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L Manuelidis

Publications and source records attributed to L Manuelidis.

At least 37 records · Page 2Linked to original sources
PubMed1995

Viral particles are required for infection in neurodegenerative Creutzfeldt-Jakob disease.

Several models have been proposed for the infectious agents that cause human Creutzfeldt-Jakob disease (CJD) and sheep scrapie. Purified proteins and extracted nucleic acids are not infectious. To further identify the critical molecular components of the CJD agent, 120S infectious material with reduced prion protein (PrP) was treated with guanidine hydrochloride or SDS. Particulate and soluble components were then separated by centrifugation and molecularly characterized. Conditions that optimally solubilized residual PrP and/or nucleic acid-protein complexes were used to produce subfractions that were assayed for infectivity. All controls retained > 90% of the 120S titer (approximately 15% of that in total brain) but lost > 99.5% of their infectivity after heat-SDS treatment (unlike scrapie fractions enriched for PrP). Exposure to 1% SDS at 22 degrees C produced particulate nucleic acid-protein complexes that were almost devoid of host PrP. These sedimenting complexes were as infectious as the controls. In contrast, when such complexes were solubilized with 2.5 M guanidine hydrochloride, the infectious titer was reduced by > 99.5%. Sedimenting PrP aggregates with little nucleic acid and no detectable nucleic acid-binding proteins had negligible infectivity, as did soluble but multimeric forms of PrP. These data strongly implicate a classical viral structure, possibly with no intrinsic PrP, as the CJD infectious agent. CJD-specific protective nucleic acid-binding protein(s) have already been identified in 120S preparations, and preliminary subtraction studies have revealed several CJD-specific nucleic acids. Such viral candidates deserve more attention, as they may be of use in preventing iatrogenic CJD and in solving a fundamental mystery.

Animals↗
PubMed1994

Dementias, neurodegeneration, and viral mechanisms of disease from the perspective of human transmissible encephalopathies.

Our transmission experiments with human CJD emphasize the centrality of an exogenous infectious pathogen that can exist in symbiosis with its host for extended periods. Many latent or persistent viruses can cause neurodegenerative disease and may have a role in late onset dementias. There are reasons to believe that CJD infections may share properties with some of these latent viruses in causing dementia, and several retroviral mechanisms may be operative in CJD. In order to clarify viral-like attributes of the CJD agent we have closely followed infectivity and find the following: 1) the CJD agent has a virus-like size and density, and is biochemically separable from most host-encoded prion protein (PrP); 2) Endogenous retroviral IAP RNA sequences of 5,000 bases, as well as several gag-like nucleic acid binding proteins, co-purify with infectivity in preparations treated with high concentrations of anionic detergents and exhaustive nuclease digestion. They signify the purification of true viral cores rather than aggregation artifacts, and diminish claims that there are no protected nucleic acids of > 50 bases in highly purified infectious preparations; 3) In established hamster CJD, temporal studies show the agent has an effective doubling time of approximately 7.5 days in brain, consistent with complex host-viral interactions common to slow viral infections; 4) PrP-res does not correspond to titered levels of infectivity either in a biochemical or an in vivo setting but may function as a viral receptor that can modulate disease expression. Interestingly, functional changes in glial cells occur earlier than PrP-res changes, and indicate an important role for glial cells in evolving infections; 5) Human-rodent transmission studies suggest that CJD, or a CJD-like variant can be a common but latent infection of humans, with relatively infrequent expression of neurological disease. Susceptibility to disease can rest on host attributes and possibly age-related co-factors. Nonetheless, fundamental viral principles are also operative. Agent strain variants, viral burden, and the routes of infection are critical parameters for latency and disease expression. The properties described above have led me to return to the inclusion of CJD (and scrapie) in the panorama of conventional slow viral infections of the brain, as originally proposed by Sigurdsson. Identification of virus-specific molecules are essential for elucidating the role of these agents in the spectrum of human dementias.

Brain↗
PubMed1994

Endogenous viral complexes with long RNA cosediment with the agent of Creutzfeldt-Jakob disease.

A class of viruslike agents that induces Creutzfeldt-Jakob Disease (CJD) and scrapie remains undefined at the molecular level. Several investigators believe this infectious agent is constituted by a single host protein or 'prion', and have emphasized data that would seem to exclude the presence of any viral nucleic acids. However, more rigorous evaluations in scrapie have shown reasonably abundant nucleic acids. Additionally, in highly purified 120S CJD preparations that have been treated with nucleases, RNAs as long as 6,000 bases have been detected. Few nucleic acids have been characterized in either scrapie or CJD, but previous cloning experiments delineated relatively short LTR regions of the endogenous IAP retrovirus in 120S CJD preparations. We therefore used specific primers encompassing the entire IAP genome to test for the presence of long viral RNAs, and here show approximately 5,000 contiguous bases of the IAP RNA genome can be recovered from reasonable amounts of starting brain. The 3' env region of IAP is comparably truncated in CJD and normal preparations, and we find no evidence for IAP transduction of CJD-specific sequences. Because IAP cores can coencapsidate unrelated sequences, and are unusually resistant to physical and chemical treatments, it was relevant to find if cosedimenting cognate proteins of the IAP core, such as gag, could be detected. The predicted approximately 65 kd acidic gag protein, showing appropriate antigenic and nucleic acid binding features, was apparent in both one and 2-D Western blots. This data strongly indicates specific viral complexes cofractionate with the CJD agent. Interestingly, these nuclease resistant IAPs do not appear to be in morphologically recognizable 'R' particles. This cosedimenting viral assembly therefore provides a paradigm for non-particulate CJD complexes in infectious preparations. In developing strategies to identify a CJD specific sequence, cosedimenting IAPs can be used to assess the quality, length and recovery of RNAs extracted from highly resistant viral complexes.

Animals↗
PubMed1994

Genomic stability and instability in different neuroepithelial tumors. A role for chromosome structure?

Selected childhood and adult neoplasm exemplify fundamental differences in their propensity for genomic change. DNA replication is essential for the formation of neuroepithelial tumors, probably because the genome can be remodeled. Nonetheless, several differentiated and stable childhood neoplasms retain their nuclear controls for differentiation. In contrast, rapidly arising gliomas often show a variety of phenotypic changes. Genomic plasticity and instability allow gliomas to flexibly adapt to new environments. Gene changes (in DNA) can be limited in childhood tumors whereas more widespread genetic changes in malignant gliomas indicate a fundamental alteration in many chromosome regions. Can such regions be defined? We used one repeated DNA sequence (TTAGGG)n, present at the end of all normal human chromosomes, to investigate chromosome termini in more detail. Pulsed-field gel electrophoresis showed this region can be unusually variable, as several other multilocus probes did not reveal comparable changes. Because telomeres form unique chromosomal structures, and are thought to provide essential signals to position chromosomes in the interphase nucleus, it was pertinent to assess these regions by in situ hybridization. Many telomeric domains localized at variable as well as interior nuclear positions in glioma cells. These positions, which are presumably abnormal, may be generated by the DNA variants observed. Such position changes may contribute to the more general 'disorder' observed in glioma nuclei. Other chromosome domains with a unique DNA-protein structure may define additional genomic loci that are preferentially modified in neoplasia. A fundamental understanding of chromosome structure should clarify the problem of multilocus instability in glioblastoma.

Age of Onset↗
PubMed1993

A transmissible Creutzfeldt-Jakob disease-like agent is prevalent in the human population.

The etiology of most human dementias is unknown. Creutzfeldt-Jakob disease (CJD), a relatively uncommon human dementia, is caused by a transmissible virus-like agent. Molecular markers that are specific for the agent have not yet been defined. However, the infectious disease can be transmitted to rodents from both brain and infected buffy coat (blood) samples. To determine whether human CJD infections are more widespread than is apparent from the low incidence of neurological disease, we attempted to transmit CJD from buffy coat samples of 30 healthy volunteers who had no family history of dementing illness. Primary transmissions from 26 of 30 individuals produced CJD-like spongiform changes in the brains of recipient hamsters at 200-500 days postinoculation. This positive evidence of viremia was found for individuals in all age groups (20-30, 40-50, and 61-71 years old), whereas 12 negatively scored brain samples failed to produce similar changes in hamsters observed for > 900 days in the same setting. We suggest that a CJD agent endemically infects humans but only infrequently produces an infectious dementia. Disease expression is likely to be influenced by several host factors in combination with viral variants that have altered neurovirulence.

Adult↗
PubMed1993

Nucleic acid binding proteins in highly purified Creutzfeldt-Jakob disease preparations.

The nature of the infectious agent causing human Creutzfeldt-Jakob disease (CJD), a slowly progressive dementia, is controversial. As in scrapie, no agent-specific proteins or nucleic acids have been identified. However, biological features of exponential replication and agent strain variation, as well as physical size and density data, are most consistent with a viral structure--i.e., a nucleic acid-protein complex. It is often assumed that nuclease treatment, which does not reduce infectious titer, leaves no nucleic acids of > 50 bp. However, nucleic acids of 500-6000 bp can be extracted from highly purified infectious complexes with a mass of approximately 1.5 x 10(7) daltons. It was therefore germane to search for nucleic acid binding proteins that might protect an agent genome. We here use Northwestern blotting to show that there are low levels of nonhistone nucleic acid binding proteins in highly purified infectious 120S gradient fractions. Several nucleic acid binding proteins were clearly host encoded, whereas others were apparent only in CJD, but not in parallel preparations from uninfected brain. Small amounts of residual host Gp34 (prion protein) did not bind any 32P-labeled nucleic acid probes. Most of the minor "CJD-specific" proteins had an acidic pI, a characteristic of many viral core proteins. Such proteins deserve further study, as they probably contribute to unique properties of resistance described for these agents. It remains to be seen if any of these proteins are agent encoded.

Animals↗
PubMed1993

Protected endogenous retroviral sequences copurify with infectivity in experimental Creutzfeldt-Jakob disease.

Scrapie and Creutzfeldt-Jakob disease (CJD) are caused by infectious agents that are defined phenomenologically. No agent-specific molecules or particles have been identified. Biological properties, such as exponential agent replication and strain variation, as well as physical characteristics of infectivity indicate a protected viral structure. A host membrane glycoprotein of 34 kDa ("prion" protein) that aggregates at end stages of disease is clearly important in pathology and susceptibility to infection, but has no demonstrable infectivity in any purified or recombinant form. Thus a characterization of more viral-like molecules is important. In order to identify viral-like nucleic acids we previously developed methods to substantially purify the human CJD agent from experimentally infected hamster brains, and demonstrated selected retroviral-like LTR bands at pg levels that were insufficient for sequencing. To further define these and other viral-like sequences we cloned nucleic acids from highly infectious CJD fractions, and tested the efficacy of our methods using a selected retroviral probe. RNA extracted from an infectious 120 S Gaussian peak, which is reproducibly purified approximately 100,000 fold with respect to starting nucleic acids, and contains approximately 20% of the initial brain infectivity, was used to generate a cDNA library in a sequence independent amplification strategy for low levels of RNA (< 6 ng). Reconstituted strong stop experiments using several retroviral tRNA primers had indicated that Syrian hamster IAP (SHIAP) sequences should be present in both CJD and uninfected control fractions. Because SHIAP particles are extremely resistant to denaturation, their representation in a cDNA library would imply adequate extraction of other protected RNAs of viral origin. At least 900 bases of the Syrian hamster retroviral IAP genome were unambiguously identified in the cDNA library, and in independent PCR walks with selected primers, all of which were based on our cloned sequences. Sequencing confirmed the presence of protected LTR and adjacent retroviral motifs. Because these sequences were also present in control preparations they may represent normal endogenous viral contaminants that cosediment with infectivity in size and density gradients. On the other hand, LTRs can drive the expression of many diverse sequences, and it remains to be seen if CJD specific sequences are either transduced, or copackaged with, protected IAP complexes. The effective extraction and amplification of highly protected SHIAP nucleic acids of significant length sets the stage for identifying additional protected viral elements that may specify the CJD agent.

Animals↗
PubMed1992

Analysis of Creutzfeldt-Jakob disease infectious fractions by gel permeation chromatography and sedimentation field flow fractionation.

Gel permeation chromatography and sedimentation field flow fractionation (SF3) were used to further analyze highly infectious fractions from Creutzfeldt-Jakob disease (CJD) infected hamster brain. These analyses defined the relative molecular mass and physical size of the Creutzfeldt-Jakob disease (CJD) agent with greater precision than previously possible. Highly purified disaggregated fractions yielded single, homogeneous Gaussian peaks with both methods. The relevant analytical peaks contained protein-nucleic acid complexes with an M(r) of approximately 1.5 x 10(7) daltons and a mean radius of approximately 30 nm. The experimental evidence further solidifies the concept of an infectious agent that resembles a viral core rather than a simple protein.

Animals↗
PubMed1991

Heterochromatic features of an 11-megabase transgene in brain cells.

Transgenic mice provide a remarkable experimental setting for the study of nuclear architecture. The three-dimensional localization and fine structure of a foreign DNA within the mouse genome can be conveniently followed by using high-resolution in situ hybridization. Foreign DNAs designed with specific characteristics, such as base bias, sequence motif(s), and size can stably integrate into finite positions on host chromosomes. Thus the relative importance of each of these characteristics in determining the three-dimensional nuclear position and the detailed morphology of the transgene can be evaluated in different cell types. The aim of this study was to evaluate a transgene with sequence characteristics that might contribute to the de novo formation of heterochromatin in interphase nuclei. The structure of a phenotypically silent 11-megabase transgene, containing tandem repeats of beta-globin-pBR sequences integrated into the peritelomeric region of both mouse chromosome 3 homologs, was determined in adult brain cells. Neurons that are largely euchromatic were especially informative in three-dimensional studies of transgene position. The two transgenic loci behaved much like centromeric or paracentromeric A + T-rich satellite DNAs of comparable length from a single chromosome; one or both transgene domains localized together with centromeric satellite DNA on the nucleolus. This is an unusual nuclear position for a telomeric or chromosome arm region that does not contain a substantial amount of constitutively heterochromatic satellite DNA. G + C richness did not prevent these regions from assembling as dense heterochromatic bodies of approximately 1 micron3 in volume. Ultrastructurally, transgenic domains were often intimately connected with constitutive heterochromatin and were highly condensed. Labeled supercoils, formed by a discrete approximately 250-nm-wide fiber, were observed in oblique thin sections through the center of the domain. The structural data were consistent with negligible transcriptional activity detected for this locus, as well as the predicted conformation of constitutive heterochromatin. Interestingly, in transgenic but not control mice, a substantial number of large neurons, including approximately 30% of cerebellar Purkinje cells, showed excessive invaginations of the nuclear membrane.

Animals↗
PubMed1991

Integration site preferences of endogenous retroviruses.

Retroviruses have the ability to integrate into the genome of their host, in many cases with little apparent sequence or site specificity. However, relatively few studies have addressed more general features of chromosomal integration. In this study we directly visualized the chromosomal organization of three representative endogenous retroviruses by in situ hybridization. Because there are 50-1000 copies of each of these retroviruses in the genome, it was possible to evaluate repeated integration events. Each retroviral sequence exhibited a unique and markedly different integration pattern. In order to characterize more precisely the chromosomal domains targeted by each retrovirus, later replicating domains were differentially labeled. Additionally, prototypic SINES and LINES (short and long interspersed reiterated sequences), which are inhomogeneously distributed on chromosome arms, were simultaneously detected. Retroviral copies of greater than or equal to 2 kb were found (i) exclusively in a discrete set of later replicating domains, most of which have the staining characteristics of constitutive heterochromatin, (ii) widely represented in disparate types of chromosome domains, or (iii) almost completely confined to CpG Alu-rich regions that are known to be early replicating. Retroviral elements in Alu-rich domains would be expected to be actively transcribed in all cells. Surprisingly, hybridization to blots of brain RNA showed an approximately 25 fold lower level of transcripts from these Alu associated elements than from retroviral sequences restricted to later replicating, heterochromatic domains. Retroviral insertions may subvert more typical transcriptional characteristics of a domain. The present results indicate that there are highly specific integration patterns for each endogenous retrovirus that do not readily relate to their sequence or particle classification. Each host genome may utilize these elements for contrary, and possibly beneficial functions.

Adult↗
PubMed1990

A view of interphase chromosomes.

Metaphase chromosomes are dynamically modified in interphase. This review focuses on how these structures can be modified, and explores the functional mechanisms and significance of these changes. Current analyses of genes often focus on relatively short stretches of DNA and consider chromatin conformations that incorporate only a few kilobases of DNA. In interphase nuclei, however, orderly transcription and replication can involve highly folded chromosomal domains containing hundreds of kilobases of DNA. Specific "junk" DNA sequences within selected chromosome domains may participate in more complex levels of chromosome folding, and may index different genetic compartments for orderly transcription and replication. Three-dimensional chromosome positions within the nucleus may also contribute to phenotypic expression. Entire chromosomes are maintained as discrete, reasonably compact entities in the nucleus, and heterochromatic coiled domains of several thousand kilobases can acquire unique three-dimensional positions in differentiated cell types. Some aspects of neoplasia may relate to alterations in chromosome structure at several higher levels of organization.

Animals↗
PubMed1990

Detection of monosomy 7 in interphase cells of patients with myeloid disorders.

Six patients, five with acute myeloid leukemia (AML) and one with a myelodysplastic syndrome (MDS), were found to have monosomy 7 by conventional cytogenetics at diagnosis. Repetitive DNA sequences from the heterochromatic region of human chromosomes 1 and 7 were used as probes for in situ hybridization experiments on interphase cells of these patients. A double hybridization protocol was used to reveal the particular chromosomes as distinct spots or clusters of signals within interphase nuclei. The chromosome 1 sequence served as an internal control. Simultaneous detection of the sequences showed the presence of two normal number 1 chromosomes and a missing 7 chromosome from individual cells. While cytogenetic preparations showed only -7 metaphases in 3 AML and 1 MDS patients, in situ hybridization of interphase cells showed many normal cells as well as the presence of -7 in fully mature granulocytes. One AML patient studied in remission showed only normal metaphases yet had 9% interphase cells with a missing 7 and relapsed within 3 months. We conclude that examination of interphase cells by in situ hybridization provides clinically useful data since every cell including mature granulocytes can be examined, the lineage of a cell can be determined, and efficacy of differentiation therapy can be evaluated.

Chromosome Deletion↗
PubMed1990

A unified model of eukaryotic chromosomes.

A revised model of DNA packaging into chromosomes is presented. Its features are consistent with observed structural dimensions and the molecular periodicities related to transcription, replication and matrix attachment domains. The transitions between euchromatic, heterochromatic and metaphase states are explained simply. Molecular and physical properties of chromosomal bands, and their correlation with specific DNA sequence motifs are discussed.

Animals↗
PubMed1990

P0 glycoprotein mRNA distribution in myelin-forming Schwann cells of the developing rat trigeminal ganglion.

A biotinylated P0 cDNA was hybridized in situ to aldehyde-fixed vibratome sections of trigeminal ganglia from day 2, day 7, day 15, day 30 and adult rats. Nickel-enhanced horseradish peroxidase (HRP) was used in an antibody sandwich method to detect hybridization. After postfixation in osmium tetroxide, the sections were dehydrated in ethanol and embedded in epon. At each age, some vibratome sections were used to count the HRP-positive and HRP-negative myelin-forming Schwann cells. The percentage of HRP-positive myelin-forming Schwann cells in ganglia from day 2, 7, 15, 30, and adult rats were 31%, 56%, 47%, 12% and 3%. In sections of ganglia from 2-day-old rats, studied by light and electron microscopy, peroxidase reaction product localizing hybridized P0 mRNA was found on profiles of granular (rough) endoplasmic reticulum (RER) in perinuclear regions of Schwann cells which had formed two to three compact myelin lamellae. Peroxidase deposits were larger and more numerous in the cytoplasm cells with thicker myelin sheaths. At day 7, some Schwann cells had long external mesaxons; the cytoplasm between these mesaxons and the cell surface often contained abundant HRP-stained profiles of RER. In sections from day 7 and day 15 ganglia, substantially more reaction product was found. In each myelin-forming Schwann cell, the amount was generally proportional to the size of the newly formed myelin sheath. HRP deposits were observed all along the outer surfaces of myelin segments at these ages, and their distribution corresponded to that of the RER.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Nuclease treatment results in high specific purification of Creutzfeldt-Jakob disease infectivity with a density characteristic of nucleic acid-protein complexes.

Representative preparations of partially purified Creutzfeldt-Jakob disease (CJD), including disaggregated density gradient fractions, were treated with a variety of nucleases. RNases as well as exhaustive digestions with micrococcal nuclease did not significantly diminish infectivity, but resulted in an approximately 7,000-fold specific purification of infectivity with respect to nucleic acid. Protected nucleic acids included species of up to 2,000 bases in length. After nuclease treatment, infectivity co-migrated with nucleic acid-protein complexes at a density of 1.27 g/cm3 in sucrose. Substantial specific protein purification were also achieved in the gradient step (approximately 11,000-fold), where 70% the host Gp34 ("prion protein") as well as other free proteins separated from infectivity. These CJD purifications are better than those previously attained in scrapie, and may be useful for further studies of non-host protein and nucleic acid species. The data are consistent with the hypothesis that CJD-like agents are composed of nucleic acid-protein complexes.

Animals↗
PubMed1990

Nuclease-resistant polyadenylated RNAs of significant size are detected by PCR in highly purified Creutzfeldt-Jakob disease preparations.

The molecular nature of the 'unconventional viruses' that cause slow, progressive brain deterioration is still poorly understood. As part of a reinvestigation of potential agent-specific nucleic acids, we developed a protocol for enriching agent-specific sequences. This protocol uses extensive micrococcal nuclease digestion followed by rate zonal sucrose sedimentation. Most of the infectivity in the gradient (84%) had a characteristic mean size of approximately 120S, and was resolved from 70% of a host glycoprotein (PrP) that can cosediment with infectivity. In infectious size fractions, nucleic acids were reduced approximately one million-fold with respect to starting brain homogenate, and specific purification of infectivity was approximately 100,000-fold with respect to nucleic acid. Using a novel polymerase chain reaction strategy, we were able to amplify RNA species in these fractions. Remarkably, host polyadenylated sequences of 1 to over 4 kb were detected in the nuclease-protected infectious fractions. These strategies set the stage for the identification of similar nucleic acids that may be specific for the CJD agent.

Base Sequence↗