Biomedical subjects

S E Bloom

Publications and source records attributed to S E Bloom.

At least 37 records · Page 2Linked to original sources

PubMed1992

Distribution and inducibility of a P450I activity in cellular components of the avian immune system.

The level of expression of the cytochrome P450 system in an immune tissue could influence the sensitivity of that immune tissue to damage by xenobiotics. The capacity of immune organs and their cellular components for P450I-catalyzed metabolism was assayed in the 4-week-old chicken using the P450I-specific ethoxyresorufin-O-deethylase (EROD) assay and the P450I-inducer, 3,4,3',4'-tetrachlorobiphenyl (TCB). After induction by TCB, EROD was detectable in microsomes from whole thymus, bursa and in peritoneal exudate cells (containing primarily macrophages) at levels of 28.3, 7.2 and 1.3 pmol/mg microsomal protein/min, respectively; the level in control liver was 89.9 pmol/mg microsomal protein/min. No activity was detected in these immune tissues without induction. The P450I specific in vitro inhibitor, alpha-naphthoflavone (NF) inhibited the TCB-induced liver and immune tissue EROD by 50% at concentrations in the range of 0.07-0.1 microM. The cellular distribution of EROD in the bursa and thymus was studied in lymphocytes and supporting tissue cells after their separation by density gradient centrifugation. Much higher TCB-induced EROD was detected in immune tissue supporting cells than in lymphocytes, particularly in the thymus. The P450I in the supporting tissue of the bursa and thymus at 1 week post-hatch was also measured after eradication of the lymphocytes in both immune tissues by in ovo administration of CP. TCB-induced EROD was 12-fold higher in the lymphocyte-depleted thymus than in normal thymus, with a less marked but similar pattern in the bursa.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Cytogenetic mechanisms in the selective toxicity of cyclophosphamide analogs and metabolites towards avian embryonic B lymphocytes in vivo.

Cyclophosphamide (CP) is selectively toxic to avian and mammalian B lymphocytes, but the mechanisms of action are incompletely understood. We used a structure-activity approach to determine the cytogenetic mechanisms underlying the selective lymphoid toxicity in chicken embryos at 18-19 days of incubation. Two doses of 5-bromo-2'-deoxyuridine (BrdU; 3 mg/200 microliters x 2) were pipetted onto the inner shell membrane to label lymphocyte DNA over 20 h. A single dose of the CP analogs or metabolites was given 1 h after the initial BrdU application. After a terminal 3-h exposure to demecolcine to block cells in metaphase, the embryos were sacrificed at hour 20, and their bursae and thymi were removed for cytogenetic processing. Microscope slide preparations of metaphases were stained by the fluorescence-plus-Giemsa technique to differentiate the sister chromatids for an assessment of sister-chromatid exchange (SCE) induction and cell cycle progression based on replication cycle-specific staining patterns. Isophosphamide (1.25-40 mg/kg), phosphoramide mustard (0.7-45.7 mg/kg), and 4-methylcyclophosphamide (1.3-42.1 mg/kg) selectively damaged B cells as shown by dose-related reductions in the mitotic activity, inhibition of cell cycle kinetics, and approximately 9-15-fold increases in the SCE frequency above control. B cells were up to 392 times more susceptible to the toxicity of these three bifunctional alkylating agents compared to T cells based on reductions in the mitotic activity. At most of the drug doses tested, the T-cell mitotic index was not depressed significantly and was usually higher than the control value by as much as 50-60%. Importantly, monochloroethylcyclophosphamide (70-245 mg/kg; monofunctional alkylation) did not induce differential lymphoid toxicity, although a 9-fold increase in the SCE frequency of B cells was observed at the highest dose. Didechlorocyclophosphamide (181-422 mg/kg; acrolein generation only) was a weak SCE inducer (approximately 1.8-fold increase) and was not selectively toxic to B cells. Our data show that selective toxicity to B lymphocytes is strongly associated with bifunctional alkylation via the chloroethyl groups rather than with monofunctional alkylation and acrolein-mediated damage. In addition, the results with phosphoramide mustard and 4-methylcyclophosphamide emphasize that aldehyde dehydrogenase activity is not the primary determinant in the relative sparing of T lymphocytes in vivo.

Animals↗

PubMed1992

Assessment of neonatal avian inflammatory macrophage function following embryonic cyclophosphamide exposure.

Inflammatory macrophage recruitment and function were examined in 4-5-week-old chickens which had received two doses of cyclophosphamide (CP) or vehicle (dH2O) during late embryogenesis (18 and 19 days of incubation). Mononuclear leukocyte chemotaxis to N-formyl-methionyl-leucyl-phenylalanine (FMLP) and hematological parameters were unchanged in CP-treated vs control chickens. Peritoneal exudate cells (PECs) elicited in response to intraperitoneal (i.p.). Sephadex injection did not differ in CP-treated vs control chickens with respect to cell number, cell type, superoxide anion production, or cell surface expression of Ia and transferrin receptor (TfR) antigens. The CP-treated chickens did exhibit the expected decrease in bursa weight; male chickens exposed to CP also had inhibited testes growth. Although embryonic exposure to CP at this dose results in irreversible bursal damage and subsequent impaired humoral immunity, it appears that there are no long-lasting effects on avian inflammatory macrophage function.

Animals↗

PubMed1991

Roles of dosage, pharmacokinetics, and cellular sensitivity to damage in the selective toxicity of cyclophosphamide towards B and T cells in development.

Cyclophosphamide (CP) is a known immunomodulating agent. When presented to either late stage chick embryos (e.g. 18 days of incubation (DI] or neonatal chicks. CP induces selective B cell damage resulting in humoral immunosuppression in chickens. The present study was undertaken in order to provide further insights into CP's selective immunotoxic effects. We investigated the influences of age, CP-dose, and drug distribution on CP-induced cytotoxicity in the B and T cell compartments of the developing chick. In this test system, differential immunotoxicity was strongly dosage-dependent; at all ages tested, B cell depletion predominated at low CP dosages (50 and 100 mg/kg) whereas equally extensive lymphocyte toxicity was observed in both thymus and bursa at 200 mg/kg drug levels. Furthermore, while immunotoxicity profiles were similar at the two later developmental timepoints (18 DI and 1 day post-hatch), significantly higher CP dosages were required to induce lymphoid damage at 12 DI. Pharmacokinetic studies with radiolabeled CP revealed that approximately two-fold higher levels of CP and its metabolites are taken up in bursal tissue as compared to the thymus. Experiments concerning the possible inherent differences in susceptibility to CP-induced mitotic inhibition and cell killing mediating selective toxicity towards B cells versus T cells showed that B cell mitosis was inhibited at CP dosages as low as 5 mg/kg. No such inhibition of T cell mitosis was observed at this same low dosage. However, mitosis was completely arrested in both B and T cells at 50 mg/kg CP. Observations of cellularity in immune organs shortly after CP exposure revealed that the bursa is at least ten times more sensitive than the thymus to CP-induced mitotic inhibition and killing of resident lymphocytes. These studies reveal that multiple factors are involved in modulating CP selective immunotoxicity in the developing embryo. These include the dosage level, preferential distribution of the drug to the bursa, and a much greater sensitivity of B cells to CP-mediated mitotic inhibition and cell killing.

Animals↗

PubMed1991

Cyclophosphamide metabolism in the primary immune organs of the chick: assays of drug activation, P450 expression, and aldehyde dehydrogenase.

Several diagnostic catalytic assays were used to determine whether organ-specific metabolic activation or detoxification of cyclophosphamide (CP) contributes to the selective toxicity of CP directed towards differentiating B cells as compared to T cells in the developing chicken. An assay for the alkylation of 4-[p-nitrobenzyl] pyridine (NBP) was used to assess comparative levels of CP activation products generated from microsomal preparations from liver, bursa of Fabricius (B cells), and thymus (T cells) of day-old chicks. Three catalytic assays were used to characterize and compare cytochrome P450-associated enzyme activities in neonatal hepatic and lymphoid tissues. Aldrin epoxidase (AE) was used to detect phenobarbital (PB)-inducible P450 activity. Ethoxyresorufin-O-deethylase (EROD) and aryl hydrocarbon hydroxylase (AHH) were used for the evaluation of polycyclic aromatic hydrocarbon (PAH)-inducible P450 activities in control and PB- or 3,3',4,4'-tetrachlorobiphenyl (TCB)-induced animals. Using the NBP assay, basal and PB-induced CP activation were observed using chick liver microsomes. However, no evidence of CP activation from immune organ microsomes was observed in control, PB-, or TCB-induced chicks. Basal and PB-induced AE activities were observed in thymus, but not bursa, and represented less than 1% of basal liver activity. EROD activity was detected in TCB-induced samples from both thymus and bursa, the thymus having the greater activity. Activities of aldehyde dehydrogenase (ALDH), an enzyme involved in CP detoxification, were about equal in cytosolic fractions from the bursa and thymus. These studies suggest strongly that tissue-specific differences in metabolic capacities are not the major factors governing the selective toxicity of CP directed towards differentiating B lymphocytes in vivo.

Aldehyde Dehydrogenase↗

PubMed1991

A cytogenetic approach for detecting the selective toxicity of drugs in avian embryonic B and T lymphocytes.

The developing immune system of late stage embryos and neonates may be particularly susceptible to the toxicity of drugs and environmental contaminants due to high rates of cell proliferation and ongoing processes of differentiation. We have developed a cytogenetic assay to study the mechanisms of the selective targeting of cyclophosphamide (CP) to B lymphocytes compared to T lymphocytes in chicken embryos at days 18-19 of incubation. 5-Bromo-2'-deoxyuridine (BrdU; 3 mg/200 microliters PBS; two doses; 3-h interval) was pipetted onto the inner shell membrane in order to label DNA of replicating lymphoid cells. CP (1.25-40 mg/kg) was injected 1 h after the initial BrdU dose, and the embryos were exposed to colcemid (10 micrograms/100 microliters H2O) at hour 17. Three hours later, the bursa and thymus were removed, and the lymphocytes were swollen in hypotonic solution, fixed, and processed through a fluorescence-plus-Giemsa technique to differentiate sister chromatids. Based on reductions in mitotic indices, B cells were approximately 213 times more susceptible than T cells to the cytotoxicity of CP. Because the mitotic indices of B and T cells were comparable (21.3 +/- 3.7%, vs. 25.5 +/- 6.9%), the differential toxicity cannot be ascribed to greater numbers of B cells being in mitosis. CP induced a dose-related increase in the sister-chromatid exchange frequency in B cells of up to 10.4-fold above controls, representing one of the most sensitive vertebrate systems for detecting the genotoxicity of CP. The average generation time was slowed from 9.8 +/- 0.3 h in control B cells to 19.4 +/- 0.9 h in embryos exposed to 10 mg CP/kg. Furthermore, an analysis of control SCE data from 56 embryos indicated that there was a significant overdispersion of B cells exhibiting relatively high SCE frequencies compared to a Poisson distribution. Our data indicate that the chicken embryo in the late developmental stage is a good model for detecting the presence and selective toxicity of drugs and environmental toxins in differentiating B and T lymphocytes in vivo.

Animals↗

PubMed1990

Dynamics of avian inflammatory response to cross-linked dextran. Changes in avian blood leukocyte populations.

A model inflammatory compound, cross-linked dextran (Sephadex), which preferentially recruits macrophages, was used to examine shifts in chicken leukocyte distribution and monocyte functional status during the inflammatory response. Following intraperitoneal Sephadex administration, total leukocyte counts were transiently elevated because of a fivefold increase in circulating heterophils (granulocytes). While both heterophils and monocytes may become demarginated, primarily monocytes enter the peritoneal cavity in response to Sephadex. Since leukocyte chemotaxis is an important feature of the inflammatory response, this parameter was analyzed during the recruitment period. The optimum conditions for in vitro chicken mononuclear leukocyte chemotaxis to N-formyl-methionyl-leucyl-phenyl-alanine (FMLP) were defined and compared with those for the chicken macrophage-like cell line, HD11. Mononuclear leukocytes from birds injected 6 or 12 h previously with Sephadex were significantly decreased in chemotactic ability when compared with controls; at 24 h after injection, the response to FMLP had returned to normal. This system offers an opportunity to examine differential effects of inflammatory stimuli upon leukocyte function and distribution.

Animals↗

PubMed1989

Toxic effects of methyl methanesulfonate (MMS) on activated macrophages from chickens.

Adherent peritoneal exudate cells rich in macrophages were harvested from Cornell K-strain chickens 42 hr after i.p. stimulation with Sephadex G-50. Glass-adherent monolayers were obtained on coverslips and subjected to in vitro exposure to methyl methanesulfonate (MMS) at various doses for 1 hr. Solvent (0.17% ethanol final concentration) and sham (RPMI 1640 growth media) exposures were also performed. At selected times after exposure, the macrophages were analyzed for cell viability, adherence, DNA damage, and functional activity. Although MMS doses of 5 x 10(-3) M and 1 x 10(-3) M concentrations resulted in significant cytoxicity, 2 x 10(-4) M had no significant cytotoxic effect. However, this exposure resulted in DNA damage as measured by alkaline elution. Concomitant with the DNA damage was a significant decrease in the phagocytic activity of macrophages. Repair of MMS-induced DNA lesions in macrophages was indicated by a normal DNA alkaline elution profile 10 hr postrecovery. Functional activity of cells also returned to normal levels. In contrast, the incidence of Fc receptor-positive cells detected by rosetting increased immediately after MMS exposure, and phagocytosis of opsonized SRBCs was not affected by 2 x 10(-4) M MMS treatment. Similarly, MMS treatment did not alter the acid phosphatase activity of macrophages. However, bactericidal ability of MMS-treated macrophages for unopsonized Escherichia coli was significantly depressed. These results suggest that the avian macrophage is a useful target cell for examining possible relationships between genotoxic and immunotoxic effects of environmental mutagens.

Animals↗

PubMed1989

Effects of increased major histocompatibility complex dosage on chicken monocyte-macrophage function.

The influence of major histocompatibility complex (B complex) dosage on monocyte-macrophage function was examined using 4- to 6-week-old trisomic strain chickens. Di- (B15B15), tri- (B15B15B15), and tetrasomic (B15B15B15B15) progeny were produced from trisomic x trisomic crosses. Although mononuclear leukocytes from tetrasomics exhibited enhanced chemotactic activity in response to both f-met-leu-phe and Enterobacter cloacae culture supernatant as compared with that of cells from other groups, the ability to generate peritoneal exudate cells in response to intraperitoneal Sephadex stimulation was similar in all groups. Among peritoneal exudate cells, tetrasomic birds produced a significantly lower percentage of adherent macrophages with a higher proportion of Fc receptor-positive and CMTD-2-reactive macrophages than either disomic or trisomic chickens. Both tetrasomic and trisomic peritoneal macrophages exhibited a reduced phagocytic activity for unopsonized but not opsonized SRBC than was found with disomic macrophages. Thus, the number of major histocompatibility complex copies present in cells appears to influence monocyte-macrophage function.

Aneuploidy↗

PubMed1989

Evidence for a PCN-P450 enzyme in chickens and comparison of its development with that of other phenobarbital-inducible forms.

Of four monoclonal antibodies to purified rat liver cytochrome P450s, including those from 3-methylcholanthrene-, phenobarbital-, ethanol-, and pregnenolone-16-alpha-carbonitrile-treated rats, only the monoclonal antibody against pregnenolone-16-alpha-carbonitrile-inducible P450 immunodetected proteins in chicken liver microsomes after blotting from sodium dodecyl sulfate-polyacrylamide gels. This protein migrated identically with the pregnenolone-16-alpha-carbonitrile-inducible P450 detected in microsomes from dexamethasone-treated rats. It was most predominant in liver microsomes from chickens at 1 day posthatching, whereas much lower levels were observed in the embryo and at 36 days posthatch. Phenobarbital and dexamethasone were both effective inducers of this protein. The developmental profile and induction by phenobarbital and dexamethasone of several cytochrome P450-associated catalytic activities were compared with those of the immunodetected protein. Chicken liver microsomal erythromycin demethylase, a characteristic activity of rat pregnenolone-16-alpha-carbonitrile-inducible P450, was similar in developmental profile and induction to the immunodetected protein, with a high degree of augmentation at 1 day posthatch compared with that in the embryo and at 36 days posthatch; aldrin epoxidase, benzphetamine demethylase, ethylmorphine demethylase, and aminopyrine demethylase were more similar to each other in development and induction and were less well correlated with the immunodetected protein. This evidence suggests the presence in chicken liver of at least two types of P450, one a form related to the pregnenolone-16-alpha-carbonitrile-inducible P450 family. All of the catalytic activities were induced after pretreatment of chickens with phenobarbital but aldrin epoxidase was most effectively induced. Aldrin epoxidase was also detected in microsomes from untreated embryos as early as 7 days of incubation. Erythromycin demethylase was the only catalytic activity induced by dexamethasone. There was a trend of increased specific activity toward all the substances after hatching, indicating a more efficient P450 system, possibly due to a sharp increase in some isozymes, including the form from the pregnenolone-16-alpha-carbonitrile-inducible P450 family. This evidence for a pregnenolone-16-alpha-carbonitrile-inducible P450 in chickens agrees with sequence information that suggests the early evolution of this form and demonstrates the suitability of the chicken for studies of P450 evolution.

Animals↗

PubMed1988

Isolation of a cDNA clone from the B-G subregion of the chicken histocompatibility (B) complex.

The B-G antigens are highly polymorphic antigens encoded by genes located within the major histocompatibility complex (MHC) of the chicken, the B system. The B-G antigens of the chicken MHC are found only on erythrocytes and correspond to neither MHC class I nor class II antigens. Several clones were selected from a lambda gt11 erythroid cell expression library by means of rabbit antisera prepared against a purified, denatured B-G antigen. One clone chosen for further study, lambda bg28, was confirmed as a B-G subregion cDNA clone by the results obtained through using it as a nucleic acid hybridization probe. In Northern hybridizations lambda bg28 anneals specifically with erythroid cell mRNA. In Southern blot analyses the lambda bg28 clone could be assigned to the B system-bearing microchromosome of the chicken karyotype on the basis of its hybridization to DNA from birds disomic, trisomic, and tetrasomic for this microchromosome. The cDNA clone was further mapped to the B-G subregion on the basis of its pattern of hybridization with DNA from birds of known B region recombinant haplotypes. Southern blot analyses of the hybridization of lambda bg28 with genomic DNA from birds of known haplotypes strongly suggest that the B-G antigens are encoded by a highly polymorphic multigene family.

Animals↗

PubMed1988

MHC-chromosome dosage effects: evidence for increased expression of Ia glycoprotein and alteration of B cell subpopulations in neonatal aneuploid chickens.

Quantitative variation in the expression of MHC-encoded class II (Ia) glycoproteins has been associated with stages of lymphocyte development and a number of disease conditions. We have used an avian MHC dosage model to study the regulation of Ia expression and the effects of quantitative variation in membrane Ia on B-cell development. Lymphocyte membrane expression of Ia glycoprotein molecules and the frequency of small-versus-large lymphocytes were examined in trisomic line chickens containing either two (disomic), three (trisomic), or four (tetrasomic) copies of the microchromosome encoding the MHC. This was accomplished by quantitative laser flow cytometry analysis of bursa-resident B lymphocytes from neonatal trisomic line chickens. The aneuploids (trisomics and tetrasomics) expressed more cell surface Ia than did normal disomic birds. Furthermore, the aneuploids exhibited a greater frequency of small B lymphocytes as compared to disomic chickens. Dual parameter analysis of Ia quantity and cell size was undertaken to study B lymphocyte subpopulations in these birds. It was observed that the aneuploids had altered frequencies of two distinct subpopulations of cells: (1) an increased percentage of small cells which express high levels of Ia antigen and (2) a decreased percentage of large cells which express medium levels of Ia antigen. These findings support the view that MHC class II genes are regulated and expressed in a dosage-dependent manner. Therefore, increases in the number of MHC copies per cell result in the increased expression of Ia glycoprotein on bursa-resident B cells. The stepwise increase in membrane Ia on trisomic and tetrasomic B cells is correlated, and perhaps casually linked, with progressive degrees of alteration of developing B cell subpopulations in the bursa of aneuploid chicks. These events may ultimately alter the humoral immunity of the aneuploid animals.

Aneuploidy↗

PubMed1987

Ontogeny of the chicken cytochrome P-450 enzyme system. Expression and development of responsiveness to phenobarbital induction.

The sensitivity of the developing embryo to toxins and drugs is highly dependent on the state of development of the cytochrome P-450 system. Previous work in this laboratory has demonstrated the genotoxicity of aflatoxin B1 (AFB1) to the chicken embryo at 3 days of incubation (DI) and induction of AFB1 genotoxicity by phenobarbital at 7 DI. In this study, the basal and 24-hr phenobarbital (PB) induced levels of aminopyrine-N-demethylase (AMPD) and cytochrome P-450 were assayed in hepatic microsomes from 7 DI to 36 days posthatching (PH) and in microsomes from whole embryos at 5 DI. A dose-response for induction by PB was observed in embryonic hepatic microsomes as early as 7 DI, whereas a low level of cytochrome P-450 was detected in control 7 DI microsomes using the reduced CO vs oxidized CO difference spectrum. Basal levels of AMPD and cytochrome P-450 in hepatic microsomes increased steadily throughout development as did the responsiveness of the embryonic liver to induction with PB. Hepatic microsomes from control and PB-induced chickens had the highest AMPD activities posthatching particularly from 1 to 3 days PH. Maximal induced levels, which were 2- to 3-fold over control throughout development, ranged from 1.22 at 7 DI to 12.72 nmol HCHO/mg protein/min at 2 days PH. The potency of PB as an inducer increased about 1000-fold between 7 DI and hatching. PB induction did not increase the specific activity of AMPD at any period of development. The specific activity of AMPD posthatching increased about 3-fold above embryonic levels, indicating the development of a cytochrome P-450 complex more active toward aminopyrine in the neonatal period.

Aminopyrine N-Demethylase↗

PubMed1987

Targeting of chemical mutagens to differentiating B-lymphocytes in vivo: detection by direct DNA labeling and sister chromatid exchange induction.

In vivo systems for analyzing mutagen interactions with a specific differentiating cell population are rare. Taking advantage of the unique anatomical features of the bursa of Fabricius in the chicken, we explored the possibility of targeting chemical mutagens to a defined differentiating cell population in the animal, namely, the B-lymphocytes series. Such cells are known to be the targets for the oncogene-activating avian leukosis virus. Targeting of chemicals to cells of the bursa was demonstrated by application of the DNA-specific fluorochrome 4'-6-diamidino-2-phenylindole (DAPI) to the anal lips of neonatal chicks. Bright nuclear fluorescence of cells in the bursa was demonstrated to occur within minutes after the application of 500 microliters of DAPI. DAPI labeling of nuclei was detected up to several days after a single application. No nuclear labeling was exhibited in cells of neighboring tissues. Methyl methanesulfonate (MMS) (10 microliters) was applied to the anal lips of day-old chicks to study dose-response kinetics for mutagen targeting to DNA of dividing B-lymphocytes in the bursa. Since the mitotic index was found to be quite high (25-30%) in the bursa, chromosome analysis was used to assay for genome damage. Sister chromatid exchange frequencies of 3.9, 7.3, and 9.0 (baseline 2.5) per cell were obtained at MMS dosages per animal of 50 micrograms, 100 micrograms, and 200 micrograms respectively. These indicate the rapid and quantitative localization of DNA-binding chemicals to cells of the bursa, particularly the resident B-lymphocytes. The bursa should be a useful system for studying mutagen-DNA interactions in the differentiating B-lymphocyte and subsequent influences on the development of immunity and lymphoproliferative disease.

Animals↗

PubMed1987

Cellular expression of MHC glycoproteins on erythrocytes from normal and aneuploid chickens.

The major histocompatibility complex (MHC) in the chicken (B-complex) encodes glycoproteins homologous in function and distribution to the mammalian MHC. These are the B-F (class I) and B-L (class II) glycoproteins. In addition, a third glycoprotein (B-G) is also encoded by the chicken MHC. We are interested in examining gene regulation and cellular expression of these MHC gene products in the chicken. The trisomic line of chickens is being developed as an animal model for this purpose. Birds from this line contain either 2, 3, or 4 MHC-encoding chromosomes. In this study, we investigated the hypothesis that the quantities of B-complex glycoproteins on the membranes of fully differentiated erythrocytes are proportional to the number of MHC-encoding chromosomes present in particular birds. Hemagglutination final titer and quantitative adsorption assays were carried out using erythrocytes from disomic and aneuploid chickens homozygous for the B15 haplotype. The average hemagglutination final titers were higher for tetrasomic cells as compared to disomic cells. Furthermore, in adsorption assays, employing a B15 cross-reacting alloantiserum, trisomic and tetrasomic erythrocytes displayed increased adsorption capabilities (1.6 and 3.1 fold, respectively) compared to disomic control cells. These results indicate a step-wise increase in the amounts of erythrocyte surface glycoproteins per cell in the trisomics and tetrasomics, respectively. Such findings are consistent with a MHC-dosage-dependent model of gene expression in homeothermic vertebrates.

Adsorption↗

PubMed1987

Characterization of ribosomal RNA synthesis in a gene dosage mutant: the relationship of topoisomerase I and chromatin structure to transcriptional activity.

The genes encoding 18S, 5.8S, and 28S ribosomal RNA (rRNA) are tandemly repeated at the nucleolus organizer region (NOR). The NORs in the chicken map to one pair of microchromosomes. A line of chickens that contains individuals that are either disomic, trisomic, or tetrasomic for this chromosome, and have two, three, or four nucleoli and NORs, per cell, respectively, has been described previously. Aneuploid animals display a proportional increase in the rRNA gene copy number per cell. But, despite an increase in rDNA dosage, the levels of mature rRNA are regulated to normal levels in cells from aneuploid chickens (Muscarella, D.E., V.M. Vogt, and S.E. Bloom, 1985, J. Cell Biol., 101:1749-1756). This paper addresses the question of how regulation of mature rRNA synthesis occurs in cells with elevated levels of rDNA. An analysis of rRNA transcription in chicken embryo fibroblasts (CEFs) revealed that the relative rates of rRNA synthesis and processing and the amounts of precursor rRNA per cell are similar for all three genotypes. A comparison of chromatin structure, as determined by sensitivity of rDNA in nuclei from CEFs to digestion by DNase I, revealed that some of the rRNA genes from aneuploid cells are more resistant to digestion than corresponding sequences in the disomic cells. A determination of the distribution of topoisomerase I on rDNA has also been performed using the compound camptothecin, which introduces single- and double-strand breaks in topoisomerase-DNA complexes. Quantitation of camptothecin-induced cleavages revealed that a larger proportion of the rRNA genes in aneuploid cells was resistant to cleavage than in disomic cells, and therefore have no detectable amounts of topoisomerase I. These results suggest that the regulation of rRNA synthesis in CEFs with elevated levels of rDNA is achieved by the use of a subset of the rRNA genes.

Aneuploidy↗

PubMed1987

Chromosomal localization of the major histocompatibility (B) complex (MHC) and its expression in chickens aneuploid for the major histocompatibility complex/ribosomal deoxyribonucleic acid microchromosome.

By the cytogenetic method of trisomy mapping, the major histocompatibility complex (MHC) (B) was located on the microchromosome that also contains all of the ribosomal ribonucleic acid (rDNA) genes that are detected as a nucleolar organizer region. Crosses involving aneuploid chickens homozygous or heterozygous for particular B haplotypes yield an F1 of disomic, trisomic, and tetrasomic offspring suitable for studies of gene dosage effects and gene regulation. Studies to date show that MHC genes are expressed on each chromosome in aneuploid cells unlike the rDNA gene cluster, which is regulated to produce only diploid levels of mature rRNA. Biological effects of extra MHC chromosome dosage range from altered cell surface content of glycoproteins to altered growth potential of chickens. In addition, enhanced MHC-encoded cell surface products may influence the progression of B-cell differentiation and cell population dynamics in the developing bursa of Fabricius. Recent research shows a possible mechanism to account for the formation of unequal products in meiosis within the rDNA and MHC gene clusters. Unequal recombinational events may be promoted in the meiotic process in trisomic chickens.

Aneuploidy↗

PubMed1986

Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin: ontogeny in chick embryo liver.

Aryl hydrocarbon hydroxylase (AHH, cytochrome P1-450) is induced in chick liver very early during embryonic development if embryos are treated with 3-methylcholanthrene-type compounds such as 3,4,3'4'-tetrachlorobiphenyl. In mammals, AHH induction is known to be mediated by the Ah receptor. Liver from embryonic and newly hatched chicks was found to contain a cytosolic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) which has properties that are very similar to properties of the Ah receptor previously characterized in mammalian tissues. In chick embryo liver, cytosolic binding sites for TCDD were of high affinity (Kd for [3-H]-TCDD = 0.2 nM) and were specific for 3-methylcholanthrene-type inducers. The specific binding component sedimented at about 9S on sucrose density gradients prepared at low ionic strength. A high level of Ah receptor was detected in chick embryo liver by the fifth day of incubation (5 DI); this is at least 24 hours prior to the onset of AHH inducibility. The Ah receptor concentration increased from 5 DI to 8 DI, the period when chick liver is undergoing early morphological differentiation. After 8 DI, Ah receptor levels dropped substantially and remained low into the posthatching period. In contrast, AHH inducibility was high by 7 DI and remained high throughout embryonic development and into the posthatching period. The discrepancy between Ah receptor levels and the degree of AHH inducibility suggests that only a small fraction of the Ah receptor population is required for maximal AHH induction.

Animals↗