Monoclonal Antibodies Against the Human Metalloprotease EC 3.4.24.15 Label Neurofibrillary Tangles in Alzheimer's Disease Brain

Abstract: Alzheimer's disease is characterized neuropathologically by the presence of neuritic and amyloid plaques, vascular amyloid, and neurofibrillary tangles in specific brain areas. The main constituent of amyloid deposits is amyloid β protein, a 40–42 amino acid proteolytic product of the amyloid β-precursor protein. In our search for proteases that can generate the N-terminus of amyloid β protein (β-secretases), we discovered a thiol-dependent metalloprotease that was identified, by peptide sequencing, as metalloendopeptidase EC 3.4.24.15. In vitro, the metalloprotease cleaves the methionine-aspartic acid bond in a 10 amino acid synthetic peptide, indicating that it could generate the N-terminus of amyloid β protein, and generates amyloidogenic fragments from full-length recombinant amyloid β-precursor protein. Mouse monoclonal antibodies produced against a unique synthetic peptide from the metalloprotease labeled various monkey tissues as detected by western blots and immunohistochemistry. Unexpectedly, two monoclonal antibodies, IVD6 and IIIF3, immunolabeled strongly intracellular neurofibrillary tangles, neurites of senile plaques, and neuropil threads, but not "ghost" tangles or amyloid in sections taken from Alzheimer's disease brain. This finding provides further evidence for the metalloprotease's relevance to Alzheimer's disease pathology, although the connection between tangle staining and the formation of amyloid β protein remains to be elucidated.     

The extended replicator

This paper evaluates and criticises the developmental systems conception of evolution and develops instead an extension of the gene's eye conception of evolution. We argue (i) Dawkin's attempt to segregate developmental and evolutionary issues about genes is unsatisfactory. On plausible views of development it is arbitrary to single out genes as the units of selection. (ii) The genotype does not carry information about the phenotype in any way that distinguishes the role of the genes in development from that other factors. (iii) There is no simple and general causal criterion which distinguishes the role of genes in development and evolution. (iv) There is, however, an important sense in which genes but not every other developmental factor represent the phenotype. (v) The idea that genes represent features of the phenotype forces us to recognise that genes are not the only, or almost the only, replicators. Many mechanisms of replication are involved in both development and evolution. (vi) A conception of evolutionary history which recognises both genetic and non-genetic replicators, lineages of replicators and interactors has advantages over both the radical rejection of the replicator/interactor distinction and the conservative restriction of replication to genetic replication.     

Mutational analysis of VAMP domains implicated in Ca2+-induced insulin exocytosis.

Vesicle-associated membrane protein-2 (VAMP-2) and cellubrevin are associated with the membrane of insulin-containing secretory granules and of gamma-aminobutyric acid (GABA)-containing synaptic-like vesicles of pancreatic beta-cells. We found that a point mutation in VAMP-2 preventing targeting to synaptic vesicles also impairs the localization on insulin-containing secretory granules, suggesting a similar requirement for vesicular targeting. Tetanus toxin (TeTx) treatment of permeabilized HIT-T15 cells leads to the proteolytic cleavage of VAMP-2 and cellubrevin and causes the inhibition of Ca2+-triggered insulin exocytosis. Transient transfection of HIT-T15 cells with VAMP-1, VAMP-2 or cellubrevin made resistant to the proteolytic action of TeTx by amino acid replacements in the cleavage site restored Ca2+-stimulated secretion. Wild-type VAMP-2, wild-type cellubrevin or a mutant of VAMP-2 resistant to TeTx but not targeted to secretory granules were unable to rescue Ca2+-evoked insulin release. The transmembrane domain and the N-terminal region of VAMP-2 were not essential for the recovery of stimulated exocytosis, but deletions preventing the binding to SNAP-25 and/or to syntaxin I rendered the protein inactive in the reconstitution assay. Mutations of putative phosphorylation sites or of negatively charged amino acids in the SNARE motif recognized by clostridial toxins had no effect on the ability of VAMP-2 to mediate Ca2+-triggered secretion. We conclude that: (i) both VAMP-2 and cellubrevin can participate in the exocytosis of insulin; (ii) the interaction of VAMP-2 with syntaxin and SNAP-25 is required for docking and/or fusion of secretory granules with the plasma membrane; and (iii) the phosphorylation of VAMP-2 is not essential for Ca2+-stimulated insulin exocytosis.     

Growth Physiology of the Hyperthermophilic Archaeon Thermococcus litoralis: Development of a Sulfur-Free Defined Medium, Characterization of an Exopolysaccharide, and Evidence of Biofilm Formation

Nutritional characteristics of the hyperthermophilic archaeon Thermococcus litoralis have been investigated with emphasis on the development of a sulfur-free, defined growth medium, analysis of an exocellular polysaccharide, and formation of a biofilm. An artificial-seawater-based medium, containing 16 amino acids, adenine, uracil, vitamins, and trace elements, allowed T. litoralis to attain growth rates and cell densities similar to those found with complex media. Four amino acids (alanine, asparagine, glutamine, and glutamate) were not included due to their lack of effect on growth rates and cell yields. In this medium, cultures reached densities of 10(sup8) cells per ml, with doubling times of 55 min (without maltose) or 43 min (with maltose). Neither the addition of elemental sulfur nor the presence of H(inf2) significantly affected cell growth. A sparingly soluble exopolysaccharide was produced by T. litoralis grown in either defined or complex media. Analysis of the acid-hydrolyzed exopolysaccharide yielded mannose as the only monosaccharidic constituent. This exopolysaccharide is apparently involved in the formation of a biofilm on polycarbonate filters and glass slides, which is inhabited by high levels of T. litoralis. Biofilm formation by hyperthermophilic microorganisms in geothermal environments has not been examined to any extent, but further work in this area may provide information related to the interactions among high-temperature organisms.     

The evolutionary history of the amylase multigene family in Drosophila pseudoobscura

In Drosophila pseudoobscura, the amylase (Amy) multigene family is contained within a series of inversions, or gene arrangements, on the third chromosome. The Standard (ST), Santa Cruz (SC), and Tree Line (TL) inversions are central to the phylogeny of arrangements, and have clusters of other arrangements derived from them. The gene arrangements belonging to each of these three clusters have a characteristic number of Amy genes, ranging from three in ST to two in SC to one in TL. This distribution pattern can reflect a history of either duplications or deletions, although the data available in the past did not permit a decision between these alternatives. We provide unambiguous evidence that three Amy genes were present before the divergence of the ST, SC, and TL arrangements. Thus, the current status of the Amy multigene family is the result of deletions in the TL and SC arrangements, which created three new pseudogenes: TL Amy2-psi, TL Amy3-psi, and SC Amy3- psi. Analysis of pseudogene sequences revealed that, in the SC and ST arrangements, pseudogene evolution has been retarded, most likely due to the homogenization effect of gene conversion. Finally, by determining the original copy number, we have reconstructed the evolutionary history of the Amy multigene family and linked it with the evolution of the central gene arrangements.      

Prairie plant guilds: a multivariate analysis of prairie species based on ecological and morphological traits

An ecomorphological analysis of the tallgrass prairie of central North America divided representative species of the native grassland flora into eight guilds or groups of species with similar life-form, phenology, and ecology. The guilds, segregated by multivariate analysis, are: (1) warm-season graminoids with Kranz anatomy and the Hatch-Slack photosynthetic pathway (C4 grasses); (2) cool-season graminoids without Kranz anatomy, but with the common Calvin or C3 photosynthetic pathway (C3 grasses and sedges); (3) annuals and biennial forbs; (4) ephemeral spring forbs; (5) spring forbs; (6) summer/fall forbs; (7) legumes; and (8) woody shrubs. The study was based on 158 plant species indigenous to three upland prairie sites in northeastern Kansas. Each species was scored for 32 traits which fall into five broad categories: plant habit, leaf characteristics, stem structures, root structures, and reproductive traits, including phenology. A multivariate, detrended correspondence analysis sorted the 158 species into the eight principal groups or guilds. These groups were further supported by a cluster analysis and discriminant function analysis of the same data set. The discriminant function analysis determined that 94.3% of the species were correctly classified in their respective guilds, and that the guilds were statistically different. Results indicate that guild analysis offers a basis for detailed classification of grassland vegetation that is more ecologically focused than species composition, as the myriad of species (about 1,000 prairie species on the central plains of North America) vary in presence, cover, and importance with their individualistic distribution.Abbreviations C3= C3 photosynthesis - C4= C4 photosynthesis - LSD= least significant difference     

The evolutionary ecology of mast seeding

The past seven years have seen a revolution in understanding the causes of mast seeding In perennial plants. Before 1987, the two main theories were resource matching (i.e. plants vary their reproductive output to match variable resources) and predator satiation (i.e. losses to predators are reduced by varying the seed crop). Today, resource matching is restricted to a proximate role, and predator satiation is only one of many theories for the ultimate advantage of masting. Wind pollination, prediction of favourable years for seedling establishment, animal pollination, animal dispersal of fruits, high accessory costs of reproduction and large seed size have all been advanced as possible causes of masting. Of these, wind pollination, predator satiation and environmental prediction are important in a number of species, but the other theories have less support. In future, Important advances seem likely from quantifying synchrony within a population, and examining species with very constant reproduction between years.     

Purification and properties of the cyclodextrinase of Bacillus sphaericus ATCC 7055

Bacillus sphaericus ATCC 7055 produced an intracellular cyclodextrinase (EC 3.2.1.54). It was purified by solublilising with Triton X-100, Q-Sepharose ion-exchange chromatography, phenyl-Sepharose CL-4B hydrophobic interaction chromatography and Superose-12 gel filtration and gave a single band on SDS-PAGE and preparative isoelectric focusing. The maxima for pH and temperature of the purified enzyme were pH 6.0–6.5 and 40°C. The enzyme had a relative molecular mass of 91 200–95 000 and an isoelectric point of 5.3. The amino-linked pseudotetrasaccharide, acarbose, inhibited activity. As well as cyclodextrins the enzyme was active on a broad range of substrates ranging in size from maltooligosaccharides (G3) to polysaccharides such as starch and pullulan, and branched cyclodextrins. End-product profiles of the cyclodextrinase on various substrates revealed that, upon hydrolysis of 1% (w/v) -, - and -cyclodextrin and maltoheptaose, glucose and maltose were the dominant end-products. Pullulan degradation resulted in panose (92%, w/v) as the main end-product, and glucose (27%, w/v) and maltose (37%, w/v) were the sole products formed from starch degradation.     

Transport of glycine-betaine by Listeria monocytogenes

Uptake of [¹⁴C]glycine-betaine by Listeria monocytogenes was stimulated by NaCl with optimal stimulation at 0.4–0.5 M. The glycine-betaine transport system had a K _m of 22 M and a V _max of 11.7 nmol^-1 min^-1 mg^-1 protein when grown in the absence of NaCl. When grown in the presence of 0.8 M NaCl the V _max increased to 27.0 nmol^-1 min^-1 mg^-1 protein in 0.8 M NaCl. At NaCl concentrations above 0.5 M the uptake rate of glycine-betaine was reduced. Measurement of intracellular K⁺ concentrations and fluorescent dye quenching indicated that higher NaCl concentrations also led to a decrease in the electrochemical potential difference across the cytoplasmic membrane. Uptake of glycine was also observed, but this was not stimulated by NaCl.