Magnetic anisotropy and magnetization dynamics of individual atoms and clusters of Fe and Co on Pt(111).

The recently discovered giant magnetic anisotropy of single magnetic Co atoms raises the hope of magnetic storage in small clusters. We present a joint experimental and theoretical study of the magnetic anisotropy and the spin dynamics of Fe and Co atoms, dimers, and trimers on Pt(111). Giant anisotropies of individual atoms and clusters as well as lifetimes of the excited states were determined with inelastic scanning tunneling spectroscopy. The short lifetimes due to hybridization-induced electron-electron scattering oppose the magnetic stability provided by the magnetic anisotropies.

[Lichen ruber planus after hepatitis B vaccination]. / Lichen ruber planus nach Hepatitis-B-Impfung.

Lichen planus is a T cell mediated papular skin disease. Chronic hepatic disorders, especially chronic hepatitis, have been found to be associated with lichen planus. Recently, it has been recognized that lichen planus can be triggered by hepatitis B vaccination. Here we report on cases of lichen planus triggered by vaccination and review the current literature. Hepatitis B vaccination is being increasingly used and should be considered as a possible trigger of lichen planus.

The beta 3-adrenergic receptor gene and obesity in a population sample of African Americans.

OBJECTIVE: To examine the role of the Trp64Arg polymorphism in the beta 3-adrenergic receptor gene and the beta 3-adrenergic receptor gene locus in obesity-related traits in African Americans. SUBJECTS: A total of 687 individuals representing 193 African American families who were residents of metropolitan Chicago. MEASUREMENTS: Genotyping of the Trp64Arg polymorphism in the beta 3-adrenergic receptor gene and three microsatellite markers flanking the beta 3-adrenergic receptor gene (ADRB3) locus and measuring various obesity-related traits, including body mass index (BMI), fat-free mass, fat mass, percentage fat mass, waist circumference and serum lipid levels. RESULTS: The prevalence of obesity (defined as body mass index > or = 30 kg/m(2)) in the population was 27.3% and 51.2% in men and women, respectively. The frequency of the Arg64 allele was 10.0%. Multivariate regression analyses confirmed the existence of a significant contribution of familial variance to each of the five obesity-related traits noted above. Likelihood ratio statistics computed in a multivariate regression analysis failed to demonstrate a significant association between the Arg64 allele and any of the five obesity-related traits. Single and multipoint analyses using extended Haseman--Elston regression analyses failed to demonstrate suggestive evidence of linkage of three microsatellite markers that flank the beta 3-adrenergic receptor gene to BMI, percentage body fat, waist circumference or serum leptin levels. CONCLUSION: Given the contribution of familial variance to obesity-related traits in this population, neither the null finding for the Arg64 allele nor the lack of evidence of linkage of the ADRB3 locus to obesity-related traits could be attributed to lack of transmissibility of the traits suggesting that neither the Arg64 variant of the beta 3-adrenergic receptor gene nor another genetic variant in or near the ADRB3 locus contribute significantly to familial aggregation of obesity-related traits in African Americans. International Journal of Obesity (2001) 25, 54-60

Lack of association between the -308 polymorphism of the tumor necrosis factor-alpha gene and the insulin resistance syndrome.

BACKGROUND: Previous studies have demonstrated a role for tumor necrosis factor-alpha (TNF-alpha) in insulin resistance. A polymorphic variant of the TNF-alpha gene, the TNF2 allele, which is a guanine to adenine polymorphism at position -308 in the TNF-alpha promoter, is associated with higher basal and inducible promoter activity. The present study examined whether the TNF2 allele was associated with altered levels of different components of the insulin resistance syndrome, clustering of these components, or the 10-year change in the level of these components. METHODS: Components of the insulin resistance syndrome included insulin resistance, as determined by fasting insulin levels, body mass index, systolic blood pressure, triglycerides, uric acid, and high density lipoprotein-cholesterol. The study population was a subsample of participants from the Coronary Artery Risk Development in (Young) Adults (CARDIA) study, which included African American and white men and women aged 18-30. The sample included 243 black women, 142 black men, 392 white women, and 386 white men. Subjects were typed at the TNF-alpha locus. RESULTS: The frequency of the TNF2 allele was 12% in blacks and 16% in whites. Age-adjusted levels of the different components examined were not different at either baseline or year 10 in carriers of the TNF2 allele versus homozygotes for the wild-type allele, and the 10-year change in the level of different components was not different between the two genotype groups. There also was no evidence of increased clustering of components of the insulin resistance syndrome in carriers of the TNF2 allele. Moreover, there was no evidence of an association between the TNF2 allele and clustering across quartiles of BMI or quartiles of dietary fat intake (i.e., Key's score). CONCLUSIONS: In African Americans and whites, neither the TNF2 allele nor another polymorphism in the TNF-alpha gene or a neighboring gene with which the TNF2 allele is in linkage disequilibrium is associated with differences in the level of or increased clustering of components of the insulin resistance syndrome.

DNA vaccination against La Crosse virus.

For the development of effective conventional vaccines or DNA vaccines against viruses, the availability of suitable animal models is an essential prerequisite. For many recently emerging zoonotic viruses, suitable animal models are still missing. We have established a novel small animal model for DNA vaccines using mice lacking a functional interferon-alpha/beta receptor (IFNAR-1). IFNAR-1-deficient mice are highly susceptible to many different viruses despite their ability to mount a normal humoral and cellular immune response. Taking advantage of this animal model, we show that mice can be completely protected from lethal challenge with a single injection of plasmid DNA encoding the viral envelope proteins G1 and G2. By contrast, vaccination with a plasmid encoding the internal nucleocapsid protein N had little effect. In an effort to enhance the protective immune response to N we assessed the efficacy of vaccination with plasmid DNA encoding N in combination with a plasmid encoding the cytokine IL-12 as adjuvant. IL-12 enhanced the survival of mice following viral challenge, but the effect was independent of N indicating the involvement of components of the innate immune system such as NK cells.

DNA-based vaccine against La Crosse virus: protective immune response mediated by neutralizing antibodies and CD4+ T cells.

La Crosse virus (LACV)-mediated encephalitis is the most frequently reported arboviral disease in the United States, but to date no vaccine against this virus is available. We have established a new animal model, genetically targeted mice lacking a functional interferon type I receptor (IFNAR-1). These mice show an age-independent susceptibility to LACV and develop an acute encephalitis within 6 days of infection, thereby allowing the evaluation of vaccines against LACV. Taking advantage of this knockout mouse model, we have assessed the feasibility of DNA vaccination against this viral disease. Plasmid DNAs, encoding either the virus surface glycoproteins G1 and G2 or the internal nucleocapsid protein N, were used to immunize IFNAR-1-deficient mice. Mice vaccinated with DNA encoding the glycoproteins G1 and G2 produced neutralizing antibodies and exhibited a high degree of protection against challenge with high doses of LACV. Depletion of CD4+ T cells in mice vaccinated with DNA encoding G1/G2 reduced their capacity to control the infection. Virus titration and immunohistological analysis revealed that the protected mice showed no evidence of LACV particles in the brain. This indicates that the vaccine-induced immune response efficiently blocked viral spreading from the primary replication site to the brain. In contrast, immunization with DNA encoding protein N yielded only a partial protective effect that can be attributed to the cellular immune response. Taken together, this study shows that DNA vaccines can be designed to efficiently induce a protective immune response based on neutralizing antibodies and CD4+ T cells.

Enhanced protection against viral infection by co-administration of plasmid DNA coding for viral antigen and cytokines in mice.

BACKGROUND: DNA vaccines have been shown to induce protective immunity against viral infections in different animal models. We have recently demonstrated that DNA vaccine induced protective immunity against influenza A virus and La Crosse virus (LACV) is primarily mediated by humoral immune response. OBJECTIVE: The goal of this study was to investigate whether administration of DNA coding for cytokines such as interleukin 12 (IL-12) and granulocyte-macrophage colony-stimulating factor (GM-CSF) could increase the protective immune response induced by vaccination with DNA coding for viral antigens. STUDY DESIGN: For the influenza A virus or LACV model, C57BL/6 or interferon-alpha/beta receptor (IFNAR-1)-deficient mice, respectively, were vaccinated once or twice with 100 micrograms of DNA encoding viral antigens. At the same time plasmid DNAs (100 micrograms) coding either for mouse GM-CSF or mouse IL-12 were administered. The mice were subsequently challenged with a lethal dose of influenza A virus or LACV and monitored for clinical symptoms (weight loss) and survival. RESULTS: To achieve a high degree of protection (70% survival) two injections of DNA encoding the influenza A virus surface protein hemagglutinin (HA) were required. Intriguingly, administration of DNA coding for IL-12 alone also led to a pronounced protective effect against virus challenge. Co-administration of DNAs encoding IL-12 and HA significantly increased the protective immunity against influenza A virus, while IL-12 expression did not improve protection upon vaccination with DNA coding for the internal nucleocapsid protein N of LACV. Co-injection of DNA coding for mouse GM-CSF and HA also showed an adjuvant effect. CONCLUSIONS: The data clearly indicate that co-administration of DNA encoding cytokines such as IL-12 and GM-CSF with DNA coding for viral antigens has adjuvant effects on the protective immune response against different viral pathogens.

COUP-TFI is a potential regulator of retinoic acid-modulated development in Xenopus embryos.

Isomers of retinoic acid are considered likely regulators of developmental pattern formation in vertebrate embryos. The orphan receptor COUP-TFI, which can alter cellular responses to retinoic acid in cultured cells, is expressed in distinct regions of the developing zebrafish and mouse anterior central nervous system. We asked if COUP-TFI can modulate retinoic acid signaling and anterior neural development in a vertebrate embryo by examining: (1) whether COUP-TFI could alter transcriptional responses to retinoic acid in Xenopus embryonic explants, and (2) whether misexpression of COUP-TFI could regulate anterior neural gene expression during early Xenopus development. The results from these studies show that COUP-TFI is a potent regulator of retinoic acid-induced gene expression in Xenopus embryonic cells, and that misexpression of COUP-TFI causes deficiencies in anterior neural structures and head development in Xenopus embryos with a concomitant change in anterior neural gene expression. These results support the proposition that COUP-TFI has a role in the elaboration and patterning of anterior neural gene expression in vertebrates, possibly via effects on the retinoic acid signaling pathways.

Temporal distribution, localization and metabolism of all-trans-retinol, didehydroretinol and all-trans-retinal during Xenopus development.

Recently, the temporal and spatial distribution patterns of the retinoid receptor ligands 9-cis-retinoic acid and all-trans-retinoic acid were described in Xenopus embryos during early development [Creech Kraft, Schuh, Juchau and Kimelman (1994) Proc. Natl. Acad. Sci. U.S.A., in the press]. The present study demonstrates the presence and distribution of their likely precursors, all-trans-retinol, didehydroretinol, didehydroretinal and all-trans-retinal, as well as the occurrence of 4-oxo metabolites, in Xenopus embryos. The temporal and spatial distribution patterns of all-trans-retinol, didehydroretinol and all-trans-retinal did not coincide with that observed for 9-cis-retinoic acid but, in certain regards, were similar to the patterns delineated for all-trans-retinoic acid and all-trans-retinoyl beta-glucuronide. Evidence is presented that 9-cis-retinoic acid can be synthesized from both all-trans-retinoic acid and all-trans-retinol in Xenopus embryos, suggesting that the difference between the distributions of 9-cis-retinoic acid and the other retinoids may be caused by selective synthesis and/or protein binding of the 9-cis isomer.

The retinoid X receptor ligand, 9-cis-retinoic acid, is a potential regulator of early Xenopus development.

Endogenous retinoids are potential regulators of vertebrate embryogenesis that have been implicated in early anterior-posterior patterning and limb-bud development. We have characterized the temporal and spatial distribution of 9-cis-retinoic acid in the Xenopus embryo and compared it to two other retinoids, all-trans-retinoic acid and all-trans-retinoyl-beta-glucuronide. 9-cis-Retinoic acid is first detected after the midblastula transition and by the end of gastrulation is localized primarily within the anterior and posterior dorsal regions of the embryo. Since 9-cis-retinoic acid is a 6-fold more potent dysmorphogen than trans-retinoic acid, we suggest that it is involved in the early specification of the Xenopus anterior-posterior axis.

v-erbA and citral reduce the teratogenic effects of all-trans retinoic acid and retinol, respectively, in Xenopus embryogenesis.

Treatment of late blastula/early gastrula stage Xenopus embryos with all-trans retinoic acid results in disruption of the primary body axis through effects on both mesoderm and neuroectoderm. This effect of retinoic acid, coupled with the known presence of retinoic acid in Xenopus embryos has led to the proposal that retinoic acid may be an endogenous morphogen providing positional information in early development. To further elucidate the role of retinoic acid in early Xenopus development, we have attempted to interfere with the retinoic acid signalling pathway both at the level of retinoic acid formation, by treatment with citral (3,7-dimethy-2,6-octadienal), and at the level of nuclear retinoic acid receptor function, by microinjection of v-erbA mRNA. The feasibility of this approach was demonstrated by the ability of citral treatment and v-erbA mRNA injection to reduce the teratogenic effects of exogenous retinol and retinoic acid, respectively, in early Xenopus development. Interestingly, v-erbA mRNA injection and citral treatment of gastrula stage embryos resulted in tadpoles with a similar set of developmental defects. The defects were chiefly found in tissues that received a contribution of cells from the neural crest, suggesting that at least a subset of neural crest cells may be sensitive to the endogenous level of retinoic acid. In accord with this proposal, it was found that the expression patterns of two early markers of cranial neural crest cells, Xtwi and XAP-2, were altered in embryos injected with v-erbA mRNA. These results indicate that structures in addition to the primary axis are regulated by retinoic acid signalling during early Xenopus development.

Estrogen receptor-dependent formation of two distinct multiprotein complexes on the human pS2 gene regulatory segment. Participation of a c-fos related protein.

DNA-protein interactions around the regulatory region of the pS2 gene were studied to gain insight into the mechanisms that operate in the estrogen receptor regulated expression of this gene in the MCF-7 human breast cancer cell. Using a revised photocrosslinking technology in combination with gel retardation assays, two distinct multiprotein DNA complexes were shown to assemble in an estrogen receptor-dependent process. Immunological analysis demonstrated the participation of both the estrogen receptor and a c-fos related protein in the formation of these complexes. The results support a model of estrogen receptor function in which the receptor facilitates the formation of multiprotein complexes at DNA sites that can regulate the transcription of a hormone responsive gene by RNA polymerase II and any additional general transcription machinery. These receptor-containing complexes are referred to as "receptorsomes."

A ligand-induced conformational change in the estrogen receptor is localized in the steroid binding domain.

Upon binding estrogen, the estrogen receptor (ER) is proposed to undergo some form of conformational transition leading to increased transcription from estrogen-responsive genes. In vitro methods used to study the transition often do not separate heat-induced effects on the ER from estrogen-induced effects. The technique of affinity partitioning with PEG-palmitate was used to study the change in the hydrophobic surface properties of the ER upon binding ligand with and without in vitro heating. Upon binding estradiol (E2), the full-length rat uterine cytosolic ER undergoes a dramatic decrease in surface hydrophobicity. The binding of the anti-estrogen 4-hydroxytamoxifen (4-OHT) results in a similar decrease in surface hydrophobicity. These effects are independent of any conformational changes induced by heating the ER to 30 degrees C for 45 min. The use of the human ER steroid binding domain overproduced in Escherichia coli (ER-C) and the trypsin-generated steroid binding domain from rat uterine cytosolic ER demonstrates that the decrease in surface hydrophobicity upon binding E2 or 4-OHT is localized to the steroid binding domain. Gel filtration analysis indicates that the change in surface hydrophobicity upon binding ligand is an inherent property of the steroid binding domain and not due to a ligand-induced change in the oligomeric state of the receptor. The decrease in surface hydrophobicity of the steroid binding domain of the ER upon binding E2 or 4-OHT represents an early and possibly a necessary event in estrogen action and may be important for "tight" binding of the ER in the nucleus.

Overproduction of full-length and truncated human estrogen receptors in Escherichia coli.

The full-length human estrogen receptor (hER) as well as two overlapping peptides of hER were overproduced in Escherichia coli JM109 cells, using the inducible pIC vector system. The N-terminal receptor peptide contains the DNA-binding domain as well as the hinge region, whereas the C-terminal peptide contains the same hinge region and the hormone-binding domain. Typically, 1-6 mg of estrogen receptor (ER) peptides can be recovered from 1 L E. coli cell cultures. The majority of the overexpressed proteins are found in inclusion bodies, which allow the isolation of ER peptides in high yields and of 50-80% purity. Induction for short time periods at 10 microM inducer yielded up to 50% of the ER peptides in soluble form with full biological activity. Both the intact receptor and the C-terminal fragment specifically bound estrogens and antiestrogens, whereas ER peptides that contained the DNA-binding domain were retained on a DNA-agarose resin.

Polyclonal antibodies from rabbits and chickens against the estrogen receptor and related peptides. Use in the affinity isolation of estrogen receptors and the retrieval of chromatin fragments associating with estrogen receptors.

Polyclonal antibodies from chickens and rabbits have been prepared against polypeptides representing two regions of the human estrogen receptor (hER). The estrogen receptor (ER) peptides used as antigens were overproduced in Escherichia coli. When indicated, the antibodies were affinity purified using resins to which the antigens contained in bacterial inclusion bodies had been coupled in high yield to epoxy-activated agarose. The antibodies recognize denatured human, bovine, rat, and rabbit ER in immunoblotting experiments. Immuno-precipitation of native ER protein was readily accomplished using rabbit antisera and immobilized protein A. The chicken antibodies, available in larger quantities, were also useful for immunoisolation after coupling to agarose. With the use of these reagents, the selective retrieval of chromatin fragments from MCF-7 cells that interact with ER has been achieved.