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Biomedical subjects

M G Anderson

Publications and source records attributed to M G Anderson.

At least 19 recordsLinked to original sources
PubMed1999

Nuclear extracts lacking DNA-dependent protein kinase are deficient in multiple round transcription.

We have compared levels of in vitro transcription in nuclear extracts from DNA-dependent protein kinase (DNA-PK)-deficient and DNA-PK-containing Chinese hamster ovary cell lines. DNA-PK-deficient cell lines are radiosensitive mutants lacking either the catalytic subunit or the 80-kDa subunit of the Ku protein regulatory component. Extracts from DNA-PK-deficient cell lines had a 2-7-fold decrease in the level of in vitro transcription when compared with matched controls. This decrease was observed with several promoters. Transcription could be restored to either of the deficient extracts by addition of small amounts of extract from the DNA-PK-containing cell lines. Transcription was not restored by addition of purified DNA-PK catalytic subunit, Ku protein, or individually purified general transcription factors. We conclude that extracts from DNA-PK-deficient cells lack a positively acting regulatory factor or a complex of factors not readily reconstituted with individual proteins. We have also investigated the mechanistic defect in the deficient extracts and have found that the observed differences in transcription levels between Ku-positive and Ku-negative cell lines can be attributed solely to a greater ability of the Ku-positive nuclear extracts to carry out secondary initiation events subsequent to the first round of transcription.

Androstadienes↗
PubMed1999

Interacting loci cause severe iris atrophy and glaucoma in DBA/2J mice.

Glaucomas are a major cause of blindness. Visual loss typically involves retinal ganglion cell death and optic nerve atrophy subsequent to a pathologic elevation of intraocular pressure (IOP). Some human glaucomas are associated with anterior segment abnormalities such as pigment dispersion syndrome (PDS) and iris atrophy with associated synechiae. The primary causes of these abnormalities are unknown, and their aetiology is poorly understood. We recently characterized a mouse strain (DBA/2J) that develops glaucoma subsequent to anterior segment changes including pigment dispersion and iris atrophy. Using crosses between mouse strains DBA/2J (D2) and C57BL/6J (B6), we now show there are two chromosomal regions that contribute to the anterior segment changes and glaucoma. Progeny homozygous for the D2 allele of one locus on chromosome 6 (called ipd) develop an iris pigment dispersion phenotype similar to human PDS. ipd resides on a region of mouse chromosome 6 with conserved synteny to a region of human chromosome 7q that is associated with human PDS. Progeny homozygous for the D2 allele of a different locus on chromosome 4 (called isa) develop an iris stromal atrophy phenotype (ISA). The Tyrpl gene is a candidate for isa and likely causes ISA via a mechanism involving pigment production. Progeny homozygous for the D2 alleles of both ipd and isa develop an earlier onset and more severe disease involving pigment dispersion and iris stromal atrophy.

Age Factors↗
PubMed1998

Ripped pocket and pickpocket, novel Drosophila DEG/ENaC subunits expressed in early development and in mechanosensory neurons.

Drosophila melanogaster has proven to be a good model for understanding the physiology of ion channels. We identified two novel Drosophila DEG/ ENaC proteins, Pickpocket (PPK) and Ripped Pocket (RPK). Both appear to be ion channel subunits. Expression of RPK generated multimeric Na+ channels that were dominantly activated by a mutation associated with neurodegeneration. Amiloride and gadolinium, which block mechanosensation in vivo, inhibited RPK channels. Although PPK did not form channels on its own, it associated with and reduced the current generated by a related human brain Na+ channel. RPK transcripts were abundant in early stage embryos, suggesting a role in development. In contrast, PPK was found in sensory dendrites of a subset of peripheral neurons in late stage embryos and early larvae. In insects, such multiple dendritic neurons play key roles in touch sensation and proprioception and their morphology resembles human mechanosensory free nerve endings. These results suggest that PPK may be a channel subunit involved in mechanosensation.

Amiloride↗
PubMed1996

Antigenic variation of SIV: mutations in V4 alter the neutralization profile.

Antigenic variation is a characteristic feature of lentiviral infection. The SIV/macaque model of AIDS provides an ideal system in which to investigate the molecular basis of antigenic variation. The purpose of this study was to genetically map the nucleotide changes in env that alter the neutralization phenotype of SIV. Serum taken from an SIVmac239-infected macaque (2D) at 30 weeks postinoculation was found to neutralize the input virus (SIVmac239) and an isolate, P9, obtained at 10 weeks p.i., but did not neutralize two other isolates, P13 and P23, obtained at 20 and 52 weeks, respectively. Sequence analysis of these virus variants revealed clustered amino acid changes in V1 and single base pair changes in V2-V4 of P13 and P23. Infectious recombinant viruses in which the V1 and V1-V3 sequences of SIVmac239 were replaced with those of P13 or P23 retained the neutralization profile of SIVmac239; both were neutralized by macaque 2D serum. Recombinants containing the entire surface glycoprotein (gp120) (V1-V5) and the 5' portion of gp41 of P13 and P23 and those containing gp120 sequences from V4 through the 5' portion of the transmembrane glycoprotein (gp41) were not neutralized by 2D serum. Using a panel of monoclonal antibodies in radioimmunoprecipitation assays, P23 and recombinants containing V4 and V5 of P23 were shown to be antigenically distinct from P13 and SIVmac239. The majority of the amino acid changes in the antigenically distinct viruses were clustered in V4 (amino acids 413-418) and these changes created new potential N-linked glycosylation sites. This study demonstrates that a small number of specific amino acid changes (amino acids 412 to 418 in the env gene) in the V4 region of the SIV envelope glycoprotein can alter antibody recognition and neutralization and that these phenotypic changes may be associated with altered glycosylation of the envelope.

Amino Acid Sequence↗
PubMed1996

Distinct variant DNA-binding sites determine cell-specific autoregulated expression of the Drosophila POU domain transcription factor drifter in midline glia or trachea.

Transcriptional regulators utilizing the POU domain DNA-binding motif have been shown to form multi-protein complexes dependent on the POU domain itself and its flexible recognition of various octamer sequence elements. We have identified two variant POU domain recognition elements DFRE1 and DFRE2, which are found within a 514-bp autoregulatory enhancer of the Drosophila melanogaster POU domain gene drifter (dfr). Both elements are capable of binding bacterially produced full-length DFR protein with high affinity, although they differ in the 5'-to-3' orientation of POU-specific and POU homeodomain subelements. When placed in dfr loss-of-function genetic backgrounds, all expression of dfr-lacZ fusion genes under control of the autoregulatory enhancer is dependent on DFR activity levels. However, the complete enhancer sequence directs beta-galactosidase expression in only a subset of cells which normally express the endogenous DFR protein, including the middle pair of midline glias of the ventral nerve cord, the oenocyte clusters, and all tracheal cells. In addition, DFRE1 and DFRE2 exhibit separable tissue-specific functions when independently disrupted or deleted. Disruption of DFRE1 function specifically abolishes beta-galactosidase expression in the middle pair of midline glias. Deletion of DFRE causes a specific loss of tracheal expression, leaving oenocyte and midline glia expression intact. These results suggest that dfr cell-specific autoregulation is determined by the context of DFR POU domain binding within the enhancer, which is possibly mediated by the formation of recognition element-specific heteromultimeric complexes containing additional tissue-specific factors.

Animals↗
PubMed1996

Function of the Drosophila POU domain transcription factor drifter as an upstream regulator of breathless receptor tyrosine kinase expression in developing trachea.

Organogenesis of the Drosophila tracheal system involves extensive directed cell migrations leading to a stereotypic series of interconnected tubules. Although numerous gene products have been shown to be essential for tracheal morphogenesis, direct functional relationships between participants have not been previously established. Both the breathless gene, encoding a Drosophila fibroblast growth factor receptor tyrosine kinase homologue, and the POU-domain transcription factor gene, drifter, are expressed in all tracheal cells and are essential for directed cell migrations. We demonstrate here that ubiquitously expressed Breathless protein under control of a heterologous heat-shock promoter is able to rescue the severely disrupted tracheal phenotype associated with drifter loss-of-function mutations. In the absence of Drifter function, breathless expression is initiated normally but transcript levels fall drastically to undetectable levels as tracheal differentiation proceeds. In addition, breathless regulatory DNA contains seven high affinity Drifter binding sites similar to previously identified Drifter recognition elements. These results suggest that the Drifter protein, which maintains its own expression through a tracheal-specific autoregulatory enhancer, is not necessary for initiation of breathless expression but functions as a direct transcriptional regulator necessary for maintenance of breathless transcripts at high levels during tracheal cell migration. This example of a mechanism for maintenance of a committed cell fate offers a model for understanding how essential gene activities can be maintained throughout organogenesis.

Animals↗
PubMed1995

drifter, a Drosophila POU-domain transcription factor, is required for correct differentiation and migration of tracheal cells and midline glia.

The Drosophila drifter (dfr) gene, previously referred to as Cf1a, encodes a POU-domain DNA-binding protein implicated as a neuron-specific regulator in the developing central nervous system (CNS). We have isolated full-length dfr cDNA clones that encode a 46-kD protein containing the conserved POU-domain DNA-binding domain. The use of alternate polyadenylation sites produces two dfr mRNA transcripts that are first expressed in stage 10 embryos at 5- to 6-hr of development. A specific anti-dfr polyclonal antiserum generated against a dfr-glutathione S-transferase fusion protein recognizes a 46-kD protein on Western blots and has been used to analyze the cell-specific distribution of dfr protein during embryonic development. dfr protein is distributed in a complex expression pattern including the tracheal system, the middle pair of midline glia, and selected CNS neurons. We have carried out a genetic characterization of the dfr locus, previously localized to region 65D of the third chromosome, by generating a series of overlapping deficiencies between 65A and 65E1 that were used to isolate dfrE82, an EMS-induced lethal allele. Analysis of dfrE82 mutant embryos shows a disruption of the developing tracheal tree as well as commissural defects in the developing CNS. Based on an examination of a cell-specific marker for tracheal cells and midline glia, these defects appear to be caused by a failure of these cells to follow their characteristic routes of migration. The dfrE82 tracheal phenotype is rescued by a dfr minigene present as a P-element transposon expressing wild-type dfr protein in tracheal cells. These results suggest that the dfr protein plays a fundamental role in the differentiation of tracheal cells and midline glia possibly by regulating the expression of essential cell-surface proteins required for cell-cell interactions involved in directed cell migrations.

Animals↗
PubMed1995

Structures and absolute configurations of antibiotics of the oligosporon group from the nematode-trapping fungus Arthrobotrys oligospora.

Spectroscopic data define the structures of three new antibiotics, 4',5'-dihydro-oligosporon (4), hydroxyoligosporon (5) and 10',11'-epoxyoligosporon (6) from the nematode-trapping fungus Arthrobotrys oligospora, and confirm the structures of the recently reported antibiotics oligosporon (1) and oligosporol B (3). The absolute configuration of the substituted 7-oxabicyclo[4.1.0]hept-3-ene nucleus of these metabolites is determined by circular dichroic spectroscopy. Oligosporon (1) and its dihydro-derivative (4) represent the second and most complex structural type of nematocidal metabolite to be characterised from cultures of nematophagous fungi.

Animals↗
PubMed1994

Quantitative studies of the effect of HTLV-I Tax protein on CREB protein--DNA binding.

The human T-cell leukemia virus type I (HTLV-I) Tax protein increases the DNA binding activity of a number of different host cell transcription factors, including the cyclic AMP response element binding protein (CREB). We have performed quantitative studies of CREB binding in the presence and absence of Tax in an attempt to gain insight into the mechanism of the Tax effect. Enhancement of binding occurred over a wide range of CREB concentrations, but sharply increased at the lowest concentrations tested. The data are best explained by a two-step binding model where Tax changes the apparent equilibrium constants for both a CREB-CREB dimerization step and a (CREB)2-DNA binding step. We used the model to perform a quantitative analysis of the binding of CREB to DNA that had been mutated at positions flanking the core CREB recognition site. Results suggest that there are altered or more extensive DNA-protein contacts at these positions in the presence of Tax. We also used the model to analyze differences in the interaction of Tax with nonphosphorylated and protein kinase A-phosphorylated CREB protein. There was no significant change in the behavior of CREB upon phosphorylation.

Base Sequence↗
PubMed1993

Antigenic variation of molecularly cloned SIVmac239 during persistent infection in a rhesus macaque.

Eight rhesus macaques inoculated with molecularly cloned SIVmac239 developed viremia and virus-binding antibodies, but only one (macaque 2D) developed neutralizing antibodies to the virus. Viremia persisted in macaque 2D even in the presence of neutralizing antibodies. Neutralizing antibodies in the plasma collected from macaque 2D late in infection neutralized virus isolated early in infection. In contrast, these antibodies failed to neutralize the plasma viruses isolated after the appearance of neutralizing antibodies. Only antigenic variants were isolated from blood, spleen, and lymph nodes. Viruses isolated from other macaques that did not develop neutralizing antibodies were neutralized by 2D serum and were of the parental (SIVmac239) phenotype. The variant viruses maintained their strict tropism for lymphocytes, similar to the parental virus.

Animals↗
PubMed1993

Analysis of envelope changes acquired by SIVmac239 during neuroadaption in rhesus macaques.

Nucleotide sequence analyses of the env genes of two neurotropic variants of SIVmac239 were performed to determine whether molecular changes in these genes could be correlated with neurotropism. Biological characterization of virus from the infectious molecular clone of SIVmac239 had shown that it is highly lymphocyte-tropic and poorly macrophage-tropic. This virus failed to replicate in the brain after intracerebral inoculation, but passage of this virus in macaques resulted in development of viral variants that had acquired cell tropism for macrophages and were neurovirulent (D. P. Sharma, M. C. Zink, H. Anderson, R. J. Adams, J. E. Clements, S. V. Joag, and O. Narayan, J. Virol., 66, 3550-3556, 1992). The neurotropic virus SIVmac239/R71 was obtained from the brain of a monkey after the third in vivo passage of SIVmac239. Inoculation of this virus into another macaque leads to CNS disease and the isolation of another neurotropic virus SIVmac239/17E. The viral env sequences obtained by polymerase chain reaction amplification directly from DNA obtained from the brain of R71 and 17E macaques had a limited number of changes dispersed throughout the env gene when compared to the parental virus, SIVmac239. The most important finding was that there was a common set of nucleotide changes in the env gene of both R71 and 17E. This suggested that viruses containing these changes had a selective growth advantage in the brain and were the predominant species present in the central nervous system of macaques R71 and 17E. Analysis of individual clones containing the R71 env gene revealed that different env genes were present, but all had the changes that were conserved in both R71 and 17E but not present in the original lymphocyte-tropic parental virus, SIVmac239. Construction of an infectious recombinant virus containing the tat, rev, and env genes from 17E and the remainder of the genome from the parental virus SIVmac239 resulted in a virus that had the macrophage-tropism of 17E virus isolated from brain. This demonstrates that the env gene of 17E confers the cellular tropism of the virus on the parental virus, SIVmac239.

Amino Acid Sequence↗
PubMed1992

Two strains of SIVmac show differential transactivation mediated by sequences in the promoter.

Two infectious molecular clones of simian immunodeficiency virus, SIVmac251 and SIVmac239, have very different in vivo properties, SIVmac239 being much more pathogenic than SIVmac251. To assess whether the in vivo differences between the two viruses would be reflected in transcriptional rates in vitro, transcriptional activity in the presence of the transactivation protein tat was analyzed by transient transfection assays in HUT-78 and U937 cells. Whereas the two promoters had similar basal activities (Anderson and Clements, 1991, J. Virol. 65, 51-60) the promoter of SIVmac239 was transactivated to a greater extent. Removal of sequences 5' to -225 and 3' to +18 maintained the basal activity, yet made the promoter unresponsive to tat. Addition of bases +19 to +149 reconstituted transactivation and decreased basal activity. Analysis of deletion mutants with reconstituted transactivation response region determined that differences between the two strains were maintained even when only the proximal sequences, -225 to +18 of the U3 and R region were placed upstream of the TAR sequences. This region contains four nucleotide differences and the potential Sp-1-binding sites, where there are an additional 11 bases in SIVmac239 that create a third potential Sp-1 site, compared to only 2 in SIVmac251. Transactivation in this assay system was found to correlate better to RNA differences shortly after transfection (12 hr) than later (46 hr).

Base Sequence↗
PubMed1992

Differences in neutralization of simian lentivirus (SIVMAC) in lymphocyte and macrophage cultures.

Comparison of neutralization of SIVmac251 in primary macrophage cultures with neutralization in lymphocytes (CEM174 cells) showed that neutralizing antibodies induced by SIV251 in infected rhesus macaques protected both macrophages and T lymphocytes against infection when the virus was preincubated with the antibodies. In macrophages, the neutralizing antibodies also protected against infection when added 1 hour after the virus. Addition of antisera to macrophages between 24 and 48 hours after virus inoculation resulted in infection with continuous release of small amounts of p24 into the supernatant fluids but these antibody-treated cultures failed to exhibit cytopathic virus replication. In contrast, the same neutralizing antisera did not protect lymphocytes against infection and subsequent cytopathic replication of the virus when added only 1 hour after virus inoculation. This distinction in the effect that neutralizing antibodies had on the development of cytopathic infection in lymphocytes and macrophages when added after virus inoculation, suggests that they could alter the dynamics of virus replication and therefore the pathogenesis of disease.

Animals↗
PubMed1992

Neutralizing antibodies modulate replication of simian immunodeficiency virus SIVmac in primary macaque macrophages.

Cultured macaque macrophages are permissive for the replication of SIVmac251, and inoculation with virus is followed by the production of viral p27. Neutralizing macaque polyclonal and murine monoclonal antibodies preincubated with the virus prevented infection but did not prevent cytopathic virus replication when added more than 3 days after inoculation with virus. However, application of the neutralizing antibodies to macrophages 24 h after inoculation with virus resulted in sustained, low-level production of viral antigen. Cell lysates and individual macrophages from treated cultures contained less viral protein by Western blot (immunoblot) and immunocytochemistry than untreated controls. In situ hybridization and polymerase chain reaction procedures for detecting and estimating relative amounts of viral RNA and DNA showed that both viral nucleic acids failed to increase beyond the levels obtained before the addition of neutralizing antibodies. The data suggest that macrophages may need to be infected with a minimum threshold of virus particles in order to reach their full potential for virus replication and that their exposure to neutralizing antibodies prior to reaching this threshold resulted in limited virus replication.

Animals↗
PubMed1991

Safety of alcohol after viral hepatitis.

To test the validity of the generally held belief that moderate consumption of alcohol during convalescence from acute viral hepatitis adversely affects outcome, 87 adults recovering from acute viral hepatitis (hepatitis A in 36, hepatitis B in 34, hepatitis non-A, non-B in 17) were studied. Criteria for entry to the study attempted to ensure that no patient was a chronic hepatitis B carrier. Patients were randomised either to a moderate alcohol intake, or to continued complete abstention. Drinkers consumed 26 g alcohol daily (mean) and none remained abstinent. At 3 months all patients were well, with normal liver function tests. There were no significant differences between the two groups at anytime. The findings suggest that moderate alcohol intake during convalescence from acute viral hepatitis does not seem to be harmful.

Acute Disease↗