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AbstractExposure to the endocrine disruptor bisphenol A (BPA) is ubiquitous and associated with health abnormalities that persist in subsequent generations. However, transgenerational effects of BPA on metabolic health are not widely studied. In a maternal C57BL/6J mice (F0) exposure model using BPA doses that are relevant to human exposure levels (10 μg/kg/day, LowerB; 10 mg/kg/day, UpperB), we showed male- and dose-specific effects on pancreatic islets of the first (F1) and second generation (F2) offspring relative to controls (7% corn oil diet; control). In this study, we determined the transgenerational effects (F3) of BPA on metabolic health and pancreatic islets in our model. Adult F3 LowerB and UpperB male offspring had increased body weight relative to Controls, however glucose tolerance was similar in the three groups. F3 LowerB, but not UpperB, males had reduced β-cell mass and smaller islets which was associated with increased glucose-stimulated insulin secretion. Similar to F1 and F2 BPA male offspring, staining for markers of T-cells and macrophages (CD3 and F4/80) was increased in pancreas of F3 LowerB and UpperB male offspring, which was associated with changes in cytokine levels. In contrast to F3 BPA males, LowerB and UpperB female offspring had comparable body weight, glucose tolerance and insulin secretion as Controls. Thus, maternal BPA exposure resulted in fewer metabolic defects in F3 than F1 and F2 offspring, and these were sex- and dose-specific.

Coronene is one of the basic polycyclic aromatic hydrocarbons (PAHs) used to test the reliabilty of a multidimensional potential energy surface (PES) and to assess its influence on the formation dynamics of PAH clusters with defined physical and chemical properties. We report an analytical potential energy surface for modeling the coronene–coronene interaction, whose parameters were fine-tuned on dispersion-corrected DFT estimations performed within the generalized gradient PBE approximation, that is suitable for describing molecular aggregates involving aromatic species. This model was used to build a potential function for coronene clusters (Corn) that was then applied in a detailed global geometry optimization study with an evolutionary algorithm. A large variety of low-energy structures were obtained for the Corn(n= 2–15) clusters ranging from columnar-type to two-stacked in a handshake association motifs. Moreover, it was found that a transition from a single-stack columnar regime to other more complex shapes occurs at n= 6, whereas previous results based on a simpler coarse-grained potential pointed to a transition at n= 8. Geometry reoptimizations were also performed at the DFT level for the most representative low-energy structures of Corn(n= 3–6), which confirmed the reliability of the present findings.

The chemical interactions and spin structure of (O2)4in its ground singlet state are analyzed by means of Quantum Chemical Topology descriptors. The energetic contributions of the Interacting Quantum Atoms approach are used to obtain information about the class of interactions displayed along the dissociation path of (O2)4. The exchange-correlation contribution to the binding energy is non-negligible for the O2–O2interactions at intermolecular distances close to those found for the pressure induced ε phase of solid (O2) and this strengthening of the intermolecular bonding is built up from a simultaneous weakening of the intramolecular bond. This result is of interest in connection with the observed softening of the IR vibron frequency in the lower pressure range of the ε phase. The spin structure in the real space along the dissociation process is interpreted with the help of the so-called electron number distribution functions. At large distances, the four triplet O2molecules are arranged in a way consistent with an antiferromagnetic structure, whereas at short distances, a significant spin redistribution is driven by the exchange process and it involves a propensity toward a null magnetic moment per molecule. Such probability behavior can be related with the magnetic evolution of solid oxygen across the δ → ε phase transition. Additional calculations of (O2)4excited states support the conclusion that the relative stabilization and magnetic features of the ground singlet state are due to the onset of the new intermolecular bonds, and not to an exclusive modification of the electronic character within the O2molecules.

SS receptors are overexpressed in many tumors, mainly of neuroendocrine origin, thus enabling the treatment with SS analogs. The clinical experience of receptor radionuclide therapy with the new analog [90Y-DOTA0-Tyr3]-octreotide [90Y-DOTATOC] has been developed over the last decade and is gaining a pivotal role in the therapeutic workout of these tumors. It is well known that some procedures performed in diagnostic and therapeutic management of endocrine tumors, such as agobiopsy and hepatic chemoembolization, can be associated with the occurrence of symptoms related to the release of vasoactive amines and/or hormonal peptides from tumor cell lysis. This is the first report of a severe carcinoid crisis developed after receptor radionuclide therapy with 90Y-DOTATOC administered in a patient affected by liver metastases from bronchial neuroendocrine tumor (atypical carcinoid). Despite protection with H1 receptor antagonists, octreotide and corticosteroids, few days after the therapy the patient complained of persistent flushing of the face and upper trunk, severe labial and periocular oedema, diarrhoea and loss of appetite. These symptoms increased and required new hospitalisation. The patient received iv infusion of octreotide associated with H1 and H2 receptor antagonists and corticosteroid therapy, which induced symptom remission within few days. The case here reported confirms that radionuclide therapy is highly effective in determining early rupture of metastatic tissue and also suggests that pre-medication should be implemented before the radiopeptide administration associated with a close monitoring of the patient in the following days.

Molecular beam scattering measurements of total cross sections have been performed at a sufficiently low energy in the thermal range and at an angular resolution high enough to permit for the first time the observation of the “glory” interference effect in collisions of a benzene molecule with He, Ne, and Ar. Information on range, strength, and anisotropy of the interaction in the C6H6− rare gas dimers has been obtained from the analysis of the energy dependence of the total cross sections. In benzene−He, −Ne, and −Ar dimers bond energies are 0.98, 1.95, and 4.20 kJ/mol, respectively, for the most stable geometry, in all cases an out-of-plane configuration with the rare gas atom located on the 6-fold symmetry axis of benzene, at distances of 0.323, 0.331, and 0.359 nm, respectively. The results of the present investigation show that well depths for all three systems decrease by a factor 2 or 5 and corresponding distances increase by 40% or 70% for planar rare gas approaches respectively perpendicular to a C−C bond or collinear to a C−H bond (estimated uncertainties of 10% for bond energies and 3% for bond lengths). These experimental findings provide a crucial test of correlation formulas recently proposed (Chem. Phys. Lett. 2001, 350, 286−296) to estimate van der Waals minimum well depths and distances at selected approach geometries of rare gases on hydrocarbons.

Somatostatin receptors type 2 (sst2) are expressed in high concentration on numerous neudoendocrine tumors. The successful use of radiolabeled somatostatin analogs in imaging promoted further studies in utilizing them in radiopeptide therapy. The somatostatin analog [⁹⁰Y–DOTA–D-Phe¹–Try3]octreotide (DOTATOC) (DOTA: 1,4,7,10-tetraazacyclododecane-N,N',N",N‴-tetraacetic acid) possesses favorable characteristic for its therapeutic use; shows high affinity for sst2, moderately high affinity for sst5, and intermediate affinity for sst3; high hydrophilicity; stable and facile labeling with ¹¹¹ In and ⁹⁰ Y. In this article we report our experience with ⁹⁰Y–DOTATOC in neuroendocrine tumors. Eighty-seven patients with neuroendocrine tumors were treated with a cumulated activity ranging from 7.4 to 20.2 GBq. Most patients responded with stabilization of disease (48%); however, objective responses were observed in 28% of patients (5% complete response). No major acute reactions were observed up to the activity of 5.55 GBq per cycle. The dose limiting was bone marrow toxicity and the maximal tolerated dose was defined as 5.18 GBq. © 2003 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 66: 393–398, 2002

Accurate potential energy surfaces for the N₂–N₂ and N₂–O₂ dimers have been obtained from the analysis of scattering experiments from our laboratory, and of available second vi rial coefficient data. A harmonic expansion functional form describes the geometries of the dimers and accounts for the relative contributions to the intermolecular interaction from components of different nature. Together with the previously obtained singlet, triplet and quintet O₂–O₂ surfaces, where the role of spin–spin coupling was also considered, the new surfaces allow the full characterization of structure and internal dynamics of the clusters, whose bound states and eigenfunctions are obtained by exact quantum mechanics. Besides the information on the nature of the bond, these results can be of use in modelling the role of dimers in air. The calculated rotovibrational levels also provide a guidance for the analysis of spectra, thus establishing the ground for atmospheric monitorings.

The aim of this study was to determine the maximum-tolerated dose, of a pre-targeting three-step (3-S) method employing 90Y-biotin in the locoregional radioimmunotherapy (RIT) of recurrent high grade glioma, and to investigate the antitumor efficacy of this new treatment.Twenty-four patients with recurrent glioma underwent second surgical debulking and implantation of a catheter into the surgical resection cavity (SRC), in order to introduce the radioimmunotherapeutic agents [biotinylated monoclonal antibody (MoAb), avidin and 90Y-biotin].Eight patients with anaplastic astrocytoma (AA) and 16 patients with glioblastoma (GBM) were injected with biotinylated anti-tenascin MoAb (2 mg), then with avidin (10 mg; 24 h later) and finally 90Y-biotin (18 h later). Each patient received two of these treatments 8-10 weeks apart. The injected activity ranged from 0.555 to 1.110 GBq (15-30 mCi). Dosage was escalated by 0.185 GBq (5 mCi) in four consecutive groups. The treatment was well tolerated without acute side effects up to 0.740 GBq (20 mCi). The maximum tolerated activity was 1.110 GBq (30 mCi) limited by neurological toxicity. None of the patients developed hematologic toxicity. In three patients infection occurred around the catheter. The average absorbed dose to the normal brain was minimal compared with that received at the SRC interface.At first control (after 2 months), partial (PR) and minor (MR) responses were observed in three GBM (1 PR; 2 MR) and three AA patients (1 PR; 2 MR) with an overall objective response rate of 25%. Stable disease (SD) was achieved in seven GBM and five AA patients (50%). There was disease progression in six GBM patients (25%), but in none of the AA patients.At the dosage of 0.7-0.9 GBq per cycle, locoregional 3-S-RIT was safe and produced an objective response in 25% of patients. Based on these encouraging results, phase II studies employing 3-S-RIT soon after first debulking are justified.

The molecular mechanism leading to the imprinted expression of genes is poorly understood. While no conserved cis-acting elements have been identified within the known loci, many imprinted genes are located near directly repetitive sequence elements, suggesting that such repeats might play a role in imprinted gene expression. The maternally expressed mouse H19gene is located approximately 1.5 kb downstream from a 461-bp G-rich repetitive element. We have used a transgenic model to investigate whether this element is essential for H19imprinting. Previous results demonstrated that a transgene, which contains 14 kb of H19sequence, exhibits parent-of-origin specific expression and methylation analogous to the endogenous H19imprinting pattern. Here, we have generated transgenes lacking the G-rich repeat. One transgene, containing a deletion of the G-rich repetitive element but which includes an additional 1.7 kb of 5’H19sequence, is imprinted similarly to the endogenous H19gene. To determine whether the G-rich repeat is conserved in other imprinted mammalian H19homologues, additional 5’ flanking sequences were cloned from the rat and human. This element is conserved in the rat but not in human DNA. These results suggest that the 461-bp G-rich repetitive element is not essential for H19imprinting.

Inclusion complex formation between pyridoxine and β- and γ-cyclodextrins in aqueous solution has been investigated by1H NMR and UV spectroscopy. Both complexes exhibited a 1:1 stoichiometry and the inclusion process has been shown to perturb the equilibrium between the lactim and the lactam tautomer of pyridoxine, with a preferential inclusion of the former, less polar tautomer.

Differentially methylated sequences associated with imprinted genes are proposed to control genomic imprinting. A 2-kb region located 5' to the imprinted mouse H19 gene is hypermethylated on the inactive paternal allele throughout development. To determine whether this differentially methylated domain (DMD) is required for imprinted expression at the endogenous locus, we have generated mice harboring a 1.6-kb targeted deletion of the DMD and assayed for allelic expression of H19 and the linked, oppositely imprinted Igf2 gene. H19 is activated and Igf2 expression is reduced when the DMD deletion is paternally inherited; conversely, upon maternal transmission of the mutation, H19 expression is reduced and Igf2 is activated. Consistent with the DMD's hypothesized role of setting up the methylation imprint, the mutation also perturbs allele-specific methylation of the remaining H19 sequences. In conclusion, these experiments show that the H19 hypermethylated 5' flanking sequences are required to silence paternally derived H19. Additionally, these experiments demonstrate a novel role for the DMD on the maternal chromosome where it is required for the maximal expression of H19 and the silencing of Igf2. Thus, the H19 differentially methylated sequences are required for both H19 and Igf2 imprinting.

Background and objective: The clinical effectiveness of noninjectable routes for specific immunotherapy has been demonstrated in many studies, but no data are available on the kinetics of allergens administered by these routes. Therefore we studied the kinetics of the radiolabeled purified major Parietaria judaica allergen (Par j 1) after sublingual, oral, and intranasal administration to healthy human beings. Methods: After tracer administration (10 to 12.5 @mg of Par j 1 labeled with iodine 123) to nonallergic volunteers, scintigraphic images were recorded at various times. Blood samples were also obtained at serial intervals to evaluate the absorption and distribution of radioactivity in plasma and to identify circulating radioactive species by molecular exclusion gel chromatography. Results: When the sublingual route was used, no circulating radioactivity was detected until the tracer was kept under the tongue. The labeled allergen was rapidly degraded and absorbed in the gastrointestinal tract after swallowing. Plasma radioactivity peaked at about 1.5 to 3 hours and was mostly represented by free radioiodine and small radiolabeled peptides. Some activity not caused by free ^1^2^3I remained associated with the oral mucosa up to 18 to 20 hours after administration. When the oral route was used, the results were similar to those observed after swallowing the sublingually administered allergen but without any persistence of the tracer in the mouth. When the intranasal route was used, the pattern of plasma radioactivity mimicked that of the sublingual and oral routes, with absorption of activity from the radiolabeled allergen occurring in the gastrointestinal tract after transport to the pharynx by mucociliary cle arance. A relevant fraction of the tracer was retained on the nasal mucosa up to 48 hours after administration. Conclusion: The data in this study provide the first experimental basis for exploring the in vivo kinetics of allergen administered through noninjectable routes for specific immunotherapy in human beings. (J Allergy Clin Immunol 1997;100:122-9.)

The gene encoding the largest subunit of RNA polymerase II was mapped to mouse chromosome 11 by Southern blotting analysis of mouse-Chinese hamster somatic cell hybrids and by in situ hybridization. This assignment extends the previously defined homology between mouse chromosome 11 and human chromosome 17.

The imprinted mouse H19 gene is hypermethylated on the inactive paternal allele in somatic tissues and sperm. Previous observations from a limited analysis have suggested that methylation of a few CpG dinucleotides in the region upstream from the start of transcription may be the mark that confers parental identity to the H19 alleles. Here we exploit bisulfite mutagenesis coupled with genomic sequencing to derive the methylation status of 68 CpGs that reside in a 4-kb region 5' to the start of transcription. This method reveals a 2-kb region positioned between 2 and 4 kb upstream from the start of transcription that is strikingly differentially methylated in midgestation embryos. At least 12 of the cytosine residues in this region are exclusively methylated on the paternal allele in blastocysts. In contrast, a 350-bp promoter-proximal region is less differentially methylated in midgestation embryos and, like most of the genome, is largely devoid of methylation on both alleles in blastocysts. We also demonstrate exclusive expression of the maternal H19 allele in the embryos that exhibit paternal methylation of the upstream 2-kb region. These data suggest that the 2-kb differentially methylated region acts as a key regulatory domain for imprinted H19 expression.

RNA polymerase II is inhibited by the mushroom toxin alpha-amanitin. A mouse BALB/c 3T3 cell line was selected for resistance to alpha-amanitin and characterized in detail. This cell line, designated A21, was heterozygous, possessing both amanitin-sensitive and -resistant forms of RNA polymerase II; the mutant form was 500 times more resistant to alpha-amanitin than the sensitive form. By using the wild-type mouse RNA polymerase II largest subunit (RPII215) gene (J.A. Ahearn, M.S. Bartolomei, M. L. West, and J. L. Corden, submitted for publication) as the probe, RPII215 genes were isolated from an A21 genomic DNA library. The mutant allele was identified by its ability to transfer amanitin resistance in a transfection assay. Genomic reconstructions between mutant and wild-type alleles localized the mutation to a 450-base-pair fragment that included parts of exons 14 and 15. This fragment was sequenced and compared with the wild-type sequence; a single AT-to-GC transition was detected at nucleotide 6819, corresponding to an asparagine-to-aspartate substitution at amino acid 793 of the predicted protein sequence. Knowledge of the position of the A21 mutation should facilitate the study of the mechanism of alpha-amanitin resistance. Furthermore, the A21 gene will be useful for studying the phenotype of site-directed mutations in the RPII215 gene.

The carboxyl-terminal domain (CTD) of the mouse RNA polymerase II largest subunit consists of 52 repeats of a seven-amino-acid block with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. A genetic approach was used to determine whether the CTD plays an essential role in RNA polymerase function. Deletion, insertion, and substitution mutations were created in the repetitive region of an alpha-amanitin-resistant largest-subunit gene. The effects of these mutations on RNA polymerase II activity were assayed by measuring the ability of mutant genes to confer alpha-amanitin resistance after transfection of susceptible rodent cells. Mutations that resulted in CTDs containing between 36 and 78 repeats had no effect on the transfer of alpha-amanitin resistance, whereas mutations with 25 or fewer repeats were inactive in this assay. Mutations that contained 29, 31, or 32 repeats had an intermediate effect; the number of alpha-amanitin-resistant colonies was lower and the colonies obtained were smaller, indicating that the mutant RNA polymerase II was defective. In addition, not all of the heptameric repeats were functionally equivalent in that repeats that diverged in up to three amino acids from the consensus sequence could not substitute for the conserved heptamer repeats. We concluded that the CTD is essential for RNA polymerase II activity, since substantial mutations in this region result in loss of function.

The mouse H19 gene is expressed exclusively from the maternal allele. The imprinted expression of the endogenous gene can be recapitulated in mice by using a 14-kb transgene encompassing 4 kb of 5'-flanking sequence, 8 kb of 3'-flanking sequence, which includes the two endoderm-specific enhancers, and an internally deleted structural gene. We have generated multiple transgenic lines with this 14-kb transgene and found that high-copy-number transgenes most closely follow the imprinted expression of the endogenous gene. To determine which sequences are important for imprinted expression, deletions were introduced into the transgene. Deletion of the 5' region, where a differentially methylated sequence proposed to be important in determining parental-specific expression is located, resulted in transgenes that were expressed and hypomethylated, regardless of parental origin. A 6-kb transgene, which contains most of the differentially methylated sequence but lacks the 8-kb 3' region, was not expressed and also not methylated. These results indicate that expression of either the H19 transgene or a 3' DNA sequence is key to establishing the differential methylation pattern observed at the endogenous locus. Finally, methylation analysis of transgenic sperm DNA from the lines that are not imprinted reveals that the transgenes are not capable of establishing and maintaining the paternal methylation pattern observed for imprinted transgenes and the endogenous paternal allele. Thus, the imprinting of the H19 gene requires a complex set of elements including the region of differential methylation and the 3'-flanking sequence.

The genomic locus (RPII215) encoding the largest subunit of mouse RNA polymerase II has been cloned by low stringency hybridization to a Drosophila RPII215 probe. The mouse gene consists of 28 exons which span 30 kilobases. Analysis of the nucleotide and predicted protein sequences indicates that the protein is comprised of two domains. There is a 1500 residue amino-terminal domain which contains seven regions strikingly similar to those in the beta' subunit of Escherichia coli RNA polymerase, and a carboxyl-terminal domain comprised of 52 repeats of a 7-amino-acid consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Among the seven highly conserved regions are a strongly basic domain consistent with a DNA-binding site and a consensus sequence characteristic of a potential zinc-binding domain. The 5' upstream region contains three tandem sequences similar to binding sites for the transcription factor SP1. Two of the introns in this gene splice at donor GC dinucleotides as opposed to previously described invariant GT sites. The identification of regions which are highly conserved as compared with bacterial and yeast RNA polymerase and other regions which are unique to the mouse protein suggests which domains of RNA polymerase large subunits are involved in aspects of transcription common to both procaryotes and eucaryotes and which are characteristic of transcription in higher organisms.

One hundred normal Caucasian eyes and 29 eyes with restrictive disorders of ocular motility were studied in order to delineate intraocular pressure changes in secondary positions of gaze. Applanation tonometry was performed in the primary position and at an angle of 22°. In patients with restrictive syndromes the changes of intraocular pressure in the secondary positions of gaze were significantly higher (p < 0.0001) than in normal subjects.

The application of a thin, transparent, polyvinyl-chloride film on the double prism of Goldmann's tonometer is proposed, so as to prevent bacterial and viral infection during tonometry. This film, mounted in such a way as to facilitate its application, is supplied in disposable sterile packaging. The use of the film does not alter the calibration of the instrument and does not substantially modify readings thereby obtained.

Weakly interacting molecules leading to collisional complexes, and to either stable or metastable dimers, potentially play an important role in molecular and surface physics, in astrophysics, in atmospheric photochemistry and physics, and climate. Accurate intermolecular potential energy surfaces for the major components of the atmosphere, leading to the characterization of the O2-O2, N2-N2 and N2-O2 dimers, have been obtained from the analysis of scattering experiments from our laboratory, also exploiting where available second virial coefficient data. A spherical harmonic expansion functional form describes the geometries of the dimers and accounts for the relative contributions to the intermolecular interaction from components of different nature. For O2-O2, singlet, triplet and quintet surfaces are obtained accounting for the role of spin-spin coupling. The new surfaces allow the full characterization of structure and internal dynamics of the clusters, whose bound states and eigenfunctions are obtained by exact quantum mechanics. Besides the information on the nature of the bond, these results can be of use in modelling the role of dimers in air and the calculated rotovibrational levels provide a guidance for the analysis of spectra, thus establishing the ground for atmospheric monitorings. Reference is also briefly made to recent insight on the role of water.