Osteocyte calcium signaling response to bone matrix deformation

Osteocytes embedded in calcified bone matrix have been widely believed to play important roles in mechanosensing to achieve adaptive bone remodeling in a changing mechanical environment. In vitro studies have clarified several types of mechanical stimuli such as hydrostatic pressure, fluid shear stress, and direct deformation influence osteocyte functions. However, osteocyte response to mechanical stimuli in the bone matrix has not been clearly understood. In this study, we observed the osteocyte calcium signaling response to the quantitatively applied deformation in the bone matrix. A novel experimental system was developed to apply deformation to cultured bone tissue with osteocytes on a microscope stage. As a mechanical stimulus to the osteocytes in bone matrix, in-plane shear deformation was applied using a pair of glass microneedles to bone fragments, obtained from 13-day-old embryonic chick calvariae. Deformation of bone matrix and cells was quantitatively evaluated using an image correlation method by applying for differential interference contrast images of the matrix and fluorescent images of immunolabeled osteocytes, together with imaging of the cellular calcium transient using a ratiometric method. As a result, it was confirmed that the newly developed system enables us to apply deformation to bone matrix and osteocytes successfully under the microscope without significant focal plane shift or deviation from the observation view field. The system could be a basis for further development to investigate the mechanosensing mechanism of osteocytes in bone matrix through examination of various types of rapid biochemical signaling responses and intercellular communication induced by matrix deformation.     

Integrin-linked kinase is required for radial sorting of axons and Schwann cell remyelination in the peripheral nervous system

During development, Schwann cells (SCs) interpret different extracellular cues to regulate their migration, proliferation, and the remarkable morphological changes associated with the sorting, ensheathment, and myelination of axons. Although interactions between extracellular matrix proteins and integrins are critical to some of these processes, the downstream signaling pathways they control are still poorly understood. Integrin-linked kinase (ILK) is a focal adhesion protein that associates with multiple binding partners to link integrins to the actin cytoskeleton and is thought to participate in integrin and growth factor–mediated signaling. Using SC-specific gene ablation, we report essential functions for ILK in radial sorting of axon bundles and in remyelination in the peripheral nervous system. Our in vivo and in vitro experiments show that ILK negatively regulates Rho/Rho kinase signaling to promote SC process extension and to initiate radial sorting. ILK also facilitates axon remyelination, likely by promoting the activation of downstream molecules such as AKT/protein kinase B.     

Alzheimer Disease A�� Production in the Absence of S-Palmitoylation-dependent Targeting of BACE1 to Lipid Rafts

Alzheimer disease β-amyloid (Aβ) peptides are generated via sequential proteolysis of amyloid precursor protein (APP) by BACE1 and γ-secretase. A subset of BACE1 localizes to cholesterol-rich membrane microdomains, termed lipid rafts. BACE1 processing in raft microdomains of cultured cells and neurons was characterized in previous studies by disrupting the integrity of lipid rafts by cholesterol depletion. These studies found either inhibition or elevation of Aβ production depending on the extent of cholesterol depletion, generating controversy. The intricate interplay between cholesterol levels, APP trafficking, and BACE1 processing is not clearly understood because cholesterol depletion has pleiotropic effects on Golgi morphology, vesicular trafficking, and membrane bulk fluidity. In this study, we used an alternate strategy to explore the function of BACE1 in membrane microdomains without altering the cellular cholesterol level. We demonstrate that BACE1 undergoes S-palmitoylation at four Cys residues at the junction of transmembrane and cytosolic domains, and Ala substitution at these four residues is sufficient to displace BACE1 from lipid rafts. Analysis of wild type and mutant BACE1 expressed in BACE1 null fibroblasts and neuroblastoma cells revealed that S-palmitoylation neither contributes to protein stability nor subcellular localization of BACE1. Surprisingly, non-raft localization of palmitoylation-deficient BACE1 did not have discernible influence on BACE1 processing of APP or secretion of Aβ. These results indicate that post-translational S-palmitoylation of BACE1 is not required for APP processing, and that BACE1 can efficiently cleave APP in both raft and non-raft microdomains.Alzheimer disease-associated β-amyloid (Aβ)³ peptides are derived from the sequential proteolysis of β-amyloid precursor protein (APP) by β- and γ-secretases. The major β-secretase is an aspartyl protease, termed BACE1 (β-site APP-cleaving enzyme 1) (1–4). BACE1 cleaves APP within the extracellular domain of APP, generating the N terminus of Aβ. In addition, BACE1 also cleaves to a lesser extent within the Aβ domain between Tyr¹⁰ and Glu¹¹ (β′-cleavage site). Processing of APP at these sites results in the shedding/secretion of the large ectodomain (sAPPβ) and generating membrane-tethered C-terminal fragments +1 and +11 (β-CTF) (5). The multimeric γ-secretase cleaves at multiple sites within the transmembrane domain of β-CTF, generating C-terminal heterogeneous Aβ peptides (ranging in length between 38 and 43 residues) that are secreted, as well as cytosolic APP intracellular domains (6). In addition to BACE1, APP can be cleaved by α-secretase within the Aβ domain between Lys¹⁶ and Leu¹⁷, releasing sAPPα and generating α-CTF. γ-Secretase cleavage of α-CTF generates N-terminal truncated Aβ, termed p3.Genetic ablation of BACE1 completely abolishes Aβ production, establishing BACE1 as the major neuronal enzyme responsible for initiating amyloidogenic processing of APP (4, 7). Interestingly, both the expression and activity of BACE1 is specifically elevated in neurons adjacent to senile plaques in brains of individuals with Alzheimer disease (8). In the past few years additional substrates of BACE1 have been identified that include APP homologues APLP1 and APLP2 (9), P-selectin glycoprotein ligand-1 (10), β-galactoside α2,6-sialyltransferase (11), low-density lipoprotein receptor-related protein (12), β-subunits of voltage-gated sodium channels (13), and neuregulin-1 (14, 15), thus extending the physiological function of BACE1 beyond Alzheimer disease pathogenesis.BACE1 is a type I transmembrane protein with a long extracellular domain harboring a catalytic domain and a short cytoplasmic tail. BACE1 is synthesized as a proenzyme, which undergoes post-translational modifications that include removal of a pro-domain by a furin-like protease, N-glycosylation, phosphorylation, S-palmitoylation, and acetylation, during the transit in the secretory pathway (16–20). In non-neuronal cells the majority of BACE1 localizes to late Golgi/TGN and endosomes at steady-state and a fraction of BACE1 also cycles between the cell surface and endosomes (21). The steady-state localization of BACE1 is consistent with the acidic pH optimum of BACE1 in vitro, and BACE1 cleavage of APP is observed in the Golgi apparatus, TGN, and endosomes (22–25). BACE1 endocytosis and recycling are mediated by the GGA family of adaptors binding to a dileucine motif (⁴⁹⁶DISLL) in its cytoplasmic tail (21, 26–31). Phosphorylation at Ser⁴⁹⁸ within this motif modulates GGA-dependent retrograde transport of BACE1 from endosomes to TGN (21, 26–31).Over the years, a functional relationship between cellular cholesterol level and Aβ production has been uncovered, raising the intriguing possibility that cholesterol levels may determine the balance between amyloidogenic and non-amyloidogenic processing of APP (32–34). Furthermore, several lines of evidence from in vitro and in vivo studies indicate that cholesterol- and sphingolipid-rich membrane microdomains, termed lipid rafts, might be the critical link between cholesterol levels and amyloidogenic processing of APP. Lipid rafts function in the trafficking of proteins in the secretory and endocytic pathways in epithelial cells and neurons, and participate in a number of important biological functions (35). BACE1 undergoes S-palmitoylation (19), a reversible post-translational modification responsible for targeting a variety of peripheral and integral membrane proteins to lipid rafts (36). Indeed, a significant fraction of BACE1 is localized in lipid raft microdomains in a cholesterol-dependent manner, and addition of glycosylphosphatidylinositol (GPI) anchor to target BACE1 exclusively to lipid rafts increases APP processing at the β-cleavage site (37, 38). Antibody-mediated co-patching of cell surface APP and BACE1 has provided further evidence for BACE1 processing of APP in raft microdomains (33, 39). Components of the γ-secretase complex also associate with detergent-resistant membrane (DRM) fractions enriched in raft markers such as caveolin, flotillin, PrP, and ganglioside GM1 (40). The above findings suggest a model whereby APP is sequentially processed by BACE1 and γ-secretase in lipid rafts.Despite the accumulating evidence, cleavage of APP by BACE1 in non-raft membrane regions cannot be unambiguously ruled out because of the paucity of full-length APP (APP FL) and BACE1 in DRM isolated from adult brain and cultured cells (41). Moreover, it was recently reported that moderate reduction of cholesterol (<25%) displaces BACE1 from raft domains, and increases BACE1 processing by promoting the membrane proximity of BACE1 and APP in non-raft domains (34). Nevertheless, this study also found that BACE1 processing of APP is inhibited with further loss of cholesterol (>35%), consistent with earlier studies (32, 33). Nevertheless, given the pleiotropic effects of cholesterol depletion on membrane properties and vesicular trafficking of secretory and endocytic proteins (42–47), unequivocal conclusions regarding BACE1 processing of APP in lipid rafts cannot be reached based on cholesterol depletion studies.In this study, we explored the function of BACE1 in lipid raft microdomains without manipulating cellular cholesterol levels. In addition to the previously reported S-palmitoylation sites (Cys⁴⁷⁸/Cys⁴⁸²/Cys⁴⁸⁵) within the cytosolic tail of BACE1 (19), we have identified a fourth site (Cys⁴⁷⁴) within the transmembrane domain of BACE1 that undergoes S-palmitoylation. A BACE1 mutant with Ala substitution of all four Cys residues (BACE1-4C/A) fails to associate with DRM in cultured cells, but is not otherwise different from wtBACE1 in terms of protein stability, maturation, or subcellular localization. Surprisingly, APP processing and Aβ generation were unaffected in cells stably expressing the BACE1-4C/A mutant. Finally, we observed an increase in the levels of APP CTFs in detergent-soluble fractions of BACE1-4C/A as compared with wtBACE1 cells. Thus, our data collectively indicate a non-obligatory role of S-palmitoylation and lipid raft localization of BACE1 in amyloidogenic processing of APP.     

Biochemical characterization of a novel metalloendopeptidase from Streptomyces aureofaciens TH-3 with post-proline hydrolysis activity

Streptomyces aureofaciens TH-3 secretes a protease termed ‘kibilysin’, for which we showed unique substrate specificity and preference for Tyr, Pro, and Leu at the P₁ position using fluorescence energy transfer substrate (FRETS) combinatorial libraries. Using (7-methoxycoumarin-4-yl) acetyl-Lys-Pro-Leu-Gly-Leu-d-2,3-diamino propionic acid (2,4-dinitrophenyl)-Ala-Arg-NH₂, we confirmed that kibilysin digests the substrate between Pro and Leu. Its gene was cloned and sequenced. The primary structure of the enzyme showed 40, 66, and 61% identity, respectively, with those of thermolysin from Bacillus thermoproteolyticus, and metalloendopeptidases from Streptomyces cinamoneus TH-2 and S. griseus. Its deduced amino acid sequence contained an HEXXH consensus sequence for zinc binding, which is a common motif of the peptidase family M4. Moreover, we succeeded in over-expression of kibilysin using Streptomyces lividans.     

Creation of cross-linked electrospun isotypic-elastin fibers controlled cell-differentiation with new cross-linker

Effective application of elastin materials for vascular grafts in tissue engineering requires these materials to retain the elastic and biological properties of native elastin. To clarify the influence of soluble elastin isotypes on vascular smooth muscle cells (VSMCs), soluble elastin was prepared from insoluble elastin by hydrolysis with oxalic acid. Its fractions were separated and classified into three isotypes. Elastin retaining 2.25 mol% of cross-linked structures exhibited significant differentiation of VSMCs, which adhered to the elastin with contraction phenotypes similar to that of native elastin, causing proliferation to cease. This trend was more strongly demonstrated in cotton-like elastin fibers with a new cross-linker. The results suggest that elastin isotypes could be applied as new effective biomaterials for suppressing intimal hyperplasia in vascular grafts.     

Deep-sea mystery solved: astonishing larval transformations and extreme sexual dimorphism unite three fish families

The oceanic bathypelagic realm (1000–4000m) is a nutrient-poor habitat. Most fishes living there have pelagic larvae using the rich waters of the upper 200m. Morphological and behavioural specializations necessary to occupy such contrasting environments have resulted in remarkable developmental changes and life-history strategies. We resolve a long-standing biological and taxonomic conundrum by documenting the most extreme example of ontogenetic metamorphoses and sexual dimorphism in vertebrates. Based on morphology and mitogenomic sequence data, we show that fishes currently assigned to three families with greatly differing morphologies, Mirapinnidae (tapetails), Megalomycteridae (bignose fishes) and Cetomimidae (whalefishes), are larvae, males and females, respectively, of a single family Cetomimidae. Morphological transformations involve dramatic changes in the skeleton, most spectacularly in the head, and are correlated with distinctly different feeding mechanisms. Larvae have small, upturned mouths and gorge on copepods. Females have huge gapes with long, horizontal jaws and specialized gill arches allowing them to capture larger prey. Males cease feeding, lose their stomach and oesophagus, and apparently convert the energy from the bolus of copepods found in all transforming males to a massive liver that supports them throughout adult life.     

Convergence of multisensory inputs in Xenopus tadpole tectum

The integration of multisensory information takes place in the optic tectum where visual and auditory/mechanosensory inputs converge and regulate motor outputs. The circuits that integrate multisensory information are poorly understood. In an effort to identify the basic components of a multisensory integrative circuit, we determined the projections of the mechanosensory input from the periphery to the optic tectum and compared their distribution to the retinotectal inputs in Xenopus laevis tadpoles using dye‐labeling methods. The peripheral ganglia of the lateral line system project to the ipsilateral hindbrain and the axons representing mechanosensory inputs along the anterior/posterior body axis are mapped along the ventrodorsal axis in the axon tract in the dorsal column of the hindbrain. Hindbrain neurons project axons to the contralateral optic tectum. The neurons from anterior and posterior hindbrain regions project axons to the dorsal and ventral tectum, respectively. While the retinotectal axons project to a superficial lamina in the tectal neuropil, the hindbrain axons project to a deep neuropil layer. Calcium imaging showed that multimodal inputs converge on tectal neurons. The layer‐specific projections of the hindbrain and retinal axons suggest a functional segregation of sensory inputs to proximal and distal tectal cell dendrites, respectively. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Presence of Thrombin-Activatable Fibrinolysis Inhibitor in Helicobacter pylori-Associated Gastroduodenal Disease

Thrombin-activatable fibrinolysis inhibitor (TAFI) plays a role in the regulation of coagulation and inflammation. In addition to inhibiting the fibrinolytic system, TAFI may also regulate the bradykinin and complement systems. We hypothesized that TAFI also plays a role in defense mechanisms of the gastric mucosa during Helicobacter pylori infection. This study comprised 65 patients with gastroduodenal disorders: 41 patients with H. pylori infection, 13 without, and 11 patients with cured H. pylori infection. The gastric intramucosal concentrations of TAFI were measured by enzyme immunoassay. The gastric levels of TAFI and plasminogen activator inhibitor-1 were significantly increased in patients with H. pylori compared to those without infection or cured H. pylori . The presence of TAFI was detected in gastric mucosal epithelial cells. The concentration of TAFI was correlated with the degree of gastric mucosal atrophy, inflammation, and disease activity. These results show that TAFI is present in the gastric mucosa and that it may play a role in the pathogenesis of H. pylori infection-associated gastroduodenal disorders.