Alzheimer's disease-like phosphorylation of the microtubule-associated protein tau by glycogen synthase kinase-3 in transfected mammalian cells

Background: Paired helical filaments (PHFs) are a characteristic pathological feature of Alzheimer's disease; their principal component is the microtubule-associated protein tau. The tau in PHFs (PHF-tau) is hyperphosphorylated, but the cellular mechanisms responsible for this hyperphosphorylation have yet to be elucidated. A number of kinases, including mitogen-activated protein (MAP) kinase, glycogen synthase kinase (GSK)-3α, GSK-3β and cyclin-dependent kinase-5, phosphorylate recombinant tau in vitro so that it resembles PHF-tau as judged by its reactivity with a panel of antibodies capable of discriminating between normal tau and PHF-tau, and by a reduced electrophoretic mobility that is characteristic of PHF-tau. To determine whether MAP kinase, GSK-3α and GSK-3β can also induce Alzheimer's disease-like phosphorylation of tau in mammalian cells, we studied the phosphorylation status of tau in primary neuronal cultures and transfected COS cells following changes in the activities of MAP kinase and GSK-3.Results Activating MAP kinase in cultures of primary neurons or transfected COS cells expressing tau isoforms did not increase the level of phosphorylation for any PHF-tau epitope investigated. But elevating GSK-3 activity in the COS cells by co-transfection with GSK-3α or GSK-3β decreased the electrophoretic mobility of tau so that it resembled that of PHF-tau, and induced reactivity with eight PHF-tau-selective monoclonal antibodies.Conclusion Our data indicate that GSK-3α and/or GSK-3β, but not MAP kinase, are good candidates for generating PHF-type phosphorylation of tau in Alzheimer's disease. The involvement of other kinases in the generation of PHFs cannot, however, be eliminated. Our results suggest that aberrant regulation of GSK-3 may be a pathogenic mechanism in Alzheimer's disease.     

Production of a membrane-bound proteins,the human gamma-glutamyl transferase,by CHO cells cultivated on microcarriers,in aggregates and in suspension

Recombinant Chinese Hamster Ovary (CHO) cells, engineered for the production of human gamma-glutamyl transferase (GGT), have been grown on Cytodex 1 microcarriers, as aggregates, or as single cells in suspension after adaptation. GGT is a membrane bound enzyme which was not secreted during the culture period. The maximal enzyme activity was found to be directly related to the achieved maximal cell density. Culture of CHO on microcarriers yielded the fastest growth, with a specific growth rate of 0.04 h^–1, the highest cell density (near 1.3×10⁶ cells ml^–1), and the highest enzyme activity around 300 mU ml^–1, which corresponded to a specific cellular level of 20 mU 10^–5 cells. GGT could also be produced by growing CHO cells in suspension as single cells or as aggregates. Under these conditions, however, the specific CHO growth rate was significantly slower and the GGT level per cell was divided by a factor 6. Growing CHO cells without microcarriers also resulted in differences in cell metabolism, with a higher conversion yield of glutamine into ammonia, and a higher cell lysis. The catalytic kinetic constants of the enzyme were found identical for the three culture systems.     

Microbial transformation of ethylpyridines

Pyridine and its derivatives have been found as pollutants in the environment. Although alkylpyridines constitute the largest class of pyridines contaminating the environment, little information is available concerning the fate and transformation of these compounds. In this investigation ethylpyridines have been used as model compounds for investigating the biodegradability of alkylpyridines. A mixed culture of ethylpyridine-degrading microorganisms was obtained from a soil that had been exposed to a variety of pyridine derivatives for several decades. The enrichment culture was able to degrade 2-, 3-, and 4-ethylpyridine (100 mg/L) at 28° C and pH 7 within two weeks under aerobic conditions. The degradation rate was greatest for 2-ethylpyridine and least for 3-ethylpyridine. Transformation of ethylpyridines was dependent on substrate concentration, pH, and incubation temperature. Studies on the metabolic pathway of 4-ethylpyridine revealed two products; these chemicals were identified by MS and NMR analyses as 4-ethyl-2(1H)-pyridone and 4-ethyl-2-piperidone. 6-Ethyl-2(1H)-pyridone was determined to be a product of 2-ethylpyridine degradation. These results indicate that the transformation mechanism of ethylpyridines involves hydroxylation and reduction of the aromatic ring before ring cleavage.     

The Presence of (+)-S-Adenosyl-l-Methionine in the Rat Brain and Its Lack of Effect on Phenylethanolamine N-Methyltransferase Activity

Abstract: (+)-S-Adenosyl- l -methionine [(+)-SAM] was isolated from rat brain and was quantified by HPLC followed by UV spectrophotometric measurements and by ¹H-NMR. Its estimated ratio in brain is 3% of total SAM. Because of its commercial unavailability, (+)-SAM was also prepared from chemically synthesized SAM by separation of the two diastereoisomers on a preparative reverse-phase Nucleosil C8 column. The (+) diastereoisomer thus obtained was then assayed in vitro both as an inhibitor and a substrate of phenylethanolamine N -methyltransferase. Enzymatic activity was measured by HPLC analysis. It was shown that (+)-SAM has no effect on phenylethanolamine N -methyltransferase activity; therefore, it is unlikely that (+)-SAM plays any possible role in regulation of adrenaline synthesis in the brain.     

Nucleotide substitutions at the -6 position in the promoter region of the factor IX gene result in different severity of hemophilia B Leyden: consequences for genetic counseling

Mutations in the promoter region of the factor IX gene result in hemophilia B Leyden, which is characterized by considerable improvement in the disease after puberty. We have found that distinct nucleotide substitutions at the -6 position in the Leyden-specific (LS) region are associated with a different severity of hemophilia B. The proband (aged 2) from one family is a severe hemophiliac with factor IX activity (F.IXC) and antigen (F.IXAg) levels less than 1.0U/dl. F.IXC and F.IXAg levels in two affected uncles are approximately 30% of normal levels. The LS region was targeted for analysis because the phenotypes suggested the inheritance of a factor IX Leyden gene. An abnormal TaqI digestion pattern was found in amplified DNA from the proband, and sequencing showed a G (-6) to C transversion that was linked to the disease in the family. In another family, two brothers (aged 8 and 9) suffer from mild hemophilia with F.IXC ranging from 7 to 10 U/dl and F.IXAg from 3 to 4 U/dl. They are the only documented members of the family with a bleeding tendency. Denaturing gradient gel electrophoresis on amplified fragments from one of the patient's genomic DNA corresponding to the 8 exons and flanking sequences of the factor IX gene suggested a defect only in a segment from the 5 region. This segment showed an altered TaqI digestion pattern, and sequencing demonstrated a G(-6) to A transition that was traced to the patients's mother and a grandmother. The different phenotypes associated with the G (-6) to A purine nucleotide transition compared with a G(-6) to C transversion provide evidence that this area is directly involved in the regulation of the human factor IX gene expression in vivo by binding of regulatory factors. The ability to predict that the conditions of a hemophilia B patient will improve with age has important implications for genetic counseling of the family. Therefore, the LS region should always be included when scanning the factor IX gene for mutations.     

Response of a raptor community to shrinking area and degradation of tropical rain forest in the south western Ghâts (India)

The influence of irregular or incomplete fragmentation and increasing degradation of natural rain forest on diurnal raptor community was studied in the northern part of the western Ghâts in southwestern India A census of mainly territorial breeding pairs on 400 ha sample quadrats was associated with a measure of the percent cover of the main habitat types and a degree of forest fragmentation including irregular patch shape and habitat heterogeneity
Four groups of 3-4 species were defined according to their decreasing tolerance to forest fragmentation and disturbance, from mostly open grassland species to interior forest specialists Habitat selection, density and sensitivity to landscape structure were investigated The community composition and dynamic of each habitat were the sum of these specific reactions The distribution of species along the succession of increasing fragmentation and forest degradation was thus found to be non random, but did not follow a nested subset pattern The density of forest species declined with forest patch size possibly because of the irregular patch shape, the increased edge effects and the consequent increase of linear distance for a bird to cover within its territory However, sensitivity to habitat structure and disturbance was found to be even higher than sensitivity to area per se The need to conserve the largest patches of little disturbed forest is emphasized, as well as the conservation value of woodlots-open habitat mosaics that are suitable for a different set of species     

Actin and spectrin-like (Mr= 260–240 000) proteins in gregarines

In Gregarina blaberae a M_r = 47 000 and a M_r = 260–240 000 doublet polypeptides reacted in immunoblotting: i) with a polyclonal monospecific rabbit antibody to frog muscular actin, a monoclonal anti-actin antibody against chicken gizzard; and ii) with polyclonal and monoclonal antibodies to human erythrocyte β-spectrin, respectively. The M_r = 47 000 actin-like protein is associated with the ghost and a contractille cytoplasmic extract. The presence of an actin-like protein in Gregarina and Lecudina and its cellular distribution in the cortex indicated that the gliding movement might involve an actin-myosin system in contrast to previous studies. Immunofluorescence showed clear differences between the anterior part of Gregarina and Lecudina which illustrated the high cell polarity of these protozoa. The M_r = 260–240 000 doublet was detected in SDS-PAGE from G. blaberae trophozoite ghosts but not in the cytoplasmic extracts or in extracts from sexual stages, indicating that the presence of these spectrin-like proteins is stage-dependent. Visualization of the M_r = 260–240 000 by immunofluorescence showed clear species differences, with rings arranged perpendicular to the longitudinal narrow folds of G. blaberae, with longitudinal lines underlying the folds of L. pellucida and with lines separating the large folds of Selenidium pendula. The cellular distribution is consistent with a stabilizer function of the spectrin-like proteins in the scaffolding of the cortex of gregarines according to the high diversity of the cell-shape and the cell motility systems in gregarines. The presence of spectrin-like proteins in protozoa and particularly in parasites from primitive arthropods indicated that ancestral spectrin genes could the M_r = 260–240 000 form.     

A kinetic model of the branch-point between the methionine and threonine biosynthesis pathways in Arabidopsis thaliana.

This work proposes a model of the metabolic branch-point between the methionine and threonine biosynthesis pathways in Arabidopsis thaliana which involves kinetic competition for phosphohomoserine between the allosteric enzyme threonine synthase and the two-substrate enzyme cystathionine gamma-synthase. Threonine synthase is activated by S-adenosylmethionine and inhibited by AMP. Cystathionine gamma-synthase condenses phosphohomoserine to cysteine via a ping-pong mechanism. Reactions are irreversible and inhibited by inorganic phosphate. The modelling procedure included an examination of the kinetic links, the determination of the operating conditions in chloroplasts and the establishment of a computer model using the enzyme rate equations. To test the model, the branch-point was reconstituted with purified enzymes. The computer model showed a partial agreement with the in vitro results. The model was subsequently improved and was then found consistent with flux partition in vitro and in vivo. Under near physiological conditions, S-adenosylmethionine, but not AMP, modulates the partition of a steady-state flux of phosphohomoserine. The computer model indicates a high sensitivity of cystathionine flux to enzyme and S-adenosylmethionine concentrations. Cystathionine flux is sensitive to modulation of threonine flux whereas the reverse is not true. The cystathionine gamma-synthase kinetic mechanism favours a low sensitivity of the fluxes to cysteine. Though sensitivity to inorganic phosphate is low, its concentration conditions the dynamics of the system. Threonine synthase and cystathionine gamma-synthase display similar kinetic efficiencies in the metabolic context considered and are first-order for the phosphohomoserine substrate. Under these conditions outflows are coordinated.     

EPR and multi-field magnetisation of reduced forms of the binuclear iron centre in ribonucleotide reductase from mouse

Mouse ribonucleotide reductase is composed of a 1?:?1 complex of two homodimeric subunits and catalyses the first unique step on the biochemical pathway to DNA synthesis. The small subunit, protein R2, contains dinuclear iron-oxygen clusters and a tyrosyl free radical required for catalytic activity. We have studied the mixed valent and fully reduced forms of the diiron oxygen cluster from mouse R2 protein by low-temperature EPR. EPR signals of the mixed-valent states of proteins R2 reconstituted with ferrous iron and oxygen in normal and deuterated water, using the same buffers, show apparent g values of 1.92, 1.73, and 1.60 for the mixed-valent state in H₂O and 1.93, 1.73, and 1.62 in D₂O. These g values are typical for diiron-oxygen proteins, while the effect of D₂O is unprecedented for this class of proteins. We estimate the coupling constant J for the Heisenberg exchange (H?=?2J*S₁*S₂) to be J?=?–7.5±1?cm^–1 for the mixed-valent form. The diferrous R2 protein shows an integer spin EPR signal in the presence of azide or 20% glycerol. Variable temperature variable field saturation magnetisation measurements show that only in the azide-complexed R2 protein does a weak ferromagnetic coupling occur (J?=?0.26±0.05?cm^–1), while R2 protein in the absence or presence of 20% glycerol contains non-coupled mononuclear ferrous iron (S?=?2) sites.