c) salience network structures (ACC and IFG/AI) (d) somatosensory network structures (postcentral gyrus, MCC, anterior cerebellum, cerebellar lobule VIII), and (e) a structure within the central executive network (DLPFC). The identification of these associations may enhance future investigations into how exposure to oscillating environments can modulate brain function and affect motion perception as well cognitive and affective control.     

Quantitative variability of 342 plasma proteins in a human twin population

The degree and the origins of quantitative variability of most human plasma proteins are largely unknown. Because the twin study design provides a natural opportunity to estimate the relative contribution of heritability and environment to different traits in human population, we applied here the highly accurate and reproducible SWATH mass spectrometry technique to quantify 1,904 peptides defining 342 unique plasma proteins in 232 plasma samples collected longitudinally from pairs of monozygotic and dizygotic twins at intervals of 2–7 years, and proportioned the observed total quantitative variability to its root causes, genes, and environmental and longitudinal factors. The data indicate that different proteins show vastly different patterns of abundance variability among humans and that genetic control and longitudinal variation affect protein levels and biological processes to different degrees. The data further strongly suggest that the plasma concentrations of clinical biomarkers need to be calibrated against genetic and temporal factors. Moreover, we identified 13 cis‐SNPs significantly influencing the level of specific plasma proteins. These results therefore have immediate implications for the effective design of blood‐based biomarker studies.     

Hepatic Stellate Cells Express Thymosin Beta 4 in Chronically Damaged Liver

Although the various biological roles of thymosin β4 (Tβ4) have been studied widely, the effect of Tβ4 and Tβ4-expressing cells in the liver remains unclear. Therefore, we investigated the expression and function of Tβ4 in chronically damaged livers. CCl4 was injected into male mice to induce a model of chronic liver disease. Mice were sacrificed at 6 and 10 weeks after CCl4 treatment, and the livers were collected for biochemical analysis. The activated LX-2, human hepatic stellate cell (HSC) line, were transfected with Tβ4-specific siRNA and activation markers of HSCs were examined. Compared to HepG2, higher expression of Tβ4 in RNA and protein levels was detected in the activated LX-2. In addition, Tβ4 was up-regulated in human liver with advanced liver fibrosis. The expression of Tβ4 increased during mouse HSC activation. Tβ4 was also up-regulated and Tβ4-positive cells were co-localized with α-smooth muscle actin (α-SMA) in the livers of CCl4-treated mice, whereas such cells were rarely detected in the livers of corn-oil treated mice. The suppression of Tβ4 in LX-2 cells by siRNA induced the down-regulation of HSC activation-related genes, tgf-β, α-sma, collagen, and vimentin, and up-regulation of HSC inactivation markers, ppar-γ and gfap. Immunofluorescent staining detected rare co-expressing cells with Tβ4 and α-SMA in Tβ4 siRNA-transfected cells. In addition, cytoplasmic lipid droplets were observed in Tβ4 siRNA-treated cells. These results demonstrate that activated HSCs expressed Tβ4 in chronically damaged livers, and this endogenous expression of Tβ4 influenced HSC activation, indicating that Tβ4 might contribute to liver fibrosis by regulating HSC activation.     

A Genome-Wide Association Study Uncovers a Genetic Locus Associated with Thoracic-to-Hip Ratio in Koreans

The thoracic-to-hip circumference ratio (THR) is an anthropometric marker recently described as a predictor of type 2 diabetes. In this study, we performed a genome-wide association study (GWAS) followed by confirmatory analyses to identify genetic markers associated with THR. A total of 7,240 Korean subjects (4,988 for the discovery stage and 2,252 for the confirmatory analyses) were recruited for this study, and genome-wide single nucleotide polymorphism (SNP) genotyping of the initial 4,988 individuals was performed using Affymetrix Human SNP array 5.0. Linear regression analysis was then performed to adjust for the effects of age, sex, and current diabetes medication status on the THR of the study subjects. In the initial discovery stage, there was a statistically nominal association between minor alleles of SNP markers on chromosomes 4, 8, 10, and 12, and THR changes (p < 5.0 × 10⁻⁶). The subsequent confirmatory analyses of these markers, however, only detected a significant association between two SNPs in the HECTD4 gene and decreased THRs. Notably, this association was detected in male (rs11066280: p = 1.14 × 10⁻²; rs2074356: p = 1.10 × 10⁻²), but not in female subjects. Meanwhile, the combined results from the two analyses (initial and confirmatory) indicated that minor alleles of these two intronic variants exhibited a significant genome-wide association with decreased THR in the male subjects (n = 3,155; rs11066280: effect size = −0.008624, p = 6.19 × 10⁻⁹; rs2074356: effect size = −0.008762, p = 1.89 × 10⁻⁸). Furthermore, minor alleles of these two SNPs exhibited protective effects on patients’ risks for developing type 2 diabetes. In conclusion, we have identified two genetic variations in HECTD4 that are associated with THR, particularly in men.