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.     

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.     

Flash-lamp annealing of semiconductor materials—Applications and process models

Flash-lamp annealing (FLA) on a millisecond time scale has been shown to be a promising tool in the preparation of high-quality semiconducting materials. The process imposes time varying through-thickness temperature profiles on the substrates being processed, and consequently thermal stresses. A combined thermal and optical model has been developed to predict the substrate temperature distribution and this model has been linked to a structural model to compute stresses and deflections. The paper shows how these models can be used to explore process conditions in flash lamp annealing, with particular regard to the annealing of ion implants in silicon and the crystallization of amorphous silicon layers on glass substrates.     

The amphiphysin family of proteins and their role in endocytosis at the synapse

Clathrin-mediated endocytosis at the plasma membrane is a major pathway of synaptic vesicle recycling in neurones, but little is known about the molecular machinery that orchestrates the process. The amphiphysin protein has recently emerged into the limelight since its discovery in 1992 as a synaptic vesicle-associated protein. It was subsequently found to interact in vitro with the GTPase dynamin through its SH3 domain. However, only in the past year has its role in endocytosis been confirmed, with the demonstration that the introduction of dominant-negative-acting SH3 domains into living cells causes a potent blockade of clathrin-mediated endocytosis. This, together with the discovery by several groups of a second nerve terminal-enriched amphiphysin isoform, and the finding that the two proteins heterodimerize, further suggests that the amphiphysins are closely connected with dynamin-mediated vesicle budding. This review summarizes current views in the field, and draws on data that suggest intriguing alternative roles--including possible involvement in the cytoskeleton and in tumour suppression--for certain members of the amphiphysin family.     

Hip impact velocities and body configurations for voluntary falls from standing height

OBJECTIVE: To determine prevalence of various pheno- and genotypes of Serpulina sp in young pigs in relation to diarrhea and feed medication in Swedish pig-rearing herds. DESIGN: Isolation of spirochetes. Phenotypical and genotypical classification. SAMPLE POPULATION: Young pigs (n = 358) in 19 pigrearing herds. PROCEDURE: Serpulina isolates were classified according to a biochemical scheme based on hemolysis, indole production, hippurate hydrolysis, and alpha-galactosidase, alpha-glucosidase, and beta-glucosidase activities. The 16S rRNA sequences for 10 of the field strains and 2 type strains of Serpulina spp were aligned and compared. Minimum inhibitory concentrations of olaquindox for 9 of the strains were determined. RESULTS: Weakly beta-hemolytic intestinal spirochetes (WBHIS) were isolated from 17 of the herds and 65% of the samples. More than 1 phenotype of WBHIS was found in 12 of the 19 herds. S hyodysenteriae was not isolated in any of the herds. Hippurate-positive WBHIS were isolated in 6 of 7 herds affected by diarrhea, but in only 1 of 8 herds without diarrhea. Hippurate-positive strains were closely related to the pathogenic strain P43 if judged from sequence comparisons. Strains with the same biochemical profile isolated within a herd had identical sequences, but when isolated from different herds, sequence differences were observed. The prevalence of WBHIS was reduced in herds medicated with olaquindox. Investigated field strains had minimum inhibitory concentration values < or = 1 microgram/ml for olaquindox. CONCLUSION: The presence of WBHIS, with the ability to hydrolyze hippurate, was related to diarrhea in pig herds. CLINICAL RELEVANCE: Potentially pathogenic WBHIS can be distinguished from nonpathogenic strains by the hippurate hydrolysis test.     

Chronic Q fever. Ninety-two cases from France, including 27 cases without endocarditis

OBJECTIVE: Chronic Q fever is seldom recognized; before 1989, only 234 cases had been reported in the literature. The 92 cases of chronic Q fever collected at the French National Reference Center for Rickettsioses from 1982 through 1990 represent the largest series ever reported. PATIENTS: The patients included in the study were diagnosed between July 31, 1982, and August 1, 1990, at the French National Reference Center for Rickettsioses as having chronic Q fever by the following criteria: presence of antibody against Coxiella burnetii phase I antigen at a titer greater than or equal to 800 for IgG and 50 for IgA by the indirect immunofluorescence test. Epidemiologic, clinical, laboratory, and treatment data were collected from 39 different collaborative hospitals throughout France. MAIN OUTCOME MEASURE: For each serologically selected patient, a computerized questionnaire was utilized to record 188 different items of demographic, epidemiologic, clinical, laboratory, and therapeutic data, which were analyzed. RESULTS: Chronic Q fever occurs more frequently in city dwellers than in rural inhabitants, and exposure to domestic ruminants and raw milk is an important feature. Immunocompromising conditions (20.2%) and underlying heart disease (88.4%) or vascular disease are the most important risk factors to consider in potential cases of chronic Q fever. The mortality in these patients with endocarditis was high (23.5%). The clinical spectrum of 84 patients included 57 cases of endocarditis, three cases of vascular prosthesis infection, three cases of aneurysmal infection, three cases of osteoarthritis, four cases with lung localizations, nine asymptomatic cases, three cases of hepatitis, and two cases with cutaneous forms of the disease. CONCLUSIONS: In patients with unexplained fever, negative blood cultures, and a history of underlying vascular or cardiac disease, Q fever should be considered.     

Platinum concentrations in urban road dust and soil, and in blood and urine in the United Kingdom

Increasing Pt concentrations from vehicle catalysts have been reported from a number of countries. Analysis of Pt and Pd in soils and road dusts taken from areas of high and low traffic flows in SE England show concentrations of Pt in the range < 0.30-40.1 ng g-1 and Pd in the range < 2.1-57.9 ng g-1. Higher concentrations of Pt are associated with high traffic densities. Samples taken from streets of lower traffic flows were found to contain the lower concentrations of the ranges. Pilot studies of Pt concentrations in blood and urine using ICP-MS have been carried out. Platinum concentrations in whole blood were: precious metal workers, 780-2170, mean 1263 pmol l-1 (0.152-0.423, mean 0.246 microgram l-1); motorway maintenance workers, 645-810, mean 744 pmol l-1 (0.126-0.158, mean 0.145 microgram l-1); Imperial College staff, 590-713, mean 660 pmol l-1 (0.115-0.139, mean 0.129 microgram l-1). Platinum concentrations in urine in pmol Pt per mmol creatinine were: precious metal workers, 122-682, mean 273 [0.21-1.18, mean 0.47 microgram Pt (g creatinine)-1]; motorway maintenance workers, 13-78, mean 33.7 [0.022-0.135, mean 0.058 microgram Pt (g creatinine)-1]; Imperial College staff, 28-130, mean 65.6 [0.048-0.224, mean 0.113 microgram Pt (g creatinine)-1]. Detection limits were 0.03 microgram l-1 for both blood and urine. The possible health effects of increasing Pt in the environment are discussed. Platinum provides an excellent example of the significance of speciation in metal toxicity. Platinum allergy is confined to a small group of charged compounds that contain reactive ligand systems, the most effective of which are chloride ligand systems. Metallic Pt is considered to be biologically inert and non allergenic and since the emitted Pt is probably in the metallic or oxide form, the sensitising potential is probably very low. Platinum from road dusts, however, can be solubilised, and enter waters, sediments, soils and the food chain. There is at present no evidence for any adverse health effects from Pt in the general environment, particularly allergic reactions.     

A novel DNA duplex. A parallel-stranded DNA helix with Hoogsteen base pairing

We show here for the first time that a stable parallel double helix with Hoogsteen pairing can exist independently of the triple helix of which it is a component part. The experiments employ DNA oligonucleotides with mixed sequences of normal bases. These duplexes are distinct from previously reported ribopolynucleotide helices containing bulky substituents which prevent Watson-Crick pairing as well as from parallel duplexes with Donohue, or reversed Watson-Crick, pairing. Stoichiometry is established by mixing curves and gel electrophoresis. Tm depends linearly upon pH, increasing with acidity because of the need to protonate N3 of C. The Tm of the 20-mer studied here is 52 degrees C at pH 5.2 and 0.1 M NaCl. At pH above 6, the molecule rearranges to form an antiparallel duplex with imperfect Watson-Crick pairing and loops, and the Tm is then independent of pH. The CD spectrum of the parallel duplex is very similar to that of the corresponding triple helix but quite different from that of the Watson-Crick helix. The infrared spectrum in the double bond region closely resembles that of the triple helix but, as with the CD, is quite different from that of the Watson-Crick duplex. The infrared spectra of the duplex and triple helix are also nearly identical in the region form 800 to 1000 cm-1, which is sensitive to backbone conformation. The only symmetry element present is a pseudorotational axis coincident with the helix axis of the parallel duplex as well as with the axis of the corresponding triple helix.(ABSTRACT TRUNCATED AT 250 WORDS)