A further insight into the binding of blood clotting factors to membranes

The active site of factor Xa, labelled with dansylglutamylglycylarginine (DnsEGR) is sensitive to association with Ca2+, factor Va and phospholipids. When bound to factor Va, DnsEGR-factor-Xa does not change the composition of the binding site of factor Va, as shown by fluorescence energy-transfer experiments between the Trp residues of factor Va and pyrene-labelled phospholipids. Prothrombin was cleaved by alpha-chymotrypsin into two parts: N-terminal residues 1-41 (peptide 1-41) containing the gamma-carboxyglutamic acid residues (Gla), and des-(1-41)-prothrombin; their membrane association was investigated. Peptide 1-41 contains the aromatic residues Tyr and Trp in positions 24 and 41, respectively, and is suitable for fluorescence spectroscopy. The absence of fluorescence energy transfer between these residues suggests that they are more than 2.8 nm apart. Binding of Ca2+ and of phospholipids involves essentially the Tyr residue, while the C-terminal characteristics of the Trp residue remain unchanged. The conformational change which takes place on binding does not shorten the distance between Tyr and Trp beyond 2.8 nm. Our conclusion is that peptide 1-41 has an extended conformation. This result is compatible with the disordered character of the Gla region found in the crystalline structure of fragment 1 of prothrombin. Ca2+ induces a greater fluorescence energy transfer between prothrombin and membranes labelled with pyrene but has no influence on the binding of des-(1-41)-prothrombin. Moreover, the binding curves of des(1-41)-prothrombin are similar to those of prothrombin in the absence of Ca2+. It is concluded that the Ca2+-independent association of prothrombin with membranes involves essentially that part of the prothrombin molecule deleted in the Gla region.     

Genetic heterogeneity in tuberous sclerosis

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by widespread hamartosis. Preliminary evidence of linkage between the TSC locus and markers on chromosome 9q34 was established, but subsequently disputed. More recently, a putative TSC locus on chromosome 11 has been suggested and genetic heterogeneity seems likely. Here we describe an approach combining multipoint linkage analysis and heterogeneity tests that has enabled us to obtain significant evidence for locus heterogeneity after studying a relatively small number of families. Our results support a model with two different loci independently causing the disease. One locus (TSC1) maps in the vicinity of the Abelson oncogene at 9q34 and a second locus (TSC2) maps in the region of the anonymous DNA marker Lam L7 and the dopamine D2 receptor gene at 11q23.     

Non-carrier-mediated uptake of the mannosidase I inhibitor 1-deoxymannojirimycin by K562 erythroleukemic cells

A 3H label was introduced at the C-1 position of the mannosidase I inhibitor 1-deoxymannojirimycin (dMM) by catalytic hydrogenolysis of benzyl-2,3-O-isopropylidene-5-N-benzyl-6-O-benzyl-alpha-D-mannofurano side with 3H2. 1-[3H]dMM as well as its precursor 1-[3H]2,3-O-isopropylidene-dMM had identical Rf as the nonradioactive compounds on TLC. Furthermore, alpha 1-antitrypsin secreted by HepG2 cells was modified indistinguishably by treatment of the cells with dMM and 1-[3H]dMM. Thus, 1-[3H]dMM had chemical and biological properties identical with authentic dMM. Uptake of [14C]mannose by K562 cells could be inhibited by glucose but not by the mannose analogue dMM. Thus, dMM does not enter the cell through hexose transporter(s). Uptake of 1-[3H]dMM by K562 cells could not be inhibited by increasing concentrations of nonradioactive dMM (from 1-32,000 microM), showing transport of dMM into cells through nonfacilitated diffusion. Furthermore, uptake of 1-[3H]dMM by K562 cells was observed at 0 degrees C.     

Seven New Mutations in hMSH2, an HNPCC Gene, Identified by Denaturing Gradient-Gel Electrophoresis

Hereditary nonpolyposis colorectal cancer (HNPCC) is a relatively common autosomal dominant cancer-susceptibility condition. The recent isolation of the DNA mismatch repair genes (hMSH2, hMLH1, hPMS1, and hPMS2) responsible for HNPCC has allowed the search for germ-line mutations in affected individuals. In this study we used denaturing gradient-gel electrophoresis to screen for mutations in the hMSH2 gene. Analysis of all the 16 exons of hMSH2, in 34 unrelated HNPCC kindreds, has revealed seven novel pathogenic germ-line mutations resulting in stop codons either directly or through frameshifts. Additionally, nucleotide substitutions giving rise to one missense, two silent, and one useful polymorphism have been identified. The proportion of families in which hMSH2 mutations were found is 21%. Although the spectrum of mutations spread at the hMSH2 gene among HNPCC patients appears extremely heterogeneous, we were not able to establish any correlation between the site of the individual mutations and the corresponding tumor spectrum. Our results indicate that, given the genomic size and organization of the hMSH2 gene and the heterogeneity of its mutation spectrum, a rapid and efficient mutation detection procedure is necessary for routine molecular diagnosis and presymptomatic detection of the disease in a clinical setup.     

Follicular dendritic cells and apoptosis: Life and death in the germinal centre

Summary The germinal centre forms a specialized microenvironment thought to play a key role in the induction of antibody synthesis, affinity maturation of B cells and memory B cell formation. Clonal-expanded follicular B lymphocytes with mutated antigen receptors (centrocytes) have to be selected on the basis of their capacity to compete for binding to antigen held in limited amounts on the follicular dendritic cells. In this way, only high-affinity B cells are selected. Binding to a follicular dendritic cell is an unconditional prerequisite for centrocytes to survive. Cells that do not succeed in binding to a follicular dendritic cell die rapidly by apoptosis. Apoptosis is a common form of cell death characterized by the activation of an endonuclease culminating in nuclear destruction. The pathway by which apoptosis is triggered varies from cell type to cell type. However, for germinal centre B cells this process is still poorly understood.     

Enzymatic Engineering of Polysialic Acid on Cells in Vitro and in Vivo Using a Purified Bacterial Polysialyltransferase

In vertebrates, polysialic acid (PSA) is typically added to the neural cell adhesion molecule (NCAM) in the Golgi by PST or STX polysialyltransferase. PSA promotes plasticity, and its enhanced expression by viral delivery of the PST or STX gene has been shown to promote cellular processes that are useful for repair of the injured adult nervous system. Here we demonstrate a new strategy for PSA induction on cells involving addition of a purified polysialyltransferase from Neisseria meningitidis (PST_Nm) to the extracellular environment. In the presence of its donor substrate (CMP-Neu5Ac), PST_Nm synthesized PSA directly on surfaces of various cell types in culture, including Chinese hamster ovary cells, chicken DF1 fibroblasts, primary rat Schwann cells, and mouse embryonic stem cells. Similarly, injection of PST_Nm and donor in vivo was able to produce PSA in different adult brain regions, including the cerebral cortex, striatum, and spinal cord. PSA synthesis by PST_Nm requires the presence of the donor CMP-Neu5Ac, and the product could be degraded by the PSA-specific endoneuraminidase-N. Although PST_Nm was able to add PSA to NCAM, most of its product was attached to other cell surface proteins. Nevertheless, the PST_Nm-induced PSA displayed the ability to attenuate cell adhesion, promote neurite outgrowth, and enhance cell migration as has been reported for endogenous PSA-NCAM. Polysialylation by PST_Nm occurred in vivo in less than 2.5 h, persisted in tissues, and then decreased within a few weeks. Together these characteristics suggest that a PST_Nm-based approach may provide a valuable alternative to PST gene therapy.     

The Cf-ECP2 gene is linked to, but not part of, the Cf-4/Cf-9 cluster on the short arm of chromosome 1 in tomato

A gene has been identified in tomato, which confers resistance to Cladosporium fulvum through recognition of the pathogenicity factor ECP2. Segregation analysis of F₂ and F₃ populations showed monogenic dominant inheritance, as for previously reported Cf resistances. The gene has been designated Cf-ECP2. Using several mapping populations, Cf-ECP2 was accurately mapped on chromosome 1, 7.7 cM proximal to TG236 and 6.0 cM distal to TG184. Although Cf-ECP2 is linked to Cf-4, it is not located in the Hcr9 cluster “Milky Way”. Therefore, Cf-ECP2 is the first functional Cf homologue on chromosome 1 that does not belong to this Hcr9 cluster. No recombination events between Cf-ECP2 and CT116 have been observed in three populations tested, representing 282 individuals. The low value for the physical distance per cM around CT116 reported previously and the high probability that Cf-ECP2 is also a member of a Hcr9 cluster will facilitate cloning of the locus. Received: 15 June 1999 / Accepted: 24 August 1999     

Using lanthanide ions to align troponin complexes in solution: order of lanthanide occupancy in cardiac troponin C

The potential for using paramagnetic lanthanide ions to partially align troponin C in solution as a tool for the structure determination of bound troponin I peptides has been investigated. A prerequisite for these studies is an understanding of the order of lanthanide ion occupancy in the metal binding sites of the protein. Two-dimensional [(1)H, (15)N] HSQC NMR spectroscopy has been used to examine the binding order of Ce(3+), Tb(3+), and Yb(3+) to both apo- and holo-forms of human cardiac troponin C (cTnC) and of Ce(3+) to holo-chicken skeletal troponin C (sTnC). The disappearance of cross-peak resonances in the HSQC spectrum was used to determine the order of occupation of the binding sites in both cTnC and sTnC by each lanthanide. For the lanthanides tested, the binding order follows that of the net charge of the binding site residues from most to least negative; the N-domain calcium binding sites are the first to be filled followed by the C-domain sites. Given this binding order for lanthanide ions, it was demonstrated that it is possible to create a cTnC species with one lanthanide in the N-domain site and two Ca(2+) ions in the C-domain binding sites. By using the species cTnC.Yb(3+).2 Ca(2+) it was possible to confer partial alignment on a bound human cardiac troponin I (cTnI) peptide. Residual dipolar couplings (RDCs) were measured for the resonances in the bound (15)N-labeled cTnI(129-148) by using two-dimensional [(1)H, (15)N] inphase antiphase (IPAP) NMR spectroscopy.     

Effects of T142 phosphorylation and mutation R145G on the interaction of the inhibitory region of human cardiac troponin I with the C-domain of human cardiac troponin C

Cardiac troponin I (cTnI) is the inhibitory component of the troponin complex, and its interaction with cardiac troponin C (cTnC) plays a critical role in transmitting the Ca(2+) signal to the other myofilament proteins in heart muscle contraction. The switch between contraction and relaxation involves a movement of the inhibitory region of cTnI (cIp) from cTnC to actin-tropomyosin. This region of cTnI is prone to missense mutations in heart disease, and a specific mutation, R145G, has been associated with familial hypertrophic cardiomyopathy. It also contains the unique cardiac PKC phosphorylation site at residue T142. To determine the structural consequences of the mutation R145G and the T142 phosphorylation on the interaction of cIp with cTnC, we have utilized 2D [(1)H, (15)N]-HSQC NMR spectroscopy to monitor the binding of native cIp, cIp-R (R145G), and cIp-P (phosphorylated T142), respectively, to the Ca(2+)-saturated C-domain of cTnC (cCTnC.2Ca(2+)). We also report a strategy for cloning, expression, and purification of cTnI peptide, and both synthetic and recombinant peptides are used in this study. NMR chemical shift mapping indicates that the binding epitope of cIp on cCTnC.2Ca(2+) is not greatly affected, but the affinity is reduced by approximately 14-fold by the T142 phosphorylation and approximately 4-fold by the mutation R145G, respectively. This suggests that these modifications of cIp have an adverse effect on the binding of cIp to cCTnC.2Ca(2+). These perturbations may correlate with the impairment or loss of cTnI function in heart muscle contraction.     

QTL mapping provides evidence for lack of association of the avoidance of leaf rust in Hordeum chilense with stomata density

In cereals, rust fungi are among the most harmful pathogens. Breeders usually rely on short-lived hypersensitivity resistance. As an alternative, "avoidance" may be a more durable defence mechanism to protect plants to rust fungi. In Hordeum chilense avoidance is based on extensive wax covering of stomata, which interferes with the induction of appressorium formation by the rust fungi. High avoidance levels are associated with a higher stoma density on the abaxial leaf epidermis. The avoidance level was assessed as the percentage of germ tube/stoma encounters that did not result in appressorium differentiation by Puccinia hordei, the barley leaf rust fungus. One hundred F(2) individuals from the cross between two H. chilense accessions with contrasting levels of avoidance showed a continuous distribution for avoidance of the rust fungus and for stoma density, indicating quantitative inheritance of the traits. No significant correlation was found between avoidance and stoma density in the segregating F(2) population. In order to map quantitative trait loci (QTLs) for both traits, an improved molecular marker linkage map was constructed, based on the F(2) population. The resulting linkage map spanned 620 cM and featured a total of 437 AFLP markers, thirteen RFLPs, four SCARs, nine SSRs, one STS and two seed storage protein markers. It consisted of seven long and two shorter linkage groups, and was estimated to cover 81% of the H. chilense genome. Restricted multiple interval mapping identified two QTLs for avoidance and three QTLs for stoma density in the abaxial leaf surface. The QTLs for avoidance were mapped on chromosome 3 and 5; those for stoma density on chromosomes 1, 3 and 7. Only the two QTLs regions located on chromosome 3 (one for avoidance and the other for stoma density) overlapped. The wild barley H. chilense has a high crossability with other members of the Triticeae tribe. The knowledge on the location of the QTLs responsible for the avoidance trait is a prerequisite to transfer this favourable agronomic trait from H. chilense to cultivated cereal genomes.