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Contains fulltext : 235824.pdf (Publisher’s version ) (Closed access) AIM: Tumour budding ('attacker') and CD8(+) T cells ('defender') are recognised as important parameters for risk stratification in colon cancers and, combined, may have an even stronger clinical impact. Here, we determine the value of tumour budding and CD8(+) in rectal cancer patients treated with/without neoadjuvant therapy. METHODS AND RESULTS: Using digital scans of all tumour slides/case, we analysed CD8(+) T cell counts in two patient cohorts: 45 neoadjuvantly treated and 47 primarily surgically treated (totalling n = 543 slides) after double-staining of the surgical resection specimen for pan-cytokeratin and CD8(+) . Tumour buds in hot-spots were manually counted (area = 0.785 mm(2) ) and CD8(+) T cell counts were analysed separately both in tumour budding hot-spots and the densest CD8(+) regions throughout the tumour. In neoadjuvantly treated patients, only tumour budding and not CD8(+) T cells was associated with tumour features, including more advanced ypT (P = 0.0062), venous invasion (P = 0.002), lymphatic invasion (P = 0.0003) and perineural invasion (P = 0.0017), as well as higher American Joint Committee on Cancer (AJCC) tumour regression score (P = 0.0035), indicating less tumour response. Overall survival was also worse in patients with high-grade budding in univariate analysis only. In contrast, all three variables, namely tumour budding (P = 0.0347), CD8(+) T cells in budding hot-spots (P = 0.0382) and CD8(+) T cells in the densest areas (P = 0.0117) were also associated with worse (budding) and better (CD8) survival time in the multivariate setting. CONCLUSION: In rectal cancer, tumour budding has clinical relevance in both primarily surgically treated patients and in those with neoadjuvantly treated patients, where it characterises highly aggressive residual disease. CD8(+) T cell counts appear not to have prognostic relevance in the neoadjuvant context.

We report measurements of the exclusive neutral pion electroproduction cross section off protons at large values of $x_B$ (0.36, 0.48 and 0.60) and $Q^2$ (3.1 to 8.4 GeV$^2$) obtained from Jefferson Lab Hall A experiment E12-06-014. The corresponding structure functions $d\sigma_L/dt+\epsilon d\sigma_T/dt$, $d\sigma_{TT}/dt$, $d\sigma_{LT}/dt$ and $d\sigma_{LT'}/dt$ are extracted as a function of the proton momentum transfer $t-t_{min}$. The results suggest the amplitude for transversely polarized virtual photons continues to dominate the cross-section throughout this kinematic range. The data are well described by calculations based on transversity Generalized Parton Distributions coupled to a helicity flip Distribution Amplitude of the pion, thus providing a unique way to probe the structure of the nucleon.

Empirical findings consistently suggest that an external focus of attention leads to superior performance when compared to an internal focus by encouraging more automatic processing. However, for certain skills (e.g., gymnastics routines) it can be challenging to identify a meaningful external focus cue. A related line of research suggests that focusing on the general feeling of a movement (i.e., holistic focus) may also be useful in avoiding conscious control of movements. The purpose of this study was to determine how an internal focus (INT), external focus (EXT), and holistic focus (HOL) impact the performance of a standing long jump. Participants (N = 27) completed two baseline jumps followed by two jumps in each focus condition. Jump distance was analyzed in a 6 (Order) × 4 (Focus) mixed ANOVA. Results indicated a significant main effect of focus (p p-values

International audience; In this paper, the problem of optimizing the deployment of a fleet of Unmanned Aerial Vehicles (UAVs) for the inspection of networks of linear infrastructures is addressed. In this optimization problem, two levels need to be considered: the UAVs need to follow a trajectory allowing them to fly over linear infrastructures, with a limited endurance, while ground vehicles need to be deployed to act as platforms that can launch and retrieve the UAVs from fixed parking positions. Both parking positions of ground vehicles and the UAVs fleet routes along the linear infrastructures have to be optimized to improve the inspection efficiency, which is related to multiple contradictory objectives such as minimizing operational cost and maximizing inspection performance. The algorithm proposed in this paper was developed by expanding upon several algorithms from literature. More precisely, we propose to solve a Two-Echelon Vehicle Routing Problem (2E-VRP) with a custom multi-objective Hybrid Genetic Algorithm (HGA) based on a Capacitated Arc Routing Problem (CARP). Simulation results are provided on a benchmark of networks of linear infrastructures along with a sensitivity analysis on the parameters of the algorithm, showing promising results.

Tumour budding ('attacker') and CD8Using digital scans of all tumour slides/case, we analysed CD8In rectal cancer, tumour budding has clinical relevance in both primarily surgically treated patients and in those with neoadjuvantly treated patients, where it characterises highly aggressive residual disease. CD8

This study evaluated the use of molecular imaging of fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) as a discriminatory marker for intraoperative tumor border identification in a murine glioma model.2-NBDG was assessed in GL261 and U251 orthotopic tumor-bearing mice. Intraoperative fluorescence of topical and intravenous 2-NBDG in normal and tumor regions was assessed with an operating microscope, handheld confocal laser scanning endomicroscope (CLE), and benchtop confocal laser scanning microscope (LSM). Additionally, 2-NBDG fluorescence in tumors was compared with 5-aminolevulinic acid-induced protoporphyrin IX fluorescence.Intravenously administered 2-NBDG was detectable in brain tumor and absent in contralateral normal brain parenchyma on wide-field operating microscope imaging. Intraoperative and benchtop CLE showed preferential 2-NBDG accumulation in the cytoplasm of glioma cells (mean [SD] tumor-to-background ratio of 2.76 [0.43]). Topically administered 2-NBDG did not create sufficient tumor-background contrast for wide-field operating microscope imaging or under benchtop LSM (mean [SD] tumor-to-background ratio 1.42 [0.72]). However, topical 2-NBDG did create sufficient contrast to evaluate cellular tissue architecture and differentiate tumor cells from normal brain parenchyma. Protoporphyrin IX imaging resulted in a more specific delineation of gross tumor margins than intravenous or topical 2-NBDG and a significantly higher tumor-to-normal-brain fluorescence intensity ratio.After intravenous administration, 2-NBDG selectively accumulated in the experimental brain tumors and provided bright contrast under wide-field fluorescence imaging with a clinical-grade operating microscope. Topical 2-NBDG was able to create a sufficient contrast to differentiate tumor from normal brain cells on the basis of visualization of cellular architecture with CLE. 5-Aminolevulinic acid demonstrated superior specificity in outlining tumor margins and significantly higher tumor background contrast. Given the nontoxicity of 2-NBDG, its use as a topical molecular marker for noninvasive in vivo intraoperative microscopy is encouraging and warrants further clinical evaluation.

This document presents the technical layout and the performance of the CLAS12 Forward Tagger (FT). The FT, composed of an electromagnetic calorimeter based on PbWO4 crystals (FT-Cal), a scintillation hodoscope (FT-Hodo), and several layers of Micromegas trackers (FT-Trk), has been designed to detect electrons and photons scattered at polar angles from 2 ∘ to 5 ∘ and to meet the physics goals of the hadron spectroscopy program and other experiments running with the CLAS12 spectrometer in Hall B.

The Micromegas Vertex Tracker was designed to improve upon the tracking capabilities of the baseline design of the CLAS12 spectrometer in Hall B at Jefferson Laboratory. A Barrel Micromegas Tracker made with six concentric cylinders, each made of three 120 ∘ -sector tiles, surrounds the Silicon Vertex Tracker, and a Forward Micromegas Tracker composed of 6 disks is placed 30 cm downstream of the liquid-hydrogen target. Both trackers sit in a 5 T solenoid magnetic field. All Micromegas elements are based on resistive technology to withstand luminosities up to 1 0 35 cm−2 s−1, as well as on bulk technology to enforce gain uniformity and mechanical robustness. Due to the high magnetic field, dedicated electronics have been designed and displaced ∼ 2 m away from the detectors. The electronics readout is based on the DREAM ASICs that allow sustained operation up to 20 kHz trigger rate at the maximum luminosity.

Fluorescence imaging is an emerging clinical technique for real-time intraoperative visualization of tumors and their boundaries. Though multiple fluorescent contrast agents are available in the basic sciences, few fluorescence agents are available for clinical use. Of the clinical fluorophores, delta aminolevulinic acid (5ALA) is unique for generating visible wavelength tumor-specific fluorescence. In 2017, 5ALA was FDA-approved for glioma surgery in the United States. Additionally, clinical studies suggest this agent may have utility in surgical subspecialties outside of neurosurgery. Data from dermatology, OB/GYN, urology, cardiothoracic surgery, and gastrointestinal surgery show 5ALA is helpful for intraoperative visualization of malignant tissues in multiple organ systems. This review summarizes data from English-language 5ALA clinical trials across surgical subspecialties. Imaging systems, routes of administration, dosing, efficacy, and related side effects are reviewed. We found that modified surgical microscopes and endoscopes are the preferred imaging devices. Systemic dosing across surgical specialties range between 5 and 30 mg/kg bodyweight. Multiple studies discussed potential for skin irritation with sun exposure, however this side effect is infrequently reported. Overall, 5ALA has shown high sensitivity for labeling malignant tissues and providing a means to visualize malignant tissue not apparent with standard operative light sources.

We studied the direct effect of reactive hydroxyl precursors and inhibitors on CD4+ T-cell function. We used hydrogen peroxide plus ferrous chloride as the hydroxyl radical-generating system and di-methyl sulphourea, di-methyl sulfoxide, pyrrolidine dithiocarbonate, methanol, and ethanol, at a noncytotoxic concentration, as inhibitors. The immune parameter studies were proliferation and interleukin-2 production by peripheral blood lymphocytes stimulated with anti-CD3 antibody, phytohemagglutinin and alloantigens; proliferation, interleukin-2 production and mRNA expression of interleukin-4 and interferon gamma by allogeneic CD4+ T-cell clones stimulated with alloantigens. The results show that lymphocytes produce significant amounts of reactive oxygen species as measured by malondialdehyde produced in cultures. The hydroxyl radical-generating system did not change any of the cellular responses studied although it doubled Malondialdehyde production. Hydroxyl radical scavengers signi tly inhibited all responses at doses that didn't significantly decrease malondialdehyde production. DNA analysis failed to show evidence for apoptosis. Conclusion: Hydroxyl radical scavengers inhibit lymphocyte mitogenesis by a process that is independent of scavenging hydroxyl radicals.

Histidine-containing protein (HPr) is a phosphocarrier protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system. HPr is phosphorylated at the active site residue, His15, by phosphoenolpyruvate-dependent enzyme I in the first enzyme reaction in the process of phosphoryl transfer to sugar. In many Gram-positive bacterial species HPr may also be phosphorylated at Ser46 by an ATP-dependent protein kinase but not in the Gram-negative Escherichia coli and Salmonella typhimurium. One effect of the phosphorylation at Ser46 is to make HPr a poor acceptor for phosphorylation at His15. In Bacillus subtilis HPr, the mutation Ser46Asp mimics the effects of phosphorylation. A series of mutations were made at Ser46 in E. coli HPr: Ala, Arg, Asn, Asp, Glu, and Gly. The two acidic replacements mimic the effects of phosphorylation of Ser46 in HPrs from Gram-positive bacteria. In particular, when mutated to Asp46, the His 15 phosphoacceptor activity (enzyme I Km/Kcat) decreases by about 2000-fold (enzyme I Km, 4 mM HPr; Kcat, approximately 30%). The alanine and glycine mutations had near-wild-type properties, and the asparagine and arginine mutations yielded small changes to the Km values. The crystallographic tertiary structure of Ser46Asp HPr has been determined at 1.5 A resolution, and several changes have been observed which appear to be the effect of the mutation. There is a tightening of helix B, which is demonstrated by a consistent shortening of hydrogen bond lengths throughout the helix as compared to the wild-type structure. There is a repositioning of the Gly54 residue to adopt a 3(10) helical pattern which is not present in the wild-type HPr. In addition, the higher resolution of the mutant structure allows for a more definitive placement of the carbonyl of Pro11. The consequence of this change is that there is no torsion angle strain at residue 16. This result suggests that there is no active site torsion angle strain in wild-type E. coli HPr. The lack of substantial change at the active center of E. coli HPr Ser46Asp HPr suggests that the effect of the Ser46 phosphorylation in HPrs from Gram-positive bacteria is due to an electrostatic interference with enzyme I binding.

The goal of the work was to establish the toxicity and biodistribution of the superparamagnetic protein cationized ferritin (CF) after intravenous injection. Intravenously injected CF has been used to target the extracellular matrix with high specificity in the kidney glomerulus, allowing measurements of individual glomeruli using T2*-weighted MRI. For the routine use of CF as an extracellular matrix-specific tracer, it is important to determine whether CF is toxic. In this work, we investigated the renal and hepatic toxicity, leukocyte count, and clearance of intravenously injected CF. Furthermore, we studied CF labeling in several organs using MRI and immunohistochemistry. Serum measurements of biomarkers suggest that intravenous injection of CF is neither nephrotoxic nor hepatotoxic and does not increase leukocyte counts in healthy rats at a dose of 5.75 mg/100 g. In addition to known glomerular labeling, confocal and MRI suggest that intravenously injected CF labels the extracellular matrix of the hepatic sinusoid, extracellular glycocalyx of alveolar endothelial cells, and macrophages in the spleen. Liver T2* values suggest that CF is cleared by 7 days after injection. These results suggest that CF may serve as a useful contrast agent for detection of a number of structures and functions with minimal toxicity.

Usually manufacturer's specifications do not deal with the ability of linear sheet polarizers to have a constant transmittance function over their geometric area. These parameters are fundamental for developing low cost polarimetric sensors(for instance rotation, torque, displacement) specifically for hybrid car (thermic + electricity power). It is then necessary to specially characterize commercial polarizers sheets to find if they are adapted to this kind of applications. In this paper, we present measuring methods and bench developed for this purpose, and some preliminary characterization results. We state conclusions for effective applications to hybrid car gearbox control and monitoring.

Histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli, when incubated at elevated temperatures forms many species of protein. The two major species are HPr-1 and HPr-2, which have been shown to lack one or two amides, respectively (Anderson, B., Weigel, N., Kundig, W., and Roseman, S. (1971) J. Biol. Chem. 246, 7023-7033). The formation of HPr-1 and HPr-2 is shown to be pH-dependent and does not occur readily below pH 6. Investigation of the identities and properties of the two residues that deamidate involved creation of site-directed mutants at the 6 glutamine and 2 asparagine residues of HPr; description of their deamidation species by isoelectric focusing; determination of their relative antibody binding properties; assay of their phosphoacceptor and phosphodonor activities; characterization of tryptic and V8-protease peptides; obtaining two-dimensional nuclear magnetic resonance spectra of HPr, HPr-1, and several mutants. It was determined that the sequential deamidation of Asn-38 and Asn-12 yields HPr-1 and HPr-2. Both residues exist as Asn-Gly pairs, and both deamidations probably form isoaspartyl acid. HPr from Bacillus subtilis and Staphylococcus carnosus which also have Asn-Gly at residues 38 and 39 form HPr-1 species presumably by deamidation. HPr from Streptococcus faecalis which does not have Asn-38 does not form a HPr-1 species. The E. coli mutant HPrs, N12D and Q51E, residues that may be involved in the active site, had impaired phosphohydrolysis properties and decreased phosphoenolpyruvate:sugar phosphotransferase system activity.

Histidine-containing protein, HPr, of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has an active site His-15 that is phosphorylated to form N delta 1-P-histidine. The nearby conserved residue, Arg-17, has been replaced by: lysine, histidine, glutamate, glycine, serine, and cysteine. All mutations resulted in impairment of the phosphoacceptor function of HPr with enzyme I: kcat/Km values between 6% (Ser-17) and 0.1% (Glu-17), relative to wild type. Several sugar-specific enzymes II had different responses. Both the Vmax and Km of enzyme IIN-acetylglucosamine were altered, while for enzyme IImannose only Km was affected, except for R17E. For both enzymes, kcat/Km values were between 0.5 and 3%, with R17E being 10-fold lower. Except for R17E, minimal effects were observed for enzyme IImannitol. These results suggest that there are different rate-limiting steps in the enzymes II. Phosphohydrolysis properties and the pKa values for His-15 and phosphorylated His-15 determined by NMR for both wild type and mutant HPrs suggest that Arg-17 is partly responsible for the instability of P-His-15 and the depressed pKa values in wild type HPr. Other feature(s) of the tertiary structure influence the protonation of His-15 and the phosphohydrolysis properties of phosphorylated His-15.

Thirty-four of the 85 residues of the histidine-containing protein HPr of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system have been changed by site-directed mutagenesis. Many of the mutations have wild-type activity suggesting an unaltered tertiary structure but have altered binding to three monoclonal antibodies: Jel42, Jel44, and Jel323. This altered binding defines the residues that are involved in the epitopes of HPr. At present, two different three-dimensional structures have been determined for HPr, one from two-dimensional nuclear magnetic resonance spectra and the other from x-ray diffraction of HPr crystals. The epitope mapping for Jel42 does not distinguish between the tertiary structures. However, only the HPr structure derived from two-dimensional nuclear magnetic resonance spectra is consistent with a contiguous surface binding site that can be defined as the epitope for Jel44. Thus the x-ray structure may represent a partially unfolded HPr.

To evaluate the efficacy of intravenous immunoglobulin (IVIg) in the treatment of Birdshot retinochoroiditis.Eighteen patients were followed-up prospectively in a total number of 37 patients recorded. Inclusion criteria were birdshot retinochoroiditis (BRC) according to the criteria defined by Ryan et al., and a decrease in visual acuity (VA). Efficacy was assessed by measurements of visual acuity and a decrease in inflammation and macular edema on fluorescein angiograms.Sex ratio=1, mean age was 51 (range 29 to 72 years), HLA A29 was positive in 100% of the patients. VA at baseline was 0.6+/-2.4 (range 0.25 to 0.9). Angiography showed retinal vasculitis in 32 patients (86.48%) and cystoid macular edema in 16 patients (43.24%). IVIg was administered at a dosage of 0.4g/kg/d for 4 days then 0.6g/kg/d for 2 days every 4 weeks. Follow-up lasted a mean of 2.7+/-2.0 years (range, 4 months to 5.6 years). Visual acuity of 35 out of 66 eyes (53%) increased by 2.6+/-1.5 (range 1 to 5). In 19 eyes out of 66 (29%), VA remained stable and in 12 eyes out of 66 (18%), VA decreased by -1.8+/-0.8 (ranges-4 to -1). Inflammation on fluorescein angiography improved in 17 patients (81%) and cystoid macular edema decreased in 65% of the cases. Side effects were rare. Treatment was discontinued for side effects in only 3 patients.BRC is a chronique disease threatening vision. Treatments including systemic corticosteroids and cyclosporin A may result in severe long-term side effects. IVIg is well tolerated and may be therapeutic alternative for the treatment of BRC.

The c-fos gene is expressed in the central nervous system in response to various neuronal stimuli. Using in situ hybridization, we examined the effects of chronic morphine treatment and withdrawal on c-fos mRNA in the rat brain, and particularly within identified striatal neurons. Morphine dependence was induced by subcutaneous implantation of two pellets of morphine for 6 days and withdrawal was precipitated by administration of naltrexone. Placebo animals and morphine-dependent rats showed a very weak c-fos mRNA expression in all the structures studied. Our study emphasized the spatial variations in c-fos mRNA expression, and also revealed a peak expression of c-fos mRNA at 1 h after naltrexone-precipitated withdrawal in the projection areas of dopaminergic neurons, noradrenergic neurons and in several regions expressing opiate receptors. Interestingly, morphine withdrawal induces c-fos mRNA expression in the two efferent populations of the striatum (i.e. striatonigral and striatopallidal neurons) both in the caudate putamen and nucleus accumbens. Moreover, the proportions of activated neurons during morphine withdrawal are different in the caudate putamen (mostly in striatopallidal neurons) and in the shell and core parts of the nucleus accumbens (mostly in striatonigral neurons). The activation of striatopallidal neurons suggests a predominant dopaminergic regulation on c-fos gene expression in the striatum during withdrawal. On the contrary, c-fos induction in striatonigral neurons during withdrawal seems to involve a more complex regulation like opioid-dopamine interactions via the mu opioid receptor and the D1 dopamine receptor coexpressed on this neuronal population or the implication of other neurotransmitter systems.

Enzyme I mutants of the Salmonella typhimurium phosphoenolpyruvate:sugar phosphotransferase system (PTS), which show in vitro intragenic complementation, have been identified as Arg126Cys (strain SB1690 ptsI34), Gly356Ser (strain SB1681 ptsI16), and Arg375Cys (strain SB1476 ptsI17). The mutation Arg126Cys is in the N-terminal HPr-binding domain, and complements Gly356Ser and Arg375Cys enzyme I mutations located in the C-terminal phosphoenolpyruvate(PEP)-binding domain. Complementation results in the formation of unstable heterodimers. None of the mutations alters the K(m) for HPr, which is phosphorylated by enzyme I. Arg126 is a conserved residue; the Arg126Cys mutation gives a V(max) of 0.04% wild-type, establishing a role in phosphoryl transfer. The Gly356Ser and Arg375Cys mutations reduce enzyme I V(max) to 4 and 2%, respectively, and for both, the PEP K(m) is increased from 0.1 to 3 mM. It is concluded that this activity was from the monomer, rather than the dimer normally found in assays of wild-type. In the presence of Arg126Cys enzyme, V(max) for Gly356Ser and Arg375Cys enzymes I increased 6- and 2-fold, respectively; the K(m) for PEP decreased to

The structure of the N-terminal domain of enzyme I complexed with histidine-containing protein (HPr) has been described by multi-dimensional NMR. Residues in HPr involved in binding were identified by intermolecular nuclear Overhauser effects (Garrett et al. 1999). Most of these residues have been mutated, and the effect of these changes on binding has been assessed by enzyme I kinetic measurement. Changes to Thr16, Arg17, Lys24, Lys27, Ser46, Leu47, Lys49, Gln51, and Thr56 result in increases to the HPr Km of enzyme I, which would be compatible with changes in binding. Except for mutations to His15 and Arg17, very little or no change in Vmax was found. Alanine replacements for Gln21, Thr52, and Leu55 have no effect. The mutation Lys40Ala also affects HPr Km of enzyme I; residue 40 is contiguous with the enzyme I binding site in HPr and was not identified by NMR. The mutations leading to a reduction in the size of the side chain (Thr16Ala, Arg17Gly, Lys24Ala, Lys27Ala, and Lys49Gly) caused relatively large increases in Km (5-fold) indicating these residues have more significant roles in binding to enzyme I. Acidic replacement at Ser46 caused very large increases (100-fold), while Gln51Glu gave a 3-fold increase in Km. While these results essentially concur with the identification of residues by the NMR experiments, the apparent importance of individual residues as determined by mutation and kinetic measurement does not necessarily correspond with the number of contacts derived from observed intermolecular nuclear Overhauser effects.

The influence of chronic morphine and spontaneous withdrawal on the expression of dopamine receptors and neuropeptide genes in the rat striatum was investigated. Morphine dependence was induced by subcutaneous implantation of two morphine pellets for 6 days. Rats were made abstinent by removal of the pellets 1, 2 or 3 days before they were killed. The mRNA levels coding for D1- and D2-dopamine receptors, dynorphin, preproenkephalin A and substance P were determined by quantitative in situ hybridization. The caudate putamen and the nucleus accumbens showed equivalent modifications in dopamine receptor and neuropeptide gene expression. After 6 days of morphine, a decrease in D2-dopamine receptor and neuropeptide mRNA levels was observed (-30%), but there was no change in D1-dopamine receptor mRNA. In abstinent rats, both D1- and D2-dopamine receptor mRNA levels were decreased 1 day after withdrawal (-30% compared with chronic morphine). In contrast, neuropeptide mRNA levels were unaffected when compared with those observed after 6 days of morphine. During the second and third day of withdrawal, there was a gradual return to the levels seen in the placebo-treated group, for both dopamine receptor and neuropeptide mRNAs. Phenotypical characterization of striatal neurons expressing mu and kappa opioid receptor mRNAs showed that, in striatonigral neurons, both mRNAs were colocalized with D1-receptor and Dyn mRNAs. Our results suggest that during morphine dependence, dopamine and morphine exert opposite effects on striatonigral neurons, and that effects occurring on striatopallidal neurons are under dopaminergic control. We also show that withdrawal is associated with a down regulation of the postsynaptic D1 and D2 receptors.

We studied the direct effect of reactive hydroxyl precursors and inhibitors on CD4+ T-cell function. We used hydrogen peroxide plus ferrous chloride as the hydroxyl radical-generating system and di-methyl sulphourea, di-methyl sulfoxide, pyrrolidine dithiocarbonate, methanol, and ethanol, at a noncytotoxic concentration, as inhibitors. The immune parameter studies were proliferation and interleukin-2 production by peripheral blood lymphocytes stimulated with anti-CD3 antibody, phytohemagglutinin and alloantigens; proliferation, interleukin-2 production and mRNA expression of interleukin-4 and interferon gamma by allogeneic CD4+ T-cell clones stimulated with alloantigens. The results show that lymphocytes produce significant amounts of reactive oxygen species as measured by malondialdehyde produced in cultures. The hydroxyl radical-generating system did not change any of the cellular responses studied although it doubled Malondialdehyde production. Hydroxyl radical scavengers significantly inhibited all responses at doses that didn't significantly decrease malondialdehyde production. DNA analysis failed to show evidence for apoptosis.Hydroxyl radical scavengers inhibit lymphocyte mitogenesis by a process that is independent of scavenging hydroxyl radicals.

Histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli, when incubated at elevated temperatures forms many species of protein. The two major species are HPr-1 and HPr-2, which have been shown to lack one or two amides, respectively (Anderson, B., Weigel, N., Kundig, W., and Roseman, S. (1971) J. Biol. Chem. 246, 7023-7033). The formation of HPr-1 and HPr-2 is shown to be pH-dependent and does not occur readily below pH 6. Investigation of the identities and properties of the two residues that deamidate involved creation of site-directed mutants at the 6 glutamine and 2 asparagine residues of HPr; description of their deamidation species by isoelectric focusing; determination of their relative antibody binding properties; assay of their phosphoacceptor and phosphodonor activities; characterization of tryptic and V8-protease peptides; obtaining two-dimensional nuclear magnetic resonance spectra of HPr, HPr-1, and several mutants. It was determined that the sequential deamidation of Asn-38 and Asn-12 yields HPr-1 and HPr-2. Both residues exist as Asn-Gly pairs, and both deamidations probably form isoaspartyl acid. HPr from Bacillus subtilis and Staphylococcus carnosus which also have Asn-Gly at residues 38 and 39 form HPr-1 species presumably by deamidation. HPr from Streptococcus faecalis which does not have Asn-38 does not form a HPr-1 species. The E. coli mutant HPrs, N12D and Q51E, residues that may be involved in the active site, had impaired phosphohydrolysis properties and decreased phosphoenolpyruvate:sugar phosphotransferase system activity.

Histidine-containing protein, HPr, of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has an active site His-15 that is phosphorylated to form N delta 1-P-histidine. The nearby conserved residue, Arg-17, has been replaced by: lysine, histidine, glutamate, glycine, serine, and cysteine. All mutations resulted in impairment of the phosphoacceptor function of HPr with enzyme I: kcat/Km values between 6% (Ser-17) and 0.1% (Glu-17), relative to wild type. Several sugar-specific enzymes II had different responses. Both the Vmax and Km of enzyme IIN-acetylglucosamine were altered, while for enzyme IImannose only Km was affected, except for R17E. For both enzymes, kcat/Km values were between 0.5 and 3%, with R17E being 10-fold lower. Except for R17E, minimal effects were observed for enzyme IImannitol. These results suggest that there are different rate-limiting steps in the enzymes II. Phosphohydrolysis properties and the pKa values for His-15 and phosphorylated His-15 determined by NMR for both wild type and mutant HPrs suggest that Arg-17 is partly responsible for the instability of P-His-15 and the depressed pKa values in wild type HPr. Other feature(s) of the tertiary structure influence the protonation of His-15 and the phosphohydrolysis properties of phosphorylated His-15.

Histidine-containing protein, HPr, of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has an active site that involves His-15, which is phosphorylated to form a N delta 1-P-histidine, Arg-17, and the carboxy-terminal residue Glu-85. Mutant HPrs with alterations to the three C-terminal residues, Glu-85, Leu-84, and Glu-83, were produced by site-directed mutagenesis. The properties of these mutants were assessed by kinetic analysis of enzyme I, enzyme IImannose, enzyme IIN-acetylglucosamine, and enzyme IImannitol, and the phosphohydrolysis properties of the HPr mutants. The results show that it is the C-terminal alpha-carboxyl of Glu-85 that is involved in the active site, and this involvement may be restricted to the phosphoryl donor action of HPr. The contribution of this alpha-carboxyl group is modest as the deletion of Glu-85 resulted in the reduction of the enzyme II activity (kcat/Km) to about 33%. Removal of both Glu-85 and Leu-84 yields an HPr that is an impaired substrate of both the enzyme I and enzyme II reactions. Glu-83 appears to have no role in the active site.

The isotopic dilution method is really suitable for the determination of blood volume in small animals. The main purpose of this study is to find the limits of measuring errors made during the injection procedures (injected volumes about one microliter) and the sampling of blood (blood samples less than one milliliter). After evaluation of the extravascular rate of diffusion of the radioactive iodinated human serum-albumin, the blood volume of the 17 days incubated chick embryo is measured by this method for control purposes.

This paper deals with an interactive flying spot scanner connected to a digital picture processing system. Its structure is discussed. One of the characteristics is analyzed in detail: the possibility of proceeding to interactive geometrical deformations with a programmable scanning system. A type of deformation was selected, and the principle of its implementation is outlined. The procedure which enables the execution of a geometric matching of a topographic map and a Landsat digital image is given as an application example. The main advantages of this method are pointed out.

We have studied oxygen transport and haemodynamics in 19 clinically stable chronic bronchitis split into two groups with comparable blood gases but different haematocrits (Group I = 33%, Group II = 51.5%). The aim of the study was to assess the suitability of bringing the blood volume down to the predicted normal value by repeated venesection. The respiratory function was moderately altered in these patients. The pulmonary and systemic vascular resistance were significantly more elevated in Group II. The arterial oxygen transport was comparable in the two groups. On the other hand for Group I the systolic work of the right ventricle was greater on account of the increased cardiac output. We have not observed any difference in blood volume between the two groups of patients. These results suggest that a return to the predicted blood volume is not recommended without some doubts in chronic bronchitis with polycythaemia.

Very few studies have been described comparing the value of exercise myocardial scintigraphy and left ventricular angioscintigraphy. The authors designed a study comparing these two investigations with conventional exercise stress testing and coronary angiography. The isotopic investigations were carried out within 48 hours of coronary angiography. A total of 143 patients undergoing coronary angiography (35 normal, 108 coronary patients: 36 single vessel, 36 double vessel and 36 triple vessel disease) were included in this study. The lesions were located of the LAD (77 cases), left circumflex (77 cases) and right coronary arteries (62). The sensitivity and specificity of both radionuclide investigations were evaluated to assess their diagnostic value; the best results were obtained with myocardial scintigraphy (sensitivity 86 p. 100; specificity 100 p. 100); angioscintigraphy had a sensitivity of 71 p. 100 and specificity of 97 p. 100, and conventional exercise stress testing of 42 p. 100 and 70 p. 100 respectively. The sensitivity seemed to increase with the degree of stenosis; although the sensitivity of myocardial scintigraphy increased progressively, that of angioscintigraphy doubled in cases of stenosis 90 p. 100 (stenosis less than 90 p. 100, sensitivity = 37 p. 100; stenosis greater than 90 p. 100, sensitivity = 73 p. 100). The sensitivity of myocardial scintigraphy with respect to the severity of the coronary artery disease was best in cases of right coronary artery stenosis (sensitivity in cases of RCA stenosis = 74 p. 100; sensitivity in LAD stenosis = 58 p. 100; sensitivity in left circumflex stenosis = 43 p. 100). The sensitivity of left ventricular angioscintigraphy was best in LAD stenosis (RCA stenosis = 50 p. 100, LAD stenosis = 64 p. 100, left circumflex stenosis = 36 p. 100). The sensitivity of both investigations was poor in left circumflex artery stenosis even when severely diseased. The sensitivity of both investigations was better in diffuse coronary artery disease: myocardial scintigraphy (single vessel disease: 72 p. 100, double vessel disease: 92 p. 100, triple vessel disease: 94 p. 100), left ventricular angioscintigraphy (61 p. 100, 69 p. 100, and 83 p. 100 respectively). Although the association of these two radioisotopic investigations does not improve diagnostic sensitivity, it does provide more information about the localisation and extension of the coronary artery disease especially in LAD and right coronary artery stenosis. These results suggest that these investigations are complementary in the evaluation of patients with coronary artery disease.(ABSTRACT TRUNCATED AT 400 WORDS)

Transient expression of chloramphenicol acetyl transferase gene has been detected in cultured barley (Hordeum vulgare L. cv. Heartland) cells and freshly isolated immature zygotic embryos (cv. Ellice) following the introduction of the gene by microprojectile bombardment. The DNA expression vector used to introduce the CAT gene, pCaMVI1CN, is a pUC8 derivative and consisted of a CaMV35S promoter, a fragment of alcohol dehydrogenase intron1, a CAT coding region and NOS polyadenylation region. The inclusion of the Adh1 intron1 was essential for the expression of CAT activity in cultured cells as well as immature zygotic embryos. Expression of CAT activity, which was dependent upon the DNA concentration used, could be detected as early as 20 h after bombardment. The results also suggested that the recipient cells have to be in an active state of cell division in order for the introduced gene to be expressed since mature zygotic as well as somatic embryos failed to reveal any gene expression. The effect of other parameters which influence the expression of the introduced gene as well as the potential of this novel technology for cereal transformation are also discussed.