Stroke research in GenomEUtwin.

Stroke is one of the leading causes of severe disability and death in the world. In the present article we outline possibilities and limitations for future stroke research within the GenomEUtwin. The combined sample of twins born before 1958 from Denmark, Finland, and Sweden, and available for follow-up into the second millennium for non-fatal and fatal stroke events through national inpatient and death registers exceeds 70,000 twin pairs. This sample size will enable the study of genetic influences on stroke and major stroke subtypes. Large samples of twins in GenomEUtwin have been followed up repeatedly through interviews and questionnaires concerning a variety of exposures and potential risk factors for stroke. We briefly outline how this information can be combined with the health register information for epidemiologic and genetic epidemiologic studies of stroke. We also present the number of twin pairs concordant and discordant for stroke in Denmark, Finland and Sweden, and time lags between events for twins concordant for stroke. This information illustrates that the number of affected sib pairs for linkage studies is relatively limited, but the sample sizes are promising for association studies.     

Organization of the origins of replication of the chromosomes of Mycobacterium smegmatis, Mycobacterium leprae and Mycobacterium tuberculosis and isolation of a functional origin from M. smegmatis

The genus Mycobacterium is composed of species with widely differing growth rates ranging from approximately three hours in Mycobacterium smegmatis to two weeks in Mycobacterium leprae. As DNA replication is coupled to cell duplication, it may be regulated by common mechanisms. The chromosomal regions surrounding the origins of DNA replication from M. smegmatis, M. tuberculosis, and M. leprae have been sequenced, and show very few differences. The gene order, rnpA-rpmH-dnaA-dnaN-recF-orf-gyrB-gyrA, is the same as in other Gram-positive organisms. Although the general organization in M. smegmatis is very similar to that of Streptomyces spp., a closely related genus, M. tuberculosis and M. leprae differ as they lack an open reading frame, between dnaN and recF, which is similar to the gnd gene of Escherichia coli. Within the three mycobacterial species, there is extensive sequence conservation in the intergenic regions flanking dnaA, but more variation from the consensus DnaA box sequence was seen than in other bacteria. By means of subcloning experiments, the putative chromosomal origin of replication of M. smegmatis, containing the dnaA-dnaN region, was shown to promote autonomous replication in M. smegmatis, unlike the corresponding regions from M. tuberculosis or M. leprae.     

Two independent pathways of expression lead to self-assembly of the rabbit hemorrhagic disease virus capsid protein.

The rabbit hemorrhagic disease virus capsid protein was expressed in insect cells either as an individual protein species, from a mRNA analogous to the viral subgenomic RNA, or as part of a polyprotein that included the viral 3C-like protease and the RNA polymerase. Both pathways of expression led to the assembly of viruslike particles morphologically and antigenically similar to purified virus.     

Insertion mutations at the maizeOpaque2 locus induced by transposable element familiesAc, En/Spm andBg

Eight independently isolated unstable alleles of theOpaque2 (O2) locus were analysed genetically and at the DNA level. The whole series of mutations was isolated from a maize strain carrying a wild-typeO2 allele and the transposable elementActivator (Ac) at thewx-m7 allele. Previous work with another unstable allele of the same series has shown that it was indeed caused by the insertion of anAc element. Unexpectedly, the remaining eight mutations were not caused by the designatedAc element, but by other insertions that are structurally similar or identical to one of two different autonomous transposable elements. Six mutations were caused by the insertion of a transposable element of theEnhancer/Suppressor-Mutator (En/Spm) family. Two mutations were the result of the insertion of a transposable element of theBergamo (Bg) family. Genetic tests carried out with plants carrying the unstable mutations demonstrated that all were caused by the insertion of an autonomous transposable element.     

Role of fungal patchiness on vegetal detritus in the trophic interactions between two brackish detritivores,Idotea baltica and Gammarus insensibilis

We investigated patterns of resource partitioning between two brackish crustaceans (I. baltica and G. insensibilis) on trophic mosaics of fungal species colonizing vegetal detritus. Laboratory feeding experiments were carried out to assess consumption rates and diet selection of single individuals belonging to five populations. Adults of two co-occurring population of both G. insensibilis and I. baltica and one population of G. insensibilis, occurring alone in another habitat patch, were studied. Each individual were offered both fragments of Cymodocea nodosa conditioned by 8 fungal species and sterilized fragments as sole food source for 7 days. Both species preferred "conditioned" detritus but potential resource use was greater in G. insensibilis than in I. baltica. Individual niche breadth increased and phenotypic variability reduced in the allotopic sample. Trophic similarities were lower between individuals of co-occuring populations than between I. baltica and the allotopic G. insensibilis. The results suggest that co-occurrence is an important factor contributing to the enhancement of phenotypic variability and, consequently, to a trophic generalization at population level in G. insensibilis. It emphasizes the role of fungal patchiness on detritus in regulating resource partitioning between the two species.     

Modulated red blood cell survival by membrane protein clustering

Human and murine blood cells treated with ZnCl₂ and bis(sulfosuccinimidyl)suberate (BS³) (a cross linking agent) undergo band 3 clustering and binding of hemoglobin to red blood cell membrane proteins. These clusters induce autologous IgG binding and complement fixation, thus favouring the phagocytosis of ZnCl₂/BS³ treated cells by macrophages. The extension of red blood cell opsonization can be easily modulated by changing the ZnCl₂ concentration in the 0.1–1.0 mM range thus providing an effective way to affect blood cell recognition by macrophages. In fact, murine erythrocytes treated with increasing ZnCl₂ concentrations have proportionally reduced survivals when reinjected into the animal. Furthermore, the organ sequestration of ZnCl₂/BS³ treated cells strongly resembles the typical distribution of the senescent cells. Since the ZnCl₂/BS³ treatment can also be performed on red blood cells loaded with drugs or other substances, this procedure is an effective drug-targeting system to be used for the delivery of molecules to peritoneal, liver and spleen macrophages.     

Leaf characteristics and optical properties of different woody species

 Light partition has been examined and evaluated on five woody species (Olea europaea, Ficus carica, Pittosporum tobira, Hedera helix maculata, Persica vulgaris) in relation to their leaf morpho-histological characteristics, water and chlorophyll contents. Leaf parameters and optical properties (reflectance, transmittance, absorbance) in PAR, FR and NIR wavebands (400–1100 nm) were preliminarily submitted to a canonical correlation analysis where lamina thickness and water content showed a leading role in determining all the optical properties, while chlorophyll, influential in the PAR region, was remarkably effective only in an extreme pigment situation when green and albino patches of ivy leaves were compared. Transmittance appeared inversely related to lamina thickness in accordance with the Lambert Beer law. Significant correlations were found also between mesophyll water content and both transmittance (positive) and reflectance (negative). Olive leaves showed peculiar optical patterns because of the dense and continuous trichome layer on their abaxial surface. Received: 3 January 1997 / Accepted: 5 May 1997     

Use of sequence characterised amplified region and RAPD markers in the identification of the white truffle Tuber magnatum Pico

Isolates of white truffles were identified as Tuber magnatum Pico species using a pair of primers selected from a sequence characterised amplified region (SCAR) and a specific random amplified polymorphic DNA (RAPD) marker. The present study reveals that PCR-fragment-pattern polymorphisms, the construction of probes and couples of primers from one or more of these polymorphic fragments may provide a useful and rapid tool for identifying species of ectomycorrhizal fungi in addition to conventional methods (morphological parameters).     

Gene pho-2 codes for the multiple active forms of Pi-repressible alkaline phosphatase in the mould Neurospora crassa

The mycelial Pi-repressible alkaline phosphatase of the wild-type strain 74A of Neurospora crassa was separated into at least ten isoforms by isoelectric focusing. The components visualized by activity with sodium -naphthyl phosphate as the substrate were predominantly acidic proteins with isoelectric points ranging from pH 4.5 to 7.6. The number of these isoforms was a function of growth pH. Strain pho-2A did not produce active Pi-repressible alkaline phosphatase (the pho-2 gene codes for its amino acid sequence), which gives an indication that these isoforms are encoded by the same structural gene.     

The membrane topology of the amino-terminal domain of the red cell calcium pump.

A systematic study of the membrane-associated regions in the plasma membrane Ca2+ pump of erythrocytes has been performed by hydrophobic photolabeling. Purified Ca2+ pump was labeled with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazirine ([125I]TID), a generic photoactivatable hydrophobic probe. These results were compared with the enzyme labeled with a strictly membrane-bound probe, [3H]bis-phosphatidylethanolamine (trifluoromethyl) phenyldiazirine. A significant light-dependent labeling of an M(r) 135,000-140,000 peptide, corresponding to the full Ca2+ pump, was observed with both probes. After proteolysis of the pump labeled with each probe and isolation of fragments by SDS-PAGE, a common pattern of labeled peptides was observed. Similarly, labeling of the Ca2+ pump with [125I]TID, either in isolated red blood cell membranes or after the enzyme was purified, yields a similar pattern of labeled peptides. Taken together, these results validate the use of either probe to study the lipid interface of the membrane-embedded region of this protein, and sustain the notion that the conformation of the pump is maintained throughout the procedures of solubilization, affinity purification, and reconstitution into proteoliposomes. In this work, we put special emphasis on a detailed analysis of the N-terminal domain of the Ca2+ pump. A labeled peptide of M(r) 40,000 belonging to this region was purified and further digested with V8 protease. The specific incorporation of [125I]TID to proteolytic fragments pertaining to the amino-terminal region indicates the existence of two transmembrane stretches in this domain. A theoretical analysis based on the amino acid sequence 1-322 predicts two segments with high probability of membrane insertion, in agreement with the experimental data. Each segment shows a periodicity pattern of hydrophobicity and variability compatible with alpha-helical structure. These results strongly suggest the existence of a transmembrane helical hairpin motif near the N-terminus of the Ca2+ pump.