Apolipoprotein E promotes astrocyte colocalization and degradation of deposited amyloid-beta peptides

We have previously shown that apolipoprotein E (Apoe) promotes the formation of amyloid in brain and that astrocyte-specific expression of APOE markedly affects the deposition of amyloid-beta peptides (Abeta) in a mouse model of Alzheimer disease. Given the capacity of astrocytes to degrade Abeta, we investigated the potential role of Apoe in this astrocyte-mediated degradation. In contrast to cultured adult wild-type mouse astrocytes, adult Apoe(-/-) astrocytes do not degrade Abeta present in Abeta plaque-bearing brain sections in vitro. Coincubation with antibodies to either Apoe or Abeta, or with RAP, an antagonist of the low-density lipoprotein receptor family, effectively blocks Abeta degradation by astrocytes. Phase-contrast and confocal microscopy show that Apoe(-/-) astrocytes do not respond to or internalize Abeta deposits to the same extent as do wild-type astrocytes. Thus, Apoe seems to be important in the degradation and clearance of deposited Abeta species by astrocytes, a process that may be impaired in Alzheimer disease.     

Chromatographic behavior of Bothrops erythromelas phospholipase and other venom constituents on Superdex 75

In a chromatographic method modification intended to preserve protease activity in Bothrops erythromelas venom, 2 mM CaCl2 was added to the gel filtration buffer [50mM Tris/HCl/150mM NaCl (pH 8.0)], in lieu of an equimolar portion of NaCl. This minor compositional change induced significant differences in the venom elution profile on Superdex 200. For this reason, the influence of buffer composition on chromatographic behavior was investigated using an analytical Superdex 75 HR 10/30 column. Phospholipase (PLA) was used as a marker because Naja atra PLA had previously been observed to interact hydrophobically with this resin. PLA elution volumes generally increased as buffer pH decreased. Addition of 20% acetonitrile to the Tris buffer with CaCl2, reduced hydrophobic interaction of the PLA so significantly that its elution was non-overlapping in the two buffers. Other venom constituents, including bradykinin-potentiating peptides and probable hemorrhagic metalloproteases, were similarly affected. Buffer calcium, bound by vicinal dextran hydroxyl groups, appears to retard elution of this acidic PLA.     

Cancer immunotherapy: moving beyond current vaccines

Great progress has been made in the field of tumor immunology in the past decade, but optimism about the clinical application of currently available cancer vaccine approaches is based more on surrogate endpoints than on clinical tumor regression. In our cancer vaccine trials of 440 patients, the objective response rate was low (2.6%), and comparable to the results obtained by others. We consider here results in cancer vaccine trials and highlight alternate strategies that mediate cancer regression in preclinical and clinical models.     

Ribosomal proteins Rps0 and Rps21 of Saccharomyces cerevisiae have overlapping functions in the maturation of the 3' end of 18S rRNA

The Rps0 proteins of Saccharomyces cerevisiae are components of the 40S ribosomal subunit required for maturation of the 3′ end of 18S rRNA. Drosophila and human homologs of the Rps0 proteins physically interact with Rps21 proteins, and decreased expression of both proteins in Drosophila impairs control of cellular proliferation in hematopoietic organs during larval development. Here, we characterize the yeast RPS21A/B genes and show that strains where both genes are disrupted are not viable. Relative to the wild type, cells with disrupted RPS21A or RPS21B genes exhibit a reduction in growth rate, a decrease in free 40S subunits, an increase in the amount of free 60S subunits, and a decrease in polysome size. Ribosomal RNA processing studies reveal RPS21 and RPS0 mutants have virtually identical processing defects. The pattern of processing defects observed in RPS0 and RPS21 mutants is not a general characteristic of strains with suboptimal levels of small subunit ribosomal proteins, since disruption of the RPS18A or RPS18B genes results in related but distinct processing defects. Together, these data link the Rps0 and Rps21 proteins together functionally in promoting maturation of the 3′ end of 18S rRNA and formation of active 40S ribosomal subunits.     

Comparative wood anatomy of epacrids (Styphelioideae,Ericaceae s.L.)

The wood anatomy of 16 of the 37 genera within the epacrids (Styphelioideae, Ericaceae s.l.) is investigated by light and scanning electron microscopy. Several features in the secondary xylem occur consistently at the tribal level: arrangement of vessel-ray pits, distribution of axial parenchyma, ray width, and the presence and location of crystals. The primitive nature of Prionoteae and Archerieae is supported by the presence of scalariform perforation plates with many bars and scalariform to opposite vessel pitting. The wood structure of Oligarrheneae is similar to that of Styphelieae, but the very narrow vessel elements, exclusively uniseriate rays and the lack of prismatic crystals in Oligarrheneae distinguish these two tribes. The secondary xylem of Monotoca tamariscina indicates that it does not fit in Styphelieae; a position within Oligarrheneae is possible. Like most Cosmelieae, all Richeeae are characterized by exclusively scalariform perforation plates with many bars, a very high vessel density and paratracheal parenchyma, although they clearly differ in ray width (exclusively uniseriate rays in Cosmelieae vs. uniseriate and wide multiseriate rays in Richeeae). Several wood anatomical features confirm the inclusion of epacrids in Ericaceae s.l. Furthermore, there are significant ecological implications. The small vessel diameter and high vessel frequency in many epacrids are indicative of a high conductive safety to avoid embolism caused by freeze-thaw cycles, while the replacement of scalariform by simple vessel perforation plates and an increase in vessel diameter would suggest an increased conductive efficiency, which is especially found in mesic temperate or tropical Styphelieae.     

Differential modulation of glucose,lactate, and pyruvate oxidation by insulin and dichloroacetate in the rat heart

Despite the fact that lactate and pyruvate are potential substrates for energy production in vivo, our understanding of the control and regulation of carbohydrate metabolism is based principally on studies where glucose is the only available carbohydrate. Therefore, the purpose of this study was to determine the contributions of lactate, pyruvate, and glucose to energy production in the isolated, perfused rat heart over a range of insulin concentrations and after activation of pyruvate dehydrogenase with dichloroacetate (DCA). Hearts were perfused with physiological concentrations of [1-13C]glucose, [U-13C]lactate, [2-13C]pyruvate, and unlabeled palmitate for 45 min. Hearts were freeze clamped, and 13C NMR glutamate isotopomer analysis was performed on tissue extracts. Glucose, lactate, and pyruvate all contributed significantly to myocardial energy production; however, in the absence of insulin, glucose contributed only 25-30% of total pyruvate oxidation. Even under conditions where carbohydrates represented >95% of substrate entering the tricarboxylic acid (TCA) cycle, we found that glucose contributed at most 50-60% of total carbohydrate oxidation. Despite being present at only 0.1 mM, pyruvate contributed between approximately 10% and 30% of total acetyl-CoA entry into the TCA cycle. We also found that insulin and DCA not only increased glucose oxidation but also exogenous pyruvate oxidation; however, lactate oxidation was not increased. The differential effects of insulin and DCA on pyruvate and lactate oxidation provide further evidence for compartmentation of cardiac carbohydrate metabolism. These results may have important implications for understanding the mechanisms underlying the beneficial effects of increasing cardiac carbohydrate metabolism.     

Expression and functional characterization of SCaMPER: a sphingolipid-modulated calcium channel of cardiomyocytes

Calciumchannels are important in a variety of cellular events including musclecontraction, signaling, proliferation, and apoptosis.Sphingolipids have been recognized as mediators of intracellularcalcium release through their actions on a calcium channel,sphingolipid calcium release-mediating protein of the endoplasmicreticulum (SCaMPER). The current study investigates the expression andfunction of SCaMPER in cardiomyocytes. Northern analyses and RT-PCRcloning and sequencing revealed SCaMPER expression in both human andrat cardiac tissue. Immunofluorescence and Western blot analysesdemonstrated that SCaMPER is abundant in cardiac tissue and islocalized to the sarcotubular junction. This was confirmed by thecolocalization of SCaMPER with dihydropyridine and ryanodine receptorsby confocal microscopy. Purified T tubules were shown to containSCaMPER and immunoelectron micrographs suggested that SCaMPER islocated to the junctional T tubules, but a junctional SR localizationcannot be ruled out. The sphingolipid ligand for SCaMPER,sphingosylphosphorylcholine (SPC), initiated calcium release from thecardiomyocyte SR. Importantly, antisense knockdown of SCaMPER mRNAproduced a substantial reduction of sphingolipid-induced calciumrelease, suggesting that SCaMPER is a potentially important calciumchannel of cardiomyocytes.

     

Hyperoxia induces retinal vascular endothelial cell apoptosis through formation of peroxynitrite

Hyperoxia exposure induces capillary endothelial cell apoptosis in the developing retina, leading to vaso-obliteration followed by proliferative retinopathy. Previous in vivo studies have shown that endothelial nitric oxide synthase (NOS3) and peroxynitrite are important mediators of the vaso-obliteration. Now we have investigated the relationship between hyperoxia, NOS3, peroxynitrite, and endothelial cell apoptosis by in vitro experiments using bovine retinal endothelial cells (BREC). We found that BREC exposed to 40% oxygen (hyperoxia) for 48 h underwent apoptosis associated with activation of caspase-3 and cleavage of the caspase substrate poly(ADP-ribose) polymerase. Hyperoxia-induced apoptosis was associated with increased formation of nitric oxide, peroxynitrite, and superoxide anion and was blocked by treatment with uric acid, nitro-L-arginine methyl ester, or superoxide dismutase. Analyses of the phosphatidylinositol 3-kinase/Akt kinase survival pathway in cells directly treated with peroxynitrite revealed inhibition of VEGF- and basic FGF-induced activation of Akt kinase. These results suggest that hyperoxia-induced formation of peroxynitrite induces BREC apoptosis by crippling key survival pathways and that blocking peroxynitrite formation prevents apoptosis. These data may have important clinical implications for infants at risk of retinopathy of prematurity. oxygen-induced retinopathy; vaso-obliteration; superoxide; nitric oxide     

Strategies for structural proteomics of prokaryotes: Quantifying the advantages of studying orthologous proteins and of using both NMR and X-ray crystallography approaches

Only about half of non-membrane-bound proteins encoded by either bacterial or archaeal genomes are soluble when expressed in Escherichia coli (Yee et al., Proc Natl Acad Sci USA 2002;99:1825-1830; Christendat et al., Prog Biophys Mol Biol 200;73:339-345). This property limits genome-scale functional and structural proteomics studies, which depend on having a recombinant, soluble version of each protein. An emerging strategy to increase the probability of deriving a soluble derivative of a protein is to study different sequence homologues of the same protein, including representatives from thermophilic organisms, based on the assumption that the stability of these proteins will facilitate structural analysis. To estimate the relative merits of this strategy, we compared the recombinant expression, solubility, and suitability for structural analysis by NMR and/or X-ray crystallography for 68 pairs of homologous proteins from E. coli and Thermotoga maritima. A sample suitable for structural studies was obtained for 62 of the 68 pairs of homologs under standardized growth and purification procedures. Fourteen (eight E. coli and six T. maritima proteins) samples generated NMR spectra of a quality suitable for structure determination and 30 (14 E. coli and 16 T. maritima proteins) samples formed crystals. Only three (one E. coli and two T. maritima proteins) samples both crystallized and had excellent NMR properties. The conclusions from this work are: (1) The inclusion of even a single ortholog of a target protein increases the number of samples for structural studies almost twofold; (2) there was no clear advantage to the use of thermophilic proteins to generate samples for structural studies; and (3) for the small proteins analyzed here, the use of both NMR and crystallography approaches almost doubled the number of samples for structural studies.