The study of progesterone action in human myometrial explants.

STUDY HYPOTHESIS: Myometrial explants represent a superior model compared with cell culture models for the study of human myometrial progesterone (P4) signalling in parturition. STUDY FINDING: Gene expression analysis showed myometrial explants closely resemble the in vivo condition and the anti-inflammatory action of P4 is not lost with labour onset. WHAT IS KNOWN ALREADY: Circulating P4 levels decline before the onset of parturition in most animals, but not in humans. This has led to the suggestion that there is a functional withdrawal of P4 action at the myometrial level prior to labour onset. However, to date, no evidence of a loss of P4 function has been provided, with studies hampered by a lack of a physiologically relevant model. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Myometrial biopsies obtained at Caesarean section were dissected into explants after a portion was immediately snap frozen (t = 0). Microarray analysis was used to compare gene expression of t = 0 with paired (i) explants, (ii) passage 4 myometrial cell cultures or (iii) the hTERT myometrial cell line. Western blotting and chemokine/cytokine assays were used to study P4 signalling in myometrial explants. MAIN RESULTS AND THE ROLE OF CHANCE: Gene expression comparison of t = 0 to the three models demonstrated that explants more closely resemble the in vivo status. At the protein level, explants maintain both P4 receptor (PR) and glucocorticoid receptor (GR) levels versus t = 0 whereas cells only maintain GR levels. Additionally, treatment with 1 µM P4 led to a reduction in interleukin-1 (IL-1) ß-driven cyclooxygenase-2 in explants but not in cells. P4 signalling in explants was PR-mediated and associated with a repression of p65 and c-Jun phosphorylation. Furthermore, the anti-inflammatory action of P4 was maintained after labour onset. LIMITATIONS/REASONS FOR CAUTION: There is evidence of basal inflammation in the myometrial explant model. WIDER IMPLICATIONS OF THE FINDINGS: Myometrial explants constitute a novel model to study P4 signalling in the myometrium and can be used to further elucidate the mechanisms of P4 action in human labour. LARGE SCALE DATA: Data deposited at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=gvmpggkurbgxfqf&acc=GSE77830. STUDY FUNDING AND COMPETING INTEREST: This work was supported by grants from the Joint Research Committee of the Westminster Medical School Research Trust, Borne (No. 1067412-7; a sub-charity of the Chelsea and Westminster Health Charity) and the Imperial NIHR Biomedical Research Centre. The views expressed are those of the author(s) and not necessarily those of the NHS or the Department of Health. The authors have no conflict of interest.

The role of mesenchymal stem cells in veterinary therapeutics - a review.

Adult mammalian tissue contains a population of cells known as mesenchymal stem cells (MSC), that possess the capability to secrete regenerative cytokines and to differentiate into specialised cell types. When transplanted to a site of injury MSC embed in damaged tissue and repair and regenerate the tissue by secreting cytokines. The immuno-privileged and immuno-regulatory capabilities of MSC enhance their therapeutic potential not only in autologous but also allogeneic recipients. Studies have demonstrated the beneficial effects of MSC in the treatment of a variety of clinical conditions including osteoarthritis, tendon injuries, and atopic dermatitis in domestic animals. Studies using animal models have shown promising results following MSC or MSC secretion therapy for induced injury in musculoskeletal and nervous systems and some organ diseases. This review describes the stem cell types relevant to regenerative medicine and the procedures used for isolation, identification, expansion, enrichment and differentiation of these cells. We also review the use of MSC in animal models of disease as well as diseases in the clinical veterinary setting.

Challenges, current status and future perspectives of proteomics in improving understanding, diagnosis and treatment of vascular disease.

Technical advances have seen the rapid adoption of genomics and multiplex genetic polymorphism identification to research on vascular diseases. The utilization of proteomics for the study of vascular diseases has been limited by comparison. In this review we outline currently available proteomics techniques, the challenges to using these approaches and modifications which may improve the utilization of proteomics in the study of vascular diseases.

What place for polyacrylamide in proteomics?

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) continues to deliver high quality protein resolution and dynamic range for the proteomics researcher. To remain as the preferred method for protein separation and characterization, several key steps need to be implemented to ensure quality sample preparation and speed of analysis. Here, we describe the progress made towards establishing 2D-PAGE as the optimal separation tool for proteomics research.

Proteomic analysis of the Escherichia coli outer membrane.

Outer membrane proteins (OMPs) of Gram-negative bacteria are key molecules that interface the cell with the environment. Traditional biochemical and genetic approaches have yielded a wealth of knowledge relating to the function of OMPs. Nonetheless, with the completion of the Escherichia coli genome sequencing project there is the opportunity to further expand our understanding of the organization, expression and function of the OMPs in this Gram-negative bacterium. In this report we describe a proteomic approach which provides a platform for parallel analysis of OMPs. We propose a rapid method for isolation of bacterial OMPs using carbonate incubation, purification and protein array by two-dimensional electrophoresis, followed by protein identification using mass spectrometry. Applying this method to examine E. coli K-12 cells grown in minimal media we identified 21 out of 26 (80%) of the predicted integral OMPs that are annotated in SWISS-PROT release 37 and predicted to separate within the range of pH 4-7 and molecular mass 10-80 kDa. Five outer membrane lipoproteins were also identified and only minor contamination by nonmembrane proteins was observed. Importantly, this research readily demonstrates that integral OMPs, commonly missing from 2D gel maps, are amenable to separation by two-dimensional electrophoresis. Two of the identified OMPs (YbiL, YeaF) were previously known only from their ORFs, and their identification confirms the cognate genes are transcribed and translated. Furthermore, we show that like the E. coli iron receptors FhuE and FhuA, the expression of YbiL is markedly increased by iron limitation, suggesting a putative role for this protein in iron transport. In an additional demonstration we show the value of parallel protein analysis to document changes in E. coli OMP expression as influenced by culture temperature.

Proteomics: capacity versus utility.

Until recently scientists studied genes or proteins one at a time. With improvements in technology, new tools have become available to study the complex interactions that occur in biological systems. Global studies are required to do this, and these will involve genomic and proteomic approaches. High-throughput methods are necessary in each case because the number of genes and proteins in even the simplest of organisms are immense. In the developmental phase of genomics, the emphasis was on the generation and assembly of large amounts of nucleic acid sequence data. Proteomics is currently in a phase of technological development and establishment, and demonstrating the capacity for high throughput is a major challenge. However, funding bodies (both in the public and private sector) are increasingly focused on the usefulness of this capacity. Here we review the current state of proteome research in terms of capacity and utility.

High-throughput mass spectrometric discovery of protein post-translational modifications.

The availability of genome sequences, affordable mass spectrometers and high-resolution two-dimensional gels has made possible the identification of hundreds of proteins from many organisms by peptide mass fingerprinting. However, little attention has been paid to how information generated by these means can be utilised for detailed protein characterisation. Here we present an approach for the systematic characterisation of proteins using mass spectrometry and a software tool FindMod. This tool, available on the internet at http://www.expasy.ch/sprot/findmod.html , examines peptide mass fingerprinting data for mass differences between empirical and theoretical peptides. Where mass differences correspond to a post-translational modification, intelligent rules are applied to predict the amino acids in the peptide, if any, that might carry the modification. FindMod rules were constructed by examining 5153 incidences of post-translational modifications documented in the SWISS-PROT database, and for the 22 post-translational modifications currently considered (acetylation, amidation, biotinylation, C-mannosylation, deamidation, flavinylation, farnesylation, formylation, geranyl-geranylation, gamma-carboxyglutamic acids, hydroxylation, lipoylation, methylation, myristoylation, N -acyl diglyceride (tripalmitate), O-GlcNAc, palmitoylation, phosphorylation, pyridoxal phosphate, phospho-pantetheine, pyrrolidone carboxylic acid, sulphation) a total of 29 different rules were made. These consider which amino acids can carry a modification, whether the modification occurs on N-terminal, C-terminal or internal amino acids, and the type of organisms on which the modification can be found. We illustrate the utility of the approach with proteins from 2-D gels of Escherichia coli and sheep wool, where post-translational modifications predicted by FindMod were confirmed by MALDI post-source decay peptide fragmentation. As the approach is amenable to automation, it presents a potentially large-scale means of protein characterisation in proteome projects.

Extraction of Escherichia coli proteins with organic solvents prior to two-dimensional electrophoresis.

Compared to soluble proteins, hydrophobic proteins, in particular membrane proteins, are an underrepresented protein species on two-dimensional (2-D) gels. One possibility is that many hydrophobic proteins are simply not extracted from the sample prior to 2-D gel separation. We attempted to isolate hydrophobic proteins from Escherichia coli by extracting with organic solvents, then reconstituting the extracted proteins in highly solubilising sample solution amenable to 2-D electrophoresis using immobilized pH gradients (IPGs). This was conducted by an extraction with a mixture of chloroform and methanol, followed by solubilisation using a combination of urea, thiourea, sulfobetaine detergents and tributyl phosphine. Peptide mass fingerprinting assisted in the identification of 13 proteins, 8 of which have not previously been reported on 2-D gels. Five of these new proteins possess a positive hydropathy plot. These results suggest that organic solvent extractions may be useful for selectively isolating some proteins that have previously been missing from proteome maps.

Identification and quantitation of cysteine in proteins separated by gel electrophoresis.

A simple technique is introduced to identify and quantitate cysteine (Cys) after acid hydrolysis of protein. The technique involves using 9-fluorenylmethyl chloroformate (Fmoc)-based amino acid analysis that recovers all of the amino acids (asparagine and glutamine are recovered in their acidic forms) except tryptophan. Cys adducts with acrylamide and iodoacetamide have been observed in hydrolysates of gel-separated proteins. To enable quantitation of Cys by amino acid analysis, different conditions of reduction [dithiothreitol (DTT) and tributylphosphine] and alkylation [vinylpyridine, acrylamide and iodoacetamide] were compared. Optimal conditions for on-blot reduction (125 mM of DTT, pH 8.5, at 80 degrees C) and alkylation (0.25 M iodoacetamide, pH 8.5, at 37 degrees C) of proteins which have been separated by gel electrophoresis and blotted onto polyvinylidenedifluoride (PVDF) membrane were established to achieve complete recovery of alkylated Cys. Even with the optimal on-blot iodoacetamide alkylation, there may still be some acrylamide adducts present and these were able to be separated by HPLC along with the other 16 amino acids. The Cys content has been successfully determined by Fmoc-amino acid analysis of PVDF-blotted proteins separated by 1D or 2D gel electrophoresis. Lysine alkylation with iodoacetamide and acrylamide has also been characterised. Protein identification using amino acid composition including Cys has been introduced.

Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis.

We describe the extraction and enrichment of membrane proteins for separation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) after differential solubilization of an Escherichia coli cell lysate. In a simple three-step sequential solubilization protocol applicable for whole cell lysates, membrane proteins are partitioned from other cellular proteins by their insolubility in solutions conventionally used for isoelectric focusing (IEF). As the first step, Tris-base was used to solubilize many cytosolic proteins. The resultant pellet was then subjected to conventional solubilizing solutions (urea, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, dithiothreitol, Tris, carrier ampholytes). Following the completion of this step, 89% of the initial E. coli sample mass was solubilized. Finally, the membrane protein rich pellet was partially solubilized using a combination of urea, thiourea, tributyl phosphine and multiple zwitterionic surfactants. Using N-terminal sequence tagging and peptide mass fingerprinting we have identified 11 membrane proteins from this pellet. Two of these outer membrane proteins (Omp), OmpW and OmpX, have previously been known only as an open reading frame in E. coli, while OmpC, OmpT and OmpTOLC have not previously been identified on a 2-D gel. The prefractionation of an entire cell lysate into multiple fractions, based on solubility, results in simplified protein patterns following 2-D PAGE using broad-range pH 3.5-10 immobilized pH gradients (IPGs). Additional advantages of sample prefractionation are that protein identification and gel matching, for database construction, is a more manageable task, the procedure requires no specialized apparatus, and the sequential extraction is conducted in a single centrifuge tube, minimizing protein loss.

Improved protein solubility in two-dimensional electrophoresis using tributyl phosphine as reducing agent.

In this study, dithiothreitol was replaced by tributyl phosphine as the reducing agent in both the sample solution for the first-dimensional isoelectric focusing and during the immobilised pH gradient (IPG) equilibration procedure. Tributyl phosphine improves protein solubility during isoelectric focusing, which results in shorter run times and increased resolution. Tributyl phosphine is nonionic and thus does not migrate in the IPG, therefore maintaining reducing conditions during the course of the first-dimensional separation. The increased solubility provided by the maintenance of reducing conditions gives improved focusing and decreased horizontal streaking on the subsequent second-dimension gel. The use of tributyl phosphine in the equilibration step allows the procedure to be simplified, incorporating reduction and alkylation in a single step. This is possible because, in direct contrast to dithiothreitol (DTT), tributyl phosphine does not contain a free thiol and therefore does not react with thiol-specific alkylating reagents.

Identification of wallaby milk whey proteins separated by two-dimensional electrophoresis, using amino acid analysis and sequence tagging.

Micropreparative two-dimensional polyacrylamide gel electrophoresis has been used to separate milk whey proteins from the Tammar wallaby (Macropus eugenii). We have used a combination of amino acid analysis and N-terminal sequence tagging as a rapid and sensitive method to identify the major whey proteins. Using these techniques, we confidently identified alpha-lactalbumin and late lactation protein. While these are the only two M. eugenii whey proteins with a corresponding SWISS-PROT entry, we demonstrate that by using amino acid analysis and matching across species boundaries, we can identify previously unsequenced conserved wallaby whey proteins including beta-lactoglobulin and serum albumin.

Prefractionation of protein samples prior to two-dimensional electrophoresis.

Thousands of proteins may be visualised on a two-dimensional (2-D) gel, but only hundreds are present at levels sufficient for chemical analysis. Therefore, prefractionation of protein samples prior to 2-D polyacrylamide gel electrophoresis (PAGE) will be important for the investigation of proteins that are present at sub-picogram levels in physiological samples. We describe an approach to prefractionate protein samples prior to 2-D PAGE using the Gradiflow, which is a new (preparative) electrokinetic membrane apparatus designed to fractionate proteins in a number of different ways. We have fractionated human serum under nonreducing conditions using the 'reflux' mode, in which proteins are fractionated according to their relative mobility under controlled electrophoretic conditions, where the current is periodically reversed. We describe how fractionation occurs and present examples of enrichment of specific proteins.

Characterisation of wool intermediate filament proteins separated by micropreparative two-dimensional electrophoresis.

Wool intermediate filament proteins (IFP) are a subclass of the cytokeratins, a group of structural proteins which form intermediate filaments in many cell types. Post-translational modifications, such as phosphorylation, play an important role in the control of intermediate filament assembly. Two-dimensional electrophoresis has previously been used to study the IFP distribution in wools with different physical characteristics. Charge heterogeneity has been observed in Type I and Type II IFP. In a previous study, two-dimensional electrophoresis of alkaline phosphatase-treated wool protein extracts was used to show that Type II IFP are phosphorylated. To facilitate post-separation analysis, micropreparative two-dimensional electrophoresis was used to separate milligram quantities of wool protein. Direct phosphoamino acid analysis has confirmed the presence of phosphorylation on serine residues on Type II IFP, whose identity was confirmed by amino acid compositional analysis. The isoelectric points of Type I IFP are very similar and they do not separate completely on the commercially available pH 4-7 immobilized pH gradients (IPG) used in this study. In situ tryptic digestion followed by automated Edman sequencing of the high performance liquid chromatography (HPLC)-separated peptides was used to confirm the identity of this group as Type I IFP. To improve the separation of the Type I IFP it will be necessary to use narrow range IPGs such as Immobiline DryPlates which are available from Pharmacia Biotech, in the pH ranges 4.2-4.9, 4.5-5.4, 5.0-6.0 and 5.6-6.6.

Establishment of the human reflex tear two-dimensional polyacrylamide gel electrophoresis reference map: new proteins of potential diagnostic value.

To understand the changes in protein expression associated with various physiological states as well as the development of pathological eye disease, we have begun to map the protein components of normal human reflex tears. An analytical reference map of normal human reflex tears was created using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) with pH 3.5-10 immobilized pH gradients (IPGs). Micropreparatively loaded gels were transferred to polyvinylidene difluoride (PVDF) and analysed by a combination of N-terminal sequence tagging and amino acid compositional analysis. Thirty spots were sequence tagged, resulting in identification of six different proteins (lipocalin, lysozyme, lactotransferrin, zinc-alpha-2 glycoprotein, cystatin S, cystatin SN) that matched to entries in the SWISS-PROT database. A group of N-terminally blocked proteins was clearly identified from SWISS-PROT by amino acid analysis, isoelectric point (pI) and molecular weight (Mr). A number of highly expressed protein components remain unidentified despite being subjected to amino acid analysis and Edman sequencing. A majority of the abundant proteins showed varying degrees of charge heterogeneity attributed to post-translational processing such as glycosylation and N-terminal truncation. We have identified a previously undescribed protein that we have named lacryglobin. This protein displays strong homology with mammaglobin, a protein overexpressed in breast cancer. The discovery of this homologue in tears offers the potential for disease diagnosis by screening tear fluid proteins.

Investigation of wool protein heterogeneity using two-dimensional electrophoresis with immobilised pH gradients.

The heterogeneity of intermediate filament proteins (IFP) from wool has been investigated using two-dimensional polyacrylamide gel electrophoresis with immobilised pH gradients in the first dimension. The charge heterogeneity has been confirmed with some of the IFP separated as a string of spots with similar molecular weight, but markedly different in isoelectric point (pI). The molecular weight and pI distribution of the string of IFP changed after treatment with alkaline phosphatase, indicating that some of the heterogeneity is caused by phosphorylation.

Immobilised pH gradient isoelectric focusing of wool proteins.

Conventional carrier ampholyte-isoelectric focusing is unsuitable for separating wool and hair proteins. The inability to form stable narrow pH gradients at acidic pH values does not allow good resolution of the many acidic wool and hair proteins. To evaluate the usefulness of immobilised pH gradient-isoelectric focusing (IPG-IEF) for wool proteins, S-amidomethyl wool proteins were analysed using IPG-IEF. Over twenty bands were visible on a pH 4-7 IPG-IEF gel. IPG-IEF was combined with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to give a two dimensional separation, and over 50 protein spots were observed. IPG-IEF appears to be ideally suited to the separation of wool proteins. The resolution of two-dimensional electrophoresis using IPG-IEF was greater than previously obtained using acid or alkaline PAGE in the first dimension. Good reproducibility of spot position was observed.