Effect of Kainic Acid, Glutamate, and Aspartate on CO2 Production by Goldfish Tectal Slices

For a study of the excitatory effect of kainate, glutamate, and aspartate in the goldfish optic tectum, these substances were tested on the production of CO2 from radioactive glucose in tectal slices incubated in Krebs-Ringer medium for fish. Kainate increased the rate of CO2 production for up to 30 min in a dose-related manner, the effect being maximum at 0.1 mM concentration and decreasing at higher doses. The effect was blocked by ouabain (1 mM) as well as by the substitution of choline for Na+ in the incubation medium. Glutamate and aspartate exerted a less pronounced excitatory effect on CO2 production at higher concentration than kainate. This effect was also abolished by ouabain. Glutamate, added to the medium at a concentration at least 100-fold higher than kainate, partially reversed the increase in CO2 production induced by kainic acid. No similar effect was noticed for aspartate. The supposed glutamate antagonists glutamic acid diethylester (1 mM) and proline (5 mM) did not affect the excitatory action of kainic acid or exert an antagonistic effect towards glutamate. At higher concentration (10 mM) glutamic acid diethylester increased CO2 production, an effect that was, however, ouabain insensitive. Methyltetrahydrofolic acid (1 mM), a substance reported to compete for the kainate receptor, did not inhibit the effect of kainic acid or increase CO2 production.     

Amino acid transport in pig lymphocytes Enhanced activity of transport system asc following mitogenic stimulation

Changes in neutral amino acid transport activity caused by addition of phytohaemagglutinin-P to quiescent peripheral pig lymphocytes have been evaluated by measurements of ¹⁴C-labelled neutral and analogue amino acids under conditions approaching initial entry rates. Utilizing methylaminoisobutyric acid, the best model substrate of System A, we confirmed our previous report (Borghetti, A.F., Kay, J.E. and Wheeler, K.P. (1979) Biochem. J. 182, 27–32) on the absence of this transport system in quiescent cells and its emergence following stimulation. Furthermore, we demonstrated the presence in quiescent cells of an Na⁺-dependent transport system for neutral amino acids that has been characterized as System ASC by several criteria including intolerance to methylaminoisobutyric acid, strict Na⁺-dependence, the property of transtimulation and specificity for pertinent substrates such as alanine, serine, cysteine and threonine. Analysis of the relationship between influx and substrate concentration revealed that two independent saturable components contribute to entry of alanine in quiescent cells: a low affinity ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= ≈4}\text{mM}$) and a high affinity ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= ≈0.2}\text{mM}$) component. The high affinity component could be inhibited in a competitive way by serine, cysteine and threonine, but methylaminoisobutyric acid did not change appreciably its constants. The enhanced activity of alanine transport through the ASC system observed in activated cells resulted from a large increase in the capacity ($\text{V}$) of the high affinity component without any substantial change in the apparent affinity constant ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}$).     

Etude de la dynamique des populations de cilies d'un torrent experimental pendant deux annees

The sampling of Ciliates performed in a little experimental torrent through two years, showed a great similarity of the two annual cycles. This similarity concerns both the species composition as well as the dynamics of the Ciliates populations. The basic species which affect the community structure are as follows: Ctedoctema acanthocrypta, Mesodinium acarus, Cyclidium glaucoma, Cinetochilum margaritaceum, Pleuronema coronatum, Lacrymaria pupula, Aspidisca costata and Holosticha vernalis. The dynamics of the ciliated Protozoa living in this environment seem to be influenced by the speed of the current, the temperature and the amount of dissolved organic matter in the water.

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Etude de la dynamique des populations de cilies d'un torrent experimental pendant deux annees

     

On the fungitoxicity of some new thiocyanatopyrazole derivatives: Electron microscopical study in trichophyton mentagrophytes

Four thiocyanatopyrazole derivatives were synthesized and their fungistatic activity was demonstrated in vitro against a number of dermatophytic fungi. In Trichophyton mentagrophytes, the most active compound induced an unusual increase of the plasma membrane with production of intra and extracytoplasmic complexes, a deterioration of nuclear and mitochondrial membranes and a formation of autophagic-like vacuoles. Plasmolysis, accompanied by an almost complete disorganization of cytoplasmic structures, seemed to be the final event. A possible mechanism of action of the compounds was discussed.Investigation supported by a grant from Consiglio Nazionale delle Ricerche of Italy (Contract No. 7500536).     

Optogenetic actuator – ERK biosensor circuits identify MAPK network nodes that shape ERK dynamics

Combining single‐cell measurements of ERK activity dynamics with perturbations provides insights into the MAPK network topology. We built circuits consisting of an optogenetic actuator to activate MAPK signaling and an ERK biosensor to measure single‐cell ERK dynamics. This allowed us to conduct RNAi screens to investigate the role of 50 MAPK proteins in ERK dynamics. We found that the MAPK network is robust against most node perturbations. We observed that the ERK‐RAF and the ERK‐RSK2‐SOS negative feedback operate simultaneously to regulate ERK dynamics. Bypassing the RSK2‐mediated feedback, either by direct optogenetic activation of RAS, or by RSK2 perturbation, sensitized ERK dynamics to further perturbations. Similarly, targeting this feedback in a human ErbB2‐dependent oncogenic signaling model increased the efficiency of a MEK inhibitor. The RSK2‐mediated feedback is thus important for the ability of the MAPK network to produce consistent ERK outputs, and its perturbation can enhance the efficiency of MAPK inhibitors.     

Nuclear localization of TRK-A in liver cells

The liver represents a site of expression of neurotrophins and their receptors. We have characterized the expression and intracellular localization of the nerve growth factor (NGF) receptor, Trk-A, in liver cells in vivo and in vitro. In both normal and fibrotic liver tissue, Trk-A immunostaining was present in different cell types, including parenchymal cells and cells of the inflammatory infiltrate. In hepatocytes and activated stellate cells (HSC), Trk-A showed a predominant nuclear localization, both in the presence and absence of injury. In cultured HSC, Trk-A was found to be functional, because exposure of the cells to recombinant NGF resulted in stimulation of cell migration and activation of intracellular signaling pathways, including Ras-ERK and PI3K/Akt. Remarkably, in cultured HSC, Trk-A staining was found constitutively in the nucleus. In these cells, Trk-A could be stained only by antibodies directed against the intracellular domain but not by those recognizing the extracellular portion of Trk-A suggesting that the intracellular portion of the receptor is the major determinant of nuclear Trk-A staining. In contrast to HSC, freshly isolated hepatocytes did not show any nuclear localization of the intracellular portion of Trk-A. In pheocromocytoma cells, nuclear staining for Trk-A was not present in conditions of serum deprivation, but could be induced by exposure to NGF or to a mixture of soluble mediators. We conclude that nuclear localization of the intracellular domain of Trk-A is observed constitutively in liver cells such as HSC, while in other cell types it could be induced in response to soluble factors.     

Effect of CMV-immunoglobulins (cytotect biotest) prophylaxis on CMV pneumonia after lung transplantation

Lung transplant (LT) recipients among solid organ transplant recipients are at high risk for cytomegalovirus (CMV) infections. We evaluated the effect of CMV-Immunoglobulins (CMV-IG) (Cytotect Biotest) on CMV pneumonia diagnosed in 303 follow-up transbronchial biopsies (TBB) of lung transplant recipients. 24 patients (control group, 155 TBB from 1999 to 2002) received acyclovir for 24 months and 33 recipients (study group, 148 TBB from 2003 to 2008) received a combined CMV prophylaxis consisting of CMV-IG (Cytotect Biotest) for 12 months and a short Ganciclovir or Valganciclovir therapy from 21th to 42th postoperative day followed by acyclovir up to 24 months. In our study the percentage of pneumonia at first month TBB was similar in the study group vs the control group, 9.1% (3/33) vs 8.3% (2/24), p=0.9 ns, but after the first month the percentage was significantly lower in the study group in the first year at follow-up TBB, 1% (1/99) vs 6.4% (5/78), p=0.048, and in first two years follow-up TBB, 0.8% (1/122) vs 6.5% 8/124), p=0.018 (Statistical analysis: Chi-square test for proportion differences). Our data suggest a strong efficacy of CMV-IG prophylaxis in reducing CMV pneumonia after first month in lung transplant recipients.     

Molecular Genetics of Cystinuria: Identification of Four New Mutations and Seven Polymorphisms, and Evidence for Genetic Heterogeneity

A cystinuria disease gene (rBAT) has been recently identified, and some mutations causing the disease have been described. The frequency of these mutations has been investigated in a large sample of 51 Italian and Spanish cystinuric patients. In addition, to identify new mutated alleles, genomic DNA has been analyzed by an accurate and sensitive method able to detect nucleotide changes. Because of the lack of information available on the genomic structure of rBAT gene, the study was carried out using the sequence data so far obtained by us. More than 70% of the entire coding sequence and 8 intron-exon boundaries have been analyzed. Four new mutations and seven intragenic polymorphisms have been detected. All mutations so far identified in rBAT belong only to cystinuria type I alleles, accounting for ~44% of all type I cystinuric chromosomes. Mutation M467T is the most common mutated allele in the Italian and Spanish populations. After analysis of 70% of the rBAT coding region, we have detected normal sequences in cystinuria type II and type III chromosomes. The presence of rBAT mutated alleles only in type I chromosomes of homozygous (type I/I) and heterozygous (type I/III) patients provides evidence for genetic heterogeneity where rBAT would be responsible only for type I cystinuria and suggests a complementation mechanism to explain the intermediate type I/type III phenotype.     

Organization and possible origin of the Bari-1 cluster in the heterochromatic h39 region of Drosophila melanogaster

The molecular organization of the heterochromatic h39 region of the Drosophila melanogaster second chromosome has been investigated by studying two BAC clones identified both by Southern blotting and by FISH experiments as containing tandem arrays of Bari1, a transposable element present only in this region. Such BAC clones appear to contain different portions of the h39 region since they differ in the DNA sequences flanking the Bari1 repeats on both sides. Thus, the 80 Bari1 copies estimated to be present in the h39 region are split into at least two separated subregions. On the basis of the analysis of the flanking sequences a possible mechanism depending on an aberrant activity of the Bari1 transposase is proposed for the genesis of the heterochromatic tandem arrays of the element.