The ability of Vibrio vulnificus to use a synthetic hydrophilic heme compound, Fe-TPPS, as a single iron source

Vibrio vulnificus, an opportunistic human pathogen, can obtain iron from a variety of heme proteins. This process involves the digestion of heme proteins by an exoprotease to liberate protoheme (iron-protoporphyrin IX). In the present study, we tested whether this pathogen also uses a synthetic heme compound, Fe-alpha,beta,gamma,delta-tetraphenylporphine tetrasulfonic acid (Fe-TPPS), as an iron source. When inoculated into a medium containing Fe-TPPS, V. vulnificus L-180 multiplication was seen to be dependent on the concentration of the synthetic heme compound; a mutant lacking the ability to utilize protoheme did not multiply. Cells of the strain grown under the iron-restricted condition showed time-dependent uptake of Fe-TPPS. The ability to use either protoheme or Fe-TPPS was significantly reduced by the addition of an excess amount of free TPPS or Cu-TPPS. The data suggest that, V. vulnificus may assimilate Fe-TPPS, at least partially, through the same system as that for protoheme.     

Software for precise tracking of cell proliferation

We have developed a multi-target cell tracking program TADOR, which we applied to a series of fluorescence images. TADOR is based on an active contour model that is modified in order to be free of the problem of locally optimal solutions, and thus is resistant to signal fluctuation and morphological changes. Due to adoption of backward tracing and addition of user-interactive correction functions, TADOR is used in an off-line and semi-automated mode, but enables precise tracking of cell division. By applying TADOR to the analysis of cultured cells whose nuclei had been fluorescently labeled, we tracked cell division and cell-cycle progression on coverslips over an extended period of time.     

Targeted disruption of exogenous EGFP gene in medaka using zinc-finger nucleases

Zinc-finger nucleases (ZFNs) are artificial enzymes that create site-specific double-strand breaks and thereby induce targeted genome editing. Here, we demonstrated successful gene disruption in somatic and germ cells of medaka (Oryzias latipes) using ZFN to target exogenous EGFP genes. Embryos that were injected with an RNA sequence pair coding for ZFNs showed mosaic loss of green fluorescent protein fluorescence in skeletal muscle. A number of mutations that included both deletions and insertions were identified within the ZFN target site in each embryo, whereas no mutations were found at the non-targeted sites. In addition, ZFN-induced mutations were introduced in germ cells and efficiently transmitted to the next generation. The mutation frequency varied (6-100%) in the germ cells from each founder, and a founder carried more than two types of mutation in germ cells. Our results have introduced the possibility of targeted gene disruption and reverse genetics in medaka.     

IgM-type GM-CSF autoantibody is etiologically a bystander but associated with IgG-type autoantibody production in autoimmune pulmonary alveolar proteinosis

The granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibody (GMAb) is the causative agent underlying autoimmune pulmonary alveolar proteinosis (aPAP). It consists primarily of the IgG isotype. At present, information on other isotypes of the autoantibody is limited. We detected serum the IgM isotype of GMAb (IgM-GMAb) in more than 80% of patients with aPAP and 22% of healthy subjects, suggesting that a continuous antigen pressure may be present in most patients. Levels of the IgM isotype were weakly correlated with IgG-GMAb levels but not IgA-GMAb, suggesting that its production may be associated with that of IgG-GMAb. The mean binding avidity to GM-CSF of the IgM isotype was 100-fold lower than the IgG-GMAb isotype, whereas the IC(50) value for neutralizing capacity was 20,000-fold higher than that of IgG-GMAb, indicating that IgM-GMAb is only a very weak neutralizer of GM-CSF. In bronchoalveolar lavage fluid from nine patients, IgG-GMAb was consistently detected, but IgM-GMAb was under the detection limit in most patients, confirming that IgM-GMAb is functionally a bystander in the pathogenesis of aPAP. It rather may be involved in the mechanism for development of IgG-GMAb in vivo.     

Low-dose lipopolysaccharide pretreatment suppresses choroidal neovascularization via IL-10 induction

Recent studies have suggested that some kinds of microbial infection may have a crucial role in the development of many diseases such as autoimmune diseases and certain types of cancer. It has been reported that some chronic infections, such as Chlamydia pneumoniae, and immunological dysfunctions are associated with age-related macular degeneration (AMD), a leading cause of blindness. To evaluate the association between systemic low-level inflammation induced by infection and AMD pathogenesis, we investigated whether intraperitoneal injection of lipopolysaccharide (LPS) can modulate the development of laser-induced choroidal neovascularization (CNV), a key feature of AMD. Contrary to our expectations, the sizes of CNV in mice with LPS pretreatment were approximately 65% smaller than those of the control mice. After LPS pretreatment, serum IL-10 concentration and IL-10 gene expression in peritoneal macrophages and in the posterior part of the eye increased. Peritoneal injection of anti-IL10 antibody reduced CNV suppression by LPS pretreatment. Moreover, adoptive transfer of the resident peritoneal macrophages from LPS-treated mice into control littermates resulted in an approximately 26% reduction in the size of CNV compared with PBS-treated mice. We concluded that CNV formation was suppressed by low-dose LPS pretreatment via IL-10 production by macrophages.     

IL-17 is a critical component of vaccine-induced protection against lung infection by lipopolysaccharide-heterologous strains of Pseudomonas aeruginosa

In a murine model of acute fatal pneumonia, we previously showed that nasal immunization with a live-attenuated aroA deletant of Pseudomonas aeruginosa strain PAO1 elicited LPS serogroup-specific protection, indicating that opsonic Ab to the LPS O Ag was the most important immune effector. Because P. aeruginosa strain PA14 possesses additional virulence factors, we hypothesized that a live-attenuated vaccine based on PA14 might elicit a broader array of immune effectors. Thus, an aroA deletant of PA14, denoted PA14DeltaaroA, was constructed. PA14DeltaaroA-immunized mice were protected against lethal pneumonia caused not only by the parental strain but also by cytotoxic variants of the O Ag-heterologous P. aeruginosa strains PAO1 and PAO6a,d. Remarkably, serum from PA14DeltaaroA-immunized mice had very low levels of opsonic activity against strain PAO1 and could not passively transfer protection, suggesting that an antibody-independent mechanism was needed for the observed cross-serogroup protection. Compared with control mice, PA14DeltaaroA-immunized mice had more rapid recruitment of neutrophils to the airways early after challenge. T cells isolated from P. aeruginosa DeltaaroA-immunized mice proliferated and produced IL-17 in high quantities after coculture with gentamicin-killed P. aeruginosa. Six hours following challenge, PA14DeltaaroA-immunized mice had significantly higher levels of IL-17 in bronchoalveolar lavage fluid compared with unimmunized, Escherichia coli-immunized, or PAO1DeltaaroA-immunized mice. Antibody-mediated depletion of IL-17 before challenge or absence of the IL-17 receptor abrogated the PA14DeltaaroA vaccine's protection against lethal pneumonia. These data show that IL-17 plays a critical role in antibody-independent vaccine-induced protection against LPS-heterologous strains of P. aeruginosa in the lung.     

Intramembrane processing by signal peptide peptidase regulates the membrane localization of hepatitis C virus core protein and viral propagation

Hepatitis C virus (HCV) core protein has shown to be localized in the detergent-resistant membrane (DRM), which is distinct from the classical raft fraction including caveolin, although the biological significance of the DRM localization of the core protein has not been determined. The HCV core protein is cleaved off from a precursor polyprotein at the lumen side of Ala(191) by signal peptidase and is then further processed by signal peptide peptidase (SPP) within the transmembrane region. In this study, we examined the role of SPP in the localization of the HCV core protein in the DRM and in viral propagation. The C terminus of the HCV core protein cleaved by SPP in 293T cells was identified as Phe(177) by mass spectrometry. Mutations introduced into two residues (Ile(176) and Phe(177)) upstream of the cleavage site of the core protein abrogated processing by SPP and localization in the DRM fraction. Expression of a dominant-negative SPP or treatment with an SPP inhibitor, L685,458, resulted in reductions in the levels of processed core protein localized in the DRM fraction. The production of HCV RNA in cells persistently infected with strain JFH-1 was impaired by treatment with the SPP inhibitor. Furthermore, mutant JFH-1 viruses bearing SPP-resistant mutations in the core protein failed to propagate in a permissive cell line. These results suggest that intramembrane processing of HCV core protein by SPP is required for the localization of the HCV core protein in the DRM and for viral propagation.     

Possible involvement of CD81 in acrosome reaction of sperm in mice

Tetraspanin CD81 is closely homologous in amino acid sequence with CD9. CD9 is well known to be involved in sperm-egg fusion, and CD81 has also been reported to be involved in membrane fusion events. However, the function of CD81 as well as that of CD9 in membrane fusion remains unclear. Here, we report that disruption of the mouse CD81 gene led to a reduction in the fecundity of female mice, and CD81-/- eggs had impaired ability to fuse with sperm. Furthermore, we demonstrated that when CD81-/- eggs were incubated with sperm, some of the sperm that penetrated into the perivitelline space of CD81-/- eggs had not yet undergone the acrosome reaction, indicating that the impaired fusibility of CD81-/- eggs may be in part caused by failure of the acrosome reaction of sperm. In addition, we showed that CD81 was highly expressed in granulosa cells, somatic cells that surround oocytes. Our observations suggest that there is an interaction between sperm and CD81 on somatic cells surrounding eggs before the direct interaction of sperm and eggs. Our results may provide new clues for clarifying the cellular mechanism of the acrosome reaction, which is required for sperm-egg fusion.     

Multiple roles of phosphoinositide-specific phospholipase C isozymes

Phosphoinositide-specific phospholipase C is an effector molecule in the signal transduction process. It generates two second messengers, inositol-1,4,5-trisphosphate and diacylglycerol from phosphatidylinositol 4,5-bisphosphate. Currently, thirteen mammal PLC isozymes have been identified, and they are divided into six groups: PLC-beta, -gamma, -delta, -epsilon, -zeta and -eta. Sequence analysis studies demonstrated that each isozyme has more than one alternative splicing variant. PLC isozymes contain the X and Y domains that are responsible for catalytic activity. Several other domains including the PH domain, the C2 domain and EF hand motifs are involved in various biological functions of PLC isozymes as signaling proteins. The distribution of PLC isozymes is tissue and organ specific. Recent studies on isolated cells and knockout mice depleted of PLC isozymes have revealed their distinct phenotypes. Given the specificity in distribution and cellular localization, it is clear that each PLC isozyme bears a unique function in the modulation of physiological responses. In this review, we discuss the structural organization, enzymatic properties and molecular diversity of PLC splicing variants and study functional and physiological roles of each isozyme.