Pro-inflammatory effect of freshly solubilized beta-amyloid peptides in the brain

It has recently been shown that the level of soluble beta-amyloid (Abeta) peptides correlates well with the severity of synaptic loss and the density of neurofibrillary tangles observed in Alzheimer's disease (AD) brain. However, the biological activity of soluble forms of Abeta peptides in the brain remains to be determined. We have investigated ex vivo the effect of freshly solubilized Abeta1-40 peptides (fsAbeta) on prostaglandin E2 (PGE2) production in rat brain slices. PGE2 levels increased rapidly following treatment with fsAbeta, an effect that was prevented by SB202190, a selective inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), and by NS-398, which preferentially inhibits cyclooxygenase-2 (COX-2) compared to COX-1. In an attempt to determine the cellular systems of the brain responsible for prostaglandin production in response to fsAbeta, the effect of fsAbeta was tested on isolated brain microvessels, primary cultures of brain smooth muscle cells/pericytes and endothelial cells, and a human neuron-like cell line (IMR32). Our data show that fsAbeta ex vivo can stimulate prostaglandin accumulation in incubates of isolated rat brain microvessels. In addition, fsAbeta appears to cause a concentration-dependent enhancement of prostaglandin accumulation in primary cultures of brain microvessel-derived smooth muscle cells/pericytes but not of brain endothelial cells. Finally, fsAbeta also stimulated PGF2alpha accumulation in cultures of differentiated IMR32 cells, but to a lesser extent than in brain smooth muscle cell/pericyte cultures. Deposition of aggregated forms of Abeta in the brain has been thought to trigger an inflammatory response which accompanies the neuropathologic events of AD. Our data provide evidence that fsAbeta triggers a pro-inflammatory reaction in rat brain, and suggest that the cerebrovasculature may constitute an important source of pro-inflammatory eicosanoids.     

Rapamycin promotes β-amyloid production via ADAM-10 inhibition

Rapamycin is a well known immunosuppressant drug for rejection prevention in organ transplantation. Numerous clinical trials using rapamycin analogs, involving both children and adults with various disorders are currently ongoing worldwide. Most recently, rapamycin gained much attention for what appears to be life-span extending properties when administered to mice. The risk for Alzheimer disease (AD) is strongly and positively correlated with advancing age and is characterized by deposition of β-amyloid peptides (Aβ) as senile plaques in the brain. We report that rapamycin (2.5 μM), significantly increases Aβ generation in murine neuron-like cells (N2a) transfected with the human “Swedish” mutant amyloid precursor protein (APP). In concert with these observations, we found rapamycin significantly decreases the neuroprotective amino-terminal APP (amyloid precursor protein) cleavage product, soluble APP-α (sAPP-α) while increasing production of the β-carboxyl-terminal fragment of APP (β-CTF). These cleavage events are associated with decreased activation of a disintegrin and metallopeptidase domain-10 (ADAM-10), an important candidate α-secretase which opposes Aβ generation. To validate these findings in vivo, we intraperitoneal (i.p.) injected Tg2576 Aβ-overproducing transgenic mice with rapamycin (3 mg/kg/day) for 2 weeks. We found increased Aβ levels associated with decreased sAPP-α at an average rapamycin plasma concentration of 169.7 ± 23.5 ng/mL by high performance liquid chromatography (HPLC). These data suggest that although rapamycin may increase the lifespan in some mouse models, it may not decrease the risk for age-associated neurodegenerative disorders such as AD.     

Arachidonic acid stimulates DNA synthesis in brown preadipocytes through the activation of protein kinase C and MAPK

Arachidonic acid (AA) is a polyunsaturated fatty acid that stimulates the proliferation of many cellular types. We studied the mitogenic potential of AA in rat brown preadipocytes in culture and the signaling pathways involved. AA is a potent mitogen which induces 4-fold DNA synthesis in brown preadipocytes. The AA mitogenic effect increases by NE addition. AA also increases the mitogenic action of different growth factor combinations. Other unsaturated and saturated fatty acids do not stimulate DNA synthesis to the same extent as AA. We analyzed the role of PKC and MEK/MAPK signaling pathways. PKC inhibition by bisindolilmaleimide I (BIS) abolishes AA and phorbol ester stimulation of DNA synthesis and reduces the mitogenic activity of different growth factors in brown preadipocytes. Brown preadipocytes in culture express PKC α, δ, ε and ζ isoforms. Pretreatment with high doses of the phorbol ester PDBu, induces downregulation of PKCs ε and δ and reproduces the effect of BIS indicating that AA-dependent induction of DNA synthesis requires PKC activity. AA also activates MEK/MAPK pathway and the inhibition of MEK activity inhibits AA stimulation of DNA synthesis and brown adipocyte proliferation. Inhibition of PKC δ by rottlerin abolishes AA-dependent stimulation of DNA synthesis and MAPK activation, whereas PKC ε inhibition does not produce any effect. In conclusion, our results identify AA as a potent mitogen for brown adipocytes and demonstrate the involvement of the PDBu-sensitive PKC δ isoform and MEK/MAPK pathway in AA-induced proliferation of brown adipocytes. Increased proliferative activity might increase the thermogenic capacity of brown fat.     

Influence of level of zilpaterol chlorhydrate supplementation on growth performance and carcass characteristics of feedlot lambs

Forty-eight Pelibuey × Katahdin (38.8 ± 0.67 kg) crossbred male lambs were used in a 32-day feeding trial (four pens per treatment in a randomized complete block design), to evaluate the influence of zilpaterol (β₂-agonist) supplementation level on growth performance and carcass characteristics. Lambs were fed a dry-rolled corn-based finishing diet (3.04 Mcal/kg of ME) supplemented with 0, 0.15, 0.20, or 0.25 mg/kg of live weight d⁻¹ zilpaterol (as zilpaterol chlorhydrate, Zilmax^®, Intervet México, México City). DM intake averaged 1.099 ± 0.042 kg/d and was not affected (P = 0.40) by treatments. Compared with control lambs, zilpaterol supplementation increased gain efficiency (15.8%, P < 0.03), apparent energy retention per unit DMI (10.9%, P = 0.03), and tended to increased daily gain (16%, P < 0.07) and total gain (17.7%, P < 0.08). Zilpaterol supplementation did not affect (P = 0.20) carcass weight, longissimus muscle area (LM), or fat thickness, but increased (2.3%, P = 0.04) carcass dressing percentage and reduced (36%, P < 0.01) kidney-pelvic fat. Increasing level of zilpaterol supplementation increased total weight gain (linear component, P < 0.05), gain:feed (linear component, P < 0.01), and dressing percentage (linear component, P < 0.02), and decreased (linear component, P < 0.01) kidney-pelvic fat. We conclude that zilpaterol supplementation enhances growth performance and dressing percentage in lambs in a manner comparable to that of cattle (greater muscle accretion, reduced body fat). Responses to zilpaterol was optimal when supplemented at 0.20 mg of zilpaterol/kg of live weight d⁻¹.     

Numerous transposed sequences of mitochondrial cytochrome oxidase I-II in aphids of the genus Sitobion (Hemiptera: Aphididae)

Polymerase chain reaction (PCR) products corresponding to 803 bp of the cytochrome oxidase subunits I and II region of mitochondrial DNA (mtDNA COI-II) were deduced to consist of multiple haplotypes in three Sitobion species. We investigated the molecular basis of these observations. PCR products were cloned, and six clones from one individual per species were sequenced. In each individual, one sequence was found commonly, but also two or three divergent sequences were seen. The divergent sequences were shown to be nonmitochondrial by sequencing from purified mtDNA and Southern blotting experiments. All seven nonmitochondrial clones sequenced to completion were unique. Nonmitochondrial sequences have a high proportion of unique sites, and very few characters are shared between nonmitochondrial clones to the exclusion of mtDNA. From these data, we infer that fragments of mtDNA have been transposed separately (probably into aphid chromosomes), at a frequency only known to be equalled in humans. The transposition phenomenon appears to occur infrequently or not at all in closely related genera and other aphids investigated. Patterns of nucleotide substitution in mtDNA inferred over a parsimony tree are very different from those in transposed sequences. Compared with mtDNA, nonmitochondrial sequences have less codon position bias, more even exchanges between A, G, C and T, and a higher proportion of nonsynonymous replacements. Although these data are consistent with the transposed sequences being under less constraint than mtDNA, changes in the nonmitochondrial sequences are not random: there remains significant position bias, and probable excesses of synonymous replacements and of conservative inferred amino acid replacements. We conclude that a proportion of the inferred change in the nonmitochondrial sequences occurred before transposition. We believe that Sitobion aphids (and other species exhibiting mtDNA transposition) may be important for studying the molecular evolution of mtDNA and pseudogenes. However, our data highlight the need to establish the true evolutionary relationships between sequences in comparative investigations.      

Diversity and vertical distribution of epiphytic macrolichens in lowland rain forest and lowland cloud forest of French Guiana

Recent work on bryophyte diversity in lowland forests of northern South America has suggested the existence of a new type of cloud forest, the “tropical lowland cloud forest” (LCF). LCF occurs in river valleys with high air humidity and radiation fog, and is rich in epiphytes. We explored the lichenological characteristics of putative LCF in a lowland area (200–400 m a.s.l.) near Saül, central French Guiana, using macrolichens (including large crustose species) as indicator taxa. We analyzed macrolichen diversity on 16 trees in two 1 ha plots, in LCF and in lowland rain forest without fog (LRF). Sampling efficiency was ca. 80% in both forest types. Canopies of both LRF and LCF were richer in lichen species than understory trunks. Species richness of macrolichens was rather similar in the two forest types but species composition was significantly different. Cyanolichen richness in LCF was ca. 2.5 times higher than in LRF; in contrast, LRF had 4 times more species of green-algal Parmeliaceae. Our study suggests that cyanolichens except for Coccocarpiaceae serve as indicators of LCF. We explain the detected diversity patterns by differences in water availability due to fog precipitation and higher humidity. This is indicated by the higher relative air humidity in the lowland cloud forest, which was >6% higher than in the rain forest.     

HIV-1 p24-immunoglobulin fusion molecule: a new strategy for plant-based protein production

We describe the engineering of a human immunodeficiency virus-1 (HIV-1) p24-immunoglobulin A (IgA) antigen-antibody fusion molecule for therapeutic purposes and its enhancing effect on fused antigen expression in tobacco plants. Although many recombinant proteins have been expressed in transgenic plants as vaccine candidates, low levels of expression are a recurring problem. In this paper, using the HIV p24 core antigen as a model vaccine target, we describe a strategy for increasing the yield of a recombinant protein in plants. HIV p24 antigen was expressed as a genetic fusion with the alpha2 and alpha3 constant region sequences from human Ig alpha-chain and targeted to the endomembrane system. The expression of this fusion protein was detected at levels approximately 13-fold higher than HIV p24 expressed alone, and a difference in the behaviour of the two recombinant proteins during trafficking in the plant secretory pathway has been identified. Expressing the antigen within the context of alpha-chain Ig sequences resulted in the formation of homodimers and the antigen was correctly recognized by specific antibodies. Furthermore, the HIV p24 elicited T-cell and antibody responses in immunized mice. The use of Ig fusion partners is proposed as a generic platform technology for up-regulating the expression of antigens in plants, and may represent the first step in a strategy to design new vaccines with enhanced immunological properties.