Ionic mechanism of mechano-perception in Characeae

Characean internodal cells generate receptor potential in response to mechanical stimuli. We studied responses of internodal cells to a long-lasting stimulus and the results were as follows. (i) The cell generated receptor potential at the moment of both compression and decompression. (ii) The receptor potential (DeltaE (m)) was significantly larger at the moment of decompression than at compression. (iii) The longer the duration of the stimulus, the larger was the magnitude of DeltaE (m) at the moment of decompression. (iv) Aequorin studies revealed that the increase in [Ca(2+)](c) (Delta[Ca(2+)](c)) took place at the moment of both compression and decompression. (v) The amplitude of Delta[Ca(2+)](c) was larger at the moment of decompression than at compression, as was the case for DeltaE (m). It was suggested that the amplitude of the receptor potential had a tight correlation with the degree of membrane deformation. We discussed the ionic mechanism of mechano-perception under a long-lasting stimulus in relation to mechanosensitive activation of Ca(2+) channels at the plasma membrane.     

Enzyme-linked sensitive fluorometric imaging of glutamate release from cerebral neurons of chick embryos

This paper describes a method for imaging the endogenous release of glutamate from cerebral neurons. This method is based on the reactions of glutamate oxidase and peroxidase, and on the detection of hydrogen peroxide by a fluorescent substrate of peroxidase. Glutamate has been sensitively measured in vitro in the range of 20 nM to 1 microM. We used two types of Ca(2+) channel inhibitors, MK-801 and omega-Conotoxin GVIA, which act to suppress Ca(2+) transport at postsynaptic and presynaptic neurons, respectively. MK-801 did not inhibit the increase in glutamate release after KCl stimulation, while there was no increase in glutamate release after KCl stimulation when omega-Conotoxin GVIA was used, probably due to the inhibition of voltage-activated Ca(2+) channels in the presynapse. Glutamate release and Ca(2+) flow in the synaptic regions were imaged using a laser confocal fluorescence microscope. KCl-evoked glutamate release was localized around cell bodies linked to axon terminals. This procedure allows imaging that can be sensitively detected by the fluorometric enzymatic assay of endogenous glutamate release in synapses.     

Potential role for 53BP1 in DNA end-joining repair through direct interaction with DNA

Upon DNA damage, p53-binding protein 1 (53BP1) relocalizes to sites of DNA double-strand breaks and forms discrete nuclear foci, suggesting its role in DNA damage responses. We show that 53BP1 changed its localization from the detergent soluble to insoluble fraction after treatment of cells with x-ray, but not with ultraviolet or hydroxyurea. Either DNase or phosphatase treatment of the insoluble fraction released 53BP1 into the soluble fraction, showing that 53BP1 binds to chromatin in a phosphorylation-dependent manner after X-irradiation of cells. 53BP1 was retained at discrete nuclear foci in X-irradiated cells even after detergent extraction of cells, showing that the chromatin binding of 53BP1 occurs at sites of DNA double-strand breaks. The minimal domain for focus formation was identified by immunofluorescence staining of cells ectopically expressed with 53BP1 deletion mutants. This domain consisted of conserved Tudor and Myb motifs. The Tudor plus Myb domain possessed chromatin binding activity in vivo and bound directly to both double-stranded and single-stranded DNA in vitro. This domain also stimulated end-joining by DNA ligase IV/Xrcc4, but not by T4 DNA ligase in vitro. We conclude that 53BP1 has the potential to participate directly in the repair of DNA double-strand breaks.     

Crystal structure of human 53BP1 BRCT domains bound to p53 tumour suppressor

The BRCT (BRCA1 C-terminus) is an evolutionary conserved protein-protein interacting module found as single, tandem or multiple repeats in a diverse range of proteins known to play roles in the DNA-damage response. The BRCT domains of 53BP1 bind to the tumour suppressor p53. To investigate the nature of this interaction, we have determined the crystal structure of the 53BP1 BRCT tandem repeat in complex with the DNA-binding domain of p53. The structure of the 53BP1-p53 complex shows that the BRCT tandem repeats pack together through a conserved interface that also involves the inter-domain linker. A comparison of the structure of the BRCT region of 53BP1 with the BRCA1 BRCT tandem repeat reveals that the interdomain interface and linker regions are remarkably well conserved. 53BP1 binds to p53 through contacts with the N-terminal BRCT repeat and the inter-BRCT linker. The p53 residues involved in this binding are mutated in cancer and are also important for DNA binding. We propose that BRCT domains bind to cellular target proteins through a conserved structural element termed the 'BRCT recognition motif'.     

Synthesis and insecticidal activity of N-oxydihydropyrroles: 4-hydroxy-3-mesityl-5,5-dimethyl derivatives with various substituents at the 1-position

A new series of N-oxydihydropyrrole derivatives was synthesized and evaluated for insecticidal activity against Nilaparvata lugens and Myzus persicae. Various substituents were introduced to the 1-position of the dihydropyrrole ring, and the derivatives obtained exhibited systemic and/or contact insecticidal activity. The structure-activity relationship revealed that small alkyoxy and alkoxyalkoxy groups were more favorable than alkylcarbonyloxy, alkoxycarbonyloxy, or sulfonyloxy groups as substituents at the 1-position.     

Selective priming of syntactic processing by event-related transcranial magnetic stimulation of Broca's area

It remains controversial whether Broca's aphasia is an articulatory deficit, a lexical-access problem, or agrammatism. In spite of recent neuroimaging studies, the causal link between cortical activity and linguistic subcomponents has not been elucidated. Here we report an experiment with event-related transcranial magnetic stimulation (TMS) to clarify the role of Broca's area, more specifically, the left inferior frontal gyrus (F3op/F3t), in syntactic processing. An experimental paradigm contrasted sentences requiring syntactic decisions with those requiring semantic decisions. We found selective priming effects on syntactic decisions when TMS was administered to the left F3op/F3t at a specific timing, but not to the left middle frontal gyrus (F2). Our results provide direct evidence of the involvement of the left F3op/F3t in syntactic processing.     

Thermoinduction of genes encoding the enzymes of gibberellin biosynthesis and a putative negative regulator of gibberellin signal transduction in<Emphasis Type="Italic"> Eustoma grandiflorum</Emphasis>

Eustoma grandiflorum Shinn requires vernalization for the induction of stem elongation and flowering. To investigate the role of gibberellins (GAs) in vernalization, the expression levels of genes encoding enzymes of GA biosynthesis, copalyl diphosphate synthetase, GA 20-oxidase and GA 3-hydroxylase, were examined using two culitvars that show different responses to vernalization. The three genes were induced in a vernalization- and a cultivar-dependent manner. EgSPY, a putative negative regulator of GA signal transduction, was also induced during the vernalization period. The results suggest that the expression of the genes encoding GAs biosynthesis is regulated by vernalization. We postulate that EgSPY functions as a negative regulator of GA signal transduction during vernalization, inhibiting adventitious shoot elongation during vernalization.Communicated by K.K. Kamo     

Purification, characterization cloning, and sequencing of metalloendopeptidase from Streptomyces septatus TH-2

Streptomyces septatus TH-2 secretes a large amount of a protease when cultured on a medium containing K(2)HPO(4) and glucose. The enzyme was purified to homogeneity by a three-step procedure. This enzyme had a molecular mass of approximately 35kDa, and was particularly inhibited by EDTA and phosphoramidon. Its substrate specificity was investigated using novel fluorescence energy transfer combinatorial libraries. The protease was found to prefer Phe and Tyr at the P(1) position, a hydrophobic or basic residue at the P(2) position, and a basic or small residue at the P(3) position. Its gene was cloned and sequenced, and its deduced amino acid sequence contained an HEXXH consensus sequence for zinc binding, confirming that it encodes metalloendopeptidase. The primary structure of the enzyme showed 40 and 69% identities with that of thermolysin from Bacillus thermoproteolyticus and that of a metalloendopeptidase from Streptomyces griseus, respectively.     

Distribution and transport of cholesterol-rich membrane domains monitored by a membrane-impermeant fluorescent polyethylene glycol-derivatized cholesterol

Cholesterol-rich membrane domains function in various membrane events as diverse as signal transduction and membrane traffic. We studied the interaction of a fluorescein ester of polyethylene glycol-derivatized cholesterol (fPEG-Chol) with cholesterol-rich membranes both in cells and in model membranes. Unlike filipin and other cholesterol probes, this molecule could be applied as an aqueous dispersion to various samples. When added to live cells, fPEG-Chol distributed exclusively in the outer plasma membrane leaflet and was enriched in microdomains that dynamically clustered by the activation of receptor signaling. The surface-bound fPEG-Chol was slowly internalized via clathrin-independent pathway into endosomes together with lipid raft markers. Noteworthy, fPEG-Chol could be microinjected in the living cells in which we found Golgi apparatus as the sole major organelle to be labeled. PEG-Chol, thus, provides a novel, sensitive probe for unraveling the dynamics of cholesterol-rich microdomains in living cells.