Schedules with due-date assignment and position-dependent processing times

Single machine scheduling problems are considered, in which the processing of jobs depend on positions of the jobs in a schedule and the due-dates are assigned either according to the CON rule (a due-date common to all jobs is chosen) or according to the SLK rule (the due-dates are computed by increasing the actual processing times of each job by a slack, common to all jobs). Polynomial-time dynamic programming algorithms are proposed for the problems with the objective functions that include the cost of assigning the due-dates, the total cost of disgarded jobs (which are not scheduled) and, possibly, the total earliness of the scheduled jobs.

Seven 3-methylidene-1H-indol-2(3H)-ones related to the multiple-receptor tyrosine kinase inhibitor sunitinib

The solid-state structures of a series of seven substituted 3-methylidene-1H-indol-2(3H)-one derivatives have been determined by single-crystal X-ray diffraction and are compared in detail. Six of the structures {(3Z)-3-(1H-pyrrol-2- ylmethylidene)-1H-indol-2(3H)-one, C13H10N2O, (2a); (3Z)-3-( 2-thienylmethylidene)-1H-indol-2(3H)-one, C13H9NOS, (2b); (3E)-3-(2-furylmethylidene)-1H-indol-2(3H)-one monohydrate, C13H9NO2 center dot H2O, (3a); 3-(1-methylethylidene)-1H-indol- 2(3H)-one, C11H11NO, (4a); 3-cyclohexylidene-1H-indol- 2(3H)-one, C14H15NO, (4c); and spiro[1,3-dioxane-2,3'-indolin]- 2'-one, C11H11NO3, (5)} display, as expected, intermolecular hydrogen bonding (N-H center dot center dot center dot O=C) between the 1H-indol-2(3H)-one units. However, methyl 3-(1-methylethylidene)- 2-oxo-2,3-dihydro-1H-indole-1-carboxylate, C13H13NO3, (4b), a carbamate analogue of (4a) lacking an N-H bond, displays no intermolecular hydrogen bonding. The structure of (4a) contains three molecules in the asymmetric unit, while (4b) and (4c) both contain two independent molecules.

Identification of Genes Involved in the C. elegans VAB-1 Eph Receptor Tyrosine Kinase Signaling Pathway

The generation of a functional nervous system requires that neuronal cells and axons navigate precisely to their appropriate targets. The Eph Receptor Tyrosine Kinases (RTKs) and their ephrin ligands have emerged as one of the important guidance cues for neuronal and axon navigation. However, the molecular mechanisms of how Eph RTKs regulate these processes are still incomplete. The purpose of this work was to contribute to the understanding of how Eph receptors regulate axon guidance by identifying and characterizing components of the Caenorhabditis elegans Eph RTK (VAB-1) signaling pathway. To achieve this objective I utilized a hyper active form of the VAB-1 Eph RTK (MYR-VAB-1) that caused penetrant axon guidance defects in the PLM mechanosensory neurons, and screened for suppressors of the MYR-VAB-1 phenotype. Through a candidate gene approach, I identified the adaptor NCK-1 as a downstream effector of VAB-1. Molecular and genetic analysis revealed that the nck-1 gene encodes for two isoforms (NCK-1A and NCK-1B) that share similar expression patterns in parts of the nervous system, but also have independent expression patterns in other tissues. Genetic rescue experiments showed that both NCK-1 isoforms can function in axon guidance, but each isoform also has specific functions. In vitro binding assays showed that NCK-1 binds to VAB-1 in a kinase dependent manner. In addition to NCK-1, WSP-1/N-WASP was also identified as an effector of VAB-1 signaling. Phenotypic analysis showed that nck-1 and wsp-1 mutants had PLM axon over extension defects similar to vab-1 animals. Furthermore, VAB-1, NCK-1 and WSP-1 formed a complex in vitro. Intriguingly, protein binding assays showed that NCK-1 can also bind to the actin regulator UNC-34/Ena, but genetic experiments suggest that unc-34 is an inhibitor of nck-1 function. Through various genetic and biochemical experiments, I provide evidence that VAB-1 can disrupt the NCK-1/UNC-34 complex, and negatively regulate UNC-34. Taken together, my work provides a model of how VAB-1 RTK signaling can inhibit axon extension. I propose that activated VAB-1 can prevent axon extension by inhibiting growth cone filopodia formation. This is accomplished by inhibiting UNC-34/Ena activity, and simultaneously activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex.

Inositol 1,4,5-trisphosphate receptors modulate Ca2+ sparks and Ca2+ store content in vas deferens myocytes.

Spontaneous Ca2+ sparks were observed in fluo 4-loaded myocytes from guinea pig vas deferens with line-scan confocal imaging. They were abolished by ryanodine (100 microM), but the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) blockers 2-aminoethoxydiphenyl borate (2-APB; 100 microM) and intracellular heparin (5 mg/ml) increased spark frequency, rise time, duration, and spread. Very prolonged Ca2+ release events were also observed in approximately 20% of cells treated with IP3R blockers but not under control conditions. 2-APB and heparin abolished norepinephrine (10 microM; 0 Ca2+)-evoked Ca2+ transients but increased caffeine (10 mM; 0 Ca2+) transients in fura 2-loaded myocytes. Transients evoked by ionomycin (25 microM; 0 Ca2+) were also enhanced by 2-APB. Ca2+ sparks and transients evoked by norepinephrine and caffeine were abolished by thimerosal (100 microM), which sensitizes the IP3R to IP3. In cells voltage clamped at -40 mV, spontaneous transient outward currents (STOCs) were increased in frequency, amplitude, and duration in the presence of 2-APB. These data are consistent with a model in which the Ca2+ store content in smooth muscle is limited by tonic release of Ca2+ via an IP3-dependent pathway. Blockade of IP3Rs elevates sarcoplasmic reticulum store content, promoting Ca2+ sparks and STOC activity.

Carbachol triggers RyR-dependent Ca(2+) release via activation of IP(3) receptors in isolated rat gastric myocytes.

Possible interactions between different intracellular Ca(2+) release channels were studied in isolated rat gastric myocytes using agonist-evoked Ca(2+) signals. Spontaneous, local Ca(2+) transients were observed in fluo-4-loaded cells with linescan confocal imaging. These were blocked by ryanodine (100 microM) but not by the inositol 1,4,5-trisphosphate receptor (IP(3)R) blocker, 2-aminoethoxydiphenyl borate (100 microM), identifying them as Ca(2+) sparks. Caffeine (10 mM) and carbachol (10 microM) initiated Ca(2+) release at sites which co-localized with each other and with any Ca(2+) spark sites. In fura-2-loaded cells extracellular 2-aminoethoxydiphenyl borate and intracellular heparin (5 mg ml(-1)) both inhibited the global cytoplasmic [Ca(2+)] transient evoked by carbachol, confirming that it was IP(3)R-dependent. 2-Aminoethoxydiphenyl borate and heparin also increased the response to caffeine. This probably reflected an increased Ca(2+) store content since 2-aminoethoxydiphenyl borate more than doubled the amplitude of transients evoked by ionomycin. Ryanodine completely abolished carbachol and caffeine responses but only reduced ionomycin transients by 30 %, suggesting that blockade of carbachol transients by ryanodine was not simply due to store depletion. Double labelling of IP(3)Rs and RyRs demonstrated extensive overlap in their distribution. These results suggest that carbachol stimulates Ca(2+) release through co-operation between IP(3)Rs and RyRs, and implicate IP(3)Rs in the regulation of Ca(2+) store content.

Impaired functioning of thermolabile methylenetetrahydrofolate reductase is dependent on riboflavin status: implications for riboflavin requirements.

BACKGROUND: Methylenetetrahydrofolate reductase (MTHFR; EC 1.7.99.5) supplies the folate needed for the metabolism of homocysteine. A reduction in MTHFR activity, as occurs in the homozygous state for the 677C-->T (so-called thermolabile) enzyme variant (TT genotype), is associated with an increase in plasma total homocysteine (tHcy). OBJECTIVE: In vitro studies suggest that the reduced activity of thermolabile MTHFR is due to the inappropriate loss of its riboflavin cofactor. We investigated the hypothesis that MTHFR activity in the TT genotype group is particularly sensitive to riboflavin status. DESIGN: We studied tHcy and relevant B-vitamin status by MTHFR genotype in a cross-sectional study of 286 healthy subjects aged 19-63 y (median: 27 y). The effect of riboflavin status was examined by dividing the sample into tertiles of erythrocyte glutathionine reductase activation coefficient, a functional index of riboflavin status. RESULTS: Lower red blood cell folate (P = 0.0001) and higher tHcy (P = 0.0082) concentrations were found in the TT group than in the heterozygous (CT) or wild-type (CC) groups. However, these expected relations in the total sample were driven by the TT group with the lowest riboflavin status, whose mean tHcy concentration (18.09 micromol/L) was almost twice that of the CC or CT group. By contrast, adequate riboflavin status rendered the TT group neutral with respect to tHcy metabolism. CONCLUSIONS: The high tHcy concentration typically associated with homozygosity for the 677C-->T variant of MTHFR occurs only with poor riboflavin status. This may have important implications for governments considering new fortification policies aimed at the prevention of diseases for which this genotype is associated with increased risk.

Role of p190RhoGAP in beta2 Integrin Regulation of RhoA in Human Neutrophils

We found that engagement of beta 2 integrins on human neutrophils induced activation of RhoA, as indicated by the increased ratio of GTP:GTP 1 GDP recovered on RhoA and translocation of RhoA to a membrane fraction. The clustering of beta 2 integrins also induced a time-dependent increase in GDP bound to RhoA, which correlated with beta 2 integrin-induced activation of p190RhoGAP. The activation of p190RhoGAP was completely blocked by [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] (PP1), a selective inhibitor of Src family tyrosine kinases. However, clustering of beta 2 integrins did not increase the basal tyrosine phosphorylation of p190RhoGAP, nor did it affect the amount of p120RasGAP bound to p190RhoGAP. Instead, the beta 2 integrin-induced activation of p190RhoGAP was accompanied by increased tyrosine phosphorylation of a p190RhoGAP-associated protein, p120RasGAP, and accumulation of both p120RasGAP and p190RhoGAP in a membrane fraction. PP1 blocked the beta 2 integrin-induced phosphorylation of p120RasGAP, as well as the translocation of p190RhoGAP and p120RasGAP, but it did not affect the accumulation of RhoA in the membrane fraction. In agreement with the mentioned findings, PP1 also increased the GTP:GTP 1 GDP ratio recovered on RhoA immunoprecipitated from beta2 integrin-stimulated cells. Thus, in neutrophils, beta 2 integrin-induced activation of p190RhoGAP requires a signal from a Src family tyrosine kinase, but it does not occur via the signaling pathway responsible for activation of RhoA.

Tyrosine phosphorylated SOCS-3 inhibits STAT activation but binds p120-RasGAP and activates Ras

Suppressors of cytokine signalling (SOCS, also known as CIS and SSI) are encoded by immediate early genes that act in a feedback loop to inhibit cytokine responses and activation of 'signal transducer and activator of transcription' (STAT). Here we show that SOCS-3 is strongly tyrosine-phosphorylated in response to many growth factors, including interleukin-2 (IL-2), erythropoietin (EPO), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). The principal phosphorylation sites on SOCS-3 are residues 204 and 221 at the carboxy terminus, and upon phosphorylation tyrosine 221 interacts with the Ras inhibitor p120 RasGAP. After IL-2 stimulation, phosphorylated SOCS-3 strongly inhibits STAT5 activation but, by binding to RasGAP, maintains activation of extracellular-signal-regulated kinase (ERK). A tyrosine mutant of SOCS-3 still blocks STAT phosphorylation, but also strongly inhibits IL-2-dependent activation of ERK and cell proliferation. Moreover, it also inhibits EPO- and PDGF-induced proliferation and ERK activation. Therefore, although SOCS proteins inhibit growth-factor responses, tyrosine phosphorylation of SOCS-3 can ensure cell survival and proliferation through the Ras pathway.

Nonapical and Cytoplasmic Expression of Interleukin-8, CXCR1, and CXCR2 Correlates with Cell Proliferation and Microvessel Density in Prostate Cancer

Purpose: We characterized interleukin-8 (IL-8) and IL-8 receptor expression (CXCR1 and CXCR2) in prostate cancer to address their significance to this disease. Experimental Design: Immunohistochemistry was conducted on 40 cases of human prostate biopsy containing histologically normal and neoplastic tissue, excised from patients with locally confined or invasive androgen-dependent prostate cancer, and 10 cases of transurethral resection of the prostate material from patients with androgen-independent disease. Results: Weak to moderate IL-8 expression was strictly localized to the apical membrane of normal prostate epithelium. In contrast, membranous expression of IL-8, CXCR1, and CXCR2 was nonapical in cancer cells of Gleason pattern 3 and 4, whereas circumferential expression was present in Gleason pattern 5 and androgen-independent prostate cancer. Each of IL-8, CXCR1, and CXCR2 were also increasingly localized to the cytoplasm of cancer cells in correlation with advancing stage of disease. Cytoplasmic expression (but not apical membrane expression) of IL-8 in Gleason pattern 3 and 4 cancer correlated with Ki-67 expression (R = 0.79; P <0.001), cyclin D1 expression (R = 0.79; P <0.001), and microvessel density (R = 0.81; P <0.001). In vitro studies on androgen-independent PC3 cells confirmed the mitogenic activity of IL-8, increasing the rate of cell proliferation through activation of both CXCR1 and CXCR2 receptors. Conclusions: We propose that the concurrent increase in IL-8 and IL-8 receptor expression in human prostate cancer induces autocrine signaling that may be functionally significant in initiating and promoting the progression of prostate cancer by underpinning cell proliferation and angiogenesis.

Glucose-potentiated chemotaxis in human vascular smooth muscle is dependent on cross-talk between the P13K and MAPK signalling pathways

Atheroma formation involves the movement of vascular smooth muscle cells (VSMC) into the subendothelial space. The aim of this study was to determine the involvement of PI3K and MAPK pathways and the importance of cross-talk between these pathways, in glucose-potentiated VSMC chemotaxis to serum factors. VSMC chemotaxis occurred in a serum gradient in 25 mmol/L glucose (but not in 5 mmol/L glucose) in association with increased phosphorylation (activation) of Akt and ERK1/2 in PI3K and MAPK pathways, respectively. Inhibitors of these pathways blocked chemotaxis, as did an mTOR inhibitor. VSMC expressed all class IA PI3K isoforms, but microinjection experiments demonstrated that only the p110beta isoform was involved in chemotaxis. ERK1/2 phosphorylation was reduced not only by MAPK pathway inhibitors but also by PI3K and mTOR inhibitors; when PI3K was inhibited, ERK phosphorylation could be induced by microinjected activated Akt, indicating important cross-talk between the PI3K and ERK1/2 pathways. Glucose-potentiated phosphorylation of molecules in the p38 and JNK MAPK pathways inhibited these pathways but did not affect chemotaxis. The statin, mevinolin, blocked chemotaxis through its effects on the MAPK pathway. Mevinolin-inhibited chemotaxis was restored by farnesylpyrophosphate but not by geranylgeranylpyrophosphate; in the absence of mevinolin, inhibition of farnesyltransferase reduced ERK phosphorylation and blocked chemotaxis, indicating a role for the Ras family of GTPases (MAPK pathway) under these conditions. In conclusion, glucose sensitizes VSMC to serum, inducing chemotaxis via pathways involving p110beta-PI3K, Akt, mTOR, and ERK1/2 MAPK. Cross-talk between the PI3K and MAPK pathways is necessary for VSMC chemotaxis under these conditions.

Enhancement of Ca2+-dependent outward current in sheep bladder myocytes by evans blue dye.

Whole-cell and inside-out patch-clamp techniques were used to assess the action of a well-known dye, Evans blue, on membrane currents in bladder isolated smooth muscle cells from sheep. In whole cells Evans blue dose-dependently increased the outward current by up to fivefold. In contrast, Evans blue had no effect on inward Ca2+ current. The effect on outward current was abolished or reduced if the cells were bathed in Ca2+-free solution, iberiotoxin (5 x 10(-8) M), or charybdotoxin (5 x 10(-8) M), but was unaffected by externally applied caffeine (5 mM) or in cells exposed to heparin (1 mg/ml) via the patch pipette. In inside-out patches bathed in a Ca2+ concentration of 5 x 10(-7) M, Evans blue (10(-4) M) increased the open probability of large-conductance (298-pS) Ca2+-dependent K+ channels (BK channels), shifting the half maximal-activation voltage by -70 mV. We conclude that Evans blue dye acts as an opener of BK channels.

Rasch Analysis of the Daily Living Tasks Dependent on Vision (DLTV)

PURPOSE. To examine internal consistency, refine the response scale, and obtain a linear scoring system for the visual function instrument, the Daily Living Tasks Dependent on Vision (DLTV). METHODS. Data were available from 186 participants with a clinical diagnosis of AMD who completed the 22-item DLTV (DLTV-22) according to four-point ordinal response scale. An independent group of 386 participants with AMD were administered a reduced version of the DLTV with 11 items (DLTV-11), according to a five-point response scale. Rasch analysis was performed on both datasets and used to generate item statistics for measure order, response odds ratios per item and per person, and infit and outfit mean square statistics. The Rasch output from the DLTV-22 was examined to identify redundant items and for factorial validity and person item measure separation reliabilities. RESULTS. The average rating for the DLTV-22 changed monotonically with the magnitude of the latent person trait. The expected versus observed average measures were extremely close, with step calibrations evenly separated for the four-point ordinal scale. In the case of the DLTV-11, step calibrations were not as evenly separated, suggesting that the five-point scale should be reduced to either a four- or three-point scale. Five items in the DLTV-22 were removed, and all 17 remaining items had good infit and outfit mean squares. PCA with residuals from Rasch analysis identified two domains containing 7 and 10 items each. The domains had high person separation reliabilities (0.86 and 0.77 for domains 1 and 2, respectively) and item measure reliabilities (0.99 and 0.98 for domains 1 and 2, respectively). CONCLUSIONS. With the improved internal consistency, establishment of the accuracy and precision of the rating scale for the DLTV and the establishment of a valid domain structure we believe that it constitutes a useful instrument for assessing visual function in older adults with age-related macular degeneration.

Measles virus minigenomes encoding two autofluorescent proteins reveal cell-to-cell variation in reporter expression dependent on viral sequences between the transcription units.

Transcription from morbillivirus genomes commences at a single promoter in the 3' non-coding terminus, with the six genes being transcribed sequentially. The 3' and 5' untranslated regions (UTRs) of the genes (mRNA sense), together with the intergenic trinucleotide spacer, comprise the non-coding sequences (NCS) of the virus and contain the conserved gene end and gene start signals, respectively. Bicistronic minigenomes containing transcription units (TUs) encoding autofluorescent reporter proteins separated by measles virus (MV) NCS were used to give a direct estimation of gene expression in single, living cells by assessing the relative amounts of each fluorescent protein in each cell. Initially, five minigenomes containing each of the MV NCS were generated. Assays were developed to determine the amount of each fluorescent protein in cells at both cell population and single-cell levels. This revealed significant variations in gene expression between cells expressing the same NCS-containing minigenome. The minigenome containing the M/F NCS produced significantly lower amounts of fluorescent protein from the second TU (TU2), compared with the other minigenomes. A minigenome with a truncated F 5' UTR had increased expression from TU2. This UTR is 524 nt longer than the other MV 5' UTRs. Insertions into the 5' UTR of the enhanced green fluorescent protein gene in the minigenome containing the N/P NCS showed that specific sequences, rather than just the additional length of F 5' UTR, govern this decreased expression from TU2.