Over-expression of protein tyrosine phosphatase 1 (PTP1) alters IL-3-dependent growth and tyrosine phosphorylation

Interleukin-3 (IL-3) is a hematopoietic growth factor receptor which stimulates the proliferation of multilineage progenitor cells. It is known that IL-3 stimulates tyrosine phosphorylation while transducing a mitogenic signal. The signal transduction pathways activated by the IL-3 receptor, however, are not fully understood. In this study a protein tyrosine phosphatase has been over-expressed in the IL-3 dependent, murine myeloid progenitor cell line, 32D cl3 in order to test whether altering the levels of tyrosine phosphorylation would change IL-3 stimulated proliferation. These cells were transfected with a metal-inducible expression vector containing a rat cDNA encoding PTP1. A low basal level of rat PTP1 message and protein was detected in cells transfected with the PTP1 vector, and zinc treatment resulted in a three- to fourfold increase in the amount of PTP1 message, protein and catalytic activity. Over-expression of PTP1 resulted in a two- to threefold decrease in IL-3 stimulated proliferation. Cells over-expressing PTP1 also exhibited decreased levels of tyrosine phosphorylation; phosphorylation of the IL-3 receptor beta subunit and the Shc protein were both dramatically decreased. Thus, PTP1 over-expression negatively modulated IL-3 signal transduction. To identify potential substrates of PTP1, 32D cl3 cells were transfected with a catalytically inactive PTP1 mutant, PTP1(C/S). Three tyrosine-phosphorylated proteins of MW 140, 79 and 69 k coprecipitated with PTP1(C/S). We believe that the 140 kDa protein represents the beta subunit of the IL-3 receptor. In addition, a GST-fusion protein containing active PTP1 dephosphorylated the beta-subunit in an in vitro assay. By immunofluorescent microscopy over-expressed PTP1(C/S) co-localized largely with calnexin, an endoplasmic reticulum-associated protein. Immunofluorescent microscopy also indicated that PTP1(C/S) and the beta subunit co-localized at discrete sites at the plasma membrane and around a cytoplasmic organelle where most of the beta subunit was located. These observations suggest PTP1 over-expression may down-regulate the growth response to IL-3 through dephosphorylation of the IL-3 receptor, perhaps in an intracellular compartment, thereby inhibiting propagation of the IL-3 mitogenic signal.     

Novel aliphatic epoxide hydrolase activities from dematiaceous fungi

Epoxide hydrolases were found to be constitutively expressed in dematiaceous fungi coincident with secondary metabolite pigment production in stationary or idiophase. Washed-cell preparations of two fungi, Ulocladium atrum CMC 3280 and Zopfiella karachiensis CMC 3284, exhibited affinity for 2,2-dialkylated oxiranes, for which contrasting enantioselectivities were observed, but not for aromatic styrene oxide or alicyclic cyclohexene oxide type substrates. Lyophilised preparations of soluble epoxide hydrolase activities proved to be effective catalysts for the mild hydrolysis of aliphatic epoxides.     

Defining “Engineer:” How To Do It and Why It Matters

Controversy concerning whether “software engineers” are, or should be, engineers provides an opportunity to think about how to define “engineer” and what effect different definitions may have on our understanding of engineering. The standard definitions of engineering are shown to generate more confusion than insight. Engineering should be defined historically, as an occupation, and ethically, as a profession. An engineer is a member of the engineering profession, that is, a member both of an occupation that is engineering by “birth,” “adoption,” or “marriage” and of the profession committed to engineering's code of ethics. Today, few “software engineers” satisfy either of these conditions. It is an open question whether they should.     

Receptor activity modifying proteins regulate the activity of a calcitonin gene-related peptide receptor in rabbit aortic endothelial cells

In Xenopus oocytes with an endogenous calcitonin gene-related peptide (CGRP) receptor, a receptor activity modifying protein (RAMP1) enhancing CGRP stimulated chloride currents of the cystic fibrosis transmembrane regulator was recently cloned [McLatchie, L.M. et al. (1998) Nature 393, 333-339]. Here, transient expression of RAMP1 in rabbit aortic endothelial cells (RAEC) brought about stimulation of cAMP accumulation by human (h) alphaCGRP with an EC50 of 0.41 nM. This was antagonized by a CGRP receptor antagonist alphaCGRP(8-37). Co-expression of RAMP3 together with RAMP1 reduced the maximal cAMP response to h alphaCGRP by 47% (P < 0.05). The cells also express RAMP2 encoding mRNA and an adrenomedullin (ADM) receptor coupled to stimulation of cAMP formation by hADM (EC50 0.18 nM). The latter was antagonized by an ADM receptor antagonist hADM(22-52). In conclusion, expression of a CGRP receptor in RAEC requires RAMP1. The same receptor presumably recognizes ADM making use of endogenous RAMP2. The results reveal competition between the different RAMPs in the regulation of CGRP/ADM receptor activity.     

Cloning and overexpression in Escherichia coli of the gene encoding citrate synthase from the hyperthermophilic Archaeon Sulfolobus solfataricus

A transurethral resection of the prostate is a good operation to relieve bladder outflow obstruction and has a low incidence of complications. However, recent work suggests that many men with symptoms may not require an operation, and it can probably be delayed in a majority for many years. This may be particularly important in old and frail patients. Many men with outflow obstruction have irritative symptoms such as urgency, frequency and nocturia; these could be treated with anticholinergics, provided they have normal flow rates and small or absent residual urine volumes. Pharmacological treatment to relieve outflow obstruction is disappointing. There may be some benefit from alpha-adrenoreceptor antagonists, but the place for 5 alpha-reductase inhibitors is still unsure. All drugs have side effects which are unacceptable in patients who are not bothered by their urinary symptoms and can wait for active treatment.     

The CTFA Evaluation of Alternatives Program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (Phase III) surfactant-based formulations

The CTFA Evaluation of Alternatives Program is an evaluation of the relationship between data from the Draize primary eye irritation test and comparable data from a selection of promising in vitro eye irritation tests. In Phase III, data from the Draize test and 41 in vitro endpoints on 25 representative surfactant-based personal care formulations were compared. As in Phase I and Phase II, regression modelling of the relationship between maximum average Draize score (MAS) and in vitro endpoint was the primary approach adopted for evaluating in vitro assay performance. The degree of confidence in prediction of MAS for a given in vitro endpoint is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curve. Prediction intervals reflect not only the error attributed to the model but also the material-specific components of variation in both the Draize and the in vitro assays. Among the in vitro assays selected for regression modeling in Phase III, the relationship between MAS and in vitro score was relatively well defined. The prediction bounds on MAS were most narrow for materials at the lower or upper end of the effective irritation range (MAS = 0-45), where variability in MAS was smallest. This, the confidence with which the MAS of surfactant-based formulations is predicted is greatest when MAS approaches zero or when MAS approaches 45 (no comment is made on prediction of MAS > 45 since extrapolation beyond the range of observed data is not possible). No single in vitro endpoint was found to exhibit relative superiority with regard to prediction of MAS. Variability associated with Draize test outcome (e.g. in MAS values) must be considered in any future comparisons of in vivo and in vitro test results if the purpose is to predict in vivo response using in vitro data.