Role of ERAB/L-3-hydroxyacyl-coenzyme A dehydrogenase type II activity in Abeta-induced cytotoxicity

Endoplasmic reticulum-associated amyloid beta-peptide (Abeta)-binding protein (ERAB)/L-3-hydroxyacyl-CoA dehydrogenase type II (HADH II) is expressed at high levels in Alzheimer's disease (AD)-affected brain, binds Abeta, and contributes to Abeta-induced cytotoxicity. Purified recombinant ERAB/HADH II catalyzed the NADH-dependent reduction of S-acetoacetyl-CoA with a Km of approximately 68 microM and a Vmax of approximately 430 micromol/min/mg. The contribution of ERAB/HADH II enzymatic activity to Abeta-mediated cellular dysfunction was studied by site-directed mutagenesis in the catalytic domain (Y168G/K172G). Although COS cells cotransfected to overexpress wild-type ERAB/HADH II and variant beta-amyloid precursor protein (betaAPP(V717G)) showed DNA fragmentation, cotransfection with Y168G/K172G-altered ERAB and betaAPP(V717G) was without effect. We thus asked whether the enzyme might recognize alcohol substrates of which the aldehyde products could be cytotoxic; ERAB/HADH II catalyzed oxidation of a variety of simple alcohols (C2-C10) to their respective aldehydes in the presence of NAD+ and NAD-dependent oxidation of 17beta-estradiol. Addition of micromolar levels of synthetic Abeta(1-40) to purified ERAB/HADH II inhibited, in parallel, reduction of S-acetoacetyl-CoA (Ki approximately 1.6 microM), as well as oxidation of 17beta-estradiol (Ki approximately 3.2 microM) and (-)-2-octanol (Ki approximately 2.6 microM). Because micromolar levels of Abeta were required to inhibit ERAB/HADH II activity, whereas Abeta binding to ERAB/HADH II occurred at much lower concentrations (Km approximately 40-70 nM), the latter more closely simulating Abeta levels within cells, Abeta perturbation of ERAB/HADH II was likely to result from mechanisms other than the direct modulation of enzymatic activity. Cells cotransfected to overexpress ERAB/HADH II and betaAPP(V717G) generated malondialdehyde-protein and 4-hydroxynonenal-protein epitopes, which were detectable only at the lowest levels in cells overexpressing either ERAB/HADH II or betaAPP(V717G) alone. Generation of such toxic aldehydes was not observed in cells contransfected to overexpress Y168G/K172G-altered ERAB and betaAPP(V717G). We conclude that the generalized alcohol dehydrogenase activity of ERAB/HADH II is central to the cytotoxicity observed in an Abeta-rich environment.     

Cyclic stretch down-regulates calcium transporter gene expression in neonatal rat ventricular myocytes

Abnormal intracellular Ca2+ handling in hypertrophied and failing hearts is partly due to changes in Ca2+ transporter gene expression, but the mechanisms responsible for these alterations remain largely unknown. We previously showed that intrinsic mechanical load (i.e. spontaneous contractile activity) induced myocyte hypertrophy, and down-regulated SR Ca2+ ATPase (SERCA2) gene expression in cultured neonatal rat ventricular myocytes (NRVM). In the present study, we examined whether extrinsic mechanical load (i.e. cyclic stretch) also induced NRVM hypertrophy, and led to down-regulation of SERCA2 and other Ca2+ transporter genes which have been associated with cardiac hypertrophy and failure in vivo. NRVM were maintained in serum-free culture medium under control conditions, or subjected to cyclic mechanical deformation (1.0 Hz, 20% maximal strain, 48 h). Under these conditions, cyclic stretch induced NRVM hypertrophy, as evidenced by significant increases in total protein/DNA ratio, myosin heavy chain (MHC) content, and atrial natriuretic factor (ANF) secretion. Cyclic stretch also induced the MHC isoenzyme "switch" which is characteristic of hemodynamic overload of the rat heart in vivo. Cyclic stretch significantly down-regulated SERCA2 and ryanodine receptor (RyR) mRNA and protein levels, while simultaneously increasing ANF mRNA. In contrast, Na+-Ca2+ exchanger and phospholamban mRNA levels were unaffected. Load-dependent SERCA2 and RyR down-regulation was independent of Ca2+ influx via voltage-gated, L-type Ca2+ channels, as cyclic stretch down-regulated SERCA2 and RyR mRNA levels in both control and verapamil-treated NRVM. These results indicate that extrinsic mechanical load (in the absence of other exogenous stimuli) induces NRVM hypertrophy and causes down-regulation of Ca2+ transporter gene expression. This in vitro model system should prove useful to dissect the intracellular signaling pathways responsible for transducing this phenotype during cardiac hypertrophy and heart failure in vivo.     

Software development for the 8052AH-basic microcontroller

Application software for Intel's 8052AH-basic microcontroller can be developed in MCS basic-52, an enhanced version of basic, using the onchip basic interpreter. The paper explores the 8052AH-basic device and its language in comparison with the standard Microsoft basic and presents some software routines and other guidelines for programmers. Control-oriented applications, as opposed to conventional number-crunching applications, are emphasized. The paper also discusses how the resources of the Intellec development system can be used to facilitate the development of application software.     

The photovoltage of macaque cone photoreceptors: adaptation, noise, and kinetics

Whole-cell voltage and current recordings were obtained from red and green cone photoreceptors in isolated retina from macaque monkey. It was demonstrated previously that the cone photovoltage is generated from two sources, phototransduction current in the cone outer segment and photocurrent from neighboring rods. Rod signals are likely transmitted to cones across the gap junctions between rods and cones. In this study, the "pure" cone and rod components of the response were extracted with rod-adapting backgrounds or by subtracting the responses to flashes of different wavelength equated in their excitation of either rods or cones. For dim flashes, the pure cone component was similar in waveform to the cone outer segment current, and the rod component was similar to the photovoltage measured directly in rods. With bright flashes, the high frequencies of the rod signal were filtered out by the rod/cone network. The two components of the cone photovoltage adapted separately to background illumination. The amplitude of the rod component was halved by backgrounds eliciting approximately 100 photoisomerizations sec-1 per rod; the cone component was halved by backgrounds of 8700 photoisomerizations sec-1 per cone. Coupling between rods and cones was not modulated by either dim backgrounds or dopamine. Voltage noise in dark-adapted cones was dominated by elementary events other than photopigment isomerizations. The dark noise was equivalent in magnitude to a steady light eliciting approximately 3800 photoisomerizations sec-1 per cone, a value significantly higher than the psychophysical estimates of cone "dark light."     

Biochemical basis for a cholesterol-lowering activity of 2-[2"-(1",3"-dioxolane)]-2-methyl-4-(2'-oxo-1'-pyrrolidinyl)-6- nitro-2H-1-benzopyran (SKP-450), a novel antihypertensive agent

Administration (p.o.) of SKP-450, 2-[2"-(1",3"-dioxolane)]-2-methyl-4-(2'-oxo-1'-pyrrolidinyl)-6-nitro-2H- 1-benzopyran, a novel antihypertensive agent, to hypercholesterolemic Syrian hamsters led to a significant reduction in plasma lipids in a dose-dependent manner, i.e., a 10.8% to 29% reduction in low-density lipoprotein cholesterol at doses of 0.3 to 10 mg/kg of SKP-450. SKP-450 was found to specifically inhibit the hepatic microsomal lanosterol 14alpha-methyl demethylase (14alpha-DM) in a competitive manner (Ki:2.65 microM). Furthermore, a dose-dependent decrease in the 14alpha-DM activity by SKP-450 parallelled the cholesterol synthetic rate in vitro in both the rat hepatic S10 fractions (supernatants at 10,000 g; IC50:20 microM) and Chinese hamster ovary cells (IC50:23 microM). However, this phenomenon was not seen in AR45 cells, which are deficient in 14alpha-DM, suggesting that 14alpha-DM is the major target for the inhibitory action of SKP-450 in regard to cholesterol biosynthesis.     

Germ-line mutations in the neurofibromatosis 2 gene: correlations with disease severity and retinal abnormalities

Neurofibromatosis 2 (NF2) features bilateral vestibular schwannomas, other benign neural tumors, and cataracts. Patients in some families develop many tumors at an early age and have rapid clinical progression, whereas in other families, patients may not have symptoms until much later and vestibular schwannomas may be the only tumors. The NF2 gene has been cloned from chromosome 22q; most identified germ-line mutations result in a truncated protein and severe NF2. To look for additional mutations and clinical correlations, we used SSCP analysis to screen DNA from 32 unrelated patients. We identified 20 different mutations in 21 patients (66%): 10 nonsense mutations, 2 frameshifts, 7 splice-site mutations, and 1 large in-frame deletion. Clinical information on 47 patients from the 21 families included ages at onset and at diagnosis, numbers of meningiomas, spinal and skin tumors, and presence of cataracts and retinal abnormalities. We compared clinical findings in patients with nonsense or frameshift mutations to those with splice-site mutations. When each patient was considered as an independent random event, the two groups differed (P < or = .05) for nearly every variable. Patients with nonsense or frameshift mutations were younger at onset and at diagnosis and had a higher frequency and mean number of tumors, supporting the correlation between nonsense and frameshift mutations and severe NF2. When each family was considered as an independent random event, statistically significant differences between the two groups were observed only for mean ages at onset and at diagnosis. A larger data set is needed to resolve these discrepancies. We observed retinal hamartomas and/or epiretinal membranes in nine patients from five families with four different nonsense mutations. This finding, which may represent a new genotype-phenotype correlation, merits further study.     

XR-C1, a new CHO cell mutant which is defective in DNA-PKcs, is impaired in both V(D)J coding and signal joint formation

DNA-dependent protein kinase (DNA-PK) plays an important role in DNA double-strand break (DSB) repair and V(D)J recombination. We have isolated a new X-ray-sensitive CHO cell line, XR-C1, which is impaired in DSB repair and which was assigned to complementation group 7, the group that is defective in the XRCC7 / SCID ( Prkdc ) gene encoding the catalytic subunit of DNA-PK (DNA-PKcs). Consistent with this complementation analysis, XR-C1 cells lackeddetectable DNA-PKcs protein, did not display DNA-PK catalytic activity and were complemented by the introduction of a single human chromosome 8 (providing the Prkdc gene). The impact of the XR-C1 mutation on V(D)J recombination was quite different from that found in most rodent cells defective in DNA-PKcs, which are preferentially blocked in coding joint formation, whereas XR-C1 cells were defective in forming both coding and signal joints. These results suggest that DNA-PKcs is required for both coding and signal joint formation during V(D)J recombination and that the XR-C1 mutant cell line may prove to be a useful tool in understanding this pathway.