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G A Held

Publications and source records attributed to G A Held.

13 recordsLinked to original sources
PubMed2000

Glucocorticoid receptor regulation in the rat embryo: a potential site for developmental toxicity?

Glucocorticoids play a key role in controlling numerous cellular processes during embryogenesis and fetal development. Excess glucocorticoids during development have been linked to dysmorphogenesis and/or intrauterine growth impairment in rodents. The actions of glucocorticoids are mediated by interaction with their receptors. Negative feedback regulation of glucocorticoid receptor (GR) is important for limiting cellular sensitivity to the hormones. Hence, acute exposure of the adult rat to the synthetic glucocorticoid dexamethasone (DEX) reduced both GR mRNA and protein in a variety of tissues that include hippocampus and liver, in a dose- and time-dependent fashion. Reduction in GR mRNA and protein were observable when DEX was given repeatedly at doses as low as 0. 05 mg/kg. In the control whole rat embryo, GR mRNA was low but measurable at as early as gestational day (GD) 10, but underwent rapid ontogenetic increase in the ensuring days. In contrast to the adult, neither GR mRNA nor protein in the whole rat embryo was affected by acute or repeated DEX administration to pregnant rats on GD10-13, even at doses as high as 0.8 mg/kg. Similar results were obtained in embryonic palate and liver, tissues known to be glucocorticoid targets. These data suggest that GR autoregulation does not occur during organogenesis in the rat. Accordingly, hormonal elevations from stress or chemical insults can be transduced unrestrictedly, ultimately leading to aberrant cell function and development. The unique mode of GR regulation seen in the embryonic cells may provide a potential common mechanism for developmental perturbation and toxicity for a variety of insults.

Analysis of Variance↗
PubMed1999

Adverse reproductive outcomes in the transgenic Ah receptor-deficient mouse.

The aryl hydrocarbon receptor (AHR) is a transcriptional regulatory protein that binds to upstream DNA response elements of target genes. Activation of the AHR by binding of ligands such as polyhalogenated dioxins, furans, and PCBs is associated with a wide range of adverse biological outcomes, including cancer, immune deficiencies, embryo/fetotoxicity, and reproductive toxicity. Investigations of the diverse biological responses mediated by the AHR led to production of a transgenic mouse in which the gene coding for the AhR was inactivated. AHR-deficient mice were fertile and at maturity exhibited immune system impairment and hepatic fibrosis. Our laboratory received several of these homozygous knockout (-/-) mice and mated them with wild-type (+/+) C57BL/6N mice to generate large numbers of heterozygotes (+/-). The -/- males were then mated with a total of 45 heterozygous +/- females. Offspring of these matings were genotyped and mated in all genotypic combinations. Although male and female -/- adults were fertile, the -/- females had difficulty maintaining conceptuses during pregnancy, surviving pregnancy and lactation, and rearing pups to weaning. Only 46% of the 39 pregnant -/- females successfully raised pups to weaning. The -/- pups showed poor survival during lactation (average death rate per litter was 16%) and after weaning (26.5% of the 230 weaned -/- pups died within 2 weeks). Only 39% of the implantations in uteri of -/- dams resulted in offspring surviving to Postnatal Day 45. Across all litters the sex ratios and genotypic frequencies were comparable to expected values. Reproductive success was adversely affected in Ahr-null females and conceptuses. Additional study is needed to reveal the etiology of these effects.

Animals↗
PubMed1999

AhR, ARNT, and CYP1A1 mRNA quantitation in cultured human embryonic palates exposed to TCDD and comparison with mouse palate in vivo and in culture.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is developmentally toxic in many species and induces cleft palate in the C57BL/6N mouse embryo. Palatogenesis in mouse and human embryos involves homologous processes at the morphological, cellular, and molecular levels. In organ culture, mouse and human palates respond similarly to TCDD. The present study quantitates the expression of AhR, ARNT, and CYP1A1 mRNA in human embryonic palates in organ culture. Palatal tissues were exposed to 1 x 10(-10), 1 x 10(-9), or 1 x 10(-8) M TCDD or control medium and sampled at 0, 2, 4, and 6 hours for quantitative RT-PCR using a synthetic RNA internal standard. Similar measurements of CYP1A1 gene expression were collected for mouse palates cultured in this model. In human palates, AhR expression correlated with ARNT and CYP1A1 mRNA expression. TCDD induction of CYP1A1 was time- and concentration-dependent. The expression of these genes presented a uniform and continuous distribution across the group of embryos, with no subset of either high or low expressors/responders. The ratio of AhR to ARNT was approximately 4:1. AhR mRNA increased during the culture period in both treated and control subjects; however, ARNT expression was relatively constant. TCDD did not alter either AhR or ARNT expression in a consistent dose- or time-related manner. Comparison of human and mouse data showed a high correlation across species for the induction of CYP1A1. Human embryos expressed approximately 350 times less AhR mRNA than the mouse, and in earlier studies it was shown that human palates required 200 times more TCDD to produce the same effects. When the morphological, cellular, and molecular responses to TCDD between mouse and human are compared, it seems highly unlikely that human embryos could be exposed to sufficient TCDD to achieve changes in palatal differentiation that would lead to cleft palate.

Animals↗
PubMed1999

RT-PCR quantification of AHR, ARNT, GR, and CYP1A1 mRNA in craniofacial tissues of embryonic mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and hydrocortisone.

C57BL/6N mouse embryos exposed to hydrocortisone (HC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) develop cleft palate. An interaction between these agents produces clefts at doses which alone are not teratogenic. The glucocorticoid receptor (GR) and dioxin receptor (AhR) mediated these responses and their gene expression was altered by TCDD and/or HC in palates examined on gestation day (GD) 14 by Northern blot analysis and in situ hybridization. The present study quantifies AhR, AhR nuclear translocator (ARNT), and GR mRNA at 4, 12, 24, and 48 h after exposure (time 0 = dose administration at 8 A.M. on gestation day 12) on GD12 to TCDD (24 micrograms/kg), HC (100 mg/kg) or HC (25 mg/kg) + TCDD (3 micrograms/kg). The induction of CYP1A1 mRNA was also quantified at 2, 4, 6, 12, 24, and 48 h for control and TCDD-exposed samples. Total RNA was prepared from midfacial tissue of 4-6 embryos/litter at each time and dose. An RNA internal standard (IS) for each gene was synthesized, which included the gene's primer sequences separated by a pUC19 plasmid sequence. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on total RNA + IS using a range of 5-7 IS concentrations across a constant level of total RNA. PCR products were separated in gels (mRNA and IS-amplified sequences differed by 30-50 bases), ethidium bromide-stained, imaged (Hamamatsu Photonics Systems, Bridgewater, NJ), and quantified with NIH Image. CYP1A1 mRNA was significantly induced in the TCDD-exposed samples at all time points examined (p = 0.005 at 2 h and 0.001 after 2 h). During palatal shelf outgrowth on GD12, AhR mRNA levels increased significantly and this was not affected by treatment with TCDD or HC + TCDD. A significant increase in GR was detected at 24 h (p < 0.05) and this was unaffected by any of the exposures. Expression of ARNT increased at 12 h (p < 0.001); however, treatment with HC or HC + TCDD blocked this increase (p < 0.05). At 24 h, the TCDD-treated embryos had significantly lower ARNT mRNA compared with controls (p < 0.001). The relative overall expression level of the genes was AhR > ARNT > GR. Within individuals, expression of AhR and/or ARNT was highly correlated with GR level. In conclusion, CYP1A1 mRNA was expressed in developing craniofacial tissue and was highly induced by TCDD exposure. AhR, ARNT, and GR mRNA are upregulated in early palatogenesis, although not on the same schedule. The TCDD-induced decrease in ARNT at 24 h after dosing and the HC and HC + TCDD-induced delay in upregulation of ARNT may affect the dynamics of heterodimer formation between AhR and ARNT. The changes in ARNT mRNA level could also affect availability of this transcriptional regulator to interact with other potential partners, and these effects, separately or in combination, may be involved in disruption of normal embryonic development.

Animals↗
PubMed1998

Differentially expressed genes associated with 5-Aza-2'-deoxycytidine-induced hindlimb defects in the Swiss Webster mouse.

5-Aza-2'-deoxycytidine (d-AZA) inhibits methylation of DNA, a process that serves as an epigenetic regulator of gene expression. We have shown that d-AZA causes temporally related defects in mice. Gestational day (GD) 10 treatment induced severe long-bone defects of the hindlimb but not the forelimb. Exposure of younger embryos (GD 8 or 9) does not induce similar defects in forelimbs. This limb-dependent response suggests that methylation alterations in genes specific for fore- or hindlimbs may contribute to the observed pattern of defects. Subtraction hybridization (SH) studies were conducted to identify differential expression of DNA subsequent to the administration of d-AZA to mice on GD 10. Hindlimb buds collected from both treated and untreated embryos at 4, 12, and 24 hours post-treatment were used. A clone isolated from the untreated sample (down-regulation in treated tissue) was identified as a member of the murine B1 family of repetitive sequences. The two other clones isolated from the treated tissue (up-regulation) were homologous to avian myogenic regulatory protein mRNA and activin receptor type II gene. Both species are active during embryogenesis. These findings suggest that the isolated clones may have roles in abnormal embryonic development when inappropriately expressed.

Animals↗
PubMed1990

Characterization of the parasporal inclusion of Bacillus thuringiensis subsp. kyushuensis.

Electron microscopy of Bacillus thuringiensis subsp. kyushuensis revealed that the parasporal inclusions are composed of a homogeneous center surrounded by a thick, electron-dense coating. Antibodies directed against the 135- and 65-kilodalton B. thuringiensis subsp. israelensis peptides cross-reacted with the 70- and 26-kilodalton peptides, respectively, of B. thuringiensis subsp. kyushuensis.

Animals↗
PubMed1989

Characterization of the mammalian toxicity of the crystal polypeptides of Bacillus thuringiensis subsp. israelensis.

Solubilized crystal polypeptide preparations of Bacillus thuringiensis subsp. israelensis (BTI) were fractionated by immunoaffinity chromatography using a bound monoclonal antibody formed against the 28K crystal polypeptide. The 28K polypeptide was confirmed to be hemolytic and to possess low mosquitocidal activity against Aedes aegypti larvae. By comparison, the 28K polypeptide was more potent than the solubilized BTI crystals in male Swiss Webster mice, as the LD50 values were (p less than 0.05) 0.77 and 2.33 mg protein/kg body wt, respectively. Acute administration of the 28K polypeptide (mg/kg, ip) produced severe hypothermia and bradycardia in the mouse. No evidence for cooperativity between the 28K and other crystal polypeptides was observed. Preliminary histological examination of the mouse hearts exposed to the 28K polypeptide did not reveal any specific lesion, suggesting that the deficient cardiac performance might be a secondary physiological response. Gross pathological examination of mice as well as Sprague-Dawley rats acutely treated with equivalent doses of solubilized BTI crystal preparations revealed focal to segmental reddened and edematous areas within the small intestine. Histopathology indicated that the major lesion was in the jejunum. Contrary to expectations from in vitro hemolysis assays, cytolysis of mouse red and white blood cells was not detectable after in vivo exposure to the BTI solubilized proteins. The present results indicate that the 28K polypeptide is the mammalian toxic component of BTI crystals.

Aedes↗
PubMed1986

Effect of removal of the cytolytic factor of Bacillus thuringiensis subsp. israelensis on mosquito toxicity.

Solubilized crystal protein of Bacillus thuringiensis subsp. israelensis was fractionated by affinity chromatography using a monoclonal antibody directed against the crystal's 28 kDa peptide. The 28 kDa peptide was found to be relatively nontoxic to mosquito larvae although it does contain the hemolytic activity of the crystals. The crystal protein fraction depleted of the 28 kDa peptide was found to be nonhemolytic and to retain nearly full toxicity to mosquito larvae. These results suggest that the 28 kDa peptide is not required for the toxicity of solubilized crystal protein.

Aedes↗
PubMed1985

Only part of the protoxin gene of Bacillus thuringiensis subsp. berliner 1715 is necessary for insecticidal activity.

Escherichia coli strains harboring deletion mutations of the insecticidal protoxin gene of Bacillus thuringiensis subsp. berliner 1715 were constructed. Although these strains did not produce intact protoxin, cell extracts from one of the mutants were extremely toxic to tobacco hornworm (Manduca sexta) larvae, indicating that only a part of the protoxin gene is required for insecticidal activity.

Bacillus thuringiensis↗
PubMed1983

Evidence for plasmid-associated crystal toxin production in Bacillus thuringiensis subsp. israelensis.

Three crystalliferous (Cry+) strains of Bacillus thuringiensis subsp. israelensis (serotype 14) that produce parasporal protein crystals toxic to dipteran larvae and several acrystalliferous (Cry-) mutants, either induced or spontaneously derived from a single Cry+ parent, were examined for the presence of covalently closed circular (CCC) DNA in attempts to correlate toxin production with the presence of a specific plasmid. The plasmid profiles of both Cry+ and Cry- variants were analyzed by both a cleared lysate- and a modified Eckhardt lysate-electrophoresis technique. All of the Cry- mutants derived from the Cry+ parental strain had lost a 4.0- to 4.4-megadalton (Mdal) plasmid. Bioassay data confirmed loss of toxin production by the Cry- variants. All three Cry+ strains, including the parent of the Cry- strains, contained CCC plasmids DNAs of the following approximate molecular weights: 4.0 to 4.4, 5.2 to 6.0, and 11.4 to 13.0 Mdal. One Cry+ strain contained an additional CCC plasmid of 6.7 to 7.2 Mdal. The plasmid patterns for several Cry- derivatives differed in other respects from the pattern for their parent strain. The various Cry+ and Cry- strains could be distinguished either by phenotypical differences in antibiotic sensitivity, crystal production, and toxicity, or by differences in their plasmid profiles.

Bacillus thuringiensis↗
PubMed1983

Toxicity of Bacillus thuringiensis subsp. israelensis to adult Aedes aegypti mosquitoes.

Adult female Aedes aegypti mosquitoes were killed by the parasporal crystals of Bacillus thuringiensis subsp. israelensis (ONR-60A) when the crystals were introduced into the insect midgut as an enema. The 50% lethal dose for intact parasporal crystals was 0.21 microgram/mg of mosquito (wet weight), and for solubilized crystals the 50% lethal dose was 0.04 microgram/mg. These values were compared with 50% lethal concentrations in a free-feeding larval mosquito bioassay of 0.018 and 1.28 microgram/ml for intact and solubilized crystals, respectively. Preparations from B. thuringiensis subsp. kurstaki were ineffective against both adult and larval mosquitoes. An adult mosquito bioassay is suggested as a direct means of screening potential mosquito control agents.

Aedes↗
PubMed1982

Cloning and localization of the lepidopteran protoxin gene of Bacillus thuringiensis subsp. kurstaki.

Bacillus thuringiensis subsp. kurstaki produces a proteinaceous crystalline inclusion that is toxic for lepidopteran larvae. There are several size classes of plasmids in this organism and the presence of one or more has been correlated with production of this protein, defined as a protoxin. DNA fragments of B. thuringiensis subsp. kurstaki, obtained by EcoRI digestion, were cloned into the vector Charon 4A. Recombinant phage were screened immunologically for the production of protoxin. Cells infected with one phage, C4K6c, produced antigen that was the same size as the protoxin and was toxic to Manduca sexta larvae. A 4.6-kilobase-pair (kbp) EcoRI fragment from C4K6c was subcloned into pBR328 and in both orientations in pHV33. Both Escherichia coli and Bacillus subtilis containing these recombinant plasmids produced antigen that crossreacted with antibody directed against the protoxin. The various sized plasmids of B. thuringiensis were purified and only an EcoRI fragment from the 45-kbp plasmid hybridized to phage C4K6c. One of the pHV33 subclones, pSM36, hybridized to the same size EcoRI/HindIII restriction fragments from plasmid or chromosomal DNA. The cloned EcoRI fragment contained a 0.9-kbp Pvu II fragment that was also present in chromosomal but not in plasmid digests. The original clone was therefore of chromosomal origin, although very similar or identical protoxin genes were present in both the 45-kbp plasmid and the chromosome. Several acrystalliferous nontoxic mutants have been isolated that lacked the 45-kbp plasmid and in some cases all plasmids. All of the mutants contained the chromosomal gene but did not produce protoxin antigen.

Bacillus thuringiensis↗