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.     

Studies on the total synthesis of an A7,B7-dicarbainsulin. III. Assembly of segments and generation of biological activity

As a further contribution to the synthesis of an insulin analogue with a stable A7-B7 interchain bond, the synthesis of A(8-21) by solution methods, and of B(9-25) as well as [7-(2,7-diaminosuberic acid)]B(1-8) by solid phase methods is described. In the latter compound, the amino group of the diaminosuberic acid residue was acylated with A(1-6), and the resulting "U-peptide" sequentially elongated with the C-terminal A- and finally B-chain sequences. The conversion of the product into the disulfide moiety gave a mixture which could not be resolved by currently available methods. However, the low biological activity of the crude product indicates that the A7-B7 disulfide bond is not crucially important for the activity of insulin.     

“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.     

Identification of residues in the insulin molecule important for binding to insulin-degrading enzyme

Insulin-degrading enzyme (IDE) hydrolyzes insulin at a limited number of sites. Although the positions of these cleavages are known, the residues of insulin important in its binding to IDE have not been defined. To this end, we have studied the binding of a variety of insulin analogues to the protease in a solid-phase binding assay using immunoimmobilized IDE. Since IDE binds insulin with 600-fold greater affinity than it does insulin-like growth factor I (25 nM and approximately 16,000 nM, respectively), the first set of analogues studied were hybrid molecules of insulin and IGF I. IGF I mutants [insB1-17,17-70]IGF I, [Tyr55,Gln56]IGF I, and [Phe23,Phe24,Tyr25]IGF I have been synthesized and share the property of having insulin-like amino acids at positions corresponding to primary sites of cleavage of insulin by IDE. Whereas the first two exhibit affinities for IDE similar to that of wild type IGF I, the [Phe23,Phe24,Tyr25]IGF I analogue has a 32-fold greater affinity for the immobilized enzyme. Replacement of Phe-23 by Ser eliminates this increase. Removal of the eight amino acid D-chain region of IGF I (which has been predicted to interfere with binding to the 23-25 region) results in a 25-fold increase in affinity for IDE, confirming the importance of residues 23-25 in the high-affinity recognition of IDE. A similar role for the corresponding (B24-26) residues of insulin is supported by the use of site-directed mutant and semisynthetic insulin analogues. Insulin mutants [B25-Asp]insulin and [B25-His]insulin display 16- and 20-fold decreases in IDE affinity versus wild-type insulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Functional definition of ABA-response complexes: the promoter units necessary and sufficient for ABA induction of gene expression in barley (Hordeum vulgare L.)

Abscisic acid (ABA)-response promoter complexes (ABRCs), consisting of an ACGT core-containing element (ACGT box) and a coupling element (CE), have been shown to be necessary and sufficient for ABA induction of gene expression in cereal plants. In this work, the component elements of two ABRCs are defined in terms of base sequence, orientation, and distance from each other. The ACGT element requires the sequence 5-ACGTGGC-3 and the elements CE1 and CE3 require the sequences CCACC and GCGTGTC, respectively. The ACGT element and CE3 are next to each other in the barley ABA-inducible gene HVA1, and lengthening the distance between them gradually decreases their activity in conferring ABA response. On the other hand, the ACGT element and CE1 are separated by about 20 bp in the promoter of another ABA-inducible gene, HVA22, and need to be separated by multiples of 10 bp in order to confer high ABA induction, suggesting that these two elements have to be located in the same side of the DNA double helix. Although the coupling between an ACGT box and a CE is sufficient for ABA induction, two copies of the ACGT element are equally active. However, two copies of CE3 appear to be less active. Specific interactions between ABRC and nuclear proteins have been detected. In vitro binding activities of nuclear proteins to an ABRC and to its mutant forms appear to be proportional to the biological activities of these sequences in vivo. Our data suggest that the specific response to ABA is determined by the presence of two ACGT boxes or an ACGT box plus a CE as well as by the flanking sequences of the ACGT boxes and the CEs.