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The Amyloid Precursor Protein (APP) and its homologues are transmembrane proteins required for various aspects of neuronal development and activity, whose molecular function is unknown. Specifically, it is unclear whether APP acts as a receptor, and if so what its ligand(s) may be. We show that APP binds the Wnt ligands Wnt3a and Wnt5a and that this binding regulates APP protein levels. Wnt3a binding promotes full-length APP (flAPP) recycling and stability. In contrast, Wnt5a promotes APP targeting to lysosomal compartments and reduces flAPP levels. A conserved Cysteine-Rich Domain (CRD) in the extracellular portion of APP is required for Wnt binding, and deletion of the CRD abrogates the effects of Wnts on flAPP levels and trafficking. Finally, loss of APP results in increased axonal and reduced dendritic growth of mouse embryonic primary cortical neurons. This phenotype can be cell-autonomously rescued by full length, but not CRD-deleted, APP and regulated by Wnt ligands in a CRD-dependent manner., Competing Interests: TL, TZ, MN, LB, HR, AS, AC, IP, LF, BD, MP, BH No competing interests declared, (© 2021, Liu et al.)

Women with an abnormal Pap smear are often referred to colposcopy, a procedure during which endocervical curettage (ECC) may be performed. ECC is a scraping of the endocervical canal lining. Our goal was to compare the performance of a naïve Poisson (NP) regression model with that of a zero-inflated Poisson (ZIP) model when identifying predictors of the number of distress/pain vocalizations made by women undergoing ECC. Data on women seen in the colposcopy clinic at a medical school in El Paso, Texas, were analyzed. The outcome was the number of pain vocalizations made by the patient during ECC. Six dichotomous predictors were evaluated. Initially, NP regression was used to model the data. A high proportion of patients did not make any vocalizations, and hence a ZIP model was also fit and relative rates (RRs) and 95% CIs were calculated. AIC was used to identify the best model (NP or ZIP). Of the 210 women, 154 (73.3%) had a value of 0 for the number of ECC vocalizations. NP identified three statistically significant predictors (language preference of the subject, sexual abuse history and length of the colposcopy), while ZIP identified one: history of sexual abuse (yes vs no; adjusted RR=2.70, 95% CI 1.47 to 4.97). ZIP was preferred over NP. ZIP performed better than NP regression. Clinicians and epidemiologists should consider using the ZIP model (or the zero-inflated negative binomial model) for zero-inflated count data., Competing Interests: Competing interests: None declared., (© American Federation for Medical Research 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

We determined the effect of attaching palmitate, tocopherol or cholesterol to PS ASOs and their effects on plasma protein binding and on enhancing ASO potency in the muscle of rodents and monkeys. We found that cholesterol ASO conjugates showed 5-fold potency enhancement in the muscle of rodents relative to unconjugated ASOs. However, they were toxic in mice and as a result were not evaluated in the monkey. In contrast, palmitate and tocopherol-conjugated ASOs showed enhanced potency in the skeletal muscle of rodents and modest enhancements in potency in the monkey. Analysis of the plasma-protein binding profiles of the ASO-conjugates by size-exclusion chromatography revealed distinct and species-specific differences in their association with plasma proteins which likely rationalizes their behavior in animals. Overall, our data suggest that modulating binding to plasma proteins can influence ASO activity and distribution to extra-hepatic tissues in a species-dependent manner and sets the stage to identify other strategies to enhance ASO potency in muscle tissues., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The elastodynamic geometrical theory of diffraction (GTD) has proved to be useful in ultrasonic nondestructive testing (NDT) and utilizes the so-called diffraction coefficients obtained by solving canonical problems, such as diffraction from a half-plane or an infinite wedge. Consequently, applying GTD as a ray method leads to several limitations notably when the scatterer contour cannot be locally approximated by a straight infinite line: when the contour has a singularity (for instance, at a corner of a rectangular scatterer), the GTD field is, therefore, spatially nonuniform. In particular, defects encountered in ultrasonic NDT have contours of complex shape and finite length. Incremental models represent an alternative to standard GTD in the view of overcoming its limitations. Two elastodynamic incremental models have been developed to better take into consideration the finite length and shape of the defect contour and provide a more physical representation of the edge diffracted field: the first one is an extension to elastodynamics of the incremental theory of diffraction (ITD) previously developed in electromagnetism, while the second one relies on the Huygens principle. These two methods have been tested numerically, showing that they predict a spatially continuous scattered field and their experimental validation is presented in a 3-D configuration.

Background: Shift work has been associated with increased body mass index (BMI), metabolic disruption and increased chronic disease risk. Typically, these reports compare individuals who work the day shift to those who work the night shift. Because shift assignment is not random, differences may reflect other, unmeasured characteristics that account for outcome differences., Objective: To compare dietary intake on days on which the participant worked the night shift to days on which she worked the day shift in a population of female nurses who work rotating shifts at a hospital., Methods: This cross-sectional study recruited 132 female registered nurses who work rotating shifts in surgical or internal medicine departments. Dietary intake was ascertained using food diaries and analyzed on Tzameret Nutrition Analysis Software (Israel Ministry of Health). Demographic and anthropometric variables were also recorded., Results: Compared to dietary intake on a day the nurse worked the day shift, intake of the following nutrients increased significantly on the day she worked the night shift: energy; protein; carbohydrates; total fat; saturated fat; and calcium., Discussion: A significant increase in calorie, macronutrient and calcium intake on days the night shift was worked compared to days the day shift was worked among female nurses who work rotating shifts was demonstrated. These findings could be extended to other professionals who work rotating shifts, including physicians and allied healthcare personnel. It appears that the difference detected may be influenced by the food supplied by the hospital as well as by increased food intake in general.

Wnt ligands are secreted glycoproteins that control many developmental processes and are crucial for homeostasis of numerous tissues in the adult organism. Signal transduction of Wnts involves the binding of Wnts to receptor complexes at the surface of target cells. These receptor complexes are commonly formed between a member of the Frizzled family of seven-pass transmembrane proteins and a co-receptor, which is usually a single-pass transmembrane protein. Among these co-receptors are several with structural homology to receptor tyrosine kinases, including Ror, PTK7, Ryk and MUSK. In vertebrates, Ror-2 and PTK7 are important regulators of planar cell polarity (PCP). By contrast, PCP phenotypes were not reported for mutations in off-track ( otk ) and off-track2 ( otk2 ), encoding the Drosophila orthologs of PTK7. Here we show that Drosophila Ror is expressed in the nervous system and localizes to the plasma membrane of perikarya and neurites. A null allele of Ror is homozygous viable and fertile, does not display PCP phenotypes and interacts genetically with mutations in otk and otk2 We show that Ror binds specifically to Wingless (Wg), Wnt4 and Wnt5 and also to Frizzled2 (Fz2) and Otk. Our findings establish Drosophila Ror as a Wnt co-receptor expressed in the nervous system., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

We developed a multifiber optical probe for oblique polarized reflectance spectroscopy (OPRS) in vivo and evaluated its performance in detection of dysplasia in the oral cavity. The probe design allows the implementation of a number of methods to enable depth resolved spectroscopic measurements including polarization gating, source–detector separation, and differential spectroscopy; this combination was evaluated in carrying out binary classification tasks between four major diagnostic categories: normal, benign, mild dysplasia (MD), and severe dysplasia (SD). Multifiber OPRS showed excellent performance in the discrimination of normal from benign, MD, SD, and MD plus SD yielding sensitivity/specificity values of 100%/93%, 96%/95%, 100%/98%, and 100%/100%, respectively. The classification of benign versus dysplastic lesions was more challenging with sensitivity and specificity values of 80%/93%, 71%/93%, and 74%/80% in discriminating benign from SD, MD, and SD plus MD categories, respectively; this challenge is most likely associated with a strong and highly variable scattering from a keratin layer that was found in these sites. Classification based on multiple fibers was significantly better than that based on any single detection pair for tasks dealing with benign versus dysplastic sites. This result indicates that the multifiber probe can perform better in the detection of dysplasia in keratinized tissues.

Ultrasonic telemetry imaging systems are used to monitor such immersed structures as main vessels of nuclear reactors. The interaction between acoustic beams and targets involves scattering phenomena, mainly specular reflection and tip diffraction. In order to assist in the design of imaging systems, a simulation tool is required for the accurate modeling of such phenomena. Relevant high-frequency scattering models have been developed in electromagnetic applications, in particular, the geometrical optics (GO), Geometrical Theory of Diffraction (GTD) and its uniform corrections (UAT and UTD), Kirchhoff approximation (KA) and Physical Theory of Diffraction (PTD). Before adopting any of them for simulation of scattering of acoustic waves by edged immersed rigid bodies, it is important to realize that in acoustics the characteristic dimension to the wave length ratio is usually considerably smaller than in electromagnetics and a further study is required to identify models' advantages, disadvantages and regions of applicability. In this paper their numerical comparison is carried out. As the result, the most suitable algorithm is identified for simulating ultrasonic telemetry of immersed rigid structures., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Simulation of ultrasonic Non Destructive Testing (NDT) is helpful for evaluating performances of inspection techniques and requires the modelling of waves scattered by defects. Two classical flaw scattering models have been previously usually employed and evaluated to deal with inspection of planar defects, the Kirchhoff approximation (KA) for simulating reflection and the Geometrical Theory of Diffraction (GTD) for simulating diffraction. Combining them so as to retain advantages of both, the Physical Theory of Diffraction (PTD) initially developed in electromagnetism has been recently extended to elastodynamics. In this paper a PTD-based system model is proposed for simulating the ultrasonic response of crack-like defects. It is also extended to provide good description of regions surrounding critical rays where the shear diffracted waves and head waves interfere. Both numerical and experimental validation of the PTD model is carried out in various practical NDT configurations, such as pulse echo and Time of Flight Diffraction (TOFD), involving both crack tip and corner echoes. Numerical validation involves comparison of this model with KA and GTD as well as the Finite-Element Method (FEM)., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Diffraction phenomena studied in electromagnetism, acoustics, and elastodynamics are often modeled using integrals, such as the well-known Sommerfeld integral. The far field asymptotic evaluation of such integrals obtained using the method of steepest descent leads to the classical Geometrical Theory of Diffraction (GTD). It is well known that the method of steepest descent is inapplicable when the integrand's stationary phase point coalesces with its pole, explaining why GTD fails in zones where edge diffracted waves interfere with incident or reflected waves. To overcome this drawback, the Uniform geometrical Theory of Diffraction (UTD) has been developed previously in electromagnetism, based on a ray theory, which is particularly easy to implement. In this paper, UTD is developed for the canonical elastodynamic problem of the scattering of a plane wave by a half-plane. UTD is then compared to another uniform extension of GTD, the Uniform Asymptotic Theory (UAT) of diffraction, based on a more cumbersome ray theory. A good agreement between the two methods is obtained in the far field.

We study two canonical problems, diffraction of a plane elastic wave by a thin crack and diffraction of a plane elastic wave by a wedge, both in the high-frequency regime. In applications this regime is usually treated using the so-called Kirchhoff approximation. It is very easy to implement but there are situations when it is known to give distorted results. We discuss an easy correction procedure, which is applicable not only in geometrical regions but inside penumbras too. The procedure involves a version of the Physical Theory of Diffraction that relies on the Geometrical Theory of Diffraction rather than the full solution of the corresponding canonical problem., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We study the guided waves in a structure which consists of two overlapping steel plates, with the overlapping section grouted. This geometry is often encountered in support structures of large industrial offshore constructions, such as wind turbine monopiles. It has been recognized for some time that the guided wave technology offers distinctive advantages for the ultrasonic inspections and health monitoring of structures of this extent. It is demonstrated that there exist advantageous operational regimes of ultrasonic transducers guaranteeing a good inspection range, even when the structures are totally submerged in water, which is a consideration when the wind turbines are deployed off shore., (Copyright © 2010 Elsevier B.V. All rights reserved.)

High-frequency approximations that can be interpreted in terms of the Uniform Geometrical Theory of Diffraction (UGTD) and Uniform Kirchhoff Approximation (UKA) are used to develop a code for modeling ultrasonic scatter of a plane compressional wave by an elliptic crack in the radiating near field. The approximations are intercompared and partially validated against a direct numerical code based on an FD (Finite-Difference) scheme. At present, in many realistic situations the approximate codes of the type described here offer the only viable simulation tool; purely numeric codes are not only much slower, they still require too much computer memory to simulate the complex structure of the radiating near fields., (Copyright 2009 Elsevier B.V. All rights reserved.)

The near field underneath the ultrasonic probe fluid coupled to an isotropic solid is approximated in the frequency domain by a closed form asymptotic solution. The approximation is based on the problem decomposition and uses the stationary phase method evaluating the response to an equivalent surface source. This results in a sum of contributions, each dominating in a specific geometrical region, the main beam or a side lobe. The transitional zones are also described. The pulse trains are computed using the harmonic synthesis and compared with the results obtained by direct calculation of Fourier integrals. It is shown that the asymptotic approach permits us to elucidate the physics of problem and leads to a numerical algorithm which is about 10(4) times faster than the direct computations.

An investigation of the optical and magnetic properties of a unique hydrogen-linked conjugate nanostructure, comprised of superparamagnetic gamma-Fe(2)O(3) nanoparticles (NPs) and near-infrared PbSe nanocrystal quantum dot (NQD) chromophores, is reported. The results show retention of the NQDs' emission quantum efficiency and radiative lifetime, and only a small red shift of its band energy, upon conjugation to the dielectric surroundings of gamma-Fe(2)O(3) NPs. The study also shows the sustainability of the superparamagnetism of the NPs after conjugation, with only a slight decrease of the ferromagnetic-superparamagnetic transition temperature with respect to that of the individual NPs. Thus, the conjugate nanostructure can be considered as a useful medical platform when PbSe NQDs act as fluorescent tags, while the gamma-Fe(2)O(3) NPs are used as a vehicle driven by an external magnetic field for targeted delivery of tags or drugs.

New insights on the molecular level details of the recently reported light-assisted injection of positive charge into single conjugated polymer chains are reported. Extensive new fluorescence-voltage single molecule spectroscopy (FV-SMS) measurements were performed on single chains of the archetypical conjugated polymer MEH-PPV embedded in a capacitor device to complement previous studies of the influence of the bias scan rate and optical excitation intensity. The use of a vacuum microscope allowed for the precise control of the device atmosphere, demonstrating the influence of triplet states in the MEH-PPV on the FV-SMS modulation. For identical device conditions, little variation was observed in the rate and yield of charging from molecule to molecule. Through the use of thicker supporting matrices and insulating polymer "blocking layers", it was determined that good electrical contact between the hole transport layers and the single molecules was necessary for charge injection. The results demonstrate the complexity of charge transfer processes at the interface of organic semiconductors and highlight the ability of single molecule methods to advance the understanding of such processes at the nanoscale.

The injection of positive charge carriers (holes) into a single conjugated polymer chain was observed to be light-assisted. This effect may underlie critical, poorly understood organic electronic device phenomena such as the build-up of functional deeply trapped charge layers in polymer light emitting diodes. The charging/discharging dynamics were investigated indirectly by a variety of single molecule electro-optical spectroscopic techniques, including an "image-capture" approach.

Neural function is dependent upon the proper formation and development of synapses. We show here that Wnt5 regulates the growth of the Drosophila neuromuscular junction (NMJ) by signaling through the Derailed receptor. Mutations in both wnt5 and drl result in a significant reduction in the number of synaptic boutons. Cell-type specific rescue experiments show that wnt5 functions in the presynaptic motor neuron while drl likely functions in the postsynaptic muscle cell. Epistatic analyses indicate that drl acts downstream of wnt5 to promote synaptic growth. Structure-function analyses of the Drl protein indicate that normal synaptic growth requires the extracellular Wnt inhibitory factor domain and the intracellular domain, which includes an atypical kinase. Our findings reveal a novel signaling mechanism that regulates morphology of the Drosophila NMJ.

Gene delivery by intravenous injection of cationic liposome-DNA complexes (LDC) can generate efficient transgene expression in the lungs and other organs, but the duration of expression is typically short. Previous studies have suggested a major role for interferon-gamma (IFN-gamma) and TNF in this process. However, plasmid DNA is also capable of eliciting production of type I IFNs. Therefore, we assessed the ability of LDC to elicit production of type I IFNs in vivo and assessed the effects of type I IFNs on suppression of transgene expression following in vivo gene delivery with LDC. Injection of LDC was found to induce production of high levels of both IFN-alpha and IFN-beta in vivo. Moreover, the levels of transgene expression following in vivo gene delivery were markedly increased in mice lacking functional type I IFN receptor genes, compared to wild-type mice or mice lacking IFN-gamma or TNF receptors. Addition of recombinant IFN-alpha and IFN-beta inhibited transgene expression by in vitro-transfected endothelial cells, and incubation of macrophages with LDC in vitro triggered production of both IFN-alpha and IFN-beta. Therefore, type I IFNs appear to play a key role in suppressing transgene expression in vivo following systemic nonviral gene delivery using LDC.

The review describes the studies of the magneto-optical properties of II-VI and III-V semiconductor nanocrystals (NCs) capped with organic or inorganic epitaxial shells. The investigations focused on the chemical identification of localization sites (core, shell, or interface) of photogenerated carriers in spherical NCs and elucidated the influence of the surface/interface quality on the optical properties of the materials. Optically detected magnetic resonance (ODMR) spectroscopy was used for the study of the proposed physical properties. The ODMR method provides the means to identify the surface/interface sites and correlate them with specific optical transition. In addition, this method reveals information about the spin multiplicity of band edge and trapped states and the electron-hole exchange interaction, determines the spectroscopic g-factors, distinguishes between the radiative and nonradiative characteristic of a trapping site, and evaluates the spin-lattice relaxation times.

The synthesis and magneto-optical properties of HgTe nanocrystals capped with HgxCd1-xTe(S) alloyed shells have been investigated. The magneto-optical measurements included the use of optically detected magnetic resonance (ODMR) and circular polarized photoluminescence (CP-PL) spectroscopy. The PL spectra suggest the existence of luminescence events from both the core HgTe and the HgxCd1-xTe(S) shells. The continuous-wave (cw) and time-resolved ODMR measurements revealed that the luminescence at the shell regime is associated with a trap-to-band recombination emission. The electron trap is comprised of a Cd-Hg mixed site, confirming the existence of an alloyed HgxCd1-xTe(S) composition. The ODMR data and the CP-PL measurements together revealed the g-values of the trapped electron and the valence band hole.

We develop a theory of elastic waves in oriented monodomain nematic elastomers. The effect of soft elasticity, combined with the Leslie-Ericksen version of dissipation function, results in an unusual dispersion and anomalous anisotropy of shear acoustic waves. A characteristic time scale of nematic rotation determines the crossover frequency, below which waves of some polarizations have a very strong attenuation while others experience no dissipation at all. We study the anisotropy of low-frequency Poynting vectors and wave fronts, and discuss a "squeeze" effect of energy transfer nonparallel to the wave vector. Based on these theoretical results, an application, the acoustic polarizer, is proposed.

The magneto-optical properties of chromophore/semiconductor nanocrystalline superstructures were examined using optically detected magnetic resonance (ODMR) and time-resolved photo-luminescence (PL) spectroscopy. The samples consisted of CdS or PbS nanocrystals separated by conjugated organic chains, forming three-dimensional superstructures. The ODMR measurements revealed that the magnetooptical properties of the superstructures are mainly controlled by the individual characteristic of the nanocrystals. The ODMR spectra were compared with simulated curves generated by the diagonalization of a spin Hamiltonian, indicating the existence of the following luminescence events. a) Recombination between electron-hole pairs trapped at a stoichiometric defect (metal or sulfur vacancies) or oxygen adatom sites at the surface of the nanocrystals. These electron-hole pairs showed anistropic g-factors and weak exchange interactions. b) Bound exciton emission, from strongly coupled electron-hole pairs trapped at intrinsic stoichiometric or structural defects at the core of the nanocrystals. The existence of the two overlapping luminescence events is further confirmed by the acceptance of biexponential PL decay processes.

Local expression of cytokine genes by ex vivo transfection or intratumoral gene delivery can control the growth of cutaneous tumors. However, control of tumor metastases by conventional nonviral gene therapy approaches is more difficult. Intravenous injection of lipid-DNA complexes containing noncoding plasmid DNA can significantly inhibit the growth of early metastatic lung tumors. Therefore, we hypothesized that delivery of a cytokine gene by lipid-plasmid DNA complexes could induce even greater antitumor activity in mice with established lung metastases. The effectiveness of treatment with lipid-DNA complexes containing the IL-2 or IL-12 gene was compared with the effectiveness of treatment with complexes containing noncoding (empty vector) DNA. Treatment effects were evaluated in mice with either early (day 3) or late (day 6) established lung tumors. Lung tumor burdens and local intrapulmonary immune responses were assessed. Treatment with either noncoding plasmid DNA or with the IL-2 or IL-12 gene significantly inhibited the growth of early tumors. However, only treatment with the IL-2 or IL-12 gene induced a significant reduction in lung tumor burden in mice with more advanced metastases. Furthermore, the reduction in tumor burden was substantially greater than that achieved by treatment with recombinant cytokines. Treatment with the IL-2 or IL-12 gene was accompanied by increased numbers of NK cells and CD8+ T cells within lung tissues, increased cytotoxic activity, and increased local production of IFN-gamma by lung tissues, compared with treatment with noncoding DNA. Thus, cytokine gene delivery to the lungs by means of intravenously administered lipid-DNA complexes may be an effective method of controlling lung tumor metastases.

An assay measuring RNA expression levels of a gene-encoded therapeutic must distinguish between endogenous mRNA and mRNA transcribed from the transgene. Specificity for the delivered transgene is especially critical when the treatment involves genes that are expressed in the target tissue. To facilitate uniform detection of transgene RNA without interference from endogenous mRNA, we have engineered expression vectors that include a 5' untranslated region (5' UTR) containing a synthetic intron (PGL3). The synthetic intron splice junction was the target sequence for a quantitative reverse transcription (RT)-PCR assay utilizing Taq-Man technology. In this study, we demonstrate that a quantitative RT-PCR assay designed to recognize an engineered intron splice site in the 5'UTR of expression constructs effectively measures the expression level of in vivo-delivered gene therapeutics.

Cationic lipid-DNA complexes (CLDC) are reported to be safe and effective for systemic gene delivery, particularly to the lungs. However, we observed that i.v. injection of CLDC induced immunologic effects not previously reported. We found that even very low doses of CLDC administered i.v. induced marked systemic immune activation. This response included strong up-regulation of CD69 expression on multiple cell types and systemic release of high levels of Th1 cytokines, from both lung and spleen mononuclear cells. CLDC were much more potent immune activators on a per weight basis than either LPS or poly(I:C). The remarkable potency of CLDC appeared to result from enhancement of the immune stimulatory properties of DNA, since cationic lipids alone were without immune stimulatory activity. Systemic treatment with CLDC controlled tumor growth and significantly prolonged survival times in mice with metastatic pulmonary tumors. NK cells accumulated to high levels in the lungs of CLDC-treated mice, were functionally activated, and released high levels of IFN-gamma. The antitumor activity induced by CLDC injection was dependent on both NK cells and IFN-gamma. Thus, DNA complexed to cationic liposomes becomes highly immunostimulatory and capable of inducing strong antitumor activity when administered systemically.

As the sequencing of the human genome proceeds, the need for a new screen for in vivo function is becoming apparent. Many investigators are turning to various transgenic models as a means of studying function. However, these approaches are very time consuming, with a transgene-expressing mouse model often taking months to establish. We have developed an efficient system for delivering genes in vivo, which allows the gene product to be studied as early as 24 h after introduction into the mouse model. The delivery system employs a novel cationic lipid, 1-[2-(9-(Z)-octadecenoyloxy)ethyl]-2-(8-(Z)-heptadecenyl)-3- (hydroxyethyl)imidazolinium chloride (DOTIM), and a neutral lipid, cholesterol, complexed with an expression vector containing the reporter gene chloramphenicol acetyl transferase (CAT). After a single intravenous injection of these complexes, several tissues were seen to express the transgene. High, persistent expression in the vascular endothelial cells in the mouse lung was obtained. Delivery of DNA in vivo has been evaluated by quantitative polymerase chain reaction and protein expression by CAT activity assays. In vivo studies showed reproducible expression in more than 500 mice injected via the tail vein. An early peak of expression was followed by lower, but sustained, expression for > 50 days. Transgene expression of CAT could also be identified by immunohistochemistry staining in mouse lung and appeared to be located within the capillaries. The pattern of in vivo expression could be modulated and targeted to specific organs by altering the lipid-DNA formulation. New expression vectors with altered introns and polyadenylation sites further improved expression. The expression reported here may be sufficient in magnitude, duration, and flexibility to be an attractive alternative, in some cases, to establishing transgenic animals by stable gene transfer.

The Wnt gene family encodes highly conserved cysteine-rich proteins which appear to act as secreted developmental signals. Both the mouse Wnt-1 gene and the Drosophila wingless (wg) gene play important roles in central nervous system (CNS) development. wg is also required earlier, in the development of the embryonic metameric body pattern. We have begun to characterize the developmental expression and role of another member of the Drosophila Wnt gene family, DWnt-3. Using antisera raised to the DWnt-3 protein, we show that the protein is secreted in vivo. The early protein expression domains include the limb and appendage primordia. Late expression domains comprise the ventral cord and supraesophageal ganglia of the CNS. Notably, DWnt-3 protein accumulates on the commissural and longitudinal axon tracts of the CNS. Ectopic expression of DWnt-3 in transgenic embryos bearing a HS-DWnt-3 construct leads to specific disruption of the commissural axon tracts of the CNS. We also show that DWnt-3 does not functionally replace wg in an in vivo assay. Experiments with a tissue culture cell line transfected with a construct encoding the DWnt-3 gene show that DWnt-3 protein is efficiently synthesized, glycosylated, proteolytically processed, and transported to the extracellular matrix and medium. DWnt-3, therefore, encodes a secreted protein, which is likely to play a role in development of the Drosophila CNS.

Stepwise reconstitution of the subunits of DNA polymerase III holoenzyme of Escherichia coli offers insights into the organization and function of this multisubunit assembly. A highly processive, holoenzyme-like activity can be generated when the gamma complex, in the presence of ATP and a primed template, activates the beta subunit to form a preinitiation complex, and this is then followed by addition of the core polymerase. Further analysis of early replicative complexes has now revealed: 1) that the gamma complex can stably bind a single-stranded DNA binding protein (SSB)-coated template, 2) that neither SSB coating of the template nor a proper primer terminus is required to form the preinitiation complex, and 3) that the gamma complex stabilizes the preinitiation complex in the presence of ATP and destabilizes it in the presence of adenosine 5'-O-(thiotriphosphate). Based on these findings, a sequence of stages can be formulated for an activation of the beta subunit that enables it to bind the template-primer and thereby interact with the core to create a processive polymerase.

The gene encoding the protein that binds the three 13-mers in the origin (oriC) of Escherichia coli to block initiation of replication in vitro has been cloned, sequenced, and overexpressed. The gene possesses an open reading frame for 297 amino acids (mass of 33,471 Da). The protein has a motif for DNA-binding (helix-turn-helix) and has homology to a diverse set of prokaryotic regulatory proteins, known as the LysR family. The protein, previously referred to as the 33-kDa protein, has been named IciA (for inhibitor of chromosome initiation). The iciA gene is at 62.8 min on the chromosomal map. Cells with enhanced levels of the protein grow at a normal rate but generally exhibit a pronounced lag upon transfer to a fresh medium.

Quantitative health risk assessments have been performed for a number of proposed municipal waste combustor (MWC) facilities over the past several years. This article presents the results of a comparative analysis of a total of 21 risk assessments, focusing on seven of the most comprehensive methodologies. The analysis concentrates on stack emissions of noncriteria pollutants and is comparative rather than critical in nature. Overall, the risk assessment methodologies used were similar whereas the assumptions and input values used varied from study to study. Some of this variability results directly from differences in site-specific characteristics, but much of it is due to absence of data, lack of field validation, lack of specific guidelines from regulatory agencies, and reliance on professional judgment. The results indicate that carcinogenic risks are more significant than chronic non-carcinogenic risks. In most instances polychlorodibenzodioxins, polychlorodibenzofurans, and cadmium contribute more significantly to the total carcinogenic risk from MWC stack emissions than other contaminants. In addition, the contribution to total risk of all indirect routes of exposure (ingestion and dermal contact) exceeds that of the direct inhalation route for most studies reviewed.

Transcription factor TFIIIC2 derived from human cells is required for tRNA-type gene transcription and binds with high affinity to the essential B-box promoter element of tRNA-type genes. Although 5S rRNA genes contain no homology with the tRNA-type gene B box, we show that TFIIIC2 is also required for Xenopus laevis 5S rRNA gene transcription. TFIIIC2 protected an approximately 30-base-pair (-10 to +18) region of a Xenopus 5S rRNA gene from DNase I digestion. This region, which spanned the transcription start site, included sequences that are highly conserved among eucaryotic 5S rRNA genes and have no homology with the B-box sequence of tRNA genes. Mutation of the TFIIIC2-binding site reduced transcription of the 5S rRNA gene by a factor of 10 in HeLa cell extracts. Methylation of C residues within the TFIIIC2-binding site interfered with binding of TFIIIC2. These results suggest a role of the TFIIIC2-binding sequence in 5S rRNA gene transcription. In addition, the 5S rRNA gene binding site and the tRNA-type gene B-box sequence did not compete with each other for binding to TFIIIC2 any better than did an unrelated DNA sequence, indicating that TFIIIC2 interacts with 5S rRNA genes and tRNA-type genes through separate DNA-binding domains or polypeptides.

An unusual set of submembrane particles is obtained from digitonintreated barley chloroplasts as five gel-electrophoretic zones. Four of them are photochemically active, whereas the most mobile fifth zone has essential traits of the light-harvesting complexes. All of the particles contain the well-known chlorophyll-protein complexes and represent an intermediate level of membrane organization. When isolated from plants fed delta-aminolevulinate in the dark, the fifth zone is characterized by a high level of protochlorophyllide, which is also present to a lesser extent in all the other zones. When [(14)C]aminolevulinate was fed in the dark, followed by exposing the plants to light, the same pattern of the distribution was observed for [(14)C]chlorophyll a. Thus, particles of all the types are involved in chlorophyll formation and the fifth zone is the most distinct in this respect. Its material seems to originate from the most intensely developing areas of the metabolically heterogeneous chloroplast membrane system.

It is shown that Mg-protoporphyrin monomethyl ester as well as immediate chlorophyll precursors are localized in chloroplast submembrane particles, some of them being especially enriched. A conclusion is made that the centers of chlorophyll biosynthesis coupled with chloroplast submembrane particles perform a wide range of biosynthetic reactions beginning at least with Mg-protoporphyrin IX monomethyl ester transformation.

An ambient air sampling study was conducted around a municipal waste combustor; a primary goal was to develop procedures and methods to evaluate the emissions of organic mutagens resulting from incomplete combustion of municipal waste. The products of incomplete combustion from incineration include complex mixtures of organics, particularly polycyclic aromatic compounds, which are present after atmospheric dilution and cooling in emissions as semi-volatile or particle bound organic compounds. Combustion emissions are generally recognized as a potential cancer risk since they contain many carcinogenic and mutagenic polycyclic aromatic hydrocarbons. Analyzing such a complex mixture for the presence of even a few selected chemicals is difficult and provides risk information on only a fraction of the chemicals present. Bioassay methods, however, may be directly applied to evaluate the mutagenic and potential carcinogenic activity of the complex organics from combustion emissions. The Salmonella (Ames) assay was used to determine the mutagenicity associated with particles from ambient air collected near a municipal waste combustor. Dose-response data was generated, and mutagenicity concentrations were calculated to demonstrate the utility of bioassay in assessing the potential impact of emissions from municipal waste combustion. This phase of study quantified mutagenicity concentrations in ambient air but did not detect organic mutagens that could be attributed to incinerator emissions.

The chromosomal translocation associated with many tumors of immunoglobulin-producing cells frequently results in the joining of the immunoglobulin heavy-chain locus and the c-myc oncogene. This translocation of c-myc has profound structural and functional consequences for the oncogene, including loss of the 5' end of the gene and transcriptional deregulation. We report in this communication that translocation results in a new methylation pattern of c-myc. In normal kidney and liver tissue, the c-myc gene is methylated at its 3' end. The translocated gene in plasmacytoma DNA is extensively demethylated. On the other hand, the nonrearranged c-myc gene in plasmacytoma DNA (which is transcriptionally silent) is extensively methylated. In addition, we confirm the nucleotide sequence (with 19 discrepancies out of 1400 bp) 5' to the murine c-myc gene, as reported by Corcoran et al. [Cell 40 (1985) 71-79].

The inhibition of transcription by RNA polymerase III in poliovirus-infected cells was studied. Experiments utilizing two different cell lines showed that the initiation step of transcription by RNA polymerase III was impaired by infection of these cells with the virus. The observed inhibition of transcription was not due to shut-off of host cell protein synthesis by poliovirus. Among four distinct components required for accurate transcription in vitro from cloned DNA templates, activities of RNA polymerase III and transcription factor TFIIIA were not significantly affected by virus infection. The activity of transcription factor TFIIIC, the limiting component required for transcription of RNA polymerase III genes, was severely inhibited in infected cells, whereas that of transcription factor TFIIIB was inhibited to a lesser extent. The sequence-specific DNA-binding of TFIIIC to the adenovirus VA1 gene internal promoter, however, was not altered by infection of cells with the virus. We conclude that (i) at least two transcription factors, TFIIIB and TFIIIC, are inhibited by infection of cells with poliovirus, (ii) inactivation of TFIIIC does not involve destruction of its DNA-binding domain, and (iii) sequence-specific DNA binding by TFIIIC may be necessary but is not sufficient for the formation of productive transcription complexes.

A sensitive assay for detection of eukaryotic RNA polymerase II has been developed. This assay depends on the ability of polymerase II to elongate a small RNA primer, oligo(U), hybridized to a single-stranded homopolymeric DNA template, poly(dA). The poly(dA).oligo(U)-dependent RNA polymerase II from calf thymus has been purified approximately 10,000-fold using this assay. The purified enzyme contains four polypeptides of apparent Mr 180,000, 140,000, 24,000, and 16,000 and is fully active in accurate initiation of transcription from the adenovirus major late promoter in the presence of transcription factors from HeLa cells. The poly(dA).oligo(U)-dependent RNA polymerase activity can be detected in crude cell extracts from a variety of tissue culture cells and appears to be largely due to polymerase II, since 90-95% of this activity is inhibited by alpha-amanitin at a concentration of 1 microgram/ml.