Macrophages play a unique role in the plaque calcification by enhancing the osteogenic signals exerted by vascular smooth muscle cells

Vascular calcification is a major risk factor for the cardiovascular disease, yet its underlying molecular mechanisms remain to be elucidated. Recently, we identified that osteogenic signals via bone morphogenetic protein (BMP)-2 exerted by vascular smooth muscle cells (VSMCs) play a crucial role in the formation of atherosclerotic plaque calcification. Here we report a synergistic interaction between macrophages and VSMCs with respect to plaque calcification. Treatment with conditioned medium (CM) of macrophages dramatically enhanced BMP-2 expression in VSMCs, while it substantially reduced the expression of matrix Gla-protein (MGP) that inhibits the BMP-2 osteogenic signaling. As a result, macrophages significantly accelerated the osteoblastic differentiation of C2C12 cells induced by VSMC-CM. In contrast, macrophage-CM did not enhance the osteoblastic gene expressions in VSMCs, indicating that macrophages unlikely induced the osteoblastic trans-differentiation of VSMCs. We then examined the effect of recombinant TNF-α and IL-1β on the VSMC-derived osteogenic signals. Similar to the macrophage-CM, both cytokines enhanced BMP-2 expression and reduced MGP expression in VSMCs. Nevertheless, only the neutralization of TNF-α but not IL-1β attenuated the effect of macrophage-CM on the expression of these genes in VSMCs, due to the very low concentration of IL-1β in the macrophage-CM. On the other hand, VSMCs significantly enhanced IL-1β expression in macrophages, which might in turn accelerate the VSMC-mediated osteogenic signals. Together, we identified a unique role of macrophages in the formation of plaque calcification in coordination with VSMCs. This interaction between macrophages and VSMCs is a potential therapeutic target to treat and prevent the atherosclerotic plaque calcification.     

Direct transplantation of mesenchymal stem cells into the knee joints of Hartley strain guinea pigs with spontaneous osteoarthritis

Introduction

Mesenchymal stem cells (MSCs) can differentiate into various connective tissue cells. Several techniques have been used for the clinical application of MSCs in articular cartilage repair; however, there are many issues associated with the selection of the scaffold material, including its ability to support cell viability and differentiation and its retention and degradation in situ. The application of MSCs via a scaffold also requires a technically demanding surgical procedure. The aim of this study was to test the outcome of intra-articular transplantation of mesenchymal stem cells suspended in hyaluronic acid (HA) in the knee joints of Hartley strain guinea pigs with spontaneous osteoarthritis (OA).

Methods

Commercially available human MSCs were cultured, labeled with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), suspended in either PBS or HA, and injected into the knee joints of 7-month-old animals. The control animals were injected with either PBS or HA alone. The animals were sacrificed at 1, 3, and 5 weeks post transplantation, the knee joints harvested, and fluorescent microscopic analysis was performed. Histological and immunohistochemical analysis were performed at 5 weeks post transplantation.

Results

At 5 weeks post transplantation, partial cartilage repair was noted in the HA-MSC group but not in the other groups. Examination of CFDA-SE-labeled cells demonstrated migration, differentiation, and proliferation of MSC in the HA-MSC group. There was strong immunostaining for type II collagen around both residual chondrocytes and transplanted MSCs in the OA cartilage.

Conclusion

This scaffold-free and technically undemanding technique appears to result in the regeneration of articular cartilage in the spontaneous OA animal model. Although further examination of the long-term effects of transplantation is necessary, the findings suggest that intra-articular injection of HA-MSC mixture is potentially beneficial for OA.     

Borna disease virus RNA detected in Japanese macaques (Macaca fuscata)

We have examined the seroprevalence of BDV in wild Japanese macaques (Macaca fuscata) in the peninsula (Chiba prefecture), Japan. Serum samples from macaques were examined by the ELISA, Western blot and immunofluorescence assays to detect the presence of serum antibodies that react specifically to BDV antigens. Among 49 investigated individuals, 6 (12.2%) showed positive reaction to BDV antigens. RT-PCR studies detected BDV sequences in brain tissue of one case among four seropositive cases examined. Sequence analysis revealed a high degree of genetic conservation between BDV sequences derived from Japanese macaques and those documented for other animal species. Nevertheless, phylogenetic analysis revealed unique differences between macaque and other species derived BDV sequences.     

Characterization of dual substrate binding sites in the homodimeric structure of Escherichia coli mRNA interferase MazF

MazF and MazE constitute a so-called addiction module that is critical for bacterial growth arrest and eventual cell death in response to stress. The MazF toxin was recently shown to possess mRNA interferase (MIase) activity, and acts as a protein synthesis inhibitor by cleaving cellular mRNA. As a cognate regulator, the short-lived antitoxin, MazE, inhibits MazF MIase activity and hence maintains the delicate homeostasis between these two components. In the present study, we have shown that the MazF homodimer contains two symmetric binding sites, each of which is capable of interacting with a MazE C-terminal peptide, MazEp(54-77). The slow exchange phenomenon between free and peptide-bound MazF on the NMR timescale indicates relatively high affinities for MazEp(54-77) at both sites (Kd,K'd < 10(-7) M). However, the observed sequential binding behavior suggests a negative cooperativity between the two sites (Kd < K'd). A 13 base single-stranded DNA, employed as an uncleavable RNA substrate analog, can also bind to both sites on the MazF homodimer with moderate affinity (Kd approximately 10(-5) -10(-6) M). Chemical shift perturbation data deduced from NMR experiments indicates that the two binding sites for the MazEp peptide coincided with those for the single-stranded DNA competitive inhibitor. These dual substrate-binding sites are located on the concave interface of the MazF homodimer, consisting of a highly basic region underneath the S1-S2 loop and two hydrophobic regions containing the H1 helix of one subunit and the S3-S4 loop of the opposing subunit. We show that the MazF homodimer is a bidentate endoribonuclease equipped with two identical binding sites for mRNA processing and that a single MazE molecule occupying one of the binding sites can affect the conformation of both sites, hence efficiently hindering the activity of MazF.     

Genetic variants of the IgA Fc receptor (FcαR, CD89) promoter in chronic hepatitis C patients

Fc receptor for IgA (FcαR, CD89) is capable of triggering IgA-mediated immune responses to pathogens and has been proposed to function in circulating IgA clearance. Because inheritable variations modifying individual immune responses or immunoglobulin catabolism may affect the chronicity of viral infection, we investigated whether promoter polymorphisms of the FcαR gene (FCAR) affect chronic hepatitis C virus (HCV) infection and its disease progression. The two −311T/C and −142T/C single-nucleotide polymorphisms (SNPs) were studied by direct DNA sequencing in 177 Japanese patients with chronic hepatitis C (CHC). Both −311CC and −142CC genotypes were more frequent in CHC patients (15.9 and 18.6%) compared with 210 healthy controls (5.7 and 10.0%) [p = 0.001, odds ratio (OR) = 3.10, 95% confidence interval CI) = 1.53–6.30 and p = 0.014, OR = 2.06, 95% CI = 1.14–3.72, respectively], and were associated with infection with HCV genotype 2a/2b (p = 0.019 and p = 0.005, respectively). Conversely, −311CC and −142CC were decreased in 59 patients at advanced stages of disease as assessed on the basis of hepatic fibrosis markers such as decreased platelet count (PLT) ( < 150,000/μl) (5.1 and 8.5%) compared with 91 patients with normal PLT ( ≥ 150,000/μl) (24.2 and 26.4%) (p = 0.006 and p = 0.005, respectively). Moreover, among the patients with normal PLT (but not with decreased PLT), −311CC or −142CC was significantly associated with decreased serum IgA levels (p = 0.023 or p = 0.007, respectively). These results suggest that the FCAR promoter SNPs may be related to chronic HCV infection and disease progression in Japanese CHC, which might be explained by altered FcαR expression affecting IgA-mediated immune responses and/or IgA catabolism.     

Local adherent technique for transplanting mesenchymal stem cells as a potential treatment of cartilage defect

Introduction  

Current cell therapy for cartilage regeneration requires invasive procedures, periosteal coverage and scaffold use. We have developed a novel transplantation method with synovial mesenchymal stem cells (MSCs) to adhere to the cartilage defect.     

Ontogenetic and seasonal changes in the diet of the halfbeak <Emphasis Type="Italic">Zenarchopterus dunckeri</Emphasis> at Iriomote Island,southern Japan

Ontogenetic and seasonal changes in the diet of the halfbeak Zenarchopterus dunckeri were examined in the Urauchi River estuary, Iriomote Island, southern Japan, using gut content analysis. The diet of this species changed dramatically with growth. Smaller juveniles fed mostly on small zooplankton and small gastropods, with a shift to the consumption of terrestrial insects, such as ants and flies, by larger individuals. Seasonal dietary differences were also apparent in adults. Ants were the most dominant prey item in the spring to fall months, whereas flies were abundant in the diet in the winter and early summer months.     

Identification and characterization of subfamily-specific signatures in a large protein superfamily by a hidden Markov model approach

Background  

Most profile and motif databases strive to classify protein sequences into a broad spectrum of protein families. The next step of such database studies should include the development of classification systems capable of distinguishing between subfamilies within a structurally and functionally diverse superfamily. This would be helpful in elucidating sequence-structure-function relationships of proteins.