The band areas of proteins determined by fluorescent scanning in the commercial automated gel electrophoresis apparatus

An automated gel electrophoresis apparatus, recently available commercially, allows one to follow the band during electrophoresis in real time, and lends itself therefore to an evaluation of bandwidth as a function of migration time (the dispersion coefficient), resolution and band shape. These determinations assume the constancy of band area with migration time and at various gel concentrations. The purpose of the present study was to verify these assumptions. Representative proteins and sodium dodecyl sulfate (SDS)-proteins, either natively fluorescent or fluorescein carboxylate labeled, were found to exhibit band areas which approach constancy as a function of migration time in both agarose and polyacrylamide gel electrophoresis, provided that (i) the protein concentration under the band was low enough to obviate self-quenching of fluorescence; (ii) the separation of the protein of interest from contaminants had progressed sufficiently during the time at which band areas were measured; (iii) the baseline under the peak was sufficiently well defined. However, band areas decrease with increasing gel concentration. Protein peaks exhibited leading and trailing tails. The ratio of the combined tail area to total area appeared to be near-constant at varying migration times. However, that ratio increases with increasing gel concentration. The tail area does not appear to be an artifact of fluorometric detection since it is reproduced upon fluorimetric analysis of the protein eluted from gel slices after electrophoresis. However, it may be due to photochemical destruction under the conditions of repetitive fluorometric peak detection.     

Engagement of T cell receptor triggers its recruitment to low-density detergent-insoluble membrane domains

T-cell receptors (TCRs) upon binding to peptide-MHC ligands transduce signals in T lymphocytes. Tyrosine phosphorylations in the cytoplasmic domains of the CD3 (gammadeltaepsilon) and zeta subunits of the TCR complex by Src family kinases initiate the signaling cascades via docking and activation of ZAP-70 kinase and other signaling components. We examined the role of the low-density detergent-insoluble membranes (DIMs) in TCR signaling. Using mouse thymocytes as a model, we characterized the structural organization of DIMs in detail. We then demonstrated that TCR engagement triggered an immediate increase in the amount of TCR/CD3 present in DIMs, which directly involves the engaged receptor complexes. TCR/CD3 recruitment is accompanied by the accumulation of a series of prominent tyrosine-phosphorylated substrates and by an increase of the Lck activity in DIMs. Upon TCR stimulation, the DIM-associated receptor complexes are highly enriched in the hyperphosphorylated p23 zeta chains, contain most of the TCR/CD3-associated, phosphorylation-activated ZAP-70 kinases and seem to integrate into higher order, multiple tyrosine-phosphorylated substrate-containing protein complexes. The TCR/CD3 recruitment was found to depend on the activity of Src family kinases. We thus provide the first demonstration of recuitment of TCR/CD3 to DIMs upon receptor stimulation and propose it as a mechanism whereby TCR engagement is coupled to downstream signaling cascades.     

Summary of discussion on ‘Sciences related to pitch and coke usage in carbon manufacture’

This article is a brief summary of the Discussion session held after the presentation of the preceding papers at the conference organized by the Industrial Carbon and Graphite Group of the Society of Chemical Industry, London, March 1984.     

The 1987-8 Taulbee Survey report

This report describes the results of a survey completed in December 1988 on the production and employment of PhDs and faculty in PhD-granting computer science/engineering departments during the 1987-8 academic year. All 127 computer science (CS) departments (115 US and 12 Canadian) participated. In addition, 34 departments offering the PhD in computer engineering (CE) were included. Throughout the report. CE statistics are reported separately so that comparisons with previous years can be made for CS. The intent is to merge all statistics for CS and CE after several years     

Phase I study of mitoxantrone plus etoposide with multidrug blockade by SDZ PSC-833 in relapsed or refractory acute myelogenous leukemia

PURPOSE: Expression of the multidrug resistance gene (MDR1) p170 protein is frequent in leukemic blasts from patients with relapsed acute myelogenous leukemia (AML). A phase I study using the nonimmunosuppressive MDR1 blocker SDZ PSC-833 (PSC) in combination with mitoxantrone (MITO) and etoposide (VP) was performed. PATIENTS AND METHODS: Starting doses (LVL0) of MITO (3.25 mg/m2/d on days 1 and 3 to 6) and VP (210 mg/m2/d on days 1 and 3 to 5) were 40% of the maximal-tolerated dose (MTD) from a prior study. A 1.5-mg/kg loading dose of PSC was followed by a 120-hour continuous infusion of 10 mg/kg/d on days 2 to 6. Blood samples for PSC, MITO, and VP pharmacokinetics (PK) were taken on days 1 and 3, and samples for MDR1 expression were taken on day 0. RESULTS: Severe mucositis developed in all patients at LVL0; therefore, MITO and VP doses were reduced to 2.5 and 170 mg/m2 (LVL-1) for the next seven patients, and this dose proved to be MTD. All LVL0 and three LVL-1 patients had transient elevations in the serum bilirubin level to > or = 4 mg/dL. Serum creatinine level increased to greater than 2 mg/dL in one case. There were no other grade 3 or 4 nonhematologic toxicities observed. The peripheral blood was cleared of leukemia in three LVL0 and four LVL-1 patients. The marrow was cleared of leukemic cells in one LVL0 and five LVL-1 patients, and a significant reduction in marrow leukemic infiltrate was observed in eight of 10. No patient achieved complete remission (CR), and all died of progressive disease (n = 8) or infection (n = 2). MDR1 expression was detected by fluorescent-activated cell sorter (FACS) analysis in five of seven cases. An elevated MDR1 mRNA level was detected by quantitative polymerase chain reaction (Q-PCR) in six of eight cases studied. Clearing of leukemia cells from the marrow occurred in four of six MDR1-positive and one of three MDR1-negative patients. Despite the fact that LVL0 doses had to be reduced due to toxicity, coadministration of PSC did not produce a consistent effect on MITO PK; however, it did repeatedly lead to increased levels of VP in the serum. CONCLUSION: We conclude that PSC-MITO-VP is a tolerable regimen with antileukemic activity. Addition of PSC necessitated a 66% reduction in MITO and VP doses from a prior study without PSC.     

A dislocation shielding prediction of the toughness transition during cleavage of semibrittle crystals

An interconnected set of observations assesses current equilibrium models of the ductile-brittle-transition temperature (DBTT). This involvesin situ transmission electron microscopy (TEM) studies of crack-tip dislocations in single and polycrystals and bulk fracture toughness tests at various temperatures. Beyond K_I values of 8 MPa · m^1/2 in both iron-base single and polycrystals, large numbers of redundant dislocations are created, as postulated recently by Weertman. [38] Still, the necessary shielding dislocations, as required by equilibrium, can be detected at values as high as 20 and 40 MPa · m^1/2 byex situ TEM and electron channeling, respectively. In addition, the close approach of dislocations to the crack tip in some of the studies, as opposed to others, suggests that large dislocation free zones (DFZ) are a thin-film artifact. However, a failure criterion based partly on the Rice-Thomson model’21 is both consistent with the absence of a large DFZ and observed fracture toughness variations with test temperature. It is emphasized that this toughness transition is entirely in the semibrittle regime where cleavage is the failure mode. Nevertheless,K _lc values increase from 3 to 60 MPa·m^1/2 with an increase in test temperature. This article is based on a presentation made in the symposium “Quasi-Brittle Fracture” presented during the TMS fall meeting, Cincinnati, OH, October 21–24, 1991, under the auspices of the TMS Mechanical Metallurgy Committee and the ASM/MSD Flow and Fracture Committee.     

Experimental densities for compressed R134a

This paper reports densities of compressed R134a (1,1,1,2-tetrafluoroethane) determined by using a contiuously weighed pycnometer at 20 K intervals between 180 and 380 K at pressures from slightly greater than the vapour pressure to 70 MPa. The results are accurate to within ±0.1%. Saturated liquid densities derived by extrapolation from the experimental values agree with other reported values to within ±0.3%.