Genetic studies of B-lymphocyte deficiency and mastocytosis in strain A/WySnJ mice

The A/WySnJ mouse, but not the related A/J strain, has peripheral B-lymphocyte deficiency and mastocytosis. Minimally, two quantitative trait loci (QTLs) control the B-cell deficiency in (A/WySnJ x CAST/Ei)F2 intercross mice; one of them, Bcmd-1, mapped to Chromosome (Chr) 15. Several QTLs controlled the mastocytosis in this intercross, and it was not possible to determine whether any of them co-segregated with Bcmd-1. We have now mapped a second QTL controlling the B-cell deficiency, Bcmd-2, to Chr 4. Furthermore, we narrowed the map position of Bcmd-1 to <2.0 cM. Both QTLs have been confirmed through the construction of AW. Bcmd-1(c), AW. Bcmd-2(c), and AW. Bcmd-1(c)Bcmd-2(c) recombinant congenic strains. The Bcmd-1 locus is the major regulator of B-cell homeostasis, while Bcmd-2 is the minor regulator, and their effects are additive, as shown by splenic B-cells analysis in these congenic strains. In addition, Bcmd-2 or a linked locus controls mastocytosis, while Bcmd-1 does not, as indicated by splenic mast cell analysis in the congenic strains. Thus, the major genetic controls on B-cell homeostasis and mast cell homeostasis in A/WySnJ mice are asserted by distinct genes.     

Chemiluminescent measurement of telomere DNA content in biopsies

Telomeres are nucleoprotein complexes that protect the ends of chromosomes from fusion and degradation. They are typically shorter in tumor cells than in paired normal cells, and shorter telomeres are associated with poor outcome in cancer. We previously described a slot blot-based methodfor measuring telomere DNA content, a proxy for telomere length. Although this method represented an improvement over existing methods, its 30-ng limit of sensitivity was insufficient for use with biopsy or other scant tissues. Here we describe a chemiluminescent slot blot assay for telomere DNA content that has the sensitivity required for use with biopsy materials. The results obtained with DNA derived from human placental, HeLa, human peripheral blood lymphocytes, sham-needle core prostate biopsies, and archival prostatectomy tissues demonstrated that telomere DNA content can be reliably and reproducibly measured in 5 ng, and sometimes as little as 2 ng, genomic DNA. Sham-needle core prostate biopsy and prostatectomy specimens processed in parallel produced comparable results. The contribution of truncated telomeres in admixtures containing as much as 75% normal placental DNA could be established. We also demonstrated that the treatment of tissue with formalin before DNA purification does not decrease the efficacy of the assay.     

Adriamycin-induced senescence in breast tumor cells involves functional p53 and telomere dysfunction

Direct experimental evidence implicates telomere erosion as a primary cause of cellular senescence. Using a well characterized model system for breast cancer, we define here the molecular and cellular consequences of adriamycin treatment in breast tumor cells. Cells acutely exposed to adriamycin exhibited an increase in p53 activity, a decline in telomerase activity, and a dramatic increase in beta-galactosidase, a marker of senescence. Inactivation of wild-type p53 resulted in a transition of the cellular response to adriamycin treatment from replicative senescence to delayed apoptosis, demonstrating that p53 plays an integral role in the fate of breast tumor cells treated with DNA-damaging agents. Stable introduction of hTERT, the catalytic protein component of telomerase, into MCF-7 cells caused an increase in telomerase activity and telomere length. Treatment of MCF-7-hTERT cells with adriamycin produced an identical senescence response as controls without signs of telomere shortening, indicating that the senescence after treatment is telomere length-independent. However, we found that exposure to adriamycin resulted in an overrepresentation of cytogenetic changes involving telomeres, showing an altered telomere state induced by adriamycin is probably a causal factor leading to the senescence phenotype. To our knowledge, these data are the first to demonstrate that the mechanism of adriamycin-induced senescence is dependent on both functional p53 and telomere dysfunction rather than overall shortening.     

Channel openings are necessary but not sufficient for use-dependent block of cardiac Na(+) channels by flecainide: evidence from the analysis of disease-linked mutations

Na(+) channel blockers such as flecainide have found renewed usefulness in the diagnosis and treatment of two clinical syndromes arising from inherited mutations in SCN5A, the gene encoding the alpha subunit of the cardiac voltage-gated Na(+) channel. The Brugada syndrome (BrS) and the LQT-3 variant of the Long QT syndrome are caused by disease-linked SCN5A mutations that act to change functional and pharmacological properties of the channel. Here we have explored a set of SCN5A mutations linked both to BrS and LQT-3 to determine what disease-modified channel properties underlie distinct responses to the Na(+) channel blocker flecainide. We focused on flecainide block that develops with repetitive channel activity, so-called use-dependent block (UDB). Our results indicate that mutation-induced changes in the voltage-dependence of channel availability (inactivation) may act as determinants of flecainide block. The data further indicate that UDB by flecainide requires channel opening, but is not likely due to open channel block. Rather, flecainide appears to interact with inactivation states that follow depolarization-induced channel opening, and mutation-induced changes in channel inactivation will alter flecainide block independent of the disease to which the mutation is linked. Analysis of flecainide block of mutant channels linked to these rare disorders has provided novel insight into the molecular determinants of drug action.     

Embryonic hatching enzyme strypsin/ISP1 is expressed with ISP2 in endometrial glands during implantation

Embryo hatching and outgrowth are the first critical steps on the way to a successful pregnancy. It is generally held that serine proteases are responsible for this process, although the exact mechanisms of action are not clearly understood. Recently, we described two novel implantation serine proteinase (ISP) genes that are expressed during the implantation period. The ISP1 gene encodes the embryo-derived enzyme strypsin, which is necessary for blastocyst hatching in vitro and the initiation of invasion. The ISP2 gene, which encodes a related tryptase, is expressed in endometrial glands and is regulated by progesterone during the peri-implantation period. Based on similarities between ISP2 gene expression and that of a progesterone-regulated lumenal serine proteinase activity associated with lysis of the zona pellucida, we have suggested that the strypsin related protein, ISP2, may encode a zona lysin proteinase. As tryptases naturally assemble to form tetrameric structures, we have hypothesized that ISP1 and ISP2 tetramerize to form strypsin and lysin, respectively. In this study, we demonstrate that like ISP2, the ISP1 gene is also expressed in endometrial glands and is positively regulated by progesterone during implantation. Using in situ hybridization of adjacent tissue sections, we show that the ISP1 and ISP2 genes are co-expressed within the endometrial gland. Following evidence that ISP1 and 2 can efficiently form homotetramers and heterotetramers in silico, we suggest that ISP heterotetramers may be also be secreted into the uterine lumen during the implantation period. That the embryonic hatching enzyme, may also be secreted into the uterine lumen from uterus, may provide insight into the mechanisms of hatching and implantation initiation.     

Angiogenic factors and alveolar vasculature: development and alterations by injury in very premature baboons

Proper formation of the pulmonary microvasculature is essential for normal lung development and gas exchange. Lung microvascular development may be disrupted by chronic injury of developing lungs in clinical diseases such as bronchopulmonary dysplasia. We examined microvascular development, angiogenic growth factors, and endothelial cell receptors in a fetal baboon model of chronic lung disease (CLD). In the last third of gestation, the endothelial cell marker platelet endothelial cell adhesion molecule (PECAM)-1 increased 7.5-fold, and capillaries immunostained for PECAM-1 changed from a central location in airspace septa to a subepithelial location. In premature animals delivered at 67% of term and supported with oxygen and ventilation for 14 days, PECAM-1 protein and capillary density did not increase, suggesting failure to expand the capillary network. The capillaries of the CLD animals were dysmorphic and not subepithelial. The angiogenic growth factor vascular endothelial growth factor (VEGF) and its receptor fms-like tyrosine kinase receptor (Flt-1) were significantly decreased in CLD. Angiopoietin-1, another angiogenic growth factor, and its receptor tyrosine kinase with immunoglobulin and epidermal growth factor homology domains were not significantly changed. These data suggest that CLD impairs lung microvascular development and that a possible mechanism is disruption of VEGF and Flt-1 expression.     

Analysis of similarity within 142 pairs of orthologous intergenic regions of Caenorhabditis elegans and Caenorhabditis briggsae

Patterns of similarity between genomes of related species reflect the distribution of selective constraint within DNA. We analyzed alignments of 142 orthologous intergenic regions of Caenorhabditis elegans and Caenorhabditis briggsae and found a mosaic pattern with regions of high similarity (phylogenetic footprints) interspersed with non-alignable sequences. Footprints cover ~20% of intergenic regions, often occur in clumps and are rare within 5′ UTRs but common within 3′ UTRs. The footprints have a higher ratio of transitions to transversions than expected at random and a higher GC content than the rest of the intergenic region. The number of footprints and the GC content of footprints within an intergenic region are higher when genes are oriented so that their 5′ ends form the boundaries of the intergenic region. Overall, the patterns and characteristics identified here, along with other comparative and experimental studies, suggest that many footprints have a regulatory function, although other types of function are also possible. These conclusions may be quite general across eukaryotes, and the characteristics of conserved regulatory elements determined from genomic comparisons can be useful in prediction of regulation sites within individual DNA sequences.     

A method for assaying the sensitivity of Drosophila replication checkpoint mutants to anti-cancer and DNA-damaging drugs.

In multi-cellular organisms, failure to properly regulate cell-cycle progression can result in inappropriate cell death or uncontrolled cell division leading to tumor formation. To guard against such events, conserved regulatory mechanisms called "checkpoints" block progression into mitosis in response to DNA damage and incomplete replication, as well as in response to other signals. Checkpoint mutants in organisms as diverse as yeast and humans are sensitive to various chemical agents that inhibit DNA replication or cause DNA damage. This phenomenon is the primary rationale for chemotherapy, which uses drugs that preferentially target tumor cells with compromised checkpoints. In this study, we demonstrate the use of Drosophila checkpoint mutants as a system for assaying the effects of various DNA-damaging and anti-cancer agents in a developing multicellular organism. Dwee1, grp and mei-41 are genes that encode kinases that function in the DNA replication checkpoint. We tested zygotic mutants of each gene for sensitivity to the DNA replication inhibitor hydroxyurea (HU), methyl methanosulfonate (MMS), ara-C, cisplatin, and the oxygen radical generating compound paraquat. The mutants show distinct differences in their sensitivity to each of the drugs tested, suggesting an underlying complexity in the responses of individual checkpoint genes to genotoxic stress.     

BMP treatment of C3H10T1/2 mesenchymal stem cells induces both chondrogenesis and osteogenesis

The molecular mechanisms by which bone morphogenetic proteins (BMPs) promote skeletal cell differentiation were investigated in the murine mesenchymal stem cell line C3H10T1/2. Both BMP-7 and BMP-2 induced C3H10T1/2 cells to undergo a sequential pattern of chondrogenic followed by osteogenic differentiation that was dependent on both the concentration and the continuous presence of BMP in the growth media. Differentiation was determined by the expression of chondrogenesis and osteogenesis associated matrix genes. Subsequent experiments using BMP-7 demonstrated that withdrawal of BMP from the growth media led to a complete loss of skeletal cell differentiation accompanied by adipogenic differentiation of these cells. Continuous treatment with BMP-7 increased the expression of Sox9, Msx 2, and c-fos during the periods of chondrogenic differentiation after which point their expression decreased. In contrast, Dlx 5 expression was induced by BMP-7 treatment and remained elevated throughout the time-course of skeletal cell differentiation. Runx2/Cbfa1 was not detected by ribonuclease protection assay (RPA) and did not appear to be induced by BMP-7. The sequential nature of differentiation of chondrocytic and osteoblastic cells and the necessity for continuous BMP treatment to maintain skeletal cell differentiation suggests that the maintenance of selective differentiation of the two skeletal cell lineages might be dependent on BMP-7-regulated expression of other morphogenetic factors. An examination of the expression of Wnt, transforming growth factor-beta (TGF-beta), and the hedgehog family of morphogens showed that Wnt 5b, Wnt 11, BMP-4, growth and differentiation factor-1 (GDF-1), Sonic hedgehog (Shh), and Indian hedgehog (Ihh) were endogenously expressed by C3H10T1/2 cells. Wnt 11, BMP-4, and GDF-1 expression were inhibited by BMP-7 treatment in a dose-dependent manner while Wnt 5b and Shh were selectively induced by BMP-7 during the period of chondrogenic differentiation. Ihh expression also showed induction by BMP-7 treatment, however, the period of maximal expression was during the later time-points, corresponding to osteogenic differentiation. An interesting phenomenon was that BMP-7 activity could be further enhanced twofold by growing the cells in a more nutrient-rich media. In summary, the murine mesenchymal stem cell line C3H10T1/2 was induced to follow an endochondral sequence of chondrogenic and osteogenic differentiation dependent on both dose and continual presence of BMP-7 and enhanced by a nutrient-rich media. Our preliminary results suggest that the induction of osteogenesis is dependent on the secondary regulation of factors that control osteogenesis through an autocrine mechanism.     

Microbial degradation of high and low molecular weight polyaromatic hydrocarbons in a two-phase partitioning bioreactor by two strains of Sphingomonas sp.

A mixture of six polyaromatic hydrocarbons (naphthalene, phenanthrene, fluoranthene, pyrene, chyrysene and benzo[a]pyrene), varying in size from 2 to 5 rings, was dissolved in dodecane, and used as the delivery phase of a partitioning bioreactor. Two species of Sphingomonas were then used individually, and as a consortium, to determine which of the PAHs were degraded. Only low molecular weight PAHs (naphthalene, phenanthrene and fluoranthene) were degraded by the individual strains, but the consortium degraded all substrates either to completion or near completion.