Antibody to beta-amyloid protein increases acetylcholine in the hippocampus of 12 month SAMP8 male mice

Amyloid beta protein (Abeta) is the primary constituent of plaque seen in Alzheimer's disease. Abeta is proposed to play an etiological role in Alzheimer's disease and to be a cause of the decrease in the level of acetylcholine in the hippocampus. The SAMP8 strain of mouse develops age-related increases in Abeta and deficits in learning and memory by 12 months of age. We examined in 12 month old SAMP8 mice the effects of giving antibody to Abeta by septal or intracerebroventricular (ICV) injection on acetylcholine levels in the hippocampus. Antibody to Abeta increased acetylcholine in the hippocampus over 100% after ICV injection and over 200% after septal injection. Injection of rabbit serum, antibody directed towards mouse IgG, or a blocking antibody directed towards human interleukin-1beta were without effect. These results suggest that antagonism of Abeta increases acetylcholine concentrations in the hippocampus, an area important for learning and memory.     

Theanine,gamma-glutamylethylamide,is metabolized by renal phosphate-independent glutaminase

The distribution of theanine-degrading activity in Wistar rats was examined and this activity was detected only in the kidney. Judging from polyacrylamide gel electrophoresis, theanine-degrading enzyme from rat kidney was purified almost to homogeneity. Theanine-degrading activity was co-purified with glutaminase activity, and the relative activity for theanine was about 85% of that for L-glutamine throughout purification. Substrate specificity of purified enzyme preparation coincided well with the data of phosphate-independent glutaminase [EC 3.5.1.2], which had been previously reported. It was very curious that gamma-glutamyl methyl and ethyl esters were more effectively hydrolyzed than theanine and L-glutamine, in view of relative activity and K(m) value. It was suggested that gamma-glutamyl moiety in theanine molecule was transferred to form gamma-glutamylglycylglycine with relative ease in the presence of glycylglycine. On the other hand, purified phosphate-dependent glutaminase did not show theanine-degrading activity at all. Thus, it was concluded that theanine was hydrolyzed by phosphate-independent glutaminase in kidney and suggested that, as for the metabolic fate of theanine, its glutamyl moiety might be transferred by means of gamma-glutamyl transpeptidase reaction to other peptides in vivo.     

Hydrolysis of diadenosine polyphosphates by nucleotide pyrophosphatases/phosphodiesterases.

Diadenosine polyphosphates (ApnAs) act as extracellular signaling molecules in a broad variety of tissues. They were shown to be hydrolyzed by surface-located enzymes in an asymmetric manner, generating AMP and Apn-1 from ApnA. The molecular identity of the enzymes responsible remains unclear. We analyzed the potential of NPP1, NPP2, and NPP3, the three members of the ecto-nucleotide pyrophosphatase/phosphodiesterase family, to hydrolyze the diadenosine polyphosphates diadenosine 5',5"'-P1,P3-triphosphate (Ap3A), diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A), and diadenosine 5',5"'-P1,P5-pentaphosphate, (Ap5A), and the diguanosine polyphosphate, diguanosine 5',5"'-P1,P4-tetraphosphate (Gp4G). Each of the three enzymes hydrolyzed Ap3A, Ap4A, and Ap5A at comparable rates. Gp4G was hydrolyzed by NPP1 and NPP2 at rates similar to Ap4A, but only at half this rate by NPP3. Hydrolysis was asymmetric, involving the alpha,beta-pyrophosphate bond. ApnA hydrolysis had a very alkaline pH optimum and was inhibited by EDTA. Michaelis constant (Km) values for Ap3A were 5.1 micro m, 8.0 micro m, and 49.5 micro m for NPP1, NPP2, and NPP3, respectively. Our results suggest that NPP1, NPP2, and NPP3 are major enzyme candidates for the hydrolysis of extracellular diadenosine polyphosphates in vertebrate tissues.     

Site-directed mutagenesis of benzalacetone synthase. The role of the Phe215 in plant type III polyketide synthases

Benzalacetone synthase (BAS) and chalcone synthase (CHS) are plant-specific type III polyketide synthases (PKSs) that share approximately 70% amino acid sequence identity. BAS catalyzes a one-step decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce a diketide benzalacetone, whereas CHS performs sequential condensations with three malonyl-CoA to generate a tetraketide chalcone. A homology model suggested that BAS has the same overall fold as CHS with cavity volume almost as large as that of CHS. One of the most characteristic features is that Rheum palmatum BAS lacks active site Phe-215; the residues 214LF conserved in type III PKSs are uniquely replaced by IL. Our observation that the BAS I214L/L215F mutant exhibited chalcone-forming activity in a pH-dependent manner supported a hypothesis that the absence of Phe-215 in BAS accounts for the interruption of the polyketide chain elongation at the diketide stage. On the other hand, Phe-215 mutants of Scutellaria baicalensis CHS (L214I/F215L, F215W, F215Y, F215S, F215A, F215H, and F215C) afforded increased levels of truncated products; however, none of them generated benzalacetone. These results confirmed the critical role of Phe-215 in the polyketide formation reactions and provided structural basis for understanding the structure-function relationship of the plant type III PKSs.     

Imprint cytology of epithelioid angiomyolipoma in a patient with tuberous sclerosis. A case report

BACKGROUND: Angiomyolipoma composed predominantly of epithelioid cells has been referred to as epithelioid angiomyolipoma. As this subtype shows considerable cellular atypia, it may be erroneously diagnosed as malignant epithelioid tumor, such as renal cell carcinoma and hepatocellular carcinoma. So far, only one report describing the cytologic findings of epithelioid angiomyolipoma has been documented, and epithelioid angiomyolipoma occurring in the peritoneal cavity has not been reported. CASE: Eleven years after resection of a renal epithelioid angiomyolipoma in a 34-year-old male with tuberous sclerosis, a tumor appeared in the peritoneal cavity and three masses in the liver. The intraoperative smears imprinted from part of the peritoneal mass revealed many large, atypical cells. The well-preserved atypical cells showed abundant, round to polyhedral, granular cytoplasm. Bizarre, giant nuclei with hyperchromasia and huge nucleoli were occasionally seen. Intranuclear cytoplasmic inclusions and mitotic figures were occasionally observed. As the epithelioid cells were markedly pleomorphic, we could not rule out hepatocellular carcinoma, cytologically and histologically, in the intraoperative consultation. In permanent sections the tumor was composed predominantly of epithelioid cells showing an alveolar pattern or sheetlike arrangement. Mitotic counts were zero to one per 10 high-power fields. Immunohistochemically, the epithelioid tumor cells were positive for vimentin, alpha-smooth muscle actin and HMB-45, consistent with epithelioid angiomyolipoma. MIB-1-labeling index was 1.6%. CONCLUSION: When one sees atypical epithelioid tumor cells in a tuberous sclerosis patient during an intraoperative consultation, one must consider epithelioid angiomyolipoma.     

Enhancement of protein expression in insect cells by a lobster tropomyosin cDNA leader sequence

We describe a cis element that dramatically increases the expression levels of exogenous genes in baculovirus-infected insect cells. This 21 bp sequence element is derived from a 5' untranslated leader sequence of a lobster tropomyosin cDNA (L21). By using a transfer vector carrying L21, the expression levels of tropomyosin and luciferase were 20- and seven-fold higher with L21 than without L21, respectively. L21 has both the Kozak sequence and the A-rich sequence found in the polyhedrin leader sequence. We assume that both sequence elements are essential for the enhancement of protein expression in the baculovirus-based expression system.     

A chimera of a gelatinase inhibitor peptide with streptavidin as a bifunctional tumor targeting reagent

A chimeric protein, consisting of streptavidin fused to a cyclic decapeptide with potent inhibitory activity for matrix metalloproteinases (MMP), has been produced in Escherichia coli and purified. The purified chimera formed a tetramer and showed full biotin-binding ability. The chimera was also capable of both binding to MMP-2 and inhibiting its activity. Thus, both the streptavidin moiety and the decapeptide of the chimera are fully functional. This bifunctional nature of the chimera should facilitate the application of the decapeptide since the streptavidin moiety can be used as a specific conjugation site for almost any materials upon biotinylation.     

Targeted disruption of LIG-1 gene results in psoriasiform epidermal hyperplasia

We have designed a chimeric promoter that can be stimulated by various pro-inflammatory mediators and so drive the expression of therapeutic genes under inflammatory conditions. The promoter has two parts, the [-247/+20] fragment of the human type IIA secreted phospholipase A2 gene promoter, which is stimulated by the pro-inflammatory cytokine interleukin-1beta (IL-1beta), and a double peroxisome proliferator-activated receptor response element that is activated by some eicosanoids and by non-steroidal anti-inflammatory drugs (NSAIDs). Transfection experiments using rabbit articular chondrocytes in primary culture showed that this chimeric promoter produced a low basal activity and was induced by NSAIDs, WY-14643, IL-1beta, and 15-deoxy Delta12,14 prostaglandin J2. The latter two compounds stimulated the promoter synergistically.     

Group V phospholipase A2 induces leukotriene biosynthesis in human neutrophils through the activation of group IVA phospholipase A2

We reported previously that exogenously added human group V phospholipase A(2) (hVPLA(2)) could elicit leukotriene B(4) (LTB(4)) biosynthesis in human neutrophils (Han, S. K., Kim, K. P., Koduri, R., Bittova, L., Munoz, N. M., Leff, A. R., Wilton, D. C., Gelb, M. H., and Cho, W. (1999) J. Biol. Chem. 274, 11881-11888). To determine the mechanism of the hVPLA(2)-induced LTB(4) biosynthesis in neutrophils, we thoroughly examined the effects of hVPLA(2) and their lipid products on the activity of group IVA cytosolic PLA(2) (cPLA(2)) and LTB(4) biosynthesis under different conditions. As low as 1 nm exogenous hVPLA(2) was able to induce the release of arachidonic acid (AA) and LTB(4). Typically, AA and LTB(4) were released in two phases, which were synchronized with a rise in intracellular calcium concentration ([Ca(2+)](i)) near the perinuclear region and cPLA(2) phosphorylation. A cellular PLA(2) assay showed that hVPLA(2) acted primarily on the outer plasma membrane, liberating fatty acids and lysophosphatidylcholine (lyso-PC), whereas cPLA(2) acted on the perinuclear membrane. Lyso-PC and polyunsaturated fatty acids including AA activated cPLA(2) and 5-lipoxygenase by increasing [Ca(2+)](i) and inducing cPLA(2) phosphorylation, which then led to LTB(4) biosynthesis. The delayed phase was triggered by the binding of secreted LTB(4) to the cell surface LTB(4) receptor, which resulted in a rise in [Ca(2+)](i) and cPLA(2) phosphorylation through the activation of mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2. These results indicate that a main role of exogenous hVPLA(2) in neutrophil activation and LTB(4) biosynthesis is to activate cPLA(2) and 5-lipoxygenase primarily by liberating from the outer plasma membrane lyso-PC that induces [Ca(2+)](i) increase and cPLA(2) phosphorylation and that hVPLA(2)-induced LTB(4) production is augmented by the positive feedback activation of cPLA(2) by LTB(4).