sst2 Somatostatin receptor inhibits cell proliferation through Ras-, Rap1-, and B-Raf-dependent ERK2 activation

The G protein-coupled sst2 somatostatin receptor is a critical negative regulator of cell proliferation. sstII prevents growth factor-induced cell proliferation through activation of the tyrosine phosphatase SHP-1 leading to induction of the cyclin-dependent kinase inhibitor p27Kip1. Here, we investigate the signaling molecules linking sst2 to p27Kip1. In Chinese hamster ovary-DG-44 cells stably expressing sst2 (CHO/sst2), the somatostatin analogue RC-160 transiently stimulates ERK2 activity and potentiates insulin-stimulated ERK2 activity. RC-160 also stimulates ERK2 activity in pancreatic acini isolated from normal mice, which endogenously express sst2, but has no effect in pancreatic acini derived from sst2 knock-out mice. RC-160-induced p27Kip1 up-regulation and inhibition of insulin-dependent cell proliferation are both prevented by pretreatment of CHO/sst2 cells with the MEK1/2 inhibitor PD98059. In addition, using dominant negative mutants, we show that sst2-mediated ERK2 stimulation is dependent on the pertussis toxin-sensitive Gi/o protein, the tyrosine kinase Src, both small G proteins Ras and Rap1, and the MEK kinase B-Raf but is independent of Raf-1. Phosphatidylinositol 3-kinase (PI3K) and both tyrosine phosphatases, SHP-1 and SHP-2, are required upstream of Ras and Rap1. Taken together, our results identify a novel mechanism whereby a Gi/o protein-coupled receptor inhibits cell proliferation by stimulating ERK signaling via a SHP-1-SHP-2-PI3K/Ras-Rap1/B-Raf/MEK pathway.     

Impairing both HMA4 homeologs is required for cadmium reduction in tobacco

In tobacco, the heavy metal P1B‐ATPases HMA4.1 and HMA4.2 function in root‐to‐shoot zinc and cadmium transport. We present greenhouse and field data that dissect the possibilities to impact the two homeologous genes in order to define the best strategy for leaf cadmium reduction. In a first step, both genes were silenced using an RNAi approach leading to >90% reduction of leaf cadmium content. To modulate HMA4 function more precisely, mutant HMA4.1 and HMA4.2 alleles of a Targeting Induced Local Lesions IN Genomes (TILLING) population were combined. As observed with RNAi plants, knockout of both homeologs decreased cadmium root‐to‐shoot transfer by >90%. Analysis of plants with segregating null and wild‐type alleles of both homeologs showed that one functional HMA4 allele is sufficient to maintain wild‐type cadmium levels. Plant development was affected in HMA4 RNAi and double knockout plants that included retarded growth, necrotic lesions, altered leaf morphology and increased water content. The combination of complete functional loss (nonsense mutation) in one homeologous HMA4 gene and the functional reduction in the other HMA4 gene (missense mutation) is proposed as strategy to limit cadmium leaf accumulation without developmental effects.     

Diatom Communities and Water Quality Assessment in Mountain Rivers of the Upper Segre Basin (La Cerdanya,Oriental Pyrenees)

Epilithic diatoms of mountain rivers from the upper Segre catchment (Oriental Pyrenees) were studied in 1998, during three different seasons: March, July and September. Four rivers, the river Segre and its three most important tributaries, Duran, Molina and Querol, were sampled in upstream and downstream stretches. The diatom communities were comparable in all upstream stretches of these mountain rivers draining siliceous substrates. Dominant taxa were Achnanthidium subatomus, Diatoma mesodon, Encyonema cf. minutum, E. silesiacum, Fragilaria arcus, F. capucina, Gomphonema calcifugum, G. pumilum, Meridion circulare and Nitzschia pura. Changes in water quality in the downstream stretches lead to the appearance of pollution tolerant taxa, such as Eolimna minima, Gomphoneis minuta, Navicula gregaria, and Nitzschia inconspicua. As a result, the values obtained with the diatom water quality indices (IPS Specific Polluosensitivity Index, CEE and IBD Biological Diatom Index) decreased. The diatom community composition and the derived water quality values did not change in the upstream stretches over the year. In contrast, significant changes were observed in the downstream stretches with best water␣quality in July, during high flows due to melting snow, and worst values in September, during low␣discharge. The diatom indices, especially the IPS, showed a good performance in these mountain rivers.     

Solubilization of Sodium Channel from Human Brain

[3H]Tetrodotoxin binds to a single class of receptor sites in homogenates of human brain with a KD of 9.1 nM at 0 degree C and a maximal binding capacity of 5.9 pmol/mg of protein. This tetrodotoxin receptor has been solubilized, and several parameters influencing the efficiency of this critical step have been studied. Treatment of brain membranes with 2% (wt/vol) Nonidet P-40 solubilizes up to 38% of the tetrodotoxin receptor sites. The duration of this solubilization step must not exceed 15 min at an optimal pH of 6.8. The binding activity is most stable when exogenous phosphatidylcholine is added to the soluble receptor with a phosphatidylcholine/detergent ratio of 1:5.     

MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF PLANKTONIC CYANOBACTERIA FROM BELGIUM AND LUXEMBOURG1

For the first time in Belgium and Luxembourg, the diversity and taxonomy of 95 cyanobacterial strains isolated from freshwater blooms were assessed by the comparison of phenotypes and partial 16S rRNA gene sequences. The results showed the high diversity of nanoplanktonic, picoplanktonic, and benthic–periphytic cyanobacteria accompanying the main bloom‐forming taxa. Indeed, besides 15 morphotypes of bloom‐forming taxa, seven non‐bloom‐forming planktonic morphotypes and 11 morphotypes from benthic–periphytic taxa were isolated in culture from the plankton samples of 35 water bodies. The bloom‐forming strains belonged to the genera Microcystis, Woronichinia, Planktothrix, Anabaena, and Aphanizomenon, whereas the other strains isolated from the same samples were assigned to the nanoplanktonic Aphanocapsa, Aphanothece, Snowella, and Pseudanabaena; to the picoplanktonic Cyanobium; and to the benthic–periphytic Geitlerinema, Komvophoron, Leptolyngbya, Lyngbya, Phormidium, Calothrix, Nostoc, and Trichormus. The results supported both the polyphyletism of genera such as Aphanocapsa, Aphanothece, Leptolyngbya, Geitlerinema, Anabaena, and Aphanizomenon as well as the validity of genera such as Microcystis, Planktothrix, and Pseudanabaena with gas vesicles and cells constricted at the cross wall. The results obtained showed the close relationship between Snowella and Woronichinia for which very few sequences exist. The first sequence of Komvophoron appeared poorly related to other available cyanobacterial sequences. Although in a few cases a good agreement existed between phenotypic and genotypic features, there was generally a discrepancy. Strains with identical morphotypes show small differences in the 16S rRNA sequences, which might be related to the different chemical properties of their habitats. The results showed the importance of the polyphasic approach in order to improve the taxonomy of cyanobacteria.     

Overexpression of CD39 in Mouse Airways Promotes Bacteria-Induced Inflammation

In airways, the ecto-nucleoside triphosphate diphosphohydrolase CD39 plays a central role in the regulation of physiological mucosal nucleotide concentrations and likely contributes to the control of inflammation because accelerated ATP metabolism occurs in chronic inflammatory lung diseases. We sought to determine whether constant elevated CD39 activity in lung epithelia is sufficient to cause inflammation and whether this affects the response to acute LPS or Pseudomonas aeruginosa exposure. We generated transgenic mice overexpressing human CD39 under the control of the airway-specific Clara cell 10-kDa protein gene promoter. Transgenic mice did not develop any spontaneous lung inflammation. However, intratracheal instillation of LPS resulted in accelerated recruitment of neutrophils to the airways of transgenic mice. Macrophage clearance was delayed, and the amounts of CD8(+) T and B cells were augmented. Increased levels of keratinocyte chemoattractant, IL-6, and RANTES were produced in transgenic lungs. Similarly, higher numbers of neutrophils and macrophages were found in the lungs of transgenic mice infected with P. aeruginosa, which correlated with improved bacteria clearance. The transgenic phenotype was partially and differentially restored by coinstillation of P2X(1) or P2X(7) receptor antagonists or of caffeine with LPS. Thus, a chronic increase of epithelial CD39 expression and activity promotes airway inflammation in response to bacterial challenge by enhancing P1 and P2 receptor activation.     

Medial Temporal Lobe Roles in Human Path Integration

Path integration is a process in which observers derive their location by integrating self-motion signals along their locomotion trajectory. Although the medial temporal lobe (MTL) is thought to take part in path integration, the scope of its role for path integration remains unclear. To address this issue, we administered a variety of tasks involving path integration and other related processes to a group of neurosurgical patients whose MTL was unilaterally resected as therapy for epilepsy. These patients were unimpaired relative to neurologically intact controls in many tasks that required integration of various kinds of sensory self-motion information. However, the same patients (especially those who had lesions in the right hemisphere) walked farther than the controls when attempting to walk without vision to a previewed target. Importantly, this task was unique in our test battery in that it allowed participants to form a mental representation of the target location and anticipate their upcoming walking trajectory before they began moving. Thus, these results put forth a new idea that the role of MTL structures for human path integration may stem from their participation in predicting the consequences of one''s locomotor actions. The strengths of this new theoretical viewpoint are discussed.     

The repA2 gene of the plasmid R100.1 encodes a represser of plasmid replication

We have constructed two miniplasmids, derived from the resistance plasmid R100.1. In one of these plasmids 400 bp of R100.1 DNA have been replaced by DNA from the transposon Tn1000 (gamma-delta). This substitution removes the amino-terminal end of the repA2 coding sequence of R100.1 and results in an increased copy number of the plasmid carrying the substitution. The copy number of the substituted plasmid is reduced to normal levels in the presence of R100.1. The repA2 gene thus encodes a trans-acting repressor function involved in the control of plasmid replication.     

Specific and sensitive quantitation of 2,2′-dichlorodiethyl sulphide (sulphur mustard) in water, plasma and blood: application to toxicokinetic study in the rat after intravenous intoxication

A sensitive and specific capillary gas chromatographic method has been developed to measure trace amounts of 2,2′-dichlorodiethyl sulphide (sulphur mustard) in environmental or biological samples. Sulphur mustard was isolated from water or plasma by a solid-phase extraction procedure and from blood by liquid—liquid extraction. The accuracy and precision of the methods were demonstrated using replicate analyses of spiked water, plasma or blood: within-run and between-run variabilities were less than 20%. These analytical methods were used to evaluate the rate of sulphur mustard degradation in water or plasma. Good linear calibration curves, with a detection limit of 45 ng/ml, were obtained for quantitation and determination of sulphur mustard in blood following its intravenous administration to rats. Initial toxicokinetic data were obtained.