Modulation of collagen fibrillogenesis by tenascin-X and type VI collagen

Tenascin-X (TNX) is an extracellular matrix glycoprotein. We previously demonstrated that TNX regulates the expression of type VI collagen. In this study, we investigated the binding of TNX to type I collagen as well as to type VI collagen and the effects of these proteins on fibrillogenesis of type I collagen. Full-length recombinant TNX, which is expressed in and purified from mammalian cell cultures, and type VI collagen purified from bovine placenta were used. Solid-phase assays revealed that TNX or type VI collagen bound to type I collagen, although TNX did not bind to type VI collagen, fibronectin, or laminin. The rate of collagen fibril formation and its quantity, measured as increased turbidity, was markedly increased by the presence of TNX, whereas type VI collagen did not increase the quantity but accelerated the rate of collagen fibril formation. Combined treatment of both had an additive effect on the rate of collagen fibril formation. Furthermore, deletion of the epidermal growth factor-like (EGF) domain or fibrinogen-like domain of TNX attenuated the initial rate of collagen fibril formation. Finally, we observed abnormally large collagen fibrils by electron microscopy in the skin from TNX-deficient (TNX-/-) mice during development. These findings demonstrate a fundamental role for TNX and type VI collagen in regulation of collagen fibrillogenesis in vivo and in vitro.     

2-hydroxy-4-isopropylbenzaldehyde, a potent partial tyrosinase inhibitor

Chamaecin (2-hydroxy-4-isopropylbenzaldehyde) was synthesized and tested for its tyrosinase inhibitory activity. It partially inhibits the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by mushroom tyrosinase with an IC(50) of 2.3 microM. The inhibition kinetics analyzed by Dixon plots found that chamaecin is a mixed type inhibitor. This inhibition may come in part from its ability to form a Schiff base with a primary amino group in the enzyme.     

Modeling interactions between photic and nonphotic entrainment mechanisms in transmeridian flights

In transmeridian flights, photic and nonphotic entrainment mechanisms are expected to interact dynamically in the human circadian system. In order to simulate the reentrainment process of the circadian rhythms, the photic entrainment mechanism was introduced to our previous model, which consisted of three coupled oscillators. Regardless of flight direction, a large time difference beyond 10 h tended to induce the antidromic reentrainment. The partition between the oscillators resulted for the eastward flight over a 10-h or longer time difference and the westward over 6 h or longer. The simulated reentrainment processes almost coincided with empirical knowledge. Simulated effects of physical exercise showed that some antidromic reentrainments were switched to the orthodromic ones for the eastward flight and most of the partitions between the oscillators were prevented in the westward flight. These results are due to an augmentation of the entrainment pressure of the rest–activity cycle on the oscillators. The mechanisms underlying these various reentrainment patterns were explained based on the photic response, the interactions between the oscillators, and their adaptive modification. The simulation results suggest that an appropriate selection of departure time and physical exercise could ease the jet lag caused by transmeridian flight.This research was supported by a Grant-In-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Nos. 14015205, 15014204, and 15560372).     

Hydrogen peroxide induces GADD153 in Jurkat cells through the protein kinase C-dependent pathway

Growth arrest and DNA damage-inducible gene 153 (GADD153) is a CCAAT/enhancer binding protein (C/EBP) related gene and is induced in response to various stimuli including DNA damaging agents, UV irradiation, and serum starvation. In this study, we investigated which intracellular signals contribute to the expression of GADD153 mRNA in Jurkat cells in response to oxidative stress using several kinds of kinase inhibitors. GADD153 mRNA expression was immediately enhanced following hydrogen peroxide exposure and was significantly inhibited by treatment with H-7, staurosporin, and Ro-31-8220. In particular, rottlerin, a PKCdelta specific inhibitor, markedly attenuated hydrogen peroxide-induced GADD153 mRNA expression even at 1 microM. Treatment with a potent PKC activator, phorbol-12-myristate-13-acetate (PMA), augmented GADD153 mRNA in Jurkat cells in the presence of hydrogen peroxide, although PMA alone induced GADD153 mRNA marginally. Hydrogen peroxide significantly enhanced the AP-1 binding activity of the nuclear extract from Jurkat cells to the GADD153 AP-1 binding site. AP-1 binding activity was suppressed by rottlerin treatment. These findings indicate that PKC, especially PKCdelta, plays an important role in the induction of GADD153 mRNA following oxidative stress.     

Improvement of Germ Line Transmission by Targeting β-galactosidase to Nuclei in Transgenic Mice

The Escherichia coli lacZ gene has frequently been used as a reporter in cell lineage analysis, in determining the elements regulating spatial and temporal gene expression, and in enhancer/gene trap detection of developmentally regulated genes. However, it is uncertain whether lacZ expression affects eukaryotic cell growth and development. By using a gene trap, we previously isolated the promoter, Ayu1, which is active in ES cells and in several tissues including the gonads. We used this promoter and the nuclear location signal of the SV40 large T gene to locate β-galactosidase either in the cytoplasm or the nucleus. Transgenic lines containing β-galactosidase in the cytoplasm of a wide variety of cell types did not transmit the transgene to their offspring. In contrast, transgenic mice, containing β-galactosidase in the nucleus, did transmit the transgene successfully. Interestingly, lacZ expression in the brain was more restricted when β-galactosidase activity was detected in the cytoplasm. These data suggested that cytoplasmic β-galactosidase affects certain developmental processes or gametogenesis resulting in transmission distortion of the transgene, and that this effect can be reduced by targeting β-galactosidase to the nucleus. We also found that Ayu1-driven lacZ expression in the duodenum of adult transgenic mice was sexually dimorphic, being positive in females and negative in males.     

Utilization of Proteinase K-Treated Cells as Lipopolysaccharide Antigens for the Serodiagnosis of Helicobacter pylori Infections

We have evaluated the use of proteinase K (PK)-treated cells isolated from Helicobacter pylori as lipopolysaccharide (LPS) antigens in an immunoblot assay and an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of H. pylori infection. The sera from patients with chronic gastritis, gastric ulcer, duodenal ulcer or gastric cancer, and from healthy adults with or without H. pylori infection were assayed with three commercial serodiagnostic kits (HM-CAP, Helico-G, and G.A.P. II) and novel methods relying on the use of PK-treated cells. The PK-treated cells used in these assays were selected on the basis of their possibility to possess a common epitope in the O-polysaccharides of H. pylori, which is known to be highly immunogenic in humans. Of the sera from these patients, 71-94% were positive with the commercial kits, 97% with immunoblot assay, and 90% with ELISA. On the other hand, of the healthy adults infected with H. pylori, 72-97% were positive with the commercial kits, 86% with immunoblot assay, and 72% with ELISA. PK-treated cells that did not contain the common epitope were unsuitable as an antigen for immunoblot assay or ELISA. Furthermore, the reactivity of these sera reacted specifically with H. pylori PK-treated cells but not with LPSs from other gram-negative bacteria, such as Campylobacter, Proteus, Bordetella, and Salmonella. These results demonstrate that the serological assays relying on the use of H. pylori PK-treated cells possessing a highly antigenic epitope are potentially useful as a serodiagnostic test for H. pylori infection.     

Tctex3, related to Drosophila Polycomblike, is expressed in male germ cells and mapped to the mouse t-complex

Tctex3 showing restricted expression in male germ cells has been isolated during the process of chromosome walking in the mouse t-complex region. The total sequence of Tctex3 cDNA predicts a protein of 580 amino acids with two C4HC3 type PHD fingers. The region containing this conserved motif is shared among members of the Polycomblike proteins that include the mouse M96 and Drosophila Polycomblike. A partial cDNA for a human homolog of Tctex3, HUTEX3, has also been isolated. Mouse Tctex3 gene was mapped adjacent to Tsc2 gene on mouse Chromosome (Chr) 17, and HUTEX3 was located closely to HSET gene in the HLA class II region of chromosome 6. Received: 10 April 1998 / Accepted: 22 June 1998     

Olfactory responses of chum salmon to amino acids are independent of large differences in salt concentrations between fresh and sea water

In chum salmon captured at the coastal sea and the natal river,the magnitudes of the olfactory nerve responses to the aminoacids after perfusion of the olfactory epithelium with artificialpond water (APW) were similar to those after perfusion withartificial sea water (ASW), although the concentrations of Na⁺,Cl^-, and Ca²⁺ in ASW were 986, 430 and 27 times higher thanthose in APW, respectively. The findings suggest that the permeabilityof these ions across the apical membranes of olfactory cellsdo not essentially contribute to the transduction mechanismin the salmon.