Synthesis of fluorescent substrates for protein tyrosine phosphatase assays

Two fluorescent substrates for protein tyrosine phosphatase (PTPase) reaction were prepared by conjugation of commercially available O-phosphotyrosine and dansyl chlorides. They were hydrolyzed by CD45 tyrosine phosphatase, and proved to be useful for PTPase assay.     

Automated and quantitative immunocytochemical assays of Bcl-2 protein in breast carcinomas

Expression of the bcl-2 gene was investigated in 218 human breast carcinomas by immunohistochemical analysis. Immunodetections were assessed using (1) frozen sections, (2) documented commercially available monoclonal antibody (bcl-2/124, Dako), (3) automation of immunoperoxidase technique (Ventana) and (4) quantitative evaluation of results by image analysis (SAMBA) and statistical analysis of quantitative data (BMDP software). Bcl-2 protein expression was correlated with current prognostic indicators and with molecular markers detected by the same procedure as for Bcl-2. It was shown that Bcl-2 expression is not related to patients' age, tumour size and type or lymph node status, but an inverse relationship was observed between Bcl-2 and tumour grade (P < 0.0001). An inverse relationship was also observed between Bcl-2 expression and p53 (P < 0.0001), Ki67/MIB1 antigen- (P = 0.0012), and P-gp- (P = 0.002) positive immunoreactions. In contrast, anti-Bcl-2 positive reaction was significantly associated with ER-positive (P < 0.001) and with ER/PR-positive or ER/PR/pS2-positive immunoreactions (P < or = 0.005). Bcl-2 expression was independent of CD31 and cathepsin D expression. Thus, Bcl-2 protein, thought to be antiapoptotic, exhibits parodoxical expression in human breast carcinomas. It is strongly detected in low-grade tumours (well-differentiated) with low (MIB1) growth fraction, but is independent of the tumour progression (size, node status, CD31, and cathepsin D). Bcl-2 acting on apoptosis is related to p53 gene abnormalities in breast carcinomas. Bcl-2 protein expression may also be involved in response to endocrine therapy (associated to ER/PR/pS2 positive immunoreactions) and probably with chemoresistance mechanisms (inverse relationship with P-gp).     

Activity of quinolones in the Ames Salmonella TA102 mutagenicity test and other bacterial genotoxicity assays

Eight quinolones were examined for their bacterial mutagenicity in the Ames Salmonella TA102 assay and for their effects in other bacterial genotoxicity assays. In the quantitative Ames plate incorporation assay, all eight quinolones induced His+ deletion reversion in Salmonella tester strain TA102, with maximum reversion observed at about two to eight times the MIC. The quinolones also induced the SOS response. At quinolone concentrations close to the MIC, SOS cell filamentation gene sulA was induced in sulA::lacZ fusion strain Escherichia coli PQ37. RecA-mediated cleavage of lambda repressor in lambda::lacZ fusion strain E. coli BR513 was measurable at about 10 times the MIC, though no induction occurred at 20 micrograms of nalidixic or oxolinic acid per ml. Genotoxicity of quinolones also was observed in the Bacillus subtilis DNA repair assay, in which the mutant strain M45 (recA) was more susceptible to quinolones than its parent strain, H17 (rec+). The results from these analyses indicate that quinolones induce SOS functions and are mutagenic in bacteria; these properties correspond to their antimicrobial activities.     

Use of modified functional assays for activated protein C resistance in patients with basally prolonged aPTT

Inherited resistance to activated protein C (APCr) is currently recognized as the most prevalent cause underlying venous thrombophilia, with an estimated prevalence around 20% in thrombotic patients and around 1.8-7% in the general population. A correct laboratory diagnosis of APCr is therefore essential. Two different diagnostic approaches are at present at our disposal: the semi-quantitative plasma test based on the measurement of two aPTTs (in the presence and absence of activated protein C), and the detection of the factor V Arg506 Gln mutation by DNA analysis. In this study we firstly evaluated sensitivity, specificity and diagnostic efficiency of an aPTT-based plasma clotting test (Chromogenix, Sweden) versus DNA analysis; then, since the APC resistance test is invalidated by a basally prolonged aPTT (i.e. during warfarin and heparin therapy or in patients with clotting factor deficiencies or in the presence of a lupus anticoagulant), patient plasmas were conveniently diluted in factor V deficient plasma in order to correct clotting factor abnormalities. Nevertheless, patients with a LA and an aPTT ratio range 1.8-3.17 were still all misclassified. We obtained correct diagnoses in LA positive patients by preincubating plasmas with a mixture of phospholipids; therefore we decided to perform a double modified clotting test adding a mixture of platelet derived phospholipids to samples previously diluted in factor V deficient plasma. The performance characteristics of this novel method with a different aPTT reagent (Behring, Germany) were also evaluated. With this double modified test all patients were correctly classified as negative or positive for factor V mutation in agreement with DNA analysis, irrespectfully of the basal aPTT value and the aPTT reagent employed. We propose this modified version of the APCr clotting test as an easily reproducible, reliable, very sensitive and specific screening test which possibly reduces the need for DNA analysis.     

Conversion and extraction efficiencies for ground particles in heterogeneous process reactors

Chemical conversion of particles and extraction from particles are encountered in many metallurgical processes. Equations characterizing particle size distributions resulting from grinding were coupled with the rate equations for quasi-spherical particles under several different commonly encountered rate controlling conditions to calculate conversion/extraction efficiencies for particulate feeds in batch, plug flow, and backmix flow reactors. Families of curves were computed expressing the fraction of feed unreacted as a function of a normalized average residence time parameter. Using these curves, the required sizes of process reactors to yield a selected conversion/extraction efficiency can be determined from limited laboratory kinetic data.     

Comparison of two immunoradiometric assays for parathyroid hormone-related protein in the evaluation of cancer patients with and without hypercalcemia

Systemic lupus erythematosus (SLE) predominantly affects women (9:1 compared to men) of childbearing age and often decreases its intensity in postmenopausal women, suggesting that sex hormones play a role in its pathogenesis. Comparison of steady-state levels of calcineurin mRNA using RNase protection assays revealed increased calcineurin expression in response to estradiol in cultured T cells from nine female lupus patients. Calcineurin mRNA levels did not increase significantly in T cells from eight age-matched normal control female volunteers. Estrogen-dependent calcineurin mRNA increased in a dose-dependent fashion, while progesterone and dexamethasone did not increase calcineurin mRNA in patient cells. Lupus T cell calcineurin mRNA increased in response to estradiol at 6 h but not at 3 h. Calcineurin phosphatase activity increased in lupus T cell extracts after incubation of cells with estradiol, while phosphatase activity in normal T cells was unaffected by estrogen. Calcineurin expression in T cells from patients with vasculitis and rheumatoid arthritis taking medications similar to those taken by the lupus patients was unaffected by estradiol. This study provides the first evidence for a molecular marker of estrogen action in lupus patients and suggests that estrogen-dependent changes in lupus T cell calcineurin could alter proinflammatory cytokine gene regulation and T-B cell interactions.     

Performance demands in the selection of objects for counting

This paper considers the visual processes in object counting among children. Experiment 1 presented identical objects to 7- and 8-year-old children and found that spatially random configurations were counted more quickly than linear arrays, illustrating the difficulty of isolating objects grouped together in rows. However, the younger children were more prone to miscounting these random arrays than rows. The study also established a spatial proximity effect, with a dense arrangement of items being difficult to count. Experiment 2 revealed that this proximity effect can be removed by differentiating objects by color, providing further evidence that object counting involves overcoming Gestalt grouping forces and arguing against fine-motor control as a limiting factor in counting.     

Initiation of protein synthesis in mammalian cells with codons other than AUG and amino acids other than methionine

Protein synthesis is initiated universally with the amino acid methionine. In Escherichia coli, studies with anticodon sequence mutants of the initiator methionine tRNA have shown that protein synthesis can be initiated with several other amino acids. In eukaryotic systems, however, a yeast initiator tRNA aminoacylated with isoleucine was found to be inactive in initiation in mammalian cell extracts. This finding raised the question of whether methionine is the only amino acid capable of initiation of protein synthesis in eukaryotes. In this work, we studied the activities, in initiation, of four different anticodon sequence mutants of human initiator tRNA in mammalian COS1 cells, using reporter genes carrying mutations in the initiation codon that are complementary to the tRNA anticodons. The mutant tRNAs used are aminoacylated with glutamine, methionine, and valine. Our results show that in the presence of the corresponding mutant initiator tRNAs, AGG and GUC can initiate protein synthesis in COS1 cells with methionine and valine, respectively. CAG initiates protein synthesis with glutamine but extremely poorly, whereas UAG could not be used to initiate protein synthesis with glutamine. We discuss the potential applications of the mutant initiator tRNA-dependent initiation of protein synthesis with codons other than AUG for studying the many interesting aspects of protein synthesis initiation in mammalian cells.     

Quality control of multidrug resistance assays in adult acute leukemia: correlation between assays for P-glycoprotein expression and activity

We have compared multiple assays for the P-glycoprotein (Pgp/MDR1) phenotype in fresh and thawed adult acute leukemia to validate and quantitate measures for the expression and function of Pgp. The results are related to the Pgp-expressing KB8 and KB8-5 call lines. The most sensitive assay was the measurement of modulation of the rhodamine 123 (R123) fluorescence by 2 micromol/L PSC833, followed by the modulation of the probe calcein-AM. We also found a good intralaboratory and interlaboratory correlation between the values of the R123/PSC833 assay for fresh as well as thawed samples. In addition, the affects of PSC833 on 3H-daunorubicin (DNR) accumulation, DNR fluorescence, and 3H-vincristine accumulation were very similar. The correlation between the DNR/PSC833 and R123/PSC833 test was r = .86 (N = 51). The modulation of drug accumulation by 8 micromol/L verapamil was the some as the PSC833 effect for DNR (117%, N = 21), but was higher for vincristine in every single case (161% v 121%, N = 22; P< .001), indicating additional verapamil effects, not related to Pgp. The correlation of the staining of viable cells for Pgp with the monoclonal antibody MRK16 was r = .77 (N = 52) for the R123/PSC833 functional test and r = .84 (N = 50) for the DNR/PSC833 test. From these results it could be calculated that a maximal increase of the mean DNR accumulation of about 50% can be achieved by blocking Pgp pump activity with PSC833 in leukemic blast samples with the highest mean Pgp expression. Subpopulations of blast calls with higher Pgp activity are likely to be present. Their relevance has to be studied further. The methods outlined here allow the reliable, quantitative monitoring of the Pgp/MDR1 phenotype in leukemias in multicentered, clinical Pgp modulation studies.     

Urinary assays for desmosine and hydroxylysylpyridinoline in the detection of cirrhosis

BACKGROUND/AIMS: Non-invasive markers of liver fibrosis have great potential for both the diagnosis and therapy of liver disease and cirrhosis. The aim of this study was to evaluate the potential of urinary amino acids desmosine (DES) and isodesmosine (IDES) derived from the breakdown of elastin and hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) derived from fibrillar collagen in diagnosing chronic liver disease. METHODS: We studied 48 patients with chronic liver disease who had varying degrees of liver fibrosis, graded 0-6 using a modified Knodell score, and 20 control subjects without liver disease. Urinary DES (microg/g creatinine) and HP (nmol/mmol creatinine) were measured by an isotope dilution, high performance liquid chromatography method. For liver disease patients, aminoterminal propeptide of type III procollagen (PIIINP) and alanine aminotransferase were determined. The urine and serum markers were correlated to degree of fibrosis and inflammation on liver biopsies. Differences between groups were analyzed by ANOVA and multiple linear regression was applied to determine independence of variables. Sensitivity, specificity and receiver operating curves were derived for each marker. RESULTS: In the 17 patients with liver fibrosis score of 5-6, mean urinary DES, IDES, HP and LP were all significantly greater than in the control group (p<0.05). Urinary DES and IDES correlated best with fibrosis score, r=0.61 for both markers. The correlation coefficient between serum PIIINP and fibrosis score was 0.47. Urinary DES and HP each had an overall diagnostic accuracy of 77% for fibrosis. Combining markers improved accuracy to over 80%. No correlation was seen between the urinary markers and inflammation scores. CONCLUSIONS: Urinary DES and HP are potentially useful clinical markers for liver fibrosis, especially when used in combination or in association with PIIINP.     

Detection of human immunodeficiency virus-1 DNA, RNA and antibody, and occult blood in inactivated saliva: availability of the filter paper disk method

The resistance change to different insecticides in Culex quinquesfasciatus strain select at the laboratory with doses of pyrethroid lambdacyhalothrin that would cause a larva mortality of 90% were studied. It was attained an increase of the resistance to this insecticide of 144.5 times compared with the original level, and it was obtained a resistant strain (287x). There was an increase of the levels of resistance to methyl-pyrimifos (2.4 times), propoxur (6 times), DDT (5.2 times), clorpirifos (22 time), cypermethrin (67.5 times), and deltamethrin (20.2 times). The frequencies of the genes that codify for the elevated esterases enzymes and for the modified acetylcholinesterase reached their maximum value. Significant changes were observed in the phenotypes for esterases in the electrophoresis in polyacrylamide gels. It was detected synergism of DEF and PB with lambdacyhalothrin. Therefore, the elevated esterases and the esterases of multiple function may contribute to resistance.     

Pharmacokinetic considerations in the development of labeled liposomes and micelles for diagnostic imaging

The current status of application of liposomes and micelles as carriers for diagnostic imaging agents in experimental and clinical medicine is considered. Liposomes and micelles loaded with appropriate contrast agents have been shown to be suitable for all imaging modalities, including gamma-, magnetic resonance (MR), and computed tomography (CT). The methods are briefly described to prepare liposomes loaded with various contrast agents, as well as some basic data on their in vitro and in vivo properties and biodistribution. Certain pharmacokinetic considerations associated with the use of plain and long-circulating liposomes and micelles as pharmaceutical carriers are discussed. The application of contrast-loaded liposomes in different modalities for the experimental and clinical imaging of various organs, tissues, and pathological conditions is briefly reviewed. New trends in the preparation and use of contrast-loaded liposomes and micelles are also considered, such as the application of amphiphilic polychelating polymers and polymers for steric protection of microparticulate pharmaceutical carriers.     

Estimation of tissue protein synthesis in rats fed diets labeled with (U-14C)tyrosine

The rate of liver and muscle protein synthesis has been measured in 27 rats after feeding L-[U-14C]tyrosine in L-amino acid diets prepared as agar gels. Constant specific activity of the free tyrosine pool, as indicated by constant excretion of 14CO2, was reached within 2 h of feeding and was maintained for the remaining 6 h of the 8-h experiment. Muscle protein synthesis was decreased (P less than 0.05) in rats fed a 0.3% methionine diet compared with rats fed this diet supplemented with 0.51% cystine (fractional rate of synthesis, ks: 0.098 vs. 0.121). No effect (P greater than 0.05) of these diets on liver protein synthesis was observed (ks: 0.603 vs. 0.532). Protein synthetic rate was also determined by the constant-intravenous infusion technique in 17 rats fed unlabeled diets. The two techniques gave similar estimates. Restraint of the rats or the infusion of saline had no measurable effect on the rate of protein synthesis in rats fed labeled diets. This feeding technique is essentially equivalent to the constant-infusion technique and offers an easier, more physiological approach to achieving a steady state.     

圆盘剪自动控制的改进和完善

福建三钢（集团）有限责任公司中板厂圆盘剪控制系统剪切过程中,存在着剪刃间隙调整精度不准和压紧装置、设备整体调宽运行自动化程度不高的问题,通过深入分析发现,造成问题的原因是系统程序不完善及剪刃间隙与钢板厚度设定不合理,因此采取了以下改进措施：正确确定剪刃间隙与钢板厚度的关系、完善压紧装置的控制方式、增加整体剪切宽度自动同步调整的功能,改进后运行效果较好。     

Gastro-intestinal protein loss in late survivors of Fontan surgery and other congenital heart disease

A 2,048-bp nucleotide sequence containing a gene coding for an enzyme that degraded guar gum from Bacillus circulans K-1 was identified by polymerase chain reaction walking. This G-gene consisted of 1,551 nucleotides coding for a protein with Mr 55,242. The enzyme was overexpressed in Escherichia coli JM109 cells by the cloning the G-gene downstream of the lac Z promoter of pUC19. The molecular mass of recombinant G-enzyme estimated by SDS-PAGE was 62 KDa, close to that from strain K-1. Analysis of the recombinant enzyme showed GalNAc, Xyl, GlcNAc, Man, Glc, and Gal to account for 1.7%, 14.4%, 6.1%, 3.2%, 54.2%, and 10.4%, respectively, of the total monosaccharides. Polyacrylamide gel electrophoresis of this enzyme with staining gave a red band. The results suggested that the sugars accounted for the differences in the molecular masses. The recombinant enzyme had two kinds of N-terminal sequences, Thr-Met-Ile-Thr-Pro-Ser-Phe-Ala-Ser-Gly-Phe-Tyr-Val-Ile and Ile-Thr-Pro-Ser-Phe-Ala-Ser-Gly-Phe-Tyr-Val-Ile-Gly-Thr. Comparison of these sequences with the deduced N-terminal sequence coded for the G-gene showed that the amino acid, first Met, of the lac Z gene or the next residues Thr-Met in the recombinant enzyme were absent in the native enzyme. Methionines near and at the N-terminus of the mature protein probably were digested by methionine aminopeptidases of E. coli after translation. The properties of recombinant G-enzyme were similar to those of the enzyme from K-1 cells.     

Using evolutionary trees in protein secondary structure prediction and other comparative sequence analyses

Previously proposed methods for protein secondary structure prediction from multiple sequence alignments do not efficiently extract the evolutionary information that these alignments contain. The predictions of these methods are less accurate than they could be, because of their failure to consider explicitly the phylogenetic tree that relates aligned protein sequences. As an alternative, we present a hidden Markov model approach to secondary structure prediction that more fully uses the evolutionary information contained in protein sequence alignments. A representative example is presented, and three experiments are performed that illustrate how the appropriate representation of evolutionary relatedness can improve inferences. We explain why similar improvement can be expected in other secondary structure prediction methods and indeed any comparative sequence analysis method.     

Analysis of membrane protein self-association in lipid systems by fluorescence particle counting: application to the dihydropyridine receptor

Fluorescence particle counting (FPC) is employed to analyze the distribution of a purified membrane protein, the dihydropyridine receptor (DHP-R), in detergent micelles, in lipid vesicles, and in lipid monolayers generated from the vesicles. The method was used to identify conditions for which DHP-Rs occur singly distributed in micelles and in vesicles. In monolayers, the DHP-R showed self-association, starting from monomeric distribution at concentrations (c) of typically 10 DHP-R/microm2. The average cluster size [m(t)] of associates was followed by FPC in time and the dependence of the lateral diffusion constant [D(lat)(m,pi)] of the associates on the surface pressure (pi) was determined. By studying the dependence of m(t) on c, pi, D(lat)(pi), and salt concentration (c(s)), we derived an empirical expression for the association rate constant (k(a)) and for m(t) that fits the experimental m(t) relations. Theoretical justification for these dependencies is obtained from collision theory, leading to a mechanistic picture of the aggregation process. DHP-R association is irreversible. Its rate is not diffusion-limited. A large number of collisions is required to overcome an interaction energy barrier of about 6-11 kT, depending on m and c(s) but not on pi. The increase in association rate with increasing average cluster size m is related to increasing van der Waals attraction, while the increase in rate with increasing c(s) relates to decreasing electrostatic repulsion. Van der Waals and electrostatic forces represent, however, only part of the interaction energy. The main contribution was not dependent on the variables studied and, most likely, reflects hydration forces which need to be overcome for association.