Autoregulation of Osteocyte Sema3A Orchestrates Estrogen Action and Counteracts Bone Aging

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Nutrition,metabolism, and epigenetics: pathways of circadian reprogramming

Food intake profoundly affects systemic physiology. A large body of evidence has indicated a link between food intake and circadian rhythms, and ~24‐h cycles are deemed essential for adapting internal homeostasis to the external environment. Circadian rhythms are controlled by the biological clock, a molecular system remarkably conserved throughout evolution. The circadian clock controls the cyclic expression of numerous genes, a regulatory program common to all mammalian cells, which may lead to various metabolic and physiological disturbances if hindered. Although the circadian clock regulates multiple metabolic pathways, metabolic states also provide feedback on the molecular clock. Therefore, a remarkable feature is reprogramming by nutritional challenges, such as a high‐fat diet, fasting, ketogenic diet, and caloric restriction. In addition, various factors such as energy balance, histone modifications, and nuclear receptor activity are involved in the remodeling of the clock. Herein, we review the interaction of dietary components with the circadian system and illustrate the relationships linking the molecular clock to metabolism and critical roles in the remodeling process.     

The biomechanical effect of the collar of a femoral stem on total hip arthroplasty

To investigate the biomechanical effect of collars, finite element analyses are carried out through two hip joints that are implanted using collared and collarless stems, respectively, and an intact hip joint model. For the analyses, the sacrum, coxal bone, and the cancellous and cortical bones of a femur are modelled using finite elements based on X-ray computed tomographic images taken from a 27-year-old woman. From the results, it is found that a collar with perfect calcar contact prevents stem subsidence and decreases the proximal–lateral gap and the lateral stem tilting. Therefore, it can impart reasonable biomechanical stability for total hip arthroplasty. However, its low load transmission ability and increased stem tilting effect due to the imperfect contact between the collar and the calcar are found to be serious problems that need to be solved. Results of clinical follow-up are presented for supporting the computational results.     

Cooperative Functions of ZnT1, Metallothionein and ZnT4 in the Cytoplasm Are Required for Full Activation of TNAP in the Early Secretory Pathway

The activation process of secretory or membrane-bound zinc enzymes is thought to be a highly coordinated process involving zinc transport, trafficking, transfer and coordination. We have previously shown that secretory and membrane-bound zinc enzymes are activated in the early secretory pathway (ESP) via zinc-loading by the zinc transporter 5 (ZnT5)-ZnT6 hetero-complex and ZnT7 homo-complex (zinc transport complexes). However, how other proteins conducting zinc metabolism affect the activation of these enzymes remains unknown. Here, we investigated this issue by disruption and re-expression of genes known to be involved in cytoplasmic zinc metabolism, using a zinc enzyme, tissue non-specific alkaline phosphatase (TNAP), as a reporter. We found that TNAP activity was significantly reduced in cells deficient in ZnT1, Metallothionein (MT) and ZnT4 genes (ZnT1 ^−/− MT ^−/− ZnT4 ^−/− cells), in spite of increased cytosolic zinc levels. The reduced TNAP activity in ZnT1 ^−/− MT ^−/− ZnT4 ^−/− cells was not restored when cytosolic zinc levels were normalized to levels comparable with those of wild-type cells, but was reversely restored by extreme zinc supplementation via zinc-loading by the zinc transport complexes. Moreover, the reduced TNAP activity was adequately restored by re-expression of mammalian counterparts of ZnT1, MT and ZnT4, but not by zinc transport-incompetent mutants of ZnT1 and ZnT4. In ZnT1 ^−/− MT ^−/− ZnT4 ^−/− cells, the secretory pathway normally operates. These findings suggest that cooperative zinc handling of ZnT1, MT and ZnT4 in the cytoplasm is required for full activation of TNAP in the ESP, and present clear evidence that the activation process of zinc enzymes is elaborately controlled.     

Calcium-Dependent Interaction Sites of Tropomyosin on Reconstituted Muscle Thin Filaments with Bound Myosin Heads as Studied by Site-Directed Spin-Labeling

To identify the interaction sites of Tm, we measured the rotational motion of a spin-label covalently bound to the side chain of a cysteine that was genetically incorporated into rabbit skeletal muscle tropomyosin (Tm) at positions 13, 36, 146, 160, 174, 190, 209, 230, 271, or 279. Most of the Tm residues were immobilized on actin filaments with myosin-S1 bound to them. The residues in the mid-portion of Tm, namely, 146, 174, 190, 209, and 230, were mobilized when the troponin (Tn) complex bound to the actin-Tm-S1 filaments. The addition of Ca²⁺ ions partially reversed the Tn-induced mobilization. In contrast, residues at the joint region of Tm, 13, 36, 271, and 279 were unchanged or oppositely changed. All of these changes were detected using a maleimide spin label and less obviously using a methanesulfonate label. These results indicated that Tm was fixed on thin filaments with myosin bound to them, although a small change in the flexibility of the side chains of Tm residues, presumably interfaced with Tn, actin and myosin, was induced by the binding of Tn and Ca²⁺. These findings suggest that even in the myosin-bound (open) state, Ca²⁺ may regulate actomyosin contractile properties via Tm.     

DNA adenine methylation changes dramatically during establishment of symbiosis

The DNA adenine methylation status on specific 5'-GANTC-3' sites and its change during the establishment of plant-microbe interactions was demonstrated in several species of alpha-proteobacteria. Restriction landmark genome scanning (RLGS), which is a high-resolution two dimensional DNA electrophoresis method, was used to monitor the genomewide change in methylation. In the case of Mesorhizobium loti MAFF303099, real RLGS images obtained with the restriction enzyme MboI, which digests at GATC sites, almost perfectly matched the virtual RLGS images generated based on genome sequences. However, only a few spots were observed when the restriction enzyme HinfI was used, suggesting that most GANTC (HinfI) sites were tightly methylated and specific sites were unmethylated. DNA gel blot analysis with the cloned specifically unmethylated regions (SUMs) showed that some SUMs were methylated differentially in bacteroids compared to free-living bacteria. SUMs have also been identified in other symbiotic and parasitic bacteria. These results suggest that DNA adenine methylation may contribute to the establishment and/or maintenance of symbiotic and parasitic relationships.     

Direct interactions between NEDD8 and ubiquitin E2 conjugating enzymes upregulate cullin-based E3 ligase activity

Although cullin-1 neddylation is crucial for the activation of SCF ubiquitin E3 ligases, the underlying mechanisms for NEDD8-mediated activation of SCF remain unclear. Here we demonstrate by NMR and mutational studies that NEDD8 binds the ubiquitin E2 (UBC4), but not NEDD8 E2 (UBC12). Our data imply that NEDD8 forms an active platform on the SCF complex for selective recruitment of ubiquitin-charged E2s in collaboration with RBX1, and thereby upregulates the E3 activity.     

Localization of spectrin isoforms in the adult mouse heart

The distribution of two isoforms of spectrin in the adult mouse heart was investigated by Western blotting and immunocytochemistry by use of monospecific antibodies to erythrocyte spectrin and nonerythroid brain spectrin (240/235). Western blotting revealed proteins analogous to both isoforms of -spectrin in adult heart. Light-microscopic immunocytochemistry indicated that erythroid spectrin was distributed throughout the myocardium, with immunofluorescence localized to plasma membranes, Z-lines, and intercalated discs. Antibodies to brain spectrin (240/235) exhibited staining throughout the heart, with a generally diffuse distribution except for the prominent immunoreactivity associated with the intercalated discs. Nonerythroid spectrin immunofluorescence was detected in the endothelial cells of the endocardium and the mesothelial cell lining of the epicardium. Erythrocyte spectrin was not detected in the endocardium or the epicardium. The identification and localization of spectrin isoforms in the mammalian heart suggest the importance of spectrin proteins in the structural integrity and proper function of cardiac cells and tissues. This is the first demonstration of two different -spectrin subunits in the mammalian heart.