Epithelial migration in organ culture : Effect of incubation in unsupplemented balanced salt solution

In a study of the metabolic requirements for epithelial migration earlier results had indicated that the minimal requirement for migration in organ culture would be a suitable glycolysable substrate. To test this, explants were incubated in a glucose-containing balanced salt solution (Earle's BSS). The epithelial cells consistently failed to migrate in the BSS but resumed migration when the explants were transferred to serum-supplemented medium after a 24 h incubation in BSS. The metabolism and morphology of the explants in BSS were analysed and it was found that protein and RNA synthetic capability were retained but that DNA synthetic capability was depressed. To determine the role of nucleic acid synthesis in epithelial migration explants in serum-supplemented medium were exposed to the inhibitors mitomycin C and actinomycin D. When nucleic acid synthesis was depressed below control values by low concentrations of inhibitor it was found that epithelial migration was not inhibited. It was concluded that, while direct depression of DNA synthesis by inhibitors did not inhibit migration, the results reported here indicate that failure to migrate in BSS may affect nuclear function.     

Analysis of protein networks in resting and collagen receptor (GPVI)-stimulated platelet sub-proteomes

Proteomics approaches have made important contributions to the characterisation of platelet regulatory mechanisms. A common problem encountered with this method, however, is the masking of low-abundance (e.g. signalling) proteins in complex mixtures by highly abundant proteins. In this study, subcellular fractionation of washed human platelets either inactivated or stimulated with the glycoprotein (GP) VI collagen receptor agonist, collagen-related peptide, reduced the complexity of the platelet proteome. The majority of proteins identified by tandem mass spectrometry are involved in signalling. The effect of GPVI stimulation on levels of specific proteins in subcellular compartments was compared and analysed using in silico quantification, and protein associations were predicted using STRING (the search tool for recurring instances of neighbouring genes/proteins). Interestingly, we observed that some proteins that were previously unidentified in platelets including teneurin-1 and Van Gogh-like protein 1, translocated to the membrane upon GPVI stimulation. Newly identified proteins may be involved in GPVI signalling nodes of importance for haemostasis and thrombosis.     

SIgA Binding to Mucosal Surfaces Is Mediated by Mucin-Mucin Interactions

The oral mucosal pellicle is a layer of absorbed salivary proteins, including secretory IgA (SIgA), bound onto the surface of oral epithelial cells and is a useful model for all mucosal surfaces. The mechanism by which SIgA concentrates on mucosal surfaces is examined here using a tissue culture model with real saliva. Salivary mucins may initiate the formation of the mucosal pellicle through interactions with membrane-bound mucins on cells. Further protein interactions with mucins may then trigger binding of other pellicle proteins. HT29 colon cell lines, which when treated with methotrexate (HT29-MTX) produce a gel-forming mucin, were used to determine the importance of these mucin-mucin interactions. Binding of SIgA to cells was then compared using whole mouth saliva, parotid (mucin-free) saliva and a source of purified SIgA. Greatest SIgA binding occurred when WMS was incubated with HT29-MTX expressing mucus. Since salivary MUC5B was only able to bind to cells which produced mucus and purified SIgA showed little binding to the same cells we conclude that most SIgA binding to mucosal cells occurs because SIgA forms complexes with salivary mucins which then bind to cells expressing membrane-bound mucins. This work highlights the importance of mucin interactions in the development of the mucosal pellicle.     

Proteomic analysis of resting and thrombin‐stimulated platelets reveals the translocation and functional relevance of HIP‐55 in platelets

The platelet surface is a dynamic interface that changes rapidly in response to stimuli to co‐ordinate the formation of thrombi at sites of vascular injury. Tight control is essential as loss of organisation may result in the inappropriate formation of thrombi (thrombosis) or excessive bleeding. In this paper we describe the comparative analysis of resting and thrombin‐stimulated platelet membrane proteomes and associated proteins to identify proteins important to platelet function. Surface proteins were labelled using a biotin tag and isolated by NeurtrAvidin affinity chromatography. Liquid phase IEF and SDS‐PAGE were used to separate proteins, and bands of increased intensity in the stimulated platelet fractions were digested and identified by FT‐ICR mass spectrometry. Novel proteins were identified along with proteins known to be translocated to the platelet surface. Furthermore, many platelet proteins revealed changes in location associated with function, including G6B and Hip‐55. HIP‐55 is an SH3‐binding protein important in T‐cell receptor signalling. Further analysis of HIP‐55 revealed that this adaptor protein becomes increasingly associated with both Syk and integrin β3 upon platelet activation. Analysis of HIP‐55 deficient platelets revealed reduced fibrinogen binding upon thrombin stimulation, suggesting HIP‐55 to be an important regulator of platelet function.     

Can splenocytes enhance pancreatic β-cell function and mass in 90% pancreatectomized rats fed a high fat diet?

AimsRecent studies have shown that splenocytes may act as a possible neogenic source with regard to β-cells in rodent diabetic models. Accordingly, we sought to determine whether splenocytes played an important role in promoting β-cell function and mass among type 2 diabetic rats with and without spleen.Main methodsWe randomly divided female 90% pancreatectomized (Px) Sprague Dawley rats into three groups: splenectomy (SPX), splenectomy plus the injection of male splenocytes (SPI), and no splenectomy (NSP). They were administered with 40 energy percent fat diets over the course of five weeks. At the end of the experimental period, insulin secretion capacity was measured by hyperglycemic clamp. At 6 h after BrdU⁺ injection, the pancreas was prepared with 4% paraformaldehyde in order to perform immunohistochemistry.Key findingsSPX increased and sustained serum glucose levels more than NSP and SPI during oral glucose tolerance testing. During hyperglycemic clamp, first and second phase insulin secretion decreased in the SPX rats while splenocyte injections counteracted this. Beta-cell mass in the SPX group was reduced more than among NSP and SPI. This was the result of a decrease in the number of small β-cell clusters in SPX, which is indicative of a decrease in β-cell neogenesis.SignificanceSplenocytes play an important role with regard to the neogenesis of β-cells in insulin deficient type 2 diabetic rats, although they are not critical for β-cell regeneration.     

Rapid disappearance of the medial epithelial seam during palatal fusion occurs by multifocal breakdown that is preceded by expression of alpha smooth muscle actin in the epithelium

Breakdown of the medial epithelial seam (MES) is essential to allow bridging of the mesenchyme during palatal fusion. Evidence exists for three mechanisms for this breakdown that are incompatible at the level of individual cells in the seam. To determine if breakdown of the seam was regionally restricted, 3-dimensional reconstructions were generated using volume rendering software from 1 micron serial sections in the sagittal plane of rat palates fixed during the process of fusion. The earliest break detected in electron micrographs was cell separation and in reconstructions was a discrete defect, with a rounded outline, nearer to the nasal than to the oral margin of the seam. Further breakdown produced a pattern of rounded defects along the nasal margin of the seam resulting in interconnected columns of cells preferentially attached to the oral epithelium. Computer generated slicing of reconstructed seams showed that groups of cells evident in cross-sections as islands at this stage of breakdown of the MES could be artifacts. Unequivocal islands of epithelial cells formed later in fusion had a rounded outline, an incomplete basal lamina and a halo of cells containing phagocytosed apoptotic debris. The pattern of breakdown indicated that the MES breaks down under tension. Laser confocal microscopy of sections and whole-mounts of palates demonstrated alpha-smooth muscle actin preferentially localized in the epithelial cells of the palatal shelves immediately before and during formation of the seam. Expression in epithelial cells of the isoform of actin normally restricted to smooth muscle cells engaged in tonic contraction supported an interpretation that the epithelial cells of the seam may be capable of generating tension during the palatal fusion event.     

Regulation of SHP-1 tyrosine phosphatase in human platelets by serine phosphorylation at its C terminus

SHP-1 is a Src homology 2 (SH2) domain-containing tyrosine phosphatase that plays an essential role in negative regulation of immune cell activity. We describe here a new model for regulation of SHP-1 involving phosphorylation of its C-terminal Ser591 by associated protein kinase Calpha. In human platelets, SHP-1 was found to constitutively associate with its substrate Vav1 and, through its SH2 domains, with protein kinase Calpha. Upon activation of either PAR1 or PAR4 thrombin receptors, the association between the three proteins was retained, and Vav1 became phosphorylated on tyrosine and SHP-1 became phosphorylated on Ser591. Phosphorylation of SHP-1 was mediated by protein kinase C and negatively regulated the activity of SHP-1 as demonstrated by a decrease in the in vitro ability of SHP-1 to dephosphorylate Vav1 on tyrosine. Protein kinase Calpha therefore critically and negatively regulates SHP-1 function, forming part of a mechanism to retain SHP-1 in a basal active state through interaction with its SH2 domains, and phosphorylating its C-terminal Ser591 upon cellular activation leading to inhibition of SHP-1 activity and an increase in the tyrosine phosphorylation status of its substrates.