Mitotic and post mitotic consequences of genomic instability induced by oncogenic Ha-Ras

Induced expression of a mutant human Ha-ras oncogene in NIH3T3 cells leads to the rapid production of multicentric chromosomes, acentric chromosome fragments, double minute chromosomes, increased heteroploidy, and increased capacity to undergo gene amplification. In this study we have used fluorescent-in-situ hybridization (FISH) to demonstrate that induction of the Ha-ras oncogene also leads to disruption of the mitotic machinery, resulting in aberrant mitoses and abnormal daughter cells. Cells induced to express an oncogenic Ha-ras transgene accumulate chromosomes that lag outside of the rest of the chromosomal architecture, chromosomes that form bridges between daughter nuclei at anaphase, and that form micronuclei. Many of these mitotic aberrations contain structurally abnormal chromosomes. Theseras-induced changes were suppressed by the introduction of a gene encoding the dominant negative effector ofras, raf 301. Expression ofraf301 in cells induced to express Ha-ras reduced the level of growth in soft agar, chromosome aberrations, mitotic aberrations, and frequency of gene amplification. These data provide evidence for an association between Ha-ras induced transformation, chromosome aberrations and gene amplification. Furthermore they offer insight into how the cell responds to the formation of aberrant chromosomes, and how disrupting chromosomal architecture could lead to further imbalances in the distribution of genetic material between daughter cells.     

Role played by NK2 receptor and cyclooxygenase activation in bradykinin B2 receptor mediated-airway effects in guinea pigs

We have investigated the effects of SR-48968, an NK₂ receptor antagonist, and indomethacin, a cyclooxygenase inhibitor, against bronchoconstriction and airway microvascular leakage induced by bradykinin (BK) in anesthetized guinea pigs. In addition, we have determined whether these effects were mediated via bradykinin B₂ receptor activation, using a B₂ receptor antagonist HOE 140. Lung resistance (R _L) and extravasation of Evans blue dye into airway tissues were used as indexes of airway caliber and microvascular leakage, respectively. BK (15 nmol/kg i.v.) induced a significant increase inR _L and leakage of dye at all airway levels, responses which were completely abolished by HOE 140 (0.13 mg/kg i.v.). SR-48968 (1.5 mg/kg i.v.) had no effect against BK-induced airway effects. Indomethacin (5 mg/kg i.v.) completely blocked the increase inR _L and significantly inhibited the leakage of dye in peripheral intrapulmonary airway. In conclusion, bronchoconstriction induced by i.v. BK is mediated by release of cyclooxygenase products but not by stimulation of NK₂ receptors, while the airway microvascular leakage only partly involves cyclooxygenase activation. Cyclooxygenase activation may occur following bradykinin B₂ receptor stimulation.     

Giant cell tumor of bone express p63.

p63 contributes to skeletal development and tumor formation; however, little is known regarding its activity in the context of bone and soft tissue neoplasms. The purpose of this study was to investigate p63 expression in giant cell tumor of bone and to determine whether it can be used to discriminate between other giant cell-rich tumors. Seventeen cases of giant cell tumor of bone were examined to determine the cell type expressing p63 and identify the isoforms present. Total RNA or cell protein was extracted from mononuclear- or giant cell-enriched fractions or intact giant cell tumor of bone and examined by RT-PCR or western blot, respectively. Immunohistochemistry was used to evaluate p63 expression in paraffin embedded sections of giant cell tumor of bone and in tumors containing multinucleated giant cells, including: giant cell tumor of tendon sheath, pigmented villonodular synovitis, aneurysmal bone cyst, chondroblastoma, and central giant cell granuloma. The mononuclear cell component in all cases of giant cell tumor of bone was found to express all forms of TAp63 (alpha, beta, and gamma), whereas only low levels of the TAp63 alpha and beta isoforms were detected in multinucleated cells; DeltaNp63 was not detected in these tumors. Western blot analysis identified p63 protein as being predominately localized to mononuclear cells compared to giant cells. This was confirmed by immunohistochemical staining of paraffin-embedded tumor sections, with expression identified in all cases of giant cell tumor of bone. Only a proportion of cases of aneurysmal bone cyst and chondroblastoma showed p63 immunoreactivity whereas it was not detected in central giant cell granuloma, giant cell tumor of tendon sheath, or pigmented villonodular synovitis. The differential expression of p63 in giant cell tumor of bone and central giant cell granuloma suggest that these two tumors may have a different pathogenesis. Moreover, p63 may be a useful biomarker to differentiate giant cell tumor of bone from central giant cell granuloma and other giant cell-rich tumors, such as giant cell tumor of tendon sheath and pigmented villonodular synovitis.     

T Cells Recognize Multiple GAD65 and Proinsulin Epitopes in Human Type 1 Diabetes, Suggesting Determinant Spreading

Human type 1 diabetes is thought to be mediated by autoreactive T cells specific for antigens expressed by pancreatic beta cells. However, it is unclear which autoantigens and determinants thereof are the targets of the autoimmune attack. Using comprehensive peptide libraries that cover the entire sequence of two major candidate autoantigens, GAD65 and proinsulin, we measured the in vivo frequencies of peptide-specific, IFN-gamma-producing memory T cells in 27 diabetic patients, 14 high risk individuals, and 15 partially HLA-matched healthy controls. Compared to the controls, both a higher number of determinants on the islet cell antigens were recognized and the frequencies of peptide specific cells were increased in patients and high risk individuals. Inclusion of signal enhancing anti-CD28 antibody further accentuated this difference. Considerable heterogeneity in peptide recognition was seen even in DRB1*04, DQB1*0302 matched individuals. Unlike its peptides, the GAD protein antigen did not recall a T cell memory response. The highly heterogeneous recognition of a multitude of peptide determinants on both autoantigens, occurring in the absence of protein recognition, and the low functional avidity of the memory cells involved jointly suggest that the autoimmune T cell repertoire in human type 1 diabetes primarily targets cryptic determinants engaged by determinant spreading.     

The economic costs associated with physical inactivity and obesity in Canada: an update.

The purpose of this analytical review was to estimate the direct and indirect economic costs of physical inactivity and obesity in Canada in 2001. The relative risks of diseases associated with physical inactivity and obesity were determined from a meta-analysis of existing prospective studies and applied to the health care costs of these diseases in Canada. Estimates were derived for both the direct health care expenditures and the indirect costs, which included the value of economic output lost because of illness, injury-related work disability, or premature death. The economic burden of physical inactivity was $5.3 billion ($1.6 billion in direct costs and $3.7 billion in indirect costs) while the cost associated with obesity was $4.3 billion ($1.6 billion of direct costs and $2.7 billion of indirect costs). The total economic costs of physical inactivity and obesity represented 2.6% and 2.2%, respectively, of the total health care costs in Canada. The results underscore the importance of public health efforts aimed at combating the current epidemics of physical inactivity and obesity in Canada.     

A preliminary study on the enhancement of the osteointegration of a novel synthetic hydroxyapatite scaffold in vivo

Synthetic hydroxyapatite, a bioactive calcium phosphate, is clinically used as a bone replacement bioceramic because of its similarity in composition to bone mineral, biocompatibility, and osteoconductive nature. The aim of this study was to evaluate the bioactivity of a novel synthetic porous hydroxyapatite (PHA) in vivo in rabbit and to investigate the enhancement of its bioactivity and osteointegration. In the investigation reported here, insulin-like growth factor-I (IGF-I) has been used to enhance the bioactivity of PHA. Cylindrical PHA implants with or without IGF-I were implanted bilaterally in rabbit femurs. Fluorochrome bone markers were administered at 7-day intervals. The implants with the attached bone were retrieved at postmortem, 1 and 3 weeks after implantation, for histological and histomorphometric analysis. Undecalcified sections stained with toluidine blue showed new bone formation. Mineralization of the new bone formed in the interface, surrounding trabecular bone, and within the pores of the implants was studied. Lamellar bone mineral apposition rate (MAR) was assessed and compared among treatment groups, sham, PHA alone, and PHA with IGF-I (500 ng/implant), by fluorochrome label incorporation using UVL microscopy. We report for the first time, that the supplementation of PHA implants with IGF-I significantly increased new bone formation and MAR (6.58 +/- 0.08 microm/day) compared with implantation of PHA alone (4.08 +/- 0.05 microm/day) or sham operation (3.11 +/- 0.12 microm/day). These results suggest that synthetic PHA might provide a delivery system for bioactive agents to accelerate bone healing in orthopedic procedures.     

Disruption of the Aspergillus niger argB gene: a tool for transformation

We disrupted the Aspergillus niger gene argB, encoding ornithine transcarbamylase. Full characterisation of the argB deletion was performed by Southern blot analysis, growth tests and by means of mitotic recombination, complementation and transformation. The argB locus was found to be physically removed, thus creating an auxotrophic mutation. The latter can be supplemented by addition of arginine into the culture medium. The argB gene and its disruption do not correlate to the argI13 (formerly argB13) allele described. The delta argB is on chromosome I whereas argI13 is on V. In addition, the argI13 mutation can only be complemented by the A. nidulans argB gene, whereas the new argB deletion can be complemented by both the A. niger and A. nidulans argB genes. The delta argB strain has been used to generate several strains in a breeding programme and to study the expression of important genes, such as areA and kexB.     

Chondroitin sulfate A released from platelets blocks RANTES presentation on cell surfaces and RANTES-dependent firm adhesion of leukocytes

The sequestration of chemokines on the surface of microvascular endothelium is an early event in the selective recruitment of leukocytes. The sequestration and presentation of chemokines must be tightly controlled to confine the extravasation of leukocytes and to prevent uncontrolled inflammation. We investigated whether soluble molecules released under physiological conditions could control chemokine immobilization on cell surfaces and function as regulatory chemokine binding molecules. We determined that human serum contains a molecule that suppresses RANTES (CCL5) binding to endothelial cells, PBMC and CHO cells. Using platelet-rich and platelet-free plasma, serum from patients with thrombocytopenia, and purified platelets, we identified platelets as the source of the chemokine-binding molecule and further identified it as chondroitin sulfate A. In contrast to platelet-derived fully-sulfated chondroitin sulfate A, low-sulfated chondroitin sulfate A present in plasma was almost inactive. Under physiological flow conditions chondroitin sulfate A was found to block RANTES-mediated firm adhesion of monocytes to endothelial cells. It also prevented RANTES-mediated influx of calcium in CCR5-transfected CHO cells while internalization of CCR5 was only marginally reduced. Taken together, chondroitin sulfate A released from platelets appears to act as an important regulatory molecule for cellular responses to chemokines.     

Alteration of the LRP1B gene region is associated with high grade of urothelial cancer

We have delineated regions of interest at chromosome 2q21.2, 2q36.3, and 2q37.1 by deletion mapping of 114 urothelial cancers (UC). Altogether, 17%, 18%, and 63% of the G1, G2, and G3 tumors displayed loss of heterozygosity at chromosome 2q, respectively, The region at 2q21.2 was narrowed down to the LRP1B gene (NT_005129.6). Hemi- and homozygous deletion at the LRP1B gene region was seen in 31 of 114 UCs. Only 8% of the UCs with G1 and none with G2 tumors showed loss of heterozygosity at the LRP1B gene, whereas 49% of the G3 UCs had allelic loss at this region. RT-PCR analysis of the LRP1B gene showed the lack of expression of several exons in 2 of 9 cases analyzed. Our analysis suggests that the LRP1B gene is a candidate tumor suppressor gene in UCs.