Cleavage of amyloid-beta precursor protein (APP) by membrane-type matrix metalloproteinases

Amyloid-beta precursor protein (APP) was identified on expression cloning from a human placenta cDNA library as a gene product that modulates the activity of membrane-type matrix metalloproteinase-1 (MT1-MMP). Co-expression of MT1-MMP with APP in HEK293T cells induced cleavage and shedding of the APP ectodomain when co-expressed with APP adaptor protein Fe65. Among the MT-MMPs tested, MT3-MMP and MT5-MMP also caused efficient APP shedding. The recombinant APP protein was cleaved by MT3-MMP in vitro at the A463-M464, N579-M580, H622-S623, and H685-Q686 peptide bonds, which included a cleavage site within the amyloid beta peptide region known to produce a C-terminal fragment. The Swedish-type mutant of APP, which produces a high level of amyloid beta peptide, was more effectively cleaved by MT3-MMP than wild-type APP in both the presence and absence of Fe65; however, amyloid beta peptide production was not affected by MT3-MMP expression. Expression of MT3-MMP enhanced Fe65-dependent transactivation by APP fused to the Gal4 DNA-binding and transactivation domains. These results suggest that MT1-MMP, MT3-MMP and MT5-MMP should play an important role in the regulation of APP functions in tissues including the central nervous system.     

Antinuclear antibody-keratinocyte interactions in photosensitive cutaneous lupus erythematosus

Autoimmune diseases are characterized by various circulating autoantibodies, especially antinuclear antibodies (ANA). It has been a long-standing issue as to whether and/or how ANA interact with epidermal cells to produce skin lesions. Of these ANA, the anti-SS-A/Ro antibody is the most closely associated with photosensitivity in patients with systemic lupus erythematosus (SLE) and its subgroups, including subacute cutaneous lupus erythematosus (SCLE) and neonatal lupus erythematosus (NLE). SS-A/Ro antigens are present in the nucleus and cytoplasm, and interestingly, ultraviolet B (UVB) light translocates these antigens to the surface of the cultured keratinocytes. Thus, anti-SS-A/Ro antibodies in the sera can bind to the relevant antigens expressed on the UVB-irradiated keratinocyte surface, and have been speculated to be an important inducer of antibody-dependent keratinocyte damage. This interaction between the anti-SS-A/Ro antibodies and UVB-irradiated keratinocytes may induce the skin lesions through a cytotoxic mechanism. This review will focus on the involvement of antibody-dependent cellular cytotoxicity in the pathogenesis of the skin lesions observed in photosensitive cutaneous lupus erythematosus.     

Cyclophosphamide enhances TNF-alpha-induced apoptotic cell death in murine vascular endothelial cell

Cyclophosphamide (CPA) is one of the therapeutic agents for systemic inflammatory disorders. In murine dermal endothelial cells (F-2), 4-hydroxycyclophosphamide (4-HC), which is active metabolite of CPA, enhanced TNF-alpha-induced DNA fragmentation. In addition, 4-HC was shown to elevate TNF-alpha-induced caspase-3 activation. Caspase-8 activation was identified by the treatment of TNF-alpha, whereas 4-HC was no effect. In contrast, only when treated with 4-HC, caspase-9 activation and the increase in the intracellular expression of Bax were detected. These results suggest that CPA may sensitize endothelial cells to TNF-alpha-induced apoptosis through a mitochondria-dependent pathway and clinically may contribute to the limitation of inflammatory process.     

Characterization of the NikA histidine kinase implicated in the phosphorelay signal transduction of Aspergillus nidulans, with special reference to fungicide responses

We recently compiled a complete list of phosphorelay signal transduction components in the model filamentous fungus Aspergillus nidulans. In this study, we characterized a histidine protein kinase (designated NikA) that is found in many fungi, with special reference to responses to potent fungicides (iprodione and fludioxonil). We provided evidence that not only NikA, but also two downstream response regulators (SskA and SrrA) are crucially implicated in the mode of action of these fungicides, and also that the further downstream HogA-MAPK cascade is exaggerated abnormally (or ectopically) in hyphae by the fungicides in a manner dependent on the NikA-SskA phosphorelay.     

Laminin‐421 produced by lymphatic endothelial cells induces chemotaxis for human melanoma cells

Melanoma has a high tendency to metastasize to lymph nodes, which is one of the clinicopathological factors to indicate poor prognosis. Recent investigations have shown the importance of lymphangiogenesis in lymph node metastasis in a variety of human tumors including melanoma. However, molecular mechanism of lymphatic metastasis is still poorly defined. We examined influence of interactions between normal lymphatic endothelial cells (LECs) and melanoma cells on cell migration. Medium conditioned with LEC (LEC‐CM) contained chemotactic and chemokinetic activities for human melanoma cell lines. The chemotactic activity was fractionated in more than 100 kDa, and inactivated by heat‐treatment. The chemotactic activity of LEC‐CM was abolished by immunodepletion with anti‐laminin‐1 antibody. And immunoprecipitation and Western blot analyses revealed that LEC‐CM contained laminin‐421. When melanoma C8161 cells were treated with function‐blocking antibodies to integrin α3 or α6, their chemotactic responses to LEC‐CM were markedly reduced. Furthermore, the knock‐down of tetraspanin CD151 weakened the chemotactic responses of C8161 and MeWo cells to LEC‐CM. These data suggest that laminin‐421 secreted by LEC possibly facilitates lymphatic metastasis through the induction of chemotaxis of melanoma cells.     

A green Paramecium strain with abnormal growth of symbiotic algae

Some hundred cells of Chlorella-like green algae are naturally enclosed within the cytoplasm of a single cell of green paramecia (Paramecium bursaria). Therefore, P. bursaria serves as an experimental model for studying the nature of endo-symbiosis made up through chemical communication between the symbiotic partners. For studying the mechanism of symbiotic regulations, the materials showing successful symbiosis are widely used. Apart from such successful model materials, some models for symbiotic distortion would be of great interest in order to understand the nature of successful symbiosis. Here, we describe a case of unsuccessful symbiosis causing unregulated growth of algae inside the hosting ciliates. Recently, we have screened some cell lines, from the mass of P. bursaria cells survived after paraquat treatment. The resultant cell lines (designated as KMZ series) show novel and unusual morphological features with heavily darker green colour distinguishable from the original pale green-coloured paramecia. In this type of isolates, endo-symbiotic algae are restricted within one or two dense spherical structures located at the center of the host cells' cytoplasm. Interestingly, this isolate maintains the host cells' circadian mating response which is known as an alga-dependent behaviour in the host cells. In contrast, we discuss that KMZ lacks the host-dependent regulation of algal growth, thus the algal complex often over-grows obviously exceeding the original size of the normal hosting ciliates. Additionally, possible use of this isolate as a novel model for symbiotic cell-to-cell communication is discussed.     

Alteration of the N-glycome of bovine milk glycoproteins during early lactation

Milk provides nutritional, immunological and developmental components for newborns. Whereas identification of such components has been performed by targeting proteins and free oligosaccharides, structural and functional analyses of the N-glycome of milk glycoproteins are scarce. In this study, we investigated, for the first time, the alterations of the bovine milk N-glycome during early lactation (1 day, 1, 2, 3 and 4 weeks postpartum), characterizing more than 80 N-glycans. The glycomic profile of colostrum on day 1 after calving differed substantially from that in other periods during early lactation. The proteins in colostrum obtained 1 day postpartum were more highly sialylated than milk samples obtained at other time points, and the N-glycolylneuraminic acid (Neu5Gc)/N-acetylneuraminic acid (Neu5Ac) ratio was significantly higher on day 1, showing a gradual decline with time. In order to dissect the N-glycome of colostrum, alterations of the N-glycosylation profile of major bovine milk proteins during the early lactation stage were elucidated, revealing that the alteration is largely attributable to qualitative and quantitative N-glycosylation changes of IgG, the major glycoprotein in colostrum. Furthermore, by preparing and analyzing IgGs in which the N-glycan structure and subtypes were well characterized, we found that the interaction between IgG and FcRn was not affected by the structure of the N-glycans attached to IgG. We also found that bovine FcRn binds IgG(2) better than IgG(1) , strongly suggesting that the role of FcRn in the bovine mammary gland is to recycle IgG(2) from the udder to blood, rather than to secrete IgG(1) into colostrum.     

Impaired hippocampal long-term potentiation and failure of learning in β1,4-N-acetylgalactosaminyltransferase gene transgenic mice

Gangliosides (sialic acid-containing glycosphingolipids) play important roles in many physiological functions, including synaptic plasticity in the hippocampus, which is considered as a cellular mechanism of learning and memory. In the present study, three types of synaptic plasticity, long-term potentiation (LTP), long-term depression (LTD) and reversal of LTP (depotentiation, DP), in the field excitatory post-synaptic potential in CA1 hippocampal neurons and learning behavior were examined in β1,4-N-acetylgalactosaminyltransferase (β1,4 GalNAc-T; GM2/GD2 synthase) gene transgenic (TG) mice, which showed a marked decrease in b-pathway gangliosides (GQ1b, GT1b and GD1b) in the brain and isolated hippocampus compared with wild-type (WT) mice. The magnitude of the LTP induced by tetanus (100 pulses at 100?Hz) in TG mice was significantly smaller than that in control WT mice, whereas there was no difference in the magnitude of the LTD induced by three short trains of low-frequency stimulation (LFS) (200 pulses at 1?Hz) at 20?min intervals between the two groups of mice. The reduction in the LTP produced by delivering three trains of LFS (200 pulses at 1?Hz, 20?min intervals) was significantly greater in the TG mice than in the WT mice. Learning was impaired in the four-pellet taking test (4PTT) in TG mice, with no significant difference in daily activity or activity during the 4PTT between TG and WT mice. These results suggest that the overexpression of β1,4 GalNAc-T resulted in altered synaptic plasticity of LTP and DP in hippocampal CA1 neurons and learning in the 4PTT, and this is attributable to the shift from b-pathway gangliosides to a-pathway gangliosides.     

Characteristics of rabbit ClC-2 current expressed in Xenopus oocytes and its contribution to volume regulation

In theXenopus oocyte heterologous expressionsystem, the electrophysiological characteristics of rabbit ClC-2current and its contribution to volume regulation were examined.Expressed currents on oocytes were recorded with a two-electrodevoltage-clamp technique. Oocyte volume was assessed by taking picturesof oocytes with a magnification of ×40. Rabbit ClC-2 currentsexhibited inward rectification and had a halide anion permeabilitysequence of Cl  Br  I  F. ClC-2 currents wereinhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB),diphenylamine-2-carboxylic acid (DPC), and anthracene-9-carboxylic acid(9-AC), with a potency order of NPPB > DPC = 9-AC, but were resistant to stilbene disulfonates. These characteristics are similarto those of rat ClC-2, suggesting rabbit ClC-2 as a counterpart of ratClC-2. During a 30-min perfusion with hyposmolar solution, currentamplitude at 160 mV and oocyte diameter were compared amongthree groups: oocytes injected with distilled water, oocytes injectedwith ClC-2 cRNA, and oocytes injected with ClC-2NT cRNA (an openchannel mutant with NH₂-terminaltruncation). Maximum inward current was largest in ClC-2NT-injectedoocytes (5.9 ± 0.4 µA), followed by ClC-2-injected oocytes(4.3 ± 0.6 µA), and smallest in water-injected oocytes(0.2 ± 0.2 µA), whereas the order of increase in oocytediameter was as follows: water-injected oocytes (9.0 ± 0.2%) > ClC-2-injected oocytes (5.3 ± 0.5%) > ClC-2NT-injected oocytes (1.1 ± 0.2%). The findings that oocyte swelling wassmallest in oocytes with the largest expressed currents suggest thatClC-2 currents expressed in Xenopusoocytes appear to act for volume regulation when exposed to ahyposmolar environment.