The Protein Precursors of Peptides That Affect the Mechanics of Connective Tissue and/or Muscle in the Echinoderm Apostichopus japonicus

PMCID: PMC3432112This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Designing stem cell niches for differentiation and self-renewal

Mesenchymal stem cells, characterized by their ability to differentiate into skeletal tissues and self-renew, hold great promise for both regenerative medicine and novel therapeutic discovery. However, their regenerative capacity is retained only when in contact with their specialized microenvironment, termed the stem cell niche. Niches provide structural and functional cues that are both biochemical and biophysical, stem cells integrate this complex array of signals with intrinsic regulatory networks to meet physiological demands. Although, some of these regulatory mechanisms remain poorly understood or difficult to harness with traditional culture systems. Biomaterial strategies are being developed that aim to recapitulate stem cell niches, by engineering microenvironments with physiological-like niche properties that aim to elucidate stem cell-regulatory mechanisms, and to harness their regenerative capacity in vitro. In the future, engineered niches will prove important tools for both regenerative medicine and therapeutic discoveries

In vitro and in vivo expression of foreign genes by transmissible gastroenteritis coronavirus-derived minigenomes

A helper-dependent expression system based on transmissible gastroenteritis coronavirus (TGEV) has been developed using a minigenome of 3·9 kb (M39). Expression of the reporter gene {beta}-glucuronidase (GUS) (2–8 µg per 106 cells) and the porcine respiratory and reproductive syndrome virus (PRRSV) ORF5 (1–2 µg per 106 cells) has been shown using a TGEV-derived minigenome. GUS expression levels increased about eightfold with the m.o.i. and were maintained for more than eight passages in cell culture. Nevertheless, instability of the GUS and ORF5 subgenomic mRNAs was observed from passages five and four, respectively. About a quarter of the cells in culture expressing the helper virus also produced the reporter gene as determined by studying GUS mRNA production by in situ hybridization or immunodetection to visualize the protein synthesized. Expression of GUS was detected in the lungs, but not in the gut, of swine immunized with the virus vector. Around a quarter of lung cells showing replication of the helper virus were also positive for the reporter gene. Interestingly, strong humoral immune responses to both GUS and PRRSV ORF5 were induced in swine with this virus vector. The large cloning capacity and the tissue specificity of the TGEV-derived minigenomes suggest that these virus vectors are very promising for vaccine development

Theoretical basis of the community effect in development

Peer reviewedPublisher PD

LDL receptor-related protein-1 regulates NFκB and microRNA-155 in macrophages to control the inflammatory response

LDL receptor-related protein-1 (LRP1) is an endocytic and cell-signaling receptor. In mice in which LRP1 is deleted in myeloid cells, the response to lipopolysaccharide (LPS) was greatly exacerbated. LRP1 deletion in macrophages in vitro, under the control of tamoxifen-activated Cre-ER(T) fusion protein, robustly increased expression of proinflammatory cytokines and chemokines. In LRP1-expressing macrophages, proinflammatory mediator expression was regulated by LRP1 ligands in a ligand-specific manner. The LRP1 agonists, α2-macroglobulin and tissue-type plasminogen activator, attenuated expression of inflammatory mediators, even in the presence of LPS. The antagonists, receptor-associated protein (RAP) and lactoferrin (LF), and LRP1-specific antibody had the entirely opposite effect, promoting inflammatory mediator expression and mimicking LRP1 deletion. NFκB was rapidly activated in response to RAP and LF and responsible for the initial increase in expression of proinflammatory mediators. RAP and LF also significantly increased expression of microRNA-155 (miR-155) after a lag phase of about 4 h. miR-155 expression reflected, at least in part, activation of secondary cell-signaling pathways downstream of TNFα. Although miR-155 was not involved in the initial induction of cytokine expression in response to LRP1 antagonists, miR-155 was essential for sustaining the proinflammatory response. We conclude that LRP1, NFκB, and miR-155 function as members of a previously unidentified system that has the potential to inhibit or sustain inflammation, depending on the continuum of LRP1 ligands present in the macrophage microenvironment

Oral Protein Therapy for the Future - Transport of Glycolipid-Modified Proteins: Vision or Fiction?

The reliable and early diagnosis of common complex multifactorial diseases depends on the individual determination of all (or as many as possible) polymorphisms of each susceptibility gene together with amount and type of the corresponding gene products and their downstream effects, including the synthesis and flux of metabolites and regulation of signalling processes. In addition, this system's biology-driven personalized diagnosis must be accompanied by options for personalized reliable and early therapy. In the midterm, the direct substitution or inhibition of the proteins encoded by the corresponding defective gene products of the susceptibility genes exerting lower or higher activity by administration of the `normal' proteins or inhibitory antibodies, respectively, seems to be most promising. The critical hurdle of oral bioavailability as well as transport into the cytoplasm of the target cells, if required, could be overcome by therapeutic proteins with carboxy-terminal modification by glycosylphosphatidylinositol (GPI). This may be deduced from recent experiments with rat adipocytes. Here this membrane-anchoring glycolipid structure induces the sequential transport of proteins from special regions of the plasma membrane via the surface of intracellular lipid droplets to special membrane vesicles, which are finally released from the adipocytes together with the associated GPI proteins. It remains to be studied whether similar molecular mechanisms operate in intestinal epithelial cells and may enable the transport of GPI proteins from the intestinal lumen into the blood stream. If so, modification of proteins encoded by (combinations of) susceptibility genes with GPI could significantly facilitate the personalized therapy of common diseases on the basis of `inborn' safety, efficacy, rapid realization and oral application. Copyright (C) 2010 S. Karger AG, Base

Spinning around or stagnation - what do osteoblasts and chondroblasts really like?

<p>Abstract</p> <p>Objective</p> <p>The influcence of cytomechanical forces in cellular migration, proliferation and differentation of mesenchymal stem cells (MSCs) is still poorly understood in detail.</p> <p>Methods</p> <p>Human MSCs were isolated and cultivated onto the surface of a 3 × 3 mm porcine collagen I/III carrier. After incubation, cell cultures were transfered to the different cutures systems: regular static tissue flasks (group I), spinner flasks (group II) and rotating wall vessels (group III). Following standard protocols cells were stimulated lineage specific towards the osteogenic and chondrogenic lines. To evaluate the effects of applied cytomechanical forces towards cellular differentiation distinct parameters were measured (morphology, antigen and antigen expression) after a total cultivation period of 21 days in vitro.</p> <p>Results</p> <p>Depending on the cultivation technique we found significant differences in both gen and protein expression.</p> <p>Conclusion</p> <p>Cytomechanical forces with rotational components strongly influence the osteogenic and chondrogenic differentiation.</p

Evaluating techniques in tissue clarification using CLARITY imaging and investigating where sodium is sensed in the body

OBJECTIVE: Previous studies have shown the significant contribution of sympathoinhibition in response to sodium loading to prevent increases in mean arterial blood pressure in salt resistant phenotypes. It has also been shown that brain Gαi2 protein gated signal transduction plays a major role in this pathway, however, the specific mechanisms through which this pathway is activated remain less well understood. The purpose of this study was to elucidate the relative contribution of increased sodium in either the plasma or the cerebrospinal fluid (CSF) to the regulation of mean arterial pressure and natriuresis. Additionally we explored the potential for using the novel CLARITY Imaging technique to identify the relative activity of neurons in areas of the brain thought to play a major role in body fluid homeostasis in response to salt. METHODS: Rats that were pre-treated with either scrambled or Gαi2 oligodeoxynucleotides (ODN), to selectively down regulate brain Gαi2 proteins, were challenged either peripherally or centrally with sodium. Upon sodium loading physiological parameters were measured for two hours after which the animal's brains were recovered for immunohistochemical (IHC) analysis of the paraventricular nucleus, a known regulatory center for body fluid homeostasis and blood pressure regulation. Additionally we adapted a version of the published CLARITY Imaging protocols for optically clearing tissue through application of electrophoretic tissue clearing (ETC) to a larger rat model. RESULTS: In scrambled ODN pre-treated rats we observed a temporary increase in MAP in response to both the peripheral and central sodium challenge. In the Gαi2 ODN pre-treated animals we saw some form of attenuation to this response in both studies, however, where in the peripheral challenge there was an increase in the amount of time that it took the rats to return to normotension with no alteration in natriuresis, in the central challenge there was a large attenuation in natriuresis with no differences in the time to return to baseline MAP. Our IHC analysis also showed a decrease in neuronal activation of paraventricular medial parvocellular neurons in Gαi2 pre-treated rats that were challenged peripherally vs their SCR pre-treated counterparts. No such difference was observed in either of the pre-treatment groups from the central sodium challenge study. In the CLARITY study we found that it is possible to adapt the method for optically clearing tissue to the larger model, however, we encountered several issues related to tissue swelling and peripheral tissue damage. CONCLUSION: Based on our current results it seems evident that there are at least two different mechanisms that activate the cardiovascular regulatory control centers in the brain that prevent long term increases in mean arterial pressure in response to increased salt. It also appears that these two different mechanisms are triggered either by increases in plasma or CSF salinity, though which of these two mechanisms may be directly responsible for the development of salt sensitive hypertension requires further investigation. While we had some success at optically clearing larger tissue volumes through ETC, problems we encountered with maintaining tissue integrity for investigations of intact neural networks prevented us from applying this technique, in its current form, to our investigation of salt sensitive hypertension