Exclusion mapping of the X-linked dominant chondrodysplasia punctata/ichthyosis/cataract/short stature (Happle) syndrome: possible involvement of an unstable pre-mutation

Summary Homology with the mouse bare patches mutant suggests that the gene for the X-linked dominant chondrodysplasia punctata / ichthyosis / cataract / short stature syndrome (Happle syndrome) is located in the human Xq28 region. To test this hypothesis, we performed a linkage study in three families comprising a total of 12 informative meioses. Multiple recombinations appear to exclude the Xq28 region as the site of the gene. Surprisingly, multiple crossovers were also found with 26 other markers spread along the rest of the X chromosome. Two-point linkage analysis and analysis of recombination chromosomes seem to exclude the gene from the entire X chromosome. Three different mechanisms are discussed that could explain the apparent exclusion of an X-linked gene from the X chromosome by linkage analysis: (a) different mutations on the X chromosome disturbing X inactivation, (b) metabolic interference, i.e. allele incompatibility of an X-linked gene, and (c) an unstable pre-mutation that can become silent in males. We favour the last explanation, as it would account for the unexpected sex ratio (MF) of 1.21 among surviving siblings, and for the striking clinical variability of the phenotype, including stepwise increases in disease expression in successive generations.     

Competitive exclusion of diarrheagenic Escherichia coli (ETEC) from human enterocyte-like Caco-2 cells by heat-killed Lactobacillus

Diarrheagenic Escherichia coli (ETEC) bearing CFA/I or CFA/II adhesive factors specifically adhere onto the brush border of the polarized epithelial human intestinal Caco-2 cells in culture. Heat-killed Lactobacillus acidophilus strain LB, that adheres onto Caco-2 cells, inhibits diarrheagenic Escherichia coli adhesion in a concentration-dependent manner. Since the L. acidophilus does not express ETEC-CFA adhesive factors, it can be postulated that the heat-killed L. acidophilus LB cells inhibit diarrheagenic E. coli attachment by steric hindrance of the human enterocytic ETEC receptors.     

Cyclic behavior of plum curculio,Conotrachelus nenuphar (Herbst) (Coleoptera: Curculionidae), within caged dwarf apple trees in spring

From 12 to 19 May 1987, during Morspur apple bloom, 21 radioactively labeled (⁶⁵ Zn) adult plum curculios, Conotrachelus nenuphar (Herbst), were released within a field cage containing four dwarf apple trees and located three times a day. A technique was developed for quickly obtaining (x, y, z)coordinates of location for adults foraging within apple trees. Cyclic patterns of behavior were detected using spectral analysis procedures. Over 70% of plum curculios exhibited diel periodicity with respect to activity and rate of movement, 36% exhibited such periodicity with respect to presence in the trees, and 27% with respect to movements from the center to the periphery of the canopy. Presence in fruit clusters, height in the trees, and movements along east-west and north-south axes showed little or no periodicity. Factors triggering cyclic behavior and practical implications of the results are discussed.     

Studies on NK cell purification by negative selection in human peripheral blood

We have attempted to improve negative selection procedures for the large scale purification of human CD _in3 ^– CD56⁺ NK cells. In a series of experiments, purifications of NK cells from 10⁸ PBMC were performed by T cell depletion using either direct or indirect anti-CD3 labeling and the Magnetic Activated Cell Separation (MACS) procedure. Contaminating CD3⁺ cells were still present using either one of these two different T cell depletion protocols as shown by phenotyping IL-2 supplemented cell cultures on day 12. A second cycle of purification was therefore added. When MACS and Dynabeads were compared as complementary procedures to the first MACS cycle starting with 10⁸ cells, the Dynabeads method was found to be superior to the MACS with regard to the elimination of residual T cells. Starting from 10⁹ PBMC, we showed that this MACS+Dynabeads procedure gave similar satisfactory results when compared to the scaling-up of a previously established two steps procedure using Dynabeads. These two approaches (MACS+Dynabeads and 2 cycles of Dynabeads) have been also tested in a clinical setting to purify NK cells from cancer patients prior toin vitro expansion. The results indicate that the two methods are equivalent with respect to purity and recovery rate; a slight advantage in terms of feasibility was found in favor of 2 cycles of Dynabeads.     

Detection of Pneumocystis carinii in serum of AIDS patients with Pneumocystis pneumonia by the polymerase chain reaction.

Amplification of DNA by the polymerase chain reaction (PCR) offers a highly sensitive and specific method for detecting DNA sequences in biological samples. We applied this technology to develop an assay for the P. carinii dihydrofolate reductase (DHFR) gene. This assay was found to be sensitive enough to detect as little as 1 organism-'equivalent' of DHFR DNA. In rats with experimentally-induced P. carinii pneumonia, DHFR DNA amplification demonstrated the presence of pulmonary P. carinii 2 wk prior to the onset of histopathological changes. When rat serum was analyzed by PCR, serum P. carinii DNA was found in 5 of 14 experimental rats. Finally, P. carinii DNA was detected in the serum of 7 of 18 patients (39%) with AIDS and active P. carinii pneumonia. These results suggest that circulating serum P. carinii DNA can be detected frequently in the course of pulmonary infection and may represent a blood-borne phase of infection. The PCR detection of P. carinii DNA provides a useful tool to study the natural history of P. carinii infection and may offer a non-invasive diagnostic procedure in some patients with P. carinii pneumonia.     

Recognition of HLA-B27 by mouse cytotoxic T-cell clones: a transgenic mouse

As a basis for the characterization of mouse T cells involved in the recognition of xenogeneic HLA molecules, a panel of HLA-B27-reactive cytotoxic T-cell clones was generated upon stimulation by cells from HLA-B27-transgenic mice. The HLA-B27-induced T-cell response was found to comprise two categories of clones: some recognizing HLA-B27 independent of H-2 molecules expressed by the target cells (unrestricted clones), others recognizing HLA-B27 in an H-2 restricted manner. The unrestricted clones exhibited diverse specificities, as judged from their various cross-reactivities with other xenogeneic (HLA) or allogeneic (H-2) molecules. In addition, although most of the unrestricted clones were able to react with both mouse and human HLA-B27-transgenic mice. The HLA-B27 induced T-cell which reacted only with HLA-B27-positive mouse, and not human cells. These findings illustrate that both H-2-restricted and unrestricted T cells with diverse species contribute to HLA-B27-xenorecognition.     

The role of C5 and T-cell receptor Vb genes in susceptibility to collagen-induced arthritis

Collagen-induced arthritis (CIA) is a rodent arthritis model in which immunization with heterologous type II collagen induces an inflammatory polyarthritis. Susceptibility to the disease is mediated by major histocompatibility complex (MHC) genes as well as genes at other loci. Previous studies of the SWR/J mouse strain, which is resistant to CIA despite bearing the susceptible H-2 ^q haplotype, have suggested that this resistance is the result of a deletion of T-cell receptor (Tcr) Vb gene segments which is carried by this strain. Other studies have implicated a deficiency in complement component C5 as the cause for the resistance. In order to assess the relative importance of these two genes in susceptibility to CIA, and to provide an estimate of the number of independent genes involved in the disease, we analyzed 196 F₂ progeny of a (DBA/1 × SWR/J) cross for arthritis susceptibility, and expression of both C5 and Tcr genes. Thirty of the F₂ progeny developed arthritis. All of the arthritic mice had at least one copy of the wild-type C5 allele, while the Tcr-Vb haplotypes were distributed in Mendelian fashion. These results demonstrate that C5 sufficiency is an absolute requirement for CIA, but that Tcr-Vb genes located within the SWR deletion have little influence. Genetic analysis of the incidence rate suggests that there is polygenic control of susceptibility to CIA and that in addition to H-2, 5–6 other independent loci (including C5) may be involved.     

Regulation of the maizerab17 gene promoter in transgenic heterologous systems

The maizerab17 gene is expressed in different plant parts in response to ABA and osmotic stress (J. Vilardellet al., Plant Mol Biol 14 (1990) 423–432). Here we demonstrate that 5 upstream sequences of therab17 gene confer the appropriate patterns of expression on the chloramphenicol acetyl transferase (CAT) reporter gene in transgenic tobacco plants, as well as in protoplasts derived from cultured rice cells. Specifically, a CAT construct containing a large 5 upstream fragment ofrab17 (–1330/+29) results in high levels of CAT activity in embryos, leaves and roots of transgenic plants subjected to water stress or ABA treatment. Transient expression assays in rice protoplasts transfected with CAT genes fused torab17 promoter deletions indicate that a 300 bp DNA fragment (–351/–102) is sufficient to confer ABA responsiveness upon the reporter gene. Furthermore, a 100 bp sequence (–219/–102) is capable of conferring ABA responsiveness upon a minimal promoter derived from the 35S CaMV promoter. Gel retardation experiments indicate that maize nuclear proteins bind to this fragment. This region of 100 bp contains a sequence (ACGTGGC) which has been identified as an abscisic acid response element in studies of other ABA-responsive plant genes.     

Variability and transport of suspended sediment,particulate and dissolved organic carbon in the tidal freshwater Hudson River

Measurements of suspended matter, particulate organic carbon and dissolved organic carbon were made over a three year period at stations spanning 150 km of the tidal freshwater Hudson River. Suspended matter concentrations varied from year-to-year and were not related to freshwater discharge. The increase in suspended matter with depth in vertical profiles suggests that, during medium to low flow conditions, resuspension of bottom sediments was as important a source of sediment as loadings from tributaries. Particulate organic carbon showed significant variability among stations, and both autochthonous primary production and detrital organic matter are contributing to POC standing stocks. Dissolved organic carbon represented over half of the total organic carbon in the water column and showed little variation among stations.Examining downstream changes in transport showed that there was significant production of both suspended matter and POC within the study reach during the ice-free season. Tributary loadings within the study reach do not appear to be the cause of these increases in downstream transport. Dissolved organic carbon behaved conservatively in that there was no evidence for net production or net consumption within the river.The spatial/temporal patterns and analyses of transport suggest that suspended matter and POC, but not DOC, were controlled to a significant extent by processes occurring within the river and were not simply related to loadings from outside.