Genetic structure and demographic history should inform conservation: Chinese cobras currently treated as homogenous show population divergence.

An understanding of population structure and genetic diversity is crucial for wildlife conservation and for determining the integrity of wildlife populations. The vulnerable Chinese cobra (Naja atra) has a distribution from the mouth of the Yangtze River down to northern Vietnam and Laos, within which several large mountain ranges and water bodies may influence population structure. We combined 12 microsatellite loci and 1117 bp of the mitochondrial cytochrome b gene to explore genetic structure and demographic history in this species, using 269 individuals from various localities in Mainland China and Vietnam. High levels of genetic variation were identified for both mtDNA and microsatellites. mtDNA data revealed two main (Vietnam + southern China + southwestern China; eastern + southeastern China) and one minor (comprising only two individuals from the westernmost site) clades. Microsatellite data divided the eastern + southeastern China clade further into two genetic clusters, which include individuals from the eastern and southeastern regions, respectively. The Luoxiao and Nanling Mountains may be important barriers affecting the diversification of lineages. In the haplotype network of cytchrome b, many haplotypes were represented within a "star" cluster and this and other tests suggest recent expansion. However, microsatellite analyses did not yield strong evidence for a recent bottleneck for any population or genetic cluster. The three main clusters identified here should be considered as independent management units for conservation purposes. The release of Chinese cobras into the wild should cease unless their origin can be determined, and this will avoid problems arising from unnatural homogenization.

A new genus and two new species of the subfamily Pauropodinae (Myriapoda: Pauropoda: Pauropodidae) from China.

A new genus Songius is established and two new species--Songius rugosus from Qixia Mountain and Laoshan Forest Park, Jiangsu, and Tiantangzhai, Dabie Mountain, Anhui, and Songius bicruris from Tiantangzhai--are described. A novel surface structure of the pygidial tergum was observed by scanning electron microscopy. The genus is established on the basis of the distinctive appearance of the modification of the surface structure of the pygidial tergum.

Sequence polymorphism and evolution of three cetacean MHC genes.

Sequence variability at three major histocompatibility complex (MHC) genes (DQB, DRA, and MHC-I) of cetaceans was investigated in order to get an overall understanding of cetacean MHC evolution. Little sequence variation was detected at the DRA locus, while extensive and considerable variability were found at the MHC-I and DQB loci. Phylogenetic reconstruction and sequence comparison revealed extensive sharing of identical MHC alleles among different species at the three MHC loci examined. Comparisons of phylogenetic trees for these MHC loci with the trees reconstructed only based on non-PBR sites revealed that allelic similarity/identity possibly reflected common ancestry and were not due to adaptive convergence. At the same time, trans-species evolution was also evidenced that the allelic diversity of the three MHC loci clearly pre-dated species divergence events according to the relaxed molecular clock. It may be the forces of balancing selection acting to maintain the high sequence variability and identical alleles in trans-specific manner at the MHC-I and DQB loci.

[Cloning and prokaryotic expression of major ampullate spidroin gene of spider].

RT-PCR was conducted with one degenerate primer designed according to repetitive regions' amino acid sequence of major ampullate spidroin (MaSp) in spiders and adaptor primer in the SMART cDNA Library Construction Kit. By cloning and sequencing of amplified products, one cDNA clone (GenBank Accession No. AY365017) of Argiope amoena MaSp gene was obtained. The deduced amino acid sequence can be distinctly divided into two regions: (1) Repetitive region that consists of an alternating alanine-rich and glycine-rich domain in which many prolines are present; and (2) C-terminal non-repetitive region. The region coding for 272 amino acids of MaSp gene was subcloned into prokaryotic expression vector pET28b(+) and an about 26kD recombinant protein was expressed at high levels in Escherichia coli BL21 (DE3) after induction of IPTG. After being purified with metal-affinity chromatography on Ni(2+) -IDA-Sepharose columns as well as gel filtration chromatography, the recombinant protein was confirmed to be predicted MaSp by means of amino acid composition analysis and N-terminal amino acid sequence analysis. The solubility behavior of recombinant MaSp with C-terminal non-repetitive region in the present study is similar to that of recombinant dragline silk proteins without C-terminal non-repetitive region expressed by bacteria and yeast in the other studies. The result shows that absence or presence of C-terminal non-repetitive region is not a crucial factor affecting the solubility of the recombinant MaSp.

[Antibacterial activity of water soluble fraction from Scolopendra subspinipes mutilans].

The water soluble fraction (SWSF) of centipede Scolopendra subspinipes mautilans, injected with Escherichia coli K12 D31 for 3-4 days showed broad-spectrum antimicrobial activity against Gram-positive, Gram-negative bacteria and fungi. It showed strong antibacterial activity against E. coli K12D31 at different temperatures, pH and ionic strengths. It did not show any hemolytic and agglutination activities at the concentration below 600 microg/ml. After E. coli K12 D31 treated with SWSF, the ultrastructure showed that its outer cell wall was broken, surface collapsed and intracellular substances leaked out.

Myriapod monophyly and relationships among myriapod classes based on nearly complete 28S and 18S rDNA sequences.

Myriapods play a pivotal position in the arthropod phylogenetic tree. The monophyly of Myriapoda and its internal relationships have been difficult to resolve. This study combined nearly complete 28S and 18S ribosomal RNA gene sequences (3,826 nt in total) to estimate the phylogenetic position of Myriapoda and phylogenetic relationships among four myriapod classes. Our data set consists of six new myriapod sequences and homologous sequences for 18 additional species available in GenBank. Among the six new myriapod sequences, those of the one pauropod and two symphylans are very important additions because they were such difficult taxa to classify in past molecular-phylogenetic studies. Phylogenetic trees were constructed with maximum parsimony, maximum likelihood, and Bayesian analyses. All methods yielded moderate to strong support for the monophyly of Myriapoda. Symphyla grouped strongly with Pauropoda under all analytical conditions. The KH test rejected the traditional view of Dignatha and Progoneata, and the topology obtained here, though not significantly supported, was Diplopoda versus ((Symphyla + Pauropoda) + Chilopoda).

[Molecular identification of medicinal plants: Dendrobium chrysanthum, Dendrobium fimbriatum and their morphologically allied species by PCR-RFLP analyses].

AIM: To establish a simple method for molecular identification of original plants of D. chrysanthum and D. fimbriatum using molecular marker rDNA ITS region. METHODS: Restriction patterns of ITS fragments were obtained using PCR-RFLP method. The PCR products of D. chrysanthum and its morphologically allied species were digested at 37 degrees C by Cla I and Apa LI, those of D. fimbriatum and its morphologically allied species were digested by Sph I. RESULTS: D. chrysanthum, D. fimbriatum and their morphologically allied species could be identified by predicted restriction profiles of PCR-RFLP. The botanical origin of twenty-five fresh samples of "Shihu" collected in markets was identified by this method. CONCLUSION: The results showed that PCR-RFLP analysis of the rDNA ITS region is a feasible, simple and inexpensive method for determining the botanical origin of the traditional Chinese medicine "Shihu".

Relationship between lipocalin-type prostaglandin D synthase and alpha-glucosidase in azoospermia seminal plasma.

BACKGROUND: [corrected] To determine the correlation of lipocalin-type prostaglandin D synthase (L-PGDS) and alpha-glucosidase in semen. METHODS: We analyzed 68 seminal plasmas for lipocalin-type prostaglandin D synthase (L-PGDS) and alpha-glucosidase, L-PGDS was analyzed by ELISA. The semen donors were categorized in 3 groups: normal, obstructive and non-obstructive azoospermia. We then evaluated their correlation. RESULTS: The difference of L-PGDS concentration (P<0.001) and alpha-glucosidase activity (P<0.001) among the 3 clinical groups was statistically significant. Correlation between L-PGDS concentration and alpha-glucosidase was also statistically significant. L-PGDS concentration correlated positively with alpha-glucosidase activity (r=0.882). CONCLUSIONS: L-PGDS in seminal plasma, like alpha-glucosidase, suggests an obstruction of the seminal ducts and may be a potential marker that may aid in the differential diagnosis of obstructive and non-obstructive azoospermia.

Phylogenetic placement of the spider genus Nephila (Araneae: Araneoidea) inferred from rRNA and MaSp1 gene sequences.

The family status of the genus Nephila, which belongs to Tetragnathidae currently but Araneidae formerly, was reexamined based on molecular phylogenetic analyses. In the present study, 12S and 18S rRNA gene fragments of eight species of spiders were amplified and sequenced. In addition, 3'-end partial cDNA of major ampullate spidroin-1 (MaSp1) gene of Argiope amoena was cloned and sequenced, and the 3'-end non-repetitive region's cDNA sequence of MaSp1 gene and the predicted amino acid sequence of C-terminal non-repetitive region of MaSp1 were aligned with some previously known sequences. The resulting phylogeny showed that Araneidae and Tetragnathidae are not a sister group in the superfamily Araneoidea, and the genus Nephila is closer to the genera of the family Araneidae rather than to those of Tetragnathidae. We suggest that the genus Nephila should be transferred back to Araneidae. Or the subfamily Nephilinae might be elevated to family level after it was redefined and redelimited. Furthermore, the study showed that 3'-end non-repetitive region's cDNA sequence of MaSp1 gene and C-terminal non-repetitive region's amino acid sequence of MaSp1 are useful molecular markers for phylogenetic analysis of spiders.

Aberrant expression and function of TCF4 in the proliferation of hepatocellular carcinoma cell line BEL-7402.

Wnt signaling pathway is essential for development and tumorigenesis, however, this signaling pathway in the progress of hepatocellular carcinoma (HCC) remains unclear. In this paper, we studied the function of human T-cell transcription factor-4 (TCF4), a key factor of Wnt signaling pathway, on the proliferation of HCC cell line. We showed that the expression of TCF4 mRNA in HCC cell line BEL-7402 was higher than that in immortalized normal liver cell line L02. Blockage of Wnt pathway by Delta-NTCF4, a dominant negative TCF4, could suppress BEL-7402 cells growth and decrease the expression of cyclin D1 and c-myc, two of target genes of Wnt pathway. On the other hand, stimulating Wnt pathway by introducing a degradation-resistant -catenin S37A could increase BEL-7402 cells proliferation. But all the treatments had no effect on L02 cells. Our data indicated that TCF4 might be another key factor in Wnt pathway involved in HCC cells proliferation and TCF4 could be an effective therapeutic target for suppressing the growth of hepatocellular cancers.

[Population genetic structure of Pelophylax nigromaculata in Chinese mainland based on mtDNA control region sequences].

Molecular genetic data were used to investigate population genetic structure and differentiation of Pelophylax nigromaculata (Anura: Ranidae). Frogs were collected from 12 localities across Chinese mainland. Sample sizes of up to 10 frogs per population were assayed for mitochondrial control region sequence variation. The aligned 685 bp of the 5'CR include 111 variable sites. Sixty-seven haplotypes were defined. Most of the haplotypes are unique to local populations, only 7 of the 67 are shared among a few local populations. The overall species haplotype diversity is quite high (h = 0.98 +/- 0.005), and the nucleotide diversity is also high (pi = 0.0303 +/- 0.0029). This is corresponding to huge population size and extensive distribution of Pelophylax nigromaculata throughout Palaearctic and Oriental Regions. Phylogenetic tree of mtDNA control region haplotypes based on maximum parsimony algorithm and reconstructed phylogenetic relationships among the local populations based on neighbor joining algorithm all suggest that the haplotypes of Jiling and Liaoning populations in northeastern China have a sister relationship with 10 local populations in northern, central, southern and southwestern China. The analysis of molecular variance (AMOVA), the pairwise FST values and nucleotide divergence all support significant population subdivision between the Jilin-Liaoning group and multi-population group. The most possible cause of the significant genetic subdivision between the two groups might be referred to the effect of Quaternary glaciation. The possible reasons for the lack of obvious geographic structure on the whole in the multi-population group and the genetic differentiation in some local populations of the multi-population group were also discussed.

Expression of a novel bHLH-Zip gene in human testis.

AIM: To identify specifically expressed genes in the adult and fetal testes. METHODS: A human testis cDNA microarray was established. Then the mRNA of adult and fetal testis was purified and probes were prepared by a reverse transcription reaction with the testis mRNA as template. The microarray was hybridized with probes of adult and fetal testes. The nucleic sequences of differentially expressed genes were determined and homologies were searched in the databases of the GenBank. RESULTS: When hybridized with adult or fetal testis probes, the positive clones were 96.8 % and 95.4 %, respectively. Among these genes, one was a new testis-specific gene, which was named TSP1. TSP1 was highly expressed in human adult testis. The cDNA of TSP1 was 1,484 bp in length. The cDNA sequence of this clone was deposited in the Genbank (AF333098). TSP1 was also determined as Interim Gen Symbol (Unigene, No. Hs.98266). Protein analysis showed that TSP1 contained two functional domains: an N-terminal basic helix-loop-helix (bHLH) and a C-terminal leucine zipper (Zip). Homologous analysis showed that the 430 amino acid sequences deduced from the 1293 bp open reading frame (ORF) had a homology with the human gene FLJ2509 (AK098575). TSP1 had also a sequence homology with Spz 1 protein of mouse. Expression profiles showed that TSP1 was specifically and strongly expressed in the testis. CONCLUSION: TSP1 is a gene highly expressed in adult testis. It may play an important role in spermatogenesis in the humans.

[RbcL sequence analysis of Belamcanda chinensis and related medicinal plants of Iris].

AIM: To identify "Shegan" [Belamcanda chinensis (L.) DC.] and relative medicinal plants of Iris including Iris tectorum Maxim., I. dichotoma Pall., I. germanica L. and I. japonica Thunb. by ribulose 1,5-bisphosphate carboxylase Large Gene (rbcL) sequence analysis. METHODS: General DNA was isolated from the fresh leaves of Belamcanda chinensis and 4 Iris spp. by CTAB. A pair of primers was designed to amplify the rbcL gene and PCR Preps DNA kit was used to purify the PCR products. The rbcL sequences were determined by ABI (Applied Biosystems Inco.) Prism 310 Genetic Analyzer. RESULTS: A fragment of about 750 bp of rbcL gene from Belamcanda chinensis and 4 Iris spp. were amplified and sequenced. The rbcL sequences of Iris tectorum, I. dichotoma Pall. and I. japonica were reported for the first time. The rbcL sequences of 5 species of Iridaceae were aligned and analyzed using Clustal (Version 8.0) and MEGA (Version 2.0.) programs. The nucleotide number of difference is from 1.000 to 20.000. The tranversions is from 0.000 to 9.000 and the transitions is from 0.000 to 14.000. Phylogenetic tree based on rbcL partial sequence data indicated that the eleven samples of 5 species clustered separately. CONCLUSION: The sequence variation of rbcL can be used to identify Belamcanda chinensis and 4 species of relative medicinal plants of Iris. The molecular phylogenetic tree accords with the classical taxonomy.

[Study on sequence difference and SNP pheomenon of rDNA ITS region in F type and H type population of Dendrobium officinale].

OBJECTIVE: To study rDNA ITS sequence differences between F type and that of H type of Dendrobium officinale in main habitat of China. METHOD: The population differences of the rDNA ITS region (including ITS1, ITS2, 5.8S) sequences of D. officinale were studied by the method of DNA sequences analysis. RESULT: There were two different sites between the rDNA ITS sequence of F type and that of H type. One was in ITS1 region, and the other was in 5.8S region. It was proved that there was some relativity between the character of rDNA ITS region and the life type of the populations. The phenomenon of single nucleotide polymorphism (SNP) existed in 5.8S region of rDNA ITS region between F type and H type. The sequences of rDNA ITS region of D. officinale were reported for the first time, and the sequences of ITS region ranged 634 bp (ITS1 231 bp, ITS2 240 bp, 5.8S 163 bp). CONCLUSION: The analysis of rDNA ITS of D. officinale deeply reveal the population differences of D. officinale of F type and H type.

[Molecular authentication of Dendrobium chrysanthum from its allied species of Dendrobium].

OBJECTIVE: To define molecular characters to distinguish D. chrysanthum from its allied species D. primulinum, D. lituiflorum, D. aphyllum, D. crepidatum. METHOD: The molecular characteristics of D. chrysanthum and its allied species were compared. The sequences of rDNA ITS regions were exploited to explore the evidence for authentication D. chrysanthum and its allied species. RESULT: Although the morphological difference was slight, the sequence difference of ITS regions among five rDNAs was obvious and stable. Fifteen sites of ITS region were defined as DNA character to identify D. chrysanthum from the other four allied species. CONCLUSION: The difference of rDNA ITS sequences can be used to authenticate accurately D. chrysanthum from three allied species of Dendrobium.

[Database establishment of the whole rDNA ITS region of Dendrobium species of "fengdou" and authentication by analysis of their sequences].

AIM: To establish the whole rDNA ITS region sequence database of various Dendrobium species of "Fengdou" and to authenticate exactly the inspected species of "Fengdou". METHODS: The rDNA ITS regions of various Dendrobium species of "Fengdou" were amplified and sequenced. The database of their rDNA ITS regions was established in order to authenticate the inspected species by means of the softwares of CLUSTRAL and MEGA which were used to analyze the rDNA ITS region. RESULTS: A database of the rDNA ITS sequences of 21 species of Dendrobium has been established. The notable and stable differences of the interspecies of the rDNA ITS regions have been demonstrated. The numbers of transitions and transversions among 21 species are 11-122. The variable sites are 341 while the informative sites are 195. The ITS sequence differences between the outgroup species (Pholidota yunnanensis) and species of "Fengdou" are obvious. The numbers of transitions and transversions are 131-161. The population differences of the rDNA ITS region of various species of "Fengdou" are very small (0-6). CONCLUSION: On the basis of the database of various Dendrobium species of "Fengdou" and two genetics software, the botanical origin of the inspected species of "Fengdou" has been authenticated successfully by sequencing the rDNA ITS regions.

Cloning, Expression, Purification and Activity of the hsBLyS.

The full length cDNA of human B lymphocyte stimulator (hBLyS) was amplified by using PCR method from cDNA library of human placenta. After purifying and sequencing, the DNA fragment of functional domain of hBLyS (hsDNA fragment)was amplified by using nested PCR method from the PCR product. The prokaryotic expression plasmid pET-30a( )/ hBLyS was constructed with recombinant DNA techniques after purifying and identifying the hsDNA fragment. Then the plasmid pET-30a( )/ hBLyS was transformed into lambdaDE3 cells and the recombination protein was found to be highly expressed the expression product was purified by affinity chromatography gel, Ni(2 )-IDA, made in our laboratory. The experimental results showed that the sequence of the PCR product was identical with the published hBLyS cDNA sequence and purity of the recombination protein we obtained was high. The activity of the purified recombination protein was very significant in the proliferation test of B lymphocytes.