Both the Fab and Fc domains of IgG are essential for ROS emission from TNF-α-primed neutrophils by IVIG

Intravenous immunoglobulin (IVIG) is currently a very important therapeutic used for not only infectious diseases, but also for autoimmune diseases such as idiopathic thrombocytopenic purpura (ITP). Untoward reactions of IVIG have been thought to result from complement activation by aggregated IgG in IVIG. In addition, the aggregates have been known to activate neutrophils, which may result in the untoward reactions. However, the effect and mechanism of IVIG on neutrophils remain unclear. In this study, we investigated the activation of neutrophils by IVIG in terms of their reactive oxygen species (ROS) emission to elucidate the mechanisms. IVIG-induced ROS emission from purified neutrophils was remarkably augmented by TNF-α priming of the cells. The ROS emission from TNF-α-primed neutrophils occurred by activation with whole gammaglobulin (GG) molecules, but not F(ab')(2), Fc, or a mixture of F(ab')(2) and Fc. ROS emission by GG was inhibited by the F(ab')(2) fragment and an inhibitory antibody against FcγRIII. These results suggest that binding of IVIG to not only surface antigen(s), but also FcγRIII on neutrophils, is involved in IVIG-induced ROS emission from TNF-α-primed neutrophils, and contribute to the untoward reactions of IVIG.     

Antidepressant Acts on Astrocytes Leading to an Increase in the Expression of Neurotrophic/Growth Factors: Differential Regulation of FGF-2 by Noradrenaline

Recently, multiple neurotrophic/growth factors have been proposed to play an important role in the therapeutic action of antidepressants. In this study, we prepared astrocyte- and neuron-enriched cultures from the neonatal rat cortex, and examined the changes in neurotrophic/growth factor expression by antidepressant treatment using real-time PCR. Treatment with amitriptyline (a tricyclic antidepressant) significantly increased the expression of fibroblast growth factor-2 (FGF-2), brain-derived neurotrophic factor, vascular endothelial growth factor and glial cell line-derived neurotrophic factor mRNA with a different time course in astrocyte cultures, but not in neuron-enriched cultures. Only the expression of FGF-2 was higher in astrocyte cultures than in neuron-enriched cultures. We focused on the FGF-2 production in astrocytes. Several different classes of antidepressants, but not non-antidepressants, also induced FGF-2 mRNA expression. Noradrenaline (NA) is known to induce FGF-2 expression in astrocyte cultures, as with antidepressants. Therefore, we also assessed the mechanism of NA-induced FGF-2 expression, in comparison to amitriptyline. NA increased the FGF-2 mRNA expression via α1 and β-adrenergic receptors; however, the amitriptyline-induced FGF-2 mRNA expression was not mediated via these adrenergic receptors. Furthermore, the amitriptyline-induced FGF-2 mRNA expression was completely blocked by cycloheximide (an inhibitor of protein synthesis), while the NA-induced FGF-2 mRNA was not. These data suggest that the regulation of FGF-2 mRNA expression by amitriptyline was distinct from that by NA. Taken together, antidepressant-stimulated astrocytes may therefore be important mediators that produce several neurotrophic/growth factors, especially FGF-2, through a monoamine-independent and a de novo protein synthesis-dependent mechanism.     

Sip1, a Conserved AP-1 Accessory Protein,Is Important for Golgi/Endosome Trafficking in Fission Yeast

We had previously identified the mutant allele of apm1⁺ that encodes a homolog of the mammalian μ 1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex and demonstrated that the AP-1 complex plays a role in Golgi/endosome trafficking, secretion, and vacuole fusion in fission yeast. Here, we isolated a mutant allele of its4⁺/sip1⁺, which encodes a conserved AP-1 accessory protein. The its4-1/sip1-i4 mutants and apm1 -deletion cells exhibited similar phenotypes, including sensitivity to the calcineurin inhibitor FK506, Cl⁻ and valproic acid as well as various defects in Golgi/endosomal trafficking and cytokinesis. Electron micrographs of sip1-i4 mutants revealed vacuole fragmentation and accumulation of abnormal Golgi-like structures and secretory vesicles. Overexpression of Apm1 suppressed defective membrane trafficking in sip1-i4 mutants. The Sip1-green fluorescent protein (GFP) co-localized with Apm1-mCherry at Golgi/endosomes, and Sip1 physically interacted with each subunit of the AP-1 complex. We found that Sip1 was a Golgi/endosomal protein and the sip1-i4 mutation affected AP-1 localization at Golgi/endosomes, thus indicating that Sip1 recruited the AP-1 complex to endosomal membranes by physically interacting with each subunit of this complex. Furthermore, Sip1 is required for the correct localization of Bgs1/Cps1, 1,3-β-D-glucan synthase to polarized growth sites. Consistently, the sip1-i4 mutants displayed a severe sensitivity to micafungin, a potent inhibitor of 1,3-β-D-glucan synthase. Taken together, our findings reveal a role for Sip1 in the regulation of Golgi/endosome trafficking in coordination with the AP-1 complex, and identified Bgs1, required for cell wall synthesis, as the new cargo of AP-1-dependent trafficking.     

Polymorphism of glutathione S-transferase P1 gene affects human vitamin C metabolism

There are large inter-individual differences in the metabolism of vitamin C (VC), which is composed of both ascorbic acid (AsA) and dehydroascorbic acid (DAsA). AsA is oxidized to DAsA in a series of xenobiotic reactions. Thus, the effects of polymorphism A313G (Ile105Val) in the gene for glutathione S-transferases P1 (GSTP1), one of the most active xenobiotic enzymes, on human VC metabolism were studied. The variant frequency of GSTP1 among the present subjects (n = 210) was AA 71.0%; GA 27.0% and GG 1.9%. At 24 h after administration of 1 mmol of VC to young women (n = 17; age, 21.0 ± 1.1 y), total VC excretion (46.7 ± 18.1 mg) by AA homozygotes of GSTP1 was greater (p < 0.0069) than that (28.2 ± 14.0 mg) by GA heterozygotes. One hour after administration of VC, blood total VC levels were also significantly different (p < 0.0036) between the homozygotes and heterozygotes. The effects of other polymorphisms in xenobiotic enzymes on VC metabolism were small.     

The secreted EGF-Discoidin factor xDel1 is essential for dorsal development of the Xenopus embryo

We show here that a secreted EGF-Discoidin-domain protein, Xenopus Del1 (xDel1), is an essential factor for dorsal development in the early Xenopus embryo. Knockdown of the xDel1 function causes obvious ventralization of the embryo. Conversely, overexpression of xDel1 expands dorsal-marker expression and suppresses ventral-marker expression in the gastrula embryo. Forced expression of xDel1 dorsalizes ventral marginal zone explants, whereas it weakly induces neural differentiation but not mesodermal differentiation in animal caps. The dorsalizing activity of xDel1 is dependent on the Discoidin domains and not on the RGD motif (which is implicated in its angiogenic activity) or EGF repeats. Luciferase assays show that xDel1 attenuates BMP-signaling reporter activity by interfering with the pathway downstream of the BMP receptor. Thus, xDel1 functions as a unique extracellular regulatory factor of DV patterning in early vertebrate embryogenesis.     

A tobacco calmodulin-related protein suppresses sense transgene-induced RNA silencing but not inverted repeat-induced RNA silencing

A tobacco calmodulin-related protein, rgs-CaM, interacts with viral suppressors of RNA silencing and modulates host RNA silencing. Plants overexpressing the rgs-CaM gene were crossed with plants exhibiting sense transgene-induced RNA silencing (S-PTGS) or inverted repeat-induced RNA silencing (IR-PTGS). S44 plants harboring a sense transgene encoding a tobacco microsomal ω-3 fatty acide desaturase (NtFAD3) exhibited the S-PTGS phenotype. The frequency of the S-PTGS phenotype incidence was nearly 100 % in the hemizygous S44 plants, but was reduced to 30 % in crossbred plants with an rgs-CaM-overexpressing transgenic line. The remaining 70 % of crossbred plants successfully overexpressed the NtFAD3 transgene, and the amount of NtFAD3 small interfering RNAs (siRNAs) was largely decreased. In contrast, overexpression of rgs-CaM did not suppress siRNA production in the IR-PTGS that targeted the NtFAD3 gene. These results indicated that rgs-CaM suppresses RNA silencing at a step upstream of siRNA production and does not interfere with the later steps of RNA silencing, including siRNA-mediated RNA degradation.     

Neutral Cysteine Protease Bleomycin Hydrolase Is Essential for the Breakdown of Deiminated Filaggrin into Amino Acids

Filaggrin is a component of the cornified cell envelope and the precursor of free amino acids acting as a natural moisturizing factor in the stratum corneum. Deimination is critical for the degradation of filaggrin into free amino acids. In this study, we tried to identify the enzyme(s) responsible for the cleavage of deiminated filaggrin in vitro. First, we investigated citrulline aminopeptidase activity in the extract of newborn rat epidermis by double layer fluorescent zymography and detected strong activity at neutral pH. Monitoring the citrulline-releasing activity, we purified an enzyme of 280 kDa, comprised of six identical subunits of 48 kDa. The NH₂ terminus of representative tryptic peptides perfectly matched the sequence of rat bleomycin hydrolase (BH). The enzyme released various amino acids except Pro from β-naphthylamide derivatives and hydrolyzed citrulline-β-naphthylamide most effectively. Thus, to break down deiminated filaggrin, another protease would be required. Among proteases tested, calpain I degraded the deiminated filaggrin effectively into many peptides of different mass on the matrix-assisted laser desorption/ionization-time of flight mass spectrum. We confirmed that various amino acids including citrulline were released by BH from those peptides. On the other hand, caspase 14 degraded deiminated filaggrin into a few peptides of limited mass. Immunohistochemical analysis of normal human skin revealed co-localization of BH and filaggrin in the granular layer. Collectively, our results suggest that BH is essential for the synthesis of natural moisturizing factors and that calpain I would play a role as an upstream protease in the degradation of filaggrin.The mammalian epidermal keratinocytes arise from proliferating basal cells and move outward through a series of distinct differentiation events to form the stratum corneum (1, 2). During this progressive epidermal differentiation, keratinocytes express different proteins such as keratins, profilaggrin/filaggrin, involucrin, small proline-rich proteins, loricrin, cystatin A, and elafin, which form the cornified envelope of mature corneocytes (3–7). Profilaggrin is synthesized as a large, extremely insoluble phosphoprotein that consists of a unique NH₂-terminal Ca²⁺-binding protein of the S-100 family, linked to 10–20 tandem filaggrin monomer repeats (8–10). Each individual filaggrin repeat is completely removed by proteolysis to generate the mature filaggrin monomer (a molecular mass of 37 kDa in human). Then, filaggrin is completely degraded in the uppermost layer of the stratum corneum to produce a mixture of free and modified hygroscopic amino acids that are important for maintaining epidermal hydration (2, 11–13). In addition, a number of proteins are subjected to various post-translational modifications such as disulfide bonding, N-(γ-glutamyl)-lysine isopeptide cross-linking, and deimination during the terminal differentiation of epidermal keratinocytes (4, 6, 14, 15). Deimination is catalyzed by peptidylarginine deiminase (PAD),² which converts arginine to citrulline in proteins (17–19). The modification seems essential for the processing into free amino acids including citrulline.Several proteases reportedly participate in the processing of profilaggrin. Furin, a member of the proprotein convertase family, has been proposed to cleave the NH₂ terminus of profilaggrin, facilitating the release of the NH₂-terminal S-100 protein (20, 21). In contrast, calpain I and profilaggrin endopeptidase I (PEP-I) were implicated in the processing of the linker regions between the filaggrin monomer repeats to generate the filaggrin monomer (22–25). Recently, significant results regarding the conversion of profilaggrin to filaggrin have been obtained with the knock-out of matriptase/MT-SP1, prostasin/channel-activating serine protease 1/Prss 8, and caspase 14 in mice (26–28). These proteases were a key component of the profilaggrin-processing pathway in terminal epidermal differentiation. However, although the signal initiating the degradation of profilaggrin at a defined stage of the maturation of the stratum corneum was found to be the water gradient within the stratum corneum itself (11), the proteases for the processing of filaggrin and/or the deiminated form into peptides following the breakdown of these peptides to amino acids including citrulline remain unknown.In this study, we have purified a novel aminopeptidase using a deiminated substrate from rat skin homogenate and identified it as a neutral cysteine protease, bleomycin hydrolase (BH). Furthermore, we investigated the processing of the deiminated filaggrin by calpain I or caspase 14. Based on these results, we proposed that calpain I participated preferentially in the processing of deiminated filaggrin into peptides and then BH appeared essential for the breakdown of the peptides into amino acids.     

Electrochemical microdevice with separable electrode and antibody chips for simultaneous detection of pepsinogens 1 and 2

An electrochemical microdevice with separable electrode and antibody chips has been developed and applied to detect atrophic gastritis-related proteins, pepsinogen 1 (PG1) and pepsinogen 2 (PG2), based on sandwich-type enzyme-linked immunosorbent assays (ELISAs) with horseradish peroxidase (HRP)-labeled antibody. To fabricate the electrochemical device for simultaneous analysis of several proteins, the electrode chip with eight electrode elements was assembled along with an antibody chip with eight cavities containing immobilized anti-PG1 or anti-PG2. The immunoreactions occurring in the cavities of the device were detected simultaneously by amperometry. The labeled HRP in the cavity in the presence of hydrogen peroxide catalyzed the oxidation of ferrocenemethanol (FMA) to FMA+, which was detected electrochemically by the electrode chip. The amperometric responses of respective cavities in the device increased with increasing concentration of PG1 or PG2 of 0-50 ng/ml, ensuring the simultaneous detection of PG1 and PG2. The detection limits for both PG1 and PG2 were 0.6 ng/ml (S/N=2). The electrode chip was recovered easily by disassembling the electrochemical device; thereby, it was used repeatedly, whereas the antibody chip was discarded. No marked decrease in electrochemical responses was detected after repeated use. Reuse of the electrode chip is beneficial to reduce costs of protein analysis.     

A novel oxazine ring closure reaction affording (Z)-((E)-2-styrylbenzo[b]furo[3,2-d][1,3]oxazin-4-ylideno)acetaldehydes and their anti-osteoclastic bone resorption activity

A novel oxazine ring formation method was established using the reaction of 2-acetyl-(E)-3-styrylcarbonylaminobenzo[b]furans (4) with Vilsmeier-Haack-Arnold reagent to afford (E and Z)-((E)-2-styrylbenzo[b]furo[3,2-d][1,3]oxazin-4-ylideno)acetaldehydes (5). (Z)-4-(8-Bromo-(E)-2-styrylbenzo[b]furo[3,2-d][1,3]oxazin-4-ylideno)but-(E)-2-enoic acid ethyl ester (6b), derived from (Z)-5a, showed significantly potent anti-osteoclastic bone resorption activity comparable to 17beta-estradiol (E2).     

Effects of site-directed mutagenesis of the loop residue of the N-terminal domain Gly117 of thermolysin on its catalytic activity

In the N-terminal domain of thermolysin, two polypeptide strands, Asn112-Ala113-Phe114-Trp115 and Ser118-Gln119-Met120-Val121-Tyr122, are connected by a short loop, Asn116-Gly117, to form an anti-parallel β-sheet. The Asn112-Trp115 strand is located in the active site, while the Ser118-Tyr122 strand and the Asn116-Gly117 loop are located outside the active site. In this study, we explored the catalytic role of Gly117 by site-directed mutagenesis. Five variants, G117A (Gly117 is replaced by Ala), G117D, G117E, G117K, and G117R, were produced by co-expressing in Escherichia coli the mature and pro domains as independent polypeptides. The production levels were in the order G117E > wild type > G117K, G117R > G117D. G117A was hardly produced. This result is in contrast to our previous one that all 72 active-site thermolysin variants were produced at the similar levels whether they retained activity or not (M. Kusano et al. J. Biochem., 145, 103-113 (2009)). G117E exhibited lower activity in the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and higher activity in the hydrolysis of N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester than the wild-type thermolysin. G117K and G117R exhibited considerably reduced activities. This suggests that Gly117 plays an important role in the activity and stability of thermolysin, presumably by affecting the geometries of the Asn112-Trp115 and Ser118-Tyr122 strands.