Characterization of clofibrate-induced retrograde Golgi membrane movement to the endoplasmic reticulum: clofibrate distinguishes the Golgi from the trans Golgi network

Clofibrate-induced retrograde Golgi membrane movement was blocked or retarded when NRK cells were treated with sodium azide/2-deoxyglucose, nocodazole, taxol, and destruxin B, indicating that it depends on energy, and the dynamic state of microtubules, and being acidic or vacuolar-type ATPase function. PDMP and phospholipase A2 inhibitors also blocked it. These characteristics are similar to those of brefeldin A (BFA) and nordihydroguaiaretic acid (NDGA), inducers of retrograde Golgi membrane movement. However, clofibrate was distinguished from BFA in that BFA action was insensitive to phospholipase A2 inhibitors and from NDGA in that NDGA stabilized microtubules against nocodazole and its action was almost insensitive to taxol. The trans Golgi network (TGN) was resistant to clofibrate, while BFA and NDGA dispersed it. To our knowledge, clofibrate is the first drug to show such different effects on the Golgi and TGN and, therefore, is expected to be a useful tool to distinguish their architecture and/or membrane dynamics.     

Glucosylceramide synthesis inhibitors block pharmacologically induced dispersal of the Golgi and anterograde membrane flow from the endoplasmic reticulum: implication of sphingolipid metabolism in maintenance of the Golgi architecture and anterograde membrane flow

PDMP (D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol) and PPMP (D,L-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol), inhibitors of glucosylceramide synthesis, blocked brefeldin A (BFA)- and nordihydroguaiaretic acid-induced dispersal of the Golgi and trans Golgi network, and Golgi-derived vesicles were retained in the juxtanuclear region. PDMP and PPMP did not stabilize microtubules but blocked nocodazole-induced extensive fragmentation and dispersal of the Golgi, and large Golgi vesicles were retained in the juxtanuclear region. PPMP is a stronger inhibitor of glucosylceramide synthesis than PDMP, but PDMP showed a stronger activity against BFA-induced retrograde membrane flow. However, PPMP showed a stronger activity for Golgi disruption and inhibition of anterograde trafficking from the endoplasmic reticulum, and rebuilding of the Golgi architecture. Cumulatively, these results suggest that sphingolipid metabolism is implicated in maintenance of the Golgi architecture and anterograde membrane flow from the endoplasmic reticulum but not in Golgi dispersal induced by BFA.     

Two mouse piwi-related genes: miwi and mili

Genes belonging to the piwi family are required for stem cell self-renewal in diverse organisms. We cloned mouse homologues of piwi by RT-PCR using degenerative primers. The deduced amino acid sequences of mouse homologues MIWI and MILI showed that each contains a well-conserved C-terminal PIWI domain and that each shares significant homology with PIWI and their human counterparts HIWI. Both miwi and mili were found in germ cells of adult testis by in situ hybridization, suggesting that these genes may function in spermatogenesis. Furthermore, mili was expressed in primordial germ cells (PGCs) of developing mouse embryos and may therefore play a role during germ cell formation. MIWI may be involved in RNA processing or translational regulation, since MIWI was found to possess RNA binding activity. Our data suggest that miwi and mili regulate spermatogenesis and primordial germ cell production.     

Expression of zebrafish btg-b, an anti-proliferative cofactor, during early embryogenesis

BTG/tob family proteins are thought to be a potential tumor suppressor due to their anti-proliferative activity. We cloned zebrafish btg-b, a member of the BTG1/2 subfamily, using in situ hybridization screening. The tissue-specific expression of btg-b is first observed in the organizer region at the early gastrula stage. Later in development, the forebrain, the hindbrain, the polster and the paraxial mesoderm transiently express btg-b. Recently, mouse Btg1 and Btg2 have been shown to be a cofactor for Hoxb9. Double in situ hybridization with zebrafish btg-b and hoxb9a indicates that the expression domains of these two genes overlap in the posterior paraxial mesoderm.     

HoxA and HoxB cluster genes subdivide the digestive tract into morphological domains during chick development

The digestive tract exhibits region-specific morphology and cytodifferentiation along the anteroposterior axis. We analyzed the spatial expression patterns of Hox genes belonging to the HoxA and HoxB cluster (Hoxa-4 approximately a-9, Hoxb-5 approximately b-9) in the developing chick digestive tract. The expression domains of these Hox genes correlated with morphological subdivision of the digestive tract along the anteroposterior axis.     

Rapamycin induces binding activity to the terminal oligopyrimidine tract of ribosomal protein mRNA in rats

Cathepsin G, elastase, and proteinase 3 are serine proteinases released by activated neutrophils. Cathepsin G can cleave angiotensinogen to release angiotensin II, but this activity has not been previously reported for elastase or proteinase 3. In this study we show that elastase and proteinase 3 can release angiotensin I from angiotensinogen and release angiotensin II from angiotensin I and angiotensinogen. The relative order of potency in releasing angiotensin II by the three proteinases at equivalent concentrations is cathepsin G > elastase > proteinase 3. When all three proteinases are used together, the release of angiotensin II is greater than the sum of the release when each proteinase is used individually. Cathepsin G and elastase can also degrade angiotensin II, reactions which might be important in regulating the activity of angiotensin II. The release and degradation of angiotensin II by the neutrophil proteinases are reactions which could play a role in the local inflammatory response and wound healing.     

Characterization of nuclear gangliosides in rat brain: concentration, composition, and developmental changes

Nuclear gangliosides were characterized using two distinct fractions of large (N1) and small (N2) nuclear populations from rat brain. The ganglioside concentration of N1 nuclei from adult rat brain was 0.92 microg sialic acid/mg protein, which was about 3.8 times higher than that of N2 nuclei. N1 and N2 nuclear gangliosides showed similar compositional profiles; they contained major gangliosides of GM1, GD1a, GD1b, and GT1b, with GM3 in lesser amounts. c-Series gangliosides such as GT3, GQ1c, and GP1c were also detected in both nuclear preparations. Nuclear localization of gangliosides was confirmed by immunofluorescence with anti-GM1 antibody, cholera toxin B subunit, and c-series ganglioside-specific monoclonal antibody A2B5. Developmental changes of nuclear gangliosides were examined using rats of different ages ranging from embryonic day 14 (E14) to postnatal 7 weeks. The concentration of N1 nuclear gangliosides changed only slightly during development and did not correlate with that of whole-brain gangliosides. The developmental pattern of ganglioside composition of N1 nuclei was also distinguished from that of microsomal membranes; the ganglioside changes in N1 nuclei included reduced expression of di- and polysialogangliosides at E16 and higher proportions of GM3 at early and late stages of the period. These findings suggest that gangliosides in nuclear membranes are developmentally regulated in a distinct manner in brain cells.     

The novel surveillance mechanism of the Trp53-dependent s-phase checkpoint ensures chromosome damage repair and preimplantation-stage development of mouse embryos fertilized with x-irradiated sperm

Cell cycle checkpoints and apoptosis function as surveillance mechanisms in somatic tissues. However, some of these mechanisms are lacking or are restricted during the preimplantation stage. Previously, we reported the presence of a novel Trp53-dependent S-phase checkpoint that suppresses pronuclear DNA synthesis in mouse zygotes fertilized with X-irradiated sperm (sperm-irradiated zygotes) (Shimura et al., Mol. Cell. Biol. 22, 2220-2228, 2002). Here we studied the role of the Trp53-dependent S-phase checkpoint in the early stage of development of sperm-irradiated zygotes. In the Trp53(+/+) genetic background, all of the sperm-irradiated zygotes cleaved successfully to the two-cell stage despite the fact that half of them carried a sub-2N amount of DNA. These zygotes progressed normally to the eight-cell stage and then implanted, but the subsequent fetal development was suppressed in a dose-dependent manner. In contrast, sperm-irradiated Trp53(-/-) embryos lacking an S-phase checkpoint exhibited an abnormal segregation of chromosomes at the first cleavage, even though they carried an apparently normal 2N amount of DNA. They were morphologically abnormal with numerous micronuclei, and they degenerated before reaching the eight-cell stage. As a consequence, no implants were observed for sperm-irradiated Trp53(-/-) embryos. These results suggest that the Trp53-dependent S-phase checkpoint is a surveillance mechanism involved in the repair of chromosome damage and ensures the preimplantation-stage development of sperm-irradiated embryos.     

Sulfite induces adherence of polymorphonuclear neutrophils to immobilized fibrinogen through activation of Mac-1 beta2-integrin (CD11b/CD18)

Sulfite is a major air pollutant which can cause respiratory tract inflammation characterized by an influx of polymorphonuclear neutrophils (PMN). We have previously shown that human PMN can produce sulfite either spontaneously or in response to stimulation with lipopolysaccharide. We now demonstrate that sulfite activates PMN to adhere to immobilized fibrinogen via the beta2-integrin Mac-1 (CD11b/CD18). Mac-1 expression is not altered by treatment with this agent. Although unaffected by pertussis toxin, sulfite-triggered PMN adhesion was abrogated by pretreating cells with the membrane-impermeant sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), a modifier of thiol groups on the cell surface. These results suggest that sulfite-induced PMN adhesion is dependent on a modification of thiols at the cell surface. Given its potent antioxidant and antimicrobial activities, sulfite may act as an endogenous mediator in host defense and/or inflammation.