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Biomedical subjects

M J Anderson

Publications and source records attributed to M J Anderson.

At least 19 recordsLinked to original sources
PubMed1998

Cloning and characterization of three human forkhead genes that comprise an FKHR-like gene subfamily.

Alveolar rhabdomyosarcomas are associated with unique chromosomal translocations t(2;13) and t(1;13), which arise from fusion of the genes for the paired box proteins PAX3 and PAX7, respectively, to the FKHR (forkhead in rhabdomyosarcoma) gene on chromosome 13q14. Here we report the identification and characterization of three novel human forkhead genes with similarity to FKHR. The three genes (HGMW-approved symbols FKHRP1, FKHRL1, and FKHRL1P1) map to chromosomal regions 5q35.2-q35.3, 6q21, and 17p11, respectively. Based on amino acid sequence comparisons of their forkhead domains, FKHRL1, FKHRL1P1, and FKHRP1 share 86, 84, and 68% identity, respectively, with FKHR. While FKHR and FKHRL1 are expressed in every human adult tissue examined, FKHRP1 mRNA expression could not be detected, and FKHRL1P1 expression was present only at low levels. FKHR and FKHRL1 share a similar genomic organization, each having a very large intron 1 (FKHR approximately 130 kb and FKHRL1 > 90 kb), which bisects their respective forkhead domains at identical positions, as well as a second intron just downstream of each stop codon. FKHRP1 and FKHRL1P1 lack introns and contain stop codons that prevent them from yielding full-length proteins. Thus, while FKHR and FKHRL1 represent functional genes, FKHRP1 and FKHRL1P1 probably are processed pseudogenes. These results suggest that these four genes represent an FKHR-like gene subfamily within the larger human forkhead gene family.

Amino Acid Sequence↗
PubMed1998

Variation in morphotype, karyotype and DNA type of fluconazole resistant Candida albicans from an AIDS patient.

Azole-resistant oropharyngeal and oesophageal candidiasis is a recent phenomenon observed in patients with AIDS usually previously treated with fluconazole. Some variation has been observed in antifungal susceptibility testing among separate colonies of Candida albicans from the same patient. This raises the question of whether there are multiple clones present or simply phenotypic variation in expression of azole resistance. To address this question we took 18 isolates grown from multiple swabs taken before and after experimental azole therapy from a single HIV-positive individual with fluconazole-resistant oral candidiasis and compared morphotype, karyotype, PCR-based DNA typing and azole susceptibility. Ten of the isolates were from a single 2-day period. Amongst these 10 there were seven morphotypes, five karyotypes and four polymerase chain reaction (PCR) types. Three further morphotypes, one karyotype and two PCR types were found amongst the eight isolates obtained during the subsequent 4 months. Limited variation in susceptibility to two azoles--fluconazole and D0870--was also seen. This work emphasizes both the large genotype and phenotypic variability of C. albicans isolates in the mouth of AIDS patients with fluconazole resistance, and the difficulties in interpretation of present typing methods.

AIDS-Related Opportunistic Infections↗
PubMed1998

Klebsiella endocarditis: report of two cases and review.

The rarity of endocarditis due to Klebsiella species limits its recognition and awareness of its often malignant course. We describe two recent cases of Klebsiella pneumoniae endocarditis and review the clinical context and outcomes of 48 other cases reported in the literature. At our hospital, endocarditis complicated only one of 86 consecutive episodes of bacteremia due to Klebsiella species. In 22 series of endocarditis that we reviewed, Klebsiella species caused < or =1.2% of cases of native valve endocarditis and up to 4.1% of cases of prosthetic valve endocarditis. Valves were replaced in 44% of these cases, and the mortality rate was 49% in cases for which outcome was specified. Valve replacement may be associated with improved survival. We conclude that Klebsiella species are a rare but ominous cause of complicated bacterial endocarditis that requires careful evaluation during the entire course of therapy.

Aged↗
PubMed1997

Characterization of the expression and immunogenicity of poliovirus replicons that encode simian immunodeficiency virus SIVmac239 Gag or envelope SU proteins.

The effectiveness of the poliovirus vaccines to induce both systemic and mucosal immunity has prompted the development of this virus as a vector in which to express foreign proteins. Our laboratory has previously reported on the construction and characterization of poliovirus genomes that encode HIV-1 proteins (Porter DC, et al.: J Virol 1996;70:2643-2649). To develop this system further, we have constructed poliovirus genomes, referred to as replicons, which encode the SIVmac239 Gag or Env SU in place of the poliovirus capsid gene (P1). Since the replicons do not encode capsid proteins, they are encapsidated into poliovirus by passage with a recombinant vaccinia virus, VVP1, which provides the poliovirus capsid proteins in trans. Using this system, we have derived stocks of the encapsidated replicons which encode the SIVmac239 or Env SU protein. Infection of cells with the replicon that encodes SIVmac239 Gag resulted in the expression of a 55-kDa protein that was released from the infected cells. Analysis of the sedimentation of the released proteins by sucrose density gradient centrifugation revealed that the protein was released from the cell in the form of a virus-like particle. Infection of cells with the replicons encoding the SIVmac239 Env SU resulted in the expression of a 63-kDa protein, corresponding to the molecular mass predicted for the nonglycosylated SIVmac239 SU protein. A second protein with a molecular mass greater than 160 kDa was also immunoprecipitated. After enzymatic deglycosylation, this protein migrated at a molecular mass consistent with that for an Env SU dimer. Analysis of the medium from cells infected with the replicon encoding SIVmac239 Env SU revealed the presence of a protein of molecular mass 85-90 kDa, possibly representing a fragment of the SIVmac239 or Env SU protein. To determine the immunogenicity of the replicons encoding SIVmac239 Gag or Env SU, transgenic mice that express the human receptor for poliovirus, and are thus susceptible to poliovirus, were immunized via the intramuscular route. A serum antibody response to SIV envelope was detected following booster immunization, establishing that the encapsidated replicon was immunogenic. Finally, we demonstrate that the replicons have the capacity to infect peripheral blood mononuclear monocytes/macrophages, suggesting that this cell is a possible target for in vivo infection. The results of our studies, then, lend further support for the development and application of recombinant poliovirus replicons in a vaccine strategy.

Animals↗
PubMed1997

Nerve-induced disruption and reformation of beta1-integrin aggregates during development of the neuromuscular junction.

The earliest biochemical change detected during synaptogenesis is a local elimination of muscle basal lamina proteins. To explore whether this provides signal(s) that regulate postsynaptic differentiation, we examined the effects of innervation on the distribution of beta1-integrins, which were initially present in scattered aggregates complexed with basal lamina ligands. These beta1-integrin aggregates disappear along paths of nerve contact as their basal lamina ligands are eliminated. New accumulations of these proteins then form during assembly of the postsynaptic apparatus. The new beta1-integrin aggregates at developing synapses form partly via a redistribution of mobile molecules on muscle surface. We thus consider whether (a) the removal of integrins' basal lamina ligands alters their cytoplasmic ligand-interactions, causing the dissociation of integrin clusters, and (b) this receptor modulation helps to transduce local changes in pericellular protease activity into cytoplasmic signals that control postsynaptic differentiation.

Animals↗
PubMed1997

Evaluation of critical body residue QSARs for predicting organic chemical toxicity to aquatic organisms.

The critical body residue (CBR) is the concentration of chemical bioaccumulated in an aquatic organism that corresponds to a defined measure of toxicity (e.g., mortality). The CBR can provide an alternative measure of toxicity to traditional waterborne concentration measurements (e.g., concentration in water causing 50% mortality). The CBR has been suggested as a better estimator of dose than the external water concentration and has been postulated to be constant for chemicals with the same mode of action. CBR QSARs have both theoretical and experimental support, developed primarily from studies on the acute toxicity of narcotic chemicals to small fish. CBR QSARs are less well developed for the aquatic toxicity of non-narcotic chemicals. CBRs vary substantially with the mode of action and toxicity endpoint, and may be affected by genetic, hormonal or environmental variation. CBR QSARs may not be applicable to very hydrophobic chemicals, chemicals with specific modes of action, or those with toxicity controlled by kinetic processes such as biotransformation. CBRs models have not been developed or evaluated for sediment and dietary exposure routes. Application of CBR QSARs to contaminated site assessments will require further research and development.

Animals↗
PubMed1997

The majority of dietary linoleate in growing rats is beta-oxidized or stored in visceral fat.

On a quantitative, whole-body basis, little is known about the amount of linoleate that is converted to arachidonate or the partitioning of linoleate and its longer-chain derivatives among lean and fat tissues. The aim of the present study was to examine linoleate balance and organ partitioning in rats consuming a low but adequate level of linoleate. Weanling male Sprague-Dawley rats were given free access to a semipurified diet containing 2.3% of energy as linoleate. Food intake, fecal output and body weight gain were measured for 26 d. Whole-body fatty acid balance analysis showed that 75.5% of the linoleate consumed disappeared (apparently by beta-oxidation), 18.7% was accumulated as linoleate, 3.0% was converted to (n-6) longer-chain polyunsaturated fatty acids, and 1.2% was excreted in the feces Visceral fat contained 64% of the accumulated linoleate, and 23% was in lean tissues. Comparable values for alpha-linolenate were as follows: disappearance (84.9%), accumulation (10.9%), excretion in the feces (2.2%), and conversion to (n-3) longer-chain polyunsaturated fatty acids (1.4%). Visceral fat contained 67% of the accumulated alpha-linolenate, and 23% was in lean tissues. Visceral fat also accumulated 26% of newly synthesized (n-6) longer-chain polyunsaturated fatty acids and 31% of the (n-3) longer-chain polyunsaturated fatty acids. Thus, only 6.5% of dietary linoleate consumed at a low but adequate level for rats appeared in lean tissues as linoleate or its fatty acid metabolites; the rest was beta-oxidized or stored in fat, mostly in visceral fat. These results lead us to speculate whether losses through beta-oxidation contribute to the recommended intake for linoleate in growing rats.

Adipose Tissue↗
PubMed1997

Itraconazole resistance in Aspergillus fumigatus.

Invasive aspergillosis is an increasingly frequent opportunistic infection in immunocompromised patients. Only two agents, amphotericin B and itraconazole, are licensed for therapy. Itraconazole acts through inhibition of a P-450 enzyme undertaking sterol 14alpha demethylation. In vitro resistance in Aspergillus fumigatus to itraconazole correlated with in vivo outcome has not been previously described. For three isolates (AF72, AF90, and AF91) of A. fumigatus from two patients with invasive aspergillosis itraconazole MICs were elevated. A neutropenic murine model was used to establish the validity of the MICs. The isolates were typed by random amplification of polymorphic DNA. Analysis of sterols, inhibition of cell-free sterol biosynthesis from [14C] mevalonate, quantitation of P-450 content, and [3H]itraconazole concentration in mycelial pellets were used to determine the mechanisms of resistance. The MICs for the three resistant isolates were >16 microg/ml. In vitro resistance was confirmed in vivo for all three isolates. Molecular typing showed the isolates from the two patients to be genetically distinct. Compared to the susceptible isolate from patient 1, AF72 had a reduced ergosterol content, greater quantities of sterol intermediates, a similar susceptibility to itraconazole in cell-free ergosterol biosynthesis, and a reduced intracellular [3H]itraconazole concentration. In contrast, AF91 and AF92 had slightly higher ergosterol and lower intermediate sterol concentrations, fivefold increased resistance in cell-free systems to the effect of itraconazole on sterol 14alpha demethylation, and intracellular [3H] itraconazole concentrations found in susceptible isolates. Resistance to itraconazole in A. fumigatus is detectable in vitro and is present in wild-type isolates, and at least two mechanisms of resistance are responsible.

Adult↗
PubMed1997

Species differentiation by internally transcribed spacer PCR and HhaI digestion of fluconazole-resistant Candida krusei, Candida inconspicua, and Candida norvegensis strains.

PCR amplification of the regions containing the internally transcribed spacers and 5.8S rRNA gene of Candida krusei, C. inconspicua, and C. norvegensis yielded fragments of 510, 460, and 500 bp, respectively. HhaI digestion of these fragments yielded species-specific bands. Random amplification of polymorphic DNA with primer R108 showed interspecific discriminatory band patterns. Susceptibilities to fluconazole and amphotericin B were determined.

Antifungal Agents↗
PubMed1997

Pure linoleate deficiency in the rat: influence on growth, accumulation of n-6 polyunsaturates, and [1-14C]linoleate oxidation.

Essential fatty acid deficiency has been widely studied but the extent to which its effects are attributable specifically to deficiency of linoleate as opposed to deficiency of all unsaturated fatty acids is unknown. Our objective was to evaluate the effect of pure linoleate deficiency on growth as well as changes in the metabolism and oxidation of n-6 polyunsaturates. The diets contained 20 energy % fat blended from 3 energy % pure oleate, 2 energy % linoleate (0.01 energy % in the linoleate-deficient group), 0.3 energy % pure alpha-linolenate, and the balance as palmitate and stearate from fully hydrogenated soybean oil. Thirty-five-day-old rats consumed the two diets for 84 days, after which the linoleate-deficient rats weighed 15% less than the controls (P < 0.05), had mild scaling on the paws, and visible hair loss (in a few rats). Compared with the controls, the ratio of eicosatrienoate to arachidonate after 84 days was elevated in liver (170-fold) and serum (520-fold) phospholipids of the linoleate-deficient group. In total, linoleate-deficient rats consumed 122 mg of linoleate and had a net whole body loss of 479 mg n-6 polyunsaturates compared with an intake of 24,130 mg and a net whole body gain of 7206 mg n-6 polyunsaturates in the control group. Linoleate-deficient rats oxidized 1% of an oral bolus of [1-14C]linoleate over 8 h compared with 34% in the control rats (P < 0.05). We conclude that pure linoleate deficiency has marked effects on accumulation of n-6 polyunsaturates but induces milder gross symptoms, particularly growth retardation, than classical essential fatty acid deficiency. alpha-Linolenate and possibly oleate may have a sparing effect on linoleate oxidation from body stores during linoleate deficiency.

Adipose Tissue↗
PubMed1996

Loss of oncogenic ras expression does not correlate with loss of tumorigenicity in human cells.

ras oncogenes are mutated in at variety of human tumors, which suggests that they play an important role in human carcinogenesis. To determine whether continued oncogenic ras expression is necessary to maintain the malignant phenotype, we studied the human fibrosarcoma cell line, HT1080, which contains one mutated and one wild-type N-ras allele. We isolated a variant of this cell line that no longer contained the mutated copy of the N-ras gene. Loss of mutant N-ras resulted in cells that displayed a less transformed phenotype characterized by a flat morphology, decreased growth rate, organized actin stress fibers, and loss of anchorage-independent growth. The transformed phenotype was restored following reintroduction of mutant N-ras. Although loss of the oncogenic N-ras drastically affected in vitro growth parameters, the variant remained tumorigenic in nude mice indicating that mutated N-ras expression is not necessary for maintenance of the tumorigenic phenotype. We confirmed this latter observation in colon carcinoma cell lines that have lost activated K-ras expression via targeted knockout of the mutant K-ras gene.

Animals↗
PubMed1996

Poliovirus replicons that express the gag or the envelope surface protein of simian immunodeficiency virus SIV(smm) PBj14.

Poliovirus genomes encoding the complete gag or env surface gene of the simian immunodeficiency virus SIV(smm) PBj14 (SIV-PBj14) were constructed. The in vitro-transcribed RNA from these genomes, referred to as replicons, have the capacity for self-replication when transfected into tissue culture cells. Serial passage of the replicons containing the SIV-PBj14 gag or SIV-PBj14 env (SU) genes with a recombinant vaccinia virus, VV-P1, which provides P1 in trans, resulted in the encapsidation of these replicons. Infection of cells with the encapsidated replicons that encode gag, referred to as vIC-SIV-PBj14 Gag, resulted in the production of a 55-kDa protein that was released from the infected cells. Using a sucrose density-gradient analysis, the protein was found to sediment at a density consistent with that of a virus-like particle. Infection of cells with a replicon that encodes the env SU gene, referred to as vIC-SIV-PBj14 SU, resulted in the production of two SIV-PBj14 envelope-related intracellular proteins. One of these proteins had a molecular mass consistent with that of the unglycosylated SIV-PBj14 SU protein (63 kDa); the second protein had a higher molecular mass (>160 kDa). Characterization of this larger protein revealed that it was glycosylated and possibly represented a dimer of the SU protein. A pulse-chase analysis of cells infected with vIC-SIV-PBj14 SU demonstrated that a 110- to 130-kDa protein was released, which is consistent with the molecular mass of the SIV-PBj14 SU protein. The results of these studies demonstrate that poliovirus replicons can be used to express foreign proteins, including glycoproteins, which retain many of the physical features of the native protein.

Amino Acid Sequence↗
PubMed1996

Detection of the t(2;13) chromosomal translocation in alveolar rhabdomyosarcoma using the reverse transcriptase-polymerase chain reaction.

Rhabdomyosarcoma, a small-, round-cell tumor of skeletal muscle, is the most common soft tissue sarcoma found in children. A specific and unique chromosomal translocation, t(2;13)(q35;q14), has been described cytogenetically in a subset of these tumors and is most often associated with the alveolar histologic subtype. The cloning and sequencing of complementary DNA from fusion transcripts expressed by both cell lines and tumors have shown that this chromosomal translocation results in the fusion of the PAX3 gene on chromosome 2 with a member of the forkhead gene family, FKHR, on chromosome 13. To detect this genetic abnormality we have developed a sensitive method which relies on a reverse transcriptase-polymerase chain reaction with primers designed to be specific for the chromosome 2 and chromosome 13 sides of the translocation. The utility of this approach was tested by analyzing a series of rhabdomyosarcoma cell lines and tumor samples. The data demonstrate that the transcripts derived from the t(2;13) were restricted to tumors having features of the alveolar subtype and that they could be detected with greater ease and sensitivity than with cytogenetic analysis. This approach will facilitate a large-scale group effort to determine the frequency as well as the prognostic and diagnostic significance of this chromosomal rearrangement.

Cell Line↗
PubMed1996

In vivo modulation of 17 beta-estradiol-induced vitellogenin synthesis and estrogen receptor in rainbow trout (Oncorhynchus mykiss) liver cells by beta-naphthoflavone.

Vitellogenesis or egg yolk production represents a key estrogen initiated process in oviparous vertebrates which is crucial for oocyte maturation. Previous in vitro studies have shown that cytochrome P4501A1 (CYP1A1)-inducing compounds such as beta-naphthoflavone (betaNF) modulate 17 beta-estradiol-induced vitellogenin (Vg) synthesis in primary cultures of juvenile rainbow trout liver cells. In this study, treatment of juvenile trout with 0.5 mg/kg 17 beta-estradiol plus either 25 or 50 mg/kg betaNF confirmed in vivo that betaNF may depress Vg synthesis by the liver. Alternatively, trout treated with 0.5 mg/kg 17 beta-estradiol plus 12.5 mg/kg betaNF or 5 mg/kg 17 beta-estradiol plus 12.5, 25, or 50 mg/kg betaNF showed a potentiation of Vg synthesis relative to estradiol-only injected fish. These results are significant as they suggest that changing plasma estrogen levels in sexually maturing trout will determine whether or not CYP1A1-inducing compounds may suppress, have no effect, or potentiate estrogen-induced liver Vg synthesis. Depressed Vg synthesis by trout injected with 0.5 mg/kg 17 beta-estradiol plus 50 mg/kg of betaNF correlated with depressed estrogen-binding capacity of liver, as assessed by [3H]17 beta-estradiol binding to liver nuclear protein extracts. Using gel mobility shift assay, the decrease in estrogen responsiveness of liver was not attributed to depressed estrogen response element-estrogen receptor binding. The fish liver vitellogenesis process offers a comparative model with which to further study the mechanism(s) of aryl hydrocarbon receptor-mediated antiestrogenicity and endocrine disruption.

Animals↗
PubMed1996

Molecular typing by random amplification of polymorphic DNA and M13 southern hybridization of related paired isolates of Aspergillus fumigatus.

Three forms of DNA-based typing procedures for Aspergillus fumigatus isolates have been developed over the last five years. The procedures are random amplification of polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) detection, and Southern hybridizations with various repetitive sequence-based probes. Using two of these procedures, we compared 16 selected isolates, grouped into eight pairs on the basis of epidemiology or previously assigned RFLP types. RAPD with four primers (R108, RC08, 2, and 4), including three previously used with A. fumigatus, showed that one primer, R108, gave the best discrimination (8 types). Southern hybridization of total genomic DNA digested with HindIII and probed with the total bacteriophage M13 genome resulted in the highest overall level of discrimination. Combination of the RAPD and Southern hybridization with the previously assigned RFLP types discriminated 10 isolates of 16. Isolates closely linked epidemiologically could not be distinguished from each other. In addition, three pairs of isolates previously unlinked by epidemiology had the same overall types. Two pairs were obtained from the same hospital within 2 years of each other, whereas the third pair were isolated from California and Germany. A full understanding of the epidemiology and ecology of A. fumigatus requires multiple discriminatory typing procedures.

Aspergillosis↗
PubMed1996

Proteolytic disruption of laminin-integrin complexes on muscle cells during synapse formation.

To explore whether a neural modulation of muscle integrins' extracellular ligand interactions contributes to synapse induction, we compared the distributions of beta1-integrins and basal lamina proteins on Xenopus myotomal myocytes developing in culture. beta1-Integrins formed numerous organized aggregates scattered over the entire muscle surface, with particularly dense accumulations at specialized sites resembling myotendinous and neuromuscular junctions. Integrin aggregates on muscle cells differed from those on surrounding fibroblasts and epithelial cells, both in their lack of response to cross-linking by multivalent ligands and in their consistent association with the cells' own extracellular matrices. Muscle integrin clusters were usually associated with congruent basal lamina accumulations containing laminin and a heparan sulfate proteoglycan (HSPG), sometimes including fibronectin and vitronectin acquired from the surrounding medium. Immediately prior to synaptic differentiation, any existing laminin and HSPG accumulations along the path of cell contact were eliminated, disrupting otherwise stable laminin-integrin complexes. This apparently proteolytic modulation of integrins' extracellular ligand interactions was soon followed by the accumulation of new congruent accumulations of laminin and HSPG in the developing synaptic basal lamina. Combining these results with earlier findings, we consider the possibility that postsynaptic differentiation is induced, at least in part, by the proteolytic disruption of integrin-ligand complexes at sites of nerve-muscle contact.

Animals↗
PubMed1996

Dioxinlike properties of a trichloroethylene combustion-generated aerosol.

Conventional chemical analyses of incineration by-products identify compounds of known toxicity but often fail to indicate the presence of other chemicals that may pose health risks. In a previous report, extracts from soot aerosols formed during incomplete combustion of trichloroethylene (TCE) and pyrolysis of plastics exhibited a dioxinlike response when subjected to a keratinocyte assay. To verify this dioxinlike effect, the complete extract, its polar and nonpolar fractions, some containing primarily halogenated aromatic hydrocarbons, were evaluated for toxicity using an embryo assay, for antiestrogenicity using primary liver cell cultures, and for the ability to transform the aryl hydrocarbon receptor into its DNA binding form using liver cytosol in a gel retardation assay. Each of these assays detect dioxinlike effects. Medaka (Oryzias latipes) embryos and primary liver cell cultures of rainbow trout (Oncorhynchus mykiss) were exposed to concentrations of extract ranging from 0.05 to 45 micrograms/l. Cardiotoxicity with pericardial, yolk sac, and adjacent peritoneal edema occurred after exposure of embryos to concentrations of 7 micrograms/l or greater. These same exposure levels were associated with abnormal embryo development and, at the higher concentrations, death. Some of the fractions were toxic but none was as toxic as the whole extract. In liver cells, total cellular protein and cellular lactate dehydrogenase activity were not altered by in vitro exposure to whole extract (0.05-25 micrograms/l). However, induction of cytochrome P4501A1 protein and ethoxyresorufin O-deethylase activity occurred. In the presence of whole extract, estradiol-dependent vitellogenin synthesis was reduced. Of the fractions, only fraction 1 (nonpolar) showed a similar trend, although vitellogenin synthesis inhibition was not significant. The soot extract and fractions bound to the Ah receptor and showed a significantly positive result in the gel retardation/DNA binding test. Chemical analyses using GC-MS with detection limits for 2,3,7,8-tetrachlorodibenzo-p-dioxin and dibenzofuran in the picomole range did not show presence of these compounds. Our results indicate that other chemicals associated with TCE combustion and not originally targeted for analysis may also pose health risks through dioxinlike mechanisms.

Aerosols↗