Construction of hybrid photosynthetic units using peripheral and core antennae from two different species of photosynthetic bacteria: detection of the energy transfer from bacteriochlorophyll a in LH2 to bacteriochlorophyll b in LH1

Typical purple bacterial photosynthetic units consist of supra-molecular arrays of peripheral (LH2) and core (LH1-RC) antenna complexes. Recent atomic force microscopy pictures of photosynthetic units in intact membranes have revealed that the architecture of these units is variable (Scheuring et al. (2005) Biochim Bhiophys Acta 1712:109–127). In this study, we describe methods for the construction of heterologous photosynthetic units in lipid-bilayers from mixtures of purified LH2 (from Rhodopseudomonas acidophila) and LH1-RC (from Rhodopseudomonas viridis) core complexes. The architecture of these reconstituted photosynthetic units can be varied by controlling ratio of added LH2 to core complexes. The arrangement of the complexes was visualized by electron-microscopy in combination with Fourier analysis. The regular trigonal array of the core complexes seen in the native photosynthetic membrane could be regenerated in the reconstituted membranes by temperature cycling. In the presence of added LH2 complexes, this trigonal symmetry was replaced with orthorhombic symmetry. The small lattice lengths for the latter suggest that the constituent unit of the orthorhombic lattice is the LH2. Fluorescence and fluorescence-excitation spectroscopy was applied to the set of the reconstituted membranes prepared with various proportions of LH2 to core complexes. Remarkably, even though the LH2 complexes contain bacteriochlorophyll a, and the core complexes contain bacteriochlorophyll b, it was possible to demonstrate energy transfer from LH2 to the core complexes. These experiments provide a first step along the path toward investigating how changing the architecture of purple bacterial photosynthetic units affects the overall efficiency of light-harvesting.     

Intra-strain biological and epidemiological characterization of plum pox virus

Plum pox virus (PPV) is one of the most important plant viruses causing serious economic losses. Thus far, strain typing based on the definition of 10 monophyletic strains with partially differentiable biological properties has been the sole approach used for epidemiological characterization of PPV. However, elucidating the genetic determinants underlying intra-strain biological variation among populations or isolates remains a relevant but unexamined aspect of the epidemiology of the virus. In this study, based on complete nucleotide sequence information of 210 Japanese and 47 non-Japanese isolates of the PPV-Dideron (D) strain, we identified five positively selected sites in the PPV-D genome. Among them, molecular studies showed that amino acid substitutions at position 2,635 in viral replicase correlate with viral titre and competitiveness at the systemic level, suggesting that amino acid position 2,635 is involved in aphid transmission efficiency and symptom severity. Estimation of ancestral genome sequences indicated that substitutions at amino acid position 2,635 were reversible and peculiar to one of two genetically distinct PPV-D populations in Japan. The reversible amino acid evolution probably contributes to the dissemination of the virus population. This study provides the first genomic insight into the evolutionary epidemiology of PPV based on intra-strain biological variation ascribed to positive selection.     

Secreted Nucleobindin-2 Inhibits 3T3-L1 Adipocyte Differentiation

Nucleobindin-2 is a 420 amino acid EF-hand Ca2+ binding protein that can be further processed to generate an 82 amino terminal peptide termed Nesfatin-1. To examine the function of secreted Nucleobindin-2 in adipocyte differentiation, cultured 3T3-L1 cells were incubated with either 0 or 100 nM of GST, GST-Nucleobindin-2, prior to and during the initiation of adipocyte differentiation. Nucleobindin-2 treatment decreased neutral lipid accumulation (Oil-Red O staining) and expression of several marker genes for adipocyte differentiation (PPARγ, aP2, and adipsin). When Nucleobindin- 2 was constitutively secreted into cultured medium, cAMP content and insulin stimulated CREB phosphorylation were significantly reduced. On the other hand, intracellularly overexpressed Nucleobindin-2 failed to affect cAMP content and CREB phosphorylation. Taken together, these data indicate that secreted Nucleobindin-2 is a suppressor of adipocyte differentiation through inhibition of cAMP production and insulin signal.     

Stimulation of unprimed macrophages with immune complexes triggers a low output of nitric oxide by calcium-dependent neuronal nitric-oxide synthase

Immune complexes composed of IgG-opsonized pathogens, particles, or proteins are phagocytosed by macrophages through Fcγ receptors (FcγRs). Macrophages primed with IFNγ or other pro-inflammatory mediators respond to FcγR engagement by secreting high levels of cytokines and nitric oxide (NO). We found that unprimed macrophages produced lower levels of NO, which required efficient calcium (Ca(2+)) flux as demonstrated by using macrophages lacking selenoprotein K, which is required for FcγR-induced Ca(2+) flux. Thus, we further investigated the signaling pathways involved in low output NO and its functional significance. Evaluation of inducible, endothelial, and neuronal nitric-oxide synthases (iNOS, eNOS, and nNOS) revealed that FcγR stimulation in unprimed macrophages caused a marked Ca(2+)-dependent increase in both total and phosphorylated nNOS and slightly elevated levels of phosphorylated eNOS. Also activated were three MAP kinases, ERK, JNK, and p38, of which ERK activation was highly dependent on Ca(2+) flux. Inhibition of ERK reduced both nNOS activation and NO secretion. Finally, Transwell experiments showed that FcγR-induced NO functioned to increase the phagocytic capacity of other macrophages and required both NOS and ERK activity. The production of NO by macrophages is conventionally attributed to iNOS, but we have revealed an iNOS-independent receptor/enzyme system in unprimed macrophages that produces low output NO. Under these conditions, FcγR engagement relies on Ca(2+)-dependent ERK phosphorylation, which in turn increases nNOS and, to a lesser extent, eNOS, both of which produce low levels of NO that function to promote phagocytosis.     

Gene expression profile analysis of the mouse liver during bacteria-induced fulminant hepatitis by a cDNA microarray system

Fulminant hepatic failure (FHF) is a disease characterized by sudden and severe impairment of liver function. To elucidate the mechanism involved in FHF, we adopted a murine model of FHF by administrating mice with heat-killed Propionibacterium acnes (P. acnes), followed by a low dose of lipopolysaccharide (LPS), and analyzed the dynamic change of gene expression profile of the murine liver using an in-house cDNA microarray system which contained most of the cDNAs encoding chemokines/cytokines and their receptors (33 chemokines/21 chemokine receptors, 28 cytokines/35 cytokine receptors) as well as 230 liver related proteins mostly selected by serial analysis of gene expression (SAGE). Among them, 335 genes were found to differ by more than 2-fold in at least one time point comparing with normal liver. Hierarchical cluster analysis revealed that except for a few genes, such as heme oxygenase (HO)-1 and nicotinamide N-methyltransferase (NNMT) of which expression increased, the expression of most of the genes encoding drug metabolizing enzymes decreased with the progress of the disease. The expression of the genes encoding chemokines/cytokines was dramatically changed, such as Mig, IP-10, RANTES, TNF-alpha, and IFN-gamma. In addition, the expression of those that were not previously linked to this murine model was also identified to be changed. These include endogenous IL-18 binding protein (IL-18BP), CXCL16 (the ligand of Bonzo, CXCR6) as well as ESTs. Taken together this study has shown the systemic and comprehensive gene expression profile during FHF and may contribute to better understanding of the mechanism of FHF.     

Characterization of Ca2+ channels involved in ET-1-induced transactivation of EGF receptors

The purpose of this study was to demonstrate the involvement of Ca(2+) influx through voltage-independent Ca(2+) channels (VICCs) in endothelin-1 (ET-1)-induced transactivation of epidermal growth factor receptor protein tyrosine kinase (EGFR PTK) using the Ca(2+) channel blockers LOE-908 and SK&F-96365 in rabbit internal carotid artery vascular smooth muscle cells. ET-1-induced EGFR PTK transactivation was completely inhibited by AG-1478, which is a specific inhibitor of EGFR PTK. In the absence of extracellular Ca(2+), the magnitude of EGFR PTK transactivation was near the basal level. Based on sensitivity to nifedipine, which is a specific blocker of voltage-operated Ca(2+) channels (VOCCs), VOCCs have minor roles in EGFR PTK transactivation. In contrast, Ca(2+) influx through VICCs plays an important role in EGFR PTK transactivation. Moreover, based on the sensitivity of VICCs to SK&F-96365 and LOE-908, VICCs were shown to consist of two types of Ca(2+)-permeable nonselective cation channels (NSCCs), which are designated NSCC-1 and NSCC-2, and a store-operated Ca(2+) channel. In summary, Ca(2+) influx through VICCs plays an essential role in ET-1-induced EGFR PTK transactivation in rabbit internal carotid artery vascular smooth muscle cells.     

Fabrication of Biomimetic Bone Tissue Using Mesenchymal Stem Cell-Derived Three-Dimensional Constructs Incorporating Endothelial Cells

The development of technologies to promote vascularization of engineered tissue would drive major developments in tissue engineering and regenerative medicine. Recently, we succeeded in fabricating three-dimensional (3D) cell constructs composed of mesenchymal stem cells (MSCs). However, the majority of cells within the constructs underwent necrosis due to a lack of nutrients and oxygen. We hypothesized that incorporation of vascular endothelial cells would improve the cell survival rate and aid in the fabrication of biomimetic bone tissues in vitro. The purpose of this study was to assess the impact of endothelial cells combined with the MSC constructs (MSC/HUVEC constructs) during short- and long-term culture. When human umbilical vein endothelial cells (HUVECs) were incorporated into the cell constructs, cell viability and growth factor production were increased after 7 days. Furthermore, HUVECs were observed to proliferate and self-organize into reticulate porous structures by interacting with the MSCs. After long-term culture, MSC/HUVEC constructs formed abundant mineralized matrices compared with those composed of MSCs alone. Transmission electron microscopy and qualitative analysis revealed that the mineralized matrices comprised porous cancellous bone-like tissues. These results demonstrate that highly biomimetic bone tissue can be fabricated in vitro by 3D MSC constructs incorporated with HUVECs.     

Amyloid beta protein (25-35) phosphorylates MARCKS through tyrosine kinase-activated protein kinase C signaling pathway in microglia

Myristoylated alanine-rich C kinase substrate (MARCKS) is a widely distributed specific protein kinase C (PKC) substrate and has been implicated in membrane trafficking, cell motility, secretion, cell cycle, and transformation. We found that amyloid beta protein (A beta) (25-35) and A beta (1-40) phosphorylate MARCKS in primary cultured rat microglia. Treatment of microglia with A beta (25-35) at 10 nM or 12-O-tetradecanoylphorbol 13-acetate (1.6 nM) led to phosphorylation of MARCKS, an event inhibited by PKC inhibitors, staurosporine, calphostin C, and chelerythrine. The A beta (25-35)-induced phosphorylation of MARCKS was inhibited by pretreatment with the tyrosine kinase inhibitors genistein and herbimycin A, but not with pertussis toxin. PKC isoforms alpha, delta, and epsilon were identified in microglia by immunocytochemistry and western blots using isoform-specific antibodies. PKC-delta was tyrosine-phosphorylated by the treatment of microglia for 10 min with A beta (25-35) at 10 nM. Other PKC isoforms alpha and epsilon were tyrosine-phosphorylated by A beta (25-35), but only to a small extent. We propose that a tyrosine kinase-activated PKC pathway is involved in the A beta (25-35)-induced phosphorylation of MARCKS in rat microglia.     

Spontaneous Release of γ-Aminobutyric Acid Formed from Putrescine and Its Enhanced Ca2+-Dependent Release by High K+ Stimulation in the Brains of Freely Moving Rats

The spontaneous release of [3H] gamma-aminobutyric acid ([3H]GABA) in various areas of rat brain injected with [3H]putrescine was examined using a push-pull perfusion technique. The release in a 25-min perfusate was highest in the caudate-putamen. The effect of high K+ stimulation on the release of [3H]GABA formed from [3H]putrescine was examined in the caudate-putamen. The release was enhanced by high K+ solution in a Ca2+-dependent manner.