Cell-to-cell movement of the CAPRICE protein in Arabidopsis root epidermal cell differentiation

CAPRICE (CPC), a small, R3-type Myb-like protein, is a positive regulator of root hair development in Arabidopsis. Cell-to-cell movement of CPC is important for the differentiation of epidermal cells into trichoblasts (root hair cells). CPC is transported from atrichoblasts (hairless cells), where it is expressed, to trichoblasts, and generally accumulates in their nuclei. Using truncated versions of CPC fused to GFP, we identified a signal domain that is necessary and sufficient for CPC cell-to-cell movement. This domain includes the N-terminal region and a part of the Myb domain. Amino acid substitution experiments indicated that W76 and M78 in the Myb domain are critical for targeted transport, and that W76 is crucial for the nuclear accumulation of CPC:GFP. To evaluate the tissue-specificity of CPC movement, CPC:GFP was expressed in the stele using the SHR promoter and in trichoblasts using the EGL3 promoter. CPC:GFP was able to move from trichoblasts to atrichoblasts but could not exit from the stele, suggesting the involvement of tissue-specific regulatory factors in the intercellular movement of CPC. Analyses with a secretion inhibitor, Brefeldin A, and with an rhd3 mutant defective in the secretion process in root epidermis suggested that intercellular CPC movement is mediated through plasmodesmata. Furthermore, the fusion of CPC to tandem-GFPs defined the capability of CPC to increase the size exclusion limit of plasmodesmata.     

Amino acids in positions 48, 52, and 73 differentiate the substrate specificities of the highly homologous chlorocatechol 1,2-dioxygenases CbnA and TcbC

Chlorocatechol 1,2-dioxygenase (CCD) is the first-step enzyme of the chlorocatechol ortho-cleavage pathway, which plays a central role in the degradation of various chloroaromatic compounds. Two CCDs, CbnA from the 3-chlorobenzoate-degrader Ralstonia eutropha NH9 and TcbC from the 1,2,4-trichlorobenzene-degrader Pseudomonas sp. strain P51, are highly homologous, having only 12 different amino acid residues out of identical lengths of 251 amino acids. But CbnA and TcbC are different in substrate specificities against dichlorocatechols, favoring 3,5-dichlorocatechol (3,5-DC) and 3,4-dichlorocatechol (3,4-DC), respectively. A study of chimeric mutants constructed from the two CCDs indicated that the N-terminal parts of the enzymes were responsible for the difference in the substrate specificities. Site-directed mutagenesis studies further identified the amino acid in position 48 (Leu in CbnA and Val in TcbC) as critical in differentiating the substrate specificities of the enzymes, which agreed well with molecular modeling of the two enzymes. Mutagenesis studies also demonstrated that Ile-73 of CbnA and Ala-52 of TcbC were important for their high levels of activity towards 3,5-DC and 3,4-DC, respectively. The importance of Ile-73 for 3,5-DC specificity determination was also shown with other CCDs such as TfdC from Burkholderia sp. NK8 and TfdC from Alcaligenes sp. CSV90 (identical to TfdC from R. eutropha JMP134), which convert 3,5-DC preferentially. Together with amino acid sequence comparisons indicating high conservation of Leu-48 and Ile-73 among CCDs, these results suggested that TcbC of strain P51 had diverged from other CCDs to be adapted to conversion of 3,4-DC.     

Two kinds of chlorocatechol 1,2-dioxygenase from 2,4-dichlorophenoxyacetate-degrading Sphingomonas sp. strain TFD44

Two kinds of chlorocatechol 1,2-dioxygenase (CCD), TfdC and TfdC2 were detected in Sphingomonas sp. strain TFD44. These two CCDs could be simultaneously synthesized in TFD44 during its growth with 2,4-D as the sole carbon and energy sources. The apparent subunit molecular masses of TfdC and TfdC2 estimated by SDS-PAGE analysis were 33.8 and 33.1 kDa, respectively. The genes encoding the two CCDs were cloned and expressed in Escherichia coli. The two purified CCDs showed broad substrate specificities but had different specificity patterns. TfdC showed the highest specificity constant for 3-chlorocatechol and TfdC2 showed the highest specificity constant for 3,5-dichlorocatechol. The substrate specificity difference seemed to correlate with the alternation of amino acid supposed to be involved in the interaction with substrates. Whereas phylogenetic analysis indicated that the CCDs of Sphingomonas constitute a distinctive group among Gram-negative bacteria, TfdC and TfdC2 of TFD44 have divergently evolved in terms of their substrate specificity.     

Oxidized but not acetylated low-density lipoprotein reduces preproinsulin mRNA expression and secretion of insulin from HIT-T15 cells

We examined the effect of oxidized low-density lipoprotein (oxLDL) on the insulin secretion in the culture of HIT-T15 cell line, an islet beta-cell line derived from a hamster pancreatic tumor. In order to check the uptake of modified LDL by HIT-T15 cells, we prepared DiI-labeled native LDL (nLDL), acetylated LDL (AcLDL), and oxLDL. After the addition of each LDL into the cultures of HIT-T15 cells, fluorescence microscopic study was done. It was suggested that AcLDL and oxLDL were taken up by HIT-T15 cells, as well as nLDL. mRNA expression of the LDL receptor, CD36, and SR-B1 was detected in HIT-T15 by RT-PCR. The medium insulin level was measured in the culture of HIT-T15 cells with each LDL. oxLDL significantly reduced the insulin secretion stimulated by various concentrations of glucose, the intracellular content of insulin, and the expression of preproinsulin mRNA compared to the control cultures without LDL addition. In contrast, nLDL and AcLDL had no effect on the insulin secretion, the intracellular insulin level, or the expression of preproinsulin mRNA. MTT assay findings (reflecting cell numbers) were not different between cultures with and without LDLs. These results indicated that oxLDL disturbed the insulin metabolism of HIT-T15 cells.     

Design and expression of cysteine-bearing hydrophobic polypeptides and their self-assembling properties with bacteriochlorophyll a derivatives as a mimic of bacterial photosynthetic antenna complexes. Effect of steric confinement and orientation of the polypeptides on the pigment/polypeptide assembly process

A series of cysteine-bearing hydrophobic polypeptides analogous to a light-harvesting one betapolypeptide (LH1beta) from the LH1 complex from the purple photosynthetic bacterium, Rhodobacter sphaeroides, was synthesized using an Escherichia coli expression system. The cysteine was placed in the C- or N-terminal regions of the polypeptide to investigate the influence of steric confinement and orientation of the polypeptides via disulfide linkages as they were self-assembled with zinc-substituted bacteriochlorophyll a ([Zn]-BChl a). The polypeptides were expressed as water-soluble fusion proteins with maltose-binding protein (MBP). The fusion proteins formed a subunit-type complex with the [Zn]-BChl a in an n-octyl-beta-d-glucopyranoside (OG) micellar solution regardless of the cross-links or the cleavage of the cysteines, judging from absorption, CD, and fluorescence spectra. Following treatment with trypsin, the polypeptides were detached from the MBP portion. Such trypsin-digested polypeptides formed a subunit-type LH complex at 25 degrees C, which also showed that the disulfide linkage was not crucial for the subunit formation. When a polypeptide having cysteine on the C-terminus was assembled at 4 degrees C, the Qy absorption band was remarkably red-shifted to approximately 836 nm, suggesting that the cleavage of the large MBP portion liberates the polypeptides to form the progressive type of complex similar to LH1-type complex. The trypsin-treated polypeptides bearing cysteines in both terminal regions, which are randomly cross-linked, did not form the LH1-type complex under oxidative conditions but did form the complex under reductive conditions. This observation suggests that the polypeptide orientation strongly influences the LH1-type complex formation. The progressive assembly from the subunit to the holo-LH1-type complex following cleavage of MBP portion in a lipid bilayer is also briefly discussed.     

Protection of plasminogen activator inhibitor-1-deficient mice from nasal allergy

This study was performed to clarify the relationship between fibrinolytic components and the pathology of allergy, particularly that during the development of nasal allergy and nasal tissue changes. Intranasal OVA challenge after sensitization by i.p. administration of OVA induced a higher level of excess subepithelial collagen deposition in wild-type (WT) C57BL/6J mice than in plasminogen activator inhibitor (PAI)-1-deficient (PAI-1(-/-)) mice. The excess PAI-1 induction in the nasal mucosa and higher level of active PAI-1 in the nasal lavage fluid of WT-OVA mice compared with those in WT-control mice suggested that the decrease of proteolytic activity inhibits the removal of subepithelial collagen. The frequency of sneezing, nasal rubbing, nasal hyperresponsiveness, production of specific IgG1 and IgE in the serum, and production of IL-4 and IL-5 in splenocyte culture supernatant increased significantly in WT-OVA mice. In PAI-1(-/-) mice, these reactions were absent, and specific IgG2a in serum and IFN-gamma in splenocyte culture medium increased significantly. Histopathologically, there were marked goblet cell hyperplasia and eosinophil infiltration into the nasal mucosa in WT-OVA mice, but these were absent in PAI-1(-/-) mice. These results indicate that the immune response in WT-OVA mice can be classified as a dominant Th2 response, which would promote collagen deposition. In contrast, the Th2 response in PAI-1(-/-) mice was down-regulated, and the immune response shifted from Th2-dominant reaction to a Th1-dominant one. Taken together, these findings suggest that PAI-1 plays an important role not only in thrombolysis but also in immune response.     

Ion-trap tandem mass spectrometric analysis of Amadori-glycated phosphatidylethanolamine in human plasma with or without diabetes

Peroxidized phospholipid-mediated cytotoxicity is involved in the pathophysiology of diseases [i.e., an abnormal increase of phosphatidylcholine hydroperoxide (PCOOH) in plasma of type 2 diabetic patients]. The PCOOH accumulation may relate to Amadori-glycated phosphatidylethanolamine (Amadori-PE; deoxy-D-fructosyl phosphatidylethanolamine), because Amadori-PE causes oxidative stress. However, the occurrence of lipid glycation products, including Amadori-PE, in vivo is still unclear. Consequently, we developed an analysis method of Amadori-PE using a quadrupole/linear ion-trap mass spectrometer, the Applied Biosystems QTRAP. In positive ion mode, collision-induced dissociation of Amadori-PE produced a well-characterized diglyceride ion ([M+H-303]+) permitting neutral loss scanning and multiple reaction monitoring (MRM). When lipid extract from diabetic plasma was infused directly into the QTRAP, Amadori-PE molecular species could be screened out by neutral loss scanning. Interfacing liquid chromatography with QTRAP mass spectrometry enabled the separation and determination of predominant plasma Amadori-PE species with sensitivity of approximately 0.1 pmol/injection in MRM. The plasma Amadori-PE level was 0.08 mol% of total PE in healthy subjects and 0.15-0.29 mol% in diabetic patients. Furthermore, plasma Amadori-PE levels were positively correlated with PCOOH (a maker for oxidative stress). These results show the involvement between lipid glycation and lipid peroxidation in diabetes pathogenesis.     

Molecular cloning of Mucor hiemalis endo-beta-N-acetylglucosaminidase and some properties of the recombinant enzyme

Endo-M, endo-beta-N-acetylglucosaminidase from Mucor hiemalis, is known as a useful enzyme for the synthesis of neoglycopeptides due to its transglycosylation activity. We cloned the Endo-M gene encoding a putative 744 amino acids, which shows high identity to glycoside hydrolase family 85 endo-beta-N-acetylglucosaminidases. The gene encoding Endo-M was expressed in protease-deficient Candida boidinii with a molecular mass of 85 kDa as a monomeric form. Recombinant Endo-M could liberate both high-mannose type and biantennary complex type oligosaccharides from glycopeptides, which was same as the native enzyme. The Km and Kcat values for DNS-Man6GlcNAc2Asn were 0.51 mM and 8.25 s(-1), respectively. Recombinant Endo-M also exhibited transglycosylation activity toward high-mannose type and biantennary complex type oligosaccharides, which were transferred to alcohols, monosaccharides, oligosaccharides, and glycosides. To investigate about the catalytically essential amino acids of Endo-M, site-directed mutagenesis was performed, and it was found that mutants E177G and E177Q completely abolished the hydrolytic activity and W228R partially abolished the transglycosylation activity.     

A novel HLA-A*3303-restricted minor histocompatibility antigen encoded by an unconventional open reading frame of human TMSB4Y gene

Female-to-male hemopoietic stem cell transplantation (HSCT) elicits T cell responses against male-specific minor histocompatibility (H-Y) Ags encoded by the Y chromosome. All previously identified H-Y Ags are encoded by conventional open reading frames, but we report in this study the identification of a novel H-Y Ag encoded in the 5'-untranslated region of the TMSB4Y gene. An HLA-A*3303-restricted CD8(+) CTL clone was isolated from a male patient after an HSCT from his HLA-identical sister. Using a panel of cell lines carrying Y chromosome terminal deletions, a narrow region controlling the susceptibility of these target cells to CTL recognition was localized. Minigene transfection and epitope reconstitution assays identified an 11-mer peptide, EVLLRPGLHFR, designated TMSB4Y/A33, whose first amino acid was located 405 bp upstream of the TMSB4Y initiation codon. Analysis of the precursor frequency of CTL specific for recipient minor histocompatibility Ags in post-HSCT peripheral blood T cells revealed that a significant fraction of the total donor CTL response in this patient was directed against the TMSB4Y epitope. Tetramer analysis continued to detect TMSB4Y/A33-specific CD8(+) T cells at least up to 700 days post-HSCT. This finding underscores the in vivo immunological relevance of minor histocompatibility Ags derived from unconventional open reading frame products.