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.     

Common structural features of the luxF protein and the subunits of bacterial luciferase: evidence for a (beta alpha)8 fold in luciferase.

The amino acid sequence identity and potential structural similarity between the subunits of bacterial luciferase and the recently determined structure of the luxF molecule are examined. The unique beta/alpha barrel fold found in luxF appears to be conserved in part in the luciferase subunits. From secondary structural predictions of both luciferase subunits, and from structural comparisons between the protein product of the luxF gene, NFP, and glycolate oxidase, we propose that it is feasible for both luciferase subunits to adopt a (beta alpha)8 barrel fold with at least 2 excursions from the (beta alpha)8 topology. Amino acids conserved between NFP and the luciferase subunits cluster together in 3 distinct "pockets" of NFP, which are located at hydrophobic interfaces between the beta-strands and alpha-helices. Several tight turns joining the C-termini of beta-strands and the N-termini of alpha-helices are found as key components of these conserved regions. Helix start and end points are easily demarcated in the luciferase subunit protein sequences; the N-cap residues are the most strongly conserved structural features. A partial model of the luciferase beta subunit from Photobacterium leiognathi has been built based on our crystallographically determined structure of luxF at 1.6 A resolution.     

Monte Carlo study of the effect of β2-microglobulin on the binding cleft of the HLA-A2 complex

Peptide recognition by class I products of the major histocompatibility complex requires association of the class I heavy chain with β₂-microglobulin. We present results of Monte Carlo simulations of the β-pleated sheet floor of the human class I MHC molecule, HLA-A2, with and without β₂-microglobulin. We find a significant effect of β₂-microglobulin on the side chains of residues near a region that would accommodate the C-terminus of a bound peptide. By modeling simultaneously each loop and its neighboring strand at either end of the class I cleft, we find that β₂-microglobulin restricts the conformational space of residues that are central to binding peptides. The effect is most pronounced for R97 and H114 and somewhat less important for Y99 and Y116, the latter forming strong hydrogen bonds with neighboring residues in the heavy chain itself.     

Mapping of two new markers within the smallest interval harboring the spinal muscular atrophy locus by family and radiation hybrid analysis

The locus responsible for the childhood-onset proximal spinal muscular atrophies (SMA) has recently been mapped to an area of 2–3 Mb in the region q12–13.3 of chromosome 5. We have used a series of radiation hybrids (RHs) containing distinct parts of the SMA region as defined by reference markers. A cosmid library was constructed from one RH. Thirteen clones were isolated and five of these were mapped within the SMA region. Both RH mapping and fluorescence in situ hybridization analysis showed that two clones map in the region between loci D5S125 and D5S351. One of the cosmids contains expressed sequences. Polymorphic dinucleotide repeats were identified in both clones and used for segregation analysis of key recombinant SMA families. One recombination between the SMA locus and the new marker 9Ic (D5S685) indicates that 9Ic is probably the closest distal marker. The absence of recombination between the SMA locus and marker Fc (D5S684) suggests that Fc is located close to the disease gene. These new loci should refine linkage analysis in SMA family studies and may facilitate the isolation of the disease gene.     

Differential recovery of polymorphonuclear neutrophils,B and T cell subpopulations in the thymus,bone marrow,spleen and blood of mice following split-dose polychemotherapy

In these studies, we examined the effect of a maximum-tolerated, split-dose chemotherapy protocol of cyclophosphamide, cisplatin, and 1,3-bis(2-chloroethyl)-1-nitrosourea carmustine on neutrophil and lymphocyte subpopulations in the peripheral blood (PBL), thymus, bone marrow and spleen. It was found that this protocol of polychemotherapy, modeled after the induction protocol used with autologous bone marrow transplantation for breast cancer, suppressed both B and T cell populations and T cell function at times when the absolute neutrophil count had returned to normal or supernormal numbers. In the peripheral blood, 7 days following initiation of chemotherapy, there was a twofold increase in the percentage of granulocytes as compared to the level in control animals on the basis of a differential count. The polymorphonuclear neutrophil (PMN) frequency in the bone marrow was increased on day 14 and statistically identical to that in control mice on all other days analyzed. In contrast to the bone marrow cells and PBL on day 7, the frequency of PMN in the spleen and thymus was depressed. B cells (B220+) were depressed in the PBL, spleen and bone marrow and took 18–32 days to return to their normal frequency, while the frequency of B cells in the thymus was increased owing to a loss of immature T cells. The percentage of CD3+ cells in the thymus, spleen and bone marrow was significantly increased and required 10–18 days to return to normal levels, while the absolute number of CD3+ cells in the blood varied around the normal value. The ratio of CD4+ to CD8+ cells in all the organs studied varied only slightly owing to a similar reconstitution of CD4+ and CD8+ cells. In contrast to the phenotypic recovery of the CD3+, CD4+ and CD8+ cells, the ability of the splenic lymphocytes to respond to concanavalin-A was depressed and remained depressed, despite the phenotypic reconstitution of the T cell subsets, on the basis of both percentage and absolute cell number. These results show a selective T and B cell depression following multi-drug, split-dose chemotherapy in tissue and blood leukocyte populations and a chronic depression in T cell function.     

Activation ofClostridium perfringens cytotoxic enterotoxin(s) in vivo and in vitro: Role in triggers for sudden infant death

The action ofClostridium perfringens cytotoxic enterotoxins may be activated/exacerbated both in vivo and in vitro by the addition of an activator molecule present in a brush border membrane fraction isolated from young rabbits. Increased concentrations of the activator could be induced by immunologically stimulating rabbits with Ribi adjuvant. Comparative studies suggested that the activator was interferon-gamma (IFN-). In vitro IFN- sensitized cell lines apparently by enhancement of cell permeability, which allowed a more rapid uptake of the toxins, resulting in cell death at lower toxin concentrations. Viral and/or bacterial infections are inducers of IFNs. We propose that some immunologically immature infants are predisposed to infection. In the weeks prior to death, these infants may suffer from an infection that induces the synthesis of IFNs, sensitizing the infant to a more virulent infection and possible sudden death.Florida Agricultural Experiment Station Journal Series No. R-02380     

The effect of a free radical scavenger and platelet-activating factor antagonist on FFA accumulation in post-ischemic canine brain

The effects of the platelet-activating factor antagonist BN 50739 and a free radical scavenger dimethyl sulfoxide on the accumulation of free fatty acids in post-ischemic canine brain are reported. Following 14 min of complete normothermic ischemia and 60 min of reperfusion, the total brain FFAs were approximately 150% higher than in the control group (p<0.05). Perfusion with the platelet-activating factor antagonist BN50739 in its diluent dimethyl sulfoxide during 60 min of post-ischemic reoxygenation resulted in a 61.8% (p<0.01) reduction in the total brain free fatty acid accumulation. Palmitic, stearic, oleic, linoleic, and arachidonic acids decreased by 53.8%, 63.5%, 69.0%, 47.4%, and 57.2%, respectively. Although dimethyl sulfoxide alone caused stearic and arachidonic acids to return to the normal concentration range, BN 50739 had a significant influence on recovery of palmitic, oleic, and linoleic acids and was previously shown to provide significant therapeutic protection against damage to brain mitochondria following an ischemic episode. Because free fatty acid accumulation is one of the early phenomena in cerebral ischemia, this study provides evidence to support the hypothesis that both platelet-activating factor and free radicals are involved in initiating cerebral ischemic injury.     

Spawning ofConger oceanicus andConger triporiceps (Congridae) in the Sargasso Sea and subsequent distribution of leptocephali

Synopsis Distribution of leptocephali ofConger in the Western North Atlantic Ocean was studied using specimens from our collections, specimens from other collections, and various existing collection records. The presence of leptocephali ofConger oceanicus andConger triporiceps < 30 mm long over deep water in the southwestern Sargasso Sea in autumn and winter implies a protracted spawning period there. The subtropical convergence zone, meandering east-west across the Sargasso Sea, is probably the northern limit of spawning of both species. Spawning may also occur close to the Bahamas and Antilles.C. triporiceps may spawn also in the Caribbean Sea judging by the capture of small leptocephali in the western Caribbean and of the more southerly continental distribution of its juveniles. The claim of Johannes Schmidt in 1931 that the EuropeanC. conger spawns across the North Atlantic into the western Sargasso Sea is probably incorrect, because leptocephali ofConger are rare in the eastern Sargasso Sea and becauseC. triporiceps, with myomere numbers overlapping those ofC. conger, was recently described in the western North Atlantic. With increasing size, leptocephali ofC. oceanicus and a portion ofC. triporiceps spread westward and northward in the Florida Current and Gulf Stream, but larger leptocephali especially ofC. triporiceps are found also in the Caribbean and Gulf of Mexico. Spawning ofC. oceanicus in the Sargasso Sea indicates that adults cross the Florida Current-Gulf Stream, and successful leptocephali cross the current in the opposite direction to colonize juvenile habitat on the continental shelf, a migratory pattern similar to that of the American eelAnguilla rostrata (Anguillidae).     

The barbs (Barbus spp.) of Lake Tana: a forgotten species flock?

Synopsis All living species occupy an ecological niche, and are positioned within a trophic hierarchy. Extinct organisms presumably held similar behavioral and coevolutionary characteristics in the past, and were susceptible to the same kinds of natural ecological pressures operating today. Paleoecological investigations are limited by the incompleteness of the fossil record, and particularly by a lack of behavioral data that are so fundamental to ecological studies of living communities and habitats. Opportunities to examine the coevolutionary structure of ancient communities from empirical data are extremely rare. One such opportunity is provided by the Lower Cretaceous Santana Formation of north-eastern Brazil, a series of richly fossiliferous strata approximately 110 million years old. Many fossil fishes from the Santana Formation contain identifiable prey, including decapod crustaceans and fishes. A trophic hierarchy of these organisms is reconstructed here, and their ecological relationships are discussed. Comparison is made with a similar fish fauna from the Upper Jurassic Solnhofen Limestone of Germany. Low-level, intermediate and high-level predators are identified in each fauna. Predator-prey relationships in the Santana fauna are strongly hierarchical, and are more focussed at the intermediate predator level than in Solnhofen. Comparison with a model of predator-prey relationships between fishes and benthic fauna of the Baltic Sea (which like the Araripe Basin represents a semi-enclosed environment) suggests that heavy predation on teleosts such asRhacolepis, occupying an intermediate trophic level, may have permitted benthic decapods to proliferate and exclude other benthic organisms. Less intense predation on fishes at the intermediate trophic level would allow their numbers to increase, thereby increasing the intensity of predation on the benthos at the base of the trophic hierarchy.     

Genetic diversity in and phylogenetic relationships of the Brazilian endemic cotton,Gossypium mustelinum (Malvaceae)

Gossypium mustelinum, one of five tetraploid species in the cotton genus, is geographically restricted to a few states in NE Brazil. Allozyme analysis was used to assess levels and patterns of genetic diversity inG. mustelinum and its relationship to the other tetraploid species. Genetic variation was low, with only 6 of 50 loci examined being polymorphic, a mean of 1.14 alleles per locus and a mean panmictic heterozygosity of 0.08. These estimates are low relative to other tetraploid cotton species, but are typical of island endemics. Interpopulational genetic identities were uniformly high, lending support to the concept of there being only one wild species of Brazilian cotton. The limited allelic diversity observed was correlated with geographical distribution, although variability is so limited in the species that geographically marginal populations are electrophoretically ordinary. Phylogenetic and phenetic analyses demonstrate thatG. mustelinum is isolated among polyploid cotton species, occupying one of the three basal clades resulting from an early radiation of polyploid taxa subsequent to polyploid formation. We suggest thatG. mustelinum represents a paleoendemic that presently exists as a series of widely scattered, relictual populations. Despite several centuries of sympatric cultivation ofG. barbadense andG. hirsutum, there was little evidence of interspecific introgression of alleles from cultivated cottons intoG. mustelinum.