Preparation and immunogenicity of tag-free recombinant human eppin.

Human epididymal protease inhibitor (eppin) may be effective as a male contraceptive vaccine. In a number of studies, eppin with an engineered His(6)-tag has been produced using prokaryotic expression systems. For production of pharmaceutical-grade proteins for human use, however, the His(6)-tag must be removed. This study describes a method for producing recombinant human eppin without a His(6)-tag. We constructed plasmid pET28a (+)-His(6)-tobacco etch virus (TEV)-eppin for expression in Escherichia coli. After purification and refolding, the fusion protein His(6)-TEV-eppin was digested with TEV protease to remove the His(6)-tag and was further purified by NTA-Ni(2+) affinity chromatography. Using this procedure, 2 mg of eppin without a His(6)-tag was isolated from 1 l of culture with a purity of >95%. The immunogenicity of the eppin was characterized using male Balb/c mice.

A novel pH-sensitive (±)-α-tocopherol-5-fluorouracil adduct with antioxidant and anticancer properties.

A novel pH-sensitive (±)-α-tocopherol-5-fluorouracil (VE-5-FU) adduct with antioxidant and anticancer properties for antioxidant-based cancer chemoprevention was synthesized and utilized for selective drug release in the stomach.

Synthesis of a novel hydrazone derivative and biophysical studies of its interactions with bovine serum albumin by spectroscopic, electrochemical, and molecular docking methods.

Hydrazone derivatives possess potential antitumor activities based on modulation of the iron metabolism in cancer cell. A novel hydrazone, N'-(2,4-dimethoxybenzylidene)-2-hydroxybenzohydrazide (DBH), has been synthesized and characterized, which is an analogue of 311 possessing potent anticancer activity. The interactions between DBH and bovine serum albumin (BSA) have been investigated systematically by fluorescence, molecular docking, circular dichroism (CD), UV-vis absorption, and electrochemical impedance spectroscopy (EIS) methods under physiological conditions. The fluorescence quenching observed is attributed to the formation of a complex between BSA and DBH, and the reverse temperature effect of the fluorescence quenching has been found and discussed. The primary binding pattern is determined by hydrophobic interaction occurring in Sudlow's site I of BSA. DBH could slightly change the secondary structure and induce unfolding of the polypeptides of protein. An average binding distance of ~4.0 nm has been determined on the basis of the Förster resonance energy theory (FRET). The effects of iron on the system of DBH-BSA have also been investigated. It is found that iron could compete against BSA to bind DBH. All of these results are supported by a docking study using a BSA crystal model. It is shown that DBH can efficiently bind with BSA and be transported to the focuses needed. Subsequent antitumor test and detailed anticancer mechanism are undergoing in our lab.

Fenvalerate inhibits the growth of primary cultured rat preantral ovarian follicles.

Fenvalerate is a widely used synthetic pyrethroid insecticide and is reported to disrupt reproductive function in humans and animals. However, little is known about its influence on follicular development. In this study, rat preantral follicles were primary cultured to investigate the effects of fenvalerate on follicular survival rate, morphological change, steroid hormone levels and steroidogenesis related gene mRNA expression. Follicles were cultured with 0, 1, 5 and 25 micromol/L fenvalerate for 72 h. And then the morphous was assessed by conventional light microscopy, steroid hormones were measured by RIA, and the expressions of steroidogenic acute regulatory protein (StAR) and cytochrome P450 side-chain cleavage enzyme (P450scc) were monitored by real-time quantitative PCR analysis. Results showed that fenvalerate inhibited the augmentation of follicular diameters but did not have detectable effects on follicular survival rates. The level of steroid hormones, such as progesterone, testosterone and estradiol, was inhibited. The inhibition might be due to the decreased expression levels of StAR and P450scc. These results suggested that fenvalerate restrained the follicular growth, and inhibited steroidogenesis by reducing StAR and P450scc gene expression, which might further contribute to the fenvalerate-induced reproductive dysfunction.

Localization of epididymal protease inhibitor in adult rat and its transcription profile in testis during postnatal development.

To investigate the expression pattern of rat Eppin (epididymal protease inhibitor; official symbol Spinlw1), we detected mRNA transcripts and subsequent protein translation of Eppin in several sorts of tissues by RT-PCR and western blotting. Then immunohistochemistry was performed for more detailed observation. The testicular transcription level was monitored by real-time PCR throughout postnatal development. We found that rat Eppin was specifically expressed in the testis and epididymis. The testicular transcription was slight in neonatal (1-day) and infantile stages (5-, 7- and 10-day). It increased sharply thereafter, with maximum expression level (about 38-fold compared with that of 1-day old rat) detected in prepubertal stage (15-day). Then a slightly declined but stable level (about 20-fold compared with that of 1-day old rat) was kept in pubertal-early adult (30-day) and adult (60-day) stages of postnatal maturation. In the adult rat, EPPIN protein was mainly localized in the elongated spermatids and epididymal epithelial cells. Sperm in the epididymal duct were all covered with EPPIN and its level kept constant during incubation under conditions used to achieve capacitation. Its stage-specific expression in the testis suggests that EPPIN may be important during spermatogenesis especially for the spermatid elongation. The abundant production of epididymal EPPIN indicated indirectly that it might play a role in the function of the epididymis.

Fluorescence study on the interaction of bovine serum albumin with p-aminoazobenzene.

In this paper, the interaction between p-aminoazobenzene (PAAB) and BSA was investigated mainly by fluorescence quenching spectra, circular dichroism (CD) and three-dimensional fluorescence spectra under simulative physiological conditions. It was proved that the fluorescence quenching of BSA by PAAB was mainly a result of the formation of a PAAB-BSA complex. The modified Stern-Volmer quenching constant K(a) and the corresponding thermodynamic parameters DeltaH, DeltaG and DeltaS at different temperatures were calculated. The results indicated that van der Waals interactions and hydrogen bonds were the predominant intermolecular forces in stabilizing the complex. The distance r=4.33 nm between the donor (BSA) and acceptor (PAAB) was obtained according to Förster's non-radioactive energy transfer theory. The synchronous fluorescence, CD and three-dimensional fluorescence spectral results showed that the hydrophobicity of amino acid residues increased and the losing of alpha-helix content (from 63.57 to 51.83%) in the presence of PAAB. These revealed that the microenvironment and conformation of BSA were changed in the binding reaction.