Phosphatidylinositol Transfer Protein Expression Altered by Aging and Parkinson Disease

1. Phosphatidylinositol transfer proteins (PI-TP) are responsible for the transport of phosphatidylinositol (PI) and other phospholipids from endoplasmic reticulum to the other membranes and indirectly for lipid mediated signaling. Till now little is known about PI-TPs in brain aging and neurodegeneration. The aim of this study was to investigate expression of PI-TP in the brain during aging and in animal's model of Parkinson disease (PD) induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Moreover, in vitro, effect of 1-methyl-4-phenyl-pyridine cation (MPP⁺) on PI-TP, tyrosine hydroxylase (TH) protein level, and viability of cells was investigated.2. Wistar rats 4, 24, and 36 months old and C57/BL mice and rat pheochromocytoma (PC12) cell line were used for the studies. Mice C57/BL received three injections of MPTP in saline at 2 h intervals in a total dose of 40 mg/kg and then after 3, 7, and 14 days they were used for the investigation. PC12 cells were treated with increasing concentration (50–300 μM) of MPP⁺ for 24 h at 37°C. The level of PI-TP_{α and β} and TH were determined using Western Blot analysis.3. Our data indicated that PI-TP_{α and β} level decreased in brain of 36 months old rat by 20% comparing to the control value (4 months old). In animal's model of PD, PI-TP_{α and β} level was significantly lower by 85, 69, 64% in striatum at 3, 7, and 14 days after MPTP injection, respectively, compared to the control value. MPP⁺ decreased PI-TP_{α and β}, TH expression, and viability of PC12 cells in a dose-dependent manner. H₂O₂, menadione, and NO donor significantly decreased the PI-TP level and viability of PC12 cells.4. Our results indicate the lower protein expression of PI-TP_{α and β} in aged brain and in PD and suggest that oxidative stress may be responsible for the alteration of PI-TP.     

Characterization and expression of the cDNA encoding a new kind of phospholipid transfer protein, the phosphatidylglycerol/phosphatidylinositol transfer protein from Aspergillus oryzae: evidence of a putative membrane targeted phospholipid transfer protein in fungi

The full-length cDNA of a phospholipid transfer protein (PLTP) was isolated from Aspergillus oryzae by a RACE-PCR procedure using degenerated primer pool selected from the N-terminal sequence of the purified phosphatidylinositol/phosphatidylglycerol transfer protein (PG/PI-TP). The cDNA encodes a 173 amino acid protein of 18823 Da. The deduced amino acid sequence from position 38 to 67 is 100% identical to the N-terminal sequence (first 30 amino acids) of the purified PG/PI-TP. This amino acid sequence is preceded by a leader peptide of 37 amino acids which is predicted to be composed of a signal peptide of 21 amino acids followed by an extra-sequence of 16 amino acids, or a membrane anchor protein signal (amino acid 5-29). This strongly suggests that the PG/PI-TP is a targeted protein. The deduced mature protein is 138 amino acids long with a predicted molecular mass of 14933 Da. Comparison of the deduced PG/PI-TP sequence with other polypeptide sequences available in databases revealed a homology with a protein deduced from an open reading frame coding for an unknown protein in Saccharomyces cerevisiae (36% identity and 57% similarity). Apart from this homology, the PG/PI-TP is unique and specific to the filamentous fungi on the basis of comparison of PLTP protein sequences. Northern blot analysis of RNA isolated from A. oryzae cultures grown on glucose or glucose supplemented with phospholipids suggests that the PG/PI-TP is transcribed by only one RNA species and allows us to show that expression of the protein is regulated at the messenger RNA level.     

Structure and erythroid cell-restricted expression of a chicken cDNA encoding a novel zinc finger protein of the Cys+His class

We report the cloning, sequence analysis and expression pattern of chGfi, a zinc finger protein (Zfp)-encoding cDNA that was isolated from a cDNA library constructed with RNA from avian erythroblastosis virus (AEV)-transformed primary chicken erythroblasts. The 1387-bp-long chGfi cDNA encodes a full-length 337-amino-acid (aa) protein that contains six zinc fingers (Zf) of the 2Cys+2His class at its C-terminus. Immunoblotting experiments with extracts from bone marrow cells detected a 38-kDa protein that corresponds to the M_r of 38 690 calculated for the protein deduced from chGfi. The chGfi protein is most homologous to the rat Gfi-1 showing a sequence similarity of 92% over the Zf region and only two exchanges within the N-terminal 19 aa that constitute the Gfi-1 repressor domain. Expression of chGfi is only detected in transformed primary erythroblasts, in erythroid cells of the primitive and definitive lineage and in bone marrow cells. chGfi activity does not vary significantly during differentiation of transformed primary erythroblasts of the definitive lineage. No chGfi expression is detected in cells of the myeloid and lymphoid lineages or in a total of nine different organs of adult origin. Our results indicate that chGfi expression is restricted to erythroid cells of the primitive and definitive lineage.     

Cloning and expression of a chicken α-amylase gene

We have isolated and sequenced a genomic clone for a pancreatic α-amylase gene (amy) of the chicken (Gallus gallus). The gene is interrupted by nine introns, spans over 4 kb, and encodes a protein (AMY) of 512 aa that is 83% identical to the human pancreatic α-amylase enzyme. Southern blot analysis of chicken DNA revealed two distinct pancreatic amy loci. In addition, we have generated a cDNA from chicken pancreatic RNA corresponding to the coding sequence of the genomic clone. The cDNA was inserted into a yeast expression vector, and the resulting construct used to transform Saccharomyces cerevisiae cells. Transformed yeast cells synthesized and secreted active AMY enzyme, and the gel migration pattern of the α-amylase produced by the yeast cells was identical to that of the native chicken enzyme.     

Isolation of poly(A)-containing RNA on synthetic fluorophlogopite (Mica).

Synthetic fluorophlogopite, an aluminosilicate of the same structure as naturally occurring mineral mica in which potassium ions on the basal surface have been replaced by aluminum ions, has the ability to retain polynucleotides irreversibly. This property of Al³⁺-mica was used for irreversible adsorption of poly(U) and subsequent selective adsorption of poly(A)-containing RNA from rabbit reticulocyte polysomes at high salt concentration and its elution by 50% dimethylsulfoxide. The properties of RNA isolated on poly(U)-Al³⁺-mica were studied by sucrose density gradient centrifugation and by stimulation of globin synthesis in an in vitro protein synthesizing system from wheat germ and from Krebs II-ascites cells. The preparation contained 9s RNA species which corresponds to rabbit globin messenger RNA, and under optimal conditions it stimulated protein synthesis more than 100-fold. Polyacrylamide-gel electrophoresis in sodium dodecyl sulfate showed that synthesized product was identical with rabbit globin.     

Characterization of a prion protein (PrP) gene from rabbit; a species with apparent resistance to infection by prions

The prion protein gene (PrP) encodes a cellular protein of unknown function. A conformational isoform of this protein is involved in the neurodegenerative prion diseases. To facilitate the identification of structurally and antigenically important regions within the PrP molecule, the rabbit PrP open reading frame (ORF) was cloned and characterised. There is 82–87% identity at the nucleotide sequence level and 88–93% identity at the amino acid (aa) sequence level, between the rabbit gene and PrP sequences of other mammals. The rabbit gene shares structural and organisational features common to all known PrP genes signifying that it is the rabbit PrP gene. Comparison of the rabbit PrP aa sequence with PrP aa sequences from different species revealed several potential epitopes. Two anti-ovine PrP peptide Ab raised in rabbits, 168-92 and 98-92, confirmed that two separate cross-reacting epitopes segregate with single aa differences between rabbit and sheep PrP at positions 43 and 99 of the rabbit PrP polypeptide. The presence of these epitopes correlates with the species recognition patterns of previously published Ab. The usefulness of the rabbit PrP gene sequence in predicting antigenic regions within the PrP proteins of various species is illustrated. The structure of the rabbit PrP protein in relation to rabbits' apparent resistance to infection by prions is discussed.     

RNA conversion of lymphoid cells to synthesize allogeneic immunoglobulins in vivo

Ribonucleic acid extracts (“5 day immune” and “nonimmune”-RNA) obtained from lymph nodes and spleens of rabbits homozygous for the b⁴ or b⁵ allele of light chain immunoglobulin allotypes were injected iv into nonimmunized rabbits homozygous for the alternate allele. The recipient rabbits were then given multiple iv injections of sheep red blood cells (SRBC). The spleens were assayed 13, 21, and 37 days following the RNA injection for “direct” IgM and “indirect” IgG plaque forming cells (PFC) specific for SRBC. The b4 or b5 light chain allotype and the a1, a2, and a3 heavy chain allotype of the antibody in the plaques was identified by radioautography and by inhibition of plaque formation using anti-allotype antibodies. The b light chain allotype of the RNA donor was identified in 22–32% of the IgM plaques and in 25–42% of the IgG plaques. The allotype of the host rabbit b light chain allotype was identified in 56–67% of the IgM plaques and in 57–71% of the IgG plaques. Likewise the a heavy chain allotype of the RNA donor was identified in 10–19% of the IgM plaques and in 12–19% of the IgG plaques. The allotype of the host rabbit a heavy chain allotype was identified in 51–60% of the IgM plaques and in 55–63% of the IgG plaques. The concentrated lysates of spleen and lymph node cells were also analyzed for immunoglobulins of each light chain allotype by immunodiffusion with radiolabeled antibody. The allotype of both the RNA donor rabbit and host rabbit were found in most of the lysates of lymphoid tissues and in some of the IgG isolated from the serum and concentrated.     

Glucan-phosphorylase forms in cotyledons of Pisum sativum L.: Localization,developmental change,in-vitro translation,and processing

The occurrence, location, and biosynthesis of glucan-phosphorylase (EC 2.4.1.1) isoenzymes were studied in cotyledons of developing or germinating seeds of Pisum sativum L. Type-I and type-II isoenzymes were detected, and were also localized by indirect immunofluorescence using polyclonal anti-type-I or anti-type-II phosphorylase antibodies. Type-I isoenzyme was found in the cytosol of parenchyma cells whereas the type-II enzyme form is a plastid protein which resides either in amyloplasts (in developing seeds) or in proplastids (in germinating seeds). During seed development, type-II phosphorylase was the predominant isoenzyme and the type-I isoenzyme represented a very minor compound. During germination, the latter increased whilst type-II phosphorylase remained at a constant level. In in-vitro translation experiments, type-I isoenzyme was observed as a final-size product with an apparent molecular weight of approx. 90 kDa. In contrast, type-II phosphorylase was translated as a high-molecular-weight precursor (116 kDa) which, when incubated with a stromal fraction of isolated intact pea chloroplasts, was processed to the size of the mature protein (105 kDa).Abbreviations IgG immunoglobulin G - kDa kilodalton - poly(A)⁺ RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work has been made possible by grants from the Deutsche Forschungsgemeinschaft. The authors are endebted to Mrs. Karin Niehüser for help in the immunocytochemical studies.     

Location and sequence analysis of a 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase-encoding gene (bpdF) of the biphenyl/ polychlorinated biphenyl degradation pathway in Rhodococcus sp. M5

The 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPD) hydrolase-encoding gene (bpdF) in the biphenyl (BP)/polychlorinated biphenyl (PCB)-degrading bacterium, Rhodococcus sp. M5 (M5), was found to be located within a 4.5-kb HindIII-BamHI genomic DNA that was 5.4 kb downstream from the bpdC1C2BADE gene cluster. The deduced amino acid (aa) sequence of bpdF revealed that the hydrolase contains 297 aa (32679 Da) that was verified by expression in the Escherichia coli T7 RNA polymerase/promoter system. Unlike previously known HOPD hydrolases, the aa sequence of BpdF appears unique. Interestingly, all HOPD hydrolases and related proteins from the phenol and toluene/ xylene degradation pathways, were found to have a bias in the codon usage in the catalytic Ser within the conserved VGNS(M/F)GG motif.     

Formation of reactive 1-nitropyrene metabolites by lung microsomes and isolated lung cells

The metabolism and activation of 1-nitropyrene (1-NP) to reactive intermediates by lung microsomes and isolated lung cells was studied. Mutagenicity of 1-NP metabolites was assayed in Salmonella typhimurium TA98NR, a strain lacking a major component of nitroreductase activity. In the presence of NADPH, microsomes from rabbit, rat and hamster lung metabolized 1-NP to mutagenic products to a similar degree. Pretreatment with a mixture of polychlorinated biphenyls (PCB) decreased the formation of mutagenic metabolites by rabbit lung microsomes, but did not affect the production of mutagens by rat or hamster lung microsomes. ³H-1-NP was metabolized to covalently bound protein products at a rate of 82 and 10 pmol/mg by rabbit and hamster lung microsomes, respectively, whereas no binding was detected in rat lung microsomes. PCB-pretreatment increased covalent protein binding of ³ H-1-NP in lung microsomes from hamster and rat, but decreased the binding in rabbit lung microsomes. High performance liquid chromatography analysis indicated that ³H-1-NP was readily converted to ring-hydroxylated products by rabbit and hamster lung microsomes; the rate was much lower with rat lung microsomes. ³H-1-NP was activated to metabolites that covalently bound to protein in isolated rabbit lung cells, with the following rates being observed: Clara cells > lung digest > type II cells. In contrast, covalent protein binding in cells isolated from rat lung was very low. 1-NP was not activated to products mutagenic for S. typhimurium TA 98 N R when co-incubated with cells isolated either from rabbit or rat lung.Abbreviations 1-AP 1-aminopyrene - DMSO dimethyl sulfoxide - EGTA ethylene glycol-bis(ß-aminoethyl ether) - EM electron microscopy - HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid - HPBS HEPES-phosphate-buffered-saline - HPLC high performance liquid chromatography - NBT nitroblue tetrazolium - 1-NP 1-nitropyrene - 1-NP-4,5-diol trans-4,5-dihydro-4,5-dihydroxy-1-nitropyrene - 1-NP-9,10-diol trans-9,10-dihydro-9,10-dihydroxy-1-nitropyrene - 1-NP-4,5-oxide 1-nitropyrene-4,5-oxide - 1-NP-9,10-oxide 1-nitropyrene-9,10-oxide - 3-OH-1-NP 3-hydroxy-1-nitropyrene - 6-/8-OH-1-NP a mixture of 6- and 8-hydroxy-1-nitropyrene - PBS phosphate-buffered saline - PCB a mixture of polychlorinated biphenyls (Aroclor 1254) - TLC thin layer chromatography     

Isolation and characterization of cDNA encoding rabbit reticulocyte 2,3-bisphosphoglycerate synthase

We have isolated and sequenced a cDNA clone containing the entire coding region of rabbit reticulocyte 2,3-bisphosphoglycerate (DPG) synthase. The cDNA was verified by translation of the hybridization-selected RNA and by demonstrating identity of the deduced amino acid (aa) sequence to the sequences of CNBr peptides of the purified enzyme. The aa sequence of the enzyme was homologous to the reported sequence of the human enzyme [Haggarty et al., EMBO J. 2 (1983) 1213-1220], especially in the N-terminal half (aa 1-142). Northern blot analysis of rabbit reticulocyte poly(A)+ RNA revealed a single species of mRNA with about 1700 nucleotides.