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Biomedical subjects

M C Graves

Publications and source records attributed to M C Graves.

At least 55 records · Page 3Linked to original sources
PubMed1990

Attenuated murine cytomegalovirus binds to N-acetylglucosamine, and shift to virulence may involve recognition of sialic acids.

Treatment of cells with lectins specific for N-acetylglucosamine (GlcNAc) blocked infection by mouse cytomegalovirus (MCMV), and GlcNAc pretreatment of the lectin blocked this effect. MCMV failed to infect N-acetylglucosaminidase (GlcNAcase)-treated mouse embryo fibroblasts (MEF). GlcNAc and GlcNAc-containing synthetic oligosaccharides directly inhibited viral infectivity. Ulex lectin inhibition of infection was shown to be due to inhibition of surface adsorption of 35S-labeled virus. Also, GlcNAcase eluted 35S-labeled virus adsorbed to MEF at 4 degrees C and inhibited plaque formation if added after adsorption at this temperature. These findings indicate that GlcNAc binding is involved in attachment rather than in some later step in infection. High-performance thin-layer chromatography overlay of [35S]MCMV indicated that it binds to a GlcNAc-containing asialoglycolipid. Analogous experiments indicated that MCMV made virulent by in vivo salivary gland passage binds to sialic acids in addition to GlcNAc. Treatment of MEF with sialic acid-binding lectins blocked infectivity. Incubation of virus with sialic acids also prevented infection. N-acetylneuraminic acid was 10(3)-fold more potent than N-glycolylneuraminic acid. Sialidase-treated target cells were not efficiently infected by the virus. Thus, MCMV binds to GlcNAc on the cell surface, and the shift to virulence (by in vivo salivary gland passage) correlates with viral recognition of sialic acids.

Acetylglucosamine↗
PubMed1990

Necrotizing retinopathy after intraocular inoculation of murine cytomegalovirus in immunosuppressed adult mice.

A light microscopic study was done to investigate retinal changes in healthy and immunosuppressed mice after intraocular inoculation of murine cytomegalovirus (MCMV). A 0.01-ml inoculum containing 10(5) plaque-forming units of MCMV was placed behind the lens in 138 4-week-old Swiss Webster mice. Ninety-eight mice were immunosuppressed with 0.2 mg/g of cyclophosphamide given intraperitoneally at the time of inoculation and 0.1 mg/g of cyclophosphamide every 5 days thereafter. Selected eyes were examined on postinoculation days 5, 10, 15, and 16-20. Evidence of viral infection was most prominent in uveal tissue. Uveal infection developed whether or not animals received cyclophosphamide, but retinal necrosis developed only in immunosuppressed mice. Focal retinal necrosis, primarily involving the outer retinal layers and retinal pigment epithelium, was first observed in an eye examined on day 10. Retinopathy from MCMV was present in three of five eyes (60%) examined on day 15, and in six of 16 eyes (37.5%) examined between days 16-20. Retinal disease was characterized by full-thickness retinal necrosis, scattered cytomegalic cells, intranuclear and intracytoplasmic viral inclusions, and acute and chronic inflammation. These results indicate that MCMV can produce a necrotizing retinopathy in mice and that immunosuppression facilitates infection. Although ocular MCMV infection in immunosuppressed adult mice is a potential model for study of human CMV retinopathy, many differences exist between human CMV and MCMV and between the ocular diseases they produce.

Animals↗
PubMed1990

Characterization of the recombinant Clostridium pasteurianum ferredoxin and comparison of its properties with those of the native protein.

Ferredoxins (Fds) constitute an important class of nonheme iron-sulfur proteins. One of the most studied Fds is the [8Fe-8S] Fd from Clostridium pasteurianum. The gene for this Fd has previously been cloned and sequenced. We report the expression of this Fd in Escherichia coli, and the characterization and comparison of this recombinant protein to the native Fd. We have found that the purified recombinant protein has the same enzymatic, redox, magnetic and electronic properties as the native Fd isolated from C. pasteurianum, which indicates that the two [4Fe-4S] clusters present in the Fd were correctly formed in E. coli.

Bacterial Proteins↗
PubMed1989

Effective blocking of HIV-1 proteinase activity by characteristic inhibitors of aspartic proteinases.

Inhibitory constants (Ki) between 5 and 35 nM were derived (under different conditions of pH and ionic strength) for the interaction of HIV-1 proteinase with acetyl-pepstatin and H-261, two characteristic inhibitors of aspartic proteinases. Thus this enzyme, essential for replication of the AIDS virus, may be classified unequivocally as belonging to this proteinase family.

Aspartic Acid Endopeptidases↗
PubMed1989

A placebo-controlled, randomized, double-masked, variable dosage, clinical trial of azathioprine with and without methylprednisolone in multiple sclerosis.

Ninety-eight patients with multiple sclerosis (MS) in the chronic progression phase entered a 3-year clinical trial to determine if azathioprine (AZ) alone or with adrenal cortical steroids stabilizes the course of MS. In group AM, the patients took AZ throughout and methylprednisolone (MP) for the first 36 weeks. Group AP received AZ and placebo instead of MP. Group PP took placebos for both drugs. We adjusted the AZ to maintain the total white blood cell count within 3,000 to 4,000/mm3; we gave the MP in a fixed dose "pulse" and alternate-day regimen. The "intent-to-treat" groups had no statistically significant differences in the rates of progression among the 3 treatments. Subgroup analysis suggests that patients in the AM group who completed treatment exactly according to protocol did statistically significantly better than the placebo recipients using the sum of Standard Neurological Examination scores, slightly better using the quantitative neuro-performance tests, but no better using Mickey's Illness Severity Scores or Kurtzke's Disability Status Scale. Also, the AZ-treated groups had half the relapse rate of the placebo-treated group. Adverse reactions to AZ accounted for most withdrawals. Hematologic and hepatic abnormalities were significantly associated with AZ, but serious non-MS abnormalities were uncommon and were equally distributed among the 3 groups. Addition of MP to the AZ slightly improved the efficacy of the treatment, but also increased the adverse effects. The benefits of AZ with or without steroids did not outweigh the risks, and therefore we do not recommend this treatment for patients with chronic progressive MS.

Adolescent↗
PubMed1988

Theiler's virus-associated antigens on the surfaces of cultured glial cells.

Infection of the central nervous system by Theiler's murine encephalomyelitis virus (TMEV), a picornavirus, produces chronic demyelinating disease in susceptible mice. In this immunoelectron microscopic study of TMEV infection of neonatal mouse brain cells in culture, TMEV antigen was found on the surfaces of infected oligodendrocytes and astrocytes by labeling with hyperimmune serum from TMEV-infected mice or with rabbit antiserum to purified inactivated DA strain TMEV. Brain-derived macrophages had no TMEV-specific antigen on their surfaces and were not able to maintain productive TMEV infection, even though TMEV antigen was present in the cytoplasm. The presence of TMEV antigens on the surfaces of oligodendrocytes (myelin-producing cells) was unexpected because picornaviruses are nonenveloped viruses and do not bud from cell surfaces. The finding is consistent with the hypothesis that demyelination follows damage of infected oligodendrocytes by immune cells or immunoglobulins that recognize surface virus antigen.

Animals↗
PubMed1988

An 11-kDa form of human immunodeficiency virus protease expressed in Escherichia coli is sufficient for enzymatic activity.

In order to define the protease domain of human immunodeficiency virus 1, various regions of the pol open reading frame were cloned and expressed in Escherichia coli. Antiserum directed against the conserved retroviral protease active site was used to identify pol precursor and processed species containing the presumed protease domain. The smallest product that accumulates is about 11 kDa as measured by NaDodSO4/PAGE. This size agrees with that predicted from the presence in this region of two Phe-Pro sequences, which is one of the cleavage sites recognized by HIV protease. DNA encoding only the predicted 11-kDa protein was cloned, bypassing the need for autoprocessing, and the protein was expressed to a high level in E. coli. This form is active as demonstrated by its ability to specifically cleave protease-deficient pol protein in vivo in E. coli. Extracts of E. coli containing the 11-kDa protease also process human immunodeficiency virus gag substrates in vitro. These results demonstrate that the 11-kDa protease is sufficient for enzymatic activity and are consistent with a major role for this form in virus maturation.

Cloning, Molecular↗
PubMed1988

Clinical experience with azathioprine: the pros.

We performed a randomized, placebo-controlled, double-blind, comparative clinical trial of 36 weeks of methylprednisolone and 3 years of azathioprine in 98 patients in the chronic progression phase of multiple sclerosis (MS). We demonstrated a trend in favor of the combination therapy for limiting progression. The relapse rate in the azathioprine recipients was half that of the control group, and visual evoked potential latencies were stabilized in those who received the combination. We think that a therapeutic trial of continuous use of the combination of adrenal steroids with azathioprine would be worthwhile if administered early in the course of the disease.

Azathioprine↗
PubMed1987

Isoelectric point restriction of cerebrospinal fluid and serum IgG antibodies to measles virus polypeptides in multiple sclerosis.

Thirty consecutive isoelectric point (pI)-discrete IgG fractions were isolated from multiple sclerosis (MS) cerebrospinal fluid (CSF) and used to immune precipitate measles virus (MV) polypeptides. Most basic fractions were enriched in activity against nucleocapsid protein (NP), and to a lesser extent against hemagglutinin (H) protein; intermediate fractions were enriched in activity against H and fusion (F) proteins; and more anodic pI fractions were almost exclusively enriched in activity against the large (L) protein of MV. In MS there are marked differences between CSF and autologous serum in regard to antibody activity to MV. In contrast, there were similar profiles of antibody response to MV proteins in SSPE CSF and serum.

Chemical Precipitation↗
PubMed1987

Autologous and allogeneic antibody responses to canine distemper virus isolates from dogs with chronic neurological diseases.

We describe the antibody responses to three strains of canine distemper virus (CDV) isolated from dogs with chronic neurological disease in the Los Angeles area using the naturally occurring sera and cerebrospinal fluids (CSFs) of these animals as probes for comparison. CDV/CDE-2 was derived from a dog with chronic distemper encephalitis, and CDV/ODE-8 and CDV/ODE-10 were derived from dogs with old dog encephalitis. Sera and CSFs were used in autologous (same dog) and allogeneic (different dog) combinations to immune precipitate the [35S]-methionine-labelled H, P, NP, F1, and M polypeptides of the virus-infected cell cultures. The polypeptides were separated by SDS-PAGE and detected by fluorography. There was decreased recognition by the CSF and sera of the polypeptides of the viral isolates in several autologous as well as allogeneic combinations. It is concluded that the immune responses to the CDV strains are not identical, and it is likely that viral mutations occurred after the animals were infected. Some mutations may have contributed to the pathogenesis of distemper encephalitis in these animals and some may have occurred during subsequent passage of the viruses in cell culture. This may explain the decreased recognition of the polypeptides of the viral isolates by the CSF and sera.

Animals↗
PubMed1987

Latency of mouse cytomegalovirus in sciatic nerve.

This report describes a mammalian model for exploring the role of virus in peripheral neuropathy. Schwann cells in culture were permissive for mouse cytomegalovirus (MCMV) replication. Intraperitoneal inoculation rarely led to sciatic nerve infection. Sciatic nerves infected by direct intraneural injection produced infectious virus and contained viral antigen at 4 days postinfection (pi). Nerves taken later, at 4 to 8 weeks pi, contained no infectious virus, but MCMV was present in a latent state because culture of nerve explants reactivated virus. The findings contrast the viral permissiveness of cultured Schwann cells to the latency observed in intact peripheral nerve.

Animals↗
PubMed1986

In vivo and in vitro transcription of the Clostridium pasteurianum ferredoxin gene. Evidence for "extended" promoter elements in gram-positive organisms.

Analysis of Clostridium pasteurianum genomic DNA indicates that the ferredoxin (Fd) gene is present in a single copy. The cloned Fd gene previously described (Graves, M.C., Mullenbach, G. T., and Rabinowitz, J. C. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1653-1657) was used to map in vivo and in vitro synthesized Fd transcripts. The in vivo mRNA was sized in two ways: by Northern hybridization analysis, and more directly from the known DNA sequence after the 5'- and 3'-termini were identified. The 5'-end was determined by primer extension-dideoxy sequencing and the 3'-end by S1 nuclease mapping. The monocistronic Fd mRNA contains about 255 nucleotides and, thus, is one of the shortest bacterial mRNAs yet described. We also examined the Fd transcripts produced by Escherichia coli transformed with the plasmid containing the Fd gene. E. coli RNA polymerase most likely recognizes the same promoter (P1) as the clostridial polymerase, and furthermore, efficiently uses an additional promoter (P2) that is poorly recognized by the normal host enzyme. For comparison, in vitro transcripts were generated by E. coli and Bacillus subtilis RNA polymerases. In vitro, only promoter P1 is used by either E. coli or B. subtilis RNA polymerase. The 3'-end of each of the four types of transcripts occurs essentially at the same location and maps to within a large dyad symmetry element. Comparison of the Fd promoter with other Gram-positive promoters reveals that some sequences outside of the traditional Pribnow and -35 regions are conserved. This analysis indicates that an "extended" promoter recognition site may be required in these organisms.

Base Sequence↗
PubMed1986

Theiler's virus in brain cell cultures: lysis of neurons and oligodendrocytes and persistence in astrocytes and macrophages.

The mechanisms of persistence and of demyelination in Theiler's virus (TV)-induced chronic neurologic disease (a murine model for multiple sclerosis) are, as yet, disputed. We investigated the tropism and persistence of TV in brain cell culture to better understand the pathogenesis of this disease. Using anti-genic markers to identify specific cells in culture, we have demonstrated that TV infects, lytically, neurons and oligodendrocytes and persistently astrocytes and macrophages. These results suggest that host cell factors play a key role in the mechanism of demyelination and the persistence of TV in the nervous system.

Animals↗
PubMed1985

Cloning and nucleotide sequence determination of the Clostridium pasteurianum ferredoxin gene.

We have constructed a library of Clostridium pasteurianum DNA cloned in the plasmid pBR322. Based on the known amino acid sequence for C. pasteurianum ferredoxin, a 64-fold degenerate heptadecanucleotide pool was synthesized. This mixed probe hybridized to two clones which were shown to contain greater than 6 kilobase pairs of the same genomic DNA. Sequence analysis of a common Sau3A1 0.6-kilobase-pair fragment revealed that it contains the information for the apoferredoxin structural gene. According to the DNA sequence, the only post-translational processing of this small apoprotein is the hydrolysis of the initiator methionine. Putative transcription and translation start and stop signals are present within the sequence.

Base Sequence↗
PubMed1985

Subacute sclerosing panencephalitis: measles virus matrix protein nucleic acid sequences detected by in situ hybridization.

Subacute sclerosing panencephalitis (SSPE) is characterized by a hyperimmune state toward the polypeptides of measles virus except the matrix (M) protein. Using cloned (3H)-labeled complementary DNA probes for in situ hybridization, we found the M protein and nucleocapsid (NP) protein nucleotide sequences in glial cells and neurons of cryostat sections from two SSPE brains. In one SSPE brain, M protein was lacking, but the other measles polypeptides were present. IgG and IgM antibodies eluted from that brain lacked antibodies to M protein, but antibodies to other measles polypeptides were present. In SSPE brain, the viral M-protein defect is not a deletion of the M gene, but rather a block in gene expression.

Brain Chemistry↗
PubMed1984

Multiple sclerosis.

The cause of multiple sclerosis is unknown but seems to be multifactorial. Susceptibility or resistance may be genetically determined; something in the environment interacts with the human host at the proper age to cause biochemical and structural lesions in the central nervous system. The systemic immune response and the response of the central nervous system become involved. Although multiple sclerosis cannot yet be cured, many clues are leading to an effective palliative therapy. Suppression or modulation of the immune responses may be the key to developing that treatment. If the environmental agent is one or several viruses, then antiviral regimens will be appropriate. Research must therefore continue at both basic science and clinical levels.

Adrenocorticotropic Hormone↗
PubMed1984

Subacute sclerosing panencephalitis.

SSPE is a rare, late manifestation of measles-virus infection. Discussed are its clinical features, laboratory findings, pathology, epidemiology, pathogenesis, and treatment.

Antibodies, Viral↗