Salt-induced structural changes of nucleosome core particles.

The effects of sodium chloride concentration on the structure of chicken erythrocyte nucleosome core particles have been studied by the use of fluorescently labelled histones. Histone H3 was modified with two sulfhydryl-specific dyes and reconstituted into core nucleosomes. Between 10^?4 m and 0.6 M-NaCl four different states were observed by the fluorescent techniques of collisional quenching, polarization and energy transfer. Below 5 × 10^?4 m-NaCl the nucleosome is flexible, with the single cysteine residues of the two H3 species about 48 Å apart and somewhat exposed. Between 5 × 10^?3 m and 10^?1 m-NaCl the nucleosome is rigid and non-spherical. The cysteine residues are close together and buried. Between 10^?1 m and 4 × 10^?1 m-NaCl, the cysteines become slightly more exposed but remain close together. At 6 × 10^?1 m-NaCl the nucleosome is very flexible. The cysteines are more than 70 Å apart and are quite exposed. The dramatic structural changes that are observed in core nucleosomes are consistent with the variety of functions in which they must participate in the cell.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging

A major aim of proteomics is the identification of proteins in a given proteome at a given metabolic state. This protocol describes the step-by-step labeling, purification and detection of newly synthesized proteins in mammalian cells using the non-canonical amino acid azidohomoalanine (AHA). In this method, metabolic labeling of newly synthesized proteins with AHA endows them with the unique chemical functionality of the azide group. In the subsequent click chemistry tagging reaction, azide-labeled proteins are covalently coupled to an alkyne-bearing affinity tag. After avidin-based affinity purification and on-resin trypsinization, the resulting peptide mixture is subjected to tandem mass spectrometry for identification. In combination with deuterated leucine-based metabolic colabeling, candidate proteins can be immediately validated. Bioorthogonal non-canonical amino-acid tagging can be combined with any subcellular fractionation, immunopurification or other proteomic method to identify specific subproteomes, thereby reducing sample complexity and enabling the identification of subtle changes in a proteome. This protocol can be completed in 5 days.     

Inducible expression of inflammatory chemokines in respiratory syncytial virus-infected mice: role of MIP-1alpha in lung pathology

Lower respiratory tract disease caused by respiratory syncytial virus (RSV) is characterized by profound airway mucosa inflammation, both in infants with naturally acquired infection and in experimentally inoculated animal models. Chemokines are central regulatory molecules in inflammatory, immune, and infectious processes of the lung. In this study, we demonstrate that intranasal infection of BALB/c mice with RSV A results in inducible expression of lung chemokines belonging to the CXC (MIP-2 and IP-10), CC (RANTES, eotaxin, MIP-1beta, MIP-1alpha, MCP-1, TCA-3) and C (lymphotactin) families. Chemokine mRNA expression occurred as early as 24 h following inoculation and persisted for at least 5 days in mice inoculated with the highest dose of virus (10(7) PFU). In general, levels of chemokine mRNA and protein were dependent on the dose of RSV inoculum and paralleled the intensity of lung cellular inflammation. Immunohisthochemical studies indicated that RSV-induced expression of MIP-1alpha, one of the most abundantly expressed chemokines, was primarily localized in epithelial cells of the alveoli and bronchioles, as well as in adjoining capillary endothelium. Genetically altered mice with a selective deletion of the MIP-1alpha gene (-/- mice) demonstrated a significant reduction in lung inflammation following RSV infection, compared to control littermates (+/+ mice). Despite the paucity of infiltrating cells, the peak RSV titer in the lung of -/- mice was not significantly different from that observed in +/+ mice. These results provide the first direct evidence that RSV infection may induce lung inflammation via the early production of inflammatory chemokines.     

Untersuchungen über die Osteon- und Lamellenformen im Extremitätenskelet des Erwachsenen

Zusammenfassung Auf Grund der Untersuchung von Knochenquerschnitten eines gesunden 43jährigen Mannes, bestätigt durch noch zu veröffentlichende Untersuchungen an Tibien verschiedener Individuen, werden die Strukturformen des Knochens nach ihrem Querschnittsbild beschrieben.Es wird zwischen Osteonen und Tangentiallamellen unterschieden. Zu den Tangentiallamellen mit dem mehr oder minder parallelen Verlauf zur Knochenoberfläche gehören die bisher als Generallamellen und Schaltlamellen beschriebenen Systeme.Soweit eine Lamellengliederung vorhanden ist, zeichnen sich die Tangentiallamellen durch den strengen Wechsel zwischen flach und steil gewickelten aus.Auf Grund des Querschnittsbildes werden verschiedene Osteonformen unterschieden. Die Größe des einzelnen Osteonquerschnittes wird mit Hilfe der Lamellenzahl bestimmt. Gleichzeitig wird die Steigungsfolge beachtet, d. h. der Wechsel des Kollagenfaserverlaufs von Lamelle zu Lamelle.Es ergibt sich, daß die kleineren Osteone überwiegend in der peripheren Schnitthälfte, die größeren dagegen in der zentralen liegen. Der regelmäßige Wechsel der Steigungsfolge nimmt von den kleineren zu den größeren Osteonen hin ab, die mehr steile Verlaufsweise dagegen zu. Die kleineren Größenklassen lassen häufiger die lamelläre Gliederung vermissen als die großen.Abschließend wird erörtert, daß sowohl das Osteon wie die Lamelle nur als eine besondere Lagerungsform der Kollagenfasern im Knochen angesehen werden können. Der Begriff Osteon wurde in Anlehnung an die Begriffe der überwiegend zellulären Einheiten Neuron und Chondron bzw. der sog. Entwicklungs- und Funktionseinheit Nephron gebildet. Die zirkuläre Lagerung der Kollagenfasern hat aller Voraussicht nach eine besondere festigkeitstheoretische Bedeutung. Sie ist aber abhängig vom Gefäßbaum, an dessen Verzweigungen Doppelbildungen auftreten. Diese Doppelbildungen teilen sich und begleiten die Gefäßäste. Sie werden damit zum Osteon, das sich nach Querschnittsgröße und Wicklung in benachbarten Querschnitten verschieden verhalten kann. Die zirkuläre Wicklung führt nicht zu individuellen Gebilden, die den Knochen wie Bausteine aufbauen. Sie stellt ein System dar, das den Gefäßbaum im Knochen in mehr oder minder kontinuierlichem Zusammenhang begleitet. Die zirkulären Wicklungen gehen ohne Abgrenzung in die übrigen Lamellensysteme, die Tangentiallamellen, über. Osteone und Tangentiallamellen erscheinen damit als eine übergeordnete Lagerungsform der Kollagenfasern. Die nächstniedere Stufe der Lagerung ist die Lamelle.Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Localization of a Cell Adhesion Site on Collagen XIV (Undulin)

Cell adhesion to collagen XIV is implied to be mediated by proteoglycans as cellular receptors (T. Ehniset al.,1996,Exp. Cell Res.229, 388–397). In order to define the cell binding region(s), fusion proteins expressed inEscherichia coliand covering the large noncollagenous domain NC3 of collagen XIV were used as substrates for the adhesion of skin fibroblasts. A prominent cell binding site could be localized in the N-terminal fibronectin type III repeat of collagen XIV and its immediate C-terminal extension. Since this region also mediates the binding of the small chondroitin/dermatan sulfate proteoglycan decorin (T. Ehniset al.,1997,J. Biol. Chem.272, 20414–20419), our finding could provide the molecular basis for the observation that decorin serves as inhibitor and potential modulator of cellular interactions with collagen XIV.