Stability and activity of a thermostable malic enzyme in denaturants and water-miscible organic solvents

A study was made of the effects of common protein denaturants and water-miscible organic solvents on both the stability and activity of the malic enzyme [(S)-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating); EC 1.1.1.40] from the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus. At 25 degrees C, the enzyme was not inactivated in 4 M urea or 0.05% SDS over 24 h, while the half-life was 30 min in 6 M guanidine hydrochloride and 5 h in 0.075% SDS. The enzyme stability in water-miscible organic solvents at 25 degrees C is somewhat surprising: after a 24-h incubation, the enzyme was completely active in 50% dimethylformamide; it lost 15% of its initial activity in 50% methanol or 15% ethanol. However, the resistance to organic solvents was greatly reduced at higher temperatures. The enzyme was able to catalyze the malate conversion even in the presence of 1.5% Triton X-100 or sodium deoxycholate. A number of solvents were found to stimulate the malic activity independent of time. Studies with 50% methanol revealed that the activation was reversible and inversely related to the temperature; moreover, the solvent was demonstrated to exclusively affect the maximal velocity of catalysis, the Km values for both substrates being unchanged. Investigation was made to find out whether there was a correlation between enzyme stability, as well as activation, and hydrophobicity of the organic medium. The residual malic activity after incubation in the water/organic medium correlated inversely with the logarithm of the partition coefficient in octanol/H2O of the mixture used as a hydrophobicity index. On the other hand, the extent of activation depended directly on the logarithm of the molar concentration of the organic solvent required for maximal enzymatic activation. Because of its remarkable resistance to organic solvents required for maximal enzymatic activation. Because of its remarkable resistance to organic solvents and protein denaturants in general, the malic enzyme from Sulfolobus solfataricus can be considered suitable for biotechnological applications.     

A major secretory protein from rat seminal vesicle binds ejaculated spermatozoa

The rat seminal vesicle produces large amounts of a protein-rich fluid that greatly contributes to semen volume. RSV IV, a protein abundantly secreted from this gland, binds in vitro to rat epididymal spermatozoa. However, there is no evidence that this protein may have an in vivo role as a sperm-coating antigen. We report in this paper that high-molecular-weight RSV IV immunologically related proteins can be detected on ejaculated spermatozoa, but not on epididymal spermatozoa. After incubation of purified RSV IV with ejaculated spermatozoa in freshly recovered semen or with epididymal spermatozoa in a medium containing the coagulating gland secretion, sperm-bound proteins with analogous properties were detected. These results support the hypothesis that RSV IV is modified at ejaculation to an high-molecular-weight, sperm-coating antigen.     

High-grade mucoepidermoid carcinoma of the breast. Fine needle aspiration cytology and clinicopathologic study of a case

A primary high-grade mucoepidermoid carcinoma of the breast was evaluated preoperatively by fine needle aspiration (FNA) cytology in a 72-year-old woman. The smears showed a mixed pattern consisting of clusters of poorly differentiated squamous cells, keratinized squamous cells and vacuolated mucin-secreting cells. The subsequent mastectomy specimen showed a tumor with the features of a high-grade mucoepidermoid carcinoma. Electron microscopy confirmed the diagnosis, reflecting the epidermoid and glandular differentiation of the tumor. The course was rapidly fatal, and the patient died a few months after presentation. A review of the literature indicated that mucoepidermoid carcinoma of the breast is a very rare neoplasm; the FNA cytologic features described in this report may constitute a basis to preoperatively recognize this tumor.     

Propolis Enhances 5-Fluorouracil Mediated Antitumor Efficacy and Reduces Side Effects in Colorectal Cancer: An in Vitro and in Vivo Study

In this study, we investigated the combined treatment of 5-fluorouracil (5-FU) and Anatolian propolis extract (PE) on colorectal cancer (CRC)using in vitro and in vivo studies. We exposed luciferase-transfected (Lovo-Luc CRC) cells and healthy colon cells (CCD-18Co) to varying concentrations of 5-FU and PE to assess their genotoxic, apoptotic, and cytotoxic effects, as well as their intracellular reactive oxygen species (iROS) levels. We also developed a xenograft model in nude mice and evaluated the anti-tumor effects of PE and 5-FU using various methods. Our findings showed that the combination of PE and 5-FU had selectivity against cancer cells, particularly at higher doses, and enhanced the anti-tumor effectiveness of 5-FU against colon CRC. The results suggest that PE can reduce side effects and increase the effectiveness of 5-FU through iROS generation in a dose-dependent manner.     

Dissociation by NH4Cl treatment of the enzymic activities of glutamine synthetase II from Rhizobium leguminosarum biovar viceae.

Glutamine synthetase II (GSII) was purified to homogeneity from Rhizobium leguminosarum biovar viceae and characterized. The sequence of 26 amino acid residues from the amino-terminal end of the protein showed high similarity with the sequence of GSII from Bradyrhizobium japonicum or from Rhizobium meliloti. Non-denaturing PAGE showed that GSII, either in crude extracts or in the pure state, was a mixture of an octamer and a tetramer and that under specific conditions the octamer/tetramer ratio could be modified in either direction. The pure enzyme was used to raise an antiserum which was highly specific. Addition of NH4Cl to a bacterial culture derepressed for GSII caused a specific decrease in transferase activity, faster than the one observed when the amount of immunoreactive material was measured by different methods. On the other hand, biosynthetic activity, measured as the rate of ADP or glutamine formation, paralleled the rate of decrease in immunoreactive material. A partially purified enzyme preparation retained this dissociation of kinetic parameters, strongly suggesting a post-translational modification. These findings are discussed with respect to the possible role of GSII in the Rhizobium-legume symbiosis.