Role of KIFC3 motor protein in Golgi positioning and integration

KIFC3, a microtubule (MT) minus end-directed kinesin superfamily protein, is expressed abundantly and is associated with the Golgi apparatus in adrenocortical cells. We report here that disruption of the kifC3 gene induced fragmentation of the Golgi apparatus when cholesterol was depleted. Analysis of the reassembly process of the Golgi apparatus revealed bidirectional movement of the Golgi fragments in both wild-type and kifC3-/- cells. However, we observed a markedly reduced inwardly directed motility of the Golgi fragments in cholesterol-depleted kifC3-/- cells compared with either cholesterol-depleted wild-type cells or cholesterol-replenished kifC3-/- cells. These results suggest that (a) under the cholesterol-depleted condition, reduced inwardly directed motility of the Golgi apparatus results in the observed Golgi scattering phenotype in kifC3-/- cells, and (b) cholesterol is necessary for the Golgi fragments to attain sufficient inwardly directed motility by MT minus end-directed motors other than KIFC3, such as dynein, in kifC3-/- cells. Furthermore, we showed that Golgi scattering was much more drastic in kifC3-/- cells than in wild-type cells to the exogenous dynamitin expression even in the presence of cholesterol. These results collectively demonstrate that KIFC3 plays a complementary role in Golgi positioning and integration with cytoplasmic dynein.     

Obligatory intracellular parasitism by Ehrlichia chaffeensis and Anaplasma phagocytophilum involves caveolae and glycosylphosphatidylinositol-anchored proteins

Obligatory intracellular, human ehrlichiosis agents Ehrlichia chaffeensis and Anaplasma phagocytophilum create unique replicative compartments devoid of lysosomal markers in monocytes/macrophages and granulocytes respectively. The entry of these bacteria requires host phospholipase C (PLC)-gamma2 and protein tyrosine kinases, but their entry route is still unclear. Here, using specific inhibitors, double immunofluorescence labelling and the fractionation of lipid rafts, we demonstrate that bacterial entry and intracellular infection involve cholesterol-rich lipid rafts or caveolae and glycosylphosphatidylinositol (GPI)-anchored proteins. By fluorescence microscopy, caveolar marker protein caveolin-1 was co-localized with both early and replicative bacterial inclusions. Additionally, tyrosine-phosphorylated proteins and PLC-gamma2 were found in bacterial early inclusions. In contrast, clathrin was not found in any inclusions from either bacterium. An early endosomal marker, transferrin receptor, was not present in the early inclusions of E. chaffeensis, but was found in replicative inclusions of E. chaffeensis. Furthermore, several bacterial proteins from E. chaffeensis and A. phagocytophilum were co-fractionated with Triton X-100-insoluble raft fractions. The formation of bacteria-encapsulating caveolae, which assemble and retain signalling molecules essential for bacterial entry and interact with the recycling endosome pathway, may ensure the survival of these obligatory intracellular bacteria in primary host defensive cells.     

The secretory response through electric stimulation of differentiated PC12 rat pheochromocytoma cells transfected with neuropeptide Y fused with enhanced green fluorescent protein

Exocytosis in pheochromocytoma cells was induced by electric stimulation. To chase the movement of vesicles by electric stimulation, dense-core secretory vesicles were visualized by expression of the fusion protein between neuropeptide Y and enhanced green fluorescent protein (EGFP) in these differentiated PC12 rat pheochromocytoma cells. When the cells were stimulated with constant voltage potential at –300 mV, the movement of dense-core secretory vesicles could be regulated.     

Freeze-fracture and cytochemical studies on the in vitro cyst form of reptilian Blastocystis pythoni

After cultured cysts are osmotically shocked by treating with distilled water, there is an exponential increase in the cyst form of Blastocystis pythoni; this was demonstrated by an immunofluorescence antibody assay against the culture organisms. In 11-day-old cultures of B. pythoni, 68.8% of the organisms (= 2.2 x 10(8) cysts/ml) were in the cyst form. Examination of thin sections of cysts revealed many similarities to the cyst forms of Blastocystis obtained from fecal samples in previous investigations. Freeze-fracture images of the plasma membrane of non-cyst cells also revealed a similar distribution of the intramembrane particles (IMP) when compared to non-cysts of B. hominis, while the plasma membrane of the cyst form showed practically no IMP. The size and morphology of particle-rich small depressions and smooth small protrusions observed on the P face and E face of non-cyst cells, respectively, were similar to endocytic sites reported for B. hominis. In the present study glycogen was cytochemically demonstrated at the ultrastructural level by an alkaline bismuth staining method in both cyst and non-cyst cells.     

Androgen-dependent atypical fibromas spontaneously arising in the skin of Djungarian hamsters (Phodopus sungorus)

Spontaneous atypical fibromas that arose in the thoracoabdominal skin of one aged female and 31 aged male Djungarian hamsters (Phodopus sungorus) were examined histologically, immunohistochemically, and ultrastructurally. The normal skin from both sexes obtained at various intervals until the age of 12 months was examined, as were the tumors. These tumors were composed of ganglion cell-like (GL) cells that had one or two ovoid nuclei, basophilic foamy cytoplasm, and various amount of collagen fibers between the cells. The tumor cells had positive reaction to vimentin and androgen receptor (AR); the stromal collagen fibers reacted positively with the antibody against collagen type I or III. Ultrastructurally, the tumor cells had abundant rough endoplasmic reticulum in the cytoplasm. On the other hand, small nests of the cells mimicking tumor GL cells were present in the dermal layer to the panniculus of the normal thoracoabdominal skin of adult males, but were seldom found in adult females. The morphologic and immunohistochemical features of these tumor GL cells were basically similar to those of normal skin GL cells, although the former had a certain degree of atypia. These results suggest that atypical skin fibroma in the Djungarian hamster is an androgen-dependent tumor and originates from skin GL cells.     

NMR and ICP spectroscopic analysis of the DNA-binding domain of the Drosophila GCM protein reveals a novel Zn2+ -binding motif

Drosophila GCM (glial cell missing) is a novel DNA-binding protein that determines the fate of glial precursors from the neural default to glia. The GCM protein contains the functional domain that is essential for recognition of the upstream sequence of the repo gene. In the DNA-binding region of this GCM protein, there is a cysteine-rich region with which divalent metal ions such as Zn(2+) must bind and other proteins belonging to the GCM family have a corresponding region. To obtain a more detailed insight into the structural and functional features of this DNA-binding region, we have determined the minimal DNA-binding domain and obtained inductively coupled plasma atomic emission spectra and (1)H-(15)N, (1)H-(15)N-(13)C and (113)Cd(2+) NMR spectra, with or without its specific DNA molecule. Considering the results, it was concluded that the minimal DNA-binding domain includes two Zn(2+)-binding sites, one of which is adjacent to the interface for DNA binding. Systematic mutational analyses of the conserved cysteine residues in the minimal DNA-binding domain revealed that one Zn(2+)-binding site is indispensable for stabilization of the higher order structure of this DNA-binding domain, but that the other is not.     

Dipicolinic acid prevents the copper-dependent oxidation of low density lipoprotein

Effect of dipicolinic acid (pyridine 2,6-dicarboxylic acid) and pyridine compounds on the copper-dependent oxidation of human low density lipoprotein was analyzed in relation to the inhibition of copper reduction. Dipicolinic acid inhibited copper-dependent LDL oxidation completely, but the LDL oxidation was slightly inhibited by pyridine compounds with one carboxyl group at 2 or 6-position. Reduction of copper by LDL itself and ascorbate was inhibited completely by dipicolinic acid, but only partially by picolinic acid, quinolinic acid and isocinchomeronic acid with 2- or 6-carboxylic group. Pyridine compounds without 2- or 6-carboxyl group did not show any inhibitory effect on the LDL oxidation and the copper reduction. Protective effect of dipicolinic acid on the LDL oxidation was closely correlated with the copper-reducing activity. Dipicolinic acid shows an antioxidant action by the formation of a chelation complex with copper. This may have implications in understanding mechanisms of preventing LDL oxidation during the early phase of atherosclerosis.     

Novel cap-independent translation with the 5′-noncoding region of L-A virus mRNA

A novel cap-independent translation has been performed where the ribosome entry is regulated by the 5-noncoding region (NCR) of L-A virus mRNA. Despite L-A virus mRNA containing neither cap structure nor a poly(A) tail, the reconstructed mRNA encoding the 5 NCR of L-A virus mRNA and a reporter gene (luciferase) was translated, in yeast lysate, 60 times more efficiently than control mRNA. The 5 NCR from L-A virus was effective in regulating the recruitment of ribosome in vitro. A possible mechanism in Saccharomyces cerevisiae is also suggested, whereby the ribosome entry is regulated by the 5 NCR of L-A virus mRNA.     

l-Carnitine is essential to β-oxidation of quarried fatty acid from mitochondrial membrane by PLA2

Mitochondrial β-oxidation is an important system involved in the energy production of various cells. In this system, the function of l-carnitine is essential for the uptake of fatty acids to mitochondria. However, it is unclear whether or not endogenous respiration, ADP-induced O₂ consumption without substrates, is caused by l-carnitine treatment. In this study, we investigated whether l-carnitine is essential to the β-oxidation of quarried fatty acids from the mitochondrial membrane by phospholipase A₂ (PLA₂) using isolated mitochondria from the liver of rats. Intact mitochondria were incubated in a medium containing Pi, CoA and l-carnitine. The effect of l-carnitine treatment on ADP-induced mitochondrial respiration was observed without exogenous respiratory substrate. Increase in mitochondrial respiration was induced by treatment with l-carnitine in a concentration-dependent manner. Treatment with rotenone, a complex I blocker, completely inhibited ADP-induced oxygen consumption even in the presence of l-carnitine. Moreover, the l-carnitine dependent ADP-induced mitochondrial oxygen consumption did not increase when PLA₂ inhibitors were treated before ADP treatment. The l-carnitine-dependent ADP-induced oxygen consumption did contribute to ATP productions but not heat generation via an uncoupling system. These results suggest that l-carnitine might be essential to the β-oxidation of quarried fatty acids from the mitochondrial membrane by PLA₂.     

Versatile roles of plastids in plant growth and development

Plastids, found in plants and some parasites, are of endosymbiotic origin. The best-characterized plastid is the plant cell chloroplast. Plastids provide essential metabolic and signaling functions, such as the photosynthetic process in chloroplasts. However, the role of plastids is not limited to production of metabolites. Plastids affect numerous aspects of plant growth and development through biogenesis, varying functional states and metabolic activities. Examples include, but are not limited to, embryogenesis, leaf development, gravitropism, temperature response and plant-microbe interactions. In this review, we summarize the versatile roles of plastids in plant growth and development.