Amyotrophic lateral sclerosis is associated with hypolipidemia at the presymptomatic stage in mice

Objective

To demonstrate that hypolipidemia is a typical feature of the mouse model of amyotrophic lateral sclerosis (ALS) and to assess the association between hypolipidemia and disease stage, dietary intake, and sex.

Methods

We compared daily dietary intake, body weight, and serumlipid and glucose levels in ALS mice and wild-type controls at different stages of the disease.

Findings

Total cholesterol low-density lipoprotein (LDL) and LDL/high-density lipoprotein (HDL) ratio were significantly lower in ALS mice compared with controls. Subgroup analysis revealed that the incidence of hypolipidemia was significantly greater in male, but not female, ALS mice compared with control mice and that hypolipidemia was present at the presymptomatic stage of the disease. This hypolipidemia can be found without a decrease in the serum levels of other energy sources, such as glucose, in the presymptomatic stage.

Conclusions

Hypolipidemia is present at the presymptomatic stage of the ALS mouse model in the absence of malnutrition, significant neuromuscular degeneration or regeneration, and respiratory difficulty. Our findings suggest that hypolipidemia might be associated with the pathomechanism of ALS and/or lipid-specific metabolism rather than simply an epiphenomenon of neuromuscular degeneration or energy imbalance.     

p62, Ref(2)P and ubiquitinated proteins are conserved markers of neuronal aging, aggregate formation and progressive autophagic defects

Suppression of macroautophagy, due to mutations or through processes linked to aging, results in the accumulation of cytoplasmic substrates that are normally eliminated by the pathway. This is a significant problem in long-lived cells like neurons, where pathway defects can result in the accumulation of aggregates containing ubiquitinated proteins. The p62/Ref(2)P family of proteins is involved in the autophagic clearance of cytoplasmic protein bodies or sequestosomes. These unique structures are closely associated with protein inclusions containing ubiquitin as well as key components of the autophagy pathway. In this study we show that detergent fractionation followed by western blot analysis of insoluble ubiquitinated proteins (IUP), mammalian p62 and its Drosophila homologue, Ref(2)P can be used to quantitatively assess the activity level of aggregate clearance (aggrephagy) in complex tissues. Using this technique we show that genetic or age-dependent changes that modify the long-term enhancement or suppression of aggrephagy can be identified. Moreover, using the Drosophila model system this method can be used to establish autophagy-dependent protein clearance profiles that are occurring under a wide range of physiological conditions including developmental, fasting and altered metabolic pathways. This technique can also be used to examine proteopathies that are associated with human disorders such as frontotemporal dementia, Huntington and Alzheimer disease. Our findings indicate that measuring IUP profiles together with an assessment of p62/Ref(2)P proteins can be used as a screening or diagnostic tool to characterize genetic and age-dependent factors that alter the long-term function of autophagy and the clearance of protein aggregates occurring within complex tissues and cells.     

Applications of cell-based phage display panning to proteomic analysis

In the post-genomic era, proteomics together with genomic tools have led to powerful new strategies in basic and clinical research. These combined “omics” technologies are being integrated into the drug target discovery process. Unlike the genome, the proteome is a highly dynamic entity that requires techniques capable of analyzing on selected populations of proteins in specific biological conditions that reflect the proteins’ functional characteristics. Antibodies have become one of the most important reagents for the analysis of selected populations of proteins, and the application of phage-display antibody libraries to high-throughput antibody generation against large numbers of various antigens provides a tool for proteome-wide protein expression analysis. In this review, we will discuss the utility of phage-display antibodies in proteomics applications, specifically for the discovery of novel disease markers and therapeutic targets.     

Immunochromatographic membrane strip assay system for a single-class plasma lipoprotein cholesterol, exemplified by high-density lipoprotein cholesterol measurement

In assessing risk factors of coronary heart disease, a membrane immunochromatographic system that minimizes requirements of instrument and reagent handling was investigated by utilizing high-density lipoprotein (HDL) cholesterol (HDL-C) as model analyte. The system is composed of four functional membrane strip pads connected in sequence as follows (from the bottom): immunoseparation based on the biotin-streptavidin reaction; catalytic conversion of cholesterol to hydrogen peroxide; production of a colorimetric signal; and induction of a continuous wicking of medium. For immunochromatography, a monoclonal antibody, specific to apolipoprotein B100 that is present on the surfaces of low-density lipoproteins (LDL) and very low-density lipoproteins (VLDL), with a high binding constant (5 x 10(10) L/mol), was raised and chemically conjugated to streptavidin. The conjugate was first reacted with lipoprotein particles, and this mixture was absorbed by the capillary action into the biotin pad of the system. After being transferred by medium, immunocapture of LDL and VLDL particles onto the biotin pad took place, and in situ generation of a colorimetric signal in proportion to HDL-C occurred consecutively. The capture was selective as well as effective (minimum 88% of LDL and VLDL in clinical concentration ranges), and the detection limit of the HDL-C was far lower than 20 mg per 100 mL. The same concept may also be applicable to LDL cholesterol measurement provided suitable antibodies specific to HDL and VLDL are available.     

Involvement of HDAC1 in E-cadherin expression in prostate cancer cells; its implication for cell motility and invasion

In this study, we investigate the molecular mechanism by which histone deacetylase (HDAC) inhibitors exert anti-invasiveness effect against prostate cancer cells. We first evaluate the growth inhibition effect of HDAC inhibitors in prostate cancer cells, which is accompanied by induction of p21^WAF1 expression and accumulation of acetylated histones. And we found that the migration and invasion of prostate cancer cells is strongly inhibited by treatment with HDAC inhibitors. In parallel, E-cadherin level is highly up-regulated in HDAC inhibitor-treated prostate cancer cells. And siRNA knockdown of E-cadherin significantly diminishes the anti-invasion effect of HDAC inhibitors, indicating that E-cadherin overexpression is one of possible mechanism for anti-invasion effect of HDAC inhibitors. Furthermore, specific downregulation of HDAC1, but not HDAC2, causes E-cadherin expression and subsequent inhibition of cell motility and invasion. Collectively, our data demonstrate that HDAC1 is a major repressive enzyme for E-cadherin expression as well as HDAC inhibitor-mediated anti-invasiveness.     

1,3-Propandiol production by engineered Hansenula polymorpha expressing dha genes from Klebsiella pneumoniae

Currently, 1,3-propanediol (1,3-PD) is an important chemical widely used in polymer production, but its availability is being restricted owing to its expensive chemical synthesis. A methylotrophic yeast Hansenula polymorpha was engineered by expression of dhaB1, dhaB2, dhaB3, dhaB _RA1 and dhaB _RA2 encoding glycerol dehydratase complex and dhaT encoding 1,3-PD oxidoreductase from Klebsiella pneumoniae under direction of promoter of glyceraldehyde-3 phosphate dehydrogenase (GAPDH). The engineered recombinant yeast strain can produce 1,3-PD from glucose (2.4 g L⁻¹) as well as glycerol (0.8 g L⁻¹), which might lead to a safe and cost-effective method for industrial production of 1,3-PD from various biomass resources.     

Filamin A mediates interactions between cytoskeletal proteins that control cell adhesion

Cell adhesion, spreading and migration on extracellular matrices are regulated by complex processes that involve the cytoskeleton and a large array of adhesion receptors, including the β1 integrin. Filamin A is a large, multi-domain, homodimeric actin binding protein that contributes to the mechanical stability of cells and interacts with several proteins that regulate cell adhesion including β1 integrin and several protein kinases. Here we review current data on the structure, mechanical properties and intracellular signaling functions of filamin that regulate cell adhesion. We also consider new data showing that interactions of filamin A with intermediate filaments and protein kinase C enable tight regulation of β1 integrin function and consequently early events in cell adhesion and migration on extracellular matrix proteins.     

Induction and visualization of mucosal memory CD8 T cells following systemic virus infection.

Whether CD8 T cell memory exists outside secondary lymphoid organs is unclear. Using an adoptive transfer system that enables tracking of OVA-specific CD8 T cells, we explored the antigenic requirements for inducing CD8 T cell memory and identified intestinal mucosa memory cells. Although systemic immunization with soluble OVA induced clonal expansion, memory CD8 cells were not produced. In contrast, infection with virus-encoding OVA induced memory CD8 cells in the periphery and the lamina propria and intraepithelial compartments of the intestinal mucosa. Mucosal memory cells expressed a distinct array of adhesion molecules as compared with secondary lymphoid memory cells, suggesting that there may be separate mucosal and systemic memory pools. Mucosal CD8 memory cells rapidly produced IFN-gamma after Ag stimulation. Reactivation of memory cells by Ag feeding resulted in increased cell size and up-regulation of CD28 and CD11c. CD8 mucosal memory cells exhibited ex vivo lytic activity that was up-regulated dramatically following Ag reencounter in vivo. Interestingly, reactivation of memory cells did not require CD28-mediated costimulation. The ability of the intestinal mucosa to maintain CD8 memory cells provides a potential mechanism for effective mucosal vaccination.