NADPH-cytochrome c (P-450) reductase has the activity of NADPH-linked aquacobalamin reductase in rat liver microsomes.

To elucidate the mammalian system for synthesis of cobalamin coenzymes, microsomal NADPH-linked aquacobalamin reductase was purified and characterized. The enzyme was purified about 534-fold over rat liver microsomal fraction in a yield of about 32%. The purified enzyme was homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and had a monomeric molecular weight of 79,000. The purified aquacobalamin reductase showed a high specific activity (about 55 mumol/min per mg protein) of NADPH-cytochrome c (P-450) reductase. About 33% of the NADPH-cytochrome c reductase activity found in the microsomal fraction was recovered in the final purified preparation. The activity ratio of NADPH-cytochrome c reductase/NADPH-linked aquacobalamin reductase was about 5.0 through the purification steps, indicating that the rat liver microsomal NADPH-linked aquacobalamin reductase is the NADPH-cytochrome c reductase.     

Inhibition of glutaminyl cyclase alters pyroglutamate formation in mammalian cells

Mammalian cell lines were examined concerning their Glutaminyl Cyclase (QC) activity using a HPLC method. The enzyme activity was suppressed by a QC specific inhibitor in all homogenates. Aim of the study was to prove whether inhibition of QC modifies the posttranslational maturation of N-glutamine and N-glutamate peptide substrates. Therefore, the impact of QC-inhibition on amino-terminal pyroglutamate (pGlu) formation of the modified amyloid peptides Abeta(N3E-42) and Abeta(N3Q-42) was investigated. These amyloid-beta peptides were expressed as fusion proteins with either the pre-pro sequence of TRH, to be released by a prohormone convertase, or as engineered amyloid precursor protein for subsequent liberation of Abeta(N3Q-42) after beta- and gamma-secretase cleavage during posttranslational processing. Inhibition of QC leads in both expression systems to significantly reduced pGlu-formation of differently processed Abeta-peptides. This reveals the importance of QC-activity during cellular maturation of pGlu-containing peptides. Thus, QC-inhibition should impact bioactivity, stability or even toxicity of pyroglutamyl peptides preventing glutamine and glutamate cyclization.     

The novel beta-secretase inhibitor KMI-429 reduces amyloid beta peptide production in amyloid precursor protein transgenic and wild-type mice

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid plaques and neurofibrillary tangles in the brain. The major component of the plaques, amyloid beta peptide (Abeta), is generated from amyloid precursor protein (APP) by beta- and gamma-secretase-mediated cleavage. Because beta-secretase/beta-site APP cleaving enzyme 1 (BACE1) knockout mice produce much less Abeta and grow normally, a beta-secretase inhibitor is thought to be one of the most attractive targets for the development of therapeutic interventions for AD without apparent side-effects. Here, we report the in vivo inhibitory effects of a novel beta-secretase inhibitor, KMI-429, a transition-state mimic, which effectively inhibits beta-secretase activity in cultured cells in a dose-dependent manner. We injected KMI-429 into the hippocampus of APP transgenic mice. KMI-429 significantly reduced Abeta production in vivo in the soluble fraction compared with vehicle, but the level of Abeta in the insoluble fraction was unaffected. In contrast, an intrahippocampal injection of KMI-429 in wild-type mice remarkably reduced Abeta production in both the soluble and insoluble fractions. Our results indicate that the beta-secretase inhibitor KMI-429 is a promising candidate for the treatment of AD.     

Cell Surface Expression of the Major Amyloid-β Peptide (Aβ)-degrading Enzyme, Neprilysin, Depends on Phosphorylation by Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase (MEK) and Dephosphorylation by Protein Phosphatase 1a

Neprilysin is one of the major amyloid-β peptide (Aβ)-degrading enzymes, the expression of which declines in the brain during aging. The decrease in neprilysin leads to a metabolic Aβ imbalance, which can induce the amyloidosis underlying Alzheimer disease. Pharmacological activation of neprilysin during aging therefore represents a potential strategy to prevent the development of Alzheimer disease. However, the regulatory mechanisms mediating neprilysin activity in the brain remain unclear. To address this issue, we screened for pharmacological regulators of neprilysin activity and found that the neurotrophic factors brain-derived neurotrophic factor, nerve growth factor, and neurotrophins 3 and 4 reduce cell surface neprilysin activity. This decrease was mediated by MEK/ERK signaling, which enhanced phosphorylation at serine 6 in the neprilysin intracellular domain (S6-NEP-ICD). Increased phosphorylation of S6-NEP-ICD in primary neurons reduced the levels of cell surface neprilysin and led to a subsequent increase in extracellular Aβ levels. Furthermore, a specific inhibitor of protein phosphatase-1a, tautomycetin, induced extensive phosphorylation of the S6-NEP-ICD, resulting in reduced cell surface neprilysin activity. In contrast, activation of protein phosphatase-1a increased cell surface neprilysin activity and lowered Aβ levels. Taken together, these results indicate that the phosphorylation status of S6-NEP-ICD influences the localization of neprilysin and affects extracellular Aβ levels. Therefore, maintaining S6-NEP-ICD in a dephosphorylated state, either by inhibition of protein kinases involved in its phosphorylation or by activation of phosphatases catalyzing its dephosphorylation, may represent a new approach to prevent reduction of cell surface neprilysin activity during aging and to maintain physiological levels of Aβ in the brain.     

Morphology of filamentous thalli of Analipus japonicus (Ralfsiales), temperature and irradiance effects on their formation and growth,and erect thallus formation in the field

Journal of Applied Phycology - Free-living filamentous thalli derived from an Analipus japonicus erect thallus were established in laboratory culture. The filamentous thalli were composed of...     

New embedding medium for sectioning undecalcified bone

Methylmethacrylate (MMA) is the most commonly used embedding medium for sectioning undecalcified bone; however, a number of problems exist with its use in a research laboratory. MMA requires a long infiltration time and temperature control, and it reacts with many polymers. We used Kleer Set resin? as an alternative embedding medium for sectioning undecalcified bone specimens. Fluorochrome labeled bone specimens were sectioned transversely using a ground section technique and longitudinally on a sledge macrotome. The slides were viewed using both transmitted light and epifluorescence microscopy. High quality sections were obtained using Kleer Set resin? for both sectioning techniques. We have shown that this new embedding medium is simpler, safer, quicker to use and does not interfere with visualization of fluorochromes.     

Mechanistic involvement of the calpain-calpastatin system in Alzheimer neuropathology

The mechanism by which amyloid-β peptide (Aβ) accumulation causes neurodegeneration in Alzheimer's disease (AD) remains unresolved. Given that Aβ perturbs calcium homeostasis in neurons, we investigated the possible involvement of calpain, a calcium-activated neutral protease. We first demonstrated close postsynaptic association of calpain activation with Aβ plaque formation in brains from both patients with AD and transgenic (Tg) mice overexpressing amyloid precursor protein (APP). Using a viral vector-based tracer, we then showed that axonal termini were dynamically misdirected to calpain activation-positive Aβ plaques. Consistently, cerebrospinal fluid from patients with AD contained a higher level of calpain-cleaved spectrin than that of controls. Genetic deficiency of calpastatin (CS), a calpain-specific inhibitor protein, augmented Aβ amyloidosis, tau phosphorylation, microgliosis, and somatodendritic dystrophy, and increased mortality in APP-Tg mice. In contrast, brain-specific CS overexpression had the opposite effect. These findings implicate that calpain activation plays a pivotal role in the Aβ-triggered pathological cascade, highlighting a target for pharmacological intervention in the treatment of AD.