Differential response to dexamethasone on the TXB2 release in guinea-pig alveolar macrophages induced by zymosan and cytokines

Glucocorticosteroids reduce the production of inflammatory mediators but this effect may depend on the stimulus. We have compared the time course of the effect of dexamethasone on the thromboxane B2 (TXB2) release induced by cytokine stimulation and zymosan in guinea-pig alveolar macrophages. Interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and opsonized zymosan (OZ), all stimulate TXB2 release. High concentrations of dexamethasone (1-10 microM) inhibit the TXB2 production induced by both cytokines and OZ, but the time course of this response is different. Four hours of incubation with dexamethasone reduce the basal TXB2 release and that induced by IL-1beta and TNF-alpha, but do not modify the TXB2 release induced by OZ. However, this stimulus was reduced after 24 h incubation. Our results suggest that the antiinflammatory activity of glucocorticosteroids shows some dependence on stimulus and, therefore, may have more than one mechanism involved.     

Parallel reverse genetic screening in mutant human cells using transcriptomics

Reverse genetic screens have driven gene annotation and target discovery in model organisms. However, many disease‐relevant genotypes and phenotypes cannot be studied in lower organisms. It is therefore essential to overcome technical hurdles associated with large‐scale reverse genetics in human cells. Here, we establish a reverse genetic approach based on highly robust and sensitive multiplexed RNA sequencing of mutant human cells. We conduct 10 parallel screens using a collection of engineered haploid isogenic cell lines with knockouts covering tyrosine kinases and identify known and unexpected effects on signaling pathways. Our study provides proof of concept for a scalable approach to link genotype to phenotype in human cells, which has broad applications. In particular, it clears the way for systematic phenotyping of still poorly characterized human genes and for systematic study of uncharacterized genomic features associated with human disease.     

Quantification of the kinetin effect on protein synthesis and degradation in senescing wheat leaves

Wheat leaves (Triticum aestivum L. cv San Agustin INTA) were detached when they reached maximum expansion, put individually in tubes containing water and left in darkness. After 3 days the protein content had decreased to 46% of the initial value. When the leaves were placed in 1 micromolar kinetin, they retained 60% of the initial protein content for the same period. This effect was observed only when leaves were treated with kinetin within the first 24 hours after detachment. The action of kinetin on both protein synthesis and degradation was quantitatively measured. Synthesis was estimated by the incorporation of l-[³H]leucine into proteins. It was higher in kinetin treated than in non treated leaves. It contributed to about 14 micrograms of protein retention per leaf in 3 days. Measurement of protein degradation, evaluated by the decay of radioactivity in leaf proteins previously labeled with l-[³H] leucine or as the difference between rates of protein synthesis and protein content, showed that kinetin decreased protein breakdown rates. It accounted for about 186 micrograms of protein retention per leaf in 3 days. Hence, kinetin action on protein breakdown was 13-fold average higher than its action on synthesis for the conservation of leaf protein. This difference is higher in early stages of the process.     

Mitochondrial DNA variation in maize plants regenerated during tissue culture selection

Summary Plants resistant to Helminthosporium maydis race T were obtained following selection for H. maydis pathotoxin resistance in tissue cultures of susceptible, Texas male-sterile (T) cytoplasm maize. The selected lines transmitted H. maydis resistance to their sexual progeny as an extranuclear trait. Of 167 resistant, regenerated plants, 97 were male fertile and 70 were classified male sterile for reasons that included abnormal plant, tassel, anther or pollen development. No progeny were obtained from these male-sterile, resistant plants. Male fertility and resistance to the Phyllosticta maydis pathotoxin that specifically affects T cytoplasm maize were co-transmitted with H. maydis resistance to progeny of male-fertile, resistant plants. These three traits previously were associated only with the normal (N) male-fertile cytoplasm condition in maize. Three generations of progeny testing provided no indication that the cytoplasmic association of male sterility and toxin susceptibility had been broken by this selection and regeneration procedure. Restriction endonuclease analysis of mitochondrial DNA (mtDNA) revealed that three selected, resistant lines had distinct mtDNA organization that distinguished them from each other, from T and from N cytoplasm maize. Restriction patterns of the selected resistant lines were similar to those from T cytoplasm mtDNA; these patterns had not been observed in any previous analyses of various sources of T cytoplasm. The mtDNA analyses indicated that the male-fertile, toxin-resistant lines did not originate from selection of N mitochondrial genomes coexisting previously with T genomes in the T cytoplasm line used for selection.Scientific Journal Series Article no. 11,185 of the Minnesota Agricultural Experiment Station and no. 2295 of the Florida Agricultural Experiment Station. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee of warrantly of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable     

Prolonged stability of the plantibody HB-01 directed against the hepatitis B surface antigen in cryo-preserved tobacco leaves

Since 1983, several recombinant antibodies have been expressed in important agronomic plant species. However, to date no evaluation has been published about prolonged antibody stability within plant tissues under cryo-preservation conditions. This current report presents an approach to the KDEL-plantibody HB-01 (PHB-01) stability in frozen tobacco leaves by presenting scientific evidence about the stability of a plantibody to a prolonged low temperature exposure in this biological source. Results clearly show that the PHB-01 amount is maintained during the storage of tobacco leaves at ?20 °C for 90 days. The PHB-01 recovery was not affected by any irreversible physical and/or chemical change produced in tobacco leaves after this cryo-preservation time. The amount of total soluble proteins in the clarified extract decreased in proportion with the storage time and the PHB-01 molecules isolated from frozen leaf extracts were highly pure, >95%, according to an SDS-PAGE assessment under reducing conditions. Low temperature exposure of tobacco leaves did not reveal visible changes in frozen leaves, which is essential for the further extractability of proteins. The PHB-01 is stable in tobacco leaves at ?20 °C during 90 days, which offers the possibility to overcome problems associated with detrimental climate conditions and optimize purification capabilities.     

Micronucleus assay in biomonitoring of patients undergoing excretory urography with diatrizoate and ioxaglate

In order to perform biological monitoring of exposure to radiation and contrast media, we evaluated the micronucleus count (MN) and the mitotic index (MI) in peripheral blood lymphocytes from patients undergoing excretory urography with diatrizoate (20 patients) and ioxaglate (20 patients). Three samples were taken for each patient: A (before exploration), B (immediately after exploration) and C (7 days later). There were no significant differences in the radiation doses received, nor in the dose of contrast agent, between both groups. The micronucleus count increased significantly in sample B in both groups, the increase being more statistically significant in the diatrizoate group (p less than 0.01) than in the ioxaglate group (p less than 0.05). One week later, the MN were still slightly high (p less than 0.05) in the diatrizoate group only. These results suggest a clastogenic effect which depends, to a great extent, on the nature of the contrast medium.