Independent localization of MAP2, CaMKIIα and β-actin RNAs in low copy numbers

Messenger RNA localization involves the assembly of ribonucleoprotein particles (RNPs) and their subsequent transport along the cytoskeleton to their final destination. Here, we provide new evidence that microtubule-associated protein 2 (MAP2), calcium/calmodulin-dependent protein kinase II (CaMKIIα) and β-actin RNAs localize to dendrites in distinct RNPs, which contain--unexpectedly--very few RNA molecules. The number of MAP2 molecules per particle is affected by synaptic activity and Staufen 2, indicating that RNP composition is tightly controlled. Our data suggest that the independent localization of individual RNAs in low copy numbers could contribute to tighter temporal and spatial control of expression in neurons and synapse-specific plasticity.     

Phenolics removal from transgenic Lemna minor extracts expressing mAb and impact on mAb production cost

Transgenic Lemna minor has been used successfully to produce several biotherapeutic proteins. For plant-produced mAbs specifically, the cost of protein A capture step is critical as the economic benefits of plant production systems could be erased if the downstream processing ends up being expensive. To avoid potential modification of mAb or fouling of expensive protein A resins, a rapid and efficient removal of phenolics from plant extracts is desirable. We identified major phenolics in Lemna extracts and evaluated their removal by adsorption to PVPP, XAD-4, IRA-402, and Q-Sepharose. Forms of apigenin, ferulic acid, and vitexin comprised ～ 75% of the total phenolics. Screening of the resins with pure ferulic acid and vitexin indicated that PVPP would not be efficient for phenolics removal. Analysis of the breakthrough fractions of phenolics adsorption to XAD-4, IRA-402, and Q-Sepharose showed differences in adsorption with pH and in the type of phenolics adsorbed. Superior dynamic binding capacities (DBC) were observed at pH 4.5 than at 7.5. To evaluate the cost impact of a phenolics removal step before protein A chromatography, a mAb purification process was simulated using SuperPro Designer 7.0. The economic analysis indicated that addition of a phenolics adsorption step would increase mAb production cost only 20% by using IRA-402 compared to 35% for XAD-4 resin. The cost of the adsorption step is offset by increasing the lifespan of protein A resin and a reduction of overall mAb production cost could be achieved by using a phenolics removal step.     

Molecular Evolution of Prolactin in Primates

Pituitary prolactin, like growth hormone (GH) and several other protein hormones, shows an episodic pattern of molecular evolution in which sustained bursts of rapid change contrast with long periods of slow evolution. A period of rapid change occurred in the evolution of prolactin in primates, leading to marked sequence differences between human prolactin and that of nonprimate mammals. We have defined this burst more precisely by sequencing the coding regions of prolactin genes for a prosimian, the slow loris (Nycticebus pygmaeus), and a New World monkey, the marmoset (Callithrix jacchus). Slow loris prolactin is very similar in sequence to pig prolactin, so the episode of rapid change occurred during primate evolution, after the separation of lines leading to prosimians and higher primates. Marmoset prolactin is similar in sequence to human prolactin, so the accelerated evolution occurred before divergence of New World monkeys and Old World monkeys/apes. The burst of change was confined largely to coding sequence (nonsynonymous sites) for mature prolactin and is not marked in other components of the gene sequence. This and the observations that (1) there was no apparent loss of function during the episode of rapid evolution, (2) the rate of evolution slowed toward the basal rate after this burst, and (3) the distribution of substitutions in the prolactin molecule is very uneven support the idea that this episode of rapid change was due to positive adaptive selection. In the slow loris and marmoset there is no evidence for duplication of the prolactin gene, and evidence from another New World monkey (Cebus albifrons) and from the chimpanzee and human genome sequences, suggests that this is the general position in primates, contrasting with the situation for GH genes. The chimpanzee prolactin sequence differs from that of human at two residues and comparison of human and chimpanzee prolactin gene sequences suggests that noncoding regions associated with regulating expression may be evolving differently from other noncoding regions.     

APP substitutions V715F and L720P alter PS1 conformation and differentially affect Abeta and AICD generation

The 37-43 amino acid Abeta peptide is the principal component of beta-amyloid deposits in Alzheimer's disease (AD) brain, and is derived by serial proteolysis of the amyloid precursor protein (APP) by beta- and gamma-secretase. gamma-Secretase also cleaves APP at Val50 in the Abeta numbering (epsilon cleavage), resulting in the release of a fragment called APP intracellular domain (AICD). The aim of this study was to determine whether amino acid substitutions in the APP transmembrane domain differentially affect Abeta and AICD generation. We found that the APPV715F substitution, which has been previously shown to dramatically decrease Abeta40 and Abeta42 while increasing Abeta38 levels, does not affect in vitro generation of AICD. Furthermore, we found that the APPL720P substitution, which has been previously shown to prevent in vitro generation of AICD, completely prevents Abeta generation. Using a fluorescence resonance energy transfer (FRET) method, we next found that both the APPV715F and APPL720P substitutions significantly increase the distance between the N- and C-terminus of presenilin 1 (PS1), which has been proposed to contain the catalytic site of gamma-secretase. In conclusion, both APPV715F and APPL720P change PS1 conformation with differential effects on Abeta and AICD production.     

Molecular cloning of leukocyte cell-derived chemotaxin 2 in rainbow trout

In humans, leukocyte cell-derived chemotaxin 2 (LECT2) is a 16kDa chemotactic protein that consists of 133 amino acids and three intramolecular disulphide bonds. Although it was originally demonstrated to have a chemotactic function in vitro, recent data sustain a further multifunctional role of LECT2 that extends from cell growth, differentiation, damage/repair process and carcinogenesis to autoimmune diseases. The in vivo function of LECT2 protein still remains obscure. In order to study the phylogeny of LECT2, a full-length cDNA clone of LECT2 gene, 720 bp in size, was isolated in rainbow trout (Oncorhynchus mykiss). Its deduced amino acid sequence of 156 residues, presents 40, 45 and 61% overall identity to human, mouse and carp LECT2 proteins, respectively. In contrast to mammalian LECT2 protein, trout LECT2 protein reveals two potential N-glycosylation sites. Phylogenetic analysis shows that trout LECT2 is clustered with the known homologous proteins. Trout LECT2 mRNA is predominately expressed in liver and spleen, showing lower expression in kidney, intestine, heart and brain.     

Migration and differentiation of neural precursors derived from human embryonic stem cells in the rat brain

Human embryonic stem (hES) cells provide a potentially unlimited cell source for regenerative medicine. Recently, differentiation strategies were developed to direct hES cells towards neural fates in vitro. However, the interaction of hES cell progeny with the adult brain environment remains unexplored. Here we report that hES cell-derived neural precursors differentiate into neurons, astrocytes and oligodendrocytes in the normal and lesioned brain of young adult rats and migrate extensively along white matter tracts. The differentiation and migration behavior of hES cell progeny was region specific. The hES cell-derived neural precursors integrated into the endogenous precursor pool in the subventricular zone, a site of persistent neurogenesis. Like adult neural stem cells, hES cell-derived precursors traveled along the rostral migratory stream to the olfactory bulb, where they contributed to neurogenesis. We found no evidence of cell fusion, suggesting that hES cell progeny are capable of responding appropriately to host cues in the subventricular zone.     

A role for retinal brain-derived neurotrophic factor in ocular dominance plasticity

Visual deprivation is a classical tool to study the plasticity of visual cortical connections. After eyelid closure in young animals (monocular deprivation, MD), visual cortical neurons become dominated by the open eye, a phenomenon known as ocular dominance (OD) plasticity . It is commonly held that the molecular mediators of OD plasticity are cortically derived and that the retina is immune to the effects of MD . Recently, it has been reported that visual deprivation induces neurochemical, structural, and functional changes in the retina , but whether these retinal changes contribute to the effects of MD in the cortex is unknown. Here, we provide evidence that brain-derived neurotrophic factor (BDNF) produced in the retina influences OD plasticity. We found a reduction of BDNF expression in the deprived retina of young rats. We compensated this BDNF imbalance between the two eyes by either injecting exogenous BDNF in the deprived eye or reducing endogenous BDNF expression in the nondeprived eye. Both treatments were effective in counteracting the OD shift induced by MD. Retinal BDNF could also influence OD distribution in normal animals. These results show for the first time that OD plasticity is modulated by BDNF produced in the retina.     

Abeta42 is essential for parenchymal and vascular amyloid deposition in mice

Considerable circumstantial evidence suggests that Abeta42 is the initiating molecule in Alzheimer's disease (AD) pathogenesis. However, the absolute requirement for Abeta42 for amyloid deposition has never been demonstrated in vivo. We have addressed this by developing transgenic models that express Abeta1-40 or Abeta1-42 in the absence of human amyloid beta protein precursor (APP) overexpression. Mice expressing high levels of Abeta1-40 do not develop overt amyloid pathology. In contrast, mice expressing lower levels of Abeta1-42 accumulate insoluble Abeta1-42 and develop compact amyloid plaques, congophilic amyloid angiopathy (CAA), and diffuse Abeta deposits. When mice expressing Abeta1-42 are crossed with mutant APP (Tg2576) mice, there is also a massive increase in amyloid deposition. These data establish that Abeta1-42 is essential for amyloid deposition in the parenchyma and also in vessels.