JNK3 Perpetuates Metabolic Stress Induced by Aβ Peptides

Although Aβ peptides are causative agents in Alzheimer's disease (AD), the underlying mechanisms are still elusive. We report that Aβ42 induces a translational block by activating AMPK, thereby inhibiting the mTOR pathway. This translational block leads to widespread ER stress, which activates JNK3. JNK3 in turn phosphorylates APP at T668, thereby facilitating its endocytosis and subsequent processing. In support, pharmacologically blocking translation results in a significant increase in Aβ42 in a JNK3-dependent manner. Thus, JNK3 activation, which is?increased in human AD cases and a familial AD (FAD) mouse model, is integral to perpetuating Aβ42 production. Concomitantly, deletion of JNK3 from FAD mice results in a dramatic reduction in Aβ42 levels and overall plaque loads and increased neuronal number and improved cognition. This reveals AD as a metabolic disease that is under tight control by JNK3.     

Rescue of Mtp siRNA-induced hepatic steatosis by DGAT2 siRNA silencing

Microsomal triglyceride transfer protein (Mtp) inhibitors represent a novel therapeutic approach to lower circulating LDL cholesterol, although therapeutic development has been hindered by the observed increase in hepatic triglycerides and liver steatosis following treatment. Here, we used small interfering RNAs (siRNA) targeting Mtp to achieve target-specific silencing to study this phenomenon and to determine to what extent liver steatosis is induced by changes in Mtp expression. We observed that Mtp silencing led to a decrease in many genes involved in hepatic triglyceride synthesis. Given the role of diacylglycerol O-acyltransferase 2 (Dgat2) in regulating hepatic triglyceride synthesis, we then evaluated whether target-specific silencing of both Dgat2 and Mtp were sufficient to attenuate Mtp silencing-induced liver steatosis. We showed that the simultaneous inhibition of Dgat2 and Mtp led to a decrease in plasma cholesterol and a reduction in the accumulation of hepatic triglycerides caused by the inhibition of Mtp. Collectively, these findings provide a proof-of-principle for a triglyceride synthesis/Mtp inhibitor combination and represent a potentially novel approach for therapeutic development in which targeting multiple pathways can achieve the desired response.     

Molecular identification of Taenia tapeworms by Cox1 gene in Koh Kong, Cambodia

We collected fecal samples from 21 individuals infected with Taenia tapeworms in Koh Kong Province, Cambodia, and performed nucleotide sequencing of the cox1 gene and multiplex PCR on the eggs for DNA differential diagnosis of human Taenia tapeworms. Genomic DNA was extracted from the eggs of a minimum number of 10 isolated from fecal samples. Using oligonucleotide primers Ta7126F, Ts7313F, Tso7466F, and Rev7915, the multiplex PCR assay proved useful for differentially diagnosing Taenia solium, Taenia saginata, and Taenia asiatica based on 706, 629, and 474 bp bands, respectively. All of the Taenia specimens from Kho Kong, Cambodia, were identified as either T. saginata (n=19) or T. solium (n=2) by cox1 sequencing and multiplex PCR.     

A comparison of three techniques for quantitative carbohydrate analysis used in characterization of therapeutic antibodies

A comparison of three techniques for quantitative analysis of galactosylation present on immunoglobulins is described. ESIMS, MALDI-TOF MS, and anion-exchange chromatography with fluorescence detection were evaluated in terms of repeatability, limit of quantitation, selectivity, and linearity. A recombinant monoclonal IgG was enzymatically modified in vitro to produce essentially completely galactosylated and degalactosylated forms of the immunoglobulin. Samples of known galactosylation levels were prepared by mixing the modified forms with the native form of the immunoglobulin. Good repeatability and linearity were demonstrated for all three assays (RSDs<1.0%, correlation coefficients>0.99). Differences in selectivity, sensitivity, and other performance aspects of the three techniques are also discussed in this paper.     

Improved efficacy for ezetimibe and rosuvastatin by attenuating the induction of PCSK9

Reducing circulating LDL-cholesterol (LDL-c) reduces the risk of cardiovascular disease in people with hypercholesterolemia. Current approaches to reduce circulating LDL-c include statins, which inhibit cholesterol synthesis, and ezetimibe, which blocks cholesterol absorption. Both elevate serum PCSK9 protein levels in patients, which could attenuate their efficacy by reducing the amount of cholesterol cleared from circulation. To determine whether PCSK9 inhibition could enhance LDL-c lowering of both statins and ezetimibe, we utilized small interfering RNAs (siRNAs) to knock down Pcsk9, together with ezetimibe, rosuvastatin, and an ezetimibe/rosuvastatin combination in a mouse model with a human-like lipid profile. We found that ezetimibe, rosuvastatin, and ezetimibe/rosuvastatin combined lower serum cholesterol but induce the expression of Pcsk9 as well as the Srebp-2 hepatic cholesterol biosynthesis pathway. Pcsk9 knockdown in combination with either treatment led to greater reductions in serum non-HDL with a near-uniform reduction of all LDL-c subfractions. In addition to reducing serum cholesterol, the combined rosuvastatin/ezetimibe/Pcsk9 siRNA treatment exhibited a significant reduction in serum APOB protein and triglyceride levels. Taken together, these data provide evidence that PCSK9 inhibitors, in combination with current therapies, have the potential to achieve greater reductions in both serum cholesterol and triglycerides.     

Role of peptide sequence and neighboring residue glycosylation on the substrate specificity of the uridine 5'-diphosphate-alpha-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyl transferases T1 and T2: kinetic modeling of the porcine and canine submaxillary gland mucin tandem repeats

A large family of uridine 5'-diphosphate (UDP)-alpha-N-acetylgalactosamine (GalNAc):polypeptide N-acetylgalactosaminyl transferases (ppGalNAc Ts) initiates mucin-type O-glycan biosynthesis at serine and threonine. The peptide substrate specificities of individual family members are not well characterized or understood, leaving an inability to rationally predict or comprehend sites of O-glycosylation. Recently, a kinetic modeling approach demonstrated neighboring residue glycosylation as a major factor modulating the O-glycosylation of the porcine submaxillary gland mucin 81 residue tandem repeat by ppGalNAc T1 and T2 [Gerken et al. (2002) J. Biol. Chem. 277, 49850-49862]. To confirm the general applicability of this model and its parameters, the ppGalNAc T1 and T2 glycosylation kinetics of the 80+ residue tandem repeat from the canine submaxillary gland mucin was obtained and characterized. To reproduce the glycosylation patterns of both mucins (comprising 50+ serine/threonine residues), specific effects of neighboring peptide sequence, in addition to the previously described effects of neighboring residue glycosylation, were required of the model. Differences in specificity of the two transferases were defined by their sensitivities to neighboring proline and nonglycosylated hydroxyamino acid residues, from which a ppGalNAc T2 motif was identified. Importantly, the model can approximate the previously reported ppGalNAc T2 glycosylation kinetics of the IgA1 hinge domain peptide [Iwasaki, et al. (2003) J. Biol. Chem. 278, 5613-5621], further validating both the approach and the ppGalNAc T2 positional weighting parameters. The characterization of ppGalNAc transferase specificity by this approach may prove useful for the search for isoform-specific substrates, the creation of isoform-specific inhibitors, and the prediction of mucin-type O-glycosylation sites.     

Diet-dependent shifts in ruminal butyrate-producing bacteria

The influence of a host's diet on Butyrivibrio and Pseudobutyrivibrio populations was investigated by competitive PCR. Specific primers were designed and competitive PCRs developed for both groups. Results (from 4 cows with different diets) suggested that high-fiber intake essentially increases the Butyrivibrio amounts in the rumen, whereas high-energy food additives lead to its suppression. The Pseudobutyrivibrio concentration also changed during the experiment but without any significant relation to the host's diet.     

A MALDI-TOF MS method for the simultaneous and quantitative analysis of neutral and sialylated glycans of CHO-expressed glycoproteins

The development of a MALDI-TOF MS method for the quantitative analysis of the glycosylation of CHO-expressed biotherapeutic glycoproteins shall be presented. The method utilizes a well-established chemistry, reductive amination of glycans, to derivatize glycans with either a light analog ((12)C(7) anthranilic acid) or a heavy analog ((13)C(7) anthranilic acid) to allow for the direct comparison of the alternately-labeled glycans by MALDI-TOF MS. The method allows for the simultaneous analysis of neutral and sialylated glycans and displays a linear dynamic range over two orders of magnitude with sub-picomolar sensitivity. Additionally, because the glycans are derivatized with anthranilic acid, which is a very sensitive fluorophore, the glycans can be analyzed by chromatography with fluorescence detection. The need for this type of method is highlighted by the biotechnology/biopharmaceutical industry's continuous drive towards fully understanding process control. By providing this type of quantitative data, glycosylation changes of the expressed protein can be easily observed thereby helping to further advance the understanding of a major aspect of the biopharmaceutical process.     

Brain-derived neurotrophic factor (BDNF) induces polarized signaling of small GTPase (Rac1) protein at the onset of Schwann cell myelination through partitioning-defective 3 (Par3) protein

Brain-derived neurotrophic factor (BDNF) was shown to play a role in Schwann cell myelination by recruiting Par3 to the axon-glial interface, but the underlying mechanism has remained unclear. Here we report that Par3 regulates Rac1 activation by BDNF but not by NRG1-Type III in Schwann cells, although both ligands activate Rac1 in vivo. During development, active Rac1 signaling is localized to the axon-glial interface in Schwann cells by a Par3-dependent polarization mechanism. Knockdown of p75 and Par3 individually inhibits Rac1 activation, whereas constitutive activation of Rac1 disturbs the polarized activation of Rac1 in vivo. Polarized Rac1 activation is necessary for myelination as Par3 knockdown attenuates myelination in mouse sciatic nerves as well as in zebrafish. Specifically, Par3 knockdown in zebrafish disrupts proper alignment between the axon and Schwann cells without perturbing Schwann cell migration, suggesting that localized Rac1 activation at the axon-glial interface helps identify the initial wrapping sites. We therefore conclude that polarization of Rac1 activation is critical for myelination.