Characterization of a prostate-specific tyrosine phosphatase by mutagenesis and expression in human prostate cancer cells

The cellular form of human prostatic acid phosphatase (PAcP) is a neutral protein-tyrosine phosphatase (PTP) and may play a key role in regulating the growth and androgen responsiveness of prostate cancer cells. The functional role of the enzyme is at least due in part to its dephosphorylation of c-ErbB-2, an in vivo substrate of the enzyme. In this study, we investigated the molecular mechanism of phosphotyrosine dephosphorylation by cellular PAcP. We mutated several amino acid residues including one cysteine residue that was proposed to be involved in the PTP activity of the enzyme by serving as the phosphate acceptor. The cDNA constructs of mutant enzymes were transiently transfected into C-81 LNCaP and PC-3 human prostate cancer cells that lack the endogenous PAcP expression. The phosphotyrosine level of ErbB-2 in these transfected cells was subsequently analyzed. Our results demonstrated that the phosphotyrosine level of ErbB-2 in cells expressing H12A or D258A mutant PAcP is similar to that in control cells without PAcP expression, suggesting that these mutants are incapable of dephosphorylating ErbB-2. In contrast, cells expressing C183A, C281A, or wild-type PAcP had a decreased phosphotyrosine level of ErbB-2, compared with the control cells. Similar results were obtained from in vitro dephosphorylation of immunoprecipitated ErbB-2 by these mutant enzymes. Furthermore, transient expression of C183A, C281A, or the wild-type enzyme, but not H12A or D258A, decreased the growth rate of C-81 LNCaP cells. The data collectively indicate that His-12 and Asp-258, but not Cys-183 or Cys-281, are required for the PTP activity of PAcP.     

Residue size at position 87 of cytochrome P450 BM-3 determines its stereoselectivity in propylbenzene and 3-chlorostyrene oxidation.

We report here oxidation of propylbenzene and 3-chlorostyrene by wild-type cytochrome P450 BM-3 with high turnover (479 nmol 1-phenyl-1-propanol/min/nmol P450 and 300 nmol 3-chlorostyrene oxide/min/nmol P450). Furthermore, the residue size at position 87 of P450 BM-3 was found to play critical roles in determining stereoselectivity in oxidation of propylbenzene and 3-chlorostyrene. Replacement of Phe87 with Val, Ala and Gly resulted in decreases in optical purity of produced (R)-(+)-1-phenyl-1-propanol from 90.0 to 37.4, 26.0 and -15.6% e.e., respectively, and in increases in those of produced (R)-(+)-3-chlorostyrene oxide from -61.0 to -38.0, 67.0 and 94.6% e.e., respectively.     

Crystallization and preliminary X-ray crystallographic analysis of spruce budworm antifreeze protein.

Antifreeze proteins have the ability to bind to ice with high affinity and inhibit further crystal growth. The insect antifreeze protein from spruce budworm exhibits very high thermal hysteresis activity and is implicated in the protection of overwintering larvae from freezing. This protein has been crystallized in 20-25% polyethylene glycol (Mr 6000), 0.4 M NaCl, 0.1 M Tris-HCl, pH 8.5, by vapor diffusion using the hanging drop method. The resulting crystals are very thin (typically <0.01 mm in the shortest dimension), and only after repeated seeding could crystals be grown large enough for data collection using synchrotron radiation. The crystals belong to the monoclinic space group C2, with cell dimensions a = 82.28 A, b = 62.29 A, c = 63.63 A, and beta = 113.7 degrees. Molecules in the asymmetric unit are related by a twofold axis of symmetry with two molecules present. Native data to a resolution of 2.6 A have been collected with 90.3% completeness and a Rsym of 6.9%.     

The effect of light fluence rate in photodynamic therapy of normal rat brain.

This paper reports the effect of incident light fluence rate on the depth to which necrotic lesions are produced by photodynamic therapy (PDT) in the brains of normal Fisher rats. The rats were injected intraperitoneally with Photofrin (12.5 mg kg-1) 48 h prior to PDT with a fixed incident fluence of 35 J cm-2. The treatment was performed at 10, 50, 100, and 200 mW cm-2 and also in a periodic manner (30 s "on" at 100 mW cm-2, 30 s "off"). The depth to which necrosis occurred was determined 24 h after treatment by microscopic examination of tissue sections. No differences were found in the depth to which necrosis was produced by any of the five irradiation schedules. This finding is discussed in the context of other published dose-rate experiments.     

Sprint training shortens prolonged action potential duration in postinfarction rat myocyte: mechanisms.

Two electrophysiological manifestations of myocardial infarction (MI)-induced myocyte hypertrophy are prolongation of action potential duration (APD) and reduction of transient outward current (I(to)) density. Because high-intensity sprint training (HIST) ameliorated myocyte hypertrophy and improved myocyte Ca(2+) homeostasis and contractility after MI, the present study evaluated whether 6-8 wk of HIST would shorten the prolonged APD and improve the depressed I(to) in post-MI myocytes. There were no differences in resting membrane potential and action potential amplitude (APA) measured in myocytes isolated from sham-sedentary (Sed), MI-Sed, and MI-HIST groups. Times required for repolarization to 50 and 90% APA were significantly (P < 0.001) prolonged in MI-Sed myocytes. HIST reduced times required for repolarization to 50 and 90% APA to values observed in Sham-Sed myocytes. The fast and slow components of I(to) were significantly (P < 0.0001) reduced in MI-Sed myocytes. HIST significantly (P < 0.001) enhanced the fast and slow components of I(to) in MI myocytes, although not to levels observed in Sham-Sed myocytes. There were no significant differences in steady-state I(to) inactivation and activation parameters among Sham-Sed, MI-Sed, and MI-HIST myocytes. Likewise, recovery from time-dependent inactivation was also similar among the three groups. We suggest that normalization of APD after MI by HIST may be mediated by restoration of I(to) toward normal levels.     

Establishment of an Efficient Screening System in Gene Transformation of High Oleic Acid Peanut Using AhFAD2B as a Reporter Gene

Russian Journal of Plant Physiology - During gene transformation, selection makers are important for efficient screening of the transgenic lines. The commonly used selection marker genes in plants...