Involvement of IL-1 in the Maintenance of Masseter Muscle Activity and Glucose Homeostasis

Physical exercise reportedly stimulates IL-1 production within working skeletal muscles, but its physiological significance remains unknown due to the existence of two distinct IL-1 isoforms, IL-1α and IL-1β. The regulatory complexities of these two isoforms, in terms of which cells in muscles produce them and their distinct/redundant biological actions, have yet to be elucidated. Taking advantage of our masticatory behavior (Restrained/Gnawing) model, we herein show that IL-1α/1β-double-knockout (IL-1-KO) mice exhibit compromised masseter muscle (MM) activity which is at least partially attributable to abnormalities of glucose handling (rapid glycogen depletion along with impaired glucose uptake) and dysfunction of IL-6 upregulation in working MMs. In wild-type mice, masticatory behavior clearly increased IL-1β mRNA expression but no incremental protein abundance was detectable in whole MM homogenates, whereas immunohistochemical staining analysis revealed that both IL-1α- and IL-1β-immunopositive cells were recruited around blood vessels in the perimysium of MMs after masticatory behavior. In addition to the aforementioned phenotype of IL-1-KO mice, we found the IL-6 mRNA and protein levels in MMs after masticatory behavior to be significantly lower in IL-1-KO than in WT. Thus, our findings confirm that the locally-increased IL-1 elicited by masticatory behavior, although present small in amounts, contributes to supporting MM activity by maintaining normal glucose homeostasis in these muscles. Our data also underscore the importance of IL-1-mediated local interplay between autocrine myokines including IL-6 and paracrine cytokines in active skeletal muscles. This interplay is directly involved in MM performance and fatigability, perhaps mediated through maintaining muscular glucose homeostasis.     

A series of novel indazole derivatives of Sirt 1 activator as osteogenic regulators

A series of indazole derivatives were identified as Sirt 1 activators though high-throughput screening. Optimization of each substituent on the indazole ring led to the identification of compound 13. Compound 13 appeared to give the best Sirt 1 activity of the compounds tested and also showed osteogenesis activity in a cell assay. Sirt 1 activators are therefore potential candidates for the treatment of osteoporosis.     

Recombinant sea urchin flagellar adenylate kinase

Adenylate kinase (AK) is localized in sea urchin sperm flagella and embryonic cilia. To investigate sea urchin Strongylocentrotus purpuratus AK (SpAK) enzymatic characteristics, the full-length recombinant protein of 130 kDa (SpAKr) and each of its three catalytic domains were expressed in Escherichia coli. Although the full-length SpAK had high enzymatic activity, each of the three catalytic domains had no activity. The Km for ATP synthesis from ADP was 0.23 mM and the Vmax was 4.51 mumol ATP formed per minute per milligram of protein. The specific AK inhibitor, Ap5A, blocks SpAKr enzymatic activity with an IC50 of 0.53 microM. The pH optimum for SpAKr is 8.1, as compared to 7.7 for the natural SpAK. Calcium inhibits SpAKr activity in a dose-dependent manner. Although SpAKr has three cAMP-dependent protein kinase phosphorylation sites, and can be phosphorylated in vitro, the enzymatic kinetics after phosphorylation are not significantly altered. SpAK and Chlamydomonas flagellar AKs are the only AKs with three catalytic sites. Further study of the SpAKr will aid in understanding the active site of this interesting and important ATP synthase.     

Prolyl 4-Hydroxylation of ��-Fibrinogen: A NOVEL PROTEIN MODIFICATION REVEALED BY PLASMA PROTEOMICS*

Plasma proteome analysis requires sufficient power to compare numerous samples and detect changes in protein modification, because the protein content of human samples varies significantly among individuals, and many plasma proteins undergo changes in the bloodstream. A label-free proteomics platform developed in our laboratory, termed “Two-Dimensional Image Converted Analysis of Liquid chromatography and mass spectrometry (2DICAL),” is capable of these tasks. Here, we describe successful detection of novel prolyl hydroxylation of α-fibrinogen using 2DICAL, based on comparison of plasma samples of 38 pancreatic cancer patients and 39 healthy subjects. Using a newly generated monoclonal antibody 11A5, we confirmed the increase in prolyl-hydroxylated α-fibrinogen plasma levels and identified prolyl 4-hydroxylase A1 as a key enzyme for the modification. Competitive enzyme-linked immunosorbent assay of 685 blood samples revealed dynamic changes in prolyl-hydroxylated α-fibrinogen plasma level depending on clinical status. Prolyl-hydroxylated α-fibrinogen is presumably controlled by multiple biological mechanisms, which remain to be clarified in future studies.For comprehensive analysis of plasma proteins, it is necessary to compare a sufficient number of blood samples to avoid simple interindividual heterogeneity, because the protein content of human samples varies significantly among individuals. Also, the provision of sufficient power is needed to detect protein modification because many plasma proteins undergo changes in the bloodstream (1). Even though the proteomic technologies have advanced (2, 3), there remains room for improvement. Different isotope labeling and identification-based methods have been developed for quantitative proteomics technologies (4–6), but the number of samples that can be compared by the current isotope-labeling methods is limited, and identification-based proteomics is unable to capture information regarding unknown modifications.A label-free proteomics platform developed in our laboratory, termed “Two-Dimensional Image Converted Analysis of Liquid chromatography and mass spectrometry (2DICAL)² (7), simply compares the liquid chromatography and mass spectrometry (LC-MS) data and detects a protein modification by finding changes in the mass to charge ratio (m/z) and retention time (RT). Enhanced methods for accurate MS peak alignment across multiple LC runs have enabled the successful implementation of clinical studies requiring comparison of a large number of samples (8, 9). Using 2DICAL to analyze plasma samples of pancreatic cancer patients and healthy controls, novel prolyl hydroxylation of α-fibrinogen was successfully discovered.Fibrinogen and its modification has been investigated because of its clinical importance (10, 11). On the other hand, prolyl hydroxylation has attracted attention after the discovery of the hypoxia-inducible factor 1α (HIF1α) prolyl-hydroxylase and its role in switching of HIF1α functions (12). Prolyl hydroxylation in other proteins has been energetically sought, but only a few such proteins have been identified (13). Only one study has reported prolyl hydroxylation of fibrinogen at the β chain (14).Here, we report the detection of prolyl 4-hydroxylated α-fibrinogen by plasma proteome analysis, a protein modification that dynamically changes in plasma depending on the clinical status and is a candidate plasma biomarker.     

Structure-activity relationship studies of sphingosine-1-phosphate receptor agonists with N-cinnamyl-β-alanine moiety

Structure-activity relationship of sphingosine-1-phosphate receptor agonist was examined. In terms of reducing the flexibility of molecule, hit compound 1 was modified to improve S1P₁ agonistic activity as well as selectivity over S1P₃ agonistic activity. Novel S1P agonists with cinnamyl scaffold or 1,2,5,6-tetrahydropyridine scaffold were identified.     

X-Linked Adrenoleukodystrophy: Molecular and Functional Analysis of the ABCD1 Gene in Argentinean Patients

X-linked adrenoleukodystrophy (X-ALD) is an inherited metabolic disease associated with mutations in the ABCD1 gene that encodes an ATP-binding cassette transporter protein, ALDP. The disease is characterized by increased concentrations of very long-chain fatty acids (VLCFAs) in plasma and in adrenal, testicular and nervous tissues, due to a defect in peroxisomal VLCFA β-oxidation. In the present study, we analyzed 10 male patients and 17 female carriers from 10 unrelated pedigrees with X-ALD from Argentina. By sequencing the ABCD1 we detected 9 different mutations, 8 of which were novel. These new mutations were verified by a combination of methods that included both functional (western blot and peroxisomal VLCFA β-oxidation) and bioinformatics analysis. The spectrum of novel mutations consists of 3 frameshift (p.Ser284fs*16, p.Glu380Argfs*21 and p.Thr254Argfs*82); a deletion (p.Ser572_Asp575del); a splicing mutation (c.1081+5G>C) and 3 missense mutations (p.Ala341Asp, p.His420Pro and p.Tyr547Cys). In one patient 2 changes were found: a known missense (p.His669Arg) and an unpublished amino acid substitution (p.Ala19Ser). In vitro studies suggest that p.Ala19Ser is a polymorphism. Moreover, we identified two novel intronic polymorphisms and two amino acid polymorphisms. In conclusion, this study extends the spectrum of mutation in X-ALD and facilitates the identification of heterozygous females.     

Mouse-Hamster Chimeric Prion Protein (PrP) Devoid of N-Terminal Residues 23-88 Restores Susceptibility to 22L Prions,but Not to RML Prions in PrP-Knockout Mice

Prion infection induces conformational conversion of the normal prion protein PrP^C, into the pathogenic isoform PrP^Sc, in prion diseases. It has been shown that PrP-knockout (Prnp^0/0) mice transgenically reconstituted with a mouse-hamster chimeric PrP lacking N-terminal residues 23-88, or Tg(MHM2Δ23-88)/Prnp^0/0 mice, neither developed the disease nor accumulated MHM2^ScΔ23-88 in their brains after inoculation with RML prions. In contrast, RML-inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice developed the disease with abundant accumulation of MHM2^ScΔ23-88 in their brains. These results indicate that MHM2Δ23-88 itself might either lose or greatly reduce the converting capacity to MHM2^ScΔ23-88, and that the co-expressing wild-type PrP^C can stimulate the conversion of MHM2Δ23-88 to MHM2^ScΔ23-88 in trans. In the present study, we confirmed that Tg(MHM2Δ23-88)/Prnp^0/0 mice remained resistant to RML prions for up to 730 days after inoculation. However, we found that Tg(MHM2Δ23-88)/Prnp^0/0 mice were susceptible to 22L prions, developing the disease with prolonged incubation times and accumulating MHM2^ScΔ23-88 in their brains. We also found accelerated conversion of MHM2Δ23-88 into MHM2^ScΔ23-88 in the brains of RML- and 22L-inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice. However, wild-type PrP^Sc accumulated less in the brains of these inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice, compared with RML- and 22L-inoculated Prnp^0/+ mice. These results show that MHM2Δ23-88 itself can convert into MHM2^ScΔ23-88 without the help of the trans-acting PrP^C, and that, irrespective of prion strains inoculated, the co-expressing wild-type PrP^C stimulates the conversion of MHM2Δ23-88 into MHM2^ScΔ23-88, but to the contrary, the co-expressing MHM2Δ23-88 disturbs the conversion of wild-type PrP^C into PrP^Sc.     

Preferential increase in the hippocampal synaptic vesicle protein 2A (SV2A) by pentylenetetrazole kindling

The present study evaluated the expressional levels of synaptic vesicle protein 2A (SV2A) and other secretary machinery proteins (i.e., soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, Munc18-1, N-ethylmaleimide-sensitive factor (NSF) and soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)) in a pentylenetetrazole (PTZ) kindling model. Repeated administration of sub-convulsive PTZ (40 mg/kg, i.p.) progressively increased seizure susceptibility in mice and consistently induced clonic seizures in most animals tested at 15 days after the treatment. Western blot analysis revealed that, among the secretary machinery proteins examined, hippocampal SV2A was selectively elevated by PTZ kindling. PTZ kindling-induced SV2A expression appeared region-specific and the SV2A levels in the cerebral cortex or cerebellum were unaltered. In addition, SV2A expression by PTZ kindling was prominent in the hilar region of the dentate gyrus (DG) where GABAergic interneurons are located, but not in other hippocampal regions (e.g., the stratum lucidum of the CA3 and synaptic layers surrounding CA1 or CA3 pyramidal neurons). These findings suggest that PTZ kindling preferentially elevates SV2A expression in the hippocampus probably as a compensatory mechanism to activate the inhibitory neurotransmission.     

The expense for one implantation of a banked bone allograft from a cadaveric donor and the issues affecting current advanced medical treatment in the Japanese orthopaedic field

Demand for banked bone allografts is increasing in Japan; however, there are too few bone banks and the bone bank network is not well-established. One reason for this was lack of funding for banks. Bone banks had to bear all material expenses of banked bone allografts themselves because this was not designated a covered expense. In December 2004, the Japanese government started a new “Advanced Medical Treatment” administration system which allowed an approved institution to charge the expense of authorized advanced medical treatments directly to patients. The treatment named “Cryopreserved allogenic bone and ligamentous tissue retrieved from cadaveric donor” was approved as an advanced medical treatment in March 2007. We present the calculation method and the expense per implantation of a banked bone allograft from a cadaveric donor under this treatment and raise issues which affect this advanced medical treatment and remain to be resolved in the Japanese orthopaedic field.     

Water extract of Cordyceps sinensis (WECS) inhibits the RANKL-induced osteoclast differentiation

It has been reported that Cordyceps sinensis, a traditional Chinese medicine, has various pharmacological effects. The aim of this study was to clarify the effect of water extract of Cordyceps sinensis (WECS) on osteoclast differentiation in vitro. In mouse bone marrow cells and monocyte/macrophage cell line RAW264.7, WECS dose-dependently inhibited the receptor activator of nuclear factor kappa B (NF-kappaB) ligand (RANKL)-induced osteoclast differentiation by tartrate-resistant acid phosphatase (TRAP) staining. In fact, cytotoxic effect was not observed in the RAW264.7 cells treated with WECS. Moreover, the mRNA expression of osteoclast related genes (calcitonin receptor, cathepsin K, matrix metalloprotease 9 and nuclear factor of activated T cells c1) was also inhibited by WECS. Investigation of inhibitory mechanism by using electrophoretic mobility shift assay (EMSA) and Western blot analysis revealed that WECS inhibited the activation of NF-kappaB through the prevention of IkappaBalpha phosphorylation. In conclusion, the present results demonstrate for the first time that WECS is a potent inhibitor of the RANKL-induced osteoclast differentiation through a mechanism involving the NF-kappaB pathway.