The first spindle formation in brown algal zygotes

Regulation of the first spindle formation in brown algal zygotes was described. It is well known that there are three types of sexual reproduction in brown algae; isogamy, anisogamy and oogamy. Paternal inheritance of centrioles can be observed in all these cases, similar to animal fertilization. In isogamy and anisogamy, female centrioles (= flagellar basal bodies) selectively disappear and male centrioles remain after fertilization. In a typical oogamy (e.g. fucoid members), liberated egg does not have centrioles, and sperm centrioles are introduced in zygote. Participation of sperm centrioles to the spindle formation in zygotes was also described using Fucus distichus as a model system. Sperm centrioles function as a part of centrosome, namely microtubule organizing center, in zygote. Therefore, they have a crucial role in the spindle formation. Observations on the spindle formation in polygyny and karyogamy-blocked zygotes strongly suggest that egg nucleus can form a mitotic spindle by itself without centrosome, even though the resulting spindles are of abnormal shapes. %     

Amyloid β protein activates PKC-δ and induces translocation of myristoylated alanine-rich C kinase substrate (MARCKS) in microglia

The increased accumulation of activated microglia containing amyloid β protein (Aβ) around senile plaques is a common pathological feature in subjects with Alzheimer's disease (AD). Much less is known, however, of intracellular signal transduction pathways for microglial activation in response to Aβ. We investigated intracellular signaling in response to Aβ stimulation in primary cultured rat microglia. We found that the kinase activity of PKC-δ but not that of PKC- or - is increased by stimulation of microglia with Aβ, with a striking tyrosine phosphorylation of PKC-δ. In microglia stimulated with Aβ, tyrosine phosphorylation of PKC-δ was evident at the membrane fraction without an overt translocation of PKC-δ. PKC-δ co-immunoprecipitated with MARCKS from microglia stimulated with Aβ. Aβ induced translocation of MARCKS from the membrane fraction to the cytosolic fraction. Immunocytochemical analysis revealed that phosphorylated MARCKS accumulated in the cytoplasm, particularly at the perinuclear region in microglia treated with Aβ. Taken together with our previous observations that Aβ-induced phosphorylation of MARCKS and chemotaxis of microglia are inhibited by either tyrosine kinase or PKC inhibitors, our results provide evidence that Aβ induces phosphorylation and translocation of MARCKS through the tyrosine kinase-PKC-δ signaling pathway in microglia.     

Distribution of lymphatic vessels in mouse thymus: immunofluorescence analysis

Thymic blood and lymphatic vessels in humans and laboratory animals have been investigated in morphological studies. However, occasionally a clear distinction between blood vessels and lymphatic vessels cannot be made from morphological characteristics of the vasculature. To visualize thymic lymphatics in normal adult BALB/c mice, we used antibodies against specific markers of lymphatic endothelial cells. Expression of vascular endothelial growth factor receptor–3 (VEGFR–3) was detected throughout the thymus, i.e., the capsule, cortex, and medulla. Most thymic lymphatics were present in capillaries of ~20 μm in caliber. The plexuses of lymphatic capillaries were occasionally detectable. Lymphatic vessels were frequently adjacent to CD31–positive blood vessels, and some lymphatic vessels were seen in the immediate vicinity of or within the perivascular spaces around postcapillary venules. The identity of VEGFR–3–positive vessels as lymphatics was further confirmed by staining with additional markers: LYVE–1, Prox–1, neuropilin–2, and secondary lymphoid tissue chemokine (SLC). The distributions of LYVE–1 were similar to those of VEGFR–3. Most lymphatic vessels were also identified by Prox–1. Neuropilin–2 was restricted to lymphatic vessels in the thymus. The most abundant expression of SLC in the thymus was in medullar epithelial cells; SLC was also expressed in lymphatic vessels and blood vessels. Thus, lymphatic endothelium in mouse thymus was characterized by positive staining with antibodies to VEGFR–3, LYVE–1, Prox–1, neuropilin–2, or SLC, but not with an antibody to CD31. Our results suggest the presence of lymphatic capillary networks throughout the thymus.     

Acquisition of Complement Resistance through Incorporation of CD55/Decay-Accelerating Factor into Viral Particles Bearing Baculovirus GP64

A major obstacle to gene transduction by viral vectors is inactivation by human complement in vivo. One way to overcome this is to incorporate complement regulatory proteins, such as CD55/decay accelerating factor (DAF), into viral particles. Lentivirus vectors pseudotyped with the baculovirus envelope protein GP64 have been shown to acquire more potent resistance to serum inactivation and longer transgene expression than those pseudotyped with the vesicular stomatitis virus (VSV) envelope protein G. However, the molecular mechanisms underlying resistance to serum inactivation in pseudotype particles bearing the GP64 have not been precisely elucidated. In this study, we generated pseudotype and recombinant VSVs bearing the GP64. Recombinant VSVs generated in human cell lines exhibited the incorporation of human DAF in viral particles and were resistant to serum inactivation, whereas those generated in insect cells exhibited no incorporation of human DAF and were sensitive to complement inactivation. The GP64 and human DAF were detected on the detergent-resistant membrane and were coprecipitated by immunoprecipitation analysis. A pseudotype VSV bearing GP64 produced in human DAF knockdown cells reduced resistance to serum inactivation. In contrast, recombinant baculoviruses generated in insect cells expressing human DAF or carrying the human DAF gene exhibited resistance to complement inactivation. These results suggest that the incorporation of human DAF into viral particles by interacting with baculovirus GP64 is involved in the acquisition of resistance to serum inactivation.Gene therapy is a potential treatment option for genetic diseases, malignant diseases, and other acquired diseases. To this end, safe and efficient gene transfer into specific target cells is a central requirement, and a variety of nonviral and viral vector systems have been developed (6, 44). Recombinant viruses can be used for efficient gene transfer. Retroviruses, adeno-associated viruses, and lentiviruses are able to integrate foreign genes into host genomes and are suitable for gene therapeutics by virtue of their permanent expression of the therapeutic genes, whereas adenoviruses, herpesviruses, and baculoviruses can transiently express foreign genes (6, 12, 44). Pseudotype particles bearing other viral envelope proteins have been developed to improve transduction efficiency and the safety of viral vectors, including retrovirus (4, 7), lentivirus (25), vesicular stomatitis virus (VSV) (29), and baculovirus (17, 42). Pseudotype retroviruses and lentiviruses bearing the baculovirus envelope protein GP64 of Autographa californica nucleopolyhedrosis virus (AcNPV) have been shown to exhibit efficient gene transduction into a wide variety of cells with a lower cytotoxicity compared to those bearing the VSV envelope protein G (VSVG), which is commonly used for pseudotyping (18, 32, 35, 36).However, a drawback of gene transduction by viral vectors is that human sera inactivate the vectors (11, 40). Complement is a major element of the innate immune response and serves to link innate and adaptive immunity (8). Complement activation can occur via classical, lectin, and alternative pathways (2, 8). All pathways invoke several responses, such as virus opsonization, virolysis, anaphylatoxin, and chemotaxin production, as well as others (2, 8). VSV and baculovirus are inactivated by human sera via the classical pathway (1, 11). Because complement activation also induces potential damage to host cells, the complement system is tightly regulated by the complement regulatory proteins (CRPs), including CD55/decay-accelerating factor (DAF), CD46/membrane cofactor protein (MCP), and CD59 (2, 8, 15). DAF and CD46 inhibit activation of C3/C5-converting enzymes, which regulate the activation of classical and alternative pathways, whereas CD59 regulates the assembly of the membrane attack complex (2, 8, 15).Viral vectors can be manipulated to confer resistance to the complement inactivation. Human immunodeficiency virus (HIV) is known to develop resistance to human complement through the incorporation of DAF, CD46, and CD59 to the viral particles (22, 30, 31, 38). Moloney murine leukemia virus vectors produced in HT1080 cells are resistant to complement inactivation (5). Baculovirus and lentivirus vectors bearing DAF or the fusion protein between the functional domains of human DAF and the GP64 were resistant to complement inactivation (9, 13). It has been shown that lentivirus vectors pseudotyped with the GP64 are more resistant to inactivation in the sera of mice and rats (14, 32) and are capable of executing longer expression of the transgenes in nasal epithelia compared to those pseudotyped with the VSVG (35, 36). However, the precise mechanisms underlying the resistance to complement inactivation by pseudotyping of the GP64 is not known.To clarify the molecular mechanisms underlying the resistance of the viral vectors pseudotyped with the GP64 to the complement inactivation, we produced pseudotype and recombinant VSVs bearing the GP64. The recombinant VSVs carrying the gp64 gene generated in human cells but not in insect cells exhibited incorporation of human DAF on the viral particles and were resistant to the complement inactivation. Furthermore, production of the gp64 pseudotype VSV in the DAF knockdown human cells impaired serum resistance, whereas production of the gp64 recombinant VSV in the CHO cell lines stably expressing human DAF and the recombinant baculoviruses in the insect cells stably expressing human DAF or encoding the DAF gene in the genome conferred resistance to the complement inactivation. These results suggest that DAF incorporation into viral particles bearing baculovirus GP64 confers resistance to serum inactivation.     

Decreased expression of Apaf-1 with progression of melanoma

Defects in apoptotic system may contribute in the pathogenesis and resistance of malignant melanoma cells to chemotherapy. Apoptotic protease-activating factor-1 (Apaf-1) is a cell death effector that acts with cytochrome c and caspase-9 to mediate apoptosis. Recently it was shown that metastatic melanomas often lose Apaf-1 and are concomitantly resistant to apoptosis. It is not known, however, whether Apaf-1 protein is lost during melanoma progression from localized to metastatic tumor. To this end, we evaluated Apaf-1 protein expression by immunohistochemistry in 10 cases of human nevi, 11 melanomas in situ, 26 primary melanomas and 15 metastases. Significant decreases in Apaf-1 expression was observed when comparing nevi and melanomas (chi-square = 33.719; P < 0.0001). Moreover, primary melanomas with greater tumor thickness showed lesser expression of Apaf-1 (chi-square = 16.182; P < 0.003). Intriguingly, we were unable to detect Apaf-1 expression in lesions of metastatic melanomas. These data demonstrated that there is an inverse correlation between Apaf-1 expression and pathologic stage of melanoma. This suggests that the decreased expression of Apaf-1 seen in correlation with melanoma progression renders melanoma more resistant to chemotherapy.     

Na+/Ca2+ exchange inhibition protects the rat heart from ischemia-reperfusion injury by blocking energy-wasting processes

We have recently reported that exposure of rat hearts to high Ca(2+) produces a Ca(2+) overload-induced contractile failure in rat hearts, which was associated with proteolysis of alpha-fodrin. We hypothesized that contractile failure after ischemia-reperfusion (I/R) is similar to that after high Ca(2+) infusion. To test this hypothesis, we investigated left ventricular (LV) mechanical work and energetics in the cross-circulated rat hearts, which were subjected to 15 min global ischemia and 60 min reperfusion. Sixty minutes after I/R, mean systolic pressure-volume area (PVA; a total mechanical energy per beat) at midrange LV volume (mLVV) (PVA(mLVV)) was significantly decreased from 5.89 +/- 1.55 to 3.83 +/- 1.16 mmHg.ml.beat(-1).g(-1) (n = 6). Mean myocardial oxygen consumption per beat (Vo(2)) intercept of (Vo(2)-PVA linear relation was significantly decreased from 0.21 +/- 0.05 to 0.15 +/- 0.03 microl O(2).beat(-1).g(-1) without change in its slope. Initial 30-min reperfusion with a Na(+)/Ca(2+) exchanger (NCX) inhibitor KB-R7943 (KBR; 10 micromol/l) significantly reduced the decrease in mean PVA(mLVV) and Vo(2) intercept (n = 6). Although Vo(2) for the Ca(2+) handling was finally decreased, it transiently but significantly increased from the control for 10-15 min after I/R. This increase in Vo(2) for the Ca(2+) handling was completely blocked by KBR, suggesting an inhibition of reverse-mode NCX by KBR. alpha-Fodrin proteolysis, which was significantly increased after I/R, was also significantly reduced by KBR. Our study shows that the contractile failure after I/R is similar to that after high Ca(2+) infusion, although the contribution of reverse-mode NCX to the contractile failure is different. An inhibition of reverse-mode NCX during initial reperfusion protects the heart against reperfusion injury.     

Effects of DNA polymerase inhibitory and antitumor activities of lipase-hydrolyzed glycolipid fractions from spinach

We succeeded in purifying the major glycolipid fraction in the class of sulfoquinovosyl diacylglycerol, monogalactosyl diacylglycerol and digalactosyl diacylglycerol (DGDG) from a green vegetable, spinach (Spinacia oleracea L.). This glycolipid fraction was an inhibitor of DNA polymerases and a growth inhibitor of NUGC-3 human gastric cancer cells, and, interestingly, the activities were much stronger when the fraction was hydrolyzed by lipase. Glycolipids in the hydrolyzed fraction consisted of sulfoquinovosyl monoacylglycerol (SQMG), monogalactosyl monoacylglycerol (MGMG) and DGDG. In the in vivo antitumor assay using Greene's melanoma, the fraction containing SQMG, MGMG and DGDG showed to be a promising suppressor of solid tumors. Spinach glycolipid fraction might be a potent antitumor compound if directly injected into a tumor-carrying body, and this fraction may be a healthy food material that has antitumor activity.     

Gamma-tocotrienol,a vitamin E homolog,is a natriuretic hormone precursor

2,7,8-Trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman (gamma-CEHC), a metabolite of gamma-tocopherol and gamma-tocotrienol, was identified as a new endogenous natriuretic factor. However, gamma-tocopherol and gamma-tocotrienol, both precursors of gamma-CEHC, have never directly been observed to have natriuretic potency. Thus, we investigated whether gamma-tocotrienol could cause natriuresis and diuresis in rats. The rats were divided into two groups that were given a control or a high-sodium diet for 4 weeks, and then subdivided into placebo and gamma-tocotrienol subgroups given only corn oil-removed vitamin E and oil supplemented with gamma-tocotrienol, respectively. After oral administration of three experimental doses, rat urine was collected and gamma-CEHC, urine volume, sodium, and potassium content were determined. Only in rats given a high-NaCl diet did gamma-tocotrienol accelerate and increase sodium excretion, showing no effect on potassium excretion. Sodium excretion in the high-NaCl group given gamma-tocotrienol was 5.06 +/- 2.70 g/day, and in the control group given gamma-tocotrienol, 0.11 +/- 0.06 g/day. Furthermore, gamma-tocotrienol affected urine volume in the specific condition of high-NaCl body stores and gamma-tocotrienol supplementation. In this study, we found that gamma-tocotrienol, one of the natural vitamin E homologs, stimulates sodium excretion in vivo, suggesting that gamma-tocotrienol possesses a hormone-like natriuretic function.     

Enforced expression of Bcl-2 restores the number of NK cells,but does not rescue the impaired development of NKT cells or intraepithelial lymphocytes,in IL-2/IL-15 receptor beta-chain-deficient mice

IL-2/IL-15Rbeta-deficient mice display impaired development of NK cells, NKT cells, and intraepithelial lymphocytes of the intestine and skin. To determine the role of survival signals mediated by IL-2/IL-15R in the development of these innate lymphocytes, we introduced a bcl-2 transgene into IL-2/IL-15Rbeta-deficient mice. Enforced expression of Bcl-2 restored the number of NK cells in IL-2/IL-15Rbeta-deficient mice, but the rescued NK cells showed no cytotoxic activity. The numbers of NKT cells and intestinal intraepithelial lymphocytes did not increase significantly, and skin intraepithelial lymphocytes remained undetectable in the bcl-2 transgenic IL-2/IL-15Rbeta-deficient mice. These results indicate an essential role of IL-2/IL-15R-mediated survival signals in the development of NK cells, but they also show that additional nonsurvival signals from IL-2/IL-15R are necessary for innate lymphocyte development.     

Immunosuppressant FK506 induces sustained activation of MAP kinase and promotes neurite outgrowth in PC12 mutant cells incapable of differentiating

During the continuous culturing of neural PC12 cells, a drug hypersensitive PC12 mutant cell line (PC12m3) was obtained, which demonstrated high neurite outgrowth when stimulated by various drugs. When the immunosuppressant drug FK506 and nerve growth factor (NGF) were introduced to the PC12m3 cells, the frequency of neurite outgrowth increased approximately 40-fold for NGF alone. However, the effect of FK506 on neuritogenesis in PC12 parental and drug insensitive PC12m1 mutant cells was much lower than in PC12m3 cells. The sustained activation of mitogen-activated protein (MAP) kinase plays an important role in neurite outgrowth of PC12 cells. Interestingly, the drug hypersensitive PC12m3 cells exhibited the sustained activation of MAP kinase with FK506 in comparison to low or no activities in PC12 parental or drug insensitive PC12m1 cells. These results indicate that PC12m3 cells have a novel FK506-induced MAP kinase pathway for neuritogenesis.