Mitochondrial Akt-regulated mitochondrial apoptosis signaling in cardiac muscle cells

We recently reported translocation and activation of Akt in cardiac mitochondria. This study was to determine whether activation of Akt in mitochondria could inhibit apoptosis of cardiac muscle cells. Insulin stimulation induced translocation of phosphorylated Akt to the mitochondria in primary cardiomyocytes. A mitochondria-targeted constitutively active Akt was overexpressed via adenoviral vector and inhibited efflux of cytochrome c and apoptosis-inducing factor from mitochondria to cytosol and partially prevented loss of mitochondria cross-membrane electrochemical gradient. Activation of caspase 3 was suppressed in the cardiomyocytes transduced with mitochondria-targeted active Akt, whereas a mitochondria-targeted dominant negative Akt enhanced activation of caspase 3. Terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling assay showed that mitochondrial activation of Akt significantly reduced the number of apoptotic cells. When the endogenous Akt was abolished by LY294002, the antiapoptotic actions of mitochondrial Akt remained effective. These experiments suggested that mitochondrial Akt suppressed apoptosis signaling independent of cytosolic Akt in cardiac muscle cells.     

Diverse effects of type II collagen on osteogenic and adipogenic differentiation of mesenchymal stem cells

Type II collagen is known to modulate chondrogenesis of mesenchymal stem cells (MSCs). In this study, MSCs from human bone marrow aspirates were used to study the modulating effects of type II collagen on MSC differentiation during the early stages of osteogenesis and adipogenesis. With osteogenic induction, MSCs cultured on the type II collagen-coated surface showed an enhanced calcium deposition level with increasing mRNA expressions of RUNX2, osteocalcin, and alkaline phosphatase. A synthetic integrin binding peptide, which specifically interacts with the I-domain of α(1)β(1)/α(2)β(1) integrins significantly blocks the mineralization-enhancing effect of type II collagen. MSCs attached on the type II collagen-coated plates exhibited expanded cell morphology with increasing spreading area, and the pretreatment of cells with integrin α(1)β(1) or α(2)β(1)-blocking antibody reduced the effect. The phosphorylation levels of FAK, ERK, and JNK significantly increased in the MSCs that attached on the type II collagen-coated plates. On the contrary, the mineralization-enhancing effect of type II collagen was diminished by JNK and MEK inhibitors. Furthermore, type II collagen blocked the adipogenic differentiation of MSCs, and this effect is rescued by JNK and MEK inhibitors. In conclusion, type II collagen facilitates osteogenesis and suppresses adipogenesis during early stage MSC differentiation. Such effects are integrin binding-mediated and conducted through FAK-JNK and/or FAK-ERK signaling cascades. These results inspire a novel strategy encompassing type II collagen in bone tissue engineering.     

Eps8 protein facilitates phagocytosis by increasing TLR4-MyD88 protein interaction in lipopolysaccharide-stimulated macrophages

Toll-like receptors (TLRs) are crucial in macrophage phagocytosis, which is pivotal in host innate immune response. However, the detailed mechanism is not fully defined. Here, we demonstrated that the induction of Src and Eps8 in LPS-treated macrophages was TLR4- and MyD88-dependent, and their attenuation reduced LPS-promoted phagocytosis. Confocal microscopy indicated the colocalization of Eps8 and TLR4 in the cytosol and at the phagosome. Consistently, both Eps8 and TLR4 were present in the same immunocomplex regardless of LPS stimulation. Inhibition of this complex formation by eps8 siRNA or overexpression of pleckstrin homology domain-truncated Eps8 (i.e. 261-p97(Eps8)) decreased LPS-induced TLR4-MyD88 interaction and the following activation of Src, focal adhesion kinase, and p38 MAPK. Importantly, attenuation of Eps8 impaired the bacterium-killing ability of macrophages. Thus, Eps8 is a key regulator of the LPS-stimulated TLR4-MyD88 interaction and contributes to macrophage phagocytosis.     

Autocrine stimulation by insulin-like growth factor I is involved in the growth, tumorigenicity and chemoresistance of human esophageal carcinoma cells

Insulin-like growth factor I (IGF-I) receptor (IGF-IR)-mediated signals are known to be involved in cell growth and transformation and prevention of apoptosis. In this study, we demonstrated the coexpression of IGF-I and IGF-IR in human esophageal carcinoma tissues. We also demonstrated the IGF-I autocrine system in esophageal carcinoma cell lines. Both the CE48T/VGH and CE81T/VGH cell lines showed proliferative responses to IGF-I stimulation. Autokinase activity of IGF-IR in these cells can be triggered by the exogenous addition of IGF-I. In addition, an IGF-I peptide antagonist, JB1, specifically inhibited ligand-induced receptor autophosphorylation in a dose-dependent manner. Under serum-free conditions, JB1 also reduced the degree of IGF-IR phosphorylation and cell numbers. Furthermore, the addition of JB1 decreased the number of CE81T/VGH colonies formed in methyl cellulose agar and the size and the incidence of tumors which grew in mice with severe combined immunodeficiency. These results imply that an IGF-I autocrine system in human esophageal carcinoma cells could stimulate tumor growth. Finally, we found that IGF-I prevented the apoptosis of CE81T/VGH cells induced by chemotherapeutic drugs, such as cisplatin, 5-fluorouracil and camptothecin. Thus, interruption of IGF-IR function may provide a way to retard tumor growth and increase the sensitivity of esophageal carcinoma to chemotherapy.     

Non-peptide alpha(v)beta(3) antagonists. Part 5: identification of potent RGD mimetics incorporating 2-aryl beta-amino acids as aspartic acid replacements

A series of novel, highly potent alpha(v)beta(3) receptor antagonists with favorable pharmacokinetic profiles has been identified. In this series of antagonists, 2-aryl beta-amino acids function as potent aspartic acid replacements.     

Global transcriptional response of pig brain and lung to natural infection by Pseudorabies virus

Background  

Pseudorabies virus (PRV) is an alphaherpesviruses whose native host is pig. PRV infection mainly causes signs of central nervous system disorder in young pigs, and respiratory system diseases in the adult.     

MPDA: Microarray pooled DNA analyzer

Background  

Microarray-based pooled DNA experiments that combine the merits of DNA pooling and gene chip technology constitute a pivotal advance in biotechnology. This new technique uses pooled DNA, thereby reducing costs associated with the typing of DNA from numerous individuals. Moreover, use of an oligonucleotide gene chip reduces costs related to processing various DNA segments (e.g., primers, reagents). Thus, the technique provides an overall cost-effective solution for large-scale genomic/genetic research. However, few publicly shared tools are available to systematically analyze the rapidly accumulating volume of whole-genome pooled DNA data.     

Resilience of the iron environment in heme proteins

Conformational flexibility is essential to the functional behavior of proteins. We use an effective force constant introduced by Zaccai, the resilience, to quantify this flexibility. Site-selective experimental and computational methods allow us to determine the resilience of heme protein active sites. The vibrational density of states of the heme Fe determined using nuclear resonance vibrational spectroscopy provides a direct experimental measure of the resilience of the Fe environment, which we compare quantitatively with values derived from the temperature dependence of atomic mean-squared displacements in molecular dynamics simulations. Vibrational normal modes in the THz frequency range dominate the resilience. Both experimental and computational methods find a higher resilience for cytochrome c than for myoglobin, which we attribute to the increased number of covalent links to the peptide in the former protein. For myoglobin, the resilience of the iron environment is larger than the average resilience previously determined for hydrogen sites using neutron scattering. Experimental results suggest a slightly reduced resilience for cytochrome c upon oxidation, although the change is smaller than reported in previous Mössbauer investigations on a bacterial cytochrome c, and is not reproduced by the simulations. Oxidation state also has no significant influence on the compressibility calculated for cyt c, although a slightly larger compressibility is predicted for myoglobin.     

Non-peptide alphavbeta3 antagonists. Part 6: design and synthesis of alphavbeta3 antagonists containing a pyridone or pyrazinone central scaffold

Two novel series of small-molecule RGD mimetics containing either a substituted pyridone or pyrazinone central constraint were prepared. Modification of the beta-alanine 3-substituent produced compounds that are potent and selective alpha(v)beta(3) antagonists and exhibit a range of physicochemical properties.     

Inter- and intraclade neutralization of human immunodeficiency virus type 1: genetic clades do not correspond to neutralization serotypes but partially correspond to gp120 antigenic serotypes.

We have studied genetic variation among clades A through E of human immunodeficiency virus type 1 (HIV-1) at the levels of antibody binding to gp120 molecules and virus neutralization. We are unable to identify neutralization serotypes that correspond to the genetic clades. Instead, we observe that inter- and intraclade neutralization of primary isolates by HIV-1-positive sera is generally weak and sporadic; some sera show a reasonable degree of neutralization breadth and potency whereas others are relatively sensitive to neutralization, but no consistent pattern was found. However, a few sera were able to neutralize across clades with significant potency, an observation which may have implications for the feasibility of a broadly effective HIV-1 vaccine involving humoral immunity. Serological assays measuring anti-gp120 antibody binding also failed to identify serotypes that correspond precisely to the genetic clades, but some indications of clade-specific binding were observed, notably with sera from clades B and E. A representative protein for each clade (A through E) was selected on the basis of its specificity, defined as high seroreactivity with sera from individuals infected with virus of that clade and lower reactivity with sera from individuals infected with viruses from other clades. The seroreactivity patterns against these five proteins could be used to predict the genotype of the infecting virus with moderate success.