Metabolic syndrome in an island population of the eastern Adriatic coast of Croatia

The metabolic syndrome, an assemblage of risk factors, viz., obesity, dyslipidemia, carbohydrate intolerance, and hypertension, associated with development of cardiovascular diseases and Type 2 diabetes, has become a major public health problem in the developed countries. However, data on its prevalence in worldwide populations, particularly in non-industrialized societies is sparse. We report the prevalence of metabolic syndrome in an island population of the eastern Adriatic coast of Croatia, a distinctly homogenous population living in relative isolation with a traditional way of life style pattern. The participants consist of 381 unrelated individuals (136 males, 245 females) from the island of Hvar, age 19 and above. Hvar is among the most populated Croatian islands with a total population of 11,459 individuals according to the 1991 census. Metabolic syndrome was assessed following the National Cholesterol Education Program (NCEP) criteria, with the exception of using body mass index and waist hip ratio as the predictors of obesity. Overall age-adjusted prevalence of metabolic syndrome is 26% (32% in males and 24% in females) with body mass index, and 42% (57% in males and 36% in females) with waist hip ratio as the measures of obesity. Pairwise correlations of the age and sex-adjusted individual components show that with the exception of fasting plasma glucose, the other components of metabolic syndrome are significantly associated with each other, suggesting their co-morbidity. In spite of adherence to a relatively traditional life-style pattern together with a "Mediterranean diet" and rural habitat, the prevalence of metabolic syndrome is substantially high in the population of Hvar. It is likely that factors other than nutritional practices, that might include genetic susceptibility, could potentially be important in predisposition to metabolic syndrome.     

An efficient <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated transformation protocol of <Emphasis Type="Italic">Withania somnifera</Emphasis>

This is the first report on Agrobacterium rhizogenes-mediated transformation of Withania somnifera for expression of a foreign gene in hairy roots. We transformed leaf and shoot tip explants using binary vector having gusA as a reporter gene and nptII as a selectable marker gene. To improve the transformation efficiency, acetosyringone (AS) was added in three stages, Agrobacterium liquid culture, Agrobacterium infection and co-culture of explants with Agrobacterium. The addition of 75 μM AS to Agrobacterium liquid culture was found to be optimum for induction of vir genes. Moreover, the gusA gene expression in hairy roots was found to be best when the leaves and shoot tips were sonicated for 10 and 20s, respectively. Based on transformation efficiency, the Agrobacterium infection for 60 and 120 min was found to be suitable for leaves and shoot tips, respectively. Amongst the various culture media tested, MS basal medium was found to be best in hairy roots. The transformation efficiency of the improved protocol was recorded 66.5 and 59.5?% in the case of leaf and shoot tip explants, respectively. When compared with other protocols the transformation efficiency of this improved protocol was found to be 2.5 fold higher for leaves and 3.7 fold more for shoot tips. Southern blot analyses confirmed 1–2 copies of the gusA transgene in the lines W1-W4, while 1–4 transgene copies were detected in the line W5 generated by the improved protocol. Thus, we have established a robust and efficient A. rhizogenes mediated expression of transgene (s) in hairy roots of W. somnifera.     

Type 2 Diabetes and Three Calpain-10 Gene Polymorphisms in Samoans: No Evidence of Association

Although genomewide scans have identified several potential chromosomal susceptibility regions in several human populations, finding a causative gene for type 2 diabetes has remained elusive. Others have reported a novel gene, calpain-10 (CAPN10), located in a previously identified region on chromosome 2q37.3, as a putative susceptibility gene for type 2 diabetes. Three single-nucleotide polymorphisms (SNPs) (UCSNP43, UCSNP19, and UCSNP63) were shown to be involved in increased risk of the disease among Mexican Americans. We have tested the association of these three SNPs with type 2 diabetes among the Samoans of Polynesia, who have a very high prevalence of the disease. In the U.S. territory of American Samoa, prevalence is 25% and 15% in men and women, respectively, whereas, in the independent nation of Samoa, prevalence is 3% and 5% in men and women, respectively. In our study sample, which consisted of 172 unrelated affected case subjects and 96 control subjects, we failed to detect any association between case subjects and control subjects in allele frequencies, haplotype frequencies, or haplotype combinations of UCSNP43, -19, and -63. Also, our data showed no evidence of linkage, among 201 affected sib pairs, in the region of chromosome 2 that contains these SNPs. Three plausible scenarios could explain these observations. (1) CAPN10 is a susceptibility gene only in particular ethnic groups; (2) our study lacks power to detect the effects of CAPN10 polymorphisms (but our sample size is comparable to that of earlier reports); or (3) the underlying biological mechanism is too complex and requires further research.     

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.     

Genetic variation at twentythree microsatellite loci in sixteen human populations

We have analysed genetic variation at 23 microsatellite loci in a global sample of 16 ethnically and geographically diverse human populations. On the basis of their ancestral heritage and geographic locations, the studied populations can be divided into five major groups, viz. African, Caucasian, Asian Mongoloid, American Indian and Pacific Islander. With respect to the distribution of alleles at the 23 loci, large variability exists among the examined populations. However, with the exception of the American Indians and the Pacific Islanders, populations within a continental group show a greater degree of similarity. Phylogenetic analyses based on allele frequencies at the examined loci show that the first split of the present-day human populations had occurred between the Africans and all of the non-African populations, lending support to an African origin of modern human populations. Gene diversity analyses show that the coefficient of gene diversity estimated from the 23 loci is, in general, larger for populations that have remained isolated and probably of smaller effective sizes, such as the American Indians and the Pacific Islanders. These analyses also demonstrate that the component of total gene diversity, which is attributed to variation between groups of populations, is significantly larger than that among populations within each group. The empirical data presented in this work and their analyses reaffirm that evolutionary histories and the extent of genetic variation among human populations can be studied using microsatellite loci.     

Rate and directionality of mutations and effects of allele size constraints at anonymous, gene-associated, and disease-causing trinucleotide loci.

We studied the patterns of within- and between-population variation at 29 trinucleotide loci in a random sample of 200 healthy individuals from four diverse populations: Germans, Nigerians, Chinese, and New Guinea highlanders. The loci were grouped as disease-causing (seven loci with CAG repeats), gene-associated (seven loci with CAG/CCG repeats and eight loci with AAT repeats), or anonymous (seven loci with AAT repeats). We used heterozygosity and variance of allele size (expressed in units of repeat counts) as measures of within-population variability and GST (based on heterozygosity as well as on allele size variance) as the measure of genetic differentiation between populations. Our observations are: (1) locus type is the major significant factor for differences in within-population genetic variability; (2) the disease-causing CAG repeats (in the nondisease range of repeat counts) have the highest within-population variation, followed by the AAT-repeat anonymous loci, the AAT-repeat gene-associated loci, and the CAG/CTG-repeat gene-associated loci; (3) an imbalance index beta, the ratio of the estimates of the product of effective population size and mutation rate based on allele size variance and heterozygosity, is the largest for disease-causing loci, followed by AAT- and CAG/CCG-repeat gene-associated loci and AAT-repeat anonymous loci; (4) mean allele size correlates positively with allele size variance for AAT- and CAG/CCG-repeat gene-associated loci and negatively for anonymous loci; and (5) GST is highest for the disease-causing loci. These observations are explained by specific differences of rates and patterns of mutations in these four groups of trinucleotide loci, taking into consideration the effects of the past demographic history of the modern human population.     

Identification of the essential histidine residue at the active site of Escherichia coli dehydroquinase.

The shikimate pathway enzyme 3-dehydroquinase is very susceptible to inactivation by the group-specific reagent diethyl pyrocarbonate (DEP). Inactivation follows pseudo first-order kinetics and exhibits a second-order rate constant of 148.5 M-1 min-1. An equilibrium mixture of substrate and product substantially protects against inactivation by DEP, suggesting that residues within the active site are being modified. Complete inactivation of the enzyme correlates with the modification of 6 histidine residues/subunit as determined by difference spectroscopy at 240 nm. Enzymic activity can be restored by hydroxylamine treatment, which is also consistent with the modification occurring at histidine residues. Using the kinetic method of Tsou (Tsou, C.-L. (1962) Sci. Sin. 11, 1535-1558), it was shown that modification of a single histidine residue leads to inactivation. Ligand protection experiments also indicated that 1 histidine residue was protected from DEP modification. pH studies show that the pKa for this inactivation is 6.18, which is identical to the single pKa determined from the pH/log Vmax profile for the enzyme. A single active site peptide was identified by differential peptide mapping in the presence and absence of ligand. This peptide was found to comprise residues 141-158; of the 2 histidines in this peptide (His-143 and His-146), only one, His-143, is conserved among all type I dehydroquinases. We propose that His-143 is the active site histidine responsible for DEP-mediated inactivation of dehydroquinase and is a good candidate for the general base that has been postulated to participate in the mechanism of this enzyme.     

Genetic structure of Hmong-Mien speaking populations in East Asia as revealed by mtDNA lineages

Hmong-Mien (H-M) is a major language family in East Asia, and its speakers distribute primarily in southern China and Southeast Asia. To date, genetic studies on H-M speaking populations are virtually absent in the literature. In this report, we present the results of an analysis of genetic variations in the mitochondrial DNA (mtDNA) hypervariable segment 1 (HVS1) region and diagnostic variants in the coding regions in 537 individuals sampled from 17 H-M populations across East Asia. The analysis showed that the haplogroups that are predominant in southern East Asia, including B, R9, N9a, and M7, account for 63% (ranging from 45% to 90%) of mtDNAs in H-M populations. Furthermore, analysis of molecular variance (AMOVA), phylogenetic tree analysis, and principal component (PC) analysis demonstrate closer relatedness between H-M and other southern East Asians, suggesting a general southern origin of maternal lineages in the H-M populations. The estimated ages of the mtDNA lineages that are specific to H-M coincide with those based on archeological cultures that have been associated with H-M. Analysis of genetic distance and phylogenetic tree indicated some extent of difference between the Hmong and the Mien populations. Together with the higher frequency of north-dominating lineages observed in the Hmong people, our results indicate that the Hmong populations had experienced more contact with the northern East Asians, a finding consistent with historical evidence. Moreover, our data defined some new (sub-)haplogroups (A6, B4e, B4f, C5, F1a1, F1a1a, and R9c), which will direct further efforts to improve the phylogeny of East Asian mtDNAs.     

Expanding Southwest Pacific mitochondrial haplogroups P and Q

Modern humans have occupied New Guinea and the nearby Bismarck and Solomon archipelagos of Island Melanesia for at least 40,000 years. Previous mitochondrial DNA (mtDNA) studies indicated that two common lineages in this region, haplogroups P and Q, were particularly diverse, with the coalescence for P considered significantly older than that for Q. In this study, we expand the definition of haplogroup Q so that it includes three major branches, each separated by multiple mutational distinctions (Q1, equivalent to the earlier definition of Q, plus Q2 and Q3). We report three whole-mtDNA genomes that establish Q2 as a major Q branch. In addition, we describe 314 control region sequences that belong to the expanded haplogroups P and Q from our Southwest Pacific collection. The coalescence dates for the largest P and Q branches (P1 and Q1) are similar to each other (approximately 50,000 years old) and considerably older than prior estimates. Newly identified Q2, which was found in Island Melanesian samples just to the east, is somewhat younger by more than 10,000 years. Our coalescence estimates should be more reliable than prior ones because they were based on significantly larger samples as well as complete mtDNA-coding region sequencing. Our estimates are roughly in accord with the current suggested dates for the first settlement of New Guinea-Sahul. The phylogeography of P and Q indicates almost total (female) isolation of ancient New Guinea-Island Melanesia from Australia that may have existed from the time of the first settlement. While Q subsequently diversified extensively in New Guinea-Island Melanesia, it has not been found in Australia. The only shared mtDNA haplogroup between Australia and New Guinea identified to date remains one minor branch of P.