[Polymorphism analysis of the block 5 region in merozoite surface protein-1 gene of imported and local Plasmodium vivax in Yunnan Province].

Objective: To analyze the sequence of Plasmodium vivax merozoite surface protein-1(PvMSP-1) and allele polymorphism in imported and local vivax malaria parasites in Yunnan Province. Methods: Blood samples on filter paper were collected from imported and local vivax malaria cases in Yunnan Province during August 2012 and September 2015 and information of epidemiological history was recorded. Plasmodium DNA was extracted by a DNA extraction kit, and the block 5 region in PvMSP-1 gene was amplified by PCR. The PCR products were sequenced and blasted with reference sequences M75674, AAN86237, M60807, ABJ53045, AAN86238 and BAA18977. The sequence polymorphism in block 5 region of PvMSP-1 was analyzed with MEGA 5.04 and Arlequin3.5.1 softwares. The conserved sites, genetic distances among sequences and Shannon-wiener index among alleles were calculated. The clustering tree was drawn according to the genetic distances between the amino-acid sequences. Results: A total of 847 blood samples were collected from the malaria cases, comprising of 61 samples from local cases, 66 from imported cases from Africa, and 720 from Myanmar. The block 5 region in PvMSP-1 was successfully amplified in 278 samples, and sequencing was successfully made in 206 of them. The peptide coded by the block 5 region had a length of 193 to 222 aa. The amino acid sequence alignment showed that in 206 samples the proportion of genotypes of Sal-1, Belem and Recombine was 59.2%(122/206),23.3%(48/206) and 17.5%(36/206), respectively. The proportion of Sal-1 genotype in imported cases from Myanmar and Africa and in local cases was 58.8%(104/177),73.3%(11/15) and 50%(7/14), respectively. The genotypes Sal-1, Belem and Recombine had 51, 9 and 6 different alleles. The 66 alleles had a Shannon Wiener index (H') of 0.955 and an expected heterozygosis (He) of 0.567. The 206 DNA sequences had a 665-bp homologous locus, comprising of 75 conserved sites (11.3%,75/665) and 590 variable sites (88.7%, 590/665). The genetic distances between sequences were all less than 0.4. The clustering analysis showed that the 206 sequences were clustered into two categories with three branches. The homology of Recombine with Belem genotype was 91%-92%, higher than with Sal-1 genotype (82%-83%). Conclusion: The block 5 region in PvMSP-1 gene from local and imported Plasmodium vivax in Yunnan Province has varied forms of alleles, and the Sal-1 genotype is predominant among the three genotypes.

[Polymorphism of 72-76 Coding Sequence within Exon 2 Region of Pfcrt in Yunnan Province].

Objective: To understand the endemic situation of chloroquine-resistant falciparum malaria in Yunnan Province by analyzing the polymorphism of the 72-76 amino-acid coding sequence within exon 2 region of Plasmodium falciparum chloroquine resistant transporter (Pfcrt) gene （referred to as the 72-76 coding region） in malaria patients. Methods: The filter paper blood samples and relative information of falciparum malaria cases were collected in 13 prefectures of Yunnan Province （excluding Diqing, Wenshan, Zhaotong prefectures） from August 2012 to September 2015. The source of infection was determined by epidemiological investigation and the place of case discovery was confirmed according to the endemic registration in the Infectious Diseases Reporting Manage System, Chinese Center for Disease Control and Prevention. The exon2 region of Pfcrt gene was amplified by nested PCR and sequenced. The polymorphism of the 72-76 coding region was analyzed with MEGA 5.04. The variable sites and genetic distance between sequences were calculated. The constituent ratio of the polymorphism in sub-populations was analyzed with IBM SPSS Statistics 21 software. Results: Two hundred and thirty-two blood samples were collected in the period and source of infection included Yunnan of China, Africa and Myanma. Nested-PCR resulted in positive products in 210 samples. Sequence analysis showed the presence of chloroquine-sensitive genotype（CVMNK）（15.2%, 32/210） and mutated chloroquine-resistant genotype（CVIET, SVMNT and CVMNT）（76.2%, 160/210; 6.7%, 14/210; 1.9%, 4/210） 72-76 coding regions. The proportion of the CVMNK type was 100%（32/32） in cases with the range of 19-55 years, 46.9% （15/32） in farmers, and 59.4% （19/32） in patients with infection source in Southeast Asia, all significantly higher than those of other cases in the same groups（0; 31.3%, 10/32; and 37.5%, 12/32 respectively, χ2=13.674, 8.478, 6.292, P<0.05）. The proportion of the CVIET and SVMNT genotypes in patients with infection source in Myanma and Cambodia was 81.3%（130/160） and 78.6%（11/14） respectively, significantly higher than those in patients with infection source in Yunnan Province（6.3%, 10/160; 21.4%, 3/14）（χ2=6.519 and 6.620, P<0.05）. In samples with Africa infection source, the proportion of CVIET was 12.5%（20/160）, with no detection of SVMNT. There was a 145 bp homologous locus among the 210 exon2 regions, of which the conservative sites accounted for 95.2%（138/145） and variable sites for 4.8%（7/145）. The genetic distance between the 210 sequences ranged 0.000-0.036（0.012±0.005）. The genetic distances from genotypes CVIET, SVMNT and CVMNT to the chloroquine-sensitive genotype CVMNK were（0.029±0.015）, （0.021±0.013） and （0.014±0.001） respectively. 178 cases with chloroquine-resistant P. falciparum distributed in all the 13 prefectures. Among them, the regions with top detection rate of chloroquine-resistant genotypes were Dehong（51.7%, 92/178）, Baoshan（24.7%, 44/178） and Lincang（5.6%, 10/178） bordering on Myanmar and Kunming (4.5%, 8/178). Conclusion: There are three chloroquine-resistant genotypes of the 72-76 coding region in falciparum malaria cases in Yunnan Province, which distribute in 81.3%（13/16） of prefectures in the Province.

[Polymorphism Analysis of Plasmodium falciparum K13 Gene Kelch Domain Associated with Resistance to Artemisinin in Yunnan Province].

Objective: To investigate the polymorphism of Plasmodium falciparum K13 gene kelch domain region and provide basis for understanding the artemisinin resistance of falciparum malaria in Yunnan Province. Methods: The filter blood samples and relative information of falciparum malaria cases were collected in 16 prefectures of Yunnan Province from January 2013 to December 2015. The source of infection was determined by epidemiological investigation and the place of case discovery was confirmed according to the China Information System for Disease Control and Prevention Epidemic Registration. The K13 gene kelch domain region was amplified by nested PCR, sequenced, and blasted against the reference strain 3D7（PF3D7_1343700）. The K13 gene kelch domain region polymorphism was analyzed with Mega 5.04. The variable sites and genetic distance between sequences were analyzed. The constituent ratio of amino acid mutation sites was calculated and analyzed with χ2 test. Results: A total of 202 blood samples were collected from 2013 to 2015, comprising 190 from imported cases, 12 from local cases in Yunnan Province. The constitutent ratio of infection cases were 30.7% （62/202）, 34.2% （69/202） and 35.1% （71/202） respectively, increased year by year. The K13 gene kelch domain was successfully amplified from 192 samples and 190 were successfully sequenced, detecting missense mutation of K13 gene in 66 samples, the mutation rate was 34.7% （66/190）. The detection rate of K13 gene mutation was 40.9% （27/66）, 37.9% （25/66） and 21.2% （14/66） respectively, decreased year by year. In this study, ten types of mutations were detected, which were F446I, A578S, N458Y, P574L, A676D, G449A, C469Y, V494I, E556D and S16L. The highest mutation rate occurred in F446I which was 72.7% （48/66）. The proportion of F446I mutation type was 58.3% （28/48） in an age-range of 18-56 years, 70.8% （34/48） in farmers, and 91.7% （44/48） in patients with infection source in Southeast Asia, all significantly higher than that of other groups with the same characteristics （41.7%, 20/48; 29.2%, 14/48; and 8.3%, 4/48, respectively）（χ2=4.633, 5.556 and 5.152, both P＜0.05）. There was a 248 bp homologous sequence in the 190 sequences, composed of 235 conservative sites （94.8%）, 13 variable sites （5.2%）, 5 parsim-info sites （2.0%）, and 8 singleton sites （3.2%）. The genetic distance among the 190 sequences ranged 0.000-0.036, with an average of 0.001±0.001. Conclusion: There are 10 types of mutations in the K13 kelch domain in Yunnan Province, the predominant mutation type was F446I.

[Quality Analysis of Laboratory Malaria Diagnosis in Yunnan Province during 2012-2014].

OBJECTIVE: To assess the quality of microscopy-based malaria diagnosis in Yunnan Province from August 2012 to October 2014, and analyze the relevant factors. METHODS: Blood samples were collected from patients diagnosed as malaria by microscopy in county-level laboratories of Yunnan Province. The blood smears and blood filter paper samples were prepared and submitted to the provincial malaria diagnosis reference laboratory for further confirmation by both microscopy and the genetic approach. Coincidence rates for species identification between county and provincial laboratories were analyzed using the SPSS 21.0 software. RESULTS: From August 2012 to October 2014, 1 400 malaria cases were diagnosed with microscopy in 72 counties of Yunnan Province. Among them, the cases of falciparum malaria, vivax malaria, and unclassified malaria accounted for 18.4% (252/1,400), 79.3% (1,105/1,400) and 3.1% (43/1,400), respectively. The percentage of unclassified malaria cases reached a peak in 2012 (3.5%, 9/257). The coincidence rate for species identification with microscopy between county-level and provincial-level laboratories was 70.1% (845/1,216) in 2012, being the lowest during 2012-2014, and the coincidence rate for diagnosis of positive infection was 77.6% (943/1,216). Similarly, the coincidence rates for species identification and for positive infection between county-level laboratories using microscopy and the provincial-level laboratory using the genetic approach were 81.3% (150/185) and 85.0% (157/185) respectively in 2012, being also the lowest during 2012-2014. In the provincial laboratory, the inconsistency rate for species identification between microscopy and the genetic approach was 8.7% (97/1 120), predominately the infection-negative results by microscopy versus falciparum malaria, vivax malaria or mixed infection revealed by the genetic approach (57.7%, 56/97). The sampling coverage rate in counties was the lowest in November 2012 (46.9%, 82/175). The blood smear preparation scored 69.8, 70.4 and 78.8 (P < 0.05) in 2012, 2013 and 2014, respectively. CONCLUSION: The quality of laboratory malaria diagnosis has been significantly improved in most counties of Yunnan Province since 2013.

A simple and effective method to enrich endogenous DNA from mammalian faeces.

Utilization of faeces has long been a popular approach for genetic and ecological studies of wildlife. However, the success of molecular marker genotyping and genome resequencing is often unpredictable due to insufficient enrichment of endogenous DNA in the total faecal DNA that is dominated by bacterial DNA. Here, we report a simple and cheap method named PEERS to predominantly lyse animal cells over bacteria by using sodium dodecyl sulphate so as to discharge endogenous DNA into liquid phase before bacterial DNA. By brief centrifugation, total DNA with enriched endogenous fraction can be extracted from the supernatant using routine methods. Our assessments showed that the endogenous DNA extracted by PEERS was significantly enriched for various types of faeces from different species, preservation time and conditions. It significantly improves the genotyping correctness and efficiency of genome resequencing with the total additional cost of $ 0.1 and a short incubation step to treat a faecal sample. We also provide methods to assess the enrichment efficiency of mitochondrial and nuclear DNA and models to predict the usability of faecal DNA for genotyping of short tandem repeat, single-nucleotide polymorphism and whole-genome resequencing.

In vitro silencing of the insulin receptor attenuates cellular accumulation of fibronectin in renal mesangial cells.

BACKGROUND: Insulin receptor (InsR) and insulin signaling proteins are widely distributed throughout the kidney cortex. Insulin signaling can act in the kidney in multiple ways, some of which may be totally independent of its primary role of the maintenance of whole-body glucose homeostasis. However, descriptions of the insulin signaling in renal glomerular mesangial cells (MCs) are quite limited and the roles of insulin signaling in MC functions have not been sufficiently elucidated. RESULTS: InsR silencing induced a unique phenotype of reduced fibronectin (FN) accumulation in renal glomerular MCs. Transcription level of FN was not significantly changed in the InsR silenced cells, suggesting the phenotype switching was caused by post-transcriptional modification. The decreased expression of InsR was associated with enhanced activity of insulin-like growth factor-1 receptor (IGF-1R)/PI3K/Akt signaling pathway which contributed in part to the attenuation of cellular FN accumulation. Formation of IGF-1R homodimer was increased in the InsR silenced cells. The InsR silenced cells also showed increased sensitivity to exogenous IGF-1, and increased PI3K activity was reversed significantly by incubating cells with IGF-1R specific antagonist, AG538. PI3K/Akt dependent activation of cAMP responsive element-binding protein (CREB)-1 induced expression of matrix metalloproteinase (MMP)-9 and suppressing MMP activity by doxycycline partially reversed FN accumulation in the InsR silenced cells. CONCLUSIONS: The effects of InsR silencing on cellular FN accumulation in vitro are, at least partially, mediated by increased degradation of FN by MMPs which is induced by enhanced signaling sequence of IGF-1R/PI3K/Akt/CREB-1.

An alternate and reversible method for flight restraint of cranes.

Flight restraint is important for zoos, safaris, and breeding centers for large birds. Currently used techniques for flight restraint include both surgical and non-surgical approaches. Surgical approaches usually cause permanent change to or removal of tendon, patagial membrane, or wing bones, and can cause pain and inflammation. Non-surgical approaches such as clipping or trimming feathers often alter the bird's appearance, and can damage growing blood feathers in fledglings or cause joint stiffness. We observed microstructure of primary feathers of the red-crowned crane (Grus japonensis) and found that the width of barbs is a determinative factor influencing vane stiffness and geometric parameters. We hypothesized that partial longitudinal excision of barbs on the ventral surface of the primary feathers would reduce the stiffness of the vane and render the feathers unable to support the crane's body weight during flight. Furthermore, we hypothesized that this modification of barbs would also change the aerodynamic performance of feathers such that they could not generate sufficient lift and thrust during flapping to enable the bird to fly. We tested this hypothesis on a red-crowned crane that had normal flight capability by excising the ventral margin of barbs on all 10 primaries on the left wing. The bird was unable to take off until the modified feathers were replaced by new ones. Removal of barbs proved to be a simple, non-invasive, low-cost and reversible method for flight restraint. It is potentially applicable to other large birds with similar structural characteristics of primary feathers.

Sex-Biased Gene Expression and Evolution in the Cerebrum and Syrinx of Chinese Hwamei (Garrulax canorus).

It is common that males and females display sexual dimorphisms, which usually result from sex-biased gene expression. Chinese hwamei (Garrulax canorus) is a good model for studying sex-biased gene expression because the song between the sexes is quite different. In this study, we analyze cerebrum and syrinx sex-biased gene expression and evolution using the de novo assembled Chinese hwamei transcriptome. In both the cerebrum and syrinx, our study revealed that most female-biased genes were actively expressed in females only, while most male-biased genes were actively expressed in both sexes. In addition, both male- and female-biased genes were enriched on the putative Z chromosome, suggesting the existence of sexually antagonistic genes and the insufficient dosage compensation of the Z-linked genes. We also identified a 9 Mb sex linkage region on the putative 4A chromosome which enriched more than 20% of female-biased genes. Resultantly, male-biased genes in both tissues had significantly higher Ka/Ks and effective number of codons (ENCs) than unbiased genes, and this suggested that male-biased genes which exhibit accelerated divergence may have resulted from positive selection. Taken together, our results initially revealed the reasons for the differences in singing behavior between males and females of Chinese hwamei.

An efficient pipeline for ancient DNA mapping and recovery of endogenous ancient DNA from whole-genome sequencing data.

Ancient DNA research has developed rapidly over the past few decades due to improvements in PCR and next-generation sequencing (NGS) technologies, but challenges still exist. One major challenge in relation to ancient DNA research is to recover genuine endogenous ancient DNA sequences from raw sequencing data. This is often difficult due to degradation of ancient DNA and high levels of contamination, especially homologous contamination that has extremely similar genetic background with that of the real ancient DNA. In this study, we collected whole-genome sequencing (WGS) data from 6 ancient samples to compare different mapping algorithms. To further explore more effective methods to separate endogenous DNA from homologous contaminations, we attempted to recover reads based on ancient DNA specific characteristics of deamination, depurination, and DNA fragmentation with different parameters. We propose a quick and improved pipeline for separating endogenous ancient DNA while simultaneously decreasing homologous contaminations to very low proportions. Our goal in this research was to develop useful recommendations for ancient DNA mapping and for separation of endogenous DNA to facilitate future studies of ancient DNA.

Forensic validation of a panel of 12 SNPs for identification of Mongolian wolf and dog.

Wolf (Canis lupus) is a species included in appendices of CITES and is often encountered in cases of alleged poaching and trafficking of their products. When such crimes are suspected, those involved may attempt to evade legal action by claiming that the animals involved are domestic dogs (C. l. familiaris). To respond effectively to such claims, law enforcement agencies require reliable and robust methods to distinguish wolves from dogs. Reported molecular genetic methods are either unreliable (mitogenome sequence based), or operationally cumbersome and require much DNA (un-multiplexed microsatellites), or financially expensive (genome wide SNP genotyping). We report on the validation of a panel of 12 ancestral informative single nucleotide polymorphism (SNP) markers for discriminating wolves from dogs. A SNaPshot multiplex genotyping system was developed for the panel, and 97 Mongolian wolves (C. l. chanco) and 108 domestic dogs were used for validation. Results showed this panel had high genotyping success (0.991), reproducibility (1.00) and origin assignment accuracy (0.97 ± 0.05 for dogs and 1.00 ± 0.03 for wolves). Species-specificity testing suggested strong tolerance to DNA contamination across species, except for Canidae. The minimum DNA required for reliable genotyping was 6.25 pg/µl. The method and established gene frequency database are available to support identification of wolves and dogs by law enforcement agencies.

Diversity of Antimicrobial Peptides in Three Partially Sympatric Frog Species in Northeast Asia and Implications for Evolution.

Antimicrobial peptides (AMPs) are evolutionarily ancient molecules that play an essential role in innate immunity across taxa from invertebrates to vertebrates. The evolution system of AMP system has not been well explained in the literature. In this study, we cloned and sequenced AMP transcriptomes of three frog species, namely Rana dybowskii, Rana amurensis, and Pelophylax nigromaculatus, which are partially sympatric in northeast Asia, but show different habitat preferences. We found that each species contained 7 to 14 families of AMPs and the diversity was higher in species with a large geographic range and greater habitat variation. All AMPs are phylogenetically related but not associated with the speciation process. Most AMP genes were under negative selection. We propose that the diversification and addition of novel functions and improvement of antimicrobial efficiency are facilitated by the expansion of family members and numbers. We also documented significant negative correlation of net charges and numbers of amino acid residues between the propiece and mature peptide segments. This supports the Net Charge Balance Hypothesis. We propose the Cut Point Sliding Hypothesis as a novel diversification mechanism to explain the correlation in lengths of the two segments.

Ca2+ entry via alpha1G and TRPV4 channels differentially regulates surface expression of P-selectin and barrier integrity in pulmonary capillary endothelium.

Pulmonary vascular endothelial cells express a variety of ion channels that mediate Ca(2+) influx in response to diverse environmental stimuli. However, it is not clear whether Ca(2+) influx from discrete ion channels is functionally coupled to specific outcomes. Thus we conducted a systematic study in mouse lung to address whether the alpha(1G) T-type Ca(2+) channel and the transient receptor potential channel TRPV4 have discrete functional roles in pulmonary capillary endothelium. We used real-time fluorescence imaging for endothelial cytosolic Ca(2+), immunohistochemistry to probe for surface expression of P-selectin, and the filtration coefficient to specifically measure lung endothelial permeability. We demonstrate that membrane depolarization via exposure of pulmonary vascular endothelium to a high-K(+) perfusate induces Ca(2+) entry into alveolar septal endothelial cells and exclusively leads to the surface expression of P-selectin. In contrast, Ca(2+) entry in septal endothelium evoked by the selective TRPV4 activator 4alpha-phorbol-12,13-didecanoate (4alpha-PDD) specifically increases lung endothelial permeability without effect on P-selectin expression. Pharmacological blockade or knockout of alpha(1G) abolishes depolarization-induced Ca(2+) entry and surface expression of P-selectin but does not prevent 4alpha-PDD-activated Ca(2+) entry and the resultant increase in permeability. Conversely, blockade or knockout of TRPV4 specifically abolishes 4alpha-PDD-activated Ca(2+) entry and the increase in permeability, while not impacting depolarization-induced Ca(2+) entry and surface expression of P-selectin. We conclude that in alveolar septal capillaries Ca(2+) entry through alpha(1G) and TRPV4 channels differentially and specifically regulates the transition of endothelial procoagulant phenotype and barrier integrity, respectively.

Influence of pairwise genetic distance computation and reference sample size on the reliability of species identification using Cyt b and COI gene fragments in a group of native passerines.

Species identification is fundamental to wildlife forensic practice. The desirability of molecular genetic methods is increasing rapidly. The sequence of a marker, rather than its particular diagnostic nucleotides, provides greater safety through comparisons between intra- and inter-specific pairwise genetic distances. However, it has not been well described how reliability of species assignment is influenced by distance computing methods and reference sample sizes. In this study, the influences were tested using 12 species from 4 genera of passerine birds and the sequences of partial Cytochrome b (Cyt b) and Cytochrome Oxidase subunit I (COI) genes. Results showed that different substitution types have different outcomes of pairwise genetic distance estimation and this influences the risk of false inclusion and exclusion. Transition (Ts) is the most effective substitution type to reveal optimal species resolution for both Cyt b and COI gene fragments no matter whether K2P and p-distance are used. Sample size required to accurately estimate pairwise distance is essentially determined by the genetic diversity of a species in reference to a given strictness of predefined acceptable accuracy. These findings suggest that for future forensic work on birds by use of Cyt b and COI gene fragments, transition should be used exclusively for marker validation and identification practice when targeting closely related species. Meanwhile, the reference database should sufficiently represent overall genetic diversity of the species. The minimum sample size should be estimated based on existing knowledge of genetic diversity. Special caution should be used for species assignment when only several reference data are available for animals that are considered likely to have high genetic diversity.

Signals of positive selection in mitochondrial protein-coding genes of woolly mammoth: Adaptation to extreme environments?

The mammoths originated in warm and equatorial Africa and later colonized cold and high-latitude environments. Studies on nuclear genes suggest that woolly mammoth had evolved genetic variations involved in processes relevant to cold tolerance, including lipid metabolism and thermogenesis, and adaptation to extremely varied light and darkness cycles. The mitochondria is a major regulator of cellular energy metabolism, thus the mitogenome of mammoths may also exhibit adaptive evolution. However, little is yet known in this regard. In this study, we analyzed mitochondrial protein-coding genes (MPCGs) sequences of 75 broadly distributed woolly mammoths (Mammuthus primigenius) to test for signatures of positive selection. Results showed that a total of eleven amino acid sites in six genes, namely ND1, ND4, ND5, ND6, CYTB, and ATP6, displayed strong evidence of positive selection. Two sites were located in close proximity to proton-translocation channels in mitochondrial complex I. Biochemical and homology protein structure modeling analyses demonstrated that five amino acid substitutions in ND1, ND5, and ND6 might have influenced the performance of protein-protein interaction among subunits of complex I, and three substitutions in CYTB and ATP6 might have influenced the performance of metabolic regulatory chain. These findings suggest metabolic adaptations in the mitogenome of woolly mammoths in relation to extreme environments and provide a basis for further tests on the significance of the variations on other systems.

Improving Species Identification of Ancient Mammals Based on Next-Generation Sequencing Data.

The taxonomical identification merely based on morphology is often difficult for ancient remains. Therefore, universal or specific PCR amplification followed by sequencing and BLAST (basic local alignment search tool) search has become the most frequently used genetic-based method for the species identification of biological samples, including ancient remains. However, it is challenging for these methods to process extremely ancient samples with severe DNA fragmentation and contamination. Here, we applied whole-genome sequencing data from 12 ancient samples with ages ranging from 2.7 to 700 kya to compare different mapping algorithms, and tested different reference databases, mapping similarities and query coverage to explore the best method and mapping parameters that can improve the accuracy of ancient mammal species identification. The selected method and parameters were tested using 152 ancient samples, and 150 of the samples were successfully identified. We further screened the BLAST-based mapping results according to the deamination characteristics of ancient DNA to improve the ability of ancient species identification. Our findings demonstrate a marked improvement to the normal procedures used for ancient species identification, which was achieved through defining the mapping and filtering guidelines to identify true ancient DNA sequences. The guidelines summarized in this study could be valuable in archaeology, paleontology, evolution, and forensic science. For the convenience of the scientific community, we wrote a software script with Perl, called AncSid, which is made available on GitHub.

Puerarin, an isoflavonoid derived from Radix puerariae, potentiates endothelium-independent relaxation via the cyclic AMP pathway in porcine coronary artery.

Puerarin, an isoflavonoid derived from the Chinese medicinal herb Radix puerariae, has been suggested to be useful in the management of various cardiovascular disorders. The present study examined the effect of acute exposure (30 min) to puerarin on vascular relaxation. Rings from porcine coronary artery of either sex were used. The highest concentration of puerarin (100 microM) produced a small but statistically significant relaxation of U46619-contracted rings. Vascular relaxations were also studied in the presence of lower concentrations of puerarin (0.1, 1 and 10 microM) which had no direct relaxation effect. Puerarin enhanced vasorelaxation to endothelium-independent relaxing agents, sodium nitroprusside and cromakalim. However, puerarin had no effect on vasorelaxation induced by endothelium-dependent relaxing agents, bradykinin and calcium ionophore A23187. The potentiating action of puerarin (10 microM) on sodium nitroprusside-mediated relaxation was not affected by the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME; 300 microM), or by the disruption of the endothelium with Triton X-100. The effect of puerarin was reversible following a washout period. The potentiating effects were comparable with the 3'-5'-cyclic adenosine monophosphate (cyclic AMP) analogues, 8-bromoadenosine-3'-5'-cyclic monophosphate (8-Br-cyclic AMP; 10 muM) and Sp-isomer [S nomenclature refers to phosphorus] of adenosine-3', 5'-cyclic monophosphorothioate (Sp-cyclic AMPS; 3 microM), but not the 3'-5'-cyclic guanosine monophosphate (cyclic GMP) analogue, 8-bromoguanosine-3'-5'-cyclic monophosphate (8-Br-cyclic GMP; 3 microM). The cyclic AMP antagonist, Rp-isomer [R nomenclature refers to phosphorus] of 8-bromoadenosine-3', 5'-cyclic monophosphorothioate (Rp-8-Br-cyclic AMPS; 10 microM), but not cyclic GMP antagonist, Rp-isomer of 8-bromoguanosine-3', 5'-cyclic monophosphorothioate (Rp-8-Br-cyclic GMPS; 10 microM), reversed the effects of puerarin (10 microM) on the enhancement of vasorelaxation to sodium nitroprusside. Our results demonstrated that puerarin enhanced sodium nitroprusside-induced relaxation, possibly via the cyclic AMP-dependent pathway.

Individualization of tiger by using microsatellites.

In investigating criminal cases of poaching and smuggling involving tigers (Panthera tigris), the number of tiger individuals involved is critical for determining the penalty. Morphological methodologies do not often work because tiger parts do not possess the distinctive characteristics of the individual. Microsatellite DNAs have been proved a reliable marker for the individualization of animals. Seven microsatellite loci derived from domestic cat (Felis catus) were selected to individualize tigers, namely F41, F42, F146, Fca304, Fca391, Fca441 and Fca453. A reference population containing 37 unrelated tigers were used to investigate alleles, allelic frequencies, genotypes and genotype frequencies of each locus. Consequently, the data was used to assess the validity of the combination of seven loci for tiger individualization. All loci were polymorphic and easy to amplify. Three out of the seven loci were significantly departure from the Hardy-Weinberg Equilibrium (P < 0.05). Cumulative discrimination power (DP) calculated with observed genotype frequencies was 0.99999789. Match probability of an individual in the reference population with a random individual in seven loci ranged from 7.34 x 10(-9) to 2.77 x 10(-5). This suggests that combining the seven microsatellite loci provides desirable power to individualize tigers. The combination of seven loci was applied to a case of tiger bone smuggling. Genotypes of all samples were identical in all seven loci, and the P(M) of the evidence samples in the seven loci hit 5.63 x 10(-7), provided evidence that the bones belong to a single tiger.

Effectiveness of femur bone indexes to segregate wild from captive minks, Mustela vison, and forensic implications for small mammals.

Utilization of free-living populations of endangered wildlife species is usually strictly prohibited or restricted. Farming of endangered species can provide products that are in demand as a countermeasure. A novel forensic issue arises because it becomes necessary to discriminate the origin of given wildlife products. We tested the effectiveness of five measurements and four indexes of femur bone using farmed minks (n = 40) and escapees (n = 32). Results showed all measurements, namely body mass (L(f)), body length (M(f)), femur mass (V(f)), femur length (M(b)), and femur volume (L(b)), were highly discriminatory. However, they are susceptible to the influence of nutrition level and sex. Femur length index (I(fl)), femur linear density (D(l)), and femur volume density (D(v)) eliminated the influence of level of nutrition and were highly effective. However, I(fl) and D(l) were influenced by sex (p = 0.000). Because D(v) was not influenced by sex (p = 0.683) and was highly effective, it was the preferred index.

[Chemical constituents of Geranium eristemon].

OBJECTIVE: To study the chemical constituents of Geranium eristemon. METHOD: Chromatography and spectral analysis were used to isolate the constituents and elucidate their structures. RESULT: Five compounds were isolated from acetone extract of the whole grass of G. eristemon, and identified as beta-sitosterol, protocatechuic acid, myricetin, kaempferol-7-O-alpha-L-arabifuranoside and kaempferol-3-O-alpha-L-arabifuranoside. CONCLUSION: kaempferol-7-O-alpha-L-arabifuranoside and kaempferol-3-O-alpha-L-arabifuranoside were isolated from G. genus for the first time.