The maize major allergen, which is responsible for food-induced allergic reactions, is a lipid transfer protein

BACKGROUND: Cereals are the most important nutritional component in the human diet. Food-induced allergic reactions to these substances therefore have serious implications, and exhaustive diagnosis is required. Such diagnosis is still difficult because of the incomplete knowledge about major cereal allergens. In particular, few food-induced allergic reactions to maize have been reported, and no information on the allergenic proteins is available. OBJECTIVE: Having observed several anaphylactic reactions to maize, we planned a study to identify maize major allergens and cross-reactivity with other cereals, as well as to peach because the majority of patients also reacted to Prunoideae fruits. METHODS: Twenty-two patients with systemic symptoms after maize ingestion and positive skin prick test responses and serum-specific IgE antibodies to maize were selected. The IgE-reactivity pattern was identified by SDS-PAGE and immunoblotting. The major allergen identified was then purified by HPLC and characterized by mass spectrometry, determination of the isoelectric point value, and N-terminal amino acid sequencing. RESULTS: Sera from 19 (86%) of the 22 patients recognized a 9-kd protein, thus confirming this as the maize major allergen. This protein had an isoelectric point of greater than 9, a molecular mass of 9047.0 d, and no glycosylation. Determination of its N-terminal sequence showed that it was a lipid transfer protein (LTP). By using immunoblotting-inhibition experiments, we demonstrated that the LTP cross-reacts completely with rice and peach LTPs but not with wheat or barley LTPs. N-terminal sequence of the 16-kd allergen (recognized by 36% of patients) showed it to be the maize inhibitor of trypsin. This protein cross-reacts completely with grass, wheat, barley, and rice trypsin inhibitors. CONCLUSION: The major allergen of maize is an LTP with a molecular weight of 9 kd that is highly homologous with the peach LTP, the major allergen of the Prunoideae subfamily.     

A 3-D model for the CD40 ligand predicts that it is a compact trimer similar to the tumor necrosis factors

Based on the similarity in primary structure between the newlycharacterized ligand for CD40 (CD40L) and the tumor necrosisfactors (TNFs), we have modeled a detailed 3-D structure forCD40L. We used the known structure of TNF as a template forthe generation of the CD40L model. The soundness of the model-buildingalgorithms was verified by constructing a 3-D model of TNFßand comparing it to its crystallographically determined structure.The CD40L sequence is entirety compatible with the ‘Jelly-roll’ß-strand structure characteristic of the TNFs. Likethe TNFs, CD40L is predicted to form a compact trimer, althoughthe interactions between monomers are distinct from those foundin the TNFs. The model predicts which regions of CD40L couldinteract with its receptor(s) and which amino acids are essentialfor the maintenance of its trimeric structure.     

Downregulation of Bamboo mosaic virus replication requires the 5' apical hairpin stem loop structure and sequence of satellite RNA

Satellite RNAs associated with Bamboo mosaic virus (satBaMV) exhibit different phenotypes. Some isolates could reduce the accumulation of BaMV RNA and attenuate the BaMV-induced symptoms in co-inoculated plants. The determinants of the downregulation of BaMV replication were mapped in the 5' hypervariable region of satBaMV, which folds into a conserved apical hairpin stem loop (AHSL) structure comprising an apical loop and two internal loops, as evidenced by enzymatic probing. We also demonstrated that the integrity of the AHSL structure of interfering satBaMV was essential for the interference of BaMV accumulation. Concurrent analyses of natural satBaMV isolates revealed that all of the interfering isolates contained the same structures and sequences in the internal loops. Further, refined analyses indicated that, besides the AHSL structure, specific nucleotides in the internal loops play a crucial role in the downregulation, which implies that they may be required for the interaction of viral/cellular factors in this process.     

Parkin is protective for substantia nigra dopamine neurons in a tau gene transfer neurodegeneration model

Parkin is a ubiquitin ligase involved in the ubiquitin-proteasome system. Elevating parkin expression in cells reduces markers of oxidative stress while blocking parkin expression increases oxidative stress. In parkin gene knock down mouse and fly models, mitochondria function is deficient. Parkin is neuroprotective against a variety of toxic insults, while it remains unclear which of the above properties of parkin may mediate the protective actions. One of the models for which parkin is protective is overexpression of alpha-synuclein, a protein that self-aggregates in Parkinson disease. The microtubule-associated protein tau is another protein that self-aggregates in specific neurodegenerative diseases that also involve loss of dopamine neurons such as frontotemporal dementia with parkinsonism linked to chromosome 17, progressive supranuclear palsy and corticobasal degeneration. We recently developed a tau-induced dopaminergic degeneration model in rats using adeno-associated virus vectors. In this study, we successfully targeted either a mixed tau/parkin vector or mixed tau/control vector to the rat substantia nigra. While there was significant loss of dopamine neurons in the tau/control group relative to uninjected substantia nigra, there was no cell loss in the tau/parkin group. We found no difference in total tau levels between tau/control and tau/parkin groups. Parkin therefore protects dopamine neurons against tau as it does against alpha-synuclein, which further supports parkin as a therapeutic target for diseases involving loss of dopamine neurons.     

Tryptophan 95, an amino acid residue of the Caprine arthritis encephalitis virus vif protein which is essential for virus replication

The Caprine arthritis encephalitis virus (CAEV) vif gene was demonstrated to be essential for efficient virus replication. CAEV Vif deletion mutants demonstrated an attenuated replication phenotype in primary goat cell cultures and resulted in abortive infection when inoculated into goats. In this study, we determined the in vitro replication phenotype of five CAEV Vif point mutant infectious molecular clones and the ability of the corresponding in vitro translated Vif proteins to interact with the CAEV Pr55(gag) in the glutathione S--transferase (GST) binding assay. Here we show that (i) three of the mutants (S170E, S170G, S197G) behaved as the wild-type CAEV according to virus replication and Vif--Gag interactions; (ii) one mutant (Vif 6mut) was replication incompetent and bound weakly to GST-Gag fusion proteins; and (iii) one mutant (Vif RG) was impaired for replication while retaining its interaction properties. This mutant points out the critical importance of the CAEV Vif tryptophan residue at position 95 for efficient virus replication, defining for this lentivirus a functional domain unrelated to the Gag binding region.     

Capacity of Helicobacter pylori to generate ionic gradients at low pH is similar to that of bacteria which grow under strongly acidic conditions.

Helicobacter pylori colonized the highly acidic human gastric mucosa. At pH 3.0 to 7.0, this bacterium maintained a nearly neutral internal pH. Its membrane potential changed reciprocally with the pH gradient so that a relatively constant proton motive force was maintained. Possible, the capacity to maintain an appropriate transmembrane ionic gradient at a low pH contributes to the pathogenic propensities of this bacterium.     

ATM is required for the cellular response to thymidine induced replication fork stress

Genetically distinct checkpoints, activated as a consequence of either DNA replication arrest or ionizing radiation-induced DNA damage, integrate DNA repair responses into the cell cycle programme. The ataxia-telangiectasia mutated (ATM) protein kinase blocks cell cycle progression in response to DNA double strand breaks, whereas the related ATR is important in maintaining the integrity of the DNA replication apparatus. Here, we show that thymidine, which slows the progression of replication forks by depleting cellular pools of dCTP, induces a novel DNA damage response that, uniquely, depends on both ATM and ATR. Thymidine induces ATM-mediated phosphorylation of Chk2 and NBS1 and an ATM-independent phosphorylation of Chk1 and SMC1. AT cells exposed to thymidine showed decreased viability and failed to induce homologous recombination repair (HRR). Taken together, our results implicate ATM in the HRR-mediated rescue of replication forks impaired by thymidine treatment.     

A double-loop model for the replication of eukaryotic DNA

Coordinated DNA synthesis of both strands at the replication fork by a fixed ‘replisome’ may cause dynamic and topological problems. Based upon known properties of DNA helicase, DNA primase and DNA topoisomerases, and on novel properties of DNA polymerases and DNA ligase, we propose a ‘double-loop’ model for the replication of eukaryotic DNA that could minimize such problems.     

Identification of sna41 gene, which is the suppressor of nda4 mutation and is involved in DNA replication in Schizosaccharomyces pombe

Background

The replication licensing factor limits DNA replication to once in a cell cycle and is thought to contain MCM proteins as its component parts. Six MCM subtypes have been identified in various species. These MCM proteins are thought to bind each other to make a heteromeric complex. The Nda4 protein of Schizosaccharomyces pombe is one of the MCM proteins and is involved in DNA replication.

Results

The suppressor mutant of nda4 was isolated and the mutant gene was named sna41. The sna41-912 mutant demonstrated the ts phenotype, with an elongated cell shape at the restrictive temperature. Cells with 1C DNA content accumulated 2 h after shifting up to the restrictive temperature. This result suggests that sna41 is also involved in DNA replication. The sna41 genomic clone was isolated by a complementation of the ts phenotype of the mutant strain and was sequenced. The sna41 gene encodes a protein of 638 amino acids, which has low homology with CDC45 in S. cerevisiae. The gene disruption analysis showed that sna41 gene is essential for viability.

Conclusions

The S. pombe sna41 mutation suppresses the nda4-108 mutation. Sna41 is involved in DNA replication and may play some roles in the regulation of DNA replication by the MCM proteins.

     

A replication defective mutant of Rous sarcoma virus which fails to make a functional reverse transcriptase.

ts 672 is a replication defective temperature sensitive mutant of Rous sarcoma virus which produces large yields of noninfectious viral particles (NI 672) during growth in cells at the nonpermissive temperature. This study has been directed to identifying the temperature sensitive function of ts 672 by analysis of the NI 672 particles and by comparing this mutation to other known mutations in functions affecting replication.The only structural defect observed with NI 672 was the absence of the virion-associated RNA dependent DNA polymerase activity characteristic of all infectious RNA tumor viruses. Although there are other viral mutants which exhibit an altered polymerase activity, ts 672 is unique in that the DNA polymerase is only temperature sensitive prior to or during the assembly of the virus particle. The DNA polymerase activity of the ts 672 virions produced at the permissive temperature is just as temperature stable as the wild type enzyme.Mixed infection tests performed with ts 672 and with two other DNA polymerase mutants have failed to show a complementation, thus supporting the idea that ts 672 is defective in the DNA polymerase function. Recombination studies with ts 672 and an avian leukosis virus have shown that there is a very high degree of genetic linkage between the temperature sensitive DNA polymerase function of ts 672 and the determinants for the host range of these viruses, the viral envelope proteins.     

A stem-loop structure, sequence non-specific, at the origin of DNA replication of porcine circovirus is essential for termination but not for initiation of rolling-circle DNA replication

A stem-loop structure, formed by a pair of inverted repeats during DNA replication, is a conserved feature at the origin of DNA replication among plant and animal viruses, bacteriophages and plasmids that replicate their genomes via the rolling-circle replication (RCR) mechanism. In this work, a head-to-tail tandem construct of porcine circovirus capable of generating unit-length genomic DNA in Escherichia coli was employed to examine the role of the stem-loop structure with respect to the RCR initiation and termination process. The advantage of using a head-to-tail tandem construct is that the initiation and termination sites for generation of the unit-length viral genomes are physically separated, which allows independent examination of the initiation/termination processes. Nucleotide substitution mutational analysis showed that a pair of inverted repeats capable of forming a stem-loop structure was essential for termination, but not for initiation. The results also demonstrated that it is the stem-loop configuration, not nucleotide sequence specificity, that is critical for terminating RCR DNA replication.     

Analysis of the bacterial cell cycle using strains in which chromosome replication is controlled by plasmid R1.

As an alternative approach in the study of the Escherichia coli cell cycle, we have constructed strains in which chromosome replication is under the control of various plasmid R1 derivatives, IntR1 strains. The physiological properties of such strains are described. In intR1 strains, chromosome replication can be manipulated independently of cell-cycle-related control mechanisms, and the effects on cell division can be analysed. Using this approach, we have found that the timing of replication during the cell cycle is random in intR1 strains, that overreplication of the chromosome is lethal, and that chromosome replication does not trigger cell division. Current investigations include the study of the E. coli cell cycle in the absence of the DnaA protein, the effect on cell division of a specific inhibition of the initiation of chromosome replication, and the molecular basis of uni- and bidirectional replication.     

Characterization of rotavirus replication intermediates: a model for the assembly of single-shelled particles

The segmented double-stranded (ds)RNA genome of the rotaviruses is replicated asymmetrically with viral mRNA serving as the template for minus-strand RNA synthesis. To identify intermediate structures in rotavirus replication, subviral particles (SVPs) purified from the cytoplasm of simian rotavirus SA11-infected cells were assayed for RNA polymerase activity in a cell-free system that supports viral RNA replication. Intact SVPs containing newly made RNA were resolved by electrophoresis under nondenaturing conditions on 0.6% agarose gels (50 mM Tris-glycine, pH 8.8). This gel system was found to separate without disrupting SA11 single- and double-shelled virions and virion-derived core particles. SVPs from the cell-free system that contained newly made dsRNA migrated in the agarose gels at positions between virion-derived cores and intermediate of single- and double-shelled virions. SVPs containing newly made dsRNA were eluted from the gel and analyzed for protein content by electrophoresis on polyacrylamide gels. The results showed that three distinct types of replication intermediates (RIs) were present in SA11-infected cells. The smallest intermediate (precore RI, 45 nm, 220 S) contained the structural proteins VP1, VP3, and VP9 and the nonstructural proteins NS53, NS35, and NS34. A second intermediate (core RI, 60 nm, 310 S) contained the core proteins VP1, VP2, and VP3 and the proteins VP9, NS35 and NS34. The largest RI (single-shelled RI, 75 nm, 420 S) contained the inner shell proteins VP1, VP2, VP3, and VP6 and the proteins VP9, NS35 and NS34. Analysis of the formation and turnover of RIs in infected cells pulse-labeled with 35S-amino acids supports a hypothesis that rotavirus single-shelled particles are assembled in vivo by the sequential addition of VP2 and VP6 to precore RIs consisting of VP1, VP3, VP9, NS35, and NS34.     

The primary structure of Plasmodium falciparum DNA polymerase delta is similar to drug sensitive delta-like viral DNA polymerases.

We report the isolation and sequencing of genomic DNA clones that encode the 1094-amino acid catalytic subunit of DNA polymerase delta from the human malaria parasite Plasmodium falciparum. Protein sequence comparison to other DNA polymerases revealed the presence of six highly conserved regions found in alpha-like DNA polymerases from different prokaryotic, viral, and eukaryotic sources. Five additional regions of amino acid sequence similarity that are only conserved in delta and delta-like DNA polymerases, so far, were present in P. falciparum DNA polymerase delta. P. falciparum DNA polymerase delta was highly similar to both Saccharomyces cerevisiae DNA polymerase delta (DNA polymerase III; CDC2) and Epstein-Barr virus DNA polymerase at the amino acid sequence, and the predicted protein secondary structure levels. The gene that encodes DNA polymerase delta resides as a single copy on chromosome 10, and is expressed as a 4.5-kb mRNA during the trophozoite and schizont stages when parasite chromosomal DNA synthesis is active.