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1.  Finding the Missing Link in Lovastatin Biosynthesis: LovG is the Thioesterase Required for Dihydromonacolin L Release and Lovastatin Nonaketide Synthase Turnover 
Angewandte Chemie (International ed. in English)  2013;52(25):10.1002/anie.201302406.
The cryptic thioesterase LovG is found to be responsible for product release from the lovastatin nonaketide synthase (LNKS or LovB). The same enzyme also helps improving turnover of LovB through hydrolysis of incorrectly tailored intermediates.
doi:10.1002/anie.201302406
PMCID: PMC3844545  PMID: 23653178
Lovastatin; Biosynthesis; Polyketide Synthase; Product Release; Thioesterase
2.  Assaying proline hydroxylation in recombinant collagen variants by liquid chromatography-mass spectrometry 
BMC Biotechnology  2012;12:51.
Background
The fabrication of recombinant collagen and its prescribed variants has enormous potential in tissue regeneration, cell-matrix interaction investigations, and fundamental biochemical and biophysical studies of the extracellular matrix. Recombinant expression requires proline hydroxylation, a post-translational modification which is critical for imparting stability and structure. However, these modifications are not native to typical bacterial or yeast expression systems. Furthermore, detection of low levels of 4-hydroxyproline is challenging with respect to selectivity and sensitivity.
Results
We have developed a new liquid chromatography-mass spectrometry (LC-MS) method to evaluate proline hydroxylation in recombinant collagen. This assay was tested in different Saccharomyces cerevisiae expression systems to evaluate the effect of gene ratio between prolyl-4-hydroxylase and collagen on the extent of hydroxylation. These systems used a human collagen III gene that was synthesized de novo from oligonucleotides. The LC-MS assay does not require derivatization, uses only picomoles of sample, and can measure proline hydroxylation levels in recombinant and native collagen ranging from approximately 0% to 40%. The hydroxylation values obtained by LC-MS are as accurate and as precise as those obtained with the conventional method of amino acid analysis.
Conclusions
A facile, derivatization-free LC-MS method was developed that accurately determines the percentage of proline hydroxylation in different yeast expression systems. Using this assay, we determined that systems with a higher collagen-to-hydroxylase gene copy ratio yielded a lower percentage of hydroxylation, suggesting that a specifically balanced gene ratio is required to obtain higher hydroxylation levels.
doi:10.1186/1472-6750-12-51
PMCID: PMC3443662  PMID: 22901055
Hydroxyproline; Liquid chromatography-mass spectrometry; LC-MS assay; Recombinant collagen
3.  Telomere Length Attrition, a Marker of Biological Senescence, Is Inversely Correlated with Triglycerides and Cholesterol in South Asian Males with Type 2 Diabetes Mellitus 
Experimental Diabetes Research  2012;2012:895185.
South Asians have a higher risk of type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD) than white Caucasians, for a given BMI. Premature biological ageing, assessed by reduction in telomere length (TL), may be mediated by factors resulting from altered metabolic profiles associated with obesity. We hypothesise that ethnicity and metabolic status represent detrimental factors contributing to premature biological ageing. Therefore we assessed TL in two South Asian, age and BMI-matched cohorts [T2DM (n = 142) versus non-T2DM (n = 76)] to determine the effects of BMI, gender, lipid and CVD profile on biological ageing. Genomic DNA was obtained from the UKADS cohort; biochemical and anthropometric data was collected and TL was measured by quantitative real-time PCR. Our findings indicated a gender-specific effect with reduced TL in T2DM men compared with non-T2DM men (P = 0.006). Additionally, in T2DM men, TL was inversely correlated with triglycerides and total cholesterol (r = −0.419, P < 0.01; r = −0.443, P < 0.01). In summary, TL was reduced amongst South Asian T2DM men and correlated with triglycerides and total cholesterol. This study highlights enhanced biological ageing among South Asian, T2DM men, which appears to be tracked by changes in lipids and BMI, suggesting that raised lipids and BMI may directly contribute to premature ageing.
doi:10.1155/2012/895185
PMCID: PMC3303685  PMID: 22474429
4.  A vector set for systematic metabolic engineering in Saccharomyces cerevisiae 
Yeast (Chichester, England)  2010;28(2):123-136.
A set of shuttle vectors was constructed to facilitate expression of genes for metabolic engineering in Saccharomyces cerevisiae. Selectable markers include the URA3, TRP1, MET15, LEU2-d8, HIS3 and CAN1 genes. Differential expression of genes can be achieved as each marker is available on both CEN/ARS- and 2 μ-containing plasmids. Unique restriction sites downstream of TEF1, PGK1 or HXT7-391 promoters and upstream of the CYC1 terminator allow insertion of open-reading frame cassettes for expression. Furthermore, a fragment appropriate for integration into the genome via homologous recombination can be readily generated in a polymerase chain reaction. Vector marker genes are flanked by loxP recognition sites for the CreA recombinase to allow efficient site-specific marker deletion and recycling. Expression and copy number were characterized for representative high- and low-copy vectors carrying the different marker and promoter sequences. Metabolic engineering typically requires the stable introduction of multiple genes and genomic integration is often preferred. This requires an expanded number of stable expression sites relative to standard gene expression studies. This study demonstrated the practicality of polymerase chain reaction amplification of an expression cassette and genetic marker, and subsequent replacement of endogenous retrotransposons by homologous recombination with flanking sequences. Such reporters were expressed comparably to those inserted at standard integration loci. This expands the number of available characterized integration sites and demonstrates that such sites provide a virtually inexhaustible pool of integration targets for stable expression of multiple genes. Together these vectors and expression loci will facilitate combinatorial gene expression for metabolic engineering.
doi:10.1002/yea.1824
PMCID: PMC3070743  PMID: 20936606
Saccharomyces cerevisiae; vectors; integrants; markers; Cre recombinase; chromosomal sites; metabolic engineering
5.  Visfatin Is Regulated by Rosiglitazone in Type 2 Diabetes Mellitus and Influenced by NFκB and JNK in Human Abdominal Subcutaneous Adipocytes 
PLoS ONE  2011;6(6):e20287.
Visfatin has been proposed as an insulin-mimicking adipocytokine, predominantly secreted from adipose tissue and correlated with obesity. However, recent studies suggest visfatin may act as a proinflammatory cytokine. Our studies sought to determine the significance of this adipocytokine and its potential role in the pathogenesis of T2DM. Firstly, we examined the effects of diabetic status on circulating visfatin levels, and several other adipocytokines, demonstrating that diabetic status increased visfatin*, TNF-α*** and IL-6*** compared with non-diabetic subjects (*p<0.05, **p<0.01, ***p<0.001, respectively). We then assessed the effects of an insulin sensitizer, rosiglitazone (RSG), in treatment naïve T2DM subjects, on circulating visfatin levels. Our findings showed that visfatin was reduced post-RSG treatment [vs. pre-treatment (*p<0.05)] accompanied by a reduction in HOMA-IR**, thus implicating a role for insulin in visfatin regulation. Further studies addressed the intracellular mechanisms by which visfatin may be regulated, and may exert pro-inflammatory effects, in human abdominal subcutaneous (Abd Sc) adipocytes. Following insulin (Ins) and RSG treatment, our in vitro findings highlighted that insulin (100 nM), alone, upregulated visfatin protein expression whereas, in combination with RSG (10 nM), it reduced visfatin*, IKKβ** and p-JNK1/2*. Furthermore, inhibition of JNK protein exacted a significant reduction in visfatin expression (**p<0.01), whilst NF-κB blockade increased visfatin (*p<0.05), thus identifying JNK as the more influential factor in visfatin regulation. Additional in vitro analysis on adipokines regulating visfatin showed that only Abd Sc adipocytes treated with recombinant human (rh)IL-6 increased visfatin protein (*p<0.05), whilst rh visfatin treatment, itself, had no influence on TNF-α, IL-6 or resistin secretion from Sc adipocytes. These data highlight visfatin's regulation by insulin and RSG, potentially acting through NF-κB and JNK mechanisms, with only rh IL-6 modestly affecting visfatin regulation. Taken together, these findings suggest that visfatin may represent a pro-inflammatory cytokine that is influenced by insulin/insulin sensitivity via the NF-κB and JNK pathways.
doi:10.1371/journal.pone.0020287
PMCID: PMC3111427  PMID: 21694775
6.  Complete Reconstitution of a Highly-Reducing Iterative Polyketide Synthase 
Science (New York, N.Y.)  2009;326(5952):589-592.
Highly-reducing iterative polyketide synthases are large multifunctional enzymes that make important metabolites in fungi, such as lovastatin, a cholesterol-lowering drug from Aspergillus terreus. We report efficient expression of LovB (the Lovastatin Nonaketide Synthase) from an engineered strain of Saccharomyces cerevisiae, and complete reconstitution of its catalytic function in the presence and absence of cofactors (NADPH, SAM) and its partner enzyme, the enoyl reductase LovC. The results demonstrate that LovB retains correct intermediates until completion of synthesis of dihydromonacolin L, but off-loads incorrectly processed compounds as pyrones or hydrolytic products. Experiments replacing LovC with analogous MlcG from compactin biosynthesis demonstrate a gate-keeping function for this partner enzyme. This study represents a key step in the understanding the functions and structures of this family of enzymes.
doi:10.1126/science.1175602
PMCID: PMC2875069  PMID: 19900898
7.  Elevated endotoxin levels in non-alcoholic fatty liver disease 
Background
Emerging data indicate that gut-derived endotoxin may contribute to low-grade systemic inflammation in insulin resistant states. This study aimed to examine the importance of serum endotoxin and inflammatory markers in non-alcoholic fatty liver disease (NAFLD) patients, with and without type 2 diabetes mellitus (T2DM), and to explore the effect of treatment with a lipase inhibitor, Orlistat, on their inflammatory status.
Methods
Fasted serum from 155 patients with biopsy proven NAFLD and 23 control subjects were analysed for endotoxin, soluble CD14 (sCD14), soluble tumour necrosis factor receptor II (sTNFRII) and various metabolic parameters. A subgroup of NAFLD patients were re-assessed 6 and 12 months after treatment with diet alone (n = 6) or diet plus Orlistat (n = 8).
Results
Endotoxin levels were significantly higher in patients with NAFLD compared with controls (NAFLD: 10.6(7.8, 14.8) EU/mL; controls: 3.9(3.2, 5.2) EU/mL, p < 0.001); NAFLD alone produced comparable endotoxin levels to T2DM (NAFLD: T2DM: 10.6(5.6, 14.2) EU/mL; non-diabetic: 10.6(8.5, 15.2) EU/mL), whilst a significant correlation between insulin resistance and serum endotoxin was observed (r = 0.27, p = 0.008). Both sCD14 (p < 0.01) and sTNFRII (p < 0.001) increased with severity of fibrosis. A positive correlation was also noted between sTNFRII and sCD14 in the NAFLD subjects (r = 0.29, p = 0.004).
Sub-cohort treatment with Orlistat in patients with NAFLD showed significant decreases in ALT (p = 0.006), weight (p = 0.005) and endotoxin (p = 0.004) compared with the NAFLD, non-Orlistat treated control cohort at 6 and 12 months post therapy, respectively.
Conclusions
Endotoxin levels were considerably increased in NAFLD patients, with marked increases noted in early stage fibrosis compared with controls. These results suggest elevated endotoxin may serve as an early indicator of potential liver damage, perhaps negating the need for invasive liver biopsy. As endotoxin may promote insulin resistance and inflammation, interventions aimed at reducing endotoxin levels in NAFLD patients may prove beneficial in reducing inflammatory burden.
doi:10.1186/1476-9255-7-15
PMCID: PMC2873499  PMID: 20353583
8.  Changes in endotoxin levels in T2DM subjects on anti-diabetic therapies 
Introduction
Chronic low-grade inflammation is a significant factor in the development of obesity associated diabetes. This is supported by recent studies suggesting endotoxin, derived from gut flora, may be key to the development of inflammation by stimulating the secretion of an adverse cytokine profile from adipose tissue.
Aims
The study investigated the relationship between endotoxin and various metabolic parameters of diabetic patients to determine if anti-diabetic therapies exerted a significant effect on endotoxin levels and adipocytokine profiles.
Methods
Fasting blood samples were collected from consenting Saudi Arabian patients (BMI: 30.2 ± (SD)5.6 kg/m2, n = 413), consisting of non-diabetics (ND: n = 67) and T2DM subjects (n = 346). The diabetics were divided into 5 subgroups based on their 1 year treatment regimes: diet-controlled (n = 36), metformin (n = 141), rosiglitazone (RSG: n = 22), a combined fixed dose of metformin/rosiglitazone (met/RSG n = 100) and insulin (n = 47). Lipid profiles, fasting plasma glucose, insulin, adiponectin, resistin, TNF-α, leptin, C-reactive protein (CRP) and endotoxin concentrations were determined.
Results
Regression analyses revealed significant correlations between endotoxin levels and triglycerides (R2 = 0.42; p < 0.0001); total cholesterol (R2 = 0.10; p < 0.001), glucose (R2 = 0.076; p < 0.001) and insulin (R2 = 0.032; p < 0.001) in T2DM subjects. Endotoxin showed a strong inverse correlation with HDL-cholesterol (R2 = 0.055; p < 0.001). Further, endotoxin levels were elevated in all of the treated diabetic subgroups compared with ND, with the RSG treated diabetics showing significantly lower endotoxin levels than all of the other treatment groups (ND: 4.2 ± 1.7 EU/ml, RSG: 5.6 ± 2.2 EU/ml). Both the met/RSG and RSG treated groups had significantly higher adiponectin levels than all the other groups, with the RSG group expressing the highest levels overall.
Conclusion
We conclude that sub-clinical inflammation in T2DM may, in part, be mediated by circulating endotoxin. Furthermore, that whilst the endotoxin and adipocytokine profiles of diabetic patients treated with different therapies were comparable, the RSG group demonstrated significant differences in both adiponectin and endotoxin levels. We confirm an association between endotoxin and serum insulin and triglycerides and an inverse relationship with HDL. Lower endotoxin and higher adiponectin in the groups treated with RSG may be related and indicate another mechanism for the effect of RSG on insulin sensitivity.
doi:10.1186/1475-2840-8-20
PMCID: PMC2674418  PMID: 19368716
9.  Human epicardial adipose tissue expresses a pathogenic profile of adipocytokines in patients with cardiovascular disease 
Introduction
Inflammation contributes to cardiovascular disease and is exacerbated with increased adiposity, particularly omental adiposity; however, the role of epicardial fat is poorly understood.
Methods
For these studies the expression of inflammatory markers was assessed in epicardial fat biopsies from coronary artery bypass grafting (CABG) patients using quantitative RT-PCR. Further, the effects of chronic medications, including statins, as well as peri-operative glucose, insulin and potassium infusion, on gene expression were also assessed. Circulating resistin, CRP, adiponectin and leptin levels were determined to assess inflammation.
Results
The expression of adiponectin, resistin and other adipocytokine mRNAs were comparable to that in omental fat. Epicardial CD45 expression was significantly higher than control depots (p < 0.01) indicating significant infiltration of macrophages. Statin treated patients showed significantly lower epicardial expression of IL-6 mRNA, in comparison with the control abdominal depots (p < 0.001). The serum profile of CABG patients showed significantly higher levels of both CRP (control: 1.28 ± 1.57 μg/mL vs CABG: 9.11 ± 15.7 μg/mL; p < 0.001) and resistin (control: 10.53 ± 0.81 ng/mL vs CABG: 16.8 ± 1.69 ng/mL; p < 0.01) and significantly lower levels of adiponectin (control: 29.1 ± 14.8 μg/mL vs CABG: 11.9 ± 6.0 μg/mL; p < 0.05) when compared to BMI matched controls.
Conclusion
Epicardial and omental fat exhibit a broadly comparable pathogenic mRNA profile, this may arise in part from macrophage infiltration into the epicardial fat. This study highlights that chronic inflammation occurs locally as well as systemically potentially contributing further to the pathogenesis of coronary artery disease.
doi:10.1186/1475-2840-5-1
PMCID: PMC1352345  PMID: 16412224

Results 1-9 (9)