Role of corticotropin-releasing factor on bladder function in rats with psychological stress.

Stress-related peptide corticotropin-releasing factor (CRF) and CRF-related peptides are distributed in the peripheral viscera such as the bladder. We investigated the contribution of psychological stress (PS) and CRF on bladder function. Male rats received sham stress (SS) or PS using a communication box method for 120 min every day for 7 days. One group of rats received the intraperitoneal CRF-R1 antagonist antalarmin for 7 days during stress exposure. Mean voided volume per micturition was significantly lower in PS rats compared to SS rats, which was antagonized by antalarmin treatment. Increases in plasma and bladder CRF, and mRNA expressions of bladder CRF, CRF-R1, and M2/3 muscarinic receptors, were found in PS rats. CRF did not influence bladder contraction in itself; however, stress increased the response of muscarinic contraction of bladder strips. These changes were antagonized by antalarmin treatment. In conclusion, PS reinforces M3 receptor-mediated contractions via CRF-R1, resulting in bladder storage dysfunction.

Synthesis of silk fibroin-insulin bioconjugates and their characterization and activities in vivo.

The regenerated liquid silk fibroin with an average molecular mass of about 60 kDa consists of 18 kinds of amino acids containing approximately 10% of polar amino acids with hydroxyl and amino groups such as serine and lysine. The liquid silk fibroin is coupled covalently with insulin molecules through these strongly polar side groups by using glutaraldehyde. The physicochemical properties of the silk fibroin-insulin (SF-Ins) bioconjugates were investigated by enzyme-linked immunosorbent assay for the quantitative measurement of insulin. The biological activities of the insulin bioconjugates were characterized in vitro and in vivo. The SF-Ins constructs obtained by 5 h of covalent crosslinking showed much higher recovery (about 70%) and in vitro stability in human serum than bovine serum albumin-insulin (BSA-Ins) derivatives. The results in human serum indicated that the half-life in vitro of the biosynthesized SF-Ins derivatives was 2.1 and 1.7 times more than that of BSA-Ins conjugates and native insulin, respectively. The immunogenicity of the regenerated silk fibroin and the antigenicity of silk fibroin-modified insulin were not observed in both rabbits and rats. The pharmacological activity of the SF-Ins bioconjugates in diabetic rats evidently lengthened and was about 3.5 times as long as that of the native insulin, nearly 21 h. The bioconjugation of insulin with the regenerated silk fibroin greatly improved its physicochemical and biological stability.

Synthesis, characterization and immunogenicity of silk fibroin-L-asparaginase bioconjugates.

L-asparaginase (ASNase) is one basic drug in the treatment of acute lymphoblastic leukemia (ALL). Because its half-life time is too short and it is easy to arouse allergic reaction, use in practical clinic is considerably limited. Silk fibroin (SF) with different molecular mass from 40 to 120 kDa is a natural biocompatible protein and could be used as a novel bioconjugate for enzyme modification to overcome its usual shortcomings mentioned above. When the enzyme was bioconjugated covalently with the water-soluble fibroin by glutaraldehyde, the enzyme kinetic properties and immune characteristics in vivo of the resulting silk fibroin-L-asparaginase (SF-ASNase) bioconjugates were investigated in detail. The results show that the modified ASNase was characterized by its higher residual activity (nearly 80%), increased heat and storage stability and resistance to trypsin digestion, and its longer half-life (63 h) than that of intact ASNase (33 h). The abilities of intact and modified ASNases to arouse allergic reaction are 2(4) and 2(1) antibody titers, respectively. Bioconjugation of silk fibroin significantly helps to reduce the immunogenicity and antigenicity of the enzyme. The apparent Michaelis constants of the modified ASNase (K(m(app))=0.844 x 10(-3)mol L(-1)) was approximately six times lower than that of enzyme alone, which suggests that the affinity of the enzyme to substrate l-asparagine elevated when bioconjugated covalently with silk fibroin. SF-ASNase bioconjugates could overcome the common shortcomings of the native form. Therefore, the modified ASNase coupled with silk fibroin has the potential values of being studied and developed as a new bioconjugate drug.

[Cloning and expression of Buthus martensii Karsch scorpion toxin gene (BmK IT3) in Escherichia coli].

According to the reported sequence of Buthus martensii Karsch scorpion toxin gene (BmK IT3), we synthesized two primers, which were complementary in a region. By the means of PCR, we got the gene. The gene was fused in expression vector pET-28a, which gave rise to a recombinant plasmid pET(IT3R). Then it was transformed into E. coli BL21 (DE3). With IPTG induction, the gene was efficiently expressed. And the fusion product was soluble.