Novel propargylamine-linked nucleosides for high throughput SNP genotyping by MALDI-TOF MS

The synthesis of positively charged and mass tagged nucleosides containing a quaternary ammonium functionality within the penultimate position of a primer is described. Neutralization of the sugar/thiophosphate backbone by alkylation increases the detection sensitivity in the mass spectrometric analysis by a factor of at least 100. The variable introduction of these novel compounds within the extension primers enables flexible design of multiplex genotyping reactions.     

Premature chromosome condensation in humans associated with microcephaly and mental retardation: a novel autosomal recessive condition

We report a novel autosomal recessive disorder characterized by premature chromosome condensation in the early G2 phase. It was observed in two siblings, from consanguineous parents, affected with microcephaly, growth retardation, and severe mental retardation. Chromosome analysis showed a high frequency of prophase-like cells (>10%) in lymphocytes, fibroblasts, and lymphoblast cell lines with an otherwise normal karyotype. (3)H-thymidine-pulse labeling and autoradiography showed that, 2 h after the pulse, 28%-35% of the prophases were labeled, compared with 9%-11% in healthy control subjects, indicating that the phenomenon is due to premature chromosome condensation. Flow cytometry studies demonstrate that the entire cell cycle is not prolonged, compared with that in healthy control subjects, and compartment sizes did not differ from those in healthy control subjects. No increased reaction of the cells to X-irradiation or treatments with the clastogens bleomycin and mitomycin C was observed, in contrast to results in the cell-cycle mutants ataxia telangiectasia and Fanconi anemia. The rates of sister chromatid exchanges and the mitotic nondisjunction rates were inconspicuous. Premature entry of cells into mitosis suggests that a gene involved in cell-cycle regulation is mutated in these siblings.     

Determinants for removal and degradation of transit peptides of chloroplast precursor proteins

The stromal processing peptidase (SPP) cleaves a large diversity of chloroplast precursor proteins, removing an N-terminal transit peptide. We predicted previously that this key step of the import pathway is mediated by features of the transit peptide that determine precursor binding and cleavage followed by transit peptide conversion to a degradable substrate. Here we performed competition experiments using synthesized oligopeptides of the transit peptide of ferredoxin precursor to investigate the mechanism of these processes. We found that binding and processing of ferredoxin precursor depend on specific interactions of SPP with the region consisting of the C-terminal 12 residues of the transit peptide. Analysis of four other precursors suggests that processing depends on the same region, although their transit peptides are highly divergent in primary sequence and length. Upon processing, SPP terminates its interaction with the transit peptide by a second cleavage, converting it to a subfragment form. From the competition experiments we deduce that SPP releases a subfragment consisting of the transit peptide without its original C terminus. Interestingly, examination of the ATP-dependent metallopeptidase activity responsible for degradation of transit peptide subfragments suggests that it may recognize other unrelated peptides and, hence, act separately from SPP as a novel stromal oligopeptidase.     

N-terminal residues regulate the catalytic efficiency of the Hsp90 ATPase cycle

Hsp90 is an abundant molecular chaperone involved in a variety of cellular processes ranging from signal transduction to viral replication. The function of Hsp90 has been shown to be dependent on its ability to hydrolyze ATP, and in vitro studies suggest that the dimeric nature of Hsp90 is critical for this activity. ATP binding occurs at the N-terminal domains of the Hsp90 dimer, whereas the main dimerization site resides in the very C-terminal domain. ATP hydrolysis is performed in a series of conformational changes. These include the association of the two N-terminal domains, which has been shown to stimulate the hydrolysis reaction. In this study, we set out to identify regions in the N-terminal domain that are important for this interaction. We show that N-terminal deletion variants of Hsp90 are severely impaired in their ability to hydrolyze ATP. However, nucleotide binding of these constructs is similar to that of the wild type protein. Heterodimers of the Hsp90 deletion mutants with wild type protein showed that the first 24 amino acids play a crucial role during the ATPase reaction, because their deletion abolishes the trans-activation between the two N-terminal domains. We propose that the turnover rate of Hsp90 is decisively controlled by intermolecular interactions between the N-terminal domains.     

Dimerization of the type 4 cAMP-specific phosphodiesterases is mediated by the upstream conserved regions (UCRs)

The cAMP-specific PDE4 family consists of four genes, each expressed as several splice variants. These variants are termed long and short forms depending on the presence or absence of two unique N-terminal domains called upstream conserved regions 1 and 2 (UCR1 and 2). UCR1 and UCR2 have been shown to form a module necessary for the activation of PDE4 upon phosphorylation by the cAMP-dependent kinase (PKA). Here we have uncovered PDE4 oligomerization as a novel function for the UCR1/UCR2 module. Using several different approaches including gel filtration, sucrose density gradient centrifugation, pull-down of differentially tagged PDE constructs, and yeast two-hybrid assay, we show that the long PDE4 splice variant PDE4D3 behaves as a dimer, whereas the short splice variant PDE4D2 is a monomer. Internal deletions of either the C-terminal portion of UCR1 or the N-terminal portion of UCR2 abolishes dimerization of PDE4D3 indicating that both domains are involved in this intermolecular interaction. The dimerization, however, is structurally distinguishable from a previously described intramolecular interaction involving the same domains. PKA phosphorylation and site-directed mutagenesis shown to ablate the latter do not interfere with dimerization. Therefore, dimerization of the long PDE4 forms may be an additional function of the UCR domains that further explains differences in the regulatory properties between the long and short PDE4 splice variants.     

Synthesis and hypoglycemic evaluation of substituted pyrazole-4-carboxylic acids

The synthesis and in vivo activities of a series of substituted pyrazole-4-carboxylic acids as hypoglycemic agents are described. Modelization of some potent compounds, comparatively to the metformine, presents certain analogies permitting to predict the design of some novel antidiabetic drugs.     

Vertical transmission of TnSNPV,TnCPV, AcMNPV,and possibly recombinant NPV in Trichoplusia ni

Four viruses were tested for vertical transmission in Trichoplusia ni: T. ni nucleopolyhedrovirus (TnSNPV), T. ni cypovirus (TnCPV), Autographa californica nucleopolyhedrovirus (AcMNPV), and AcMNPV engineered to express a scorpion toxin (AcMNPV.AaIT). Fifth instars were exposed to each virus, the survivors were reared and mated, and second-generation (F(1)) insects were examined for infection. TnSNPV was transmitted to offspring at a prevalence rate of 15.4%, TnCPV at 10.2%, and AcMNPV at 10.1%. Only one of 2484 F(1) insects was infected with AcMNPV.AaIT; this experiment was repeated, and none of 4774 insects was infected. Thus, vertical transmission is unlikely to contribute to AcMNPV.AaIT contacting non-target organisms after its field release. There was evidence that TnCPV and possibly TnSNPV were activated to overt infections by ingestion of a different virus. TnCPV, but not the NPVs, routinely infected 0.3-1.7% of non-treated insects, probably indicating that it is vertically transmitted at enzootic levels.     

Three camelid VHH domains in complex with porcine pancreatic alpha-amylase. Inhibition and versatility of binding topology

Camelids produce functional antibodies devoid of light chains and CH1 domains. The antigen-binding fragment of such heavy chain antibodies is therefore comprised in one single domain, the camelid heavy chain antibody VH (VHH). Here we report on the structures of three dromedary VHH domains in complex with porcine pancreatic alpha-amylase. Two VHHs bound outside the catalytic site and did not inhibit or inhibited only partially the amylase activity. The third one, AMD9, interacted with the active site crevice and was a strong amylase inhibitor (K(i) = 10 nm). In contrast with complexes of other proteinaceous amylase inhibitors, amylase kept its native structure. The water-accessible surface areas of VHHs covered by amylase ranged between 850 and 1150 A(2), values similar to or even larger than those observed in the complexes between proteins and classical antibodies. These values could certainly be reached because a surprisingly high extent of framework residues are involved in the interactions of VHHs with amylase. The framework residues that participate in the antigen recognition represented 25-40% of the buried surface. The inhibitory interaction of AMD9 involved mainly its complementarity-determining region (CDR) 2 loop, whereas the CDR3 loop was small and certainly did not protrude as it does in cAb-Lys3, a VHH-inhibiting lysozyme. AMD9 inhibited amylase, although it was outside the direct reach of the catalytic residues; therefore it is to be expected that inhibiting VHHs might also be elicited against proteases. These results illustrate the versatility and efficiency of VHH domains as protein binders and enzyme inhibitors and are arguments in favor of their use as drugs against diabetes.     

Supplementary ultraviolet-B radiation induces a rapid reversal of the diadinoxanthin cycle in the strong light-exposed diatom Phaeodactylum tricornutum

A treatment of the diatom Phaeodactylum tricornutum with high light (HL) in the visible range led to the conversion of diadinoxanthin (Dd) to diatoxanthin (Dt). In a following treatment with HL plus supplementary ultraviolet (UV)-B, the Dt was rapidly epoxidized to Dd. Photosynthesis of the cells was inhibited under HL + UV-B. This is accounted for by direct damage by UV-B and damage because of the UV-B-induced reversal of the Dd cycle and the associated loss of photoprotection. The reversal of the Dd cycle by UV-B was faster in the presence of dithiothreitol, an inhibitor of the Dd de-epoxidase. Our results imply that the reversal of the Dd cycle by HL + UV-B was caused by an increase in the rate of gross Dt epoxidation, whereas the de-epoxidation of Dd was unaffected by UV-B. This is further supported by our finding that the in vitro de-epoxidation activity and the affinity toward the cosubstrate ascorbic acid of the Dd de-epoxidase were both unaffected by UV-B pretreatment of intact cells. We provide evidence that Dt epoxidation is normally down-regulated by a high pH gradient under HL. It is proposed that supplementary UV-B affected the pH gradient across the thylakoid membrane, which disrupted the down-regulation of Dt epoxidation and led to the observed increase in the rate of Dt epoxidation.     

A proteomic approach based on peptide affinity chromatography, 2-dimensional electrophoresis and mass spectrometry to identify multiprotein complexes interacting with membrane-bound receptors

There is accumulating evidence that membrane-bound receptors interact with many intracellular proteins. Multiprotein complexes associated with ionotropic receptors have been extensively characterized, but the identification of proteins interacting with G protein-coupled receptors (GPCRs) has so far only been achieved in a piecemeal fashion, focusing on one or two protein species. We describe a method based on peptide affinity chromatography, two-dimensional electrophoresis, mass spectrometry and immunoblotting to identify the components of multiprotein complexes interacting directly or indirectly with intracellular domains of GPCRs or, more generally, any other membrane-bound receptor. Using this global approach, we have characterized multiprotein complexes that bind to the carboxy-terminal tail of the 5-hydroxytryptamine type 2C receptor and are important for its subcellular localization in CNS cells (Bécamel et al., EMBO J., 21(10): 2332, 2002). Published: December 9, 2002