Origins and Divergence of the Roma (Gypsies)

The identification of a growing number of novel Mendelian disorders and private mutations in the Roma (Gypsies) points to their unique genetic heritage. Linguistic evidence suggests that they are of diverse Indian origins. Their social structure within Europe resembles that of the jatis of India, where the endogamous group, often defined by profession, is the primary unit. Genetic studies have reported dramatic differences in the frequencies of mutations and neutral polymorphisms in different Romani populations. However, these studies have not resolved ambiguities regarding the origins and relatedness of Romani populations. In this study, we examine the genetic structure of 14 well-defined Romani populations. Y-chromosome and mtDNA markers of different mutability were analyzed in a total of 275 individuals. Asian Y-chromosome haplogroup VI-68, defined by a mutation at the M82 locus, was present in all 14 populations and accounted for 44.8% of Romani Y chromosomes. Asian mtDNA-haplogroup M was also identified in all Romani populations and accounted for 26.5% of female lineages in the sample. Limited diversity within these two haplogroups, measured by the variation at eight short-tandem-repeat loci for the Y chromosome, and sequencing of the HVS1 for the mtDNA are consistent with a small group of founders splitting from a single ethnic population in the Indian subcontinent. Principal-components analysis and analysis of molecular variance indicate that genetic structure in extant endogamous Romani populations has been shaped by genetic drift and differential admixture and correlates with the migrational history of the Roma in Europe. By contrast, social organization and professional group divisions appear to be the product of a more recent restitution of the caste system of India.     

Y-Chromosome evidence for a northward migration of modern humans into Eastern Asia during the last Ice Age

The timing and nature of the arrival and the subsequent expansion of modern humans into eastern Asia remains controversial. Using Y-chromosome biallelic markers, we investigated the ancient human-migration patterns in eastern Asia. Our data indicate that southern populations in eastern Asia are much more polymorphic than northern populations, which have only a subset of the southern haplotypes. This pattern indicates that the first settlement of modern humans in eastern Asia occurred in mainland Southeast Asia during the last Ice Age, coinciding with the absence of human fossils in eastern Asia, 50,000-100,000 years ago. After the initial peopling, a great northward migration extended into northern China and Siberia.     

The structure and function of the 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Haemophilus influenzae

The gene encoding the 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase of Haemophilus influenzae has been cloned and expressed in Escherichia coli. A complex of the purified protein with a substrate analog has been crystallized and its structure solved by multiple anomalous dispersion using phase information obtained from a single crystal of selenomethione-labeled protein. The enzyme folds into a four-stranded antiparallel beta-sheet flanked on one side by two alpha-helices and on the other by three consecutive alpha-helices, giving a novel beta1alpha1beta2beta3alpha2beta4alpha3alpha4alpha5 polypeptide topology. The three-dimensional structure of a binary complex has been refined at 2.1 A resolution. The location of the substrate analog and a sulfate ion gives important insight into the molecular mechanism of the enzyme.     

A three-dimensional model of endothelin-converting enzyme (ECE) based on the X-ray structure of neutral endopeptidase 24.11 (NEP).

Endothelin-converting enzyme 1 (ECE-1, EC 3.4.24.71) is a zinc-dependent type II mammalian membrane protein comprising the active site in the ectodomain. It exists in multiple splice variants that all catalyze the last and rate-limiting step in the activation of preproendothelin to the highly potent vasoconstrictor endothelin. There is high interest in finding small and potent inhibitors for this enzyme that could be used in numerous indications, e.g. hypertension. Since there is no structural information available for this important enzyme, we built a model of the complete ectodomain using the recently solved structure of human NEP as template. The naturally derived metalloproteinase inhibitor phosphoramidon was docked in the active site of this model and comparisons with the respective NEP complex were made.     

Three-dimensional structure of bovine pancreatic DNase I at 2.5 A resolution

The three-dimensional structure of bovine pancreatic deoxyribonuclease I (DNase I) has been determined at 2.5 A resolution by X-ray diffraction from single crystals. An atomic model was fitted into the electron density using a graphics display system. DNase I is an alpha, beta-protein with two 6-stranded beta-pleated sheets packed against each other forming the core of a 'sandwich'-type structure. The two predominantly anti-parallel beta-sheets are flanked by three longer alpha-helices and extensive loop regions. The carbohydrate side chain attached to Asn 18 is protruding by approximately 15 A from the otherwise compact molecule of approximate dimensions 45 A X 40 A. The binding site of CA2+-deoxythymidine-3',5'-biphosphate (Ca-pdTp) has been determined by difference Fourier techniques confirming biochemical results that the active centre is close to His 131. Ca-pdTp binds at the surface of the enzyme between the two beta-pleated sheets and seems to interact with several charged amino acid side chains. Active site geometry and folding pattern of DNase I are quite different from staphylococcal nuclease, the only other Ca2+-dependent deoxyribonuclease whose structure is known at high resolution. The electron density map indicates that two Ca2+ ions are bound to the enzyme under crystallization conditions.     

Comparison of derivatization and chromatographic methods for GC–MS analysis of amino acid enantiomers in physiological samples

GC–MS analysis of fluorinated and non-fluorinated chloroformate and anhydride derivatives of amino acid (AA) enantiomers on two different chiral columns was compared for the direct quantification of free l- and d-AAs in human serum and urine in a single analytical run. Best sensitivity was achieved with pentafluoropropionic anhydride/heptafluorobutanol derivatives separated on a Chirasil-l-Val column. However, the occurrence of racemization during derivatization precluded accurate quantification of AA enantiomers. Derivatization with methyl chloroformate/methanol and separation on an Rt-γDEXsa column did not exhibit racemization and yielded ten baseline separated racemates of proteinogenic AAs with resolution values greater than 2.4. However, protein and peptide hydrolysis occurred in serum and urine during the highly exothermal derivatization reaction under alkaline conditions. Removing serum proteins by precipitation before derivatization and performing the reaction at neutral pH enabled the determination of accurate free AA enantiomer concentrations. Accuracy of quantification was validated by an established nonchiral GC–MS method for AA analysis. Reliable quantification was achieved using stable-isotope labeled l-AAs as internal standards. Limits of detection (LOD) and lower limits of quantification (LLOQ) for the d-AAs were in the range of 3.2–446 nM and 0.031–1.95 μM, respectively. Relative standard deviations (N = 6) for the measurement of AAs in urine and serum ranged from 0.49–11.10% to 0.70–3.87%, respectively. The method was applied to the analysis of urine from 19 patients with renal insufficiency. In comparison to healthy probands, D-ratios of Ala, Val, Pro, Thr, Asp, and Asn were significantly increased.     

New Aspects of an Old Drug – Diclofenac Targets MYC and Glucose Metabolism in Tumor Cells

Non-steroidal anti-inflammatory drugs such as diclofenac exhibit potent anticancer effects. Up to now these effects were mainly attributed to its classical role as COX-inhibitor. Here we show novel COX-independent effects of diclofenac. Diclofenac significantly diminished MYC expression and modulated glucose metabolism resulting in impaired melanoma, leukemia, and carcinoma cell line proliferation in vitro and reduced melanoma growth in vivo. In contrast, the non-selective COX inhibitor aspirin and the COX-2 specific inhibitor NS-398 had no effect on MYC expression and glucose metabolism. Diclofenac significantly decreased glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), and monocarboxylate transporter 1 (MCT1) gene expression in line with a decrease in glucose uptake and lactate secretion. A significant intracellular accumulation of lactate by diclofenac preceded the observed effect on gene expression, suggesting a direct inhibitory effect of diclofenac on lactate efflux. While intracellular lactate accumulation impairs cellular proliferation and gene expression, it does not inhibit MYC expression as evidenced by the lack of MYC regulation by the MCT inhibitor α-cyano-4-hydroxycinnamic acid. Finally, in a cell line with a tetracycline-regulated c-MYC gene, diclofenac decreased proliferation both in the presence and absence of c-MYC. Thus, diclofenac targets tumor cell proliferation via two mechanisms, that is inhibition of MYC and lactate transport. Based on these results, diclofenac holds potential as a clinically applicable MYC and glycolysis inhibitor supporting established tumor therapies.     

Glucocorticoid-induced alterations in mitochondrial membrane properties and respiration in childhood acute lymphoblastic leukemia

Mitochondria are signal-integrating organelles involved in cell death induction. Mitochondrial alterations and reduction in energy metabolism have been previously reported in the context of glucocorticoid (GC)-triggered apoptosis, although the mechanism is not yet clarified. We analyzed mitochondrial function in a GC-sensitive precursor B-cell acute lymphoblastic leukemia (ALL) model as well as in GC-sensitive and GC-resistant T-ALL model systems. Respiratory activity was preserved in intact GC-sensitive cells up to 24h under treatment with 100 nM dexamethasone before depression of mitochondrial respiration occurred. Severe repression of mitochondrial respiratory function was observed after permeabilization of the cell membrane and provision of exogenous substrates. Several mitochondrial metabolite and protein transporters and two subunits of the ATP synthase were downregulated in the T-ALL and in the precursor B-ALL model at the gene expression level under dexamethasone treatment. These data could partly be confirmed in ALL lymphoblasts from patients, dependent on the molecular abnormality in the ALL cells. GC-resistant cell lines did not show any of these defects after dexamethasone treatment. In conclusion, in GC-sensitive ALL cells, dexamethasone induces changes in membrane properties that together with the reduced expression of mitochondrial transporters of substrates and proteins may lead to repressed mitochondrial respiratory activity and lower ATP levels that contribute to GC-induced apoptosis.     

Discrimination of steatosis and NASH in mice using nuclear magnetic resonance spectroscopy

Nonalcoholic fatty liver disease (NAFLD) is a common cause of hepatic dysfunction. The disease spectrum ranges from hepatic steatosis to nonalcoholic steatohepatitis (NASH). The aim of this study was to identify metabolic differences in murine models of simple hepatic steatosis and NASH for the distinction of these NAFLD stages. For 12 weeks, male BALB/c mice were fed either a control or two different high-fat diets leading to hepatic steatosis and NASH, respectively. Metabolic differences were determined by independent component analysis (ICA) of nuclear magnetic resonance (NMR) spectra of lipophilic and hydrophilic liver extracts, and urine specimens. The results from ICA clearly discriminated the three investigated groups. Discriminatory biomarkers in the lipophilic liver extracts were free cholesterol, cholesterol ester and lipid methylene. Discrimination of the hydrophilic liver extracts was mainly mediated by betaine, glucose, and lactate, whereas in urine taurine, trimethylamine-N-oxide, and trimethylamine were the most discriminatory biomarkers. In conclusion, NMR metabolite fingerprinting of spot urine specimens may allow the noninvasive distinction of steatosis and NASH.