P-selectin mediates adhesion of the human melanoma cell line NKI-4: identification of glycoprotein ligands

Activated endothelial cells and stimulated platelets express the cell adhesion molecule P-selectin (CD62P), which mediates adhesion to various leukocytes and certain types of cancer cells. In this study, we show Ca2+-dependent binding of P-selectin to NKI-4 cells, a cell line derived from a human melanoma. The binding is inhibited by P7 (a leukocyte adhesion blocking mAb against P-selectin), but not by PL5 (a leukocyte adhesion blocking mAb against P-selectin glycoprotein ligand-1; PSGL-1). Further, expression of PSGL-1 could not be detected on NKI-4 cells by either PL5 mAb or an Ab against a synthetic peptide corresponding to a portion of the PSGL-1 sequence. P-selectin affinity chromatography of lysates from in vivo [3H]-glucosamine-labeled NKI-4 cells resulted in the isolation of three glycoproteins, with apparent molecular masses of approximately 250, approximately 110, and approximately 100 kDa under reducing conditions and approximately 230, approximately 105, and approximately 85 kDa under nonreducing conditions. These molecules could be precipitated by P-selectin, but not by E-selectin. EDTA and the P7 mAb, but not the PL5 mAb, inhibited the binding of P-selectin to the purified ligands. Surprisingly, we found that sodium chlorate, a sulfation inhibitor, did not inhibit the binding of P-selectin to NKI-4 cells and that [35S]-sulfate did not label the NKI-4 cell ligands. We conclude that P-selectin-dependent adhesion of the human melanoma cell line NKI-4 is mediated by a novel class of glycoprotein ligands.     

Saturation intensity and time response of InGaAs-InGaP MQW opticalmodulators

We report modulation saturation and time response measurements on InGaAs-InGaP MQW modulators. The measurements yield a saturation intensity of (3.7±0.1) kW/cm² for a 0-10 V swing and switching times between 10 and 90 ns, depending on the bias voltage and incident light intensity. The observed dependence indicates that field screening due to carrier build-up is the dominant physical mechanism determining both the speed and the saturation intensity. This conclusion is supported by results of theoretical calculations     

The nitric oxide-cGMP pathway may mediate communication between sensory afferents and projection neurons in the antennal lobe of Manduca sexta

The nitric oxide (NO)-cGMP signaling system is thought to play important roles in the function of the olfactory system in both vertebrates and invertebrates. One way of studying the role of NO in the nervous system is to study the distribution and properties of NO synthase (NOS), as well as the soluble guanylyl cyclases (sGCs), which are the best characterized targets of NO. We study NOS and sGC in the relatively simple and well characterized insect olfactory system of the hawkmoth, Manduca sexta. We have cloned Manduca sexta nitric oxide synthase (MsNOS) and two sGCs (MsGCalpha1 and MsGCbeta1), characterized their basic biochemical properties, and studied their expression in the olfactory system. The sequences of the Manduca genes are highly similar to their mammalian homologs and show similar biochemical properties when expressed in COS-7 cells. In particular, we find that MsGC functions as an obligate heterodimer that is stimulated significantly by NO. We also find that MsNOS has a Ca2+-sensitive NO-producing activity similar to that of mammalian neuronal NOS. Northern and in situ hybridization analyses show that MsNOS and the MsGCs are expressed in a complementary pattern, with MsNOS expressed at high levels in the antennae and the MsGCs expressed at high levels in a subset of antennal lobe neurons. The expression patterns of these genes suggest that the NO-sGC signaling system may play a role in mediating communication between olfactory receptor neurons and projection neurons in the glomeruli of the antennal lobe.     

Characterization of arylamine N-acetyltransferase in Enterobacter aerogenes

N-acetyltransferase (NAT) activity was determined by incubation of purified Enterobacter aerogenes enzyme with 2-aminofluorene (2-AF) as the substrate, followed by high pressure liquid chromatography assays. The NAT activity from E. aerogenes was 0.58 +/- 0.08 nmol/min/mg protein for 2-AF. The values of apparent K(m) and Vmax were 0.72 +/- 0.14 mM and 2.45 +/- 0.29 nmol/min/mg protein, respectively, for 2-AF. The optimal pH value for the enzyme activity was 7.5 for the 2-AF tested. The optimal temperature for enzyme activity was 37 degrees C for the 2-AF substrate. The molecular weight of NAT from E. aerogenes was 44.9 kD. Among a series of divalent cations and salts, Zn2+, Ca2+, and Fe2+ were demonstrated to be the most potent protease inhibitors, and only ethylenediaminetetraacetic acid significantly protected the NAT. Iodoacetamide, in contrast to other agents, markedly inhibited NAT.     

Ethanol, immunomodulation and cancer

Several major physiological changes or disease states occur more frequently in alcohol abusers or prolonged high users. These include: cancer, immunosuppression, and nutritional deficiencies. They interact with potential enhancement of cancer growth or survival. Clearly alcohol abuse yielding cirrhosis is associated with immunosuppression of cellular immune functions. Severe, prolonged ethanol consumption frequently is associated with one or more nutrient deficiency. Such deficiencies are more common when there are other major problems such as liver or pancreas damage and in prolonged alcohol abusers should influence the host defenses against pathogens and tumors. The amount and frequency of nutritional and immunological deficiencies with "binge" or less severe alcohol abuse is less clear, but animal studies suggest they are frequently present. Clearly, nutrient deficiencies are common in alcohol abusers and could play significant roles in ethanol associated problems including immunosuppression. Data summarized suggest that immunosuppression caused by nutritional deficiencies and/or direct effects of ethanol and its' metabolites on lymphoid cells is a potential component of cancer in alcohol abusers. However the frequent occurrence of nutritional deficiencies, immunosuppression, and certain cancers in severe, prolonged ethanol abusers is an association, not proven cause and effect.     

Mutant p53 Mediates Sensitivity to Cancer Treatment Agents in Oesophageal Adenocarcinoma Associated with MicroRNA and SLC7A11 Expression

TP53 gene mutations occur in 70% of oesophageal adenocarcinomas (OACs). Given the central role of p53 in controlling cellular response to therapy we investigated the role of mutant (mut-) p53 and SLC7A11 in a CRISPR-mediated JH-EsoAd1 TP53 knockout model. Response to 2 Gy irradiation, cisplatin, 5-FU, 4-hydroxytamoxifen, and endoxifen was assessed, followed by a TaqMan OpenArray qPCR screening for differences in miRNA expression. Knockout of mut-p53 resulted in increased chemo- and radioresistance (2 Gy survival fraction: 38% vs. 56%, p < 0.0001) and in altered miRNA expression levels. Target mRNA pathways analyses indicated several potential mechanisms of treatment resistance. SLC7A11 knockdown restored radiosensitivity (2 Gy SF: 46% vs. 73%; p = 0.0239), possibly via enhanced sensitivity to oxidative stress. Pathway analysis of the mRNA targets of differentially expressed miRNAs indicated potential involvement in several pathways associated with apoptosis, ribosomes, and p53 signaling pathways. The data suggest that mut-p53 in JH-EsoAd1, despite being classified as non-functional, has some function related to radio- and chemoresistance. The results also highlight the important role of SLC7A11 in cancer metabolism and redox balance and the influence of p53 on these processes. Inhibition of the SLC7A11-glutathione axis may represent a promising approach to overcome resistance associated with mut-p53.     

The effect of increasing application rate of granular calcium ammonium nitrate on net nitrification in a laboratory study of grassland soils

Soil incubation studies were undertaken in controlled environment cabinets at 15°C to investigate the effect of increasing application rates of calcium ammonium nitrate (CAN) on net nitrification in two grassland soils. Granular CAN was applied to the surface of freshly collected, moist soil, at a rate equivalent to 0, 100, 200, 400, 800 and 1600µg NH ₄ ⁺ -N and NO ₃ ^- -N per gram of oven dry soil. In half the treatments finely ground CaCO₃ was incorporated into the moist soil to raise the starting pH. Changes in soil mineral N and pH were measured at weekly intervals up to six-weeks. The most probable number (MPN) technique was used to enumerate the NH ₄ ⁺ -N and NO ₂ ^- -N oxidizers at the beginning and end of the incubation.At low rates of CAN application there was considerable NH ₄ ⁺ -N oxidation to NO ₃ ^- -N during the incubation of both soils. Lime stimulated this N transformation. At high application rates (i.e. 800 and 1600 ppm) there was little change in NH ₄ ⁺ -N or NO ₃ ^- -N on either soil during the 6 week incubation, in the presence or absence of lime. The rate of NO ₃ ^- -N produced peaked at 5.6 and 3.8 mg NO ₃ ^- -N kg^–1 d^–1 on soil 1 and 2 respectively, in the presence of lime. Above a level of 400 ppm CAN (equivalent to 38 kg N ha^–1) the rate of NO ₃ ^- -N produced decreased. The higher rate of net nitrification in soil 1 compared with soil 2 was probably due to a higher number of nitrifying bacteria. Although high rates of CAN decreased the nitrifying activity of both soils there was little difference between treatments in the actual numbers of NH ₄ ⁺ -N and NO ₂ ^- -N oxidizers determined by the MPN technique.The results showed that the rate of granular CAN applied to the soil surface can influence the local activity of nitrifying bacteria and subsequent N transformations. At application rates of CAN generally used agriculturally for grass production, it is likely that net nitrification of the NH ₄ ⁺ -N in the fertilizer granule will be inhibited.     

The influence of soil properties on the effectiveness of phenylphosphorodiamidate (PPD) in reducing ammonia volatilization from surface applied urea

Urea can be an inefficient N source due to rapid hydrolysis by soil urease leading to NH₃ volatilization. The current study investigated the effect of the urease inhibitor phenylphosphorodiamidate (PPD) incorporated at two concentrations (0.5% and 1% w/w) within the fertilizer granule on NH₃ volatilization from surface applied urea. The daily rates of NH₃ loss from 20 soils of widely differing properties from Northern Ireland were measured over 14 days using ventilated enclosures under simulated spring conditions. Cumulative loss rates were calculated and fitted to a logistic model from which total NH₃ loss (Amax) and the time to maximum rate of loss (Tmax) were determined. Stepwise multiple linear regression analysis related the effectiveness of PPD in reducing NH₃ volatilization from urea to soil properties.The total cumulative loss of ammonia from unamended urea varied from 0.37 to 29.2% depending on soil type. Ammonia volatilization appeared to be greatest on a soil with a high pH (R² = 0.65), a low titratable acidity (TA) (R² = 0.63) and a soil that was drying out (R² = 0.50). Soil pH was negatively correlated with TA (r = –0.826, P < 0.001) suggesting that soils with a low TA may have received recent lime. Including cation exchange capacity (CEC) and % N as well as pH-KCl in the multiple linear regression equation explained 86% of the variance.The effectiveness of PPD in reducing Amax varied between 0% to 91% depending on soil type, the average over all 20 soils being 30 and 36% for 0.5% and 1% PPD respectively. The most important soil properties influencing the effectiveness of the urease inhibitor were soil pH-H₂O and TA accounting for 33% and 29% of the variance respectively. PPD was less effective on a soil with a high pH and low TA. These were the soil conditions that led to high NH₃ volatilization from unamended urea and may explain why PPD had limited success in reducing ammonia loss on these soils. Multiple linear regression analysis indicated that 75% of the variation in the % inhibition of NH₃ loss by PPD could be significantly accounted for by pH-H₂O, initial soil NO ₃ ^- -N concentration, % moisture content and % moisture loss.The delay in Tmax by PPD ranged from 0.19 to 7.93 days, the average over all 20 soils being 2.5 and 2.8 days for 0.5% and 1% PPD respectively. TA, % moisture content, urease activity and CEC were soil properties that significantly explained 83% of the variation in the % delay in Tmax by PPD in multiple linear regression analysis. However, none of these soil properties were significant on their own. As urea hydrolysis occurs rapidly in soil, delaying Tmax under field conditions would increase the chance of rain falling to move the urea below the soil surface and reduce NH₃ volatilization. A urease inhibitor should be more effective than PPD on soils with a high pH and low TA to be successful in reducing high NH₃ losses.     

The comparative effect of a mixed urea,ammonium nitrate,ammonium sulphate granular formulation on the efficiency of N recovery by perennial ryegrass

The¹⁵N isotope was used to study the mode of action of individual nitrogen sources in a 30% urea:30% ammonium nitrate: 10% ammonium sulphate:30% filler (w/w) granular fertilizer for perennial ryegrass in a greenhouse pot experiment. The fertilizer consisted of two types of granules, one containing 80% urea and 20% filler and the second containing 48% ammonium nitrate (AN), 16% ammonium sulphate (AS) and 36% filler. In addition the effect of dolomite compared with silica as the filler was investigated on nitrogen recovery from the 30:30:10:30 formulation.Dolomite adversely affected the recovery of nitrate N from the system and evidence suggested that MgCO₃ was the active component. Granules containing dolomite resulted in a lower dry-matter yield than those containing silica, however the difference was not significant as nitrate contributed only 20% of the N in the formulation. AN gave the greatest DM yield and urea the lowest with AS being intermediate. The¹⁵N budget in shoots, roots and soil indicated that only 65% of the N from urea was recovered at the end of the experiment compared with 86% for AN and 91% for AS. The dry-matter yield of the 30:30:10:30 formulation using silica as the filler was intermediate between urea and AN; however, the apparent N recovery was significantly higher than expected from the sum of the individual components. The use of¹⁵N labelling indicated that using separate granules for ammonium N and urea the recovery of urea was improved by 11% in the triple N mixture when both AN and AS were present in the second granule compared to the recovery on its own. The enhanced recovery of urea appeared to be a function of AN and AS acting together as neither source in double combination with urea had any effect on urea N recovery.Urea enhanced the recovery of nitrate N by 10% but decreased the recovery of AS by 6% (in the 30:30:10:30 formulation) in comparison with the single sources on their own. The results indicate that interactions can occur between N sources even when they are physically separated by being in different granules.