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.     

Expression of functional recombinant human lysozyme in transgenic rice cell culture

Using particle bombardment-mediated transformation, a codon-optimized synthetic gene for human lysozyme was introduced into the calli of rice (Oryza sativa) cultivar Taipei 309. The expression levels of recombinant human lysozyme in the transformed rice suspension cell culture approached approximately 4% of total soluble protein. Recombinant human lysozyme was purified to greater than 95% homogeneity using a two-step chromatography process. Amino acid sequencing verified that the N-terminus of the mature recombinant human lysozyme was identical to native human lysozyme. This indicates that the rice RAmy3D signal peptide was correctly cleaved off from the human lysozyme preprotein by endogenous rice signal peptidase. Recombinant human lysozyme was found to have the same molecular mass, isoelectric point and specific activity as native human lysozyme. The bactericidal activity of recombinant human lysozyme was determined by turbidimetric assay using Micrococcus lysodeikticus in 96-well microtiter plates. The bactericidal activity of lysozyme on Gram-negative bacteria was examined by adding purified lysozyme to mid-log phase cultures of E. coli strain JM109. In this study, significant bactericidal activity was observed after E.coli cells were exposed to recombinant human lysozyme for 60min. Both native and recombinant human lysozyme displayed the same thermostability and resistance to degradation by low pH. The potential for using rice-derived lysozyme as an antimicrobial food supplement, particularly for infant formula and baby foods, is discussed.     

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.     

Oligosaccharyltransferase isoforms that contain different catalytic STT3 subunits have distinct enzymatic properties

Oligosaccharyltransferase (OST) is an integral membrane protein that catalyzes N-linked glycosylation of nascent proteins in the lumen of the endoplasmic reticulum. Although the yeast OST is an octamer assembled from nonhomologous subunits (Ost1p, Ost2p, Ost3p/Ost6p, Ost4p, Ost5p, Wbp1p, Swp1p, and Stt3p), the composition of the vertebrate OST was less well defined. The roles of specific OST subunits remained enigmatic. Here we show that genomes of most multicellular eukaryotes encode two homologs of Stt3p and mammals express two homologs of Ost3p. The Stt3p and Ost3p homologs are assembled together with the previously described mammalian OST subunits (ribophorins I and II, OST48, and DAD1) into complexes that differ significantly in enzymatic activity. Tissue and cell type-specific differences in expression of the Stt3p homologs suggest that the enzymatic properties of oligosaccharyltransferase are regulated in eukaryotes to respond to alterations in glycoprotein flux through the secretory pathway and may contribute to tissue-specific glycan heterogeneity.