Generation of co-culture spheroids as vascularisation units for bone tissue engineering.

Cell spheroids represent attractive building units for bone tissue engineering, because they provide a three-dimensional environment with intensive direct cell-cell contacts. Moreover, they allow for co-culture of both osteoblasts and vessel-forming cells, which may markedly increase their survival and vascularisation after transplantation. To test this hypothesis, we generated co-culture spheroids by aggregating different combinations of primary human osteoblasts (HOB), human dermal microvascular endothelial cells (HDMEC) and normal human dermal fibroblasts (NHDF) using the liquid overlay technique. Mono-culture spheroids consisting either of HOB or HDMEC served as controls. After in vitro characterisation, the different spheroids were transplanted into dorsal skinfold chambers of CD1 nu/nu mice to study in vivo their viability and vascularisation over a 2-week observation period by means of repetitive intravital fluorescence microscopy and immunohistochemistry. In vitro, co-culture spheroids containing HDMEC rapidly formed dense tubular vessel-like networks within 72 h and exhibited a significantly decreased rate of apoptotic cell death when compared to mono-culture HDMEC spheroids. After transplantation, these networks interconnected to the host microvasculature by external inosculation. Of interest, this process was most pronounced in HOB-HDMEC spheroids and could not further be improved by the addition of NHDF. Accordingly, HOB-HDMEC spheroids were larger when compared to the other spheroid types. These findings indicate that HOB-HDMEC spheroids exhibit excellent properties to preserve viability and to promote proliferation and vascularisation. Therefore, they may be used as functional vascularisation units in bone tissue engineering for the seeding of scaffolds or for the vitalisation of non-healing large bone defects.

Impact of urbanization on the proteome of birch pollen and its chemotactic activity on human granulocytes.

BACKGROUND: Epidemiologic studies reveal a dramatic increase in allergies in the last decades. Air pollution is considered to be one of the factors responsible for this augmentation. The aim of this study was to analyze the impact of urbanization on birch pollen. The birch pollen proteome was investigated in order to identify differences in protein abundance between pollen from rural and urban areas. The allergenicity of birch pollen from both areas was evaluated by assessing its chemotactic potency as well as its protein and allergen contents. METHODS: Difference gel electrophoresis (DIGE) was used to analyze the pollen proteome. The chemotactic activity of aqueous pollen extracts was determined by migration assays of human neutrophils. RESULTS: DIGE revealed 26 differences in protein spot intensity between pollen from urban and rural areas. One of these proteins was identified by de novo sequencing as the 14-3-3 protein, which resembles a stress-induced factor in other plant species. Furthermore, extracts from pollen collected in urban areas had higher chemotactic activity on human neutrophils compared to pollen from rural sites. CONCLUSIONS: The present study points to an impact of air pollution on allergen carrier proteome and release of chemotactic substances. The increment in proinflammatory substances such as pollen-associated lipid mediators might contribute to the described urban-rural gradient of allergy prevalence. Furthermore, our study suggests that allergenicity is determined by more than the sole allergen content.

Changes in protein expression during melanoma differentiation determined by computer analysis of 2-D gels.

Cytodifferentiation in many melanocytic cells is regulated through the adenylate cyclase-cAMP pathway. To analyse the molecular changes associated with this process we have compared the proteins produced by two closely related cell lines which, though derived from a single cell line, respond very differently to modulation of this signalling pathway. The human melanoma cell line DX3 shows little change in in vitro characteristics following treatment with cAMP elevating agents; in contrast the more malignant DX3 LT5.1 variant, derived from the DX3 parental line, shows pronounced dendrification, decreased proliferation and a reduction in metastatic capacity after similar treatment. The two cell lines were treated with phosphodiesterase inhibitors for 5 days and then processed for two-dimensional gel characterization using an immobilized pH gradient for the IEF dimension. Proteins were detected by silver staining the gels and protein intensities were digitized using a laser densitometer. Two-dimensional gel patterns were edited, matched and a melanoma protein database of 637 spots constructed using PDQUEST software on an Orion 1/05 computer. Eleven proteins were lost and four new proteins were detected in both cell lines following treatment. Twenty-two proteins were present in DX3 LT5.1 after treatment but not in untreated lines or treated DX3. These differentially expressed proteins may be associated with the observed changes in differentiation patterns and metastasis. Our results illustrate the resolving power of this technique and suggest potential applications to the study of cellular differentiation.

Losartan inhibits the angiotensin II-induced stimulation of the phosphoinositide signalling system in vascular smooth muscle cells.

2-n-Butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)bip hen yl-4-yl)methyl]imidazole, potassium salt (Losartan) (previous name, DuP 753 or MK 954) is a nonpeptide angiotensin II receptor antagonist. This study was performed to investigate the ability of Losartan to inhibit the angiotensin II-induced stimulation of the phospoinositide signalling system and the angiotensin II-induced hypertrophy in aortic vascular smooth muscle cells of normotensive Wistar-Kyoto rats. 10(-7) M Losartan abolished the angiotensin II-induced formation of inositol 1,4,5-trisphosphate in vascular smooth muscle cells. 10(-6) M Losartan completely abolished the angiotensin II-induced elevation of the intracellular free Ca2+ concentration ([Ca2+]i). 10(-6) M Losartan lacked effects on the [Arg8]vasopressin-induced elevation of [Ca2+]i. In addition, 10(-6) M completely inhibited the angiotensin II-induced stimulation of Na+/H+ exchange in the vascular smooth muscle cells. 10(-10) to 10(-6) M Losartan inhibited the angiotensin II-induced cell protein synthesis in a concentration-dependent manner, yielding to an effective concentration (ED50) of 6.2 +/- 1.8 x 10(-8) M (n = 4). Losartan did not affect the platelet-derived growth factor-BB-induced increase in cell protein. These results show that Losartan is a highly specific angiotensin II receptor antagonist which inhibits angiotensin II-induced cell growth and thus may have beneficial effects on the development and regression of vascular hypertrophy.

pH, urea and substrate gradients for the optimization of ultrathin polyacrylamide gel zymograms.

The preparation of ultrathin polyacrylamide gels with different kinds of gradients (pH, substrates, inhibitors) is described. By using these gels for contact printing after isoelectric focusing with Ampholines or Immobilines and for diffusion tests, the influence of pH or increasing amounts of substrates or inhibitors on enzyme and isoenzyme activities is studied. These methods are successfully applied for the optimization of zymogram techniques and for the easy characterization of industrial microbial enzyme preparations for technological purposes. With buffer-generated pH gradient gels, the pH optimum of all isoenzyme activities is demonstrated by contact printing; the total amount of isoenzyme activities dependent on pH is determined by a diffusion test. Gels with a linear gradient between 0 and 8 M urea are used for isoelectric focusing, diffusion tests and contact printing in order to differentiate the unfolding and denaturing effects of urea on isoenzymes. Alterations in polygalacturonase isoenzyme patterns dependent on urea concentration are not caused by inhibition or denaturation but by the change of charges. In respect to band sharpness and straightness urea can be added advantageously up to 2 M without changing the isoelectric points or activities of the isoenzymes. For the reproducibility of zymograms it is interesting to see that different substrate concentrations reveal different isoenzyme patterns.

High-resolution two-dimensional electrophoresis with isoelectric focusing in immobilized pH gradients.

Due to the high reproducibility of pH gradient slope and width, immobilized pH gradients (IPG) have been used as the first dimension of two-dimensional techniques in order to generate maps of constant spot position in the pI/Mr plane. However, when coupling IPG to SDS (sodium dodecyl sulphate) gels two problems were encountered: vertical streaking, due to incomplete zone solubilization in SDS, and horizontal streaking, due to spot fusion along the pH axis caused by the electroendosmosis of the charged Immobiline gels. Two methodical modifications are herewith described to overcome these drawbacks: (a) the SDS equilibration time of the first-dimension gel has been prolonged to at least 30 min; (b) the SDS electrophoresis gel has been cast together with a starting gel, containing 2.5 mM of each Immobiline species used in the first dimension, which serves as a transition from the charged to the uncharged gel.

Steady-state two-dimensional maps of very alkaline proteins in an immobilized pH 10-12 gradient, as exemplified by histone types.

Two-dimensional steady-state patterns of histones are here reported for the first time. The first dimension run consists in nonlinear immobilized pH gradients, spanning the pH 10-12 range. The second dimension run is a standard SDS-PAGE in a constant concentration (15% T) gel slab, in presence of a 6% T stacking gel. All the histone fractions analysed (II-AS, VI-S, VII-S and VIII-S) exhibit pI values between pH 11 and 12. The M(r) values range from 13 to 32 kDa, with the heaviest distribution around 18 kDa. When running all different histone fractions in a single mixture, and analysing the 2-D gel by computerized image data acquisition, a total of 35 individual spots is detected.

Gel gradient electrophoresis, isoelectric focusing and two-dimensional techniques in horizontal, ultrathin polyacrylamide layers.

An ultrathin layer, horizontal polyacrylamide gel system for electrophoresis, isoelectric focusing and two-dimensional techniques is described. Gel slabs 240 micron thin for unidimensional, or 360 micron thin for two-dimensional runs are cast on cellophane foils as support. The sample is loaded in pockets pre-cast in the gel (2--3 microliter size) or in trenches for two-dimensional experiments. The second dimension is routinely performed in concave exponential gel gradients, spanning an acrylamide concentration from 4% to 22.5%. The sensitivity with the common Coomassie Blue stain is very high, well below 0.1 microgram protein/band. Zymogram detections can be developed within a few minutes, thus retaining the band sharpness of the focused zones or of the bands separated in pore gradient electrophoresis. Sample handling, staining and destaining and gel drying and storage are greatly simplified and performed in a fraction of the time needed for conventional, thick gels in the 1-2 mm thickness range.

Isoelectric focusing in immobilized pH gradients: principle, methodology and some applications.

A new technique for generating pH gradients in isoelectric focusing is described, based on the principle that the buffering groups are covalently linked to the matrix used as anticonvective medium. For the generation of this type of pH gradient in polyacrylamide gels, a set of buffering monomers, called Immobiline (in analogy with Ampholine), is used. The pH gradient gels are cast in the same way as pore gradient gels, but instead of varying the acrylamide content, the light and heavy solutions are adjusted to different pH values with the aid of the Immobiline buffers. Available Immobiline species make it possible to generate any narrow linear pH gradient between pH 3 and 10. The behaviour of these types of gradients in isoelectric focusing is described. Immobilized pH gradients show a number of advantages compared with carrier ampholyte generated pH gradients. The most important are: (1) the cathodic drift is completely abolished; (2) they give higher resolution and higher loading capacity; (3) they have uniform conductivity and buffering capacity; (4) they represent a milieu of known and controlled ionic strength.

Stable storage conditions of Immobiline chemicals for isoelectric focusing.

Most of the problems connected with the use of the Immobiline chemicals (a set of six, non-amphoteric, acrylamido buffers having pK values in the pH 3.5-9.5 interval) can be attributed to the alkaline species (with pK values 6.2, 7.0, 8.5 and 9.3). These compounds, to varying degrees are subjected to two degradation pathways: (a) hydrolysis of the amido bond, producing free acrylic acid and a diamine, the latter unable to be incorporated into the polyacrylamide matrix; (b) spontaneous auto-polymerization, producing a number of oligomers up to n-mers, able to aggregate and precipitate large proteins. Storage of their water solutions as frozen aliquots, a method widely employed, only partially alleviates the problem. Addition of trace-amounts of inhibitors, as lately adopted by the manufacturer, could only reduce the problem of auto-polymerization, but not block the hydrolysis of the amido bond. A new solution has been found, which abolishes both phenomena: storage in n-propanol. As demonstrated by gas chromatography, HPLC analyses and two-dimensional separations of complex samples, storage in organic solvent completely abolishes both hydrolysis and auto-polymerization and allows production of highly reproducible focusing patterns.

Expansion and differentiation of human primary osteoblasts in two- and three-dimensional culture.

Despite the regenerative capability of bone, treatment of large defects often requires bone grafts. The challenge for bone grafting is to establish rapid and sufficient vascularization. Three-dimensional (3D) multicellular spheroids consisting of the relevant cell types can be used as "mini tissues" to study the complexity of angiogenesis. We investigated two-dimensional (2D) expansion, differentiation and characterization of primary osteoblasts as steps toward the establishment of 3D multicellular spheroids. Supplementation of cell culture medium with vitamin D(3) induces the osteocalcin expression of osteoblasts. An increased osteocalcin concentration of 10.8 ± 0.58 ng/ml could be measured after 19 days in supplemented medium. Vitamin D(3) has no influence on the expression of alkaline phosphatase or the deposition of calcium. Expression of these additional osteogenic markers requires addition of a cocktail of osteogenic factors that, conversely, have no influence on the expression of osteocalcin. Supplementation of the cell culture medium with both vitamin D(3) and a cocktail of osteogenic factors is recommended to produce an osteoblast phenotype that secretes osteocalcin, expresses alkaline phosphatase and deposits calcium. In such a supplemented medium, a mean osteocalcin concentration of 11.63 ± 4.85 ng/ml was secreted by the osteoblasts. Distinguishing osteoblasts and fibroblasts remains a challenge. Neither differentiated nor undifferentiated osteoblasts can be distinguished from fibroblasts by the expression of CD90, ED-A-fibronectin or α-smooth muscle actin; however, these cell types exhibit clear differences in their growth characteristics. Osteoblasts can be arranged as 3D spheroids by coating the bottom of the cell culture device with agarose. The cellular composition of 3D multicellular spheroids can be evaluated quantitatively using vital fluorescence labeling techniques. Spheroids are a promising tool for studying angiogenic and osteogenic phenomena in vivo and in vitro.

[Electrophoretical differentiation and classification of proteins. VI. Disc-electrophoresis and isoelectrical focossing of proteins and enzymes from tomatoes, cucumber, sugar-maize and onions (author's transl)]. / Uber die elektrophoretische Differenzierung und Klassifizierung von Proteinen. VI. Disk-Elektrophorese und isoelektrische Fokussierung in Polyacrylamid-Gelen von Proteinen und Enzymen aus Tomaten, Gurken, Zuckermais und Zwiebeln

Acetone-dry powders which are excellently suited for the enrichment of proteins and partially also for enzymes, have been extracted with buffers. Separation was accomplished using disc-electrophoresis and isoelectrical focusing. The protein distribution patterns of fleshy tomatoe varieties largly correspond to each other. In the protein distribution patterns of cucumbers, sugar-maize and onions, the protein pherograms indicate sort-dependency. The zymograms partially differ very much. This can not entirely be attributed to differences of the sorts.

Application of sequential extraction procedures and glycoprotein blotting for the characterization of the 2-D polypeptide patterns of barley seed proteins.

Barley (Hordeum vulgare L.) proteins were sequentially extracted from ground seeds with Tris-HCl buffer, 55% 2-propanol, 55% 2-propanol containing 1% dithiothreitol, and 6 M urea containing 2% Nonidet P-40 and 1% dithiothreitol. The protein composition of these solubility fractions was then analyzed by high resolution two-dimensional gel electrophoresis with immobilized pH gradient 4-9 in the first dimension, followed by silver staining and glycoprotein blotting, respectively, for a more detailed characterization of the two-dimensional polypeptide pattern of barley seed proteins.

Detection of polypeptides and amylase isoenzyme modifications related to malting quality during malting process of barley by two-dimensional electrophoresis and isoelectric focusing with immobilized pH gradients.

Two cultivars ("Alexis" and "Lenka") of contrasting final attenuation values were malted, and the protein and amylase isoenzyme composition, as well as the change in protein and amylase isoenzyme composition during malting, was investigated by two-dimensional polyacrylamide gel electrophoresis of total proteins, and isoelectric focusing of amylase isoenzymes, respectively. Isoelectric focusing demonstrated that significant differences exist between the amylase isoenzyme patterns of the two cultivars, suggesting a correlation between the presence of certain amylase isoenzyme bands and final attenuation. This finding was confirmed by analysis of 36 barley cultivars with a wide range of quality. It was shown that all cultivars which are of low or, at best, moderate final attenuation values exhibit the amylase band "B" (isoelectric point approximately 6.8), whereas those cultivars which are predominantly of high malting grade do not possess this "B" isoenzyme band, but exhibit the pronounced "A" isoenzyme band (isoelectric point approximately 6.5) instead, suggesting that these isoenzymes (which we suppose to be beta-amylases) can be utilized to predict the final attenuation values of unknown barley samples or new lines. However, "final attenuation" is a complex function. Preliminary results of two-dimensional gel electrophoresis indicate that other factors, such as total amount of amylases, or a 19 kDa A hordein-like polypeptide, which was degraded faster in the low malting grade cultivar "Lenka", may also have a role in determining quality.

Qualitative and quantitative changes in barley seed protein patterns during the malting process analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with respect to malting quality.

Seeds of two barley cultivars, similar in total protein content and malt extract yield but different in their final attenuation values, were malted. Samples taken at daily intervals during the malting process were extracted sequentially with Tris-HCl buffer, aqueous 2-propanol, aqueous 2-propanol containing 0.5% dithiothreitol, and 4 M urea, containing 0.5% dithiothreitol and 1% Nonidet P-40. The protein composition of these extracts was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and computer densitometry to determine whether differences observed in the rate or extent of protein modification are related to the malting quality character final attenuation. It was found that, common to both cultivars, the albumin and globulin proteins were relatively resistant to proteolysis, whereas the hordeins suffered a dramatic breakdown during malting, with the D hordein being degraded most rapidly, followed by the B and C hordeins. Besides these similarities, differences between both cultivars were observed in the relative rates of D hordein degradation, as this rate was considerably higher in the cultivar with high malting quality. Similar, but much less distinct kinetics were seen with certain B hordeins. Since a possible relationship might exist between the rate of proteolysis of the D hordeins and the character final attenuation, we analyzed a larger number of barley cultivars with different final attenuation values with a simplified technique. For the ten cultivars examined, differences during germination were again seen in the rates of modification of the D hordeins. However, significant correlations between the D hordein breakdown and final attenuation values were not obtained, so that we propose that there exists at best a loose correlation between the relative rate of proteolysis of these proteins and the malting quality character final attenuation.

Barley cultivar discrimination: I. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and glycoprotein blotting.

Two different methods of detecting electroblotted glycoproteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Tris-buffer soluble barley seed proteins were examined for their applicability for barley cultivar discrimination. These are the highly specific, lectin-based concanavalin A/peroxidase method and the more general periodate/danyslhydrazine method. The results of the periodate/dansylhydrazine method enabled us to divide the 20 examined cultivars into three groups, whereas the more sensitive concanavalin A/peroxidase method revealed six different glycoprotein patterns. In comparison, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the alcohol-soluble barley seed proteins (hordeins) gave nine different banding patterns. A combination of hordein electrophoresis together with glycoprotein staining by the concanavalin A/peroxidase method made it possible to classify the cultivars into twelve groups, the largest of which contained four cultivars. The qualitative expression of the glycoprotein patterns seemed to be independent of growth conditions, whereas the band intensities obviously were not. As a whole, glycoprotein blotting is a valuable supplement to sodium dodecyl sulfate-polyacrylamide gel electrophoresis of hordeins in barley cultivar discrimination.