Intratracheal gene transfer of decorin reduces subpleural fibroproliferation induced by bleomycin

Decorin, a small leucin-rich proteoglycan, is a negative regulator of transforming growth factor-beta, but the antifibrotic effect of decorin gene transfer has not been examined in a mouse model of usual interstitial pneumonia (UIP). We constructed a replication-defective recombinant adenovirus harboring human decorin gene (AdCMV.DC) and administered 1 x l0(9) plaque-forming units of AdCMV.DC intratracheally or intravenously to C57BL/6 mice with intraperitoneal injection of bleomycin, which induces a subpleural fibroproliferation, mimicking UIP, by day 28. Only intratracheal administration of AdCMV.DC increased decorin mRNA expression in the lung and decreased the hydroxyproline content augmented in bleomycin-induced pulmonary fibrosis (1.13 +/- 0.02 to 0.96 +/- 0.02, P = 0.006). In contrast, intravenous administration of AdCMV.DC increased the decorin expression only in the liver, but not in the lung, and without reducing lung fibrosis. These results indicate that adenoviral decorin gene transfer is effective only by direct administration to fibrosing lungs.     

TRAIL-R2 (DR5) mediates apoptosis of synovial fibroblasts in rheumatoid arthritis

TRAIL has been proposed as an anti-inflammatory cytokine in animal models of rheumatoid arthritis (RA). Using two agonistic mAbs specific for TRAIL-R1 (DR4) and TRAIL-R2 (DR5), we examined the expression and function of these death receptors in RA synovial fibroblast cells. The synovial tissues and primary synovial fibroblast cells isolated from patients with RA, but not those isolated from patients with osteoarthritis, selectively expressed high levels of cell surface DR5 and were highly susceptible to anti-DR5 Ab (TRA-8)-mediated apoptosis. In contrast, RA synoviocytes did not show increased expression of TRAIL-R1 (DR4), nor was there any difference in expression of Fas between RA and osteoarthritis synovial cells. In vitro TRA-8 induced apoptosis of RA synovial cells and inhibited production of matrix metalloproteinases induced by pro-inflammatory cytokines. In vivo TRA-8 effectively inhibited hypercellularity of a SV40-transformed RA synovial cell line and completely prevented bone erosion and cartilage destruction induced by these cells. These results indicate that increased DR5 expression and susceptibility to DR5-mediated apoptosis are characteristic of the proliferating synovial cells in RA. As highly proliferative transformed-appearing RA synovial cells play a crucial role in bone erosion and cartilage destruction in RA, the specific targeting of DR5 on RA synovial cells with an agonistic anti-DR5 Ab may be a potential therapy for RA.     

A role of CXC chemokine ligand 12/stromal cell-derived factor-1/pre-B cell growth stimulating factor and its receptor CXCR4 in fetal and adult T cell development in vivo

The functions of a chemokine CXC chemokine ligand (CXCL) 12/stromal cell-derived factor-1/pre-B cell growth stimulating factor and its physiologic receptor CXCR4 in T cell development are controversial. In this study, we have genetically further characterized their roles in fetal and adult T cell development using mutant and chimeric mice. In CXCL12(-/-) or CXCR4(-/-) embryos on a C57BL/6 background, accumulation of T cell progenitors in the outer mesenchymal layer of the thymus anlage during initial colonization of the fetal thymus was comparable with that seen in wild-type embryos. However, the expansion of CD3(-)CD4(-)CD8(-) triple-negative T cell precursors at the CD44(-)CD25(+) and CD44(-)CD25(-) stages, and CD4(+)CD8(+) double-positive thymocytes was affected during embryogenesis in these mutants. In radiation chimeras competitively repopulated with CXCR4(-/-) fetal liver cells, the reduction in donor-derived thymocytes compared with wild-type chimeras was much more severe than the reduction in donor-derived myeloid lineage cells in bone marrow. Triple negative CD44(+)CD25(+) T cell precursors exhibited survival response to CXCL12 in the presence of stem cell factor as well as migratory response to CXCL12. Thus, it may be that CXCL12 and CXCR4 are involved in the expansion of T cell precursors in both fetal and adult thymus in vivo. Finally, enforced expression of bcl-2 did not rescue impaired T cell development in CXCR4(-/-) embryos or impaired reconstitution of CXCR4(-/-) thymocytes in competitively repopulated mice, suggesting that defects in T cell development caused by CXCR4 mutation are not caused by reduced expression of bcl-2.     

Extraordinary proliferation of microorganisms in aposymbiotic pea aphids,Acyrthosiphon pisum

Aposymbiotic pea aphids, which were deprived of their intracellular symbiotic bacterium, Buchnera, exhibit growth retardation and no fecundity. High performance liquid chromatographic (HPLC) analysis revealed that these aposymbiotic aphids, when reared on broad bean plants, accumulated a large amount of histamine. To assess the possibility of extraordinary proliferation of microorganisms other than Buchnera, we enumerated eubacteria and fungi in aphids using the real-time quantitative PCR method that targets genes encoding small-subunit rRNAs. The result showed that these microorganisms were extremely abundant in the aposymbiotic aphids reared on plants. Microbial communities in aposymbiotic aphids were further profiled by phylogenetic analysis of small-subunit rDNAs. Of 172 nonchimeric sequences of fungal 18S rDNAs, 138 (80.2%) belonged to the phylum Ascomycota. Among them, 21 clustered within a monophyletic group consisting of insect-pathogenic fungi and yeast-like symbionts of homopteran insects. Thirty-one (18.0%), two (1.2%), and one (0.6%) clones were clustered within the Basidiomycota, Zygomycota, and Oomycota, respectively. Of 167 nonchimeric sequences of eubacterial 16S rDNAs, 84 (50.3%) belonged to the gamma-subdivision of Proteobacteria to which most primary endosymbionts of insects and prolific histamine producers belong. Forty (24.0%), 25 (15.0%), 10 (6.0%), and five (3.0%) clones were clustered within alpha-Proteobacteria, Cytophaga-Flavobacterium-Bacteroides (CFB) group, Actinobacteria, and beta-Proteobacteria, respectively. Three had no phylogenetic association with known taxonomic divisions. None of the sequences studied in this study coincided exactly with those deposited in GenBank.     

A palmitoylated RING finger ubiquitin ligase and its homologue in the brain membranes

Ubiquitin (Ub) ligation is implicated in active protein metabolism and subcellular trafficking and its impairment is involved in various neurologic diseases. In rat brain, we identified two novel Ub ligases, Momo and Sakura, carrying double zinc finger motif and RING finger domain. Momo expression is enriched in the brain gray matter and testis, and Sakura expression is more widely detected in the brain white matter as well as in many peripheral organs. Both proteins associate with the cell membranes of neuronal and/or glial cells. We examined their Ub ligase activity in vivo and in vitro using viral expression vectors carrying myc-tagged Momo and Sakura. Overexpression of either Momo or Sakura in mixed cortical cultures increased total polyubiquitination levels. In vitro ubiquitination assay revealed that the combination of Momo and UbcH4 and H5c, or of Sakura and UbcH4, H5c and H6 is required for the reaction. Deletion mutagenesis suggested that the E3 Ub ligase activity of Momo and Sakura depended on their C-terminal domains containing RING finger structure, while their N-terminal domains influenced their membrane association. In agreement, Sakura associating with the membrane was specifically palmitoylated. Although the molecular targets of their Ub ligation remain to be identified, these findings imply a novel function of the palmitoylated E3 Ub ligase(s).     

Chemical identification of serine 181 at the ATP-binding site of myosin as a residue esterified selectively by the fluorescent reagent 9-anthroylnitrile

The esterification reagent 9-anthroylnitrile (ANN) reacts with a serine residue in the NH2-terminal 23-kDa peptide segment of myosin subfragment-1 heavy chain to yield a fluorescent S1 derivative labeled by the anthroyl group (Hiratsuka, T. (1989) J. Biol. Chem. 264, 18188-18194). The labeling was highly selective and accelerated by nucleotides. In the present study, to determine the exact location of the labeled serine residue, the labeled 23-kDa peptide fragment was isolated. The subsequent extensive proteolytic digestion of the peptide fragment yielded two labeled peptides, a pentapeptide and its precursor nonapeptide. Amino acid sequence and composition analyses of both labeled peptides revealed that the anthroyl group is attached to Ser-181 involved in the phosphate binding loop for ATP (Smith, C. A., and Rayment, I. (1996) Biochemistry 35, 5404-5417). We concluded that ANN can esterify Ser-181 selectively out of over 40 serine residues in the subfragment 1 heavy chain. Thus ANN is proved to be a valuable fluorescent tool to identify peptides containing the phosphate binding loop of S1 and to detect the conformational changes around this loop.     

Chemo-enzymatic synthesis of 3-(2-naphthyl)- L-alanine by an aminotransferase from the extreme thermophile, Thermococcus profundus

Hyper-thermostable aminotransferase from Thermococcus profundus (MsAT) was used to synthesize 3-(2-naphthyl)-l-alanine (Nal) by transamination between its corresponding -keto acid, 3-(2-naphthyl)pyruvate (NPA) and l-glutamate (Glu) at 70 °C. Equilibrium of this reaction was shifted toward Nal production due to its low solubility, giving rise to Nal precipitate. Optically pure Nal (>99% ee) was synthesized with 93% (mol mol^–1) yield from 180 mM NPA and 360 mM Glu.     

High-cut characteristics of the baroreflex neural arc preserve baroreflex gain against pulsatile pressure

A transfer function from baroreceptor pressure input to sympathetic nerve activity (SNA) shows derivative characteristics in the frequency range below 0.8 Hz in rabbits. These derivative characteristics contribute to a quick and stable arterial pressure (AP) regulation. However, if the derivative characteristics hold up to heart rate frequency, the pulsatile pressure input will yield a markedly augmented SNA signal. Such a signal would saturate the baroreflex signal transduction, thereby disabling the baroreflex regulation of AP. We hypothesized that the transfer gain at heart rate frequency would be much smaller than that predicted from extrapolating the derivative characteristics. In anesthetized rabbits (n = 6), we estimated the neural arc transfer function in the frequency range up to 10 Hz. The transfer gain was lost at a rate of -20 dB/decade when the input frequency exceeded 0.8 Hz. A numerical simulation indicated that the high-cut characteristics above 0.8 Hz were effective to attenuate the pulsatile signal and preserve the open-loop gain when the baroreflex dynamic range was finite.     

Increased expression of cardiotrophin-1 during ventricular remodeling in hypertensive rats

Cardiotrophin-1 (CT-1) stimulates longitudinal myocardial cell hypertrophy. We examined the expression of CT-1, leukemia inhibitory factor (LIF), and gp130 by competitive RT-PCR and Western blotting in Dahl salt-sensitive (DS) rats with a high-salt diet, which showed a distinct transition from left ventricular hypertrophy (LVH) to congestive heart failure (CHF). The expression levels of CT-1 mRNA and protein were significantly increased at the CHF stage compared with the LVH stage and age-matched Dahl salt-resistant (DR) rats (n = 6 for each group). mRNA expression of LIF was not changed in the left ventricle at any stage by RT-PCR. gp130 mRNA and protein levels of DS rats at 11 and 17 wk were significantly increased compared with age-matched DR rats. The isolated myocyte length of DS rats at 17 wk was the longest among the four groups of rats. The LV end-diastolic dimension (LVDd) of DS rats, determined by echocardiography, was significantly increased at the CHF stage. There was a significant correlation between the CT-1 protein level and LVDd. CT-1 may play a role in ventricular remodeling during transition from LVH to CHF in the rat hypertensive model.     

Potential changes with gamma-band oscillation at the frontal scalp elicited by intravenous olfactory stimulation in humans

Intravenous olfaction is a unique stimulation method often used in Japan to diagnose olfactory disturbances. Odorant is injected into a vein and transported by blood flow and respiration to the upper air tract. The intravenous olfaction might allow the potential at the frontal scalp to be recorded without contamination from electromyograms, such as those caused by sniffing. We injected Alinamin (thiamine propyldisulphide) into healthy subjects according to a standard protocol for clinical intravenous olfaction testing and we simultaneously recorded potential changes at the frontal scalp. When Alinamin was injected into the right median cubital vein over a 20 s period, the potential changes with gamma-band oscillations were detected 17.6 +/- 6.7 s (mean +/- SD) after the start of the injection. The main frequency component of this gamma-band oscillation is 30-160 Hz. The gamma-band oscillation elicited by intravenous olfactory stimulation (VOP) was similar to the induced wave of the olfactory bulb. Mapping the VOPs on the frontal scalp of a subject with less developed frontal sinuses and the relation between the thickness of the frontal sinuses and VOP amplitude suggest an intracranial source, possibly the olfactory bulb. The gamma-band potential at the frontal scalp is a useful measure of central disturbance.