Search

In this study, we analyzed serum for the presence of antibodies to gamma-synuclein in patients with amyotrophic lateral sclerosis (ALS) compared to the control group of patients with other neurological diseases and healthy control donors. As a result, antibodies against gamma-synuclein are not an ALS-specific feature and have been identified in patients with ALS as well as in the control group patients. Patients with the impaired cerebral circulation showed increased incidence of autoantibodies to gamma-synuclein, yet the difference lacks statistical representativeness due to limited sample size.

gamma-Synuclein is a small cytoplasmic protein implicated in neurodegenerative diseases and cancer. However, the mechanism of its involvement in diseases is not clear. We studied the role of gamma-synuclein in the regulation of matrix metalloproteinases in retinoblastoma cell culture. Matrix metalloproteinases play important roles in the remodeling of extracellular matrix implicated in tumor progression and in the neurodegenerative diseases. Western blot and zymography data demonstrated a moderate elevation of matrix metalloproteinases-2 and significant upregulation of matrix metalloproteinases-9 in stable cell lines overexpressing gamma-synuclein. No effect of gamma-synuclein overexpression on matrix metalloproteinases-1 level or activity was found. Chloramphenicol-acetyltransferase assay demonstrated that overexpression of gamma-synuclein increases the efficiency of the matrix metalloproteinases-9 promoter. This increment of promoter activity may be mediated by the AP-1 binding site(s), since point mutations in one of these sites (Pr18 or Pr19) and elimination of the distal AP-1 site (Pr14) reduced the increment of promoter activity.

Hypertriglyceridemia and hyperlipidemia are common disorders associated with coronary artery disease and premature death. The proteins encoded by the apolipoprotein (apo) A-I/C-III/A-IV gene cluster are involved in the metabolism of both triglycerides and cholesterol. In a large sample of individuals from the ARIC study, six polymorphic markers were typed and plasma lipid values were measured to determine whether the well-established association between the Sst I S2 allele in the 3'-untranslated region of the apo C-III gene and hypertriglyceridemia was due to disequilibrium with variation in the 5' regulatory region of the apo C-III gene. The Sst I polymorphism was significantly associated with hypertriglyceridemia (P = .006) but not with carotid artery wall thickness, plasma apo C-III levels, or elevated cholesterol. The frequency of the S2 allele was 0.14 in those with high triglyceride levels and 0.05 in those with low triglyceride levels. None of the 5' flanking polymorphisms were significantly associated with any of the plasma lipids studied. There was substantial linkage disequilibrium between the Sst I polymorphism and each of the 5' apo C-III polymorphisms; however, the significant association between the apo C-III haplotypes and hypertriglyceridemia (odds ratio, 4.0; P < .0001) was solely attributable to the effects of the Sst I polymorphism (odds ratio, 3.96). As a part of these analyses, we also defined a unique haplotype that is inversely associated with the occurrence of hypertriglyceridemia, suggesting further molecular analyses of this important gene region.

We describe two rapid non-radioactive methods for the analysis of polymorphic markers in the flanking region of the human apolipoprotein CIII gene. The polymorphic markers comprise previously described variable sites located upstream from the coding region of the gene (C-641-->A, G-630-->A, T-625-->deletion, C-482-->T, and T-455-->C) and a polymorphic SstI/SacI site in the apoC-III 3' untranslated region. The first method is allele-specific amplification (ASA) with primers complementary to the normal ("wild-type") allele or to the variable ("mutant") allele at their 3' ends. The other is allele-specific oligonucleotide hybridization (ASO hybridization) with pairs of probes labeled by digoxigenin. Comparison with sequencing data showed that both methods are reliable for polymorphism analysis.

The effect of apolipoprotein E (apo E) common genotypes on fasting retinyl palmitate (RP) level was studied in 344 white individuals, of which 130 had intimal thickening of the carotid artery ("cases") and 214 were controls. In this sample the common apo E genotypes possessed a statistically significant effect on fasting RP level in cases, while in controls the effect observed was not statistically significant. It is suggested that the effect of apo E may be expressed at the level of remnant clearance particles. Additionally, in cases other traits interact with the apo E genotype to influence fasting RP level.

The structural organization of the upstream regulatory regions of the apolipoprotein genes is discussed in relation to tissue-specific gene expression. Comparison of the sequences of the regulatory and coding parts of the genes that make up this multigene family shows that some members of the family with homologous coding regions may differ in the organization of their regulatory regions (apoE compared with apoA-IV, apoC-III, or apoC-II). On the other hand, some apolipoprotein genes with different primary structures of their coding regions and different intron-exon organization show similarities in the structural organization of their regulatory regions (e.g. apoC-III and apoB).

The results of Southern blotting demonstrate that sup2 is a unique gene in Saccharomyces cerevisiae that does not possess homologous sequences in the yeast genome. The direct hybridization of DNA fragments, containing cloned sup1 and sup2 genes, did not reveal any structural homology between these two genes. By Northern blotting analysis the sizes of the transcripts were determined to be 1.6 kb for sup1 gene and 2.5 and 1.4kb for sup2 gene. Experiments with RNA isolated from yeast mutant with impaired splicing demonstrated that sup1 and sup2 genes do not contain introns.

Recessive suppressor mutations in yeast Saccharomyces cerevisiae alter a component of the cytoplasmic ribosomes, relaxing the control of translational fidelity. As a consequence ribosomes can misread nonsense codons as amino acids (Surguchov et al. 1980a). The suppressor mutants are often respiratory deficient, being unable to grow on non-fermentable substrates. The study of the cytochrome spectra has revealed that the cytochrome b and aa3 contents were lower in the mutants than in the parent strains. Furthermore, the suppressor mutations often cause hypersensitivity to paromomycin and neomycin on media with a non-fermentable source of carbon. Some of the suppressor mutants exhibited both erythromycin and chloramphenicol-dependent growth on media containing ethanol or glycerol as a sole carbon source. These results suggest that the mutations altering cytoplasmic ribosomes may simultaneously impair the mitochondrial translation. A coupling of cytoplasmic and mitochondrial protein synthesis in yeast cells is proposed. The existence of a common protein component participating both in mitochondrial and cytoplasmic protein synthesis apparatus is discussed.

Primary structures of yeast sup2 gene and polypeptide product coded by the gene are compared with the current nucleotide and amino acid sequence data base. The amino acid sequence of the sup2 product shows homology to elongation factors from different sources. Especially high homology is found in the regions, corresponding to conservative aminoacyl-tRNA- and GTP-binding domains, described in elongation factors and other proteins. The data obtained are discussed in relation to the functions of sup2 polypeptide product in protein synthesis.

The shift of recessive suppressor mutant of yeast Saccharomyces cerevisiae from permissive to restrictive conditions is accompanied by polysome decay and accumulation of 80 S ribosomes (Smirnov et al., 1976). In this paper some properties of 80 S ribosomes are studied. It is demonstrated that polysome decay under non-permissive conditions is not the consequence of the impairment of RNA synthesis. More than 70% of 80 S ribosomes accumulated under non-permissive conditions contain bound peptidyl-tRNAs localized in P-ribosomal site. tRNA moiety of bound peptidyl-tRNA is able to accept all 20 natural amino acids after chemical deacylation. Therefore it is not a specific isoacceptor species but rather total tRNA that is bound to ribosomes. The polypeptide residues of these peptidyl-tRNAs are heterogeneous in size. Their molecular weights are comparable with the molecular weights of the completed polypeptides. Some of the 80 S ribosomes accumulated under non-permissive conditions contain poly-A+ RNA. In conclusion, possible mechanism of the impairment of translation under non-permissive conditions in recessive suppressor strain is discussed.

The influence of peptidyl-tRNA on the dissociation of yeast 80 S ribosomes into subunits was studied. For this purpose temperature-sensitive (ts) suppressor strain of yeast Saccharomyces cervisiae carrying a defect in peptide chain termination was used. It was found that peptidyl-tRNA did not influence the dissociation of ribosomes either at high salt concentration or in the presence of dissociation factor (DF) from yeast. After dissociation of yeast ribosomes in 0.5 M KCl, peptidyl-tRNA remains bound to the 60 S subunit. Some characteristics of the termination process and release of nascent polypeptides from yeast ribosomes are discussed.

The ribosomal protein patterns of recessive suppressor strain and parent strain of Saccharomyces cerevisiae were analyzed by two-dimensional polyacrylamide gel electrophoresis. About 30 proteinspots were found for ribosomal proteins of small subunit for both mutant and parent strain. These patterns do not differ from each other neither in intensity of staining, nor in mobility of spots. 41 protein spots were found in electrophoregrams of 60S ribosomal proteins both from parent strain and recessive suppressor strain. The electrophoretic picture of the 60S proteins from the parent and mutant strains is similar except the intensity of staining of the L30 spot. This protein is present in 60S subunit of suppressor strain and completely absent or only weakly stained on electrophoregrams of ribosomal proteins of parent strain. The possible relationships between the content of L30 protein and the mechanism of recessive suppression in yeast are discussed.

A conditional respiratory deficiency in yeast Saccharomyces cerevisiae is expressed as a result of a nuclear mutation in sup1 and sup2 genes (II and IV chromosomes, respectively), coding for a component of cytoplasmic ribosomes (Ter-Avanesyan et al. 1982). One such strain is studied here in detail. The strain is temperature-dependent and expresses a respiratory deficient phenotype at 20 degrees C but not at 30 degrees C. Moreover, the strain is simultaneously chloramphenicol-dependent and is able to grow on media containing glycerol or ethanol as a sole carbon source only in the presence of the drug. Chloramphenicol has a differential effect on protein synthesis in mitochondria of the parent strain and the mutant. Since chloramphenicol is a ribosome-targeting antibiotic we suggest that the differential effect of the drug on parent and mutant mitochondrial protein synthesis is due to the altered properties of mito-ribosomes of the mutant compared to those of the parent strain. Mitochondria of the mutant synthesize all the mitochondrially encoded polypeptides, however, in significantly lowered amounts. A suggestion is put forward for the existence of a common component (a ribosomal protein) for mito and cyto-ribosomes.

It was found that the phenotypic suppression induced by the paromamine-containing antibiotic paromomycin could be significantly strengthened by a ribosomal suppressor mutation in yeast Saccharomyces cerevisiae. As a result the suppressor efficient towards ochre mutations in the presence of paromomycin acquired the ability to suppress both amber and opal mutations. It is suggested that phenotypic suppression by paromomycin and genotypic suppression by sup 1 both involve a similar mechanism of misreading.

Using sucrose density centrifugation and gel filtration of a 105000 X g supernatant of Bacillus brevis two enzymic activities of glycyl-tRNA synthetase were separated. Enzyme catalyzing the aminoacylation of tRNA (E1) elutes in a high-molecular-weight region. Enzyme active in glycylhydroxamate formation (E2) elutes from a Sephadex gel column and sediments in sucrose density gradient in a region of relatively low molecular weight. The presence of two enzymic activities does not depend on the method of cell disruption; their proportion does not change when protease inhibitor (diisopropylphosphorofluoridate) is added to the extraction buffer. Both E1 and E2 were purified to a nearly homogeneous state. Sedimentation coefficients (sw,20) were found to be 8.6 S and 3.6 S and molecular weights 226000 and 66000 for E1 and E2, respectively. During storage, E1 dissociates into two components, one of which has electrophoretic mobility identical to E2. The molecular weight of the other component is about 1600000. Electrophoresis of E1 in the presence of sodium dodecylsulfate reveals two bands corresponding to molecular weights of 81000 and 30000. Under these conditions, E2 dissociates into a polypeptide with a molecular weight of 30000. Valine was found to be the N-terminal amino acid for E2 and both valine and glutamic acid were N-terminal amino acids for E1. It is concluded that E1 is a tetrameric protein consisting of two large and two small subunits (alpha2beta2). E2 is a component of E1 with a structural formula alpha2.

7-ketocholesterol, one of the major product of autoxidation of dietary cholesterol, was found to inhibit secretion of very low density lipoprotein [14C]cholesterol, [14C]triacylglycerol and [35S]apoprotein B,E,C by cultured human and rabbit hepatocytes. A parallel inhibition (about 35%) of cholesterol synthesis but not of triacylglycerol formation was observed. Incubation with 10 micrograms/ml of oxysterol also reduced the total apo-B secretion measured by ELISA and increased intracellular apo-B mRNA level. These results seem to indicate that 7-ketocholesterol decreases secretion of very low density lipoprotein (VLDL) particles and exerts inhibitory effects on apo-B production at the co-translational or posttranslational level.

It has been shown that recessive suppressor mutations in the yeast Saccharomyces cerevisiae may cause sensitivity towards low temperatures (very slow growth or lack of growth at 10 degrees C). One of the sup 1 low temperature sensitive (Lts-) mutants, 26-125A-P-2156, was studied in detail. After a prolonged period of incubation (70 h) under restrictive conditions the protein synthesis apparatus in the mutant cells was irreversibly damaged. In addition, Lts- cells incubated under restrictive conditions synthesize unequal amounts of ribosomal subunits, the level of 60 S subunit being reduced. It has been suggested that the recessive suppression is mediated by a mutation in the gene coding for 60 S subunit component, probably a ribosomal protein. The mutation leads simultaneously to a defect in the assembly of 60 S subunit and to low-temperature sensitive growth of the mutant.

A plasmid, pYsup1-1, containing a DNA fragment able to suppress the recessive mutant phenotype of the suppressor locus sup1 (allele sup1-ts36) of Saccharomyces cerevisiae was isolated from a bank of yeast chromosomal DNA cloned in cosmid p3030. The complementing gene was localized on a 2.6 kb DNA fragment by further subcloning. Evidence is presented that the cloned DNA segment codes for the sup1 structural gene (chromosome IIR).

A nucleotide sequence of the yeast Saccharomyces cerevisiae omnipotent suppressor SUP2 (SUP35) gene is presented. The sequence contains a single open reading frame (ORF) of 2055 bp, which may encode a 76.5-kDa protein. A single transcript of 2.3 kb corresponding to a complete ORF is found. Analysis of codon bias suggests that the SUP2 gene is not highly expressed. The C-terminal part of the deduced amino acid sequence shows a high homology to yeast elongation factor EF-1 alpha, whereas the N-terminal part is unique for the SUP2 protein. The N terminus contains a number of short repeating elements and possesses an unusual amino acid composition. Analysis of the nucleotide and deduced amino acid sequences indicates that three additional proteins could possibly be expressed, two of which might be initiated on internal ATG codons and a third might be formed by alternative splicing. One of these proteins is supposed to be imported into mitochondria. Possible functions of the SUP2 gene product(s), especially its putative activity as a soluble factor controlling the fidelity of translation, are discussed.

Incubation of cultured rabbit hepatocytes with beta very low density lipoproteins (beta-VLDL) induces a dose-dependent increase in cell cholesterol (CH) content and VLDL apoprotein (apo) B and E secretion without change in apo-B mRNA level. These data suggest that beta-VLDL may exert a stimulatory effect on hepatic apo-B production at the co-translational and/or posttranslational level.

Mutants of the yeast Saccharomyces cerevisiae carrying ribosomal suppressor mutations in either sup1 or sup2 genes express a higher sensitivity to paromomycin - aminoglycoside antibiotic known to induce translational errors in eukaryotes. Paromomycin also induces a phenotypic suppression of all three types of nonsense mutations (ochre, amber and opal), missense mutations and frame-shift mutations. The influence of paromomycin on the activity of ribosomal suppressors has at least two aspects: (1) the drug increases translational ambiguity in sup1 and sup2 mutants in vitro and (2) it induces the alteration (extension or restriction) of sup 1 or sup2 suppression spectra in vivo. A modification of selectivity of the mutant ribosomes towards different tRNAs in the presence of paromomycin is proposed.

Mutations in sup1 and sup2 genes may cause cycloheximide-dependent growth in yeast Saccharomyces cerevisiae. Two classes of such mutants are described in the paper: 1) high temperature sensitive mutants, which do not express their sensitivity to nonpermissive temperature in the presence of cycloheximide (conditionally dependent) and 2) mutants unable to grow in the absence of the drug (true dependent). Some of the mutants of both classes express dependence toward another antibiotic - trichodermine. The binding of H(3)-labelled cycloheximide studied by equilibrium dialysis has demonstrated that both 80S ribosomes and 60S subunits isolated from conditionally dependent mutant showed a higher affinity for the drug compared to that of a parent strain. The number of binding sites per ribosome or per 60S subunit in the cycloheximide dependent mutant remains unchanged.Circular dichroism spectra of a mutant ribosomes in the presence as well as in the absence of antibiotic revealed that sup1 and sup2 mutations alter conformation of the yeast cytoplasmic ribosomes. The binding of cycloheximide to mutant ribosomes induces a conformational shift, which presumably compensates for their functional defect.

Ribosomal proteins from the cytoplasm and mitochondria of the yeast Saccharomyces cerevisiae were compared by immunoblotting techniques. Antibodies raised against cytoplasmic ribosomal proteins cross-react with five mitochondrial ribosomal proteins, four of which are located in the large and one in the small mitochondrial subunits. The possible existence of common ribosomal proteins for cytoplasmic and mitochondrial ribosomes is discussed.