Alternative pathways for production of beta-amyloid peptides of Alzheimer's disease

This highlight article describes three Alzheimer's disease (AD) studies presented at the 5th General Meeting of the International Proteolysis Society that address enzymatic mechanisms for producing neurotoxic beta-amyloid (Abeta) peptides. One group described the poor kinetics of BACE 1 for cleaving the wild-type (WT) beta-secretase site of APP found in most AD patients. They showed that cathepsin D displays BACE 1-like specificity and cathepsin D is 280-fold more abundant in human brain than BACE 1. Nevertheless, as BACE 1 and cathepsin D show poor activity towards the WT beta-secretase site, they suggested continuing the search for additional beta-secretase(s). The second group reported cathepsin B as an alternative beta-secretase possessing excellent kinetic efficiency and specificity for the WT beta-secretase site. Significantly, inhibitors of cathepsin B improved memory, with reduced amyloid plaques and decreased Abeta(40/42) in brains of AD animal models expressing amyloid precursor protein containing the WT beta-secretase site. The third group addressed isoaspartate and pyroglutamate (pGlu) posttranslational modifications of Abeta. Results showed that cathepsin B, but not BACE 1, efficiently cleaves the WT beta-secretase isoaspartate site. Furthermore, cyclization of N-terminal Glu by glutaminyl cyclase generates highly amyloidogenic pGluAbeta(3-40/42). These presentations suggest cathepsin B and glutaminyl cyclase as potential new AD therapeutic targets.     

Two peptidases that convert 125I-Lys-Arg(Met)enkephalin and 125I-(Met)enkephalin-Arg6, respectively,to 125I-(Met)enkephalin in bovine adrenal medullary chromaffin granules

Two peptidases which convert ¹²⁵I-Lys-Arg-ME and ¹²⁵I-ME-Arg⁶, respectively, to ¹²⁵I-ME, have been identified and characterized in bovine adrenomedullary chromaffin granules. The former is referred to as a secretory granule peptidase (SGP) and the latter as a carboxypeptidase B-like enzyme (CPB-like) [7] which is here further characterized. SGP cleaved ¹²⁵I-Lys-Arg-ME to produce only ¹²⁵I-ME and was localized in chromaffin granules which contained co²⁺-stimulated CPB-like activity, ME, and catecholamines. Both the SGP and the CPB-like enzymes appear to be thiol-metalloproteases. While the CPB-like enzyme seems likely to be involved in processing the enkephalin precursors [7], SGP may function as a trypsin-like or aminopeptidase enzyme in secretory granules.     

Tritrichomonas foetus: characterization of isolates and partial purification of a secreted cytotoxin

Putative virulence factors including extracellular proteases, hemagglutinin, hemolysins, and soluble cytotoxins may play significant roles in the pathogenesis of trichomoniasis. The cytotoxicity, hemagglutinating, and hemolytic activity of Tritrichomonas foetus isolate ATCC #30003 and several field isolates were compared. All isolates were hemolytic toward mouse and bovine erythrocytes but not other tested species. The isolates varied significantly in hemagglutinating ability and cytotoxin production. A 40,000 Da soluble cytotoxin was partially purified and characterized. Chromatography separated cytotoxic activity from hemagglutinating and hemolytic activity but not from protease activity. However, protease assays indicated that protease activity was inversely correlated with cytotoxic activity. Characterization studies indicated that cytotoxic activity was destroyed by heat and acidic conditions but repeated freeze/thawing did not diminish activity. Target cell specificity assays showed Henle cells were twice as sensitive to the effects of the cytotoxin as Vero cells. These results suggest that T. foetus isolates vary in the production of virulence factors and produce a soluble relatively stable non-protease cytotoxic protein capable of killing cultured mammalian cells in vitro.     

Mannosylated saponins based on oleanolic and glycyrrhizic acids. Towards synthetic colloidal antigen delivery systems

Immunostimulatory saponin based colloidal antigen delivery systems show promise as adjuvants for subunit vaccines. For this reason, allyl oleanolate was glycosylated at the 3-position using trichloroacetimidate donors to give monodesmodic saponins following deprotection. Bisdesmodic saponins were synthesized by double glycosylation at the 3- and 28-positions of oleanolic acid. When formulated together with cholesterol and phospholipids, ring-like, helical and rod-like nanostructures were formed depending on the saponin concentrations used. As an indication of adjuvant activity, the ability of these formulations, and the saponins by themselves, to induce dendritic cell maturation was measured, but no significant activity was observed.     

Interleukin-11, an IL-6-like cytokine

The aim of this review is to collect various data on different functions of interleukin-11 (IL-11), a member of a family of IL-6-like cytokines. Numerous in vitro experiments have suggested a long list of IL-11 properties including support and control of proliferation and differentiation of hematopoietic progenitors, as well as participation in osteoclastogenensis, neurogenesis, and development of some other tissues. However, a great number of in vitro effects of IL-11 have not been confirmed in experiments using murine models, hampering an understanding of the physiological role of this cytokine. We discuss possible reasons for the divergence between in vitro and in vivo data as well as the perspectives of using conditional gene targeting to assess the role of IL-11 in ontogenesis and immune response.     

Determination of the molecular mass of bacterial genomic DNA and plasmid copy number by high-pressure liquid chromatography

Relatively rapid methods for the determination of relative genome molecular mass (Mr) and the estimation of plasmid copy number have been developed. These methods are based on the ability of the Bio-Rad high-pressure liquid chromatography hydroxylapatite column to separate and quantify single-stranded DNA, double-stranded DNA, and plasmid DNA. Genome Mr values were calculated from reassociation kinetics of single-stranded DNA as measured with the hydroxylapatite column. Bacteriophage T4 DNA was used to establish a C0t (moles of nucleotides times seconds per liter), or standard reassociation value. From this C0t value, C0t values for Escherichia coli B, Beggiatoa alba B18LD, and Streptomyces coelicolor were determined by comparative calculations. From those calculated C0t values, the Mr values of 1.96 X 10(9) for E. coli, 2.02 X 10(9) for B. alba, and 3.28 X 10(9) for S. coelicolor were estimated. Plasmid concentration was determined from cleared lysates by comparing the integrated area under the phosphate buffer-eluted plasmid peak to values obtained with known amounts of plasmid. The plasmid copy number was estimated by multiplying the ratio between the amounts of plasmid and chromosomal DNA by the ratio between the Mr values of the chromosome and the plasmid. A copy number of 29 was obtained from a culture of E. coli HB101 harboring pBR322 grown to a culture density of 1.6 X 10(9) CFU . ml-1.     

Differential recovery of peptides from sample tubes and the reproducibility of quantitative proteomic data

Differential recovery of peptides due to nonspecific adsorption can seriously compromise reproducibility and quality of proteomic data for peptide analyses by liquid chromatography-mass spectrometry (LC-MS). This study demonstrates large variations in reproducibility and quantitation of LC-MS data for peptides derived from tryptic digests of BSA upon storage in different sample tubes. Notably, we show that highly improved consistency and lower errors in quantitation of BSA tryptic peptides in replicate measurements is achieved with low-retention tubes compared to regular eppendorf tubes. Furthermore, qualitative differences in peptides detected by LC-MS were observed in the two types of storage tubes. These results illustrate the necessity for careful evaluation of storage vessels and conditions to minimize variability in sample quality for LC-MS experiments.