Isolation and expression of linked zinc finger gene clusters on human chromosome 11q

Proteins that share conserved "zinc finger" motifs represent a class of DNA-binding proteins that have been shown to play a fundamental role in regulating gene expression and to be involved in a number of human hereditary and malignant disease states. We have isolated, characterized, and mapped zinc finger-encoding genes specific to human chromosome 11q to investigate their possible association in the molecular pathogenesis of several disease loci mapped to this chromosome. An arrayed chromosome 11q cosmid library was screened using a degenerate oligonucleotide corresponding to the H/C link consensus sequence of the Drosophila Kruppel zinc finger gene, resulting in the isolation of six putative zinc finger genes. Three of the genes (ZNF123, ZNF125, and ZNF126) were analyzed and shown to contain tandemly repeated zinc finger motifs of the C2-H2 class. All three novel genes were found to be expressed in normal adult human tissues, although the tissue-specific pattern of expression differs markedly. Isolated zinc finger genes were regionally mapped on chromosome 11 using fluorescence in situ suppression hybridization and demonstrated clustering of the genes at 11q13.3-11q13.4 and 11q23.1-11q23.2. Analysis of in situ hybridization to interphase nuclei demonstrated a maximum distance of 1 Mb separating distinct finger genes. This analysis defines two linked multigene families of zinc finger genes to chromosome bands associated with a high frequency of specific translocations associated with malignancies.     

“Bacillus thermoantarcticus” sp. nov., from Mount Melbourne,Antarctica: a novel thermophilic species

 A novel thermophilic Gram-positive bacillus, “Bacillus thermoantarcticus”, isolated from geothermal soil near the crater of Mount Melbourne, is described. The organism grows at an optimal temperature of 63^°C at pH 6.0, is oxidase-positive, catalase-negative and produces an exopolysaccharide, an exocellular xylanase, an intracellular alcohol dehydrogenase and exo- and endocellular α-glucosidase(s). The sequence of 16S rDNA is very similar to that of “Bacillus thermoglucosidasius”; however, the guanine-plus-cytosine (G+C) content is 8 mol% higher. The type strain is “Bacillus thermoantarcticus” (DSM 9572). Received : 3 February 1995/Accepted : 12 May 1995     

Factors,including transforming growth factor β, released in the glioblastoma residual cavity,impair activity of adherent lymphokine-activated killer cells

Adherent lymphokine-activated killer (A-LAK) cells were obtained from peripheral blood lymphocytes of patients with recurrent glioblastoma. In vitro features of A-LAK cultures were assessed in comparison to those of non-adherent lymphokine-activated killer (NA-LAK) cells of the same patients with regard to cytotoxic activity, proliferation and surface markers. Only in a minority of cases did A-LAK cells show a markedly higher cytotoxicity on K562, Daudi and allogeneic glioblastoma cells. Nevertheless, A-LAK cells proliferated significantly better than NA-LAK and contained higher percentages of CD16⁺, CD56⁺ and CD25⁺ cells, indicating that A-LAK cells from these patients represent a subpopulation of lymphocytes enriched for activated natural killer cells. We also investigated whether immunosuppressive factor(s) were present in the tumour bed of recurrent gliomas. To this end, samples of glioblastoma cavity fluid (GCF), which accumulates in the cavity of subtotally removed tumour, were recovered and tested for the presence of immunosuppressive activity. All GCF samples analysed were shown to inhibit in vitro proliferation and antitumour cytotoxicity of 1-week-cultured A-LAK cells in a dose-dependent manner. Such GCF activity was effectively antagonized by a transforming growth factor (TGF) neutralizing antibody, indicating the involvement of TGF in lymphocyte inhibition. These results show that in the tumour cavity remaining after subtotal glioblastoma resection a marked immunosuppressive activity, probably due to local release of TGF, is present; such activity may negatively influence the therapeutic effectiveness of local cellular immunotherapy.     

Research Ethics and Intellectual Disability: Broadening the Debates

This article examines the ethical issues surrounding the inclusion of people with intellectual disabilities as research subjects. It explores subject selection, competence, risk and benefits, and authority through three tensions that emerge when considering these concepts in the context of the Disability Rights Movement and critical disability scholarship. These tensions are defined as the double dangers of inclusion and exclusion; the challenges of defining competence and risk in terms of individuals vs. groups; and the conflicts that arise when pursuing the dual goals of amelioration and elimination of disabilities. Though these tensions are not resolved, they underscore the importance of researchers engaging with critical disability perspectives in order to navigate these complex ethical questions.     

Estimating the abundance of burrow‐nesting species through the statistical analysis of combined playback and visual surveys

The conservation of elusive species relies on our ability to obtain unbiased estimates of their abundance trends. Many species live or breed in cavities, making it easy to define the search units (the cavity) yet hard to ascertain their occupancy. One such example is that of certain colonial seabirds like petrels and shearwaters, which occupy burrows to breed. In order to increase the chances of detection for these types of species, their sampling can be done using two independent methods to check for cavity occupancy: visual inspection, and acoustic response to a playback call. This double‐detection process allows us to estimate the probability of burrow occupancy by accounting for the probability of detection associated with each method. Here we provide a statistical framework to estimate the occupancy and population size of burrow‐dwelling species. We show how to implement the method using both maximum likelihood and Bayesian approaches, and test its precision and bias using simulated datasets. We subsequently illustrate how to extend the method to situations where two different species may occupy the burrows, and apply it to a dataset on wedge‐tailed shearwaters Puffinus pacificus and tropical shearwaters P. bailloni on Aride Island, Seychelles. The simulations showed that the single‐species model performed well in terms of error and bias except when detection probabilities and occupancies were very low. The two‐species model applied to shearwaters showed that detection probabilities were highly heterogeneous. The population sizes of wedge‐tailed and tropical shearwaters were estimated at 13 716 (95% CI: 12 909–15 874) and 25 550 (23 667–28 777) pairs respectively. The advantages of formulating the call‐playback sampling method statistically is that it provides a framework to calculate uncertainty in the estimates and model assumptions. This method is applicable to a variety of cavity‐dwelling species where two methods can be used to detect cavity occupancy.     

Structural analysis of the hepatitis C virus RNA polymerase in complex with ribonucleotides

We report here the results of a systematic high-resolution X-ray crystallographic analysis of complexes of the hepatitis C virus (HCV) RNA polymerase with ribonucleoside triphosphates (rNTPs) and divalent metal ions. An unexpected observation revealed by this study is the existence of a specific rGTP binding site in a shallow pocket at the molecular surface of the enzyme, 30 A away from the catalytic site. This previously unidentified rGTP pocket, which lies at the interface between fingers and thumb, may be an allosteric regulatory site and could play a role in allowing alternative interactions between the two domains during a possible conformational change of the enzyme required for efficient initiation. The electron density map at 1.7-A resolution clearly shows the mode of binding of the guanosine moiety to the enzyme. In the catalytic site, density corresponding to the triphosphates of nucleotides bound to the catalytic metals was apparent in each complex with nucleotides. Moreover, a network of triphosphate densities was detected; these densities superpose to the corresponding moieties of the nucleotides observed in the initiation complex reported for the polymerase of bacteriophage phi6, strengthening the proposal that the two enzymes initiate replication de novo by similar mechanisms. No equivalent of the protein stacking platform observed for the priming nucleotide in the phi6 enzyme is present in HCV polymerase, however, again suggesting that a change in conformation of the thumb domain takes place upon template binding to allow for efficient de novo initiation of RNA synthesis.     

Effects of analogues of inorganic phosphate and sodium ion on mineralization of matrix vesicles isolated from growth plate cartilage of normal rapidly growing chickens

The mechanism of matrix vesicle (MV) mineralization was studied using MVs isolated from normal growth plate tissue, as well as several putative intermediates in the MV mineralization pathway--amorphous calcium phosphate (ACP), calcium phosphate phosphatidylserine complex (CPLX) and hydroxyapatite (HAP). Radionuclide uptake and increase in turbidity were used to monitor mineral formation during incubation in synthetic cartilage lymph (SCL). Inhibitors of phosphate (Pi) metabolism, as well as replacing Na(+) with various cations, were used to study MV Pi transport, which had been thought to be Na(+)-dependent. MVs induced rapid mineralization approximately 3 h after addition to SCL; CPLX and HAP caused almost immediate induction; ACP required approximately 1 h. Phosphonoformate (PFA), a Pi analog, potently delayed the onset and reduced the rate of mineral formation of MV and the intermediates with IC(50)'s of 3-6 microM and approximately 10 microM, respectively. PFA:Pi molar ratios required to reduce the rate of rapid mineralization by 50% were approximately 1:30 for ACP, approximately 1:20 for HAP, approximately 1:3.3 for CPLX, and approximately 1:2.0 for MVs. MV mineralization was not found to be strictly Na(+)-dependent: substitution of Li(+) or K(+) for Na(+) had minimal effect; while N-methyl D-glucamine (NMG(+)) was totally inhibitory, choline(+) was clearly stimulatory. Na(+) substitutions had minimal effect on HAP- and CPLX-seeded mineral formation. However with ACP, NMG(+) totally blocked and choline(+) stimulated, just as they did MV mineralization. Thus, kinetic analyses indicate that ACP is a key intermediate, nevertheless, formation of CPLX appears to be the rate-limiting factor in MV mineralization.