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Biomedical subjects

L Manuelidis

Publications and source records attributed to L Manuelidis.

At least 73 records · Page 4Linked to original sources
PubMed1987

Evidence suggesting that PrP is not the infectious agent in Creutzfeldt-Jakob disease.

It has been suggested that the infectious agents of scrapie and Creutzfeldt-Jakob disease (CJD) are 'prions' constituted by a protease resistant glycopeptide, PrP. To analyze the role of PrP in CJD infectivity we re-evaluated the biochemical characteristics of infectivity. First, when the infectious agent is not aggregated, infectivity is exquisitely sensitive to proteinase K treatment, and therefore a proteinase-K-resistant molecule (e.g. PrP) is unlikely to contain information essential for agent replication. Second, removal of sugar residues from Gp34 (the major precursor of the proteolyzed PrP band) failed to reduce infectivity. Third, one-half of the PrP peptides could be separated from significant infectivity using nondenaturing conditions with practical quantitative recovery of infectivity. These studies suggest that PrP in itself is unlikely to be the replicating component of the infectious agent. We suggest that these as yet undefined agents may consist of core protein and nucleic acid that are incompletely assembled in, and protected by, cell membranes. This hypothesis would explain the absence of conventional viral particles in these diseases, account for observed membrane pathology including altered behavior of endogenous membrane proteins, and would be consistent with the replication and transforming properties of CJD that indicate there is an agent specific nucleic acid.

Animals↗
PubMed1987

Use of a biotinylated probe and in situ hybridization for light and electron microscopic localization of Po mRNA in myelin-forming Schwann cells.

A biotinylated Po glycoprotein cDNA was hybridized in situ to aldehyde-fixed vibratome sections and to aldehyde-fixed thin sections of Lowicryl-embedded trigeminal ganglia of 15 day old rats. Alkaline phosphatase and peroxidase detectors were used for light microscopic (LM) studies and peroxidase or colloidal gold were employed for electron microscopic (EM) detection. In both LM and EM sections, probe was found in cytoplasmic areas of myelin-forming Schwann cells that were enriched in granular endoplasmic reticulum, demonstrating that these regions contain Po mRNA. Interestingly, Po mRNA tended to cluster in regions close to the developing myelin sheath. Relatively simple methods are here described for EM detection of mRNA with reasonable tissue preservation and high resolution. These methods may be useful for developmental and disease-related studies of specific mRNAs in mammalian tissues.

Animals↗
PubMed1987

Astrocyte gene expression in Creutzfeldt-Jakob disease.

Gliosis (hyperplasia and hypertrophy of astrocytes), the fundamental response of the central nervous system to tissue destruction, typically becomes apparent only several weeks after injury. The biochemical hallmark of this response is a marked accumulation of the specific astrocyte intermediate filament glial fibrillary acidic protein (GFAP). To date despite its importance, the mechanisms of GFAP gene regulation have not been studied in any developmental or pathological system to our knowledge, and the molecular signals for GFAP mRNA and protein accumulation are not defined. In Creutzfeldt-Jakob disease, a progressive dementing illness caused by an "unconventional agent," we find steadily increasing elevations of GFAP mRNA throughout the later stages of disease, using two independent GFAP cDNA clones, representing the entire insert or the 3'-noncoding region (pScr-1). The accumulation of GFAP, assessed immunocytochemically, follows GFAP mRNA elevation. A 5-fold stimulation of GFAP gene expression precedes the development of florid histologic lesions in the cerebrum, and in the cerebellum 5- to 6-fold increases occurred with no detectable spongiform changes at any time during disease. Therefore, these GFAP changes cannot be simply a response to neuronal damage. These effects are directly or indirectly caused by high local concentrations of agent and possibly involve a humoral factor.

Animals↗
PubMed1987

Immortality of cell cultures derived from brains of mice and hamsters infected with Creutzfeldt-Jakob disease agent.

Isolates from six patients with Creutzfeldt-Jakob disease (CJD) were injected into various strains of hamsters and mice, and the infective agent was propagated. Serially passaged cultures were established from these CJD agent-infected brains and from uninfected control brains. All healthy cultures (21 out of 21) from CJD agent-infected brains became immortal and/or transformed. In contrast only 3 out of 13 normal brain cultures became immortal, and the rest died out with serial propagation in vitro. The fact that permanent cell lines were readily derived from multiple rodent strains and all CJD isolates tested suggests that a transforming capability is an intrinsic property of CJD agents. This conclusion is supported by demonstrations of in vitro cell transformation by CJD infectious brain fractions. Although the molecular mechanism of transformation events associated with the CJD agent is not presently known, a provocative possibility is that the CJD agent has a direct effect on the host genome by mechanisms analogous to those known for slowly oncogenic retroviruses.

Animals↗
PubMed1986

Conservation in the 5' region of the long interspersed mouse L1 repeat: implications of comparative sequence analysis.

A clone of 7.1kb corresponding to the mouse L1 interspersed repeat family was selected for homology to a human interspersed repeat. This clone fairly represents mouse genomic members. Mapping of the clone revealed one common element at both the 5' and 3' ends in a head to tail arrangement, suggesting that at least some long L1 family members are tandemly arranged; genomic studies confirmed the unexpected tandem arrangement of a minor proportion of L1 members. A short SmaI tandem repeat appears to define the 5' end of most L1 family members. SmaI repeats may maintain, via a recursive regulatory function, the transcriptional viability of L1 members after retroposition events. A 2.5kb portion of the mouse L1 repeat that has not been previously sequenced is presented. It is 55-70% homologous to a corresponding portion of the human KpnI repeat family. Comparative sequence analysis revealed that one common open reading frame may conserve potential coding function across species. A second open reading frame bears an asymmetric distribution of codon replacements unlike both genes and pseudogenes. This latter feature could be consistent with a proposed chromosome organization function that is unrelated to peptide expression.

Amino Acid Sequence↗
PubMed1986

Creutzfeldt-Jakob infection increases adenylate cyclase activity in specific regions of guinea pig brain.

Creutzfeldt-Jakob disease is a slow, infectious, progressive neurological disorder which results in human dementia. Synaptic membranes from various brain regions of guinea pigs infected with Creutzfeldt-Jakob disease show increased guanyl nucleotide- or 5-hydroxytryptamine-mediated activation of adenylate cyclase. This increased enzyme activity appears due, primarily, to facilitated 'coupling' between the GTP-binding protein which stimulates adenylate cyclase (GNs) and the catalytic moiety of that enzyme rather than increased sensitivity to 5-hydroxytryptamine. It is possible that this phenomenon is due to direct effects of the Creutzfeldt-Jakob infectious agent, or a pathological product resulting from that agent, upon synaptic membrane adenylate cyclase.

Adenylyl Cyclases↗
PubMed1986

Characterization of major peptides in Creutzfeldt-Jakob disease and scrapie.

In Creutzfeldt-Jakob disease three major peptides cosediment with the infectious agent. These distinct peptides are not present in identical fractions from uninfected brain, and bind to polyclonal antibodies raised against "prion protein" purified by protease treatment. Three similar distinct peptides are also found in scrapie-infected brain fractions purified without the use of proteases. To clarify the relationships between these distinct peptides and prion protein, peptides were analyzed on immunoblots after cleavage with various glycosidases. There are two different 34-kDa peptides. One binds to ricin and cannot be detected by nonequilibrium pH gradient electrophoresis, presumably due to its highly acidic or basic pI. A second basic 34-kDa glycopeptide (Gp34) contains multiple terminal sialic acid residues responsible for charge heterogeneity (pI values, 7.2-7.8) and is reduced to a single spot with a pI value of 7.8 after neuraminidase treatment. After (but not before) neuraminidase treatment, secondary D-galactose-like sugars are detectable on Gp34, and a small number of N-acetylglucosamine residues probably represent the third sugar residue in an N-linked chain. When virtually all sugar residues are removed with endoglycosidase H the molecular weight of Gp34 is reduced by only approximately equal to 2 kDa. The residual peptide strongly binds antibody. A third acidic 24- to 26-kDa species (p26) also binds polyclonal antibodies but, in contrast to Gp34, was unaffected by any glycosidase treatment. Protease-treated peptides showed a very broad array of pI spots, consistent with a heterogeneous protein origin. None of the nonproteolyzed peptides show a clear relationship to prion protein. The number of sugar residues on Gp34 is not consistent with those estimated for prion protein. Although p26 could be the source of the "prion sequence," p26 does not appear to be glycosylated. Regardless, it is likely that all the major peptides described thus far are accumulated or modified normal gene products and are not integral components of the infectious agent.

Animals↗
PubMed1986

In vitro transformation elicited by Creutzfeldt-Jakob-infected brain material.

Previous studies indicated that tissue culture cells derived from Creutzfeldt-Jakob disease (CJD) brains possess neoplastic properties. In order to see if CJD brain material could itself transform cells in vitro, BALB/C-3T3 and normal hamster brain monolayer cultures were exposed to whole brain homogenate or synaptosomal-mitochondrial fractions derived from CJD or control brains. Cells were exposed for seven days to CJD or control brain material, washed, and then passaged at weekly intervals. Transformation was evaluated by loss of contact inhibition, replication of cells with absent or low serum content of the medium and significant colony formation in soft agar. Seven parallel experiments were done, and six were viable. All cultures exposed to CJD material containing greater than or equal to 10(4) LD50 infectious units were positive for transformation (five of six studies); the single negative experiment contained only 2 X 10(3) infectious units. Definitive transformation could be observed at 12-16 weeks after application of both mouse and hamster CJD material. BALB/C-3T3 cells exposed to control hamster or mouse brain material were studied for greater than 120 passages in vitro and showed no comparable changes or evidence of spontaneous transformation, i.e. they required serum for replication and produced essentially no colonies in soft agar. Similarly, a normal adult hamster brain culture exposed to control brain material showed no evidence of transformation, whereas the identically passaged normal brain culture exposed to CJD material not only became a permanent line, able to grow in soft agar, but also was capable of producing tumors in nude mice. Thus CJD transformation was not confined to special potentials of "permanent" BALB/C-3T3 cells. These experiments suggest that a genomic alteration can be effected by CJD material.

Animals↗
PubMed1985

Individual interphase chromosome domains revealed by in situ hybridization.

The position and arrangement of individual chromosomes in interphase nuclei were examined in mouse-human cell hybrids by in situ hybridization of biotinylated human DNA probes. Intense and even labeling of human chromosomes with little background was observed when polyethylene glycol and Tween-20 were included in hybridization solutions. Human interphase chromosomes were separated from each other in the nucleus, and were confined to well localized domains. Hybrid cells with a single human chromosome showed a reproducible position of this chromosome in the nucleus. Some chromosomes appeared to have a characteristic folding pattern in interphase. Optical section as well as electron microscopy of labeled regions revealed the presence of 0.2 micron wide fibers in each interphase domain, as well as adjacent, locally extended 500 nm fibers. Such fibers are consistent with previously proposed structural models of interphase chromosomes.

Animals↗
PubMed1985

Specific proteins associated with Creutzfeldt-Jakob disease and scrapie share antigenic and carbohydrate determinants.

Small amounts of brain tissue (2 g) infected with Creutzfeldt-Jakob disease (CJD) can be fractionated by using a simple 1-day method that includes lysis with N-lauroylsarcosine. Unique fibrils have been identified previously in scrapie- and CJD-infected tissue. These fibrils were abundant in final fractions. Preparations from human CJD autopsy material and from experimental hamster and guinea pig CJD all displayed readily identifiable fibrils that were not seen in control preparations. Thus, these methods appear to be of value in biopsy diagnosis of suspected human cases of CJD. Lysis with N-lauroylsarcosine quantitatively solubilized infectivity from membrane-rich fractions. Significant infectivity was recovered in microfractionations. After proteinase K digestion, a diffuse band at 29 kDa was detectable on NaDodSO4/PAGE. This 29-kDa material was not present in uninfected control brain and was similar to that seen in scrapie. Protein blots of human, guinea pig, and hamster CJD fractions were tested with an antibody raised against a 29-kDa band from mouse scrapie; 29-kDa proteins were labeled in all CJD and scrapie fractions but not in controls. These results indicate that specific proteins in both these diseases share common antigenic determinants. Ricin and wheat germ agglutinin, but not concanavalin A, also labeled a portion of the 29-kDa band from hamster CJD and hamster scrapie fractions, but they did not label any bands in normal hamster fractions at the same gel protein loads. When proteinase K treatment was omitted, specific bands of approximately equal to 35 kDa were detected in CJD samples. These results are consistent with the idea that some CJD- and scrapie-specific proteins are glycoproteins or sialoglycoproteins that can reside in or possibly derive from cell membranes.

Animals↗
PubMed1985

Indications of centromere movement during interphase and differentiation.

Mouse and human DNA sequences from centromeric and ribosomal domains were labeled with biotinylated deoxynucleotides and hybridized in situ to paraformaldehyde-fixed tissue culture cells. Centromeres were widely dispersed in most of these interphase nuclei. At late G2 phases of the cell cycle, centromeres appeared to coalesce and then to align in an orderly pattern, with discrete positional assignments for individuals chromosomes in metaphase and anaphase. Ribosomal cistrons were also organized in an orderly and defined fashion during mitosis. As soon as the nuclear membrane forms in early G1, centromeres rapidly disperse throughout the nucleus. Centromere patterns during G1 and S were indistinguishable in cultured cells, as determined by double-labeling experiments. Antibodies that bind to centric chromosomal proteins revealed the same patterns in cultured cells as those obtained with DNA sequence-specific probes. Large differentiated neurons display reproducible collections of centromeres in interphase that are very different from those seen in cultured cells. Neurons in widely divergent mammalian species, despite large differences in centromeric DNA sequences, maintain similar nuclear positions for these chromosomal segments. Similarly, ribosomal cistrons are positioned in comparable nuclear locales in neurons of divergent species. It is suggested that such arrangements reflect, or are necessary for, the function of a given cell type. Studies of large cerebellar neurons at critical times in development indicated a relative "movement" of centromeric domains, away from the nuclear membrane and toward the central nucleolar region. It is possible that the orderly and temporal positioning of centromeric, as well as of other chromosomal regions, is based on protein-nucleic acid interactions. Implications for trisomy 21 and other disorders involving chromosomal rearrangements, such as transposition, are considered from this perspective.

Animals↗
PubMed1984

Chromosomal and nuclear distribution of the HindIII 1.9-kb human DNA repeat segment.

A human interspersed repetitive DNA cloned in pBR322, the HindIII 1.9-kb (kilobase pair) sequence, was labeled with biotinylated dUTP and hybridized to acid-fixed chromosomes and paraformaldehyde-fixed whole cells in situ. Using our most sensitive detection techniques this probe highlighted on the order of 200 discrete loci, in punctate or banded arrays, that resembled a Giemsa-dark band pattern on chromosome arms. Interphase cells also displayed many discrete punctate spots of hybridization along chromosome fibers. The ubiquitous Alu sequence repeat also appeared to be concentrated in specific regions of the chromosome and predominantly highlighted Giemsa-light bands. Centromeric or ribosomal spacer DNA repeats used as controls in all studies gave the expected hybridization profiles and showed no non-specific labeling of chromosome arms. Cohesive groups of centromeric DNA arrays and rDNA clusters were observed in interphase nuclei. Refinements in methods for detecting biotin-labeled probes in situ were developed during these studies and calculations indicated that about 20 kb or more of the 1.9-kb repeat were present at each hybridization site. The chromosomal distribution of the 1.9-kb repeat suggests that this sequence may reflect, or participate in defining, ordered structural domains along the chromosome.

Base Composition↗
PubMed1984

Different central nervous system cell types display distinct and nonrandom arrangements of satellite DNA sequences.

Paraformaldehyde-fixed tissue from mouse cerebellum was hybridized with biotin-labeled satellite DNA for identification of centromeres. By using avidin-peroxidase conjugates, it was possible to define the nuclear position of centromeres at the ultrastructural level. Three-dimensional analysis of well-resolved centromere arrays were aided by computer reconstruction of serial sections. Different cell types displayed distinct, nonrandom centromere locations. In Purkinje neurons, the majority of detected sequences were clustered together around the central nucleolus, whereas in granule neurons, more numerous, dispersed centromere clusters were associated with the nuclear membrane. In Purkinje cells, peroxidase-labeled regions corresponded to dense heterochromatic aggregates were detected in Purkinje cells of several different species. These observations suggest that in these highly differentiated cells, the nuclear position of centromeres is maintained in evolution despite species differences in centromeric DNA sequence. Such defined ordering of centromeres may be integral to specific functional capacities.

Animals↗
PubMed1984

Active nucleolus organizers are precisely positioned in adult central nervous system cells but not in neuroectodermal tumor cells.

Active nucleolus organizing regions (NOR) were stained with silver in isolated nuclei and chromosomes, monolayer cultures, and tissues for electron microscopy. In nuclei isolated in 0.8-1.3 M urea, only the NOR stained with silver, and round or fiber-like regions with a diameter of 200-250 nm with a substructure were delineated at appropriate nucleolar or acrocentric chromosomal sites. In whole cells, additional (non NOR) silver binding regions were noted. Electrophoresis studies on nuclear isolates indicated at least two different nuclear protein subsets were responsible for the observed silver binding; the NOR protein(s) was most tightly bound with respect to urea. Interphase cells displayed a more extensive NOR network than mitotic cells, suggesting an increase in rDNA transcription during interphase. Mature sperm cells showed no active NOR regions. Interphase neuroectodermal tumor cells generally contained large multiple NOR indicating extensive activity; the position of NOR in the nucleus was variable from cell to cell, even in cloned lines. In contrast to tumor lines, central nervous system (CNS) cells from various species all showed highly reproducible or non-random NOR locations within the nucleus of each cell type. The NOR of large neurons were always central and single, whereas small granule cell neurons displayed a few small NOR that were positioned more peripherally. These findings suggest that in highly differentiated cells, the NOR region is precisely positioned in the nucleus, regardless of non-identical chromosome locations of the NOR in different species. The variability of NOR in tumor cells indicates profound changes in nuclear structure that may be part of the neoplastic transformation.

Adult↗