Bovine explant model of degeneration of the intervertebral disc

BACKGROUND: Many new treatments for degeneration of the intervertebral disc are being developed which can be delivered through a needle. These require testing in model systems before being used in human patients. Unfortunately, because of differences in anatomy, there are no ideal animal models of disc degeneration. Bovine explant model systems have many advantages but it is not possible to inject any significant volume into an intact disc. Therefore we have attempted to mimic disc degeneration in an explant bovine model via enzymatic digestion. METHODS: Bovine coccygeal discs were incubated with different concentrations of the proteolytic enzymes, trypsin and papain, and maintained in culture for up to 3 weeks. A radio-opaque solution was injected to visualise cavities generated. Degenerative features were monitored histologically and biochemically (water and glycosaminoglycan content, via dimethylmethylene blue). RESULTS AND CONCLUSION: The central region of both papain and trypsin treated discs was macro- and microscopically fragmented, with severe loss of metachromasia. The integrity of the surrounding tissue was mostly in tact with cells in the outer annulus appearing viable. Biochemical analysis demonstrated greatly reduced glycosaminoglycan content in these compared to untreated discs. We have shown that bovine coccygeal discs, treated with proteolytic enzymes can provide a useful in vitro model system for developing and testing potential new treatments of disc degeneration, such as injectable implants or biological therapies

Evaluation of the endoplasmic reticulum-stress response in eIF2B-mutated lymphocytes and lymphoblasts from CACH/VWM patients

<p>Abstract</p> <p>Background</p> <p>Eukaryotic translation initiation factor 2B (eIF2B), a guanine nucleotide exchange factor (GEF) and a key regulator of translation initiation under normal and stress conditions, causes an autosomal recessive leukodystrophy of a wide clinical spectrum. EBV-immortalised lymphocytes (EIL) from eIF2B-mutated patients exhibit a decrease in eIF2B GEF activity. eIF2B-mutated primary fibroblasts have a hyper-induction of activating transcription factor 4 (ATF4) which is involved in the protective unfolded protein response (UPR), also known as the ER-stress response. We tested the hypothesis that EIL from eIF2B-mutated patients also exhibit a heightened ER-stress response.</p> <p>Methods</p> <p>We used thapsigargin as an ER-stress agent and looked at polysomal profiles, rate of protein synthesis, translational activation of <it>ATF4</it>, and transcriptional induction of stress-specific mRNAs (<it>ATF4, CHOP, ASNS, GRP78</it>) in normal and eIF2B-mutated EIL. We also compared the level of stress-specific mRNAs between EIL and primary lymphocytes (PL).</p> <p>Results</p> <p>Despite the low eIF2B GEF activity in the 12 eIF2B-mutated EIL cell lines tested (range 40-70% of normal), these cell lines did not differ from normal EIL in their ATF4-mediated ER-stress response. The absence of hyper-induction of ATF4-mediated ER-stress response in eIF2B-mutated EIL in contrast to primary fibroblasts is not related to their transformation by EBV. Indeed, PL exhibited a higher induction of the stress-specific mRNAs in comparison to EIL, but no hyper-induction of the UPR was noticed in the eIF2B-mutated cell lines in comparison to controls.</p> <p>Conclusions</p> <p>Taken together with work of others, our results demonstrate the absence of a major difference in ER-stress response between controls and eIF2B-mutated cells. Therefore, components of the ER-stress response cannot be used as discriminantory markers in eIF2B-related disorders.</p

Rapid Dopaminergic Modulation of the Fish Hypothalamic Transcriptome and Proteome

Background - Dopamine (DA) is a major neurotransmitter playing an important role in the regulation of vertebrate reproduction. We developed a novel method for the comparison of transcriptomic and proteomic data obtained from in vivo experiments designed to study the neuroendocrine actions of DA. // Methods and Findings - Female goldfish were injected (i.p.) with DA agonists (D1-specific; SKF 38393, or D2-specific; LY 171555) and sacrificed after 5 h. Serum LH levels were reduced by 57% and 75% by SKF 38393 and LY 171555, respectively, indicating that the treatments produced physiologically relevant responses in vivo. Bioinformatic strategies and a ray-finned fish database were established for microarray and iTRAQ proteomic analysis of the hypothalamus, revealing a total of 3088 mRNAs and 42 proteins as being differentially regulated by the treatments. Twenty one proteins and mRNAs corresponding to these proteins appeared on both lists. Many of the mRNAs and proteins affected by the treatments were grouped into the Gene Ontology categorizations of protein complex, signal transduction, response to stimulus, and regulation of cellular processes. There was a 57% and 14% directional agreement between the differentially-regulated mRNAs and proteins for SKF 38393 and LY 171555, respectively. // Conclusions - The results demonstrate the applicability of advanced high-throughput genomic and proteomic analyses in an amendable well-studied teleost model species whose genome has yet to be sequenced. We demonstrate that DA rapidly regulates multiple hypothalamic pathways and processes that are also known to be involved in pathologies of the central nervous system

Thriving under Stress: Selective Translation of HIV-1 Structural Protein mRNA during Vpr-Mediated Impairment of eIF4E Translation Activity

Translation is a regulated process and is pivotal to proper cell growth and homeostasis. All retroviruses rely on the host translational machinery for viral protein synthesis and thus may be susceptible to its perturbation in response to stress, co-infection, and/or cell cycle arrest. HIV-1 infection arrests the cell cycle in the G2/M phase, potentially disrupting the regulation of host cell translation. In this study, we present evidence that HIV-1 infection downregulates translation in lymphocytes, attributable to the cell cycle arrest induced by the HIV-1 accessory protein Vpr. The molecular basis of the translation suppression is reduced accumulation of the active form of the translation initiation factor 4E (eIF4E). However, synthesis of viral structural proteins is sustained despite the general suppression of protein production. HIV-1 mRNA translation is sustained due to the distinct composition of the HIV-1 ribonucleoprotein complexes. RNA-coimmunoprecipitation assays determined that the HIV-1 unspliced and singly spliced transcripts are predominantly associated with nuclear cap binding protein 80 (CBP80) in contrast to completely-spliced viral and cellular mRNAs that are associated with eIF4E. The active translation of the nuclear cap binding complex (CBC)-bound viral mRNAs is demonstrated by ribosomal RNA profile analyses. Thus, our findings have uncovered that the maintenance of CBC association is a novel mechanism used by HIV-1 to bypass downregulation of eIF4E activity and sustain viral protein synthesis. We speculate that a subset of CBP80-bound cellular mRNAs contribute to recovery from significant cellular stress, including human retrovirus infection

Orally Administrated Cinnamon Extract Reduces β-Amyloid Oligomerization and Corrects Cognitive Impairment in Alzheimer's Disease Animal Models

An increasing body of evidence indicates that accumulation of soluble oligomeric assemblies of β-amyloid polypeptide (Aβ) play a key role in Alzheimer's disease (AD) pathology. Specifically, 56 kDa oligomeric species were shown to be correlated with impaired cognitive function in AD model mice. Several reports have documented the inhibition of Aβ plaque formation by compounds from natural sources. Yet, evidence for the ability of common edible elements to modulate Aβ oligomerization remains an unmet challenge. Here we identify a natural substance, based on cinnamon extract (CEppt), which markedly inhibits the formation of toxic Aβ oligomers and prevents the toxicity of Aβ on neuronal PC12 cells. When administered to an AD fly model, CEppt rectified their reduced longevity, fully recovered their locomotion defects and totally abolished tetrameric species of Aβ in their brain. Furthermore, oral administration of CEppt to an aggressive AD transgenic mice model led to marked decrease in 56 kDa Aβ oligomers, reduction of plaques and improvement in cognitive behavior. Our results present a novel prophylactic approach for inhibition of toxic oligomeric Aβ species formation in AD through the utilization of a compound that is currently in use in human diet

Bone Marrow-Derived Progenitor Cells Augment Venous Remodeling in a Mouse Dorsal Skinfold Chamber Model

The delivery of bone marrow-derived cells (BMDCs) has been widely used to stimulate angiogenesis and arteriogenesis. We identified a progenitor-enriched subpopulation of BMDCs that is able to augment venular remodeling, a generally unexplored area in microvascular research. Two populations of BMDCs, whole bone marrow (WBM) and Lin−/Sca-1+ progenitor cells, were encapsulated in sodium alginate and delivered to a mouse dorsal skinfold chamber model. Upon observation that encapsulated Sca-1+ progenitor cells enhance venular remodeling, the cells and tissue were analyzed on structural and molecular levels. Venule walls were thickened and contained more nuclei after Sca-1+ progenitor cell delivery. In addition, progenitors expressed mRNA transcript levels of chemokine (C-X-C motif) ligand 2 (CXCL2) and interferon gamma (IFNγ) that are over 5-fold higher compared to WBM. Tissues that received progenitors expressed significantly higher protein levels of vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1), and platelet derived growth factor-BB (PDGF-BB) compared to tissues that received an alginate control construct. Nine days following cell delivery, tissue from progenitor recipients contained 39% more CD45+ leukocytes, suggesting that these cells may enhance venular remodeling through the modulation of the local immune environment. Results show that different BMDC populations elicit different microvascular responses. In this model, Sca-1+ progenitor cell-derived CXCL2 and IFNγ may mediate venule enlargement via modulation of the local inflammatory environment

Complete Phenotypic Recovery of an Alzheimer's Disease Model by a Quinone-Tryptophan Hybrid Aggregation Inhibitor

The rational design of amyloid oligomer inhibitors is yet an unmet drug development need. Previous studies have identified the role of tryptophan in amyloid recognition, association and inhibition. Furthermore, tryptophan was ranked as the residue with highest amyloidogenic propensity. Other studies have demonstrated that quinones, specifically anthraquinones, can serve as aggregation inhibitors probably due to the dipole interaction of the quinonic ring with aromatic recognition sites within the amyloidogenic proteins. Here, using in vitro, in vivo and in silico tools we describe the synthesis and functional characterization of a rationally designed inhibitor of the Alzheimer's disease-associated β-amyloid. This compound, 1,4-naphthoquinon-2-yl-L-tryptophan (NQTrp), combines the recognition capacities of both quinone and tryptophan moieties and completely inhibited Aβ oligomerization and fibrillization, as well as the cytotoxic effect of Aβ oligomers towards cultured neuronal cell line. Furthermore, when fed to transgenic Alzheimer's disease Drosophila model it prolonged their life span and completely abolished their defective locomotion. Analysis of the brains of these flies showed a significant reduction in oligomeric species of Aβ while immuno-staining of the 3rd instar larval brains showed a significant reduction in Aβ accumulation. Computational studies, as well as NMR and CD spectroscopy provide mechanistic insight into the activity of the compound which is most likely mediated by clamping of the aromatic recognition interface in the central segment of Aβ. Our results demonstrate that interfering with the aromatic core of amyloidogenic peptides is a promising approach for inhibiting various pathogenic species associated with amyloidogenic diseases. The compound NQTrp can serve as a lead for developing a new class of disease modifying drugs for Alzheimer's disease