The cytosolic and transmembrane domains of the beta 1,6 N-acetylglucosaminyltransferase (C2GnT) function as a cis to medial/Golgi-targeting determinant

The beta 1,6 N-acetylglucosaminyltransferase (C2GnT) has been recently mapped to the cis/medial-Golgi compartment. To analyze the Golgi-targeting determinants of C2GnT, we constructed various deletion mutants of the enzyme fused to the enhanced green fluorescent protein (EGFP) and localized these proteins by fluorescence microscopy in living cells. We found that the N-terminal peptide encompassing amino acids 1 to 32 represents the minimal Golgi-targeting signal sufficient to localize EGFP to the same compartment as the full-length C2GnT. This peptide makes up the cytoplasmic and the transmembrane domains of the enzyme and was referred to as CTd (cytoplasmic and transmembrane domains). We compared the Golgi-targeting efficiency of the C2GnT-derived CTd with its homologous domains from other glycosyltransferases, including the H-type alpha(1,2)-fucosyltransferase (FucTI), the polypeptide N-acetylgalactosaminyltransferase-I (GalNAcT-I), the alpha(1,3)-fucosyltransferase VII (FucTVII), and the alpha(2,6)-sialyltransferase (ST6Gal-I) and found that the Golgi-targeting determinants of these glycosyltransferases were also composed of their cytosolic and transmembrane domains. To investigate whether the CTd of C2GnT could serve as a cis to medial Golgi-specific signal, we tested its ability to mislocalize two late-Golgi acting glycosyltransferases FucTI and FucTVII. We show that fusing the C2GnT-derived CTd with the catalytic domain of FucTVII resulted in a complete mislocalization of the enzyme to the C2GnT compartment, with a parallel alteration of sialyl-Lewis x synthesis and P-selectin binding. The intracellular distribution and activity of FucTI, however, were not affected. Thus, CTds of either early or late-Golgi acting glycosyltransferases represent the Golgi-targeting domains of these enzymes. In addition, we show that C2GnT-derived CTd can function as a cis/medial Golgi-targeting determinant.     

Specific interactions between cryogel components: role of extra domain A containing fibronectin in cryogelation

Cryogel is a physical gel formed by heterophilic aggregation of extra domain A containing fibronectin [EDA(+)FN], plasma fibronectin (pFN), fibrinogen (Fbg) and heparin (Hep), which are found in high concentrations in the blood of patients suffering from rheumatoid arthritis. In this study, we clarify the specific interactions between cryogel components in terms of the affinity constant (K(A)), obtained by surface plasmon resonance (SPR). It is found that Fbg self-interactions occur at lower temperatures, and that K(A) of Fbg-Hep changes with temperature. Specifically, K(A) (2.0 x 10(8) [M(-1)]) of Fbg-Hep at 5 degrees C increases significantly from that (1.0x10(7) [M(-1)]) at 40 degrees C. K(A) of EDA(+)FN-Hep increases with temperature, by approximately 100-fold between 40 degrees C (K(A)=10(12) [M(-1)]) and 20 degrees C (K(A)=10(10) [M(-1)]). Although K(A) of the FN fragments of Hep-binding domain containing an EDA region [EDA(+)HBD(+)] and Hep increases with temperatures above 30 degrees C, K(A)s of HBD(+)-Hep and EDA(+)-Hep are not temperature-dependent. Therefore, EDA(+)HBD(+), formed as a special structure for high Hep affinity, exhibits temperature-dependent interaction with Hep. These results suggest that the main role of EDA(+)FN in cryogelation is to support the interaction with Hep.     

New human myelodysplastic cell line,TER-3: G-CSF specific downregulation of Ca2+/calmodulin-dependent protein kinase IV

We have established a new hematopoietic cell line from a patient with myelodysplastic syndrome (MDS), which was refractory anemia with excess blasts (RAEB). This cell line, designated TER-3, depends on several cytokines for long-term survival and growth, and requires interleukin-3 (IL-3) for continuous growth. Cytochemical analysis revealed that TER-3 cells are weakly dianisidine positive and nonspecific esterase positive, but peroxidase negative. The surface marker profile shows that the TER-3 cells are strongly positive for myeloid, lymphoid, and megakaryocytic antigens such as CD15, CD19, and CD61, and negative for some common multilineage antigens such as CD13, CD33, and CD34. Thus, this cell line has a multilineage phenotype, suggesting that the transformation event occurred in multipotent stem cells. Dianisidine- and nonspecific esterase-positive TER-3 cells increase with granulocyte-colony stimulating factor (G-CSF) rather than with IL-3. These results suggest that the cell line is useful for understanding the mechanism underlying G-CSF-associated hematopoietic cell differentiation and activation in the patient with MDS.     

Molecular and clinical study of 183 patients with conotruncal anomaly face syndrome

To investigate molecular and clinical aspects of conotruncal anomaly face (CAF), we studied the correlation between deletion size and phenotype and the mode of inheritance in 183 conotruncal anomaly face syndrome (CAFS) patients. Hemizygosity for a region of 22q11.2 was found in 180 (98%) of the patients with CAFS by fluorescence in situ hybridization (FISH) using the N25(D22S75) DiGeorge critical region (DGCR) probe. No hemizygosity was found in three (2%) of the patients with CAFS by FISH using nine DiGeorge critical region probes and a SD10P1 probe (DGA II locus). None of these three patients had mental retardation and just one had nasal intonation, which was observed in almost all of the 180 CAFS patients who carried deletions (mental retardation, 92%; nasal voice, 88%). Nineteen of 143 families (13%) had familial CAFS and 16 affected parents (84%) were mothers. Although only two of the affected parents had cardiovascular anomalies, the deletion size in the 16 affected parents and their affected family members, who were studied by FISH analysis, was the same. It indicates that extragenic factors may play a role in the genesis of phenotypic variability, especially in patients with cardiovascular anomalies. No familial cases were found among CAFS patients with absent thymus/DiGeorge anomaly (DGA). Also, in all 18 CAFS patients with completely absent thymus/DGA and all 6 CAFS patients with schizophrenia, it was revealed that the deletion was longer distally. A study of the origin of the deletion using microsatellite analyses in 48 de novo patients showed that in 65% of CAFS patients it was maternal, while in 64% of DGA patients it was paternal. The findings of this study indicated that CAF was almost always associated with the deletion of 22q11.2. As well as the major features of the syndrome, other notable extracardiac anomalies were found to be susceptibility to infection, schizophrenia, atrophy or dysmorphism of the brain, thrombocytopenia, short stature, facial palsy, anal atresia, and mild limb abnormalities. Received: 5 January 1998 / Accepted: 7 March 1998     

Regioselective deacetylation of peracetylated monosaccharide derivatives by polyethylene glycol-modified lipase for the oligosaccharide synthesis

Lipase modified with polyethylene glycol became soluble and active in organic solvents, and catalyzed regioselective deacetylation of peracetylated monosaccharide derivatives in 1,1,1-trichloroethane. The deacetylation occurred only at the positions of C-4 and C-6 of the glycopyranoside ring. Especially, peracetylated methyl -D-xylopyranoside and peracetylated L-serine--D-xylopyranoside were hydrolyzed only at the position of C-4. Subsequently, one of the resulting products, that is L-serine-2,3-di-O-acetyl--D-xylopyranoside, was coupled with galactose residue to obtain L-serine-4-O-(-D-galactopyranosyl)--D-xylopyranoside, a model compound of the carbohydrate-protein linkage region of proteoglycans.     

Early-life atomic-bomb irradiation accelerates immunological aging and elevates immune-related intracellular reactive oxygen species

Reactive oxygen species (ROS) play an important role in immune responses; however, their excessive production and accumulation increases the risk of inflammation-related diseases. Although irradiation is known to accelerate immunological aging, the underlying mechanism is still unclear. To determine the possible involvement of ROS in this mechanism, we examined 10,023 samples obtained from 3752 atomic-bomb survivors in Hiroshima and Nagasaki, who participated in repeated biennial examinations from 2008 to 2016, for the effects of aging and radiation exposure on intracellular ROS (H₂O₂ and O₂^•−) levels, percentages of T-cell subsets, and the effects of radiation exposure on the relationship between cell percentages and intracellular ROS levels in T-cell subsets. The cell percentages and intracellular ROS levels in T-cell subsets were measured using flow cytometry, with both fluorescently labeled antibodies and the fluorescent reagents, carboxy-DCFDA and hydroethidine. The percentages of naïve CD4⁺ and CD8⁺ T cells decreased with increasing age and radiation dose, while the intracellular O₂^•− levels in central and effector memory CD8⁺ T cells increased. Additionally, when divided into three groups based on the percentages of naïve CD4⁺ T cells, intracellular O₂^•− levels of central and effector memory CD8⁺ T cells were significantly elevated with the lowest radiation dose group in the naïve CD4⁺ T cells. Thus, the radiation exposure-induced decrease in the naïve CD4⁺ T cell pool size may reflect decreased immune function, resulting in increased intracellular ROS levels in central and effector memory CD8⁺ T cells, and increased intracellular oxidative stress.     

Effect of thyroxine on abnormal pancreatic proteomes of the hypothyroid rdw rat

A mutation in the thyroglobulin (Tg) gene is the primary cause of hereditary dwarfism and hypothyroidism in the rdw rat. Despite the Tg mutation that causes a Tg shortage, rdw rats survive. The present study examines the influences of this condition on the pancreatic proteome. Normal control (group 1; n = 19) and rdw rats that did not receive L-thyroxine (T4) (group 2; n = 27) were sacrificed from 4 to 56 weeks after birth. The rdw rats were supplemented either with daily intraperitoneal injections of T4 from 3 to 28 days after birth (group 3; n = 4) or with normal thyroid tissues grafted at 4 weeks of age (group 4; n = 3). Groups 3 and 4 were sacrificed 12 weeks after birth. Pancreatic proteomes analyzed by two-dimensional gel electrophoresis showed that levels of at least four pancreatic proteins were higher in group 2 than in group 1, and that those of four were lower. Cluster decomposition and principal component analysis of the eight protein contents showed that groups 1 and 2 were separated into two clusters and that pancreatic proteomes of group 4 were better normalized than those of group 3. Injecting T4 into group 3 was temporarily effective, whereas the thyroid graft to group 4 provided a continuous positive effect, which concurred with the increased body weight of the other two groups of rdw rats that received grafts of normal thyroids.     

Crystal structure of the drug discharge outer membrane protein, OprM, of Pseudomonas aeruginosa: dual modes of membrane anchoring and occluded cavity end

The OprM lipoprotein of Pseudomonas aeruginosa is a member of the MexAB-OprM xenobiotic-antibiotic transporter subunits that is assumed to serve as the drug discharge duct across the outer membrane. The channel structure must differ from that of the porin-type open pore because the protein facilitates the exit of antibiotics but not the entry. For better understanding of the structure-function linkage of this important pump subunit, we studied the x-ray crystallographic structure of OprM at the 2.56-angstroms resolution. The overall structure exhibited trimeric assembly of the OprM monomer that consisted mainly of two domains: the membrane-anchoring beta-barrel and the cavity-forming alpha-barrel. OprM anchors the outer membrane by two modes of membrane insertions. One is via the covalently attached NH(2)-terminal fatty acids and the other is the beta-barrel structure consensus on the outer membrane-spanning proteins. The beta-barrel had a pore opening with a diameter of about 6-8 angstroms, which is not large enough to accommodate the exit of any antibiotics. The periplasmic alpha-barrel was about 100 angstroms long formed mainly by a bundle of alpha-helices that formed a solvent-filled cavity of about 25,000 angstroms(3). The proximal end of the cavity was tightly sealed, thereby not permitting the entry of any molecule. The result of this structure was that the resting state of OprM had a small outer membrane pore and a tightly closed periplasmic end, which sounds plausible because the protein should not allow free access of antibiotics. However, these observations raised another unsolved problem about the mechanism of opening of the OprM cavity ends. The crystal structure offers possible mechanisms of pore opening and pump assembly.