p73 is expressed in human thymic epithelial cells.

The thymus is a heterogeneous immune organ in which immature T-cells develop and eventually specialize to make certain immune responses of their own. Among various types of stromal cells in the thymus, thymic epithelial cells (TECs) have a crucially important function for presenting self-antigens and secreting cytokines to thymocytes for their maturation into T-cells. In this study we show that the p73 gene, a homologue of the tumor suppressor gene p53, was expressed in the nucleus of the human TEC in vivo and in TEC lines in vitro. Because p73 has the capacity to be a transactivator like p53, it may contribute to T-cell development in the context of TEC biology as regulated in the cell cycle and apoptosis.     

Synthesis of polyrotaxane-biotin conjugates and surface plasmon resonance analysis of streptavidin recognition

A polyrotaxane-biotin conjugate was synthesized and its interaction with streptavidin measured using surface plasmon resonance (SPR) detection. A biodegradable polyrotaxane in whichca. 22 molecules of α-cyclodextrins (α-CDs) were threaded onto a poly(ethylene oxide) chain (M _n: 4,000) capped with benzyloxycarbonyl-L-phenylalanine was conjugated with a biotin hydorazide and 2-aminoethanol after activating the hydroxyl groups of α-CDs in the polyrotaxane usingN,N′-carbony diimidazole. The results of the high-resolution¹H-nuclear magnetic resonance (¹H-NMR) spectra and gel permeation chromatography of the conjugate showed thatca. 11 biotin molecules were actually introduced to the polyrotaxane scaffold. An SPR analysis showed that the binding curves of the biotin molecules in the conjugate on the streptavidin-deposited surface changed in a concentration dependent manner, indicating that the biotin in the conjugate was actually recognized by streptavidin. The association equilibrium constant (K _a) of the interaction between the conjugate and streptavidin tetramer was of the order 10⁷. These results suggest that polyrotaxane is useful for scaffolds as a polymeric ligand in biomedical fields.     

Epitope analysis of lipid A preparations from Pseudomonas diminuta and Pseudomonas vesicularis

Abstract In vitro antigenic reactivity of lipid A from Pseudomonas diminuta and Pseudomonas vesicularis with homologous and heterologous lipid A antibodies including monoclonal antibodies was studied by inhibition test of enzyme-linked immunosorbent assay (ELISA). The results suggest that both Pseudomonas lipid As have very similar epitopes, including species-specific and cross-reactive epitopes as compared with enterobacterial lipid A.     

Voxel Based Analysis of Surgical Revascularization for Moyamoya Disease: Pre- and Postoperative SPECT Studies

Moyamoya disease (MMD) is a chronic, progressive, cerebrovascular occlusive disease that causes abnormal enlargement of collateral pathways (moyamoya vessels) in the region of the basal ganglia and thalamus. Cerebral revascularization procedures remain the preferred treatment for patients with MMD, improving the compromised cerebral blood flow (CBF). However, voxel based analysis (VBA) of revascularization surgery for MMD based on data from pre- and postoperative data has not been established. The latest algorithm called as Diffeomorphic Anatomical Registration Through Exponentiated Lie Algebra (DARTEL) has been introduced for VBA as the function of statistical parametric mapping (SPM8), and improved registration has been achieved by SPM8 with DARTEL. In this study, VBA was conducted to evaluate pre- and postoperative single photon emission computed tomography (SPECT) images for MMD by SPM8 with DARTEL algorithm, and the results were compared with those from SPM8 without DARTEL (a conventional method). Thirty-two patients with MMD who underwent superficial temporal artery-middle cerebral artery (STA-MCA) bypass surgery as the first surgery were included and all patients underwent pre- and postoperative 3D T1-weighted imaging and SPECT. Pre- and postoperative SPECT images were registered to 3D T1-weighted images, then VBA was conducted. Postoperative SPECT showed more statistically increased CBF areas in the bypassed side cerebral hemisphere by using SPM8 with DARTEL (58,989 voxels; P<0.001), and increased ratio of CBF after operation was less than 15%. Meanwhile, postoperative SPECT showed less CBF increased areas by SPM8 without DARTEL. In conclusion, VBA was conducted for patients with MMD, and SPM8 with DARTEL revealed that postoperative SPECT showed statistically significant CBF increases over a relatively large area and with at most 15% increase ratio.     

Some Possible Evidences for an Alteration in the Actin-Myosin Interaction in Stored Muscle

A two-dimensional mapping analysis was performed by HPLC for 4 kinds of standard galactosyllactoses (GLs, trisaccharide) which were assumed to be produced from lactose (galactopyranosylβ1→4 glucopyranose) in yogurt during the fermentation of lactic acid bacteria. After the pyridylamination of GLs, they were analyzed by HPLC in the reverse-phase (RP) and anion-exchange (AE) modes. The retention times of each peak obtained were converted to glucose units (GU) in RP mode for the pyridylaminated isomaltooligosaccharides (G1-3) and to relative retention time (RRT) in AE mode against pyridylaminated-isomaltotriose, and then the address data [GU, RRT] were plotted on a graph. This two-dimensional mapping method was found useful for a rapid qualitative evaluation of the chemical structure of trisaccharides formed in yogurt.     

Increase of cyclin B by overexpression of cystatin α

Degradation of cyclin B was effectively suppressed when cells were treated with ALLN (N-acetylleucylleucylnorleucinal) which inhibits proteasome, calpain and cysteine proteinase cathepsins. In order to examine which protease degrades cyclin B, the effect of a cathepsin inhibitor, cystatin α, was investigated. The cystatin α gene was inserted into an inducible expression vector, pMSG, and transfected into NIH3T3 mouse fibroblasts. The expression of cystatin α was induced effectively in the transfected cells after treatment with dexamethasone. Overexpression of cystatin α resulted in an increase of the amount of cyclin B, suggesting that cysteine proteinase cathepsins might be involved in the degradation of cyclin B.     

Enhanced Protection Against Peroxidation-Induced Mortality of Aortic Endothelial Cells by Ascorbic Acid-2-O-Phosphate Abundantly Accumulated in the Cell as the Dephosphorylated Form

Bovine aortic endothelial BAE-2 cells exposed to the peroxidizing agent, tert-butylhydroperoxide (t-BuOOH) or 2,4-nonadienal (NDE), suffered from disruption of cell membrane integrity and from reduction of mitochondrial dehydrogenase activity as assessed by fluorometry using ethidium homodimer and photometry using WST-1, respectively. The cells were protected from t-BuOOH-induced injury more markedly by L-ascorbic acid-2-O-phosphate (Asc2P) stably masked at the 2,3-enediol moiety, which is responsible for the antioxidant ability of L-ascorbic acid (Asc), than by Asc itself. In contrast, NDE-induced membrane disruption but not mitochondrial dysfunction was prevented by Asc2P, whereas Asc exhibited no prevention against both types of injury. The amount of intracellular Asc was 7.2- to 9.0-fold larger in Asc2P-administered BAE-2 cells, where the intact form Asc2P was not detected, than in Asc-administered cells as assessed by HPLC of cell extract with detection by coulometric ECD and W. During transmembrane influx into the cell, Asc2P was concentrated as highly as 70- to 90-fold relative to the extracellular Asc2P concentration, whereas Asc was 8-to 13-fold concentrated as estimated based on an intracellular water content of 0.59 pL/cell determined by [¹⁴C]PEG/gas chromatography. Thus, Asc2P but not Asc is highly concentrated in the aqueous phase of the cell after prompt dephosphorylation, and may thereby render the cell more resistant to t-BuOOH-peroxidation assumedly via scavenging of intracellular reactive oxygen species than to peroxidation with the less hydroplulic agent NDE.     

Tandem and single genes of three membrane-bound nitrate transporters in the nar gene cluster of the moderately halophilic denitrifier, Halomonas halodenitrificans.

We identified the genes encoding the membrane-bound nitrate reductase (Nar) from the moderate halophile, Halomonas halodenitrificans, and examined the structure of the gene cluster. Screening of a H. halodenitrificans genomic DNA library in lambda EMBL3 phage by chromosome walking revealed that the region adjacent to the nor gene cluster encoding nitric oxide (NO) reductase contains three nitrate transporters: tandem narK2 and narK1.1 genes and a single narK1.2 gene encoded in opposite directions. NarK1.1 and NarK1.2 proteins, which have 12 putative membrane-spanning helices, were classified as type I NarK, whereas NarK2, which has 14 putative membrane-spanning helices, was classified as a type II NarK. NarK1.1 and NarK2 proteins were considered to be functionally and structurally linked in the cytoplasmic membrane. The systems regulating the expression of the tandem narK2K1.1 gene and the single narK1.2 gene were found to be different. Further, binding sites for NarL and Fnr-like proteins are present in the promoter region of the narK2 gene.     

Establishment of a SPF colony of human apo(a) transgenic rabbits by frozen-thawed embryo transfer.

In the present study, embryo transfer was performed using frozen-thawed embryos to establish a SPF colony of human apolipoprotein (a) (apo(a)) transgenic rabbits. Apo(a) transgenic rabbits were kept under conventional condition and were infected with Bordetella bronchiseptica. Embryos at the morula stage were collected and stored in liquid nitrogen. After thawing, the in vitro survival rate was 84.6%, and 125 morphologically normal embryos were transferred to 6 SPF recipient rabbits. Four rabbits became pregnant and 23 live pups were born. PCR and Western blot analyses revealed that 9 of 23 pups were transgenic and expressed apo(a) protein. Microbiological tests showed all rabbits were free from infections. We succeeded in establishing a SPF colony of apo(a) transgenic rabbits. These rabbits are now maintained under a barrier system and are available for medical research.     

Strain-Dependent Differences in Locomotor Activity after Local Brain Irradiation with 30 GyE of Carbon Ions.

This study investigated strain differences in brain damage among male A/J, C57BL/6JNrs and C3H/HeNrs mice after local brain irradiation. Whole brains were irradiated with a single dose of 30 GyE carbon ion beams and then locomotor activity was determined as body heat of each animal. The daily locomotor activities of untreated mice differed among strains. Non-irradiated C57BL/6JNrs mice were more active than A/J mice. This variance became more obvious immediately after irradiation, when the activity of A/J and C3H/HeNrs mice diminished, whereas that of C57BL/6JNrs mice increased at the beginning of the active phase and remained elevated for three days after irradiation. The altered activities of all three strains of irradiated mice gradually recovered to normal within three to four days.