Genetic Analysis of Hispanic Individuals with Cystic Fibrosis

We have performed molecular genetic analyses of Hispanic individuals with cystic fibrosis (CF) in the southwestern United States. Of 129 CF chromosomes analyzed, only 46% (59/129) carry ΔF508. The G542X mutation was found on 5% (7/129) of CF chromosomes. The 3849+10kbC→T mutation, detected primarily in Ashkenazi Jews, was present on 2% (3/129). R1162X and R334W, mutations identified in Spain and Italy, each occurred on 1.6% (2/129) of CF chromosomes. W1282X and R553X were each detected once. G551D and N1303K were not found. Overall, screening for 22 or more mutations resulted in detection of only 58% of CF transmembrane conductance regulator gene mutations among Hispanic individuals. Analysis of KM19/XV2c haplotypes revealed an unusual distribution. Although the majority of ΔF508 mutations are on chromosomes of B haplotypes, the other CF mutations are on A and C haplotypes at higher-than-expected frequencies. These genetic analyses demonstrate significant differences between Hispanic individuals with CF and those of the general North American population. Assessment of carrier/affected risk in Hispanic CF individuals cannot, therefore, be based on the mutation frequencies found through studies of the general population but must be adjusted to better reflect the genetic makeup of this ethnic group. Further studies are necessary to identify the causative mutation(s) in this population and to better delineate genotype/phenotype correlations. These will enable counselors to provide more accurate genetic counseling.     

Lipoprotein-associated paf (LA-paf) was found in washed human platelets and monocyte/macrophage-like U937 cells

Beyond cholesterol, inflammatory ether phospholipids such as platelet-activating factor (paf) may play a role in atherogenesis. (1) We detected a paf-like compound (‘LA-paf’) associated with human serum lipoproteins, mainly in LDL but not with the lipoprotein-poor fraction. (2) LA-paf was also found in washed human platelets, from where it was partially released during platelet aggregation in response to paf (50 nM) or thrombin (1 U). In addition, resident monocyte/macrophage-like U937 cells carried huge amounts of LA-paf (41 ng per 10⁷ cells) and metabolized added [³H]paf to a labelled compound co-eluting with the retention time of LA-paf in standard HPLC. (3) Functionally, LA-paf had a comparable potency to synthetic paf, because LA-paf aggregated washed aspirin-treated platelets in a concentration-dependent manner. The specific paf receptor antagonist WEB2086 inhibited the platelet aggregation induced by three distinct LA-paf preparations as compared with synthetic paf with similar inhibitory concentrations (IC₅₀: 35.6 ± 12.8, 24.0 ± 4.0, 38.0 ± 15.8 nM for LA-paf, and 43.6 ± 6.5 nM for synthetic paf), indicating that LA-paf interacted with paf receptors. (4) However, LA-paf had a distinct retention time using high-pressure liquid chromatography (HPLC) as compared with synthetic paf. LA-paf eluted at 9–15 min and synthetic paf at 21–24 min. In addition, total and non-specific [³H]paf binding to intact washed human platelets was affected differently by the two unlabelled agonists: while LA-paf increased total and non-specific (but not specific) binding in a significant manner (P < 0.002 and P < 0.007) as LDL did (P < 0.006 and P < 0.03), synthetic paf decreased total binding (P < 0.03). Similarly, low-density lipoproteins (LDL) increased significantly the total [³H]paf binding. In contrast, paf did not affect specific [¹²⁵I]LDL binding to human fibroblasts. Our results show the presence of LA-paf in lipoproteins,     

Isolation and characterization of two cDNA clones encoding ATP-sulfurylases from potato by complementation of a yeast mutant

Sulfur plays an important role in plants, being used for the biosynthesis of amino acids, sulfolipids and secondary metabolites. After uptake sulfate is activated and subsequently reduced to sulfide or serves as donor for sulfurylation reactions. The first step in the activation of sulfate in all cases studied so far is catalyzed by the enzyme ATP-sulfurylase (E.C. 2.7.7.4.) which catalyzes the formation of adenosine-5′-phosphosulfate (APS). Two cDNA clones from potato encoding ATP-sulfurylases were identified following transformation of a Saccharomyces cerevisiae mutant deficient in ATP-sulfurylase activity with a cDNA library from potato source leaf poly(A)⁺ RNA cloned in a yeast expression vector. Several transformants were able to grow on a medium with sulfate as the only sulfur source, this ability being strictly linked to the presence of two classes of cDNAs. The clones StMet3-1 and StMet3-2 were further analyzed. DNA analysis revealed an open reading frame encoding a protein with a molecular mass of 48 kDa in the case of StMet3-1 and 52 kDa for StMet3-2. The deduced polypeptides are 88% identical at the amino acid level. The clone StMet3-2 has a 48 amino acid N-terminal extension which shows common features of a chloroplast transit peptide. Sequence comparison of the ATP-sulfurylase Met3 from Saccharomyces cerevisiae with the cDNA StMet3-1 (StMet3-2) reveals 31% (30%) identity at the amino acid level. Protein extracts from the yeast mutant transformed with the clone StMet3-1 displayed ATP-sulfurylase activity. RNA blot analysis demonstrated the expression of both genes in potato leaves, root and stem, but not in tubers. To the best of the authors' knowledge this is the first cloning and identification of genes involved in the reductive sulfate assimilation pathway from higher plants.     

Efficient generation of chimaeric mice using embryonic stem cells after long-term culture in the presence of ciliary neurotrophic factor

The aim of our study was to evaluate whether ciliary neurotrophic factor (CNTF) can substitute for leukaemia inhibitory factor (LIF) in maintaining pluripotential embryonic stem (ES) cells in culture. Two subclones of D3 ES cells were used to assess cell proliferation and differentiation in the presence of CNTF, LIF or Buffalo rat liver (BRL) cell-conditioned medium, or in the absence of exogenous differentiation inhibiting factors. ES cells maintained in medium supplemented with CNTF for up to four weeks were injected into blastocysts to investigate theirin vivo pluripotency in terms of chimaera formation. CNTF inhibited ES cell differentiation in a dose-dependent manner. The most effective concentration was 10 ng CNTF per ml of medium. The effects of CNTF on ES cell differentiation and proliferation were comparable to those of LIF at the same concentration. BRL cell-conditioned medium was less effective at preventing ES cell differentiation but induced their proliferation very markedly. Both ES cell clones efficiently formed chimaeras after long-term culture with CNTF as the only differentiation inhibiting agent. The ability of these ES cells to colonize the germ-line is the ultimate proof that CNTF can preserve the pluripotency of ES cells.     

Complex duplications in maize lines

Rp1 is a disease resistance complex and is the terminal morphological marker on the short arm of maize chromosome 10. Several restriction fragment length polymorphisms (RFLPs), which map within 5 map units of Rp1, were examined to determine if they are also complex in structure. Two RFLP loci, which mapped distally to Rp1, BNL3.04 and PIO200075, existed in a single copy in all maize lines examined. These two loci cosegregated perfectly in 130 test cross progeny. Two RFLP loci that map proximally to Rp1 had unusual structures, which have not yet been reported for maize RFLPs; the loci were complex, with variable numbers of copies in different maize lines. One of the loci, NPI285, occasionally recombined in meiosis to yield changes in the number of copies of sequences homologous to the probe. The other proximal locus, detected by the probes NPI422, KSU3, and KSU4, was relatively stable in meiosis and no changes in the number of restriction fragments were observed. The similarity in map position between Rp1 and the complex RFLP loci indicate there may be genomic areas where variable numbers of repeated sequences are common. The structure of these complex loci may provide insight into the structure and evolution of Rp1.     

Monoclonal antibodies and idiotypic network activation for ovarian carcinoma

Antibodies can be processed by the B- and T-cell systems and may lead to a selective activation of the immune system. The network structure of the immune system implicates the possibility of a selective immunization by the activation of idiotypic cascades. In a retrospective analysis, patients with advanced ovarian carcinoma, who had received MAb, against the cancer-associated antigen CA125 for diagnostic purposes, were analyzed for the production of anti-idiotypic antibodies, survival rate, and immunological effects. Furthermore, we started a prospective and randomized study for ovarian cancer patients, using a different antigen, TAG72, for the induction of idiotypic cascades. Our first results on 58 patients with advanced ovarian carcinomas showed that the induction of anti-idiotypic-antibodies against OC125 mimicking the TAA Class III CA125 leads to a prolongation of the survival rate, and, in extended stages, to an induction of antitumoral immunity, and that the induction of idiotypic cascades is also possible for different antigens like TAG72. Summarizing the activation of idio-typic network cascades seems to be a very effective way of intervention in the immune system of patients with advanced stages of ovarian carcinoma. A prospective study of the adjuvant approach seems to be necessary.     

Adrenergic Stimulation of Cyclic GMP Formation Requires NO-Dependent Activation of Cytosolic Guanylate Cyclase in Rat Pinealocytes

Abstract: Cyclic GMP (cGMP) formation in rat pinealocytes is regulated through a synergistic dual receptor mechanism involving β-and α₁-adrenergic receptors. The effects of N -monomethyl- l -arginine (NMMA), which inhibits nitric oxide (NO) synthase and NO-mediated activation of cytosolic guanylate cyclase, and methylene blue (MB), which inhibits cytosolic guanylate cyclase, were investigated in an attempt to understand the role of NO in adrenergic cGMP formation. Both NMMA and MB inhibited β-adrenergic stimulation of cGMP formation as well as α₁-adrenergic potentiation of β-adrenergic stimulation of cGMP formation, whereas they had no effect in unstimulated pinealocytes. The inhibitory action of NMMA was antagonized by addition of l -arginine. On the basis of these findings it can be concluded that the adrenergic stimulation of cGMP formation involves NO synthesis followed by activation of cytosolic guanylate cyclase.     

Pollen Expression of Herbicide Target Site Resistance Genes in Annual Ryegrass (Lolium rigidum)

Herbicide resistance can occur either through target-site insensitivity or by nontarget site-based mechanisms. Two herbicide-resistant biotypes of Lolium rigidum Gaud., one resistant to acetolactate synthase (ALS)-inhibiting herbicides (biotype WLR1) and the other resistant to acetyl CoA carboxylase (ACCase)-inhibiting herbicides (biotype WLR96) through target-site insensitivity at the whole plant and enzymic levels, were found to express this resistance in the pollen. Pollen produced by resistant biotypes grew uninhibited when challenged with herbicide, whereas that from a susceptible biotype was inhibited. A third biotype, SLR31, resistant to ACCase-inhibiting and certain ALS-inhibiting herbicides at the whole plant level through nontarget site-based mechanisms, did not exhibit this expression in the pollen. The technique described may form the basis for a rapid screen for certain nuclear-encoded, target site-based herbicide-resistance mechanisms.     

Evidence for two different blue-light-receptor systems for the fast responses of stimulation of photosynthetic capacity and acidification of the plant surface in brown algae

Two blue-light responses of Phaeophyta that are expressed within a few seconds of a blue-light stimulus were characterized with respect to their photoreception properties. The first response is the activation of red-light-saturated photosynthesis which can be stimulated to values up to 5 times the rates in red light, depending on the species. The second response is a blue-light-induced acidification measurable at the plant surface. Both responses have similar kinetic characteristics and thus led us initially to hypothesise that they were causally connected in the same transduction mechanism. The two responses have action spectra [measured for Ectocarpus siliculosus (Dillwyn) Lyngb. and Laminaria saccharina (L.) Lamouroux] that are indistinguishable within the relatively large limits of error. However, in all species tested, the threshold sensitivity for blue light of the photosynthetic response is lower than that of the pH-shift by a factor of 2 to 150. Furthermore, stimulation of photosynthesis is sensitive to the flavin inhibitors, KI and phenylacetic acid, but the pH response is not affected by these inhibitors. Thus, the blue-light-induced pH-shift does not cause the stimulation of photosynthesis. In contrast, the different fluence-response relationships of the two responses and particularly the differential effect of the inhibitors are clear evidence for the action of two independent transduction pathways and photoreceptor systems for blue light. At least photoreception for stimulation of photosynthesis involves a flavin-or and a pterin.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PAA phenylacetic acid We thank Dr. C. A. Maggs for collecting P. pavonica. This research was supported by National Environment Research Council grant No. GR3/8102.