Determination of the spectrum of beta-thalassemia genes in Spain by use of dot-blot analysis of amplified beta-globin DNA.

We have delineated the molecular lesions causing beta-thalassemia in Spain, a country that has witnessed the passage of different Mediterranean populations over the centuries, in order to evaluate the extent of heterogeneity of these mutations and to make possible simplified prenatal diagnosis of the disorder in that country. The use of the polymerase chain-reaction (PCR) technique to preferentially amplify beta-globin DNA sequences that contain the most frequent beta-thalassemia mutations in Mediterraneans enabled us to rapidly analyze 58 beta-thalassemia alleles in a dot-blot format either by hybridization with allele-specific radiolabeled oligonucleotide probes or by direct sequence analysis of the amplification product. The Spanish population carries seven different beta-thalassemia mutations; the nonsense codon 39 is predominant (64%), whereas the IVS1 position 110 mutation, the most common cause of beta-thalassemia in the eastern part of the Mediterranean basin, is underrepresented (8.5%). The IVS1 mutation at position 6 accounts for 15% of the defects and leads to a more severe form of beta+-thalassemia than originally described in most of the patients we studied. In this study, we demonstrate further the usefulness of the dot-blot hybridization of PCR-amplified genomic DNA in both rapid population surveys and prenatal diagnosis of beta-thalassemia.     

It’s Time for Some “Site”-Seeing: Novel Tools to Monitor the Ubiquitin Landscape in Arabidopsis thaliana

Ubiquitination, the covalent binding of the small protein modifier ubiquitin to a target protein, is an important and frequently studied posttranslational protein modification. Multiple reports provide useful insights into the plant ubiquitinome, but mostly at the protein level without comprehensive site identification. Here, we implemented ubiquitin combined fractional diagonal chromatography (COFRADIC) for proteome-wide ubiquitination site mapping on Arabidopsis thaliana cell cultures. We identified 3009 sites on 1607 proteins, thereby greatly increasing the number of known ubiquitination sites in this model plant. Finally, The Ubiquitination Site tool (http://bioinformatics.psb.ugent.be/webtools/ubiquitin_viewer/) gives access to the obtained ubiquitination sites, not only to consult the ubiquitination status of a given protein, but also to conduct intricate experiments aiming to study the roles of specific ubiquitination events. Together with the antibodies recognizing the ubiquitin remnant motif, ubiquitin COFRADIC represents a powerful tool to resolve the ubiquitination maps of numerous cellular processes in plants.     

New enrichment method for isolation of pathogenic Yersinia enterocolitica serogroup O:3 from pork

A new enrichment medium for the recovery of pathogenic Yersinia enterocolitica serogroup O:3 from naturally infected meat products based on three selective agents, Irgasan, ticarcillin, and potassium chlorate (ITC), was compared with several other one- or two-step enrichments. Y. enterocolitica serogroup O:3 was recovered from 96.5% of 29 pork tongues, 24% of 50 ground pork samples, 16% of 25 masseter muscle samples, and 61% of tonsils. ITC was by far the most sensitive method for the recovery of Y. enterocolitica O:3, especially from ground meat and masseter muscles, while cold and two-step enrichments yielded better results for nonpathogenic strains. Plating of ITC enrichments onto SS-deoxycholate-calcium agar gave overall better results than plating onto cefsulodin-Irgasan-novobiocin agar for serogroup O:3.     

Mechanisms of NK Cell-Macrophage Bacillus anthracis Crosstalk: A Balance between Stimulation by Spores and Differential Disruption by Toxins

NK cells are important immune effectors for preventing microbial invasion and dissemination, through natural cytotoxicity and cytokine secretion. Bacillus anthracis spores can efficiently drive IFN-γ production by NK cells. The present study provides insights into the mechanisms of cytokine and cellular signaling that underlie the process of NK-cell activation by B. anthracis and the bacterial strategies to subvert and evade this response. Infection with non-toxigenic encapsulated B. anthracis induced recruitment of NK cells and macrophages into the mouse draining lymph node. Production of edema (ET) or lethal (LT) toxin during infection impaired this cellular recruitment. NK cell depletion led to accelerated systemic bacterial dissemination. IFN-γ production by NK cells in response to B. anthracis spores was: i) contact-dependent through RAE-1-NKG2D interaction with macrophages; ii) IL-12, IL-18, and IL-15-dependent, where IL-12 played a key role and regulated both NK cell and macrophage activation; and iii) required IL-18 for only an initial short time window. B. anthracis toxins subverted both NK cell essential functions. ET and LT disrupted IFN-γ production through different mechanisms. LT acted both on macrophages and NK cells, whereas ET mainly affected macrophages and did not alter NK cell capacity of IFN-γ secretion. In contrast, ET and LT inhibited the natural cytotoxicity function of NK cells, both in vitro and in vivo. The subverting action of ET thus led to dissociation in NK cell function and blocked natural cytotoxicity without affecting IFN-γ secretion. The high efficiency of this process stresses the impact that this toxin may exert in anthrax pathogenesis, and highlights a potential usefulness for controlling excessive cytotoxic responses in immunopathological diseases. Our findings therefore exemplify the delicate balance between bacterial stimulation and evasion strategies. This highlights the potential implication of the crosstalk between host innate defences and B. anthracis in initial anthrax control mechanisms.     

Assignment of human uroporphyrinogen decarboxylase (URO-D) to the p34 band of chromosome 1

Summary A cDNA probe corresponding to mRNA encoding human uroporphyrinogen decarboxylase (URO-D) was used to determine the chromosomal localization of the URO-D gene in the human genome. In agreement with previous studies, we have found that the locus for URO-D is located on chromosome 1 in hybrid cell mapping panels. The use of in sity hybridization allowed us to map the URO-D locus to band 1p34.Part of this work was presented as an abstract entitled Localization of the uroporphyrinogen decarboxylase gene to 1p34 band, by in situ hybridization, by M. G. Mattei, A. Dubart, D. Beaupain, M. Goossens, and J. F. Mattei, for a poster presentation at the 8th International Conference on Human Gene Mapping, Helsinki, August 4–10, 1985     

Two mild cystic fibrosis-associated mutations result in severe cystic fibrosis when combined in cis and reveal a residue important for cystic fibrosis transmembrane conductance regulator processing and function

The number of complex cystic fibrosis transmembrane conductance regulator (CFTR) genotypes identified as having double-mutant alleles with two mutations inherited in cis has been growing. We investigated the structure-function relationships of a severe cystic fibrosis (CF)-associated double mutant (R347H-D979A) to evaluate the contribution of each mild mutation to the phenotype. CFTR mutants expressed in HeLa cells were analyzed for protein biosynthesis and Cl(-) channel activity. Our data show that R347H is associated with mild defective Cl(-) channel activity and that the D979A defect leads to misprocessing. The mutant R347H-D979A combines both defects for a dramatic decrease in Cl(-) current. To decipher the molecular mechanism of this phenotype, single and double mutants with different charge combinations at residues 347 and 979 were constructed as charged residues were involved in this complex genotype. These studies revealed that residue 979, located in the third cytoplasmic loop, is critical for CFTR processing and Cl(-) channel activity highlighting the role of charged residues. These results have also important implications for CF, as they show that two mutations in cis can act in concert to alter dramatically CFTR function contributing to the wide phenotypic variability of CF disease.     

Gamete origin in relation to early embryo development

Fertilization in vivo requires a complex series of selection events to occur in order to guarantee that only the fittest gametes take part in the fusion process and give rise to a viable embryo. Conventional practice in bovine in vitro fertilization however is to select oocytes and sperm by quite crude procedures. It is therefore not inconceivable that essentially unfit gametes may drive aberrant embryo development in vitro. Abnormal embryonic cells are being removed by apoptosis, which is a physiological process in embryos. Only an excess or a lack of apoptosis can lead to embryonic death or abnormal development. Suboptimal culture conditions undoubtedly contribute to undue embryonic apoptosis, but the intrinsic quality of the oocyte may also be a causative factor. It is generally accepted that the oocyte is in control of early embryogenesis, but is it also in control of future embryonic suicide? Is a compromised follicular environment predestining the oocyte to a dire fate? What is the contribution of the cumulus cells to oocyte quality, and can they rescue it from early demise? And what can be said about the origin of the spermatozoa? Research in human in vitro fertilization has definitely shown that factors such as paternal age, smoking and other sperm stressors can contribute to abnormal embryo development and even diseased offspring. This review will address the questions raised above, and will describe what is known about the cellular and molecular biology that may account for abnormal bovine embryo development caused by gamete origin.