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Regions of space are defined to maximize the probability to find a given number of electrons within. Their chemical significance and their relationship to the electron localization function (ELF) are explored by analyzing the results for a few linear molecules: LiH, BH, N2, CO, CS, C2H2, and C4H2.
Protonacceptor properties of the HOO radical were investigated previously by means of ab initio as well as topological Atoms in Molecules (AIM) and Electron Localization Function (ELF) methods. It was pointed out that in the radical there are three nonequivalent positions most susceptible to protonation, and on this basis three structures of possible hydrogen bonded complexes were proposed. Results reported in the present article concern all possible 1:1 complexes formed by HCl and HOO molecules, and fully confirm suppositions given on the basis of the above-mentioned investigations. There are three various complexes referring to the local minima, and the transition structure predicted by topological methods has been found as well. The cyclic structure appeared to be the most stable one, which confirms conclusions given in the experimental article. Apart from structure optimization, harmonic as well as anharmonic spectra of the complexes have been simulated. Anharmonicity of H-Cl stretching vibration was of special interest, as the frequency of this vibration characterizes the Cl-H...O hydrogen bond in these complexes. To obtain values of these frequencies the one-dimensional Hamiltonian has been diagonalized numerically. The potential for this Hamiltonian has been taken from a set of single-point scanning of the part of the Potential Energy Surface (PES) connected with this vibration. The potential calculated on the MP2 level leads to the result close to the experimental value, whereas the B3LYP method is inappropriate for the purpose of PES investigation of these complexes.
Although it would be tempting to associate the Lewis structures to the maxima of the squared wave function |Psi|2, we prefer in this paper the use of domains of the three-dimensional space, which maximize the probability of containing opposite-spin electron pairs. We find for simple systems (CH4, H2O, Ne, N2, C2H2) domains comparable to those obtained with the electron localization function (ELF) or by localizing molecular orbitals. The different domains we define can overlap, and this gives an interesting physical picture of the floppiness of CH5+ and of the symmetric hydrogen bond in FHF-. The presence of multiple solutions has an analogy with resonant structures, as shown in the trans-bent structure of Si2H2. Correlated wave functions were used (MCSCF or Slater-Jastrow) in the Variational Quantum Monte Carlo framework.
We describe the development of Lewis's ideas concerning the chemical bond and in particular the concept of the electron pair bond and the octet rule. We show that the concept of the electron pair bond has endured to the present day and is now understood to be a consequence of the Pauli principle. In contrast the octet rule is now regarded as much less important than was originally generally believed, although Lewis himself knew several exceptions and regarded it as less important than what he called the rule of two (the electron pair). The octet rule was more strongly promoted by Langmuir who is also responsible for the term covalent bond. However, many more exceptions to the octet rules than were known to Lewis are now known and the terms hypervalent and hypovalent used to describe such molecules are no longer particularly useful. Today it is realized that bonding electron pairs in many molecules are not as well localized as Lewis believed, nevertheless resonance structures, i.e., plausible alternative Lewis structures, are still often used to describe such molecules. Moreover electrons are not always found in pairs, as for example in linear molecules, which can, however, be satisfactorily described by Linnett's double quartet theory. The electron density distribution in a molecule can now be analyzed using the ELF and other functions of the electron density to show where electron pairs are most probably to be found in a molecule.
In an open prospective study with matched historical controls we aimed to evaluate whether a polysaccharide fraction isolated from the herb Echinacea purpurea could counteract the undesired effects of chemotherapy. Fifteen patients with advanced gastric cancer undergoing palliative chemotherapy with etoposide, leucovorin and 5-fluorouracil (ELF) received for 10 days (beginning 3 days before chemotherapy) daily i.v. injections of 2 mg of a polysaccharide fraction isolated from Echinacea purpurea herb cell cultures (EPS-EPO VIIa). The median number of leukocytes 14-16 days after chemotherapy was 3630/microL (range 1470-5770) in the patients receiving EPS-EPO VIIa compared with 2370/microL (870-3950) in the patients of the historical control group (p = 0.015). EPS-EPO VIIa had no clinically relevant effects on phagocytic activity of granulocytes or on lymphocyte subpopulations. Sixty-eight adverse events including two deaths were observed, most likely due to chemotherapy and the general condition of the patients. However, an association with the test intervention cannot be ruled out completely. The results of this pilot study suggest that EPS-EPO VIIa might be effective in reducing chemotherapy-induced leukopenia. The efficacy and safety should be investigated in further studies.
In order to monitor the effects of drugs on interleukin-8 (IL-8) production by cells, a microtiter plate assay that determines four parameters simultaneously was established (i) levels of secreted IL-8 (supernatant ELISA), (ii) levels of intracellular IL-8 (cell ELISA), (iii) intracellular localization (fluorescence microscopy), and (iv) the amount of cellular protein (colorimetric assay). The quantitative and qualitative determination of intracellular IL-8 was achieved by immunofluorescence using the ELF-detection system (Molecular Probes, Eugene, OR), which is approximately 10 times more sensitive than conventional immunofluorescence detection systems. Thus, a 32x objective magnification (without immersion oil) is sufficient to precisely assess the subcellular localization of IL-8. Experiments were carried out with human umbilical vein endothelial cells. Drugs interfering with transcription or translation and inhibitors of protein kinase C inhibited both production and secretion of IL-8. Brefeldin A (BFA), colchicine, and the HMGCoA-reductase inhibitor fluvastatin disrupted the characteristic Golgi localization of IL-8, but only BFA inhibited its secretion. This assay can therefore be used to distinguish drugs that inhibit both IL-8 synthesis and secretion from those that inhibit IL-8 secretion only. It can easily be adapted to other cellular proteins for which a sensitive detection method is required.
In the retinotectal system, positional information has long been postulated to take the form of molecular gradients within both the retina and the tectum. Recent reports have implicated Mek4, a member of the Eph (also named class V) family of tyrosine kinase receptors (RTKs), and two ligands, RAGS and ELF-1, in this process. Here, we report the cloning and distribution pattern of QEK5, another member of the Eph family of RTKs, isolated from a quail cDNA library. During retinal differentiation, QEK5 transcripts accumulate in a ventral to dorsal gradient within the retinal neuroepithelium, where its expression becomes restricted to the ganglion and bipolar cell layers. Within the tectum, QEK5 transcripts are detectable in a posterior to anterior gradient in the ventricular layer and newly formed superficial layers. The pattern of QEK5 expression in the retina and tectum is distinct from that of Mek4, suggesting that complex patterns of Eph RTKs and their ligands may play a role in cell-cell interactions involved in retinotectal projections and differentiation of the central nervous system.
Recent studies have implicated Eph-related receptor tyrosine kinases and their membrane-bound ligands in restricting or stimulating the movement of cells and axons. Members of these large families of receptors and ligands fall into two major binding specificity classes, in which the GPI-anchored subgroup of ligands can each bind to all members of a subgroup of receptors, whereas the transmembrane ligands interact with a distinct subgroup of receptors. Analysis of expression patterns is therefore important in order to understand which receptor-ligand interactions occur in vivo. We have cloned mouse orthologues of five members of the ligand family and analysed in detail their developmental expression, in comparison with each other, and with the receptor specificity class they can interact with. We find that B61, AL-1/RAGS, LERK4, and ELF-1, members of the GPI-anchored subgroup of ligands, have both distinct and overlapping aspects to their expression in early mesoderm, somites, and branchial arches; in complex, dynamic patterns in the limb; and in spatial domains and specific neurons in the CNS. Similarly, Elk-L is expressed in hindbrain segments, the roof plate, and floor plate, which overlaps with that of other transmembrane ligands, but has distinct expression in somites. The expression domains of ligands are complementary to those of the corresponding receptors in a number of tissues, including the midbrain, hindbrain, and differentiating limbs, consistent with potential roles in restricting cell movement. In addition, we find that there are some overlaps in expression of receptors and ligands, for example in somites and the early limb. Taken together with previous studies showing that Eph-related receptors also have distinct but overlapping expression patterns, these data indicate that each ligand may have stage- and tissue-specific interactions with an individual member or multiple members of the receptor family.
Despite interpretive problems associated with studies of antibiotic concentrations in tissues, it is of major interest to correlate the presence of the drug at significant concentrations in lung tissues and fluids at a particular site with its clinical efficacy. All the models described above have their uses as well as their limitations. Measurement of antibiotic concentrations achieved in lung parenchyma, ELF, bronchial mucosa of bronchial secretions has shown variable levels for the same drug. Many respiratory infections are caused by obligate or facultative intracellular pathogens, which may be eradicated as a result of intracellular penetration and accumulation of quinolones, of macrolides, as shown in several models of phagocytic cells, and as a result of intracellular antibacterial activities. For bacterial infections located in extracellular pulmonary sites, a knowledge of achievable concentrations of beta-lactams, of aminoglycosides, of quinolones should be of value, whereas for bacteria multiplying in alveolar macrophages the high concentrations of the new macrolides or new fluoroquinolones that can be achieved in extravascular and intracellular fluids should have clinical relevance. Animal models of severe pneumonia, as well as studies in patients hospitalized in intensive care units, have provided correlation between clinical outcomes and high lung tissue and intramacrophagic concentrations of fluoroquinolones, or of macrolides in legionellosis, or of ceftazidime or imipenem in pseudomonal pneumonia.
Ozone exposure, in vitro, has been shown to activate phospholipases A2 (PLA2), C (PLC), and D (PLD) in airway epithelial cells. However, because of its high reactivity, ozone cannot penetrate far into the air/lung tissue interface. It has been proposed that ozone reacts with unsaturated fatty acids (UFA) in the epithelial lining fluid (ELF) and cell membranes to generate a cascade of lipid ozonation products (LOP) that mediate ozone-induced toxicity. To test this hypothesis, we exposed cultured human bronchial epithelial cells (BEAS-2B) to LOP (1-100 microM) produced from the ozonation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and measured the activity of PLA2, PLC, and PLD. The PLA2 isoform responsible for arachidonic acid release (AA) in stimulated cultures was also characterized. Activation of PLA2, PLC, and PLD by three oxidants, hydrogen peroxide (H2O2), tert-butyl hydroperoxide (t-BOOH) and 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) also was measured and compared to that of LOP. The derivatives of ozonized POPC at the sn-2 residue, 9-oxononanoyl (PC-ALD), 9-hydroxy-9-hydroperoxynonanoyl (PC-HHP), and 8-(-5-octyl-1,2,4-trioxolan-3-yl-) octanoyl (POPC-OZ) selectively activated PLA2 in a dose-dependent fashion. Cytosolic PLA2 (cPLA2) measured in the cytosolic fraction of stimulated cell lysates was found to be the predominant isoform responsible for AA release. PLC activation was exclusively induced by the hydroxyhydroperoxide derivatives. PC-HHP and the 9-carbon hydroxyhydroperoxide (HHP-C9) increased PLC activity. PLD activity also was induced by LOP generated from POPC. Incubation of cultures with H2O2 alone did not stimulate PLC; however, in the presence of the aldehyde, nonanal, a 62 +/- 2% increase in PLC activity was found, suggesting that the increase in activity was due to the formation of the intermediate HHP-C9. t-BOOH, and AAPH also failed to induce PLA2 activation, but did activate PLC, under conditions of exposure identical to that of LOP. Only t-BOOH activated PLD. These results suggest that biologically relevant concentrations of LOP activate PLA2, PLC, and PLD in the airway epithelial cell, a primary target to ozone exposure. The activation of these phospholipases may play a role in the development of lung inflammation during ozone exposure.
We have characterized reovirus strains that differ in the degree to which they inhibit cellular protein synthesis and used them to investigate mechanisms regulating gene expression in infected cells. A previous genetic study associated distinct effects of reovirus strains on cellular translation with polymorphisms in viral protein sigma3. In cell extracts, sigma3 sequesters double-stranded RNA (dsRNA) and blocks activation of the dsRNA-activated protein kinase (PKR), an interferon-induced enzyme that inhibits translational initiation by phosphorylating elF-2alpha. We found that in infected cells, cellular protein synthesis is translationally regulated in a strain-specific manner. Using immunoprecipitation and indirect immunofluorescence we showed that the effect of a strain on cellular translation is not determined by the level of sigma3, but appears to result from differences in sigma3 localization. In cells infected with a strain that spares cellular translation, sigma3 is present throughout the cytoplasm, whereas in cells infected with inhibitory strains, sigma3 is restricted to perinuclear viral factories. Biochemical studies suggested that diffuse localization of sigma3 is a consequence of low affinity for capsid protein mu1. Our findings are consistent with a model in which the efficiency of cellular translation is determined by the cytoplasmic level of sigma3 that is not complexed with mu1.
We examined the effects of brief exposure to weak 60 Hz extremely low frequency (ELF) magnetic fields and opioid systems on spatial behavior and learning in reproductive adult male and female deer mice, Peromyscus maniculatus. Sex differences were evident in spatial performance, with male deer mice displaying significantly better performance than female mice in the Morris water maze, whereby animals had to acquire and retain the location of a submerged hidden platform. Brief (maximum 5 min) exposure to weak (100 microT) 60 Hz magnetic fields during task acquisition significantly improved female performance, eliminating the sex differences in acquisition. The opiate antagonist, naltrexone, also improved female acquisition, though significantly less than the magnetic fields. These facilitatory effects involved alterations of "non-spatial" (task familiarization and reduction of related anxiety/aversive related behaviors) and possibly "spatial" aspects of the task. Enhancement of enkephalin activity with the enkephalinase inhibitor, SCH 34826, significantly reduced task performance by male deer mice. Both naltrexone and the 60 Hz magnetic fields attenuated the enkephalin mediated reductions of spatial performance. These findings indicate that brief exposure to 60 Hz magnetic fields can enhance water maze task acquisition by deer mice and suggest that these facilitatory effects on spatial performance involve alterations in opioid activity.
A system consisting of any array of cylindrical, polytopic membrane proteins (or protein complexes) possessed of a permanent dipole moment and immersed in a closed, spherical phospholipid bilayer sheet is considered. It is assumed that rotation of the protein (complex) in a plane normal to the membrane, if occurring, is restricted by viscous drag alone. Lateral diffusion is assumed either to be free and random or to be partially constrained by barriers of an unspecified nature. The dielectric relaxation times calculated for membrane protein rotation in a suspension of vesicles of the above type are much longer than those observed with globular proteins in aqueous solution, and fall in the mid-to-high audio frequency range. If the long range lateral diffusion of (charged) membrane protein complexes is essentially unrestricted, as in the "fluid mosaic" membrane model, dielectric relaxation times for lateral motions will lie, except in the case of the very smallest vesicles, in the sub-audio (ELF) range. If, in contrast, the lateral diffusion of membrane protein complexes is partially restricted by "barriers" or "long-range" interactions (of unspecified nature), significant dielectric dispersions may be expected in both audio- and radio-frequency ranges, the critical (characteristic) frequencies depending upon the average distance moved before a barrier is encountered. Similar analyses are given for rotational and translational motions of phospholipids. At very low frequencies, a dispersion due to vesicle orientation might in principle also be observed; the dielectrically observable extent of this rotation will depend, inter alia, upon the charge mobility and disposition of the membrane protein complexes, as well as, of course, on the viscosity of the aqueous phase. The role of electroosmotic interactions between double layer ions (and water dipoles) and proteins raised above the membrane surface is considered. In some cases, it seems likely that such interactions serve to raise the dielectric increment, relative to that which might otherwise have been expected, of dispersions due to protein motions in membranes. Depending upon the tortuosity of the ion-relaxation pathways, such a relaxation mechanism might lead to almost any characteristic frequency, and, even in the absence of protein/lipid motions, would cause dielectric spectra to be much broader than one might expect from a simple, macroscopic treatment.
Studies were carried out on the dermatoglyphics (skin ridge marks) on the hands of children with Williams syndrome; this is an inherited disease with cardiovascular pathology and a characteristic facial phenotype ("elf" facies), along with specific mental and cognitive disturbances. The results suggest a characteristic dermatoglyphic type with the presence of complex whorls on the fingers and a clear predominance of marks of greater complexity on the left hand; this is a very rare trait in normal people and in those with other inherited nervous system disorders. The features of the dermatoglyphic pattern serve as a characteristic marker of a genetically determined state of the human central nervous system, and suggests directions for neurophysiological studies of children with Williams syndrome as a unique model for analysis of higher nervous function in humans.
To study the biological effects of extremely low frequency (ELF) electric fields, a fundamental study is conducted of the human perception threshold of an electric field. The perception threshold is measured with human subjects, and the results are analysed. It is clear that field perception is based on the movement of hair and not on other sensations. Variance in the perception threshold and its causes are investigated. The perception threshold decreases by almost 30% as the relative humidity increases from 50 to 90%. The perception threshold is also dependent on the physical condition (length and density) of the hair and the psychological condition (degree of awareness) of the subject. The dependence on these is much smaller than that on relative humidity. The cause of the gender difference in the threshold is ascribed to the difference in the physical condition of the hair. Through this study, some factors to be taken into account for the safety standard are made clear.
Many reports demonstrate that extremely low frequency magnetic fields (ELF MFs, 60 Hz) may be involved in hyperalgesia. In a previous investigation, we suggested that MFs may produce hyperalgesia and such a response may be regulated by the benzodiazepine system. In order to further confirm this effect of MFs, we used diazepam and/or flumazenil with MFs exposure. When testing the pain threshold of rats using hot plate tests, MFs or diazepam (0.5 microg, i.c.v.; a benzodiazepine receptor agonist) induced hyperalgesic effects with the reduction of latency. These effects were blocked by a pretreatment of flumazenil (1.5 mg/kg, i.p.; a benzodiazepine receptor antagonist). When the rats were exposed simultaneously to MFs and diazepam, the latency tended to decrease without statistical significance. The induction of hyperalgesia by co-exposure to MFs and diazepam was also blocked by flumazenil. However, the pretreatment of GABA receptor antagonists such as bicuculline (0.1 microg, i.c.v.; a GABA(A) antagonist) or phaclofen (10 microg, i.c.v.; a GABA(B) antagonist) did not antagonize the hyperalgesic effect of MFs. These results suggest that the benzodiazepine system may be involved in MFs-induced hyperalgesia.
The effect of extremely low frequency (ELF, 60 Hz) magnetic fields (MFs) on convulsions was investigated in rats. We determined the onset and duration of convulsions induced by bicuculline alone or by co-exposure to MFs and bicuculline. In addition, we measured the GABA concentrations in the rat brains using HPLC-ECD. MFs strengthened the convulsion induced by bicuculline (0.3, 1, and 3 microg, i.c.v.), with a shortening of the onset time, but lengthening of the duration time. Co-exposure to MFs and bicuculline decreased the GABA levels in the cortex, hippocampus and hypothalamus, whereas MFs alone reduced the level of GABA only in the hippocampus. These results suggest that the exposure to MFs may modulate bicuculline-induced convulsions due to GABA neurotransmissions in rat brains.
Expression profiles of mRNAs and proteins for various transcription factors were determined for human skin mast cells (sMCs), leukemic HMC-1 MCs, monocytes and granulocytes. By quantitative RT-PCR, sMCs expressed lower levels of c-fos, PU.1, C/EBPalpha, and C/EBPepsilon than monocytes and granulocytes, but higher levels of MITF, SCL, GATA-1 and GATA-2. At the protein level, MITF, SCL, GATA-2, Elf-1 and c-fos were clearly detectable in sMCs. With the exception of c-fos, these proteins were absent or expressed only slightly in monocytes and granulocytes. The expression of NF-E2p45, GATA-1, PU.1, Ets-1, C/EBPalpha and C/EBPepsilon was below the detection limit in sMCs, but detectable in other myelocytes. The high expression of SCL and GATA-2 in sMCs is reminiscent of stem cells. The absence of C/EBPvarepsilon in sMCs, but strong expression in HMC-1, suggests it may impair MC maturation. In summary, mature human MCs can be characterized as C/EBPalpha(low), C/EBPvarepsilon-, PU.1(low), GATA-1(low), GATA-2+, SCL+, MITF(high).