Backbone dynamics of the human MIA protein studied by (15)N NMR relaxation: implications for extended interactions of SH3 domains

The melanoma inhibitory activity (MIA) protein is a clinically valuable marker in patients with malignant melanoma as enhanced values diagnose metastatic melanoma stages III and IV. Here, we report the backbone dynamics of human MIA studied by (15)N NMR relaxation experiments. The folded core of human MIA is found to be rigid, but several loops connecting beta-sheets, such as the RT-loop for example, display increased mobility on picosecond to nanosecond time scales. One of the most important dynamic features is the pronounced flexibility of the distal loop, comprising residues Asp 68 to Ala 75, where motions on time scales up to milliseconds occur. Further, significant exchange contributions are observed for residues of the canonical binding site of SH3 domains including the RT-loop, the n-Src loop, for the loop comprising residues 13 to 19, which we refer to as the"disulfide loop", in part for the distal loop, and the carboxyl terminus of human MIA. The functional importance of this dynamic behavior is discussed with respect to the biological activity of several point mutations of human MIA. The results of this study suggest that the MIA protein and the recently identified highly homologous fibrocyte-derived protein (FDP)/MIA-like (MIAL) constitute a new family of secreted proteins that adopt an SH3 domain-like fold in solution with expanded ligand interactions.     

Linkage: from particulate to interactive genetics

Genetics was established on a strictparticulate conception of heredity. Geneticlinkage, the deviation from independentsegregation of Mendelian factors, was conceivedas a function of the material allocation of thefactors to the chromosomes, rather than to themultiple effects (pleiotropy) of discretefactors. Although linkage maps wereabstractions they provided strong support forthe chromosomal theory of inheritance. DirectCytogenetic evidence was scarce until X-rayinduced major chromosomal rearrangementsallowed direct correlation of genetic andcytological rearrangements. Only with thediscovery of the polytenic giant chromosomes inDrosophila larvae in the 1930s were thevirtual maps backed up by physical maps of thegenetic loci. Genetic linkage became a pivotalexperimental tool for the examination of theintegration of genetic functions in developmentand in evolution. Genetic mapping has remaineda hallmark of genetic analysis. The location ofgenes in DNA is a modern extension of thenotion of genetic linkage.     

Intravascular ultrasound assessment of culotte stent deployment for the treatment of stenoses at major coronary bifurcations

BACKGROUND: The mechanism for the disappointing late outcome following stenting of bifurcation lesions is unclear. This prospective observational study aims to evaluate culotte stent deployment and dimensions with intravascular ultrasound (IVUS). PATIENTS AND METHODS: Patients with bifurcation stenoses were treated using two stents in a culotte configuration. After optimizing the angiographic appearance of both stents, IVUS was used to evaluate both limbs of the culotte. The main outcome measures were cross-sectional area (CSA) and minimal lumen diameter (MLD) assessed by IVUS. RESULTS: Within the culotte stent, the final mean CSA in the main limb was 6.1 mm(2) (97% of reference) and in the side-limb was 5.9 mm(2) (97% of reference). However, in each case, the minimum CSA and IVUS MLD of both limbs was at the bifurcation point. For all patients, the final mean CSA at the bifurcation point of the main limb was 4.3 mm(2) (70% of main stent) and of the side-limb was 4.4 mm(2) (75% of side stent). The IVUS MLD at the bifurcation point of the main limb was 2.1 mm (78% of main stent) and of the side-limb was 2.1 mm (84% of the side stent). Importantly, this significant residual stenosis was not detectable with quantitative coronary angiography. CONCLUSIONS: IVUS evaluation of culotte stents is feasible. The minimum IVUS CSA and MLD of both limbs of the culotte stent is at the bifurcation point. Despite an optimal angiographic appearance a significant residual stenosis was noted with IVUS at each bifurcation point.     

Halofuginone does not reduce fibrosis in bleomycin-induced lung injury

Halofuginone, a coccidiostatic alkaloid, has anti-fibrotic properties, and may be useful as a therapeutic agent in lung fibrosis. To test this hypothesis we investigated the effect of halofuginone on bleomycin-induced lung fibrosis in Sprague-Dawley rats. Treatment groups included: (1) a single intratracheal (IT) instillation of 1.2U bleomycin, and intraperitoneal (IP) injection of halofuginone (0.5 mg/dose), every other day; (2) IT 1.2U bleomycin and IP distilled water (D.W.), every other day; (3) IT 0.8U bleomycin and daily IP halofuginone (0.5 mg/dose); (4) IT 0.8U bleomycin and daily IP D.W.; (5) IT saline and IP halofuginone, every other day; (6) IT saline and daily IP D.W.; (7) IT 0.625U bleomycin and oral halofuginone (10 mg/kg rodent lab chow); (8) IT 0.625U bleomycin and standard lab chow. Animals were studied 14 days after IT instillation. Lung injury was evaluated by total and differential cell count in bronchoalveolar lavage fluid, by a semi-quantitative morphological index of lung injury, and by biochemical analysis of lung hydroxyproline content. Overt signs of lung injury were apparent in bleomycin-treated rats by all measures. These changes were not affected by treatment with halofuginone, irrespective of the treatment regimen used. This study does not support the use of halofuginone to prevent or ameliorate lung fibrosis.     

A single-chain class II MHC-IgG3 fusion protein inhibits autoimmune arthritis by induction of antigen-specific hyporesponsiveness

T cells play a central role in many autoimmune diseases. A method to specifically target the function of autoreactive T cell clones would avoid the global immunosuppression associated with current therapies. To develop a molecule capable of inhibiting autoreactive T cell responses in vivo, single-chain peptide-I-A-IgG3 fusion proteins were constructed and expressed in both mammalian and insect cells. The fusion proteins were designed with an IgG3 Fc moiety to make them divalent, allowing TCR cross-linking, while lacking FcR binding and costimulation. The fusion proteins stimulated T cell hybridomas in vitro in a peptide-specific, MHC-restricted manner but failed to do so in soluble form. In vivo administration of an I-A(q) fusion protein, containing an immunodominant collagen II peptide, significantly delayed the onset and reduced the severity of collagen-induced arthritis in DBA/1 mice by induction of Ag-specific hyporesponsiveness. Such fusion proteins may be useful to study novel therapeutic approaches for T cell-mediated autoimmune diseases.     

Energy estimation in protein design

The progress achieved by several groups in the field of computational protein design shows that successful design methods include two major features: efficient algorithms to deal with the combinatorial exploration of sequence space and optimal energy functions to rank sequences according to their fitness for the given fold.     

Oncostatin M induces tissue-type plasminogen activator and plasminogen activator inhibitor-1 in Calu-1 lung carcinoma cells

Oncostatin M (OSM) is a glycoprotein cytokine that is produced by activated T-lymphocytes, monocytes, and macrophages. In a DNA synthesis assay, OSM reduced tritiated thymidine incorporation by 53% in Calu-1 lung carcinoma cells. Radiolabeled cDNAs from untreated Calu-1 cells and 30-h OSM-treated cells were used to probe duplicate nylon membrane cDNA expression arrays. This study revealed OSM-mediated expression of mRNAs encoding tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1). Northern blot analysis showed that the steady-state level of tPA mRNA is nearly undetectable in Calu-1 cells. Exposure of these cells to OSM for 30 h increased tPA mRNA expression by 20-fold and PAI-1 mRNA expression by 5-fold. Exposure of these cells to other gp130 receptor family cytokines, including leukemia inhibitory factor (LIF), interleukin-6 (IL-6), and IL-11, do not significantly affect DNA synthesis or induction of tPA/PAI-1. Western blot studies demonstrated that OSM mediates a marked increase in secretion of the tPA protein. Secreted tPA was present in the conditioned medium almost exclusively as tPA/PAI-1 complexes. Inhibitor studies demonstrated that OSM-mediated induction of tPA and PAI-1 mRNAs is largely dependent upon activation of the MEK1/2 pathway. The JAK3/STAT3 pathway potentially serves a secondary role in these regulatory events.     

Machine scoring of Her2/neu immunohistochemical stains

OBJECTIVE: To establish the feasibility of a machine scoring method for her2/neu immunohistochemistry in samples of breast carcinoma. STUDY DESIGN: A total of 65 consecutive cases of breast carcinoma with immunohistochemical stainingfor her2/neu by the Herceptest (Dako Corp., Carpinteria, California, U.S.A.) method (DAB chromogen with hematoxylin counterstain) were analyzed using an Extended Slide Wizard (Tripath Imaging, Inc., Burlington, North Carolina, U.S.A.) workstation running prototype software. Representative fields of view from the positive control, negative control and up to 10 fields from the stained tumor sample were captured interactively with a phased alternating line 3 CCD color camera. To determine the amount of specific membrane staining, chromogen separation of nuclear counterstain and membrane-positive stain was performed based on their respective absorption coefficients in the three color channels. The amount of specific membrane staining was scored based on a training set covering the rangefrom 0 to 3 + staining scores according to Dako. Manual scores of 2 + were tested for amplification by fluorescence in situ hybridization. RESULTS: The automated scoring results correlated highly with the manual scores obtained per the Herceptest (Dako) instructions (R2>.92). The results were obtained in real time in the interactive mode. CONCLUSION: Machine scoring of immunohistochemical stains is practical, rapid and inherently reproducible, especially for samples with 1+ and 2+ manual scores.