A Cascading Auction Protocol as a Framework for Integrating Process Planning and Heterarchical Shop Floor Control

A new contract net-style auction protocol is proposed as a framework for integrating process planning and shop floor control in heterarchical manufacturing systems. Process planning is partitioned into on-line and off-line activities; off-line process planning decisions are represented in a graph format and used as input for on-line process planning activities performed by machine controllers. Triggered by the opening round of an auction, the final on-line stages of process planning are dovetailed with the resource allocation process in the shop floor control system. The auction process allows final process planning decisions to be based on timely information, relying on the distribution of static process planning information rather than the distribution of a model of dynamic shop floor status and allowing a controller to identify all the primary and secondary resources and operations that must be provided for the incremental processing of a part.     

Fine needle aspiration cytology in the management of male breast masses. Nineteen years of experience.

OBJECTIVE: To determine the utility and accuracy of fine needle aspiration cytology (FNAC) as well as its sensitivity, specificity and predictive value in the diagnosis of male breast masses. STUDY DESIGN: Data on male breast FNAC done between 1978 and 1997 were retrieved from the records of the cytopathology laboratory. FNAC diagnoses were categorized as positive, negative, inconclusive or unsatisfactory. Cytohistologic correlation was done with data from histopathology records. Sensitivity, specificity, diagnostic accuracy and predictive values of FNAC were calculated using standard statistical methods. RESULTS: Five hundred seven of 13,175 patients undergoing breast FNAC were males. Of them, 393/507 had satisfactory aspirates. Of these, 70 were positive (13.8%), 295 were negative (58%), and 29 were inconclusive (5.7%). A total of 114 FNACs (22.5%) were unsatisfactory. Histopathology was available in 97/507 cases. There were no false positive or false negative diagnoses. FNAC had a sensitivity, specificity and diagnostic accuracy of 100% for male breast lesions. CONCLUSION: This large study shows that FNAC is a very accurate tool for diagnosis of male breast lesions. It is highly sensitive and specific, with good cytohistologic correlation. FNAC should therefore be an integral part of the primary assessment of breast lumps in males.     

Optimized expression and purification of adipose triglyceride lipase improved hydrolytic and transacylation activities in vitro

Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis, the mobilization of stored triacylglycerol. This work provides an important basis for generating reproducible and detailed data on the hydrolytic and transacylation activities of ATGL. We generated full-length and C-terminally truncated ATGL variants fused with various affinity tags and analyzed their expression in different hosts, namely E.coli, the insect cell line Sf9, and the mammalian cell line human embryonic kidney 293T. Based on this screen, we expressed a fusion protein of ATGL covering residues M1-D288 flanked with N-terminal and C-terminal purification tags. Using these fusions, we identified key steps in expression and purification protocols, including production in the E. coli strain ArcticExpress (DE3) and removal of copurified chaperones. The resulting purified ATGL variant demonstrated improved lipolytic activity compared with previously published data, and it could be stimulated by the coactivator protein comparative gene identification-58 and inhibited by the protein G0/G1 switch protein 2. Shock freezing and storage did not affect the basal activity but reduced coactivation of ATGL by comparative gene identification 58. In vitro, the truncated ATGL variant demonstrated acyl-CoA–independent transacylation activity when diacylglycerol was offered as substrate, resulting in the formation of fatty acid as well as triacylglycerol and monoacylglycerol. However, the ATGL variant showed neither hydrolytic activity nor transacylation activity upon offering of monoacylglycerol as substrate. To understand the role of ATGL in different physiological contexts, it is critical for future studies to identify all its different functions and to determine under what conditions these activities occur.     

Structure-function correlation during the etioplast-chloroplast transition in cucumber cotyledonary leaves

We studied the development of chloroplasts from etioplasts in the cotyledonary leaves of 4-d-old dark-grown cucumber (Cucumis sativus) seedlings after irradiation (20 μmol m^-2 s^-1). Upon irradiation, the triggering of chlorophyll (Chl) synthesis and accumulation showed a relatively short lag phase. The irradiation of etiolated seedlings initiated the synthesis of apoproteins of pigment-protein complexes. While Chl-protein 2 (CP2) was detected at 6 h after irradiation, CP1 only after 29 h. The appearance and accumulation of some of the apoproteins were monitored by Western-blotting. LHC2 apoprotein was detected after a 6 h-irradiation. The amounts of D1 protein of photosystem (PS) 2 and PsaA/B protein of PS1 were quantitated by ELISA. Further, the thylakoid membrane function during this time period in terms of PS1- and PS2-mediated electron transfer activity and intersystem electron pool size were analyzed. While PS1 activity was detected after 4 h, PS2-mediated O₂ evolution was detected only after a 17 h-irradiation. F_v/F_m value of Chl a fluorescence measurements indicated that the photochemical efficiency of these leaves reached its maximum after 29 h of irradiation. The intersystem pool size of cotyledonary leaves was equivalent to that of the control cotyledonary leaves grown for 25 h under continuous irradiation. Thus etioplasts develop into fully functional chloroplasts after approximately 25 h when 4 d-dark grown cucumber seedlings are continuously moderately irradiated. The development of photosynthetic electron transport chain seems to be limited in time at the level of PS2, possibly at the donor side. This revised version was published online in September 2006 with corrections to the Cover Date.     

Giant platinum clusters: 2-nm covalent metal cluster labels.

A new class of covalently linkable platinum cluster reagents with core diameters close to 2 nm has been prepared. The new label offers the performance advantages and versatility of the 1.4-nm Nanogold in a larger label which is more clearly visualized against electron-dense regions of a specimen. Large platinum clusters were prepared by the reduction of platinum(II) acetate in the presence of 1,10-phenanthroline ligands which had been synthetically modified to include solubilizing and reactive cross-linkable functional groups. These were then conjugated site-specifically to antibody IgG molecules and to Fab' fragments and visualized by scanning transmission electron microscopy. The resulting conjugates may be autometallographically enhanced and show sensitivity similar to that of Nanogold conjugates in immunoblotting experiments. In preliminary experiments, they have also exhibited labeling of tissue antigens and penetrate to access nuclear targets.     

Expression of human p140trk receptors in p140trk-deficient,PC12/endothelial cells results in nerve growth factor-induced signal transduction and DNA synthesis

Nerve growth factor (NGF) regulates proliferation, differentiation, and survival of sympathetic and sensory neurons through the tyrosine kinase activity of its receptor, p140^trk. These biological effects of NGF depend upon the signal-mediating function of p140^trk substrates which are likely to differ from cell to cell. To define p140^trk receptor substrates and the details of signalling by NGF in the hybrid cell PC12EN, we stably transfected cultures with a vector encoding a full-length human p140^trk cDNA sequence. Two stably transfected clones, one expressing p140^trk with higher affinity (PC12EN-trk3; K_d 57.4 pM, B_max 9.7 pmole/mg) and one expressing p140^trk with a lower affinity (PC12EN-trk1; K_d 392.4 pM, B_max 5.7 pmole/mg) were generated. Radioreceptor assays indicate that transfected p140^trk receptors show slow NGF-dissociation kinetics, are resistant to trypsin or Triton X-100 treatment, are specific for NGF compared to other neurotrophins, and are internalized or downregulated as are native PC12 p140^trk receptors. NGF stimulates p140^trk tyrosine phosphorylation in a dose- (0.01-10 ng/ml) and time- (5-120 min) dependent manner, and tyrosine phosphorylation was inhibited by 200-1,000 nM K-252a. NGF-induced Erk stimulation for 60 min was assessed using myelin basic protein as a substrate. NGF treatment also led to an increased phosphorylation of p70^S6k, SNT, and phospholipase Cγ, demonstrating that the major NGF-stimulated signalling pathways found in other cells are activated in PC12EN-trk cells. Staurosporine (5-50 nM) rapidly and dBcAMP (1 mM) more slowly, but not NGF induced morphological differentiation in PC12EN-trk cells. Rather, NGF treatment in low-serum medium stimulated a 1.3- and 2.3-fold increase in DNA synthesis measured by [³H]thymidine incorporation in PC12EN-trk1 and PC12EN-trk3, respectively. These data highlight the functionality of the transfected p140^trk receptors and indicate that these transfected cells may serve as a novel cellular model facilitating the study of the mitogenic properties of NGF signalling and the transducing role of the p140^trk receptor substrates. J. Cell. Biochem. 66:229-244. © 1997 Wiley-Liss, Inc. This article is a U.S. Government work and, as such, is in the public domain in the United States of America.     

Fluorescent diphenylphosphonate-based probes for detection of serine protease activity during inflammation

Activity-based probes are small molecules that covalently bind to the active site of a protease in an activity-dependent manner. We synthesized and characterized two fluorescent activity-based probes that target serine proteases with trypsin-like or elastase-like activity. We assessed the selectivity and potency of these probes against recombinant enzymes and demonstrated that while they are efficacious at labeling active proteases in complex protein mixtures in vitro, they are less valuable for in vivo studies. We used these probes to evaluate serine protease activity in two mouse models of acute inflammation, including pancreatitis and colitis. As anticipated, the activity of trypsin-like proteases was increased during pancreatitis. Levels of elastase-like proteases were low in pancreatic lysates and colonic luminal fluids, whether healthy or inflamed. Exogenously added recombinant neutrophil elastase was inhibited upon incubation with these samples, an effect that was augmented in inflamed samples compared to controls. These data suggest that endogenous inhibitors and elastase-degrading proteases are upregulated during inflammation.     

Identification of Single Nucleotide Polymorphisms Associated with Brown Rust Resistance, α-Amylase Activity and Pre-harvest Sprouting in Rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.)

Rye is a crop with relatively high resistance to biotic and abiotic stresses. However, the resistance to brown rust (Puccinia recondita f. sp. secalis) and pre-harvest sprouting are still not satisfactory. High α-amylase activity is also among the main disadvantages of this species. Therefore, effective tools, e.g. molecular markers, allowing precise and environmentally independent selection of favourable alleles are desirable. In the present study, two kinds of association mapping—genome-wide association mapping (GWAM) based on sequences of DArTSeq markers and candidate gene association mapping (CGAM) based on sequences of ScBx genes—were chosen for development of molecular markers fulfilling these criteria. The analysed population consisted of 149 diverse inbred lines (DILs). Altogether, 67 and 11 single nucleotide polymorphisms (SNPs) identified in, respectively, GWAM and CGAM, were significantly associated with the investigated traits: 2 SNPs with resistance to brown rust, 71 SNPs with resistance to pre-harvest sprouting and 5 SNPs with α-amylase activity in the grain. Fifteen SNPs were stable across all environments. The highest number (13) of environmentally stable SNPs was associated with pre-harvest sprouting resistance. The test employing the Kompetitive Allele Specific PCR method proved the versatility of four markers identified in both GWAM and CGAM.