Legumain/asparaginyl endopeptidase controls extracellular matrix remodeling through the degradation of fibronectin in mouse renal proximal tubular cells

Legumain/asparaginyl endopeptidase (EC 3.4.22.34) is a novel cysteine protease that is abundantly expressed in the late endosomes and lysosomes of renal proximal tubular cells. Recently, emerging evidence has indicated that legumain might play an important role in control of extracellular matrix turnover in various pathological conditions such as tumor growth/metastasis and progression of atherosclerosis. We initially found that purified legumain can directly degrade fibronectin, one of the main components of the extracellular matrix, in vitro. Therefore, we examined the effect of legumain on fibronectin degradation in cultured mouse renal proximal tubular cells. Fibronectin processing can be inhibited by chloroquine, an inhibitor of lysosomal degradation, and can be enhanced by the overexpression of legumain, indicating that fibronectin degradation occurs in the presence of legumain in lysosomes from renal proximal tubular cells. Furthermore, in legumain-deficient mice, unilateral ureteral obstruction (UUO)-induced renal interstitial protein accumulation of fibronectin and renal interstitial fibrosis were markedly enhanced. These findings indicate that legumain might have an important role in extracellular matrix remodeling via the degradation of fibronectin in renal proximal tubular cells.     

A single enzyme catalyzes both platelet-activating factor production and membrane biogenesis of inflammatory cells. Cloning and characterization of acetyl-CoA:LYSO-PAF acetyltransferase

Platelet-activating factor (PAF) is a potent proinflammatory lipid mediator eliciting a variety of cellular functions. Lipid mediators, including PAF are produced from membrane phospholipids by enzymatic cascades. Although a G protein-coupled PAF receptor and degradation enzymes have been cloned and characterized, the PAF biosynthetic enzyme, aceyl-CoA:lyso-PAF acetyltransferase, has not been identified. Here, we cloned lyso-PAF acetyltransferase, which is critical in stimulus-dependent formation of PAF. The enzyme is a 60-kDa microsomal protein with three putative membrane-spanning domains. The enzyme was induced by bacterial endotoxin (lipopolysaccharide), which was suppressed by dexamethasone treatment. Surprisingly, the enzyme catalyzed not only biosynthesis of PAF from lyso-PAF but also incorporation of arachidonoyl-CoA to produce PAF precursor membrane glycerophospholipids (lysophosphatidylcholine acyltransferase activity). Under resting conditions, the enzyme prefers arachidonoyl-CoA and contributes to membrane biogenesis. Upon acute inflammatory stimulation with lipopolysaccharide, the activated enzyme utilizes acetyl-CoA more efficiently and produces PAF. Thus, our findings provide a novel concept that a single enzyme catalyzes membrane biogenesis of inflammatory cells while producing a prophlogistic mediator in response to external stimuli.     

Lower extremity muscle activity during different types and speeds of underwater movement

To compare the activity of lower extremity muscles during land walking (LW), water walking (WW), and deep-water running (DWR), 9 healthy young subjects were tested at self-selected low, moderate, and high intensities for 8 sec with two repetitions. Surface EMG electrodes were placed on the tibialis anterior (TA), soleus (SOL), medial gastrocnemius (GAS), rectus femoris (RF), and biceps femoris (BF). During DWR, the SOL and GAS activities were lower than LW and WW. The BF activities were higher during DWR than LW and WW. It was considered that the lower activity of SOL and GAS depended on water depth, and higher activity of BF occurred by greater flexion of the knee joint or extension of the hip joint during exercise.     

Involvement of an autocrine stromal cell derived factor-1/CXCR4 system on the distant metastasis of human oral squamous cell carcinoma

We have previously shown that a stromal cell-derived factor-1 (SDF-1; CXCL12)/CXCR4 system is involved in the establishment of lymph node metastasis, but not in that of distant metastasis, in oral squamous cell carcinoma (SCC). In this study, we investigated the role of the autocrine SDF-1/CXCR4 system, with a focus on distant metastasis in oral SCC cells. The immunohistochemical staining of SDF-1 and CXCR4 using primary oral SCCs and metastatic lymph nodes showed a significantly higher number of SDF-1-positive cases among the metastatic lymph nodes than among the primary oral SCCs, which was associated with a poor survival rate among those of the former group. The forced expression of SDF-1 in B88 cells, which exhibit functional CXCR4 and lymph node metastatic potential (i.e., the autocrine SDF-1/CXCR4 system), conferred enhanced cell motility and anchorage-independent growth potential onto the cells. Orthotopic inoculation of the transfectant into nude mice was associated with an increase in the number of metastatic lymph nodes and more aggressive metastatic foci in the lymph nodes. Furthermore, the SDF-1 transfectant (i.e., the autocrine SDF-1/CXCR4 system) exhibited dramatic metastasis to the lung after i.v. inoculation, whereas the mock transfectant (i.e., the paracrine SDF-1/CXCR4 system) did not. Under the present conditions, AMD3100, a CXCR4 antagonist, significantly inhibited the lung metastasis of the SDF-1 transfectant, ameliorated body weight loss, and improved the survival rate of tumor-bearing nude mice. These results suggested that, in cases of oral SCC, the paracrine SDF-1/CXCR4 system potentiates lymph node metastasis, but distant metastasis might require the autocrine SDF-1/CXCR4 system.     

Protein binding to amphoteric polymer brushes grafted onto a porous hollow-fiber membrane

Three kinds of ampholites, i.e., 3-aminopropionic acid (NH2C2H4COOH), (2-aminoethyl)phosphonic acid (NH2C2H4PO3H2), and 2-aminoethane-1-sulfonic acid (NH2C2H4SO3H), were introduced into an epoxy group-containing polymer brush grafted onto a porous hollow-fiber membrane with a porosity of 70% and pore size of 0.36 microm. The amphoteric group density of the hollow-fiber ranged from 0.50 to 0.72 mmol/g. Three kinds of proteins, i.e., lactoferrin (Lf), cytochrome c (Cyt c), and lysozyme (Ly), were captured by the amphoteric polymer brush during the permeation of the protein solution across the ampholite-immobilized porous hollow-fiber membrane. Multilayer binding of the protein to the amphoteric polymer brush, with a degree of multilayer binding of 3.3, 8.6, and 15 for Lf, Cyt c, and Ly, respectively, with the (2-aminoethyl)phosphonic acid-immobilized porous hollow-fiber membrane, was demonstrated with a negligible diffusional mass-transfer resistance of the protein to the ampholite immobilized. The 2-aminoethane-1-sulfonic acid-immobilized porous hollow-fiber membrane exhibited the lowest initial flux of the protein solution, 0.41 m/h at a transmembrane pressure of 0.1 MPa and 298 K, and the highest equilibrium binding capacity of the protein, e.g., 130 mg/g for lysozyme. Extension and shrinkage of the amphoteric polymer brushes were observed during the binding and elution of the proteins.     

Daily growth increments in otoliths of wild-caught honmoroko Gnathopogon caerulescens

Otolith growth increments in wild-caught alizarin complex one (ALC)-marked honmoroko Gnathopogon caerulescens were examined to verify the veracity of the age determination method in cyprinids. ALC-marked G. caerulescens recaptured from their natural environment had lapilli increment counts outside the ALC ring mark that had formed on a daily basis during the juvenile stage. This apparently being the first direct evidence of daily periodicity of otolith increment formation in wild-caught cyprinids.     

CO2-dependent migration and relocation of LCIB,a pyrenoid-peripheral protein in Chlamydomonas reinhardtii

Most microalgae overcome the difficulty of acquiring inorganic carbon (Ci) in aquatic environments by inducing a CO₂-concentrating mechanism (CCM). In the green alga Chlamydomonas reinhardtii, two distinct photosynthetic acclimation states have been described under CO₂-limiting conditions (low-CO₂ [LC] and very low-CO₂ [VLC]). LC-inducible protein B (LCIB), structurally characterized as carbonic anhydrase, localizes in the chloroplast stroma under CO₂-supplied and LC conditions. In VLC conditions, it migrates to aggregate around the pyrenoid, where the CO₂-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase is enriched. Although the physiological importance of LCIB localization changes in the chloroplast has been shown, factors necessary for the localization changes remain uncertain. Here, we examined the effect of pH, light availability, photosynthetic electron flow, and protein synthesis on the localization changes, along with measuring Ci concentrations. LCIB dispersed or localized in the basal region of the chloroplast stroma at 8.3–15 µM CO₂, whereas LCIB migrated toward the pyrenoid at 6.5 µM CO₂. Furthermore, LCIB relocated toward the pyrenoid at 2.6–3.4 µM CO₂, even in cells in the dark or treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and cycloheximide in light. In contrast, in the mutant lacking CCM1, a master regulator of CCM, LCIB remained dispersed even at 4.3 µM CO₂. Meanwhile, a simultaneous expression of LCIC, an interacting protein of LCIB, induced the localization of several speckled structures at the pyrenoid periphery. These results suggest that the localization changes of LCIB require LCIC and are controlled by CO₂ concentration with ∼7 µM as the boundary.

Algal chloroplast proteins undergo localization changes in response to CO2 concentrations, reflecting their physiological function in survival under fluctuating CO2 environments.     

A high endothelial venule secretory protein,mac25/angiomodulin,interacts with multiple high endothelial venule-associated molecules including chemokines

We previously reported that mac25/angiomodulin (AGM), a 30-kDa secretory protein, is abundantly expressed in high endothelial venules (HEVs), which play a crucial role in lymphocyte trafficking to the lymph nodes and Peyer's patches. We report that mac25/AGM interacts preferentially with certain molecules that are expressed in or around HEVs. In particular, mac25/AGM interacted with not only the extracellular matrix proteins and glycosaminoglycans that are expressed in most blood vessels including HEVs, but also with some chemokines that are implicated in the regulation of lymphocyte trafficking, such as the secondary lymphoid-tissue chemokine (SLC; CCL21), IFN-gamma-inducible protein 10 (IP-10; CXCL10), and RANTES (CCL5). The binding of mac25/AGM to SLC and IP-10 was dose-dependent and saturable. The binding to IP-10 could be inhibited by SLC but not by a non-mac25/AGM-binding chemokine, EBI1-ligand chemokine (ELC; CCL19). Interestingly, mac25/AGM failed to interact with 18 other chemokines, suggesting that it binds to certain chemokines preferentially. Immunohistochemical analysis indicated that mac25/AGM colocalizes at least partially with SLC and IP-10 at the basal lamina of HEVs. Upon binding with mac25/AGM, SLC and IP-10 retained all their Ca(2+)-signaling activity in vitro, suggesting that mac25/AGM can hold and present chemokines in the basal lamina of HEVs. These results imply that mac25/AGM plays a multifunctional role, serving not only as an adhesion protein to interact with glycosaminoglycans and extracellular matrix proteins but also as a molecule to present chemokines so that lymphocytes extravasating through HEVs receive further directional cues subsequent to the luminal encounter with lymphoid chemokines.