Directed Accumulation of Anticancer Depsipeptides by Characterization of Neoantimycins Biosynthetic Pathway and an NADPH-Dependent Reductase.

Neoantimycins (NATs) are members of antimycin-types of depsipeptides with outstanding anticancer activities. We isolated NAT-A (1) and -F (2) from the fermentation extract of Streptomyces conglobatus. The NAT biosynthetic gene cluster ( nat BGC) was identified by genome sequencing and bioinformatics analysis. nat BGC includes two nonribosomal peptide synthetase (NRPS) and one polyketide synthase (PKS) gene, and a gene cassette (10 genes), of which the encoded enzymes share high homology to the ones responsible for 3-formamidosalicylate (3-FAS) biosynthesis in the antimycin biosynthetic pathway. Heterologous expression of the partial nat BGC without the 3-FAS gene cassette in the antimycin producer, Streptomyces albus J1074, results in the production of 1 and 2, suggesting that the nat BGC indeed directs NATs biosynthesis. Targeted in-frame deletion of the reductase gene ( natE) abolished the production of 1 and 2 but accumulated two NAT derivatives, the known NAT-H (3) and a new NAT-I (4). Biochemical verification demonstrated that the recombinant NatE indeed catalyzes an NADPH-dependent reaction of 3 or 4 to 1 or 2, respectively. Compound 3 presented significantly stronger activities against eight cancer cell lines than the ones using cisplatin, the clinical chemotherapy medicine. In particular, 3 displayed 559- and 57-fold higher activity toward human melanoma and cervix epidermoid carcinoma cells, respectively, compared with cisplatin. The new derivative, 4, was 1.5- to 10.9-fold more active than cisplatin toward five cancer cell lines. The evaluation of NATs biosynthesis depicted here will pave the way to generate new NAT derivatives through rational pathway engineering.

Enzymology of Pyran Ring†A Formation in Salinomycin Biosynthesis.

Tetrahydropyran rings are a common feature of complex polyketide natural products, but much remains to be learned about the enzymology of their formation. The enzyme SalBIII from the salinomycin biosynthetic pathway resembles other polyether epoxide hydrolases/cyclases of the MonB family, but SalBIII plays no role in the conventional cascade of ring opening/closing. Mutation in the salBIII gene gave a metabolite in which ring A is not formed. Using this metabolite in vitro as a substrate analogue, SalBIII has been shown to form pyran ring A. We have determined the X-ray crystal structure of SalBIII, and structure-guided mutagenesis of putative active-site residues has identified Asp38 and Asp104 as an essential catalytic dyad. The demonstrated pyran synthase activity of SalBIII further extends the impressive catalytic versatility of α+ß barrel fold proteins.

Enzymology of Pyran Ring†A Formation in Salinomycin Biosynthesis.

Tetrahydropyran rings are a common feature of complex polyketide natural products, but much remains to be learned about the enzymology of their formation. The enzyme SalBIII from the salinomycin biosynthetic pathway resembles other polyether epoxide hydrolases/cyclases of the MonB family, but SalBIII plays no role in the conventional cascade of ring opening/closing. Mutation in the salBIII gene gave a metabolite in which ring A is not formed. Using this metabolite in vitro as a substrate analogue, SalBIII has been shown to form pyran ring A. We have determined the X-ray crystal structure of SalBIII, and structure-guided mutagenesis of putative active-site residues has identified Asp38 and Asp104 as an essential catalytic dyad. The demonstrated pyran synthase activity of SalBIII further extends the impressive catalytic versatility of α+ß barrel fold proteins.

Site-specific modification of the anticancer and antituberculosis polyether salinomycin by biosynthetic engineering.

The complex bis-spiroacetal polyether ionophore salinomycin has been identified as a uniquely selective agent against cancer stem cells and is also strikingly effective in an animal model of latent tuberculosis. The basis for these important activities is unknown. We show here that deletion of the salE gene abolishes salinomycin production and yields two new analogues, in both of which the C18C19 cis double bond is replaced by a hydroxy group stereospecifically located at C19, but which differ from each other in the configuration of the bis-spiroacetal. These results identify SalE as a novel dehydratase and demonstrate that biosynthetic engineering can be used to redirect the reaction cascade of oxidative cyclization to yield new salinomycin analogues for use in mechanism-of-action studies.

Fasting nonesterified fatty acid profiles in childhood and their relationship with adiposity, insulin sensitivity, and lipid levels.

OBJECTIVE: The objective of this study was to examine the major constituent of nonesterified fatty acids in children with respect to auxologic parameters, insulin sensitivity, and lipid levels, because nonesterified fatty acid levels are elevated in obesity and are important in the development of comorbidities. METHODS: Fasting blood samples were obtained from 73 children (43 girls; 49 obese; median [range] age: 11.4 [0.9-17.6] years). Concentrations of the major circulating nonesterified fatty acids (myristate, palmitate, oleate, stearate, and arachidate) were determined by gas chromatography mass spectrometry, alongside measurement of insulin, adiponectin, and lipid profiles. RESULTS: The sum of all nonesterified fatty acids was significantly higher in obese versus normal-weight children, although gender (but not age or puberty) was an important determinant, with the difference remaining significant only in boys. Overall, obese children had higher concentrations of myristate, palmitate, and oleate but not stearate or arachidate. Age was an important determinant of myristate and arachidate, whereas gender proved more important for palmitate and stearate. Fasting insulin concentrations were not associated with either total nonesterified fatty acid concentrations or any of the individual nonesterified fatty acids, although a positive correlation was found between adiponectin and total nonesterified fatty acid concentrations that was independent of obesity status and that seemed mediated by changes in palmitate and stearate. Serum total cholesterol and low-density lipoprotein (but not high-density lipoprotein) levels seemed to correlate positively with circulating concentrations of palmitate, oleate, and stearate, whereas serum triacylglycerols correlated with myristate, palmitate, and oleate concentrations. CONCLUSIONS: Nonesterified fatty acid concentrations are elevated in obese children, primarily as a result of increases in myristate, palmitate, and oleate. Independent effects of nonesterified fatty acids on circulating adiponectin levels and lipid parameters were observed, although we found no relationship between nonesterified fatty acid concentrations and the insulin resistance identified with obesity.

Plasma volume response to 30-s cycle ergometry: influence on lipid and lipoprotein.

PURPOSE: It has been suggested that exercise-induced changes in plasma volume (PV) confound the interpretation of biochemical data obtained during the recovery period from exercise. No studies have sought to assess the effect of short-duration, high-intensity exercise on PV change and plasma lipid and lipoprotein concentrations. The purpose of this study was to compare power profiles, changes in PV, and plasma lipid and lipoprotein concentrations immediately after and 24 h after exercise. METHODS: Subjects undertook two 30-s, high-intensity cycle ergometer protocols after optimization of resistive loads calculated from total body mass (TBM) and fat-free mass (FFM). Power output indices were recorded and blood samples were analyzed before, immediately after, and 24 h after exercise. RESULTS: Peak power outputs were significantly greater in FFM (1020+/-134 vs 953+/-114 W for FFM and TBM, respectively, P<0.05). No differences were found between TBM and FFM for mean power output, fatigue index, or work done. Significant decreases (P<0.05) in PV of 12.0+/-5.7 and 12.3+/-6.7% were recorded immediately after exercise for both TBM and FFM, respectively. At 24 h after exercise, a significant (P<0.05) increase in PV of 4.2+/-10.3% was recorded for TBM only. Significant increases (P<0.01) were recorded for serum triglyceride, cholesterol, HDL cholesterol, and LDL cholesterol immediately after exercise for both TBM and FFM. These increases disappeared when corrected for PV changes, with the exception of LDL cholesterol in TBM, which still displayed a significant increase compared with the preexercise values (2.50+/-0.74 mM (before) vs 2.72+/-0.84 mM (immediately after)). CONCLUSIONS: Our data show that short-duration, high-intensity cycle ergometer exercise tests can induce significant plasma volume decreases in untrained subjects, which may affect the interpretation of bloodborne biochemical parameters.

Delineation of large deletions of the MECP2 gene in Rett syndrome patients, including a familial case with a male proband.

Comprehensive genetic screening programs have led to the identification of pathogenic methyl-CpG-binding protein 2 (MECP2) mutations in up to 95% of classical Rett syndrome (RTT) patients. This high rate of mutation detection can partly be attributed to specialised techniques that have enabled the detection of large deletions in a substantial fraction of otherwise mutation-negative patients. These cases would normally be missed by the routine PCR-based screening strategies. Here, we have identified large multi-exonic deletions in 12/149 apparently mutation-negative RTT patients using multiplex ligation-dependent probe amplification (MLPA). These deletions were subsequently characterised using real-time quantitative PCR (qPCR) and long-range PCR with the ultimate aim of defining the exact nucleotide positions of the breakpoints and rearrangements. We detected an apparent deletion in one further patient using MLPA; however, this finding was contradicted by subsequent qPCR and long-range PCR results. The patient group includes an affected brother and sister with a large MECP2 deletion also present in their carrier mother. The X chromosome inactivation pattern of all female patients in this study was determined, which, coupled with detailed clinical information, allowed meaningful genotype-phenotype correlations to be drawn. This study reaffirms the view that large MECP2 deletions are an important cause of both classical and atypical RTT syndrome, and cautions that apparent deletions detected using high-throughput diagnostic techniques require further characterisation.

Conjugated linoleic acid impairs endothelial function.

OBJECTIVE: To determine the effect of dietary supplementation with conjugated linoleic acid (CLA) on body mass index (BMI), body fat distribution, endothelial function, and markers of cardiovascular risk. METHODS AND RESULTS: Forty healthy volunteers with BMI >27 kg/m2 were randomized to receive a CLA isomeric mixture or olive oil in a 12-week double-blind study. Subcutaneous body fat and abdominal/hepatic fat content were assessed using skin-fold thicknesses and computed tomography scanning, respectively. Endothelial function was assessed by brachial artery flow-mediated dilatation (FMD). Plasma isoprostanes were measured as an index of oxidative stress. CLA supplementation did not result in a significant change in BMI index or total body fat. There was a significant decrease in limb (-7.8 mm, P<0.001), but not torso skin-fold thicknesses or abdominal or liver fat content. Brachial artery FMD declined (-1.3%, P=0.013), and plasma F2-isoprostanes increased (+91 pg/mL, P=0.042). CONCLUSIONS: A CLA isomeric mixture had at most modest effects on adiposity and worsened endothelial function. On the basis of these results, the use of the isomeric mixture of CLA as an aid to weight loss cannot be recommended.

Fibrinogen, homocyst(e)ine, and C-reactive protein concentrations relative to sex and socioeconomic status in British young people.

This study assesses the prevalence of recently identified coronary heart disease (CHD) risk factors in young people of differing socioeconomic status (SES). From November 2001 through March 2002, 100 boys and 108 girls, of age 12.9 +/- 0.3 years, selected from differing SES were assessed for CHD risk factors. Measurements included fibrinogen (Fg), homocyst(e)ine (Hcy), and C-reactive protein (CRP). Fibrinogen was significantly greater among boys from a higher SES compared with those from a low SES (P < or = 0.05). Differences according to sex (P < or = 0.05) were identified for Fg and CRP. The data indicate the prevalence of recently identified CHD risk factors in this cohort of British schoolchildren. For the purpose of this article, the phrase "young people" embraces both children and adolescents.

Somatotype and angiographically determined atherosclerotic coronary artery disease in men.

The relationship between somatotype and somatotype components with coronary artery disease (CAD) and regional adiposity was considered in 58 males (age 60.2 +/- 9.4 years) undergoing investigative coronary angiography for suspected atherosclerotic CAD. Severity of CAD was determined in terms of both the degree of stenosis and the anatomical position of the lesions on the coronary arteries (myocardial score). Six patients had negative angiographic findings but three of these had impaired left ventricular function as determined by left ventriculography. The mean (+/-SD) somatotype of the group was 5.7 / 5.6 / 1.2 (1.7 / 1.4 / 1.0), illustrating a clear dominance of the first two somatotype components. Canonical correlation analysis showed that somatotype was not significantly related to the angiography results (P > 0.05). However, correlations between the somatotype components and the angiography results with their respective first canonical variates showed that the somatotype variate was one of high mesomorphy and low ectomorphy and the angiography variate was essentially one of a high myocardial score. After adjustment for the confounding interrelationship among the somatotype components, endomorphy was significantly correlated with abdominal circumference (r = 0.65, P < 0.001), the abdomen-to-hip ratio (r = 0.53, P < 0.001) and the abdominal sagittal diameter (r = 0.60, P < 0.001). Mesomorphy was not related to these indicators of android or abdominal adiposity following partial adjustment. Ectomorphy was inversely related to the indices of general and regional adiposity. This study suggests that adiposity and muscularity are important features in terms of increased CAD risk, whereas linearity is beneficial. Am. J. Hum. Biol. 12:128-138, 2000. Copyright 2000 Wiley-Liss, Inc.