Proteolipid/DM-20 proteins bearing the paralytic tremor mutation in peripheral nerves and transfected Cos-7 cells

Paralytic tremor (Plp-pt) is a missense mutation of the myelin proteolipid gene (Plp) in rabbits. The myelin yield in the Plp-pt brain is reduced and the protein and lipid composition of central nervous system (CNS) myelin is abnormal. We studied the intracellular transport of the normal and Plp-pt mutant PLP and DM-20 in transiently transfected Cos-7 cells. While the mutant PLP accumulates in the rough endoplasmic reticulum and does not reach the plasma membrane, the spliced isoform of PLP, mutant DM-20, is normally transported to the cell surface and integrated into the membrane. Analysis of rabbit sciatic nerves revealed that concentration of peripheral nervous system (PNS) myelin proteins is normal in Plp-pt myelin. In the PNS like in the CNS, the level of Plp gene products is subnormal. But this does not affect myelination, in the PNS where PLP, present in low concentration, is not a structural component of compact myelin. The normal level of Plp gene expression in Schwann cells is low and these results suggest that, in the Plp-pt PNS, Schwann cell function is not affected by the deficiency in PLP and/or the impairment of intracellular PLP transport. Special issue dedicated to Dr Marion E. Smith.     

Effects of Rumpshaker Mutation on CNS Myelin Composition and Structure

Abstract: Myelinated CNS tissues from homozygous/hemizygous and heterozygous jimpy rumpshaker jp ^rsh mutant mice were examined to determine the consequences on myelin structure of this mutation in the proteolipid protein (PLP) gene. Polyacrylamide gel electrophoresis and immunoblotting of brain homogenates confirmed that there was a decrease in PLP levels on the B6C3 genetic background onto which this gene was bred. We also observed an increase in level of a protein band that could correspond to the uncharacterized 10-kDa PLP previously reported in jp ^rsh mice on an Rb(1.3) 1Bnr background. High-performance TLC and densitometry of lipids from brain homogenate and isolated myelin revealed a decrease in content of cerebrosides and sulfatides. Electron microscopy on optic nerves revealed that normal radial component is retained in jp ^rsh myelin, further substantiating that PLP is not a component of this junctional complex. X-raydiffraction measurements on unfixed optic nerves showed that the jp ^rsh period is 5–10 Å larger than normal. Moreover, jp ^rsh optic nerve myelin was unstable, as evidenced by a continual increase in the period postdissection. jp ^rsh myelin that was equilibrated at varying pH and ionic strength typically had a larger than normal period under all conditions (both swelling and compacting). Our findings thus demonstrate that the biochemical abnormalities in the jp ^rsh mutant correlate with a wider periodicity and less stable packing of the myelin.     

The Myelin-Deficient Rat Has a Single Base Substitution in the Third Exon of the Myelin Proteolipid Protein Gene

Several genetic disorders that occur in animals and in humans result in an inability to synthesize normal myelin. Some of these disorders are inherited in an X-linked manner. The localization of the myelin proteolipid protein (PLP) gene to the X chromosome has directed the study of X-linked myelination disorders toward PLP. The myelin-deficient rat is one such X-linked dysmyelinating mutant. From a cDNA library constructed from myelin-deficient rat brain mRNA, we have isolated and sequenced cDNAs corresponding to PLP and its alternatively spliced isoform, DM-20. An A to C transition was detected in these cDNAs, which results in a threonine to proline change at amino acid 74 in both PLP and DM-20. No other substitutions were seen in the cDNA sequences. Polymerase chain reaction amplification and sequencing of the corresponding genomic regions were used to confirm the single base change. This substitution occurs in a highly hydrophobic portion of the protein that is thought to be an alpha-helical transmembrane segment. The presence of a helix-breaking amino acid such as proline in this segment is likely to influence the ability of the protein to interact with the membrane.     

Plasma 249Ser p53 mutation in patients with hepatocellular carcinoma residing in a high risk area

Objective : To investigate plasma p53 mutation in hepatocellular carcinoma (HCC) patients from Qidong and to define its significance. Methods: Blood samples from 25 hepatocellular carcinoma patients, 20 cirrhotic patients and 30 healthy controls in Qidong area. DNA was extracted and purified from 200μl of plasma from each sample. The 249^Ser p53 mutation was detected by restriction digestion analysis and by direct sequencing of exon-7 PCR products. Results: G→T transversion at the third base of 249 codon resulting in 249^Arg→249^Ser mutation in exon 7 of p53 gene were found in 11/25(44%) hepatocellular carcinoma cases, 4/20 (20%) cirrhotics, and 2/30 (7%) healthy controls (p<0.01). Conclusions: These data show that the 249^Ser p53 mutation in plasma is strongly associated with hepatocellular carcinoma patients in Qidong area and the mutation should be screened as a new early diagnostic marker for HCC.     

Different proteolipid protein mutants exhibit unique metabolic defects

PMD (Pelizaeus–Merzbacher disease), a CNS (central nervous system) disease characterized by shortened lifespan and severe neural dysfunction, is caused by mutations of the PLP1 (X-linked myelin proteolipid protein) gene. The majority of human PLP1 mutations are caused by duplications; almost all others are caused by missense mutations. The cellular events leading to the phenotype are unknown. The same mutations in non-humans make them ideal models to study the mechanisms that cause neurological sequelae. In the present study we show that mice with Plp1 duplications (Plp1tg) have major mitochondrial deficits with a 50% reduction in ATP, a drastically reduced mitochondrial membrane potential and increased numbers of mitochondria. In contrast, the jp (jimpy) mouse with a Plp1 missense mutation exhibits normal mitochondrial function. We show that PLP in the Plp1tg mice and in Plp1-transfected cells is targeted to mitochondria. PLP has motifs permissive for insertion into mitochondria and deletions near its N-terminus prevent its co-localization to mitochondria. These novel data show that Plp1 missense mutations and duplications of the native Plp1 gene initiate uniquely different cellular responses.     

Disproportional Expression of Proteolipid Protein and DM-20 in the X-Linked, Dysmyelinating Shaking Pup Mutant

The shaking pup is an X-linked canine mutant with a severe hypomyelination of the CNS. Proteolipid protein (PLP) and the related DM-20 protein were examined in this mutant by densitometric scanning of Western blots stained with PLP antiserum. In the spinal cord of 4-week-old mutants, PLP was reduced to less than 1% of the control level, which is a greater deficiency than was found for other myelin proteins. On Western blots of control spinal cord, PLP stained much more intensely than DM-20. However, on Western blots of the mutant spinal cord, a component with the electrophoretic mobility of DM-20 stained slightly more intensely with PLP antiserum than PLP itself. This component was shown to be DM-20 by its lack of reactivity with an antiserum raised to a synthetic peptide corresponding to part of the PLP sequence that is missing in DM-20. Thus PLP and DM-20 are expressed in approximately equal and greatly reduced amounts in the mutant spinal cord. Although PLP or DM-20 could not be detected in brain from the 4-week-old mutant, similar disproportional expression of these two proteins was demonstrated in both spinal cord and brain from a 10-week-old mutant pup. Immunostaining of tissue sections showed that the small amounts of PLP and/or DM-20 synthesized in the mutant are present in the thin myelin sheaths. The results suggest that the shaking pup could have a primary defect in the PLP gene leading to a severe deficiency of PLP and DM-20 as well as disproportional expression of these two proteins.     

Expression of Na+, K+-ATPase α-Subunit in Animalized and Vegetalized Embryos of the Sea Urchin, Hemicentrotus pulcherrimus.

A marked increase in the Na⁺, K⁺-ATPase activity of sea urchin embryos occurred following an elevation of its mRNA level, revealed by Northern blotting analysis, in developmental period between the swimming blastula and the late gastrula stage. cDNA clone of Na⁺, K⁺-ATPase α-subunit, obtained from γgt10 cDNA library of sea urchin gastrulae, was digested with EcoRl ad Hindlll. The obtained 268 bp cDNA fragment, hybridized to a 4.6 Kb RNA, was used as probe for Northern blotting analysis. The level of Na⁺, K⁺-ATPase mRNA was higher in embryo-wall cell fraction isolated from late gastrulae (ectoderm cells) than the level in the bag fraction, containing mesenchyme cells (mesoderm cells) and archenteron (endoderm cells). The activity of Na⁺, K⁺-ATPase and the level of its mRNA were higher in animalized embryos obtained by pulse treatment with A23187 for 3 hr, starting at the 8–16 cell stage and were considerably lower in vegetalized embryos induced by 3 hr treatment with Li⁺ than that in normal embryos at the post gastrula corresponidng stage. Augmentation of Na⁺, K⁺-ATPase gene expression can be regarded as a marker for ectoderm cell differentiation at the post gastrula stage, which results from determination of cell fate in prehatching period.     

Therapy of Pelizaeus-Merzbacher disease in mice by feeding a cholesterol-enriched diet

Duplication of PLP1 (proteolipid protein gene 1) and the subsequent overexpression of the myelin protein PLP (also known as DM20) in oligodendrocytes is the most frequent cause of Pelizaeus-Merzbacher disease (PMD), a fatal leukodystrophy without therapeutic options. PLP binds cholesterol and is contained within membrane lipid raft microdomains. Cholesterol availability is the rate-limiting factor of central nervous system myelin synthesis. Transgenic mice with extra copies of the Plp1 gene are accurate models of PMD. Dysmyelination followed by demyelination, secondary inflammation and axon damage contribute to the severe motor impairment in these mice. The finding that in Plp1-transgenic oligodendrocytes, PLP and cholesterol accumulate in late endosomes and lysosomes (endo/lysosomes), prompted us to further investigate the role of cholesterol in PMD. Here we show that cholesterol itself promotes normal PLP trafficking and that dietary cholesterol influences PMD pathology. In a preclinical trial, PMD mice were fed a cholesterol-enriched diet. This restored oligodendrocyte numbers and ameliorated intracellular PLP accumulation. Moreover, myelin content increased, inflammation and gliosis were reduced and motor defects improved. Even after onset of clinical symptoms, cholesterol treatment prevented disease progression. Dietary cholesterol did not reduce Plp1 overexpression but facilitated incorporation of PLP into myelin membranes. These findings may have implications for therapeutic interventions in patients with PMD.     

The proteolipid protein promoter drives expression outside of the oligodendrocyte lineage during embryonic and early postnatal development

The proteolipid protein (Plp) gene promoter is responsible for driving expression of one of the major components of myelin--PLP and its splice variant DM-20. Both products are classically thought to express predominantly in oligodendrocytes. However, accumulating evidence suggests Plp expression is more widespread than previously thought. In an attempt to create a mouse model for inducing oligodendrocyte-specific gene deletions, we have generated transgenic mice expressing a Cre recombinase cDNA under control of the mouse Plp promoter. We demonstrate Plp promoter driven Cre expression is restricted predominantly to mature oligodendrocytes of the central nervous system (CNS) at postnatal day 28. However, crosses into the Rosa26(LacZ) and mT/mG reporter mouse lines reveal robust and widespread Cre activity in neuronal tissues at E15.5 and E10.5 that is not strictly oligodendrocyte lineage specific. By P28, all CNS tissues examined displayed high levels of reporter gene expression well outside of defined white matter zones. Importantly, our study reinforces the emerging idea that Plp promoter activity is not restricted to the myelinating cell lineage, but rather, has widespread activity both during embryonic and early postnatal development in the CNS. Specificity of the promoter to the oligodendrocyte cell lineage, as shown through the use of a tamoxifen inducible Plp-CreER(t) line, occurs only at later postnatal stages. Understanding the temporal shift in Plp driven expression is of consequence when designing experimental models to study oligodendrocyte biology.     

Antiporter Gene from Hordum brevisubulatum （Trin.） Link and Its Overexpression in Transgenic Tobaccos

A vacuolar Na^ /H^ antiporter cDNA gene was successfully isolated fromHordeum brevisubulatum (Trin.) Link using the rapid amplification ofcDNA ends (RACE) method. The gene was named HbNHXI and was found to consist of 1 916 bp encoding a predicted polypeptide of 540 amino acids with a conserved amiloride-binding domain. Phylogenetic tree analysis of the Na^ /H^ antiporters showed that the HbNHXI gene shares 55.3%-74.8% similarity with the vacuolar-type Na^ /H^ antiporters. Transgenic tobaccos that contain the HbNHXI gene, integrated by forward insertion into the tobacco genome, were obtained via Agrobacterium tumerfaciens and characterized for the determination of the concentration of Na^ and K^ ions, as well as proline, in the presence of 300 mmol/L NaCl. The T1 transgenic plants showed more tolerance to salt and drought than did wild-type plants. Our data suggest that overexpression of the HbNHXI gene could improve the tolerance of transgenic tobaccos to salt and drought through the function of the vacuolar Na^ /H^ antiporter.     

Developmental Study of Proteolipids in Bovine Brain: A Novel Proteolipid and DM-20 Appear Before Proteolipid Protein (PLP) During Myelination

In a developmental study, we have shown that DM-20 is present before proteolipid protein (PLP) in the fetal bovine cerebral hemispheres. When the white matter appears (27-30 weeks of gestation), the amount of DM-20 drastically increases. DM-20 remains the major proteolipid until birth. PLP is detected only 2-4 weeks after the appearance of white matter, that is, more than 4 weeks after the appearance of DM-20. The early appearance of DM-20 at the beginning of myelination raises the question of its particular function. In the adult bovine cerebral hemispheres, PLP is the major proteolipid but DM-20 remains quantitatively important because the PLP/DM-20 ratio ranges from 1.5 to 1.7. In the same developmental study we have, in the fetal cerebral hemispheres, isolated and characterized a novel proteolipid (apparent Mr 20,000), which appears even before DM-20 and is not detected in the adult brain. It is structurally related to PLP and DM-20 because the first 31 N-terminal amino acid residues are the same. However, in immunoblot, it did not react either with the antitridecapeptide 117-129 antiserum of PLP or with the anti-C-terminal hexapeptide antiserum of PLP.     

Sodium channel gene expression in mosquitoes, Aeries albopictus （S.）

A mosquito strain of Aedes albopictus, HAmAal^G0, from Huntsville, Alabama, USA, showed a normal susceptibility and low tolerance to permethrin and resmethrin （pyrethroid insecticides） compared to a susceptible Ikaken strain, even though these pyrethroid insecticides have been used in the field for a long period of time in Alabama. Recently, we treated HAmAal^G0 in the laboratory with permethrin for five generations and detected no significant change in the level of resistance to permethrin in the selected mosquitoes, HAmAal^G0, compared with the parental strain HAmAal^G0. We then examined the allelic expression at the L-to-F kdr site of the sodium channel gene in the Aedes mosquitoes to address our hypothesis that the L-to-F kdr mutation was not present in HAmAal^G0 and HAmAal^G5 mosquitoes. We found that every tested individual in Ikaken, HAmAal^G5, and HAmAal^G5 populations expressed a codon of CTA at the L-to-F kdr site encoding Leu, strongly corresponding to their susceptibility to insecticides.     

Expression of Proteolipid Protein Gene Is Directly Associated with Secretion of a Factor Influencing Oligodendrocyte Development

Abstract: Oligodendroglial cell death in the myelin proteolipid protein (PLP) mutants can be partially rescued by the environment factor(s) supplied by the wild-type cells in vivo and in vitro. It is possible that the presence of PLP or DM-20 results in secretion of a factor or factors in the CNS influencing oligodendrocyte development. We previously showed that DM-20 mRNA is produced in G26 mouse oligodendroglioma, B104 rat neuroblastoma, and B16 mouse melanoma but not in NIH3T3 mouse fibroblast cell lines. Culture supernatants from these cell lines were added to primary glial cell cultures from embryonic day 17 mouse brain. After 4 days, the number of oligodendrocytes present in cultures with supernatants from DM-20-producing cells (G26, B104, and B16) was significantly higher than that of control cultures but not with the NIH3T3 supernatant. To investigate more directly whether the PLP gene expression is involved in this process, NIH3T3 cells (nonneural cells) were forced to produce PLP or DM-20. By addition of the supernatants from the PLP/DM-20 transformants, the number of oligodendrocytes in the mixed glial cell cultures increased. This clearly demonstrates that the expression of the PLP gene is sufficient for and directly associated with secretion of a factor, which influences the oligodendrocyte development.     

Site-directed mutagenesis of the aspartokinase gene lysC and its characterization in Brevibacterium flavum

Using overlap extension polymerase chain reaction (PCR), five transformants of Escherichia coli containing site-directed mutagenized lys Cβ gene were generated and analysed. Exchange of C to A and C to T at nucleotide 1118 of the mutated lys Cβ gene causes a substitution of serine³⁰¹ in the wild-type enzyme for tyrosine³⁰¹ and phenylalanine³⁰¹ in the mutant enzymes, respectively. Enzyme assays showed that Brevibacterium flavum cells harbouring pSUMN18 with mutated lys Cβ genes exhibited 16–20 fold lower specific activities of aspartokinase as compared to that of host containing wild-type lys C gene. The mutation introduced into lys Cβ of B. flavum CCRC18271 resulted in partial feedback-resistant aspartokinase activity.