The role of T-cadherin in axonal pathway formation in neocortical circuits.

Cortical efferent and afferent fibers are arranged in a stereotyped pattern in the intermediate zone (IZ). Here, we studied the mechanism of axonal pathway formation by identifying a molecule that is expressed in a subset of cortical axons in the rat. We found that T-cadherin (T-cad), a member of the cadherin family, is expressed in deep-layer cell axons projecting to subcortical structures, but not in upper layer callosal axons projecting to the contralateral cortex.

Potential role for purple acid phosphatase in the dephosphorylation of wall proteins in tobacco cells.

It is not yet known whether dephosphorylation of proteins catalyzed by phosphatases occurs in the apoplastic space. In this study, we found that tobacco (Nicotiana tabacum) purple acid phosphatase could dephosphorylate the phosphoryl residues of three apoplastic proteins, two of which were identified as alpha-xylosidase and beta-glucosidase. The dephosphorylation and phosphorylation of recombinant alpha-xylosidase resulted in a decrease and an increase in its activity, respectively, when xyloglucan heptasaccharide was used as a substrate.

Xyloglucan for generating tensile stress to bend tree stem.

In response to environmental variation, angiosperm trees bend their stems by forming tension wood, which consists of a cellulose-rich G (gelatinous)-layer in the walls of fiber cells and generates abnormal tensile stress in the secondary xylem. We produced transgenic poplar plants overexpressing several endoglycanases to reduce each specific polysaccharide in the cell wall, as the secondary xylem consists of primary and secondary wall layers. When placed horizontally, the basal regions of stems of transgenic poplars overexpressing xyloglucanase alone could not bend upward due to low strain in the tension side of the xylem.

CK2alpha phosphorylates BMAL1 to regulate the mammalian clock.

Clock proteins govern circadian physiology and their function is regulated by various mechanisms. Here we demonstrate that Casein kinase (CK)-2alpha phosphorylates the core circadian regulator BMAL1. Gene silencing of CK2alpha or mutation of the highly conserved CK2-phosphorylation site in BMAL1, Ser90, result in impaired nuclear BMAL1 accumulation and disruption of clock function.

Selective isolation of N-terminal peptides from proteins and their de novo sequencing by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry without regard to unblocking or blocking of N-terminal amino acids.

We have developed a new method to determine the N-terminal amino acid sequences of proteins, regardless of whether their N-termini are modified. This method consists of the following five steps: (1) reduction, S-alkylation and guanidination for targeted proteins; (2) coupling of sulfo-NHS-SS-biotin to N(alpha)-amino groups of proteins; (3) digestion of the modified proteins by an appropriate protease followed by oxidation with performic acid; (4) specific isolation of N-terminal peptides from digests using DITC resins; (5) de novo sequence analysis of the N-terminal peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using the CAF (chemically assisted fragmentation) method or tandem mass spectrometric (MS/MS) analysis according to unblocked or blocked peptides, respectively. By employing DITC resins instead of avidin resins used in our previous method (Yamaguchi et al.

Purification and characterization of a non-S-RNase and S-RNases from styles of Japanese pear (Pyrus pyrifolia).

In this study we biochemically characterized stylar ribonucleases (RNases) of Japanese pear (Pyrus pyrifolia), which exhibits S-RNase-based gametophytic self-incompatibility. We separated the RNase fractions NS-1, NS-2, and NS-3 from stylar extracts of the cultivar Nijisseiki (S(2)S(4)). The RNase in each fraction was purified to homogeneity through a series of chromatographic steps.

Specific isolation of N-terminal fragments from proteins and their high-fidelity de novo sequencing.

A new method to determine N-terminal amino acid sequences of multiple proteins at low pmol level by a parallel processing has been developed. The method contains the following five steps: (1) reduction, S-alkylation and guanidination for targeted proteins; (2) coupling with sulfosucccimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate(sulfo-NHS-SS-biotin) to N(alpha)-amino groups of proteins; (3) digestion of the modified proteins by an appropriate protease; (4) specific isolation of N-terminal fragments of proteins by affinity capture using the biotin-avidin system; (5) de novo sequence analysis of peptides by MALDI-TOF-/MALDI-TOF-PSD mass spectrometry with effective utilization of the CAF (chemically assisted fragmentation) method.1 This method is also effective for N-terminal sequencing of each protein in a mixture of several proteins, and for sequencing components of a multiprotein complex.

Simultaneous detection of N-terminal fragment ions in a protein mixture using a ruthenium(II) complex.

Use of a bis(terpyridine)ruthenium(II) derivative as an N-terminal labeling reagent resulted in the simultaneous detection and individual determination of all the N-terminal fragments of the proteins in a mixture without requiring any separation. All of the N-termini of the guanidinated proteins were labeled selectively by the ruthenium complex (-CO-labeling). After chymotryptic digestion, the fragments were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and post-source decay (PSD).

Cloning and sequencing of cDNA and genomic DNA encoding serine carboxypeptidase of Fusarium moniliforme that was copurified with phosphatase.

A previous study [Yoshida, H. et al., J.

High sequence-coverage detection of proteolytic peptides using a bis(terpyridine)ruthenium(II) complex.

The use of a bis(terpyridine)ruthenium(ii) complex for peptide labeling (Ru-CO labeling) supplied high intensity peaks in mass spectrometry (MS) analysis that overcame the contribution of protonation or sodiated adduction to peptides. Ru-CO-labeled insulin A- and B-chains were detected simultaneously in comparable peak abundance by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The mass spectra of chymotryptic peptide fragments of Ru-CO-labeled insulin also simultaneously indicated both N-terminal fragment ions, and amino acid sequences were determined easily by matrix-assisted laser desorption/ionization post-source-decay (MALDI-PSD).

Proteomics-based identification of alpha-enolase as a tumor antigen in non-small lung cancer.

Autoantibodies against tumor antigens represent one type of biomarker that may be assayed in serum for detection of cancer and monitoring of disease progression. In the present study, we used a proteomics-based approach to identify novel tumor antigens in non-small cell lung cancer (NSCLC). By combining two-dimensional electrophoresis, western blotting, mass spectrometry and enzyme-linked immunosorbent assay technology, we detected autoantibodies against alpha-enolase in a subset of NSCLC patients' sera.

Proteomic analysis of autoantigens associated with systemic lupus erythematosus: Anti-aldolase A antibody as a potential marker of lupus nephritis.

To screen for autoantibodies associated with systemic lupus erythematosus (SLE), we used proteomic approaches combining 2-D PAGE and Western blot analysis, followed by protein identification by LC-MS/MS analysis, resulting in the identification of aldolase A as a novel autoantigen in SLE. ELISA showed the prevalence of anti-aldolase A antibodies to be 29.3% in SLE, 8.

Monounsaturated fatty acid modification of Wnt protein: its role in Wnt secretion.

The secretion and extracellular transport of Wnt protein are thought to be well-regulated processes. Wnt is known to be acylated with palmitic acid at a conserved cysteine residue (Cys77 in murine Wnt-3a), and this residue appears to be required for the control of extracellular transport. Here, we show that murine Wnt-3a is also acylated at a conserved serine residue (Ser209).

Cloning and sequencing of cDNA and genomic DNA encoding PDM phosphatase of Fusarium moniliforme.

PDM phosphatase was purified approximately 500-fold through six steps from the extract of dried powder of the culture filtrate of Fusarium moniliforme. The purified preparation appeared homogeneous on SDS-PAGE although the protein band was broad. Amino acid sequence information was collected on tryptic peptides from this preparation.

High-throughput method for N-terminal sequencing of proteins by MALDI mass spectrometry.

A high-throughput method for sequencing of N termini of proteins by using postsource decay (PSD) of matrix-assisted laser desorption/ionization mass spectrometry has been developed. After a protein blotted on the PVDF membrane was successively reduced, S-alkylated, and guanidinated, its N-amino group was coupled to biotinylcysteic acid. The protein was then extracted from the membrane and digested with trypsin.

Rapid and sensitive amino-acid sequencing of cloning Thermus thermophilus HB8 ferredoxin by proteomics.

Recombinant holo Thermus thermophilus [7Fe-8S] ferredoxin was synthesized by cloning from Thermus thermophilus HB8 gene. A specific sequence (Pro-His-Val-Ile) at the N-terminus of the recombinant ferredoxin was determined by a rapid and highly sensitive mass spectral method using a novel Ru(II) Edman reagent, [(tpy)Ru(tpy-C6H4-NCS)](PF6)2 (tpy=terpyridine). The formation of the recombinant holoTtFd was established by the characteristic absorptions and CD extrema as [7Fe-8S] ferredoxin.

Enhanced responses in matrix-assisted laser desorption/ionization mass spectrometry of peptides derivatized with arginine via a C-terminal oxazolone.

We have developed a novel method for enhancing the response of a peptide in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) by activating the C-terminal carboxyl group through an oxazolone with which is coupled an amine containing a functional group to help ionize the peptide. The reactions consist of dehydration with acetic anhydride to give an oxazolone, followed by aminolysis with an appropriate amino acid derivative such as arginine methyl ester. The MALDI signal of Ac-Tyr-Gly-Gly-Phe-Leu-Arg-OMe, thus converted from leucine-enkephalin, was detected while completely excluding the responses of arginine-deficient peptides coexisting in the reaction mixture.

Prophenol oxidase A3 in Drosophila melanogaster: activation and the PCR-based cDNA sequence.

Phenol oxidase exists in Drosophila hemolymph as a prophenol oxidase, A1 and A3, that is activated in vivo with a native activating system, AMM-1, by limited proteolysis with time. The polypeptide in purified prophenol oxidase A3 has a molecular weight of approximately 77,000 Da. A PCR-based cDNA sequence coding A3 has 2501 bp encoding an open reading frame of 682 amino acid residues.

The N-terminal sequence of paratropomyosin binding fragments from beta-connectin.

In order to clarify the position where paratropomyosin binds to connectin in the A-I junction region of a sarcomere, chicken beta-connectin was digested by Staphylococcus aureus V8 protease under denaturing conditions and the digested peptides were electrophoretically separated. Five peptides, 150-kDa, 100-kDa, 70-kDa, and 43-kDa fragments, were simultaneously detected by biotinylated paratropomyosin and an anti-connectin monoclonal antibody. The N-terminal sequence of the 43-kDa fragment was found to be YQFRVYAVNK, similar to the sequence of 7556-7565 amino acids in the I51 fibronectin type 3 domain that was located at the A-I junction region of human cardiac titin/connectin.

Electrostatic role of aromatic ring stacking in the pH-sensitive modulation of a chymotrypsin-type serine protease, Achromobacter protease I.

Achromobacter protease I (API) has a unique region of aromatic ring stacking with Trp169-His210 in close proximity to the catalytic triad. This paper reveals the electrostatic role of aromatic stacking in the shift in optimum pH to the alkaline region, which is the highest pH range (8.5-10) among chymotrypsin-type serine proteases.

Lysobacter strain with high lysyl endopeptidase production.

A new lysyl endopeptidase producing strain, Lysobacter sp. IB-9374, was isolated from soil. This strain secreted the endopeptidase to culture medium at 6-12-fold higher levels relative to Achromobacter lyticus and Lysobacter enzymogenes.

Cultivated tomato has defects in both S-RNase and HT genes required for stylar function of self-incompatibility.

Cultivated tomato (Lycopersicon esculentum), a self-compatible species, evolved from self-incompatible (SI) species in the genus Lycopersicon following a breakdown of the self-incompatibility system. In order to elucidate the molecular basis of this breakdown in L. esculentum, we first analysed the stylar proteins with an in-gel assay for ribonuclease activity and 2D-PAGE.

Contribution of an imidazole-indole stack to high catalytic potency of a lysine-specific serine protease, Achromobacter protease I.

Achromobacter protease I (API), a lysine-specific serine-protease of the trypsin family, has an aromatic-ring stacking Trp 169-His 210 in close proximity to the reactive site. In order to investigate the role of this novel aromatic stacking, several mutants of the two residues were constructed and their kinetic parameters were determined. Three His 210 mutants showed lower activity by one order of magnitude than the wild-type with a peptide substrate of Ala-Ala-Lys-MCA (4-methylcoumaryl-7-amide), but 30-170% activity towards Val-Leu-Lys-MCA, suggesting that His 210 plays a role in keeping high activity toward various substrates by maintaining the active form of the substrate-binding subsite.