Anticancer effect of fermented Insampaedok-san in human colon cancer cells: a pilot study.

Insampaedok-san (IPS) is one of the East Asian traditional medicines which have been prescribed for hundreds of years to treat common cold and headache. Although many herbs and prescriptions are known to have significant activities against diseases, only a limited number of reports and scientific evidences on their efficacies are available. To identify anticancer effect against colon cancer, traditional prescription IPS and its fermented IPS (FIPS) were examined by in vitro molecular biological analysis. IPS water extract was fermented, lyophilized, and examined by cytotoxicity, cell cycle, and western blot assays, using cancer cell lines. Resultantly, FIPS showed significant cytotoxicities inducing caspase dependent apoptosis and activation of caspase-3 (CASP3) and poly (ADP-ribose) polymerase (PARP) cleavage in colon cancer cell line SW620. These findings can be useful for development of anticancer functional food or complementary and alternative medicine, with remaining in-depth molecular functional studies.

In vitro and in vivo evaluation of systemic and genetic toxicity of Citrus unshiu peel.

ETHNOPHARMACOLOGICAL RELEVANCE: The peel of Citrus unshiu Markovich fruits (CUP), called "Jinpi" in Korea, and "Chenpi" in China, has been used for the treatment of respiratory and blood circulation disorders in traditional oriental medicine (TOM). Despite its widespread uses in TOM, no information on the safety of CUP has been reported. Thus, genotoxicity and systemic toxicity of CUP were evaluated in the current studies. MATERIALS AND METHODS: We conducted a toxicological evaluation of CUP water extracts using acute and subchronic (13-week repeated-dose) toxicity tests and three genotoxicity assays (bacterial reverse mutation, mammalian chromosomal aberration, and micronuclei formation). RESULTS: In acute and subchronic toxicity tests, both the median lethal dose (LD50) and no-observed-adverse-effect level (NOAEL) were more than 4000mg/kg/day in rats. None of the genotoxicity assays revealed any mutagenicity or clastogenicity in in vitro and in vivo systems. CONCLUSION: CUP water extracts were found to be nongenotoxic under our testing conditions and had low acute and subchronic toxicity.

Acute toxicity and genotoxicity of fermented traditional medicine oyaksungi-san.

BACKGROUND: The traditional medicine oyaksungi-san (OY) has been prescribed in East Asia for hundreds of years for the treatment of stroke, paralysis, and ataxia. OY also has therapeutic effects on arthralgia, myalgia, and rheumatoid arthritis, and recent studies have shown its protective effects against apoptosis of hippocampal cells and its anti-inflammatory effects on the peripheral blood cells of patient with cerebral infarction. Many studies have explored the use of traditional medicine and herb materials in the development of safe, novel, and effective pharmaceuticals with fewer side effects. These efforts commonly adopt a bioconversion tool for fermentation with beneficial microbes. However, only pharmaceuticals with high levels of safety and low levels of toxicity can be used in healthcare system. METHODS: OY water extract was fermented with Lactobacillus and assayed for acute toxicity and genotoxicity. Single dose acute toxicity, bacterial reverse mutation, chromosome aberrations, and micronucleus were observed and assayed in rats, histidine/tryptophan auxotrophic bacteria, Chinese hamster ovary fibroblast cells, and mice bone marrow cells, respectively. RESULTS: All the experimental animals showed no abnormal behavior, clinical signs, body weight increases, or mortality. In the bacterial cultures, no revertant colonies were observed. Morphological and numerical chromosomal aberrations were not found in all metaphases examined. Frequency of induced micronuclei was not significantly increased in all doses applied. CONCLUSION: As a whole, no acute toxicity or genotoxicity were observed in all the assays examined. Therefore, fermented OY is considered to be a safe material that can be used for development of complementary and alternative medicine using bioconversion.

Single, repeated dose toxicity and genotoxicity assessment of herb formula KIOM2012H.

BACKGROUND: Traditional medicine and herbal prescriptions are becoming more popular, and they account for a large share of the world's healthcare research studies, developments, and market demands. Increasing scientific evidence of the substantive efficacies such as preventive health keeping pharmaceutical materials and dietary supplements can be found elsewhere. Above all, safety should be the critical premise for considering developmental materials such as pharmaceuticals without side effects and toxicity. METHODS: The authors formulated KIOM2012H (K2H) using four herbs that were reported to have medicinal effects-including anticancer, antiaging, antimicrobial, inflammation, and neuroprotective properties. In order to examine the toxicity, single and repeated dose toxicity, and genotoxicities of bacterial mutation, micronucleus, and chromosomal aberration assays were conducted. RESULTS: All experimental observations and results showed normal findings. Toxicities or abnormal signs were not observed in all experimental assays, including oral administration, animal behavior, clinical findings, and changes in body weight in vivo. In vitro bacterial cultures produced no revertant colonies, and no increased numbers of structural or numerical aberrant metaphases were found in the metaphase chromosomes examined. Moreover, no significant increased frequency of micronucleus was observed in any of the doses used. Overall, no acute toxicity or genotoxicity was found in all analysis parameters in all the assays conducted. CONCLUSION: Reviewing the results as a whole, K2H extract was regarded as a safe material with no toxicity, and can be applied for the research and development of complementary and alternative medicines with improved efficacy in current therapeutic healthcare, based on traditional medicine and herb resources.

Fermented Sipjeondaebo-tang Alleviates Memory Deficits and Loss of Hippocampal Neurogenesis in Scopolamine-induced Amnesia in Mice.

We investigated the anti-amnesic effects of SJ and fermented SJ (FSJ) on scopolamine (SCO)-induced amnesia mouse model. Mice were orally co-treated with SJ or FSJ (125, 250, and 500 mg/kg) and SCO (1 mg/kg), which was injected intraperitoneally for 14 days. SCO decreased the step-through latency and prolonged latency time to find the hidden platform in the passive avoidance test and Morris water maze test, respectively, and both SCO effects were ameliorated by FSJ treatment. FSJ was discovered to promote hippocampal neurogenesis during SCO treatment by increasing proliferation and survival of BrdU-positive cells, immature/mature neurons. In the hippocampus of SCO, oxidative stress and the activity of acetylcholinesterase were elevated, whereas the levels of acetylcholine and choline acetyltransferase were diminished; however, all of these alterations were attenuated by FSJ-treatment. The alterations in brain-derived neurotrophic factor, phosphorylated cAMP response element-binding protein, and phosphorylated Akt that occurred following SCO treatment were protected by FSJ administration. Therefore, our findings are the first to suggest that FSJ may be a promising therapeutic drug for the treatment of amnesia and aging-related or neurodegenerative disease-related memory impairment. Furthermore, the molecular mechanism by which FSJ exerts its effects may involve modulation of the cholinergic system and BDNF/CREB/Akt pathway.

Neuroprotective effects of Liriope platyphylla extract against hydrogen peroxide-induced cytotoxicity in human neuroblastoma SH-SY5Y cells.

BACKGROUND: Oxidative stress is involved in neuronal cell death and mitochondrial dysfunction in neurodegenerative diseases. Liriope platyphylla (LP) has been suggested to have anti-inflammation, anti-bacterial, and anti-cancer effects. However, whether LP exerts neuroprotective effects on neuronal cells is unknown. METHODS: The present study was performed to investigate the neuroprotective effects of LP extract (LPE) against hydrogen peroxide (H2O2)-induced injury in human neuroblastoma cells SH-SY5Y. To test neuroprotective effects of LPE, we performed cell viability assay, flow cytometry analysis and western blot analysis. In addition, mitochondrial membrane potential (MMP) and oxidative stress were performed to evaluate the anti-apoptotic and anti-oxidant effects. RESULTS: LPE pretreatment conferred significant protection against the H2O2-induced decrease of SH-SY5Y cell viability. H2O2-induced increases of intracellular oxidative stress and mitochondrial dysfunction were attenuated by LPE pretreatment. Therefore, LPE pretreatment prevented SH-SY5Y cell injury. Treatment with H2O2 significantly induced poly(ADP ribose) polymerase (PARP) and caspase-3 cleavage, which was blocked by LPE. We found that p38 activation was involved in the neuroprotective effects of LPE. CONCLUSIONS: Current findings suggest that LPE exerts neuroprotective effects against H2O2-induced apoptotic cell death by modulating p38 activation in SH-SY5Y cells. Therefore, LPE has potential anti-apoptotic effects that may be neuroprotective in neurodegenerative diseases and aging-related dementia.

Hepatoprotective effect of herb formula KIOM2012H against nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is a hepatic ailment with a rapidly increasing incidence due to dietary hypernutrition and subsequent obesity. Fatty liver disease can lead to steatohepatitis, fibrosis, cirrhosis, and even cancer, which is associated with various complications. Discovering effective natural materials and herbs can provide alternative and complementary medical treatments to current chemical pharmaceuticals. To develop an effective natural agent for NAFLD, we formulated a combination of four herb mixtures (KIOM2012H) and observed lipid-lowering efficacy. The inhibitory effects of KIOM2012H on free fatty acid-induced lipid accumulation, triglyceride contents, and gene expressions were analyzed in HepG2 cells. Using high fat diet-fed mice, body weight changes, gross liver appearances, hepatic triglyceride contents, and gene expressions were evaluated. KIOM2012H dose-dependently inhibited lipid accumulation and gene expressions involved in lipogenesis and related regulators. Experimental animals also showed a decrease in body weight changes and lipid-associated physiological parameters. This study shows that KIOM2012H has an alleviating effect on fatty acid and lipid accumulation, and therefore can be applied for development of new therapeutic pharmaceuticals for treatment of NAFLD using natural products and herbs.

Acute toxicity and genotoxicity study of fermented traditional herb formula Guibi-tang.

ETHNOPHARMACOLOGY RELEVANCE: Guibi-tang (Guipi-tang in Chinese and Kihi-to in Japanese) is a multi-herb traditional medicine commonly prescribed to treat psychoneurosis in East Asia. Although this medicine has been widely used, there is little available information on the safety and toxicity of Guibi-tang, especially on the fermented one. MATERIALS AND METHODS: Guibi-tang, composed of 12 herbs, was fermented with bacteria and lyophilized. Single dose acute toxicity in rats was observed for 14 days after administration. Genetic toxicity of fermented Guibi-tang was evaluated on bacterial reverse mutation in Salmonella and Escherichia spp., chromosome aberrations in Chinese hamster ovary cells, and micronucleus formation in mice. Ingredients in FGBT were identified and quantified by high performance liquid chromatography-mass spectrometry. RESULTS: In acute oral toxicity study, behavior, clinical signs and body weight changes were normal observing in all experimental animals. No revertant colonies were found in any bacterial cultures examined. Morphological or numerical anomalies and significant increased number of aberrant metaphases were not observed. Micronucleus assay showed no significant increases in the frequency of inducing micronuclei in any dose examined. Decursinol, decursin, glycyrrhizin, and 6-gingerol in fermented Guibi-tang were identified and quantitated. As a whole, no acute and genotoxic effects were found in all the assays and parameters analyzed. CONCLUSION: Fermented Guibi-tang was recognized as safe and non-toxic, and therefore can be used for applications of traditional medicine in modern complementary and alternative therapeutics and health care.

Inhibitory Effect of Arctigenin from Fructus Arctii Extract on Melanin Synthesis via Repression of Tyrosinase Expression.

To identify the active compound arctigenin in Fructus Arctii (dried seed of medicinal plant Arctium lappa) and to elucidate the inhibitory mechanism in melanogenesis, we analyzed melanin content and tyrosinase activity on B16BL6 murine melanoma and melan-A cell cultures. Water extracts of Fructus Arctii were shown to inhibit tyrosinase activity in vitro and melanin content in α -melanocyte stimulating hormone-stimulated cells to similar levels as the well-known kojic acid and arbutin, respectively. The active compound arctigenin of Fructus Arctii displayed little or no cytotoxicity at all concentrations examined and decreased the relative melanin content and tyrosinase activity in a dose-dependent manner. Melanogenic inhibitory activity was also identified in vivo with zebrafish embryo. To determine the mechanism of inhibition, the effects of arctigenin on tyrosinase gene expression and tyrosinase promoter activity were examined. Also in addition, in the signaling cascade, arctigenin dose dependently decreased the cAMP level and promoted the phosphorylation of extracellular signal-regulated kinase. This result suggests that arctigenin downregulates cAMP and the tyrosinase enzyme through its gene promoter and subsequently upregulates extracellular signal-regulated kinase activity by increasing phosphorylation in the melanogenesis signaling pathway, which leads to a lower melanin content.

In vitro and in vivo safety evaluation of Acer tegmentosum.

ETHNOPHARMACOLOGICAL RELEVANCE: Acer tegmentosum, which contains salidroside and tyrosol, has been used for the treatment of hepatic disorders in eastern Asia. However, little is known about its safety. AIM OF THE STUDY: To determine the safety of Acer tegmentosum, we evaluated its acute oral toxicity and genotoxicity profiles. MATERIALS AND METHODS: Salidroside and tyrosol present in Acer tegmentosum were quantified using high-performance liquid chromatography. Acute oral toxicity testing of Acer tegmentosum was performed in rats. Genotoxicity of Acer tegmentosum was assessed by bacterial reverse mutation, chromosomal aberration, and bone marrow micronucleus tests. All the tests were conducted in accordance with the good laboratory practices. RESULTS: The amounts of salidroside and tyrosol in Acer tegmentosum were found to be 85.01±1.21mg/g and 3.12±0.04mg/g, respectively. In the bacterial reverse mutation test, Acer tegmentosum increased the number of revertant Salmonella typhimurium TA98 colonies, regardless of metabolic activation by S9 mixture. In contrast, Acer tegmentosum application did not significantly increase the number of chromosomal aberrations in Chinese hamster ovary (CHO)-K1 cells and micronucleated polychromatic erythrocytes in mice. In the acute oral toxicity test, the median lethal dose (LD50) of Acer tegmentosum was found to be >2000mg/kg in rats. CONCLUSION: Take together, Acer tegmentosum exhibits mutagenicity, which was evident from the bacterial reverse mutation test. Further studies are needed to identify the components responsible for such an effect and the underlying mechanisms.

Simultaneous quantification of marker components in Ojeok-san by HPLC-DAD.

A systematic high-performance liquid chromatography-diode array detection (HPLC-DAD) method was developed for the rapid, accurate, and simultaneous quantification of eight marker compounds, paeoniflorin, 6-gingerol, decursin, glycyrrhizin, cinnamic acid, hesperidin, poncirin and magnolol, in Ojeok-san, a traditional Korean herbal medicine. These compounds were separated in less than 50 min using a Dionex C(18) column with a gradient elution system of water and methanol at a flow rate of 1 ml/min. All calibration curve of standard components showed excellent linearity (R(2) > 0.9922). Limits of detection (LOD) and limits of quantification (LOQ) ranged from 0.01 to 0.11 µg/ml and 0.03 to 0.34 µg/ml, respectively. The relative standard deviations (RSDs) of data of the intra- and inter-day experiments were less than 2.15 and 2.60%, respectively. The accuracy of recovery test ranged from 94.27 to 107.68% with RSD values 0.15-3.61%. The results of validation showed that the HPLC method was stable and very accurate for the quantification of eight marker components in Ojeok-san.

Reciprocal regulation of gene expression by Ephedra herba in mouse brain.

Although Ephedra herba has been used to treat a variety of diseases, the molecular mechanism has not been defined clearly. To identify the molecular events, a microarray analysis was conducted using the brain tissue of mice orally administered aqueous extracts of Ephedra herba, in which the expression level of many genes was altered depending on the duration of treatment time. In particular, temporal changes in the expressions of genes can be classified into two major reciprocal patterns in which 175 and 282 genes were included in pattern 1 and 2, respectively. Classification based on gene ontology revealed different functional implications of genes between temporal pattern 1 and 2. Through promoter analysis of temporally co-expressed genes, putative co-regulated genes could be segregated based on the similarity of the transcription factor binding sites (TFBS). Interestingly, two temporal patterns were associated with different sub-classes of TFBS similarity, which implies the presence of dual regulatory mechanisms. Finally, through interaction analysis, core node genes that could interact with many other nodes were identified. By integrating the interaction information with promoter clustering, temporally co-regulated key components could be selected in mouse brain after administration of Ephedra herba.

Pitx2, a beta-catenin-regulated transcription factor, regulates the differentiation of outer root sheath cells cultured in vitro.

BACKGROUND: Beta-catenin exerts its crucial role in hair follicle development and hair growth cycle. Although the importance of Wnt/beta-catenin is well recognized, the downstream effectors of beta-catenin have not been clearly elucidated yet. OBJECTIVE: The aim of this study is to identify the beta-catenin-regulated genes in cultured human hair outer root sheath (ORS) cells. METHODS: We transduced ORS cells with adenovirus harboring the expression cassette for constitutive active form of beta-catenin, then performed cDNA microarray. RESULTS: Overexpression of beta-catenin led to the upregulation of hair cell differentiation markers such as keratin 16 and 17. In addition, the expression of Pitx2, a bicoid-type homeodomain transcription factor, was also increased by overexpression of beta-catenin in ORS cells cultured in vitro. To investigate the potential role of Pitx2, we made the recombinant adenovirus expressing Pitx2, then transduced into the cultured ORS cells. Interestingly, Pitx2 induced the expression of keratin 16 and 17, indicating that Pitx2 activates ORS cells towards the follicular differentiation pathway preferentially. CONCLUSION: Our results implicate the potential importance of Pitx2 as a beta-catenin downstream modulator in hair growth control.

Dual localization of wild-type myocilin in the endoplasmic reticulum and extracellular compartment likely occurs due to its incomplete secretion.

PURPOSE: Wild-type myocilin is known to be secreted extracellularly, but a significant amount of the protein is also present in the endoplasmic reticulum (ER). The present study was undertaken to address whether intracellular myocilin is a true ER resident protein. METHODS: Human wild-type myocilin was adenovirally expressed in human trabecular meshwork cells, and general characteristics of both intracellular and extracellular myocilins including molecular weight, pI, glycosylation state, and cleavage site of the signal peptide were examined by biochemical analyses. Topology and decay kinetics of myocilin were also examined by protease protection assay and pulse chase analysis, respectively. The expression pattern and cytopathic effect of myocilin were analyzed in individual cells by immunocytochemistry. RESULTS: Intracellular myocilin were very similar to secreted myocilin in characteristics such as molecular weight, pI, glycosylation state, and cleavage site of the signal peptide. The intracellular protein was found to be present in the lumen of the ER where it appeared to be retained without further export to the Golgi apparatus. The kinetics of myocilin turnover clearly showed that it was intrinsically a very stable but incompletely secreted protein. The expression of myocilin was confined to a subset of cells and accompanied by the upregulation of a 78 kDa glucose-regulated protein, suggesting that it was not properly folded or processed in the ER. CONCLUSIONS: Based on these findings and the fact that myocilin has no known ER retention signals, the ER localization of wild-type myocilin is likely a consequence of its incomplete secretion due to its misfolding.

Dual origins of the Japanese: common ground for hunter-gatherer and farmer Y chromosomes.

Historic Japanese culture evolved from at least two distinct migrations that originated on the Asian continent. Hunter-gatherers arrived before land bridges were submerged after the last glacial maximum (>12,000 years ago) and gave rise to the Jomon culture, and the Yayoi migration brought wet rice agriculture from Korea beginning approximately 2,300 years ago. A set of 81 Y chromosome single nucleotide polymorphisms (SNPs) was used to trace the origins of Paleolithic and Neolithic components of the Japanese paternal gene pool, and to determine the relative contribution of Jomon and Yayoi Y chromosome lineages to modern Japanese. Our global sample consisted of >2,500 males from 39 Asian populations, including six populations sampled from across the Japanese archipelago. Japanese populations were characterized by the presence of two major (D and O) and two minor (C and N) clades of Y chromosomes, each with several sub-lineages. Haplogroup D chromosomes were present at 34.7% and were distributed in a U-shaped pattern with the highest frequency in the northern Ainu and southern Ryukyuans. In contrast, haplogroup O lineages (51.8%) were distributed in an inverted U-shaped pattern with a maximum frequency on Kyushu. Coalescent analyses of Y chromosome short tandem repeat diversity indicated that haplogroups D and C began their expansions in Japan approximately 20,000 and approximately 12,000 years ago, respectively, while haplogroup O-47z began its expansion only approximately 4,000 years ago. We infer that these patterns result from separate and distinct genetic contributions from both the Jomon and the Yayoi cultures to modern Japanese, with varying levels of admixture between these two populations across the archipelago. The results also support the hypothesis of a Central Asian origin of Jomonese ancestors, and a Southeast Asian origin of the ancestors of the Yayoi, contra previous models based on morphological and genetic evidence.

Identification of flotillin-1 as a protein interacting with myocilin: implications for the pathogenesis of primary open-angle glaucoma.

Mutations in MYOC gene encoding myocilin are responsible for primary open-angle glaucoma (POAG). In order to search for protein(s) that can interact with myocilin, we screened a human skeletal muscle cDNA library using yeast two-hybrid system and identified flotillin-1, a structural protein of lipid raft that is detergent-resistant and a liquid ordered microdomain, as a protein interacting with myocilin. The interaction was confirmed by in vitro glutathione S-transferase pulldown and in vivo co-immunoprecipitation studies. In yeast two-hybrid assay, the C-terminus of myocilin, an olfactomedin-like domain in which most mutations related to POAG are scattered, was found to be necessary and sufficient for the interaction. However, myocilins with mutations such as G364V, K423E, and Y437H on the domain failed to interact with flotillin-1. Although the physiological significance of the interaction has yet to be elucidated, our results showed that the alteration of the interaction by mutations in MYOC might be a key factor of the pathogenesis of POAG.

Identification of novel allele on the locus 47z (DXYS5) in the Korean population.

Two homogenous sequences of 47z (DXYS5) are located on the X (DXYS5X) and Y (DXYS5Y) chromosomes, and these are known to be useful polymorphic markers for tracing male-specific gene flow such as the migration routes of human populations. Using the 47z/StuI PCR-RFLP system, we found a novel allele which showed two bands, in contrast to the previous two allele types, one band (Y1) and three bands (Y2). This means that copies of PCR products derived from both the DXYS5X and DXYS5Y loci were clearly cut by the StuI enzyme, implying that the DXYS5X locus of the X chromosome is polymorphic. Allelic frequencies examined in 267 male Korean individuals showed that 95.8% had Y1, 3.4% Y2, and 0.8% had the novel allele. Our findings should contribute to a better understanding of genetic polymorphism on X and Y chromosomes, the molecular evolution mechanism of sex chromosomes, and how the migration route of Koreans is related to those of other East Asian populations.

Characterization of mouse organic anion transporter 5 as a renal steroid sulfate transporter.

A family of organic anion transporters (OAT) recently identified has important roles for the excretion or reabsorption of endogenous and exogenous compounds, and several new isoforms have been reported in this decade. Although the transepithelial transport properties of organic anions are gradually being understood, many portions of their functional characteristics in functions remain to be elucidated. A recently reported new cDNA encoding a mouse OAT5 (mOAT5) was constructed, using 3'-RACE PCR, with the total RNA isolated from a mouse kidney. When mOAT5 was expressed in Xenopus oocytes, mOAT5 transported estrone sulfate, dehydroepiandrosterone sulfate and ochratoxin A. Estrone sulfate uptake by mOAT5 displayed a time-dependent and sodium-independent manner. The Km values of estrone sulfate and dehydroepiandrosterone sulfate were 2.2 and 3.8 microM, respectively. mOAT5 interacted with chemically heterogeneous steroid or organic sulfates, such as nitrophenyl sulfate, methylumbelliferyl sulfate and estradiol sulfates. In contrast to the sulfate conjugates, mOAT5-mediated estrone sulfate uptake was not inhibited by the steroid or organic glucuronides. The mOAT5 protein having about 85 kDa molecular weight was shown to be mainly localized in the apical membrane of the proximal tubules of the outer medulla. These results suggest an important role of mOAT5 for the excretion or reabsorption of steroid sulfates in the kidney.

Forensic value of 14 novel STRs on the human Y chromosome.

We identified and characterized 14 novel short-tandem-repeats (STRs) on the Y chromosome and typed them in two samples, a globally diverse panel of 73 cell lines, and 148 individuals from a European-American population. These Y-STRs include eight tetranucleotide repeats (DYS449, DYS453, DYS454, DYS455, DYS456, DYS458, DYS459, and DYS464), five pentanucleotide repeats (DYS446, DYS447, DYS450, DYS452, and DYS463), and one hexanucleotide repeat (DYS448). Sequence data were obtained to designate a repeat number nomenclature. The gene diversities of an additional 22 Y-STRs, including the most commonly used in forensic databases, were directly compared in the cell line DNAs. Six of the 10 most polymorphic markers include the newly identified Y-STRs. Furthermore, these novel Y-STRs greatly improved the resolution of paternal lineages, above the level obtained with commonly used Y-STRs, in the European-American population.