Fitria Wahyu Andriani

Several agents have now been identified which exert their anti-tumour effects in large part via the tumour vasculature; these include TNF alpha and flavone acetic acid (FAA). More recently, Vincristine and Vinblastine have also been shown to cause a prolonged and selective decrease in tumour perfusion. Vinblastine, unlike, FAA, causes no increase in plasma TNF alpha levels in mice bearing the CaNT tumour, suggesting 2 distinct mechanisms of anti-vascular activity for these structurally diverse agents. Since FAA and Vinblastine also show quite different normal tissue toxicities, which are separately dose-limiting, we have examined the strategy of combining these 2 agents. When Vinblastine preceded FAA by 24 hr or less, tumour growth delay was significantly enhanced without a concomitant increase in toxicity. The level of enhancement was not significantly reduced by a 5-fold decrease in Vinblastine dose, though any reduction in the dose of FAA caused a rapid reduction in treatment effectiveness. Investigation of the functional vasculature of treated tumours suggested that increased anti-vascular effects may contribute to the enhanced growth inhibition of the combined treatment. Our results demonstrate the potential benefit of combining 2 different classes of anti-vascular agent, using Vinblastine and FAA (or 5,6-MeXAA) as prototype drugs.

Since triglycerides (TG) are a major independent risk factor for coronary heart disease, understanding their genetic and environmental determinants is of major importance. Mouse models indicate an inverse relationship between levels of the newly identified apolipoprotein AV (APOAV) and TG concentrations. We have examined the relative influence of human APOA5 variants on plasma lipids, compared to the impact of variation in APOC3 and APOA4 which lie in the same cluster. Single nucleotide polymorphisms (SNPs) in APOA5 (S19W, -1131T>C) and APOA4 (T347S, Q360H) and an APOA4/A5 intergenic T>C SNP were examined in a large study of healthy middle-aged men (n=2808). APOA5 19WW and -1131CC men had 52% and 40% higher TG (P<0.003) compared to common allele homozygotes, respectively, effects which were independent and additive. APOA4 347SS men had 23% lower TG compared to TT men (P<0.002). Haplotype analysis was carried out to identify TG-raising alleles and included, in addition, four previously genotyped APOC3 SNPs (-2845T>G, -482C>T,…

To study the putative precursor proteins (PreA4(695), PreA4(751), and PreA4(770] of Alzheimer's disease A4 amyloid protein, polyclonal and monoclonal antibodies were raised against a recombinant bacterial PreA4(695) fusion protein. These antibodies were used to identify the precursors in different cell lines as well as in human brain homogenates and cerebrospinal fluid (CSF). The precursors are tyrosine-sulfated, O- and N-glycosylated membrane proteins and have half-lives of 20-30 min in cells. Cells express the polypeptides at their surface but also secrete C-terminal truncated proteins into the medium. These proteins are also found in CSF of both Alzheimer's disease patients and normal individuals. The proteins are derived from their cognate membrane-associated forms by proteolysis and have apparently lost the cytoplasmic and the transmembrane domains. Since the latter contributes to the A4 amyloid sequence, it seems possible that this proteolytic cleavage represents the first step in the formation of A4 amyloid deposits.

The deposition of amyloid βA4 in the brain is a major pathological hallmark of Alzheimer's disease. Amyloid βA4 is a peptide composed of 42 or 43 amino acid residues. In brain, it appears in the form of highly insoluble, filamentous aggregates.Using synthetic peptides corresponding to the natural βA4 sequence as well as analog peptides, we demonstrate requirements for filament formation in vitro. We also determine aggregational properties and the secondary structure of βA4. A comparison of amino-terminally truncated βA4 peptides identifies a peptide spanning residues 10 to 43 as a prototype for amyloid βA4. Infrared spectroscopy of βA4 peptides in the solid state shows that their secondary structure consists of a β-turn flanked by two strands of antiparallel β-pleated sheet. Analog peptides containing a disulfide bridge were designed to stabilize different putative β-turn positions. Limited proteolysis of these analogs allowed a localization of the central β-turn at residues 26 to 29 of the entire sequence.Purified βA4 peptides are soluble in water. Size-exclusion chromatography shows that they form dimers that, according to circular dichroism spectroscopy, adopt a β-sheet conformation. Upon addition of salts, the bulk fraction of peptides precipitates and adopts a β-sheet structure. Only a small fraction of peptides remains solubilized. They are monomeric and adopt a random coil conformation. This suggests that the formation of aggregates depends upon a hydrophobic effect that leads to intra- and intermolecular interactions between hydrophobic parts of the βA4 sequence. This model is sustained by the properties of βA4 analogs in which hydrophobic residues were substituted. These peptides show a markedly increased solubility in salt solutions and have lost the ability to form filaments. In contrast, the substitution of hydrophilic residues leads only to small deviations in the shape of filaments, indicating that hydrophilic residues contribute to the specificity of interactions between βA4 peptides.

In patients with Alzheimer's disease, amyloid fibrils that are aggregates of A4 protein subunits are deposited in the brain. A similar process occurs at an earlier age in persons with Down's syndrome. To investigate the deposition of amyloid in these diseases, we used a radioimmunoassay to measure levels of the amyloid precursor (PreA4) in the serum of 17 patients with Down's syndrome, 15 patients with Alzheimer's disease, and 33 normal elderly controls. The mean (+/- SD) concentration of serum PreA4 was increased 1.5-fold in patients with Down's syndrome (2.49 +/- 1.13 nmol per liter) as compared with that in controls (1.68 +/- 0.49 nmol per liter; P less than 0.007); the levels in patients with Alzheimer's disease were similar to those in controls (1.83 +/- 0.78; P less than 0.98). We also found that the concentration of PreA4 in the brain tissue of two adults with Down's syndrome (100 and 190 pmol per gram) was higher than that in the brain tissue of either 26 patients with Alzheimer's disease (64.4 +/- 17.3 pmol per gram) or 17 elderly controls with neurologic disease (68.5 +/- 26.3 pmol per gram). Immunocytochemical studies of brain tissue from 26 patients with Down's syndrome showed that the deposition of A4 protein amyloid began in these patients approximately 50 years earlier than it began in 127 normal aging subjects studied previously, although the rate of deposition was the same. We conclude that, since the gene for PreA4 is on the long arm of chromosome 21, which is present in triplicate in Down's syndrome, overexpression of this gene may lead to increased levels of PreA4 and amyloid deposition in Down's syndrome. However, since increased levels of PreA4 are not present in Alzheimer's disease, additional factors must account for the amyloid deposition in that disorder.

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The amyloid proteins isolated from neuritic plaques and the cerebrovasculature of Alzheimer's disease are self-aggregating moieties termed A4 protein and beta-protein, respectively. A putative A4 amyloid precursor (herein termed A4(695] has been characterized by analysis of a human brain complementary DNA. We report here the sequence of a closely related amyloid cDNA, A4(751), distinguished from A4(695) by the presence of a 168 base-pair (bp) sequence which adds 57 amino acids to, and removes one residue from, the predicted A4(695) protein. The peptide predicted from this insert is very similar to the Kunitz family of serine proteinase inhibitors. The two A4-specific messenger RNAs are differentially expressed: in a limited survey, A4(751) mRNA appears to be ubiquitous, whereas A4(695) mRNA has a restricted pattern of expression which includes cells from neuronal tissue. These data may have significant implications for understanding amyloid deposition in Alzheimer's disease.

Alzheimer's disease is characterized by a widespread functional disturbance of the human brain. Fibrillar amyloid proteins are deposited inside neurons as neurofibrillary tangles and extracellularly as amyloid plaque cores and in blood vessels. The major protein subunit (A4) of the amyloid fibril of tangles, plaques and blood vessel deposits is an insoluble, highly aggregating small polypeptide of relative molecular mass 4,500. The same polypeptide is also deposited in the brains of aged individuals with trisomy 21 (Down's syndrome). We have argued previously that the A4 protein is of neuronal origin and is the cleavage product of a larger precursor protein. To identify this precursor, we have now isolated and sequenced an apparently full-length complementary DNA clone coding for the A4 polypeptide. The predicted precursor consists of 695 residues and contains features characteristic of glycosylated cell-surface receptors. This sequence, together with the localization of its gene on chromosome 21, suggests that the cerebral amyloid deposited in Alzheimer's disease and aged Down's syndrome is caused by aberrant catabolism of a cell-surface receptor.