Soluble derivatives of the beta amyloid protein precursor of Alzheimer's disease are labeled by antisera to the beta amyloid protein

The amyloid deposited in Alzheimer's disease (AD) is composed primarily of a 39-42 residue polypeptide (beta AP) that is derived from a larger beta amyloid protein precursor (beta APP). In previous studies, we and others identified full-length, membrane-associated forms of the beta APP and showed that these forms are processed into soluble derivatives that lack the carboxyl-terminus of the full-length forms. In this report, we demonstrate that the soluble approximately 125 and approximately 105 kDa forms of the beta APP found in human cerebrospinal fluid are specifically labeled by several different antisera to the beta AP. This finding indicates that both soluble derivatives contain all or part of the beta AP sequence, and it suggests that one or both of these forms may be the immediate precursor of the amyloid deposited in AD.     

Actin assembly in electropermeabilized neutrophils: role of G-proteins

Polymerization of microfilaments, one of the responses triggered in neutrophils by stimuli such as the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP), involves the conversion of actin from the monomeric to the filamentous form. The exact sequence of events responsible for this conversion remains to be defined, but its susceptibility to inhibition by pertussis toxin provides indirect evidence that GTP-binding proteins (G-proteins) are involved. In this report, electropermeabilized cells were used to obtain more direct evidence of a role for G-proteins in actin assembly. Staining with 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin and flow cytometry were used to monitor the formation of filamentous actin. GTP-gamma-S, a nonhydrolyzable analogue of GTP and aluminum fluoride, which in combination with GDP can activate G-proteins, stimulated actin assembly in electropermeabilized cells but had only marginal effects on intact cells. fMLP-induced actin polymerization in permeabilized cells was inhibited by pretreatment with GDP-beta-S, an analogue of GDP that stabilizes the inactive form of G-proteins. In contrast, stimulation by phorbol 12-myristate 13-acetate (PMA) was largely unaffected by GDP-3-S. These observations indicate that activation of G-proteins is essential for actin assembly induced by receptor-dependent stimuli such as fMLP. Moreover, GTP-binding proteins do not seem to be required in the late stages of the signalling cascade, i.e. after stimulation of protein kinase C.     

Cloning, analysis, and bacterial expression of human farnesyl pyrophosphate synthetase and its regulation in Hep G2 cells

A partial length cDNA encoding farnesyl pyrophosphate synthetase (hpt807) has been isolated from a human fetal liver cDNA library in lambda gt11. DNA sequence analysis reveals hpt807 is 1115 bp in length and contains an open reading frame coding for 346 amino acids before reaching a stop codon, a polyadenylation addition sequence, and the first 14 residues of a poly(A+) tail. Considerable nucleotide and deduced amino acid sequence homology is observed between hpt807 and previously isolated rat liver cDNAs for farnesyl pyrophosphate synthetase. Comparison with rat cDNAs suggests that hpt807 is about 20 bp short of encoding the initiator methionine of farnesyl pyrophosphate synthetase. The human cDNA was cloned into a prokaryotic expression vector and Escherichia coli strain DH5 alpha F'IQ was transformed. Clones were isolated that express an active fusion protein which can be readily observed on protein gels and specifically stained on immunoblots with an antibody raised against purified chicken farnesyl pyrophosphate phosphate synthetase. These data confirm the identity of hpt807 as encoding farnesyl pyrophosphate synthetase. Slot blot analyses of RNA isolated from Hep G2 cells show that the expression of farnesyl pyrophosphate synthetase mRNA is regulated. Lovastatin increases mRNA levels for farnesyl pyrophosphate synthetase 2.5-fold while mevalonic acid, low-density lipoprotein, and 25-hydroxycholesterol decrease mRNA levels to 40-50% of control values.     

The primary structure of human apolipoprotein A-IV

Human apolipoprotein (apo) A-IV was purified from chylous ascites fluid. Proteolytic peptides produced by trypsin and Staphylococcus aureus V8 proteinase digestions were purified by high-performance liquid chromatography and sequenced. Human apoA-IV contains 376 amino acid residues. The peptide-derived sequence generally matches two previously reported DNA-derived amino acid sequences except for discrepancies in five positions. In order to examine these discrepancies further, one complete apoA-IV cDNA clone and another partial clone were sequenced. Comparison of all the available information indicates that the peptide-derived sequence reported here is accurate. Sequencing errors probably account for some of the discrepancies between the two primary sequences predicted by earlier nucleotide analyses. In certain positions, however, bona fide sequence heterogeneity or cloning artifact cannot be excluded.     

A toxic substance produced by Nocardia otitidiscaviarum isolated from cutaneous nocardiosis

During our studies on toxic substances from clinically isolated Nocarida, a new isolate identified as Nocardia otitidiscaviarum from cutaneous nocardiosis was found to produce a toxic substance called HS-6 that had strong in vitro as well as in vivo toxicity. The mouse intraperitoneal LD₅₀ value was 1.25 mg/kg and the ED₅₀ value for L1210 cultured cells was 0.3 ng/ml. The structure of HS-6 was determined and found to belong to the 16-membered macrocyclic group with a molecular formula of C₄₃H₆₈O₁₂. HS-6 also showed activity against pathogenic fungi such as Cryptococcus neoformans.     

A 40 kilodalton rat liver nuclear protein binds specifically to apolipoprotein B mRNA around the RNA editing site.

Apolipoprotein (apo) B-48 mRNA is the product of RNA editing which consists of a C----U conversion changing a CAA codon encoding Gln-2153 in apoB-100 mRNA to a UAA stop codon in apoB-48 mRNA. In the adult rat, RNA editing occurs both in the small intestine and the liver. We have studied the ability of rat liver nuclear extracts to bind to synthetic apoB mRNA segments spanning the editing site. Using an RNA gel mobility shift assay, we found the sequence-specific binding of a protein(s) to a 65-nucleotide apoB-100 mRNA. UV crosslinking followed by T1 ribonuclease digestion and SDS-polyacrylamide gel electrophoresis demonstrated the formation of a 40 kDa protein-RNA complex when 32P-labeled apoB-100 mRNA was incubated with a rat liver nuclear extract but not with HeLa nuclear extract. Binding was specific for the sense strand of apoB mRNA, and was not demonstrated with single-stranded apoB DNA, or antisense apoB RNA. The complex also failed to form if SDS was present during the UV light exposure. Binding experiments using synthetic apoB mRNAs indicate that the 40 kDa protein would also bind to apoB-48 mRNA but not apoA-I, apoA-IV, apoC-II or apoE mRNA. Experiments using deletion mutants of apoB-100 mRNA indicate efficient binding of wildtype 65-nucleotide (W65), 40-nucleotide (W40) and 26-nucleotide (W26) apoB-100 mRNA segments, but not 10-nucleotide (or smaller) segments of apoB-100 mRNA to the 40 kDa protein. In contrast, two other regions of apoB-100 mRNA, B-5' (bases 1128-3003) and B-3' (bases 11310-11390), failed to bind to the protein. The 40 kDa sequence-specific binding protein in rat liver nuclear extract may play a role in apoB-100 mRNA editing.     

Dichogamy, Sex Allocation, and Mating System of Campanula microdonta and C. punctata

Dichogamy and sex allocation in several populations of Campanula microdonta and C. punctata were investigated with regard to their mating systems. Duration of the staminate phase differed among the populations: staminate phase was longer in self-compatible (SC) and largely outcrossing populations than in self-incompatible (SI) and outcrossing populations or in SC and largely inbreeding populations. Duration of the pistillate phase among the populations was less variable than duration of the staminate phase. Male reproductive effort decreased with increase of the estimated selfing rates. Male allocation (weight ratio of androecium to gynoecium or to total flower) may be used as an indicator of the breeding system. Within each population, small flowers allocate proportionately more resources to the androecium than to the gynoecium. Among populations, SC outcrossing populations tend to produce large ovaries, and SC inbreeding populations tend to produce small ovaries.