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A major histopathological hallmark of Alzheimer's disease (AD) is the presence of amyloid deposits in the parenchyma of the amygdala, hippocampus, and neocortex1}. The principal component of the amyloid is the /3-amyloid protein (A/?), a 39-43 amino acid peptide composedof a portion of the transmembrane domain and the extracellular domain of the amyloid precursor protein (APP)2). The neurotoxicity of the A/3 has been detected in several cell systems, including primary cultured neurons3). The A/3 having an alanine C-terminus is derived from the proteolytic cleavage of the APPby the action of the yet unidentified endoproteolytic enzymes, /3-and y-secretase4). Recent studies have suggested that prolyl endopeptidase could be involved in the processing of the C-terminal portion of the APP in AD5). The prolyl endopeptidase [PEP; EC 3.4.21,26] is a serine protease, which is known to cleave peptide substrates in the C-terminal side of proline residues6). It plays an important role in degradation of the proline-containing neuropeptides such as oxytocin, vasopressin, substance P, neurotensin and angiotensin, which were suggested to participate in learning and memory processes7'8-*. It was found that the PEP

Specific peptides of varying lengths were inserted between the two metal cluster domains of metallothionein (MT), which normally are spanned by only three amino acids, Lys-Lys-Ser. These interdomain expansions were made to test if such structural alterations would affect MT function. These constructs were engineered by inserting defined oligonucleotides of up to four tandem repeats of dodecanucleotides and hexanucleotides into an Alu-1 endonuclease cleavage site, which separates the two exonic regions of an MT-coding sequence from Chinese hamster ovary cells, MT-2. The native and altered sequences were cloned into a high expression Escherichia coli-yeast shuttle vector and used to transform yeast cells whose endogenous MT genes had been previously deleted. Using metal resistance as a biological marker, all constructs were shown to be functional in rendering the host cells resistant to either copper or cadmium. As the inserts, by nature of their amino acid sequence, could add flexibility to the otherwise compact molecule, the two domains apparently are active independently. The level of activity, however, diminished with the length of the insert. Determinations for copy number of the chimeric plasmids and MT mRNAs in the transformed cells showed that the replicational and transcriptional capacity of the long and short constructs were equivalent.

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We previously isolated a rhizobacterium (Bacillus subtilis IJ-31) and demonstrated that its associated allelochemicals could indicate plant growth retardation. However, little is known about how the growth of plants is regulated by B. subtilis IJ-31 and its allelochemicals. In this study, we investigated whether plant growth retardation in this relationship occurred through the inhibition of gibberellin (GA) biosynthesis. GA 3β-hydroxylase activity was found to be inhibited by B. subtilis IJ-31 and hydrocinnamic acid (HCA), which is one of the allelochemicals produced by B. subtilis IJ-31. Additionally, thin layer chromatography (TLC) demonstrated that B. subtilis IJ-31 culture broth and HCA both inhibit GA 3β-hydroxylase (MBP-GA4) activity. The retardation of plants by HCA was then confirmed in vivo and in vitro using a Ryegrass and Arabidopsis growth retardation assay. Furthermore, treatment with either B. subtilis IJ-31 culture extract or its allelochemicals resulted in significant down-regulation of XTR9 gene expression in Arabidopsis. Overall, we identified the functional mechanism of plant growth retardation by B. subtilis IJ-31 and its allelochemicals.

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