[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).

Effects of spider Macrothele raven venom on cell proliferation and cytotoxicity in HeLa cells.

AIM: To examine the effect of venom from the spider Macrothele raven on cell proliferation and cytotoxicity in human cervical carcinoma, HeLa cells. METHODS: Morphological and biochemical signs of apoptosis appeared using acridine orange-ethidium bromide (AO/EB) staining. Marked morphological changes in HeLa cells after treatment with spider venom were observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Cell proliferation and cytotoxicity were determined by [methyl-3H] thymidine assay ([3H]TdR) and lactate dehydrogenase (LDH) release, respectively. DNA fragmentation and cell cycle distribution were monitored using flow cytometry. In addition, Western blot analysis was used to evaluate the level of caspase-3 expression. In vivo examination of the inhibition of the size of tumors in nude mice treated with spider venom was measured. RESULTS: Marked morphological changes were observed using AO/EB staining, SEM and TEM assay. Spider venom at concentrations of 10-40 mg/L caused dose- and time-dependent inhibition of HeLa cell proliferation. The ratio of apoptosis and necrosis increased. The activity of caspase-3 was upregulated after spider venom treatment. In vivo study of tumor size revealed that tumors significantly decreased in size from controls to tumors treated for 3 weeks with spider venom (P<0.05). CONCLUSION: The inhibition of HeLa cells by the venom of the spider Macrothele raveni was carried out in three ways: induction of apoptosis, necrosis of toxicity damage and direct lysis. Spider venom is a novel anti-tumor material both in vitro and in vivo.

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.