Molecular Tensor Analysis of Third-Harmonic Scattering in Liquids.

Third-harmonic scattering is a nonlinear optical process that involves the molecular second-hyperpolarizability, γ. This work presents a rigorous quantum electrodynamical analysis of the scattering process, involving a partially index-symmetric construction of the fourth-rank γ tensor-dispensing with the Kleinman symmetry condition. To account for stochastic molecular rotation in fluids, methods of isotropic averaging must be employed to relate the molecular properties to accessible experimental quantities such as depolarization ratio. A complete eighth-rank tensor rotational average yields results for observable third-harmonic scattering rates, cast as a function of the natural-invariant γ components, and the polarization geometry of the experiment. Decomposing the tensor γ into irreducible weights allows specific predictions to be made for each molecular point group, allowing greater discrimination between the results for different molecular symmetries.

Do twisted laser beams evoke nuclear hyperpolarization?

The hyperpolarization of nuclear spins promises great advances in chemical analysis and medical diagnosis by substantially increasing the sensitivity of nuclear magnetic resonance (NMR). Current methods to produce a hyperpolarized sample, however, are arduous, time-consuming or costly and require elaborate equipment. Recently, a much simpler approach was introduced that holds the potential, if harnessed appropriately, to revolutionize the production of hyperpolarized spins. It was reported that high levels of hyperpolarization in nuclear spins can be created by irradiation with a laser beam carrying orbital angular momentum (twisted light). Aside from these initial reports however, no further experimental verification has been presented. In addition, this effect has so far evaded a critical theoretical examination. In this contribution, we present the first independent attempt to reproduce the effect. We exposed a sample of immersion oil or a fluorocarbon liquid that was placed within a low-field NMR spectrometer to Laguerre-Gaussian and Bessel laser beams at a wavelength of 514.5nm and various topological charges. We acquired (1)H and (19)F NMR free induction decay data, either during or alternating with the irradiation that was parallel to B0. We observed an irregular increase in NMR signal in experiments where the sample was exposed to beams with higher values of the topological charge. However, at no time did the effect reach statistical significance of 95%. Given the measured sensitivity of our setup, we estimate that a possible effect did not exceed a hyperpolarization (at 5mT) of 0.14-6%, depending on the assumed hyperpolarized volume. It should be noted though, that there were some differences between our setup and the previous implementation of the experiment, which may have inhibited the full incidence of this effect. To approach a theoretical description of this effect, we considered the interaction of an electron with a plane wave, which is known to be able to induce electronic (e.g. in rubidium) and subsequent nuclear hyperpolarization. Compared to the plane wave, the additional transitions caused by a twisted wave are of the order of 10(-3) less. This suggests that the twist of the laser is unlikely to be responsible for the hyperpolarization of nuclear spins, unless a new mechanism of momentum transfer is identified.

One- and two-photon absorption in solution: the effects of a passive auxiliary beam.

The efficiencies of one- and two-photon absorption by chromophores in solution may be significantly modified by a sufficiently intense beam of off-resonant light. A molecular analysis based on quantum electrodynamics (QED) fully accounts for this phenomenon of laser-modified absorption. A time-dependent perturbation-theory treatment describes the process in terms of stimulated forward Rayleigh-scattering of the auxiliary beam occurring simultaneously with the absorption interaction(s). Our formulation accommodates media modifications to the basic character of light-matter interactions, taking into account the refractive and dispersive properties of a solution-phase environment. This introduces the bulk refractive index of the solvent directly into the QED framework. The measurable electronic response of molecules freely rotating in solution is defined by an average of all orientations. We explicitly derive fixed-orientation and rotationally averaged calculations for the Fermi-rule rate of laser-modified one- and two-photon absorption. For a given beam polarization geometry, the solution-phase molecular response is expressible as a set of natural invariant scalars. These results reveal details of the dependence on the beam polarisations and on the rotationally averaged molecular response: we illustrate the breadth of variation available via geometric manipulation of beam polarization, and raise new possibilities for quantum weak measurements of laser states.

Optical emission of a molecular nanoantenna pair.

The optical emission from a pair of nanoantennas is investigated within the theoretical framework of quantum electrodynamics. The analysis of fluorescent emission from a pair of molecular antenna species in close proximity is prompted by experimental work on oriented semiconductor polymer nanostructures. Each physically different possibility for separation-dependent features in photon emission by any such pair is explored in detail, leading to the identification of three distinct mechanisms: emission from a pair-delocalized exciton state, emission that engages electrodynamic coupling through quantum interference, and correlated photon emission from the two components of the pair. Although each mechanism produces a damped oscillatory dependence on the pair separation, each of the corresponding results exhibits an analytically different form. Significant differences in the associated spatial frequencies enable an apparent ambiguity in the interpretation of experiments to be resolved. Other major differences are found in the requisite conditions, the associated selection rules, and the variation with angular disposition of the emitters, together offering grounds for experimental discrimination between the coupling mechanisms. The analysis paves the way for investigations of pair-wise coupling effects in the emission from nanoantenna arrays.

A molecular theory for two-photon and three-photon fluorescence polarization.

In the analysis of molecular structure and local order in heterogeneous samples, multiphoton excitation of fluorescence affords chemically specific information and high-resolution imaging. This report presents the results of an investigation that secures a detailed theoretical representation of the fluorescence polarization produced by one-, two-, and three-photon excitations, with orientational averaging procedures being deployed to deliver the fully disordered limits. The equations determining multiphoton fluorescence response prove to be expressible in a relatively simple, generic form, and graphs exhibit the functional form of the multiphoton fluorescence polarization. Amongst other features, the results lead to the identification of a condition under which the fluorescence produced through the concerted absorption of any number of photons becomes completely unpolarized. It is also shown that the angular variation of fluorescence intensities is reliable indicator of orientational disorder.

Orbital angular momentum exchange in the interaction of twisted light with molecules.

In the interaction of molecules with light endowed with orbital angular momentum, an exchange of orbital angular momentum in an electric dipole transition occurs only between the light and the center of mass motion; i.e., internal "electronic-type" motion does not participate in any exchange of orbital angular momentum in a dipole transition. A quadrupole transition is the lowest electric multipolar process in which an exchange of orbital angular momentum can occur between the light, the internal motion, and the center of mass motion. This rules out experiments seeking to observe exchange of orbital angular momentum between light beams and the internal motion in electric dipole transitions.

Aging and resistance to Trichinella spiralis infection following xenobiotic exposure.

Aging is accompanied by well-documented physiological changes, including alterations in the immune system that can lead to reduced resistance to a variety of infectious agents. We tested the hypothesis that immunosenescence exacerbates the immunosuppressive effect of xenobiotics. If proven true, a given dose of an immunosuppressive xenobiotic would cause greater suppression of host resistance in an aged population.

The Ustilago maydis ubc4 and ubc5 genes encode members of a MAP kinase cascade required for filamentous growth.

Ustilago maydis, the causal agent of corn smut disease, displays dimorphic growth in which it alternates between a budding haploid saprophyte and a filamentous dikaryotic pathogen. We are interested in identifying the genetic determinants of filamentous growth and pathogenicity in U. maydis. To do this we have taken a forward genetic approach. Earlier, we showed that haploid adenylate cyclase (uac1) mutants display a constitutively filamentous phenotype. Mutagenesis of a uac1 disruption strain allowed the isolation of a large number of budding suppressor mutants. These mutants are named ubc, for Ustilago bypass of cyclase, as they no longer require the production of cyclic AMP (cAMP) to grow in the budding morphology. Complementation of a subset of these suppressor mutants led to the identification of the ubc4 and ubc5 genes, which are required for filamentous growth and encode a MAP (mitogen-activated protein) kinase kinase kinase and a MAP kinase kinase, respectively. Evidence suggests that they are important in the pheromone response pathway and in pathogenicity. These results further support an important interplay of the cAMP and MAP kinase signal transduction pathways in the control of morphogenesis and pathogenicity in U. maydis.

Comparison of respiratory responses to Metarhizium anisopliae extract using two different sensitization protocols.

Metarhizium anisopliae, an entomopathogenic fungus, is a prototypic microbial pesticide licensed for indoor control of cockroaches, a major source of allergens. We have previously demonstrated allergy and asthma-like responses in BALB/c mice intraperitoneally (IP) sensitized in the presence of adjuvant and intratracheally (IT) challenged with the soluble factors from M. anisopliae crude antigen (MACA) (Ward et al., 1998, 2000). This protocol has been used frequently to establish animal models of allergenicity. However, the sensitization protocol is artificial and not representative of an environmental exposure. Concern has been raised that this protocol might produce allergic responses that would not occur under normal environmental exposure conditions. The objective of this study was to compare responses in mice to MACA by two exposure protocols: (1) exclusive respiratory exposures without adjuvant (representative of environmental exposures) and (2) intraperitoneal sensitization in the presence of adjuvant followed by IT challenge (the traditional approach). The intratracheal protocol consisted of four IT exposures of 10 microg MACA in 50 microl HBSS each over a 4-week period. A vehicle control group of mice was exposed IT to HBSS. The intraperitoneal protocol consisted of IP sensitization with 25 microg MACA in 0.2 ml of 1.3% alhydrogel (aluminum hydroxide) followed 14 days later with an IT challenge (10 microg MACA/50 microl HBSS). Airway reactivity responsiveness to methacholine was assessed, serum and bronchoalveolar lavage fluid (BALF) samples were obtained, and the lungs were fixed for histopathology at 1, 3, and 8 days following the last MACA IT challenge. Both groups exhibited immune and pulmonary responses typical of allergic asthma. In general, local responses in the lung, including inflammatory responses (eosinophils, lymphocytes, and macrophages), BALF IgE, and functional responses to methacholine were greater in the IT sensitized group compared to the IP sensitized group, whereas the systemic IgE response was greater in the IP sensitized group. The BALF IL-5 cytokine levels were elevated before and throughout the eosinophil influx. IL-4 was detected in the BALF of IP sensitized, but not IT sensitized mice. Histopathologic changes in the two groups were similar in nature but more severe in the IT mice. The results suggest that the IP sensitization protocol does not induce the level of respiratory responsiveness that results from sensitization by a physiologically relevant route of exposure. Thus total serum IgE levels, which were greater following IP sensitization, may not be the best indicator of allergen potency, at least with respect to respiratory responses.

Effects of aging on resistance to Trichinella spiralis infection in rodents exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Immune function, including resistance to infection, decreases as humans and rodents age. We have shown that preinfection exposure of young (9-11 weeks) mice or rats to TCDD decreased resistance to Trichinella spiralis (Ts) infection, expressed as delayed onset or completion of parasite elimination and as increased muscle burdens of larvae. It has also been shown that aged mice express lower constitutive levels of resistance to Ts infection, compared to young adult animals. This study tested the hypothesis that the age-related decrease in constitutive levels of resistance to Ts infection exacerbates the decreased resistance to infection that follows TCDD exposure. This hypothesis addresses the concern that TCDD may pose a greater threat to the elderly than to the population at large. Animals were given a single oral dose of 1, 10, or 30 microg TCDD/kg, 7 days before infection. Eleven days later, young (approximately 10 weeks) control rodents had eliminated a greater proportion of the original parasite burden from the intestine than aged control animals. Nevertheless, parasite elimination was decreased by TCDD exposure only in young rodents. The effect of TCDD exposure on numbers of encysted larvae was evaluated only in rats. Increased larvae burdens occurred in young rats at 30 microg TCDD/kg and at 10 or 30 microg TCDD/kg in aged rats. Parasite-specific splenocyte and lymph node cell proliferation was suppressed following dioxin exposure in young mice; cells from aged mice were markedly less responsive to stimulation, yet less sensitive to TCDD exposure. The response to parasite antigens was not affected in aged rats exposed to TCDD, although elevated mitogen-driven B-cell proliferation was observed. These results indicate that age-related constitutive immunosuppression did not exacerbate TCDD-induced suppression of T-cell mediated adult parasite expulsion; rather, advanced age provided some degree of protection. On the other hand, a lower dose of TCDD was required in aged rats to suppress the combined humoral and cellular responses that limit the burden of encysted larvae, compared to young rats. These model-dependent results preclude acceptance or rejection of the tested hypothesis.

Ethanol attenuates lactate production in hypoxic postnatal day 4 rat cerebella.

Ethanol consumption during pregnancy may lead to a low oxygen supply to the brain of the developing fetus. Such a reduction in the oxygen supply will result in changes in intra- and extracellular lactate production, which subsequently may lead to cytoplasmic acidosis, changes in cerebral metabolism, and eventually, cell death. We used a novel application of gas chromatography to measure lactate changes, on a global level, in the cerebellar tissue of postnatal day (PD) 4 and PD 10 rat pups following in vitro exposure of either hypoxia or hypoxia plus ethanol (hypoxia/ethanol). The results showed hypoxia-induced increases in lactate concentrations as a function of treatment time in both PD 4 and PD 10 cerebellar tissue. However, there was a differential response to the additional ethanol treatment between the two age groups assessed, with an attenuation of the time-dependent increase of lactate production following hypoxia treatment in PD 4 cerebellar tissue. The results also indicated that PD 4 cerebellar tissue had increased oxygen utilization when compared with PD 10 tissue exposed to the same conditions. The ethanol-induced reduction in lactate is hypothesized as being due to limitations in glucose transport and utilization under ethanol/hypoxia exposure. It is believed that such limitations in cellular function may initiate a sequence of events that produce at least some of the cerebellar neuronal loss reported in the fetal alcohol literature.

DNA fragmentation during exposure of rat cerebella to ethanol under hypoxia imposed in vitro.

To gain a better understanding into the mechanisms of damage incurred by neurons in periods following heavy alcohol exposure during development, we used an in vitro system to monitor the effects of alcohol and hypoxia on cell survival and DNA integrity. Samples representing the first few hours of exposure to alcohol and hypoxia were compared to those resulting from hypoxia alone. Measurements were taken from cell counts using Trypan blue exclusion and TUNEL assays as well as digital scans of the ethidium bromide fluorescence of genomic DNA isolated from the treated tissue. We found that DNA degradation from hypoxia was accelerated by several hours in the presence of 100 mM ethanol. This result depended on age, with adult animals (>8 months) having a similar response to 4-day postnatal animals, while the effect on 10-day postnatal animals and those of intermediate age (45 days postnatal) was increasingly delayed. Different methods of inducing the processive degradation of DNA produced laddering typical of apoptosis, a biphasic degradative process, or patterns usually associated with necrosis.

Evaluation of the potential immunotoxicity of chlorinated drinking water in mice.

Recent epidemiological studies have reported associations between the consumption of chlorinated drinking water and various types of human cancer; in addition, exposure to chlorine (Cl-) in drinking water has been reported to suppress certain immune functions in laboratory animals. The current studies were conducted to extend our knowledge of the effects of drinking water exposure to Cl-. Female C57BL/6 mice were administered hyperchlorinated drinking water (7.5, 15, or 30 ppm Cl-) for 2 weeks prior to sacrifice for evaluation of spleen and thymus weights, the plaque-forming cell (PFC) response, hemagglutination (HA) titer, and lymphocyte proliferation (LP). Significant reductions in organ weights and immune response were observed in the positive control groups (i.e. dexamethasone- or cyclophosphamide-exposed mice). No consistent differences were observed between the Cl--exposed animals and vehicle control mice for the evaluated parameters. Thus, under the conditions of these experiments, 2 weeks of exposure to hyperchlorinated drinking water had no apparent adverse effects on immune function.

Chloroplast genes are expressed during intracellular symbiotic association of Vaucheria litorea plastids with the sea slug Elysia chlorotica.

The marine slug Elysia chlorotica (Gould) forms an intracellular symbiosis with photosynthetically active chloroplasts from the chromophytic alga Vaucheria litorea (C. Agardh). This symbiotic association was characterized over a period of 8 months during which E. chlorotica was deprived of V. litorea but provided with light and CO2. The fine structure of the symbiotic chloroplasts remained intact in E. chlorotica even after 8 months of starvation as revealed by electron microscopy. Southern blot analysis of total DNA from E. chlorotica indicated that algal genes, i.e., rbcL, rbcS, psaB, psbA, and 16S rRNA are present in the animal. These genes are typically localized to the plastid genome in higher plants and algae except rbcS, which is nuclear-encoded in higher plants and green (chlorophyll a/b) algae. Our analysis suggests, however, that similar to the few other chromophytes (chlorophyll a/c) examined, rbcS is chloroplast encoded in V. litorea. Levels of psbA transcripts remained constant in E. chlorotica starved for 2 and 3 months and then gradually declined over the next 5 months corresponding with senescence of the animal in culture and in nature. The RNA synthesis inhibitor 6-methylpurine reduced the accumulation of psbA transcripts confirming active transcription. In contrast to psbA, levels of 16S rRNA transcripts remained constant throughout the starvation period. The levels of the photosystem II proteins, D1 and CP43, were high at 2 and 4 months of starvation and remained constant at a lower steady-state level after 6 months. In contrast, D2 protein levels, although high at 2 and 4 months, were very low at all other periods of starvation. At 8 months, de novo synthesis of several thylakoid membrane-enriched proteins, including D1, still occurred. To our knowledge, these results represent the first molecular evidence for active transcription and translation of algal chloroplast genes in an animal host and are discussed in relation to the endosymbiotic theory of eukaryote origins.

Identification and gene expression of anaerobically induced enolase in Echinochloa phyllopogon and Echinochloa crus-pavonis.

Enolase (2-phospho-D-glycerate hydrolase, EC 4.2.1.11) has been identified as an anaerobic stress protein in Echinochloa oryzoides based on the homology of its internal amino acid sequence with those of enolases from other organisms, by immunological reactivity, and induction of catalytic activity during anaerobic stress. Enolase activity was induced 5-fold in anoxically treated seedlings of three flood-tolerant species (E. oryzoides, Echinochloa phyllopogon, and rice [Oryza sativa L.]) but not in the flood-intolerant species (Echinochloa crus-pavonis). A 540-bp fragment of the enolase gene was amplified by polymerase chain reaction from cDNAs of E. phyllopogon and maize (Zea mays L.) and used to estimate the number of enolase genes and to study the expression of enolase transcripts in E. phyllopogon, E. crus-pavonis, and maize. Southern blot analysis indicated that only one enolase gene is present in either E. phyllopogon or E. crus-pavonis. Three patterns of enolase gene expression were observed in the three species studied. In E. phyllopogon, enolase induction at both the mRNA and enzyme activity levels was sustained at all times with a further induction after 48 h of anoxia. In contrast, enolase was induced in hypoxically treated maize root tips only at the mRNA level. In E. crus-pavonis, enolase mRNA and enzyme activity were induced during hypoxia, but activity was only transiently elevated. These results suggest that enolase expression in maize and E. crus-pavonis during anoxia are similarly regulated at the transcriptional level but differ in posttranslational regulation, whereas enolase is fully induced in E. phyllopogon during anaerobiosis.

Immunological effects of 2-methoxyethanol administered dermally or orally to Fischer 344 rats.

Exposure of rats to 2-methoxyethanol (ME) by gavage for 10 consecutive days results in immunotoxicity. To determine whether dermal exposure to ME also induces immunotoxicity, undiluted ME was applied to Fisher 344 male rats at dose levels of 150, 300, 600, 900 or 1200 mg/kg/day on shaved occluded test sites for 4 consecutive days. Decreased thymus weights were produced by all doses of ME, while reductions in spleen weight were observed at doses of 900 mg/kg/day ME or greater. The alterations in these lymphoid organ weights were produced in the absence of loss in body weight. The lymphoproliferative (LP) responses to phytohemagglutinin (PHA) and pokeweed mitogen (PWM) were enhanced at 1200 mg/kg/day ME compared with water controls. Separate groups of rats, employed for the antibody plaque-forming cell (PFC) response to either trinitrophenyl-lipopolysaccharide (TNP-LPS) or sheep red blood cells (SRBC), were exposed dermally to 150, 300 or 600 mg/kg/day ME for 4 consecutive days. A reduction in the PFC response to TNP was observed at 600 mg/kg/day ME, whereas decreases in the PFC response to SRBC were observed at dosages of 300 and 600 mg/kg/day ME. To compare the immunotoxic effects of dermally applied ME to those effects caused by ME administered orally, rats were dosed by gavage with 25, 50, 100 or 200 mg/kg/day ME in distilled water for 4 consecutive days. Reductions in thymus weights were observed at oral dosages ranging from 50-200 mg/kg/day, while spleen weights were reduced in rats dosed at 200 mg/kg/day ME. LP responses to PHA, PWM and Salmonella typhimurium were increased at the 200 mg/kg/day ME dose level. PFC responses to TNP-LPS and SRBC were suppressed at the 50, 100 and 200 mg/kg/day ME dosages. These results indicate that, like oral exposure, dermal exposure to ME compromises the ability of the immune system to mount an effective humoral immune response.

Host resistance to Trichinella spiralis infection in rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

We have previously shown decreased resistance to Trichinella spiralis (Ts) infection and reduced parasite antigen-specific responses in B6C3F1 mice exposed to TCDD before infection. The current study was done to characterize the effects of preinfection administration of 1, 10, or 30 micrograms TCDD/kg on host resistance of female F344 rats to Ts infection and to examine parasite antigen-specific responses in the spleen and mesenteric lymph nodes of infected animals. TCDD exposure did not affect adult parasite elimination from the small intestine or the numbers of encysted larvae in the muscle, although host control of newborn larvae production in female parasites isolated from the highest dose group was compromised. Proliferative responses of lymphocytes cultured with parasite antigen were enhanced in groups of rats exposed to 30 micrograms TCDD/kg. These results, which are in marked contrast to the effects obtained in B6C3F1 mice, demonstrate a clear species difference in the effects of TCDD on immune function in rodents and underscore the need to determine which species more closely reflects the potential outcome of human exposure to TCDD.

Extraction of DNA from mucilaginous tissues of a sea slug (Elysia chlorotica).

Efforts to study the cellular and molecular biology of the symbiotic association between opisthobranch molluscs and algal chloroplasts have been hampered by the copious amounts of mucus produced by the animals. We report for the first time a procedure for isolating total DNA free of contaminating mucilaginous compounds from the mollusc Elysia chlorotica Gould that harbors photosynthetically active chloroplasts from the siphonaceous alga, Vaucheria litorea C. Agardh. This method involves an initial extraction of fresh or freeze-dried Elysia tissue in absolute ethanol and differential processing of the resultant two-phase pellet. Final purification by CsCl-gradient centrifugation produces high molecular weight DNA suitable for molecular analysis.

Differential induction of mRNAs for the glycolytic and ethanolic fermentative pathways by hypoxia and anoxia in maize seedlings.

Fructose-1,6-biphosphate aldolase (ALD) and enolase (ENO) from the glycolytic pathway and pyruvate decarboxylase (PDC) and alcohol dehydrogenase 2 (ADH2) from the ethanolic fermentative pathway, are enzymes previously identified as among those synthesized selectively in O2-deficient roots of maize (Zea mays L.). The present study measured levels of transcripts representing these two pathways in 5-mm root tips, root axes (the remainder of the primary seminal root), and shoots of maize seedlings to determine how closely both pathways were co-induced and how they were modulated by changes in O2 concentration. In hypoxic seedlings with the roots in solution sparged with 5% (v/v) O2 (balance N2) and the shoots in the same gaseous atmosphere, mRNAs for Pdc1 and Adh2 in root tips both increased about 15-fold during the first 12 h, followed by a decline toward initial levels by 18 to 24h. Message levels for Ald1 and Eno1 showed only small changes during hypoxia. When expression was examined under anoxia, the extent to which all four mRNAs increased in different tissues depended on whether the seedlings had been previously acclimated to hypoxia or were anoxically shocked. The results show that although all the genes examined increased expression during hypoxia and/or anoxia, they differed in the rapidity and magnitude of the response and in the time to reach maximal message levels: there was no common pattern of change of message levels for the glycolytic or for the fermantative enzymes.