P2Y(1) receptor activation inhibits NMDA receptor-channels in layer V pyramidal neurons of the rat prefrontal and parietal cortex

In the 1st part of this study, monosynaptic excitatory postsynaptic potentials (EPSPs) in layer V of the rat prefrontal cortex (PFC) were evoked by electrical stimulation of layer I. Recordings with intracellular sharp, microelectrodes showed a concentration-dependent inhibition of the EPSP by adenosine 5'-O-(2-thiodiphosphate) (ADP-beta-S). Pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), when given alone depressed the EPSP and in addition antagonized the effect of ADP-beta-S. Exclusion of the N-methyl-D-aspartate (NMDA) component of the EPSP by D(.)-amino-5-phosphonopentanoic acid (AP-5) abolished the ADP-beta-S-induced depression. The pressure-application of both NMDA and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) caused reproducible depolarizations. ADP-beta-S inhibited the effect of NMDA, but did not alter that of AMPA. PPADS was also under these conditions antagonistic with ADP-beta-S. In the 2nd part of the study, NMDA-induced currents were measured by whole-cell patch-clamp pipettes. ADP-beta-S caused a concentration-dependent inhibition of the responses to NMDA. PPADS alone did not alter the NMDA-currents but again antagonized the action of ADP-beta-S; 2'-deoxy-N(6)-methyladenosine-3',5'-diphosphate (MRS 2179) also abolished the NMDA effect. The ADP-beta-S-induced inhibition persisted in the presence of tetrodotoxin (TTX) or guanosine 5'-O-(3-thiodiphosphate) (GDP-beta-S) applied to the external medium and the pipette solution, respectively. The 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) moderately decreased the ADP-beta-S effect. The inhibitory function of ADP-beta-S on EPSPs and the interaction with PPADS was observed also in layer V pyramidal neurons of the parietal somatosensory cortex. In conclusion, metabotropic P2Y(1) receptors appear to exert a new modulatory influence on fast excitatory amino acid transmission in the cerebral cortex.     

Cofactor Tpr2 combines two TPR domains and a J domain to regulate the Hsp70/Hsp90 chaperone system

In the eukaryotic cytosol, Hsp70 and Hsp90 cooperate with various co-chaperone proteins in the folding of a growing set of substrates, including the glucocorticoid receptor (GR). Here, we analyse the function of the co-chaperone Tpr2, which contains two chaperone-binding TPR domains and a DnaJ homologous J domain. In vivo, an increase or decrease in Tpr2 expression reduces GR activation, suggesting that Tpr2 is required at a narrowly defined expression level. As shown in vitro, Tpr2 recognizes both Hsp70 and Hsp90 through its TPR domains, and its J domain stimulates ATP hydrolysis and polypeptide binding by Hsp70. Furthermore, unlike other co-chaperones, Tpr2 induces ATP-independent dissociation of Hsp90 but not of Hsp70 from chaperone-substrate complexes. Excess Tpr2 inhibits the Hsp90-dependent folding of GR in cell lysates. We propose a novel mechanism in which Tpr2 mediates the retrograde transfer of substrates from Hsp90 onto Hsp70. At normal levels substoichiometric to Hsp90 and Hsp70, this activity optimizes the function of the multichaperone machinery.     

Carcinoembryonic antigen-related cell adhesion molecule 10 expressed specifically early in pregnancy in the decidua is dispensable for normal murine development

The carcinoembryonic antigen (CEA) family consists of a large group of evolutionarily and structurally divergent glycoproteins. The murine CEACAM9 and CEACAM11-related proteins as well as the pregnancy-specific glycoproteins (PSG) are secreted members of the CEA family which are differentially expressed in fetal trophoblast cell populations during placental development. PSG are essential for a successful pregnancy, possibly by protecting the semiallotypic fetus from the maternal immune system. In contrast, Ceacam10 mRNA, coding for a protein identical in structure with CEACAM11-related proteins, is expressed in the maternal decidua surrounding the implantation site of the conceptus only during early stages of gestation between day 6.5 and day 10.5 postcoitum. To determine its role during murine development, we inactivated Ceacam10. Ceacam10(-/-) mice developed, like the previously established Ceacam9(-/-) mice, indistinguishably from wild-type littermates with respect to sex ratio, weight gain, and fertility. However, a small but significant reduction of the litter size by 23% was observed in Ceacam10(-/-) matings. Furthermore, combining the Ceacam9 and Ceacam10 null alleles, both located on chromosome 7, by meiotic recombination and subsequent mating of heterozygotes carrying both knockout alleles on one chromosome yielded wild-type and double knockout offspring at the expected Mendelian ratio. Taken together, both Ceacam10 and Ceacam9, alone or in combination, are not essential for either murine placental and embryonic development or for adult life.     

An expression pattern screen for genes involved in the induction of the posterior nervous system of zebrafish

The posterior nervous system, including the hindbrain and the spinal cord, has been shown to be formed by the transformation of neural plate of anterior character by signals derived from non-axial mesoderm. Although secreted factors, such as fibroblast growth factors (FGFs), Wnts, retinoic acid (RA) and Nodal, have been proposed to be the posteriorizing factors, the mechanism how neural tissue of posterior character is induced and subsequently specified along the anteroposterior axis remains elusive. To identify intercellular signaling molecules responsible for posteriorization of the neural plate as well as to find molecules induced intracellularly by the posteriorizing signal in the caudal neural plate, we screened by in situ hybridization for genes specifically expressed in posterior tissues, including the posterior neural plate and non-axial mesoderm when posteriorization of the neural plate takes place. From a subtracted library differentiating anterior versus posterior neural plate, 420 cDNA clones were tested, out of which 76 cDNA fragments showed expression restricted to the posterior tissue. These clones turned out to represent 32 different genes, including one novel secreted factor and one transmembrane protein. Seven genes were induced by non-axial mesodermal implants and bFGF beads, suggesting that these are among the early-response genes of the posteriorizing signal. Thus, our approach employing cDNA subtraction and subsequent expression pattern screening allows us to clone candidate genes involved in a novel signaling pathway contributing to the formation of the posterior nervous system.     

Evidence of cytochrome P450-catalyzed cleavage of the ether bond of phenoxybutyrate herbicides in Rhodococcus erythropolis K2-3

Bacterial strain Rhodococcus erythropolis K2-3 can cleave theether bond of the phenoxybutyrate herbicides, i.e., 4-(2,4-dichlorophenoxy)butyrate(2,4-DB) and 4-(4-chloro-2-methylphenoxy)butyrate (MCPB), by anenzyme system that is constitutively expressed. The enzyme(s) involved were investigated in this study. The rate ofdisappearance of 2,4-DB determined in a whole cell assay amounted to0.6 mmol/h ¶ g_{dry mass}.Carbon monoxide difference spectra of dithionite-reduced wholecells and crude cell extracts suggested that strain K2-3 contains a soluble cytochrome P450(P450), named P450_PB-1. The addition of various phenoxybutyrate substrates to crude cell extracts resulted in typical difference spectra following the type I pattern ofsubstrate binding with P450. The rate of 2,4-DB cleavage was reduced by inhibitors of P450: 5 mM metyrapone and carbon monoxide at a CO/O₂ ratio of 10 reduced the activity by about 20%, and 70%, respectively. The ether cleaving activity completely disappearedafter disruption of the cells and could not be detected in crude extracts. To elucidate theenzymatic basis of this reaction, P450 was partially purified. With the resulting enzyme preparation,2,4-DB cleavage activity was re-established, becoming measurable after the addition of eitherphenazine methosulfate or ferredoxin and ferredoxin/NADP oxidoreductase from spinach. We detected no activities attributable to -ketoglutarate-dependent dioxygenase orNAD(P)H-dependent monooxygenase. These results collectively indicatethat cleavage of the ether bond of phenoxybutyrate herbicides is catalyzed by P450-mediated activityin this strain. One of the products derived from this reaction is dichlorophenol, and comparativechromatographic analyses suggest that the other product is a C4-carbonicacid, most likely succinic semialdehyde/succinate.     

Synthesis and preliminary evaluation of (R,R)(S,S) 5-(2-(2-[4-(2-[(18)F]fluoroethoxy)phenyl]-1-methylethylamino)-1-hydroxyethyl)-benzene-1,3-diol ([(18)F]FEFE) for the in vivo visualisation and quantification of the beta2-adrenergic receptor status in lung

The (18)F-labeled beta2-adrenergic receptor ligand (R,R)(S,S) 5-(2-(2-[4-(2-[(18)F]fluoroethoxy)phenyl]-1-methylethylamino)-1-hydroxyethyl)-benzene-1,3-diol, a derivative of the original highly selective racemic fenoterol, was synthesized in an overall radiochemical yield of 20% after 65 min with a radiochemical purity higher than 98%. The specific activity was in the range of 50-60 GBq/micromol. In vitro testing of the non-radioactive fluorinated fenoterol derivative with isolated guinea pig trachea was conducted to obtain an IC(50) value of 60 nM. Preliminary ex vivo organ distribution and in vivo experiments with positron emission tomography (PET) on guinea pigs were performed to study the biodistribution as well as the displacement of the radiotracer to prove specific binding to the beta2-receptor.     

The "typical" immunophenotype of acute promyelocytic leukemia (APL-M3): does it prove true for the M3-variant?

The immunophenotypes of 12 acute promyelocytic leukemias (APL-M3; eight hypergranular, four microgranular) with documented PML-RAR-alpha fusion gene are presented. Bone marrow mononuclear cells were immunophenotyped using a panel of 20 monoclonal antibodies. The hypergranular APLs exhibited a mature myeloid phenotype as it has been described to be typical for M3. No lineage infidelity was detectable in classic M3 cases. In contrast, among the four cases of M3 variant, all leukemias showed marked expression of CD34 and two of four cases expressed the HLA-DR antigen. The CD2 antigen was expressed in three of four cases. Furthermore, one case showed expression of the CD56 antigen, and one case was positive for the blood group H antigen. The data suggest that microgranular APL is a heterogeneous entity with regard to the immunologic phenotype.     

Comparative proteome analysis of Helicobacter pylori

Helicobacter pylori, the causative agent of gastritis, ulcer and stomach carcinoma, infects approximately half of the worlds population. After sequencing the complete genome of two strains, 26695 and J99, we have approached the demanding task of investigating the functional part of the genetic information containing macromolecules, the proteome. The proteins of three strains of H. pylori, 26695 and J99, and a prominent strain used in animal models SS1, were separated by a high-resolution two-dimensional electrophoresis technique with a resolution power of 5000 protein spots. Up to 1800 protein species were separated from H. pylori which had been cultivated for 5 days on agar plates. Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) peptide mass fingerprinting we have identified 152 proteins, including nine known virulence factors and 28 antigens. The three strains investigated had only a few protein spots in common. We observe that proteins with an amino acid exchange resulting in a net change of only one charge are shifted in the two-dimensional electrophoresis (2-DE) pattern. The expression of 27 predicted conserved hypothetical open reading frames (ORFs) and six unknown ORFs were confirmed. The growth conditions of the bacteria were shown to have an effect on the presence of certain proteins. A preliminary immunoblotting study using human sera revealed that this approach is ideal for identifying proteins of diagnostic or therapeutic value. H. pylori 2-DE patterns with their identified protein species were added to the dynamic 2D-PAGE database (http://www.mpiib-berlin.mpg.de/2D-PAGE/). This basic knowledge of the proteome in the public domain will be an effective instrument for the identification of new virulence or pathogenic factors, and antigens of potentially diagnostic or curative value against H. pylori.