To provide technical support for spider silk functional modification, we developed a simple and efficient functional platform via intein trans-splicing. Small ubiquitin-related modifier protein (SUMO) was fused to the recombinant spider silk protein (W2CT) by peptide bond via S0 split intein Ssp DnaB trans-splicing, resulting in a protein SUMOW2CT. However, incorporation of exogenous protein led to mechanical property defect and lower fiber yield, and also slowed down the fiber assembly velocity but no obvious differences in supercontraction and chemical resistance when compared with fibers from W2CT (W). SUMO protease digestion showed positive results on the fibers, indicating that the SUMO protein kept its native conformation and bioactive. Above all, this work provides a technical support for spider silk high simply and efficient functionalized modification.
Animals ; Inteins ; Protein Splicing ; Recombinant Proteins ; chemistry ; Silk ; chemistry ; Small Ubiquitin-Related Modifier Proteins ; chemistry ; Spiders ; Trans-Splicing

In the present study, 13 clinical cases of canine mammary adenocarcinoma were evaluated in order to understand the effect of Tarantula cubensis extract (TCE) on tumor tissue. Punch biopsies were taken from the tumors before treatment with TCE. Subcutaneous injections of TCE were administered three times at weekly intervals (3 mL per dog). Between days 7 and 10 after the third injection, the tumor masses were extirpated by complete unilateral mastectomy. Pre- and post-treatment tumor tissues were immunohistochemically assessed. The expression of B-cell lymphoma 2 (Bcl-2) was found to be higher in pre-treatment compared to post-treatment tissues (p < 0.01) whereas Ki-67 expression was lower in post-treatment tissues (p < 0.01). No significant differences in fibroblast growth factor or vascular endothelial growth factor expression were observed between pre- and post-treatment tissues (p > 0.05). The apoptotic index was determined to be low before treatment and increased during treatment. These results suggest that TCE may be effective for controlling the local growth of canine mammary adenocarcinoma by regulating apoptosis.
Adenocarcinoma/*drug therapy/physiopathology ; Animals ; Apoptosis/drug effects ; Dog Diseases/*drug therapy/physiopathology ; Dogs ; Female ; Mammary Neoplasms, Animal/*drug therapy/physiopathology ; Mammary Neoplasms, Experimental/*drug therapy/physiopathology ; Mitosis/drug effects ; Spiders/*chemistry

Due to its special sequence structure, spider silk protein has unique physical and chemical properties, mechanical properties and excellent biological properties. With the expansion of the application value of spider silk in many fields as a functional material, progress has been made in the studies on the expression of recombinant spider silk proteins through many host systems by gene recombinant techniques. Recombinant spider silk proteins can be processed into high performance fibers, and a wide range of nonfibrous morphologies. Moreover, for their excellent biocompatibility and low immune response they are ideal for biomedical applications. Here we review the process and mechanism of preparation in vitro, chemistry and genetic engineering modification on recombinant spider silk protein.
Animals ; Arthropod Proteins ; chemistry ; Protein Engineering ; Recombinant Proteins ; chemistry ; Silk ; chemistry ; Spiders

A total of 631 species of spiders have been reported in Korea. However, there are no spiders with noxious venom such as Latrodectus sp. Because of this, to date, no serious medical problems due to spider bites have been reported in Korea, and only two cases of spider bite were officially reported. However, as the number of earning spiders from other countries is increased, the number of cases of spider bite has also shown a recent increase. A 17-year-old man presented with numbness of both extremities after being bitten by a spider which he had as a pet. The spider is called an Indian ornamental tree spider (Poecilotheria regalis sp.), one kind of tarantula species. Herein, we report on a case of a spider bite by a tarantula.
Adolescent ; Extremities ; Humans ; Hypesthesia ; Korea ; Spider Bites* ; Spider Venoms ; Spiders ; Venoms

To study the effect of physiological conditions on spidroins, we analyzed NTR1SR2CT module secondary structure, aggregation and silk-formation influenced by different salts (in different concentration intervals). According to the full-length Araneus ventricosus MiSp sequence, NTR1SR2CT module was constructed and expressed in Escherichia coli BL21 (DE3), and the recombinant proteins were purified by denaturation method mediated by 8 mol/L urea. Random coil and helix are the main secondary structures of NTR1SR2CT and could be induced into beta-sheet by drying natively and lyophilization, where methanol can be used as a promoter. Furthermore, potassium and phosphate cations can cause significant NTR1SR2CT protein aggregation and silk-formation. The results could be a basis for the determination of silk-formation mechanism, and also useful for industrialized generation of high performance spider silk-like fibers.
Animals ; Fibroins ; chemistry ; Protein Structure, Secondary ; Salts ; chemistry ; Spiders

PURPOSE: To describe our initial experience with the second-generation Single Port Instrument Delivery Extended Reach (SPIDER) laparoendoscopic single-site surgical system in a porcine model. MATERIALS AND METHODS: In four swine weighing approximately 32 to 35 kg, five nephrectomies, four adrenalectomies, three pyeloplasties, and three partial cystectomies and closures were performed by a single surgeon. The swine were placed in the lateral flank position under general anesthesia. The SPIDER surgical system was introduced through a single incision and the various urological procures were performed by use of flexible instrumentation. RESULTS: All five nephrectomies, four adrenalectomies, three pyeloplasties, and three partial cystectomies and closures were performed successfully without additional skin incisions. The mean time to set up the SPIDER platform was 3.5 minutes. The mean operative time for the right and left nephrectomies was 45.4 minutes and 47.8 minutes, respectively. The mean operative time for the right and left adrenalectomies was 37.6 minutes and 35.4 minutes, respectively. The mean operative time for the pyeloplasties for one right and two left ureters was 45.6 minutes and 47.3 minutes, respectively. The mean operative time for the partial cystectomies and closures was 18.6 minutes. There were no noticeable intraoperative complications except for minimal urine leakage in the first pyeloplasty. CONCLUSIONS: In this initial pilot evaluation, the second-generation SPIDER surgical system offered intuitive instrument maneuverability and restored triangulation. However, retraction was challenging because of the lack of strength and the limited ability for precise manipulation of the tip. Future refinements of the technology and prospective studies are needed to optimize the application of this technology in urology.
Adrenalectomy ; Anesthesia, General ; Cystectomy ; Intraoperative Complications ; Laparoscopy ; Nephrectomy ; Operative Time ; Robotics ; Skin ; Spiders ; Swine ; Ureter ; Urologic Surgical Procedures ; Urology

A repetitive DNA fragment, named P1, was amplified by PCR with the full-length Minor Ampullate Spidroin gene sequence of Araneus ventricosus as template. P1 was ligated with pPic3.5 and PKT expression vectors and transferred into GS115 and BL21(DE3) competence cells, respectively. SDS-PAGE and Western blot were used to analyze the recombinant his-tag fusion protein. With expressed in different expression systems, soluble P1 induced proteins could be obtained as the same size. Furthermore, the expression level and purification recovery efficiency were also higher in GS115 than that of BL21(DE3). Additionally, the expression level could be improved after optimizing the incubation and induction conditions of GS115. In this research, Pichia pastoris expression system is more suitable for the native repetitive Gly/Ala-rich spider spidroin gene sequence expression than Escherichia coli system. The data can help the native full-length MiSp gene expression and large-scale exploitation of recombinant of spider silk proteins.
Animals ; Escherichia coli ; genetics ; metabolism ; Fibroins ; biosynthesis ; classification ; genetics ; Genetic Vectors ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; Spiders ; classification ; genetics ; metabolism

Huwentoxin-XI (HWTX-XI) is a protein isolated from the crude venom of spider Ornithoctonus huwena. It has 55 amino acid residues containing 6 cysteine residues forming 3 disulfide bonds. It shows potent inhibitory effect on trypsin and voltage-gated potassium channels in rat dorsal root ganglion cells. According to the structure-function relationship of HWTX-XI, we designed two mutants through mutation of potassium channel inhibition related amino acid residues (R5I, R10T,R25A and R5I,R25A) and then expressed them with high purity by using the vector pVT102U on Saccharamyces cerevisiae strain S78; The two mutants had the same trypsin inhibition activity as HWTX-XI, whereas their potassium channel inhibition activity and animal toxicity were much lower than those of HWTX-XI. This study is helpful for designing drugs of trypsin related diseases based on HWTX-XI.
Amino Acid Sequence ; Animals ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Mutant Proteins ; biosynthesis ; genetics ; pharmacology ; Potassium Channel Blockers ; pharmacology ; Rats ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Saccharomyces cerevisiae ; genetics ; metabolism ; Spider Venoms ; biosynthesis ; genetics ; pharmacology ; Spiders ; Trypsin Inhibitors ; pharmacology

In Korea, a total of 44 families, 217 genus, and 631 species of spiders are reported. However there are no spiders having noxious venom such as Latrodectus sp. or Loxoscles sp. Because of this, there have been no reported serious medical problems due to spider bites in Korea up to now. Paracoelotes spinivulva is a spider belonging to Family Amaurobiidae and Genus Paracoelotes. It is characteristically distributed in Korea and some regions of Japan, China, and Russia. A 46-year-old man presented with two painful, erythematous papules on the left index finger tip. He was bitten by a spider three days prior to the visit. Punch biopsy was done from the bite site. Although spider bites may be relatively common, there is only one official report of a spider bite in Korea. Herein, we report a case of a spider bite by a indigenous spider, Paracoelotes spinivulva.
Arachnidism ; Biopsy ; Bites and Stings ; China ; Fingers ; Humans ; Japan ; Korea ; Middle Aged ; Russia ; Spiders ; Venoms

Jingzhaotoxin-V(JZTX-V) isolated from the venom of the spider Chilobrachys jingzhao is a novel potent inhibitor that acts on tetrodotoxin-resistant and tetrodotoxin-sensitive sodium channels in adult rat dorsal root ganglion(DRG) neurons. It is a 29-residue polypeptide toxin including three disulfide bridges. To investigate the structure-function relationship of the toxin, a mutant of JZTX-V in which Arg20 was substituted by Ala, was synthesized by solid-phase chemistry method with Fmoc-protected amino acids on the PS3 automated peptide synthesizer. The synthetic linear peptide was then purified by reversed-phase high performance liquid chromatography and oxidatively refolded under the optimal conditions. The refolded product was analyzed by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry(MALDI-TOF MS) and electrophysiological experiments for its relative molecular weight and prohibitive activity of sodium channels respectively. The present findings show that the prohibitive effect of R20A-JZTX-V on TTX-S sodium channels in DRG neurons is almost the same as that of native JZTX-V, suggesting that Arg20 does not play any important role in inhibiting TTX-S sodium currents in DRG neurons. In contrast, the prohibitive level of R20A-JZTX-V on TTX-R sodium channels is reduced by at last 18.3 times, indicating that Arg20 is a key amino acid residue relative to the bioactivity of JZTX-V. It is presumed that the decrease in activity of R20A-JZTX-V is due to the changes of the property in the binding site in TTX-R sodium channels.
Amino Acid Substitution ; Animals ; Arginine ; genetics ; Ganglia, Spinal ; drug effects ; Mutagenesis, Site-Directed ; Mutant Proteins ; pharmacology ; Neurons ; drug effects ; Patch-Clamp Techniques ; Peptides ; chemistry ; genetics ; pharmacology ; Rats ; Sodium Channel Blockers ; pharmacology ; Sodium Channels ; drug effects ; Spider Venoms ; chemistry ; genetics ; isolation & purification ; pharmacology ; Spiders ; Tetrodotoxin ; pharmacology