Membrane-bound and secreted IgA contain structurally different alpha-chains

Three different forms of alpha-chains are synthesized by BF0.3 and 615.2, two cloned cell lines derived from the murine B lymphoma 1.29. The three forms of alpha-chains differ in size, pI, cellular location, and rate of turnover. They were identified by means of lactoperoxidase-catalyzed radioiodination, internal 14C or 35S labeling, and immunofluorescence techniques as membrane-bound(alpha m), secreted (alpha s), and intracellular (alpha ic) proteins. Comparison of immunoglobulin products of the two lymphoma lines with those of a hybridoma cell line, Id 150, which secretes IgA of the 1.29 idiotype but lacks membrane IgA, confirmed the assignments of alpha m, alpha s, and alpha ic. Results of biosynthetic labeling of BF0.3, 615.2, and Id 150 in the presence and absence of tunicamycin suggest that the difference in m.w. and charge observed between alpha m and alpha s can be attributed to differences in primary amino acid structure rather than different degrees of glycosylation.     

Enhancement of In Vivo Antioxidant Ability in the Brain of Rats Fed Tannin

The effect of the oral administration of mimosa tannin (MMT) on the rat intra-hippocampal antioxidant ability was examined. Wistar rats at the age of 6 weeks were reared for 8 weeks with the rodent diet (RD) consisting of 0.1 g/kg of MMT (RD–MMT). The antioxidant ability of rat brain was evaluated from the decay of a brain–blood-barrier permeable stable nitroxide, 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCAM) measured by the microdialysis-electron spin resonance system under a freely moving state. The decay rate of PCAM in the brain of rats fed RD–MMT was significantly larger than that of rats fed control rodent diet, which indicates the increase of the antioxidant ability in the brain of rats fed RD–MMT. In vitro study showed that MMT did not reduce PCAM directly but enhanced the reduction of PCAM by ascorbic acid. These results indicate that MMT is a potent antioxidant in vitro and in vivo.     

Quantification of Chitinase mRNA Levels in Human and Mouse Tissues by Real-Time PCR: Species-Specific Expression of Acidic Mammalian Chitinase in Stomach Tissues

Chitinase hydrolyzes chitin, which is an N-acetyl-D-glucosamine polymer that is present in a wide range of organisms, including insects, parasites and fungi. Although mammals do not contain any endogenous chitin, humans and mice express two active chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). Because the level of expression of these chitinases is increased in many inflammatory conditions, including Gaucher disease and mouse models of asthma, both chitinases may play important roles in the pathophysiologies of these and other diseases. We recently established a quantitative PCR system using a single standard DNA and showed that AMCase mRNA is synthesized at extraordinarily high levels in mouse stomach tissues. In this study, we applied this methodology to the quantification of chitinase mRNAs in human tissues and found that both chitinase mRNAs were widely expressed in normal human tissues. Chit1 mRNA was highly expressed in the human lung, whereas AMCase mRNA was not overexpressed in normal human stomach tissues. The levels of these mRNAs in human tissues were significantly lower than the levels of housekeeping genes. Because the AMCase expression levels were quite different between the human and mouse stomach tissues, we developed a quantitative PCR system to compare the mRNA levels between human and mouse tissues using a human-mouse hybrid standard DNA. Our analysis showed that Chit1 mRNA is expressed at similar levels in normal human and mouse lung. In contrast, the AMCase expression level in human stomach was significantly lower than that expression level observed in mouse stomach. These mRNA differences between human and mouse stomach tissues were reflecting differences in the chitinolytic activities and levels of protein expression. Thus, the expression level of the AMCase in the stomach is species-specific.     

Enhanced immune responses in transgenic mice expressing a truncated form of the lymphocyte semaphorin CD100

CD100/Sema4D is a 150-kDa transmembrane protein that belongs to the semaphorin family. Binding of CD100 to CD72 enhances the immune response by turning off the negative signaling effects of CD72. To investigate the physiological functions of CD100 in vivo, we generated transgenic mice expressing a truncated form of CD100. A large amount of the soluble form of CD100 was detected in the sera of mice expressing a truncated form of CD100, although the amount of CD100 was only slightly elevated on the surface of B cells. In the mutant mice the development of conventional B and T cells appeared normal in terms of the surface marker phenotypes, while the number of CD5(+) B-1 cells in the peritoneal cavity increased in comparison with wild-type mice. In vitro proliferation and Ig production of B cells in response to CD40 stimulation were considerably enhanced in mice expressing a truncated form of CD100. Additionally, in vivo both Ab responses against T cell-dependent Ags and generation of Ag-specific T cells were enhanced. Furthermore, introduction of the CD100-transgene could restore in vitro B cell responses as well as in vivo Ab production against T cell-dependent Ag in CD100-deficient mice. Collectively, these results not only indicate that CD100 has an important role in the immune system, but also that the soluble form of CD100 released from the cell surface can exert functions in vivo.     

Purification and characterization of α-D-galactosidase produced by ADG cell line established from abalon digestive gland

ADG cell line was established from an abalonedigestive gland and previously characterized. ADGcells have the potential to grow in protein-freeculture and secrete l3 types of glycosidases. Inthis article, we determined the origin of ADG cell line,using electron microscopy, and purified a glycosidasesecreted by these cells. The electron microscopicanalysis showed that ADG cell line contains severalnuclei, which suggests that they may be derived fromprotist cells. Moreover, -D-galactosidasethat hydrolyzes p-nitorophenyl galactopyranosidewas purified 130-fold from the spent culture medium ofADG cells. The molecular weight of the enzyme,determined by sodium dodecyl sulfate polyacrylamidegel electrophoresis and gel filtration analysis, wasshown to be 43 and 42 kDa, respectively, and itappeared to consist of a single polypeptide chain. The purified enzyme preparation was practically freefrom other glycosidases secreted from the cells. Catalytic activity was optimal at pH 5.5 and at atemperature of 37 °C. The enzyme was also the most stable at pH 5.5.     

Polyphenol‐solubility alters amyloid fibril formation of α‐synuclein

Amyloid fibril formation is associated with various amyloidoses, including neurodegenerative diseases such as Alzheimer''s and Parkinson''s diseases. Amyloid fibrils form above the solubility of amyloidogenic proteins or peptides upon breaking supersaturation, followed by a nucleation and elongation mechanism, which is similar to the crystallization of solutes. Many additives, including salts, detergents, and natural compounds, promote or inhibit amyloid formation. However, the underlying mechanisms of the opposing effects are unclear. We examined the effects of two polyphenols, that is, epigallocatechin gallate (EGCG) and kaempferol‐7─O─glycoside (KG), with high and low solubilities, respectively, on the amyloid formation of α‐synuclein (αSN). EGCG and KG inhibited and promoted amyloid formation of αSN, respectively, when monitored by thioflavin T (ThT) fluorescence or transmission electron microscopy (TEM). Nuclear magnetic resonance (NMR) analysis revealed that, although interactions of αSN with soluble EGCG increased the solubility of αSN, thus inhibiting amyloid formation, interactions of αSN with insoluble KG reduced the solubility of αSN, thereby promoting amyloid formation. Our study suggests that opposing effects of polyphenols on amyloid formation of proteins and peptides can be interpreted based on the solubility of polyphenols.     

Visualization analysis of inter-fragment interaction energies of CRP-cAMP-DNA complex based on the fragment molecular orbital method

A visualization method for inter-fragment interaction energies (IFIEs) of biopolymers is presented on the basis of the fragment molecular orbital (FMO) method. The IFIEs appropriately illustrate the information about the interaction energies between the fragments consisting of amino acids, nucleotides and other molecules. The IFIEs are usually analyzed in a matrix form called an IFIE matrix. Analyzing the IFIE matrix, we detect important fragments for the function of biomolecular systems and quantify the strength of interaction energies based on quantum chemistry, including the effects of charge transfer, electronic polarization and dispersion force. In this study, by analyzing a protein-DNA complex, we report a visual representation of the IFIE matrix, a so-called IFIE map. We comprehensively examine what information the IFIE map contains concerning structures and stabilities of the protein-DNA complex.     

Smad7 Inhibits Transforming Growth Factor-β Family Type I Receptors through Two Distinct Modes of Interaction

The inhibitory Smads (I-Smads), i.e. Smad6 and Smad7, are negative regulators of transforming growth factor-β (TGF-β) family signaling. I-Smads inhibit TGF-β family signaling principally through physical interaction with type I receptors (activin receptor-like kinases), so as to compete with receptor-regulated Smads (R-Smads) for activation. However, how I-Smads interact with type I receptors is not well understood. In the present study, we found that Smad7 has two modes of interaction with type I receptors. One is through a three-finger-like structure in the MH2 domain, consisting of residues 331–361, 379–387, and the L3 loop. The other is through a basic groove in the MH2 domain (Mochizuki, T., Miyazaki, H., Hara, T., Furuya, T., Imamura, T., Watabe, T., and Miyazono, K. (2004) J. Biol. Chem. 279, 31568–31574). We also found that Smad6 principally utilizes a basic groove in the MH2 domain for interaction with type I receptors. Smad7 thus has an additional mode of interaction with TGF-β family type I receptors not possessed by Smad6, which may play roles in mediating the inhibitory effects unique to Smad7.