Rat pheochromocytoma tyrosine hydroxylase is phosphorylated on serine 40 by an associated protein kinase

Tyrosine hydroxylase, a key enzyme in the biosynthesis of catecholamines, was previously shown to be phosphorylated on four distinct serine residues in PC12 cell cultures, each one being specific for the kinase system involved (McTigue, M., Cremins, J., and Halegoua, S. (1985) J. Biol. Chem. 260, 9047-9056). A cAMP- and Ca2+-independent protein kinase was found to be associated with tyrosine hydroxylase purified from rat pheochromocytoma tumor. The use of this activity and the availability of a large amount of purified tyrosine hydroxylase allowed identification of the site phosphorylated by this kinase activity. A peptide of 1.5 kDa (about 12 residues long), carrying the phosphorylation site, was released from 32P-labeled tyrosine hydroxylase by limited proteolysis with trypsin. This peptide was isolated from trypsinized tyrosine hydroxylase by sequential gel filtration and ion exchange chromatographies. Analysis by thin layer chromatography of an acid hydrolysate of the peptide revealed that it contained phosphoserine. The sequence determination of the peptide showed that it corresponded to the residues 38-45 in the tyrosine hydroxylase primary structure (Arg-Gln-Ser(P)-Leu-Ile-Glu-Asp-Ala). Thus, the associated kinase phosphorylated Ser-40, one of the phosphorylation sites for the cAMP-dependent protein kinase also found in rat pheochromocytoma tumors. These results are compared to those recently appearing in a report by Campbell et al. (Campbell, D. G., Hardie, D. G., and Vulliet, P. R. (1986) J. Biol. Chem. 261, 10489-10492).     

Automated protein crystallization and a new crystal form of a subtilisin:eglin complex

A procedure is described for automating labour-intensive steps of the 'hanging drop' protein crystallization method. An automatic sample changer is employed to fill the wells in a multi-well plate so that concentration gradients in various components are obtained. The sample changer is also used for preparing droplets on a second multi-well plate. Subsequently, this second plate is manually turned around and placed on top of the first multi-well plate such that a large number of chambers with different conditions is obtained simultaneously. During initial trials a new crystal form of a subtilisin:eglin complex was obtained. The crystals have space group P2(1), contain two enzyme inhibitor complexes per asymmetric unit and diffract beyond 2.2 A.     

Reliability of abundance ratios between aeropalynological taxa as indicators of the climate in France

The abundance ratio between two aeropalynological taxa can sometimes give more reliable information about climate than a single taxon, especially in poorly standardized spectra of sediments, mosses or dusts. The reliabilities of 8385 ratios have been computed from 130 taxa observed in a net of 80 annual standardized aeropalynological spectra measured mostly in France. Ten average climatic factors express cold, heat and rain. The reliability of a pollen ratio is the relative frequency (between 0 and 100%) of the ranks, in the range of the climatic factor, where the ratio is more indicative than its two parent taxa. Only 169 out of the 8,385 ratios have a reliability of 80% or more, with one thousand ratios having a reliability of 50% or more. The most reliable ratios are from the less indicative taxa, whereas ubiquitous taxa give generally unreliable ratios. The accuracy and stability of estimates are approximately the same from the ratios as from the single taxa. The use of ratios foreshadows the use of sociological relations between taxa. Sociological processing will be used to transform the intermittent information about the environment given by the observed abundance of taxa into a gradual information given by their probable abundance.     

Human Amnion Epithelial Cells Induced to Express Functional Cystic Fibrosis Transmembrane Conductance Regulator

Cystic fibrosis, an autosomal recessive disorder caused by a mutation in a gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), remains a leading cause of childhood respiratory morbidity and mortality. The respiratory consequences of cystic fibrosis include the generation of thick, tenacious mucus that impairs lung clearance, predisposing the individual to repeated and persistent infections, progressive lung damage and shortened lifespan. Currently there is no cure for cystic fibrosis. With this in mind, we investigated the ability of human amnion epithelial cells (hAECs) to express functional CFTR. We found that hAECs formed 3-dimensional structures and expressed the CFTR gene and protein after culture in Small Airway Growth Medium (SAGM). We also observed a polarized CFTR distribution on the membrane of hAECs cultured in SAGM, similar to that observed in polarized airway cells in vivo. Further, hAECs induced to express CFTR possessed functional iodide/chloride (I^−/Cl⁻) ion channels that were inhibited by the CFTR-inhibitor CFTR-172, indicating the presence of functional CFTR ion channels. These data suggest that hAECs may be a promising source for the development of a cellular therapy for cystic fibrosis.     

Modulation of Malaria Phenotypes by Pyruvate Kinase (PKLR) Variants in a Thai Population

Pyruvate kinase (PKLR) is a critical erythrocyte enzyme that is required for glycolysis and production of ATP. We have shown that Pklr deficiency in mice reduces the severity (reduced parasitemia, increased survival) of blood stage malaria induced by infection with Plasmodium chabaudi AS. Likewise, studies in human erythrocytes infected ex vivo with P. falciparum show that presence of host PK-deficiency alleles reduces infection phenotypes. We have characterized the genetic diversity of the PKLR gene, including haplotype structure and presence of rare coding variants in two populations from malaria endemic areas of Thailand and Senegal. We investigated the effect of PKLR genotypes on rich longitudinal datasets including haematological and malaria-associated phenotypes. A coding and possibly damaging variant (R41Q) was identified in the Thai population with a minor allele frequency of ~4.7%. Arginine 41 (R41) is highly conserved in the pyruvate kinase family and its substitution to Glutamine (R41Q) affects protein stability. Heterozygosity for R41Q is shown to be associated with a significant reduction in the number of attacks with Plasmodium falciparum, while correlating with an increased number of Plasmodium vivax infections. These results strongly suggest that PKLR protein variants may affect the frequency, and the intensity of malaria episodes induced by different Plasmodium parasites in humans living in areas of endemic malaria.     

Sporulation of Penicillium roqueforti in solid substrate fermentation

Summary A study of the influence of temperature, aeration rate, and substrate water content on sporulation of Penicillium roqueforti on buckwheat seeds in a fixed bed reactor is described. Use of an experimental procedure based on a 2³ factorial design allowed optimum to be determined as 23.5° C for temperature, 0.48 g/g dry matter for substrate water content and 4.42 VVH for aeration rate.     

Collagen Can Selectively Trigger a Platelet Secretory Phenotype via Glycoprotein VI

Platelets are not only central actors of hemostasis and thrombosis but also of other processes including inflammation, angiogenesis, and tissue regeneration. Accumulating evidence indicates that these “non classical” functions of platelets do not necessarily rely on their well-known ability to form thrombi upon activation. This suggests the existence of non-thrombotic alternative states of platelets activation. We investigated this possibility through dose-response analysis of thrombin- and collagen-induced changes in platelet phenotype, with regards to morphological and functional markers of platelet activation including shape change, aggregation, P-selectin and phosphatidylserine surface expression, integrin activation, and release of soluble factors. We show that collagen at low dose (0.25 µg/mL) selectively triggers a platelet secretory phenotype characterized by the release of dense- and alpha granule-derived soluble factors without causing any of the other major platelet changes that usually accompany thrombus formation. Using a blocking antibody to glycoprotein VI (GPVI), we further show that this response is mediated by GPVI. Taken together, our results show that platelet activation goes beyond the mechanisms leading to platelet aggregation and also includes alternative platelet phenotypes that might contribute to their thrombus-independent functions.     

Interaction between lambda or phi80 prophage induction and gal operon expression

When Escherichia coli cells are cultivated with fucose on a rich medium giving rise to a strong catabolite repression, λh80 or φ80 prophage thermo-induction triggers a significant escape of the gal operon activity.