The usefulness of CHAPS as a non-cytotoxic stabilizing agent in purification of growth factors

Among several detergents, a zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), was found to be least cytotoxic for cultured mammalian cells. CHAPS improved the activity recovery and elution profile of crude and purified fibroblast growth factors (FGFs) during chromatographies. Diluted preparations of FGFs were stabilized by CHAPS against the loss during storage. Amino acid sequence analysis was not disturbed by CHAPS. CHAPS was removable by reversed-phase high-performance liquid chromatography. These results indicate that CHAPS is useful as a non-cytotoxic stabilizing agent in purification of various kinds of bioactive polypeptides.Abbreviations -MEM Alpha Modification of Eagle's Minimal essential medium - CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate - CHAPSO 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane sulfonate - CS Calf Serum - EGF Epidermal Growth Factor - FGF Fibroblast Growth Factor - HPLC High-Performance Liquid Chromatography - NGF Nerve Growth Factor - NOG 1-O-n-octyl--D-glucopyranoside - NP-40 Nonidet P-40 - PBS Phosphate-Buffered Saline - SB 12 3-(dodecylmethylammonio)-1-propane sulfonate - SDS Sodium Dodecyl Sulfate - TGF- and Transforming Growth Factor type and      

Developmental alteration of nerve injury induced glial cell line-derived neurotrophic factor (GDNF) receptor expression is crucial for the determination of injured motoneuron fate

Axotomy-induced neuronal death occurs in neonatal motoneurons, but not in adult rat. Here we demonstrated that during the course of postnatal development, nerve injury induced down-regulation of the glial cell line-derived neurotrophic factor (GDNF) receptor GFRalpha1 in axotomized hypoglossal motoneurons of rat are gradually converted to the adult up-regulation pattern of response. The compensatory expression of GFRalpha1 specifically in the injured motoneurons of neonates by adenovirus succeeded in rescuing the injured neurons without an application of growth factors. To the contrary, the nuclear antisense RNA for GFRalpha1 expression accelerates the axotomy-induced neuronal death in pups. These findings suggest that the receptor expression response after nerve injury is critical for the determination of injured motoneuron fate.     

Binding of cytochrome c to cytochrome c-oxidase in intact mitochondria. A study with radioactive photoaffinity-labeled cytochrome c

[³H]-p-Azidophenacylbromide-(methyl-4-mercaptobutyrimidate)-cytochrome $\text{c}$ from $\text{Saccharomyces cerevisiae}$ was prepared and its properties determined. The radioactive photoaffinity-labeled cytochrome $\text{c}$ was linked by irradiation into a covalent complex with cytochrome $\text{c}$ oxidase. Analysis of the complex on SDS-polyacrylamide gels showed that cytochrome $\text{c}$ bound to one of the smaller subunits of cytochrome $\text{c}$ oxidase with an apparent molecular weight of 15,000.     

Identification of genes and pathways involved in the synthesis of Mead acid (20:3n − 9), an indicator of essential fatty acid deficiency

In mammals, 5,8,11-eicosatrienoic acid (Mead acid, 20:3n − 9) is synthesized from oleic acid during a state of essential fatty acid deficiency (EFAD). Mead acid is thought to be produced by the same enzymes that synthesize arachidonic acid and eicosapentaenoic acid, but the genes and the pathways involved in the conversion of oleic acid to Mead acid have not been fully elucidated. The levels of polyunsaturated fatty acids in cultured cells are generally very low compared to those in mammalian tissues. In this study, we found that cultured cells, such as NIH3T3 and Hepa1–6 cells, have significant levels of Mead acid, indicating that cells in culture are in an EFAD state under normal culture conditions. We then examined the effect of siRNA-mediated knockdown of fatty acid desaturases and elongases on the level of Mead acid, and found that knockdown of Elovl5, Fads1, or Fads2 decreased the level of Mead acid. This and the measured levels of possible intermediate products for the synthesis of Mead acid such as 18:2n − 9, 20:1n − 9 and 20:2n − 9 in the knocked down cells indicate two pathways for the synthesis of Mead acid: pathway 1) 18:1n − 9 → (Fads2) → 18:2n − 9 → (Elovl5) → 20:2n − 9 → (Fads1) → 20:3n − 9 and pathway 2) 18:1n − 9 → (Elovl5) → 20:1n − 9 → (Fads2) → 20:2n − 9 → (Fads1) → 20:3n − 9.     

Development of a series of gateway binary vectors possessing a tunicamycin resistance gene as a marker for the transformation of Arabidopsis thaliana

We made two series of Gateway binary vectors, pGWBs and R4pGWBs, possessing a UDP-N-acetylglucosamine: dolichol phosphate N-acetylglucosamine-1-P transferase (GPT) gene driven by the nopaline synthase promoter (Pnos) as a tunicamycin resistance marker for the transformation of Arabidopsis thaliana. The reporters and tags employed in this system are sGFP, GUS, LUC, EYFP, ECFP, G3GFP, mRFP, TagRFP, 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7, and TAP. Selection of transformants was successful on plates containing 0.15 mg/L of tunicamycin. These vectors were compatible with existing pGWB and R4pGWB vectors for kanamycin, hygromycin B, and BASTA? selection, and are useful new tools for making transgenic Arabidopsis.     

Mdm20 Modulates Actin Remodeling through the mTORC2 Pathway via Its Effect on Rictor Expression

NatB is an N-terminal acetyltransferase consisting of a catalytic Nat5 subunit and an auxiliary Mdm20 subunit. In yeast, NatB acetylates N-terminal methionines of proteins during de novo protein synthesis and also regulates actin remodeling through N-terminal acetylation of tropomyosin (Trpm), which stabilizes the actin cytoskeleton by interacting with actin. However, in mammalian cells, the biological functions of the Mdm20 and Nat5 subunits are not well understood. In the present study, we show for the first time that Mdm20-knockdown (KD), but not Nat5-KD, in HEK293 and HeLa cells suppresses not only cell growth, but also cellular motility. Although stress fibers were formed in Mdm20-KD cells, and not in control or Nat5-KD cells, the localization of Trpm did not coincide with the formation of stress fibers in Mdm20-KD cells. Notably, knockdown of Mdm20 reduced the expression of Rictor, an mTORC2 complex component, through post-translational regulation. Additionally, PKCα^S657 phosphorylation, which regulates the organization of the actin cytoskeleton, was also reduced in Mdm20-KD cells. Our data also suggest that FoxO1 phosphorylation is regulated by the Mdm20-mTORC2-Akt pathway in response to serum starvation and insulin stimulation. Taken together, the present findings suggest that Mdm20 acts as a novel regulator of Rictor, thereby controlling mTORC2 activity, and leading to the activation of PKCα^S657 and FoxO1.     

Fucosyl-GM1a, an endoglycoceramidase-resistant ganglioside of porcine brain

The use of bovine brain has been prohibited in many countries because of the world-wide prevalence of mad cow disease, and thus porcine brain is expected to be a new source for the preparation of gangliosides. Here, we report the presence of a ganglioside in porcine brain which is strongly resistant to hydrolysis by endoglycoceramidase, an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides of various glycosphingolipids. Five major gangliosides (designated PBG-1, 2, 3, 4, 5) were extracted from porcine brain by Folch's partition, followed by mild alkaline hydrolysis and PBA column chromatography. We found that PBG-2, but not the others, was strongly resistant to hydrolysis by the enzyme. After the purification of PBG-2 with Q-Sepharose, Silica gel 60 and Prosep-PB chromatographies, the structure of PBG-2 was determined by GC, GC-MS, FAB-MS and NMR spectroscopy as Fucalpha1-2Galbeta1-3GalNAcbeta1-4(NeuAcalpha2-3)Galbeta1-4Glcbeta1-1'Cer (fucosyl-GM1a). The ceramide was mainly composed of C18:0 and C20:0 fatty acids and d18:1 and d20:1 sphingoid bases. The apparent kcat/Km for fucosyl-GM1a was found to be 30 times lower than that for GM1a, indicating that terminal fucosylation makes GM1a resistant to hydrolysis by the enzyme. This report indicates the usefulness of endoglycoceramidase to prepare fucosyl-GM1a from porcine brain.     

Subcellular localization of human neutral ceramidase expressed in HEK293 cells

We previously reported that rat and mouse neutral ceramidases were mainly localized to plasma membranes as a type II integral membrane protein and partly detached from the cells via processing of the N-terminal/anchor sequence when expressed in HEK293 cells [M. Tani, H. Iida, M. Ito, O-glycosylation of mucin-like domain retains the neutral ceramidase on the plasma membranes as a type II integral membrane protein, J. Biol. Chem. 278 (2003) 10523-10530]. In contrast, the human homologue was exclusively detected in mitochondria when expressed in HEK293 and MCF7 cells as a fusion protein with green fluorescent protein at the N-terminal of the enzyme [S.E. Bawab, P. Roddy, T. Quian, A. Bielawska, J.J. Lemasters, Y.A. Hannun, Molecular cloning and characterization of a human mitochondrial ceramidase, J. Biol. Chem. 275 (2000) 21508-21513]. Given this discrepancy, we decided to clone the neutral ceramidase from human kidney cDNA and re-examine the intracellular localization of the enzyme when expressed in HEK293 cells. The putative amino acid sequence of the newly cloned enzyme was identical to that reported for human neutral ceramidase except at the N-terminal; the new protein was 19 amino acids longer at the N-terminal. We found that the putative full-length human neutral ceramidase was transported to plasma membranes, but not to mitochondria, possibly via a classical ER/Golgi pathway and localized mainly in plasma membranes when expressed in HEK293 cells. The N-terminal-truncated mutant, previously reported as a human mitochondrial ceramidase, was also weakly expressed in HEK293 cells but mainly released into the medium possibly due to the insufficient signal/anchor sequence.     

Impact of brood parasitism and predation on nest survival of the fan-tailed gerygone in New Caledonia

Predation and brood parasitism are common reasons for nesting failure in passerine species and the additive impact by invasive species is a major conservation concern, particularly on tropical islands. Recognising the relative contribution of the different components of nesting failure rates is important to understand co-evolutionary interactions within brood parasite–host systems. In the remote archipelago of New Caledonia, the fan-tailed gerygone Gerygone flavolateralis is the exclusive host of the brood-parasitic shining bronze-cuckoo Chalcites lucidus. Additionally, invasive rodents also possibly have an impact on breeding success. To estimate the impact of potential nest predators, we 1) video monitored nests to identify predators, 2) estimated the probability of predation based on nest visibility and predator abundance and 3) tested the possibility that the location of experimental nests and lack of odour cues decrease the predation by rodents. In addition, we estimated nest survival rates using data collected in different habitats over the course of eight breeding seasons. Nesting success of fan-tailed gerygones was relatively low and predation was the main cause of nesting failure. We recorded mainly predation by native birds, including the shining bronze-cuckoo, whereas predation by rats was rare. In open habitats predation by cuckoos was much lower than predation by other avian predators. Neither predator activity around nests nor nest visibility influenced the probability of predation. Experimental nests in more accessible locations and containing an odorous bait were more exposed to rodent predation. Apparently, the fan-tailed gerygone has either never been specifically vulnerable to predation by rats or has developed anti-predator adaptations.