Generation of 19 STS markers that can be anchored at specific sites on human chromosome 21.

Sequence-tagged sites (STSs) are short stretches of DNA that can be specifically detected by the polymerase chain reaction (PCR) and can be used to construct long-range physical maps of chromosomal DNA. These STSs can be detected by PCR assays developed by reference to data obtained from the sequencing of restriction fragment length polymorphism-DNA markers for chromosome 21, which were derived from recombinant lamba-phage and plasmid clones made from DNA of a human-hamster hybrid cell line. In this report, we describe the generation of 19 new STSs that are specific for human chromosome 21.     

Rapid Identification of Viruses by Indirect Immunofluorescence: Standardization and Use of Antiserum Pool to Nine Respiratory Viruses

The indirect fluorescent antibody test employing treated and standardized antisera and conjugated antiglobulin has been used successfully, in conjunction with a technique for growing and staining virus cell systems in situ on microscope slides, in the identification of nine respiratory viruses. By using pooled antisera in a single test, the presence or absence of these viruses was determined in 18 to 45 hr after inoculation of slide microtissue culture.     

ATP-dependent regulation of nuclear Ca(2+) levels in plant cells

Localised alterations in cytoplasmic Ca(2+) levels are an integral part of the response of eukaryotic cells to a plethora of external stimuli. Due to the large size of nuclear pores, it has generally been assumed that intranuclear Ca(2+) levels reflect the prevailing cytoplasmic Ca(2+) levels. Using nuclei prepared from carrot (Daucus carota L.) cells, we now show that Ca(2+) can be transported across nuclear membranes in an ATP-dependent manner and that over 95% of Ca(2+) is accumulated into a pool releasable by the Ca(2+) ionophore A.23187. ATP-dependent nuclear Ca(2+) uptake did not occur in the presence of ADP or ADPgammaS and was abolished by orthovanadate. Confocal microscopy of nuclei loaded with dextran-linked Indo-1 showed that the initial ATP-induced rise in [Ca(2+)] occurs in the nuclear periphery. The occurrence of ATP-dependent Ca(2+) uptake in plant nuclei suggests that alterations of intranuclear Ca(2+) levels may occur independently of cytoplasmic [Ca(2+)] changes.     

Determination of immunoreactive endothelin in medium from cultured endothelial cells and human plasma

We have developed a sensitive and selective radioimmunoassay for porcine/human endothelin (ET1). The assay has a detection limit of 0.62 pg/tube and exhibits no cross-reactivity to atrial natriuretic peptide, arginine vasopressin, or angiotensin II. Procedures were developed for extraction of endothelin from human plasma samples and samples of buffer from endothelial cell incubations using C18 Sep-Pak extraction cartridges. The mean recovery following extraction was approximately 80%. Both bovine and porcine aortic endothelial cells were found to produce immunoreactive endothelin (IR-ET) with porcine cells producing 4.7 +/- 1.1 ng of IR-ET/mg cell protein after 6 hours. Human plasma samples were extracted, assayed and found to contain a mean concentration of 2.0 +/- 0.4 pg/ml of IR-ET.     

Phylogeny of the genus Turris: correlating molecular data with radular anatomy and shell morphology

There are over 10,000 species of venomous marine molluscs, the vast majority of these, which are generally referred to as "turrids", are traditionally assigned to a single family, Turridae (Powell 1966). Here, we provide an initial molecular analysis of the type genus of the family, Turris R?ding, 1798, thought to be among the most well characterized groups in the family. We show that the type genus is not monophyletic. We analyzed specimens conventionally assigned to 9 different Turris species using molecular markers, combined with the shell morphology and radular anatomy whenever feasible. The results suggest that species assigned to the genus Turris, provisionally assigned to two different subgenera are not monophyletic. Five previously described species belong to the subgenus Turris (s.s.) R?ding 1798: Turris babylonia, (Linne, 1758), Turris grandis, (J. E. Gray, 1834), Turris dollyae, (Olivera, 1999), Turris normandavidsoni (Olivera, 1999) and Turris spectabilis (Reeve, 1843). With a change in species designation, Turris assyria (formerly T. babylonia1010) is added to a well-defined clade, which is in turn more closely related to Lophiotoma and Gemmula species than to the other five Turris species. We show that these five species conventionally assigned to Turris do not belong in the same subgenus, and form a clade provisionally designated as AnnulaturrisPowell, 1966: Turris annulata, (Reeve, 1843), Turris undosa, (Lamarck, 1816), Turris cristata, (Vera-Peláez, Vega-Luz, and Lozano-Francisco 2000) Turris cryptorrhaphe (G. B. Sowerby, 1825) and Turris nadaensis (Azuma, 1973). Implications of the molecular phylogenetic results and its correlation with radular morphology are discussed.     

Linkage disequilibrium patterns vary with chromosomal location: a case study from the von Willebrand factor region.

Linkage disequilibrium analysis has been used as a tool for analyzing marker order and locating disease genes. Under appropriate circumstances, disequilibrium patterns reflect recombination events that have occurred throughout a population's history. As a result, disequilibrium mapping may be useful in genomic regions of < 1 cM where the number of informative meioses needed to detect recombinant individuals within pedigrees is exceptionally high. Its utility for refining target areas for candidate disease genes before initiating chromosomal walks and cloning experiments will be enhanced as the relationship between linkage disequilibrium and physical distance is better understood. To address this issue, we have characterized linkage disequilibrium in a 144-kb region of the von Willebrand factor gene on chromosome 12. Sixty CEPH and 12 von Willebrand disease families were genotyped for five PCR-based markers, which include two microsatellite repeats and three single-base-pair substitutions. Linkage disequilibrium and physical distance between polymorphisms are highly correlated (rm = -.76; P < .05) within this region. None of the five markers showed significant disequilibrium with the von Willebrand disease phenotype. The linkage disequilibrium/physical distance relationship was also analyzed as a function of chromosomal location for this and eight previously characterized regions. This analysis revealed a general trend in which linkage disequilibrium dissipates more rapidly with physical distance in telomeric regions than in centromeric regions. This trend is consistent with higher recombination rates near telomeres.     

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.     

Gene expression in visceral endoderm: a comparison of mutant and wild- type F9 embryonal carcinoma cell differentiation

We have examined the abundance and cell specificity of several mRNAs that are regulated during the retinoic acid (RA)-induced differentiation of F9 embryonal carcinoma cells to visceral endoderm. The experiments confirmed the multistep nature of this process by demonstrating the expression of the ERA-1/Hox 1.6 message within 6 h after RA addition; the expression of messages specific for the extracellular matrix proteins laminin B1 and B2, and collagen IV(alpha 1) between days 4 and 12; and the expression of two visceral endoderm markers, alpha-fetoprotein (AFP) and H19, by days 8-15. In situ hybridization experiments revealed that the collagen IV(alpha 1) mRNA is restricted to the outer cell layer of F9 cell aggregates regardless of the presence or absence of RA. Laminin B1 and B2 mRNAs are concentrated in the outer cell layer of RA-treated aggregates although significant levels of message are also observed within the interior cells of the aggregates. Unexpectedly, AFP mRNA is detectable in only a subset of the outer cells of F9 cell aggregates grown 15 d in the presence of RA. The results obtained from wild-type F9 cells were compared with those from a mutant F9 cell line, RA-5-1, which was previously shown to synthesize collagen IV containing six- to ninefold less 4-hydroxyproline than that in wild-type F9 cells. RA-5-1 cells exhibit four- to sixfold less of the mRNAs encoding two visceral endoderm proteins, AFP and H19, than wild-type F9 cells after RA treatment of RA-5-1 aggregates. RA-5-1 cells, however, do exhibit an RA-associated increase in the level of ERA-1/Hox 1.6 mRNA within 6 h after adding RA. Although the collagen IV protein level is similar in wild-type F9 and RA-5-1 aggregates, the collagen IV(alpha 1) message level is 6-20-fold greater in aggregates of mutant cells than in aggregates of wild-type cells. Moreover, in situ hybridizations showed that this message is evenly distributed throughout the RA-5-1 aggregates rather than restricted to the outer cell layers as it is in wild-type F9 aggregates. These results suggest that abnormal collagen IV expression and localization are associated with decreased expression of the visceral endoderm markers, AFP and H19, in RA-5-1 cell aggregates.