Experimental studies of the NaCs 12(0+) [71Σ+] state: Spin-orbit and non-adiabatic interactions and quantum interference in the 12(0+) [71Σ+] and 11(0+) [53Π0] emission spectra.

We present results from experimental studies of the 11(0+) and 12(0+) electronic states of the NaCs molecule. An optical-optical double resonance method is used to obtain Doppler-free excitation spectra. Selected data from the 11(0+) and 12(0+) high-lying electronic states are used to obtain Rydberg-Klein-Rees and Inverse Perturbation Approach potential energy curves. Interactions between these two electronic states are evident in the patterns observed in the bound-bound and bound-free fluorescence spectra. A model, based on two separate interaction mechanisms, is presented to describe how the wavefunctions of the two states mix. The electronic parts of the wavefunctions interact via spin-orbit coupling, while the individual rotation-vibration levels interact via a second mechanism, which is likely to be non-adiabatic coupling. A modified version of the BCONT program was used to simulate resolved fluorescence from both upper states. Parameters of the model that describe the two interaction mechanisms were varied until simulations were able to adequately reproduce experimental spectra.

Rotationally inelastic collisions of excited NaK and NaCs molecules with noble gas and alkali atom perturbers.

We report measurements of rate coefficients at T ≈ 600 K for rotationally inelastic collisions of NaK molecules in the 2(A)1Σ+ electronic state with helium, argon, and potassium atom perturbers. Several initial rotational levels J between 14 and 44 were investigated. Collisions involving molecules in low-lying vibrational levels (v = 0, 1, and 2) of the 2(A)1Σ+ state were studied using Fourier-transform spectroscopy. Collisions involving molecules in a higher vibrational level, v = 16, were studied using pump/probe, optical-optical double resonance spectroscopy. In addition, polarization spectroscopy measurements were carried out to study the transfer of orientation in these collisions. Many, but not all, of the measurements were carried out in the "single-collision regime" where more than one collision is unlikely to occur within the lifetime of the excited molecule. The analysis of the experimental data, which is described in detail, includes an estimate of effects of multiple collisions on the reported rate coefficients. The most significant result of these experiments is the observation of a strong propensity for ΔJ = even transitions in collisions involving either helium or argon atoms; the propensity is much stronger for helium than for argon. For the initial rotational levels studied experimentally, almost all initial orientation is preserved in collisions of NaK 2(A)1Σ+ molecules with helium. Roughly between 1/3 and 2/3 of the orientation is preserved in collisions with argon, and almost all orientation is destroyed in collisions with potassium atoms. Complementary measurements on rotationally inelastic collisions of NaCs 2(A)1Σ+ with argon do not show a ΔJ = even propensity. The experimental results are compared with new theoretical calculations of collisions of NaK 2(A)1Σ+ with helium and argon. The calculations are in good agreement with the absolute magnitudes of the experimentally determined rate coefficients and accurately reproduce the very strong propensity for ΔJ = even transitions in helium collisions and the less strong propensity for ΔJ = even transitions in argon collisions. The calculations also show that collisions with helium are less likely to destroy orientation than collisions with argon, in agreement with the experimental results.

Experimental studies of the NaCs 5(3)Π0 and 1(a)3Σ+ states.

We report high resolution measurements of 372 NaCs 5(3)Π(0)(v, J) ro-vibrational level energies in the range 0 ≤ v ≤ 22. The data have been used to construct NaCs 5(3)Π(0) potential energy curves using the Rydberg-Klein-Rees and inverted perturbation approximation methods. Bound-free 5(3)Π(0)(v, J) → 1(a)(3)Σ(+) emission has also been measured, and is used to determine the repulsive wall of the 1(a)(3)Σ(+) state and the 5(3)Π(0) → 1(a)(3)Σ(+) relative transition dipole moment function. Hyperfine structure in the 5(3)Π(0) state has not been observed in this experiment. This null result is explained using a simple vector coupling model.

Genetic control of gastrulation in the mouse.

In the past, understanding of the process of gastrulation in the mouse has primarily been based on morphological analyses. Recently, a number of molecules have been implicated in mesoderm induction and axis formation in Xenopus, and several of these exhibit unique patterns of expression during mouse gastrulation. These gene-expression data, together with fate mapping, ectopic expression experiments and mutational analysis, will now facilitate studies on the functional aspects of gastrulation in the mouse.

Rearrangement of rat immunoglobulin E heavy-chain and c-myc genes in the B-cell immunocytoma IR162.

Mammalian B-cell lymphoid malignancies frequently display aberrant translocations involving the c-myc proto-oncogene and one of the immunoglobulin loci. We have observed and characterized such a translocation in the immunoglobulin E-producing rat immunocytoma IR162 by using recombinant DNA technology. We show here for the first time that a c-myc gene has recombined with the excluded allele of the nonfunctional epsilon heavy-chain immunoglobulin gene. This recombination has resulted in the loss of 5'-proximal DNA and, consequently, potential regulatory information from the body of the c-myc structural gene via joining to the epsilon heavy-chain switch region in a head-to-head, i.e., 5'-to-5', configuration. As discussed in this report, these results, together with the previous results of others, have important implications for immunoglobulin heavy-chain class switching mechanisms controlling normal and abnormal translocations.

Isolation and characterization of the rat beta-subunit gene of the high affinity receptor for immunoglobulin E.

The high affinity receptor for IgE, which mediates allergic reactions, is found exclusively on mast cells, basophils and epidermal Langerhans cells. It is composed of an alpha, beta and two gamma-polypeptide chains, inserted as a complex into the plasma membrane. The alpha and beta-subunits are produced only by these cell types. This work describes the isolation and characterization of the gene which encodes the rat beta-subunit. The single copy gene spans a 9 kbp region of DNA and is composed of seven exons and six introns. Transcriptional initiation begins from a single site, which is preceded by a very unique, putative transcriptional consensus sequence, the GATA box. Analysis of the 5'-region, upstream of exon 1, also reveals a unique sequence bias and a collection of repeating elements, suggesting several other potential transcriptional cis-control elements. The origins of the two, previously reported, different beta-subunit transcripts result from the same start site of this gene, but use an alternative RNA processing mechanism involving exon 3.

Clozapine for treatment-refractory mania.

OBJECTIVE: The efficacy of clozapine for treatment-resistant mania was examined in a prospective trial for patients with bipolar or schizoaffective disorder. METHOD: The subjects were 25 acutely manic patients with either bipolar disorder (N = 10) or schizoaffective disorder-bipolar subtype (N = 15) for whom lithium, anticonvulsants, and neuroleptics had been ineffective, had produced intolerable side effects, or both. After a 7-day washout, the patients were treated with clozapine monotherapy. They were evaluated over 13 weeks with the Young Mania Rating Scale and the Brief Psychiatric Rating Scale (BPRS). RESULTS: Of the 25 patients, 18 (72%) exhibited marked improvement on the Young Mania Rating Scale, and eight (32%) exhibited marked improvement on the BPRS. The bipolar patients as compared to schizo-affective patients, and the nonrapid as compared to rapid cyclers, had significantly greater improvement in total BPRS score. CONCLUSIONS: These results suggest that clozapine is an effective therapy for treatment-resistant bipolar and schizoaffective mania.

Tissue-specific expression in mouse P815 mastocytoma cells of the cloned rat alpha-subunit gene of the high-affinity receptor for immunoglobulin E.

The high-affinity multisubunit receptor for IgE, Fc epsilon RI, is expressed in vivo in a tissue-specific manner, being found only on mast cells, basophils and epidermal Langerhans cells. The expression of the rat Fc epsilon RI alpha-subunit transcript has been examined here in the mouse mastocytoma cell line, P815, which does not express the endogenous mouse Fc epsilon RI alpha- or beta-subunit transcripts. These studies indicate that an exogenously introduced rat alpha-subunit gene can be faithfully expressed in P815 cells, while no transcripts are produced in the mouse or rat B-lymphoid cell lines, SP2/0 and IR162, respectively. Therefore, in contrast to both B-cell lines, factors necessary for the tissue-specific expression of the alpha-subunit mRNA are present in the P815 cell line, yielding a correctly processed mRNA transcript; nevertheless, this tissue-specific expression is not rigorously species specific. Although the mechanism responsible for these observations is unknown, these results do imply that a specific cis-trans element interaction occurs between this transcriptional unit and factors in the P815 cells that control fidelity of tissue-specific mRNA synthesis and processing from the Fc epsilon RI alpha-subunit gene. Consequently, this reconstructed rat Fc epsilon RI alpha-subunit gene contains minimally sufficient cis elements for tissue-specific expression of mRNA, and so, the P815 cell line should be useful to define these requisite DNA cis elements of the gene, as well as the trans factors from P815 necessary for this process.

Molecular analysis of the promoter region of the Clostridium difficile toxin B gene that is functional in Escherichia coli.

Clostridium difficile is a human pathogen that produces two types of toxins, A and B, that cause a potentially lethal gastrointestinal syndrome termed pseudomembranous colitis. Virtually nothing is known about the mechanism of regulation of toxin production in this organism, and cis-regulatory regions of neither toxin have yet been identified, thus prompting this investigation. A motif homologous with the Shine-Dalgarno sequence of Escherichia coli occurs upstream from the putative initiation codon of toxin B, making this region also a candidate to contain a promoter. Therefore, a subgenomic DNA library of C. difficile in a plasmid vector was first constructed encompassing the 5'-end of the toxin B gene. A 450-bp DNA fragment was excised from the subgenomic DNA library clone and subcloned into a promoter-probe plasmid vector that contains two divergently oriented, promoterless genes to assay for promoter function. This subcloned DNA fragment directed the expression of alkaline phosphatase, a reporter gene product of the promoterless vector, thus indicating the presence of a functional promoter. To locate the promoter more precisely, a series of nested deletions of the toxin B promoter subclone was constructed with exonuclease III. The promoter that facilitates expression of the toxin B gene in E. coli was localised, based on alkaline phosphatase activity. The transcriptional initiation site of toxin B mRNA in E. coli was mapped by primer extension analysis, suggesting two closely associated tandem start sites directed by two similarly spaced promoters within this localised region.

A qualitative study of male dental hygienists' experiences after graduation.

This report is part of a larger study undertaken in 1996 and 1997 for the author's doctoral dissertation. The study's purpose was to explore the experiences of male dental hygienists--focusing on their experiences before, during, and after graduation. The researcher interviewed 14 practicing male dental hygienists from east of the Mississippi River and one participant from the Midwest. Because of the length of the study, only their experiences following graduation from a dental hygiene program are discussed. Qualitative research methods were used to evaluate the information gained from the interviews, which entails analyzing interview transcripts and developing themes from the data. Four post-graduation themes emerged: participants experienced (1) no job-search difficulties, although some participants experienced minor problems with securing a position, most had little trouble in finding a job; (2) societal gender discrimination, mainly in relation to societal stereotypes about what men and women should do; (3) mixed feelings of acceptance by the profession, although most felt the profession accepting, there were some feelings of not belonging; and (4) career satisfaction, all but one of the participants felt satisfied with his career choice.

Isolamento químico e validação analítica por cromatografia líquida de alta eficiência de quercitrina em Solidago chilensis Meyen (Asteraceae) / Chemical isolation and analytical validation by high-performance liquid chromatography of quercitrin in Solidago chilensis Meyen (Asteraceae)

RESUMO A espécie Solidago chilensis Meyen, Asteraceae é conhecida como erva-lanceta ou arnica-brasileira, sendo utilizada popularmente como antimicrobiana e para o tratamento de inflamações tópicas. No entanto, estudos fitoquímicos e farmacológicos para as partes aéreas são escassos. Neste trabalho, realizou-se a determinação de flavonoides por espectrofotometria de UV/Vis, prospecção fitoquímica da fração acetato de etila visando o isolamento do constituinte químico majoritário e validação analítica por cromatografia líquida de alta eficiência (CLAE). O teor de flavonoides totais foi de 5,42%, representados como hiperosídeo. O fracionamento químico utilizando métodos cromatográficos (cromatografia líquida em coluna gel de sílica; CHCl3:EtOH; 8:2 v/v) e espectroscópicos (1H RMN,13C RMN e ESI-MS) revelou o isolamento de quercetina-3-O-α-L-ramnosídeo(quercitrina). A sensibilidade e a linearidade (r = 0,999) da validação analítica, utilizando a quercitrina isolada do extrato hidroalcoólico da planta, revelaram um rendimento de 5,29% do analito em relação à droga vegetal. Precisão, recuperação e robustez, além dos valores estabelecidos para os limites de detecção (LOD) e de quantificação (LOQ), poderão ser utilizados como parâmetros de qualidade para extratos à base de S. chilensis.

ABSTRACT The species Solidago chilensis Meyen Asteraceae, known as “erva-lanceta” or “Brazilian arnica”, is popularly used as an antimicrobial and topical treatment for inflammations. However, phytochemical and pharmacological studies of its aerial parts are scarce. In this study, flavonoids were determined by UV/Vis spectrophotometry and phytochemical screening of the ethyl acetate fraction with the goal of isolating the major chemical constituent and analytically validating it through high performance liquid chromatography (HPLC). The total flavonoid content was 5.42%, represented as hyperoside. Chemical fractionation using chromatographic (liquid chromatography in column of silica gel, CHCl3:EtOH, 8:2 v/v) and spectroscopic methods (1H RMN, 13C RMN, and ESI-MS) revealed the isolation of quercetin-3-O-α-L-rhamnoside (quercitrin). The sensitivity and linearity (r = 0.999) using the isolated quercitrin of the hydroalcoholic extract of the plant revealed a yield of 5.29% of analyte in relation to the plant. Precision, recovery, and robustness, as well as values set for the limits of detection (LOD) and quantitation (LOQ) can be used as quality parameters for extracts based on S. chilensis.

Tetrahydrobenzothiophene inhibitors of hepatitis C virus NS5B polymerase.

A novel series of selective HCV NS5B RNA dependent RNA polymerase inhibitors has been disclosed. These compounds contain an appropriately substituted tetrahydrobenzothiophene scaffold. This communication will detail the SAR and activities of this series.

Physical and biological properties of an epsilon-heavy chain mRNA and a kappa-light chain mRNA coding for rat immunoglobulin E.

The mRNA coding for epsilon-heavy chain and kappa-light chain have been highly enriched from a rat IgE-producing myeloma, IR-162. Based on denaturing gel analyses, the 20 S epsilon-heavy chain mRNA has an estimated molecular weight of 850,000, equivalent to about 2500 nucleotides. The 14S rat kappa-light chain mRNA has an estimated molecular weight of 410,000, equivalent to about 1200 nucleotides. Only about two-thirds of the length of these mature cytoplasmic rat mRNA code for protein. The 20 S mRNA stimulates the in vitro synthesis of a single major serologically related protein which is large enough to be epsilon-heavy chain. It is unglycosylated and has an apparent molecular weight of about 62,000. The in vivo unglycosylated epsilon-heavy chain, obtained in the presence of tunicamycin, has an apparent molecular weight of about 59,000, compared with about 76,000 for the glycosylated heavy chain of the secreted rat IgE. Therefore, the in vitro synthesized epsilon-heavy chain protein is about Mr = 3000 larger than the in vivo unglycosylated epsilon-heavy chain, equivalent to about 25 extra amino acids. This is consistent with the synthesis of an epsilon-heavy chain putative precursor. Likewise, the 14 S mRNA stimulates the in vitro synthesis of a single putative precursor protein, which is serologically related to kappa chain, is unglycosylated, and is about an extra 20 amino acids. This is the first report on the physical and biological properties of an epsilon-heavy chain mRNA, as well as any rat immunoglobulin mRNA.

[Age dependence of the P2 amplitudes of visually evoked cortical responses to checkerboard pattern reversal (author's transl)]. / Altersabhängigkeit der P2-Amplituden schachbrettmusterevozierter Potentiale.

The age dependence of the P2 amplitudes of visually evoked cortical responses to checkerboard pattern reversal was examined in 72 healthy individuals of between 10 and 69 years of age. Separate examination of the right and left eye yielded no statistically significant differences in P2 amplitudes. An age dependence of the P2 amplitudes was discovered inasmuch as healthy individuals under 25 years and over 55 are--with high statistical significance--apt to show higher P2 amplitudes than healthy individuals in the remaining life span.

Positional cloning of a global regulator of anterior-posterior patterning in mice.

Anterior-posterior (A-P) patterning is of fundamental importance throughout vertebrate embryonic development. Murine members of the trithorax (trx) and Polycomb group (Pc-G) of genes regulate A-P patterning of segmented axial structures, demonstrating conserved upstream regulation of homeotic pathways between Drosophila and mouse. Here we report the positional cloning of a classical mouse mutation, eed (for embryonic ectoderm development), which is the highly conserved homologue of the Drosophila Pc-G gene esc (for extra sex combs), a long-term repressor of homeotic genes. Mutants homozygous for a null allele of eed display disrupted A-P patterning of the primitive streak during gastrulation. Mutant embryos lack a node, notochord and somites, and there is no neural induction. In contrast to absence of anterior structures, extra-embryonic mesoderm is abundant. Mice carrying a hypomorphic eed mutation exhibit posterior transformations along the axial skeleton. These results indicate that eed is required globally for A-P patterning throughout embryogenesis.

Purification of 14S messenger RNA of immunoglobulin light chain that codes for a possible light-chain precursor.

Polysomes released from microsomes of MOPC 41 mouse myeloma were used to prepare a poly(A)-containing fraction of RNA by chromatography on poly-(dT)-cellulose. From that fraction, a 14S RNA species was purified to a single peak by successive sucrose gradient centrifugations, followed by acrylamide gel electrophoresis. The RNA has an apparent molecular weight of 380,000 (1100 nucleotides), as estimated from the electrophoretic analyses. In a reticulocyte lysate this RNA directs the synthesis of a protein that migrates more slowly in sodium dodecylsulfate-acrylamide gels than does the light chain secreted by the same tumor. This difference in migration corresponds to a size difference appropriate for polypeptide chain about 20 amino acids longer than the light chain. The tryptic peptides of this protein correspond to those of the secreted light chain, except for the presence of two additional peptides from the product synthesized in vitro and for the absence of one light-chain peptide. The purified RNA is, therefore, the mRNA of the light chain, and it seems to code for a precursor protein slightly larger than the light chain. From the estimated size of the 14S mRNA, it appears that only 65% of the RNA is translated.

Physical mapping of the loci Gabra3, DXPas8, CamL1, and Rsvp in a region of the mouse X chromosome homologous to human Xq28.

Using pulsed-field gel electrophoresis, a 3 million-bp physical map containing the X-linked loci Gabra3, DXPas8, CamL1, and Rsvp has been constructed for a segment of the mouse X chromosome homologous to human Xq28. Detailed mapping was performed using single and double digestions with rare-cutter restriction enzymes. Gabra3 and DXPas8 have been shown to be physically linked within a maximal distance of 1600 kb, DXPas8 and CamL1 within 750 kb, and CamL1 and Rsvp within 450 kb. In addition, several CpG islands have been detected in the region encompassing CamL1 and Rsvp. These studies confirm a gene order of cen-Gabra3-DXPas8-CamL1-Rsvp-tel determined by genetic mapping in interspecific backcrosses (A.S. Ryder-Cook et al., 1988, EMBO J. 7: 3017-3021; G.E. Herman et al., 1991, Genomics 9: 670-677). Physical distances for the loci studied agree with the calculated genetic distances. Assuming that there is conserved linkage between man and mouse in the region, the physical mapping data presented here may help to clarify the uncertain gene order for some human Xq28 loci.

A simple and precise aberrant translocation of the rat c-myc gene into the epsilon-heavy chain switch region of the IgE-producing immunocytoma, IR162.

We report a simple type of reciprocal chromosomal translocation in the LOU rat IgE-secreting immunocytoma cell line, IR162, involving the c-myc protooncogene and the switch region of the epsilon immunoglobulin heavy chain, c-myc/S epsilon. By cloning and sequencing the translocation-associated and the homologous normal c-myc and S epsilon DNAs, we have identified the position of the translocational junction in both the c-myc 5'-flanking region and the repetitive elements of the S epsilon region. The translocational recombination was precise, and no insertion or N-addition was found in the junctional region, leaving all the c-myc exons, together with two promoter sites, intact. RNase mapping confirmed that the same promoters were utilized in IR162 and normal LOU spleen cells. No point mutation was found in the 5'-flanking region and the 3'-portion of exon 1 of the translocated c-myc gene. However, the putative silencer region was lost with the translocation. It was also noticed that a strikingly AT-rich sequence associated with S epsilon region had translocated to the 5'-flanking region of c-myc gene. We discuss the possibility that a change of DNA topology, perhaps either due to the juxtaposition of an AT-rich sequence of the S epsilon region, or to the loss of the putative silencer element, may contribute to c-myc gene deregulation in IR162.

The expression and characterization of rat IgE produced by construction of the epsilon-heavy chain gene from exon modules.

A system of exon "modules" was produced from the functionally rearranged epsilon-heavy gene isolated from the rat IgE-secreting immunocytoma IR162. The five individual exons, encoding the variable and constant region domains, were isolated and subcloned into the multiple cloning site of a pair of plasmid vectors with opposed orientation multiple cloning sites. The use of opposed orientation multiple cloning sites and the flanking restriction enzyme sites contained therein allows for the modular manipulation of the gene. These exon modules were initially used to reconstruct the epsilon-heavy chain gene into the native configuration to demonstrate the efficacy of the modular system for synthesis of IgE. Upon transfection into the rat myeloma cell line Y3, the reconstructed gene produced a polypeptide that associated with the endogenous light chain polypeptide and was secreted from the cell as tetrameric IgE. All physical and functional characterizations indicate that the IgE molecule produced is indistinguishable from native IR162 IgE. This modular system of exons will facilitate the manipulation of IgE structure through the systematic assembly of different epsilon-heavy chain mutant constructions. The resulting novel IgE proteins will be very useful to study the molecular nature of the interaction of IgE with its Fc receptor.