Simian virus 40 minichromosomes as targets for retroviral integration in vivo. We present a method for studying multiple retroviral integration events into a small DNA target in vivo. Episomal simian virus 40 (SV40) genomes established by infection of CV-1 cells served as integration targets during subsequent infection with murine leukemia virus (MLV). Using a PCR-based assay for the abundance and distribution of integration events, nonrandom integration of MLV DNA into SV40 DNA is detectable as early as 4 hr and reaches a maximum level by 8 hr after MLV infection. The level of integration but not the distribution of integration sites is sensitive to the stage in the SV40 life cycle at which MLV infection is performed. Using a temperature-sensitive tumor (T) antigen mutant SV40 strain, we observed that active replication of the target DNA is not required for efficient integration in vivo. The distribution of integration sites in vivo is closely approximately by in vitro reactions with isolated SV40 minichromosomes as integration targets. However, the degree of bias between the most and least favored sites is greater in vivo than in vitro.


Growth and differentiation of embryonic stem cells that lack an intact c-fos gene. The c-fos protooncogene encodes a transcription factor that is thought to play a critical role in proliferation and differentiation as well as in the physiological response of mature cells to their environment. To test directly the role of c-fos in growth and differentiation, we generated mouse embryonic stem cell lines in which both copies of the c-fos gene were specifically disrupted by homologous recombination. Remarkably, the disruption of both copies of c-fos in these cells has no detectable effect on embryonic stem cell viability, growth rate, or differentiation potential. Embryonic stem cells lacking c-fos can differentiate into a wide range of cell types in tissue culture and also in chimeric mice. We conclude that despite a large body of literature suggesting an important role for c-fos in cell growth and differentiation, in at least some cell types this gene is not essential for these processes.


[D-Ala2]deltorphin I binding and pharmacological evidence for a special subtype of delta opioid receptor on human and invertebrate immune cells. The effects of the opioid neuropeptide [D-Ala2]deltorphin I, isolated from amphibian skin, on immunoregulatory activities were studied in representatives of vertebrates and invertebrates. The high potency of this compound parallels that of [Met]enkephalin, which was previously demonstrated in vertebrate plasma and invertebrate hemolymph. The addition of [D-Ala2]deltorphin I at 10(-11) M to human granulocytes or immunocytes of the mollusc Mytilus edulis resulted in cellular adherence and conformational changes indicative of cellular activation. This value is in line with the concentrations obtained with [Met]enkephalin, tested in the presence of the specific neutral endopeptidase 24.11 inhibitor phosphoramidon, and this opioid's synthetic analog [D-Ala2, Met5]enkephalin which, like [D-Ala2]deltorphin I, is resistant to proteolytic degradation. Both ligands appear to be acting on the same population of immunocytes. The same relationship was estimated to exist in the insect Leucophaea maderae, in which the high viscosity of the hemolymph makes the quantification of reactive cells more difficult than in Mytilus. In addition, [D-Ala2]deltorphin I is as potent as beta-endorphin in affecting the proliferation of lymphocytes in response to mitogen. Saturation experiments with unlabeled ligands and the radioligands [3H][D-Ala2]deltorphin I and [3H][D-Ala2,Met5]enkephalinamide revealed the presence of two high-affinity binding sites on human granulocytes, one sensitive to the nonequilibrium delta opioid antagonist [D-Ala2,Leu5,Cys6]enkephalinamide and the other relatively insensitive. The results obtained with [D-Ala2]deltorphin I support the view that the special role played by endogenous [Met]enkephalin in immunobiological activities of vertebrates and invertebrates is mediated by a special subtype of delta opioid receptor.


On the origin of the enthalpy and entropy convergence temperatures in protein folding. Temperature dependence of the thermodynamics of folding/unfolding for cytochrome c has been determined as a function of moderate [0-10% (vol/vol)] concentrations of methanol. Heat capacity change (delta Cp) for unfolding decreases with increased concentrations of methanol, consistent with a higher solvent hydrophobicity. For a given transition temperature, this effect results in higher experimental enthalpy (delta H) and entropy (delta S) changes with increased methanol concentrations. When the enthalpy or entropy data sets obtained at different methanol concentrations are plotted as a function of temperature, they are seen to converge and assume common values around 100 degrees C for delta H and 112 degrees C for delta S. These convergence temperatures are similar to those obtained for different proteins in aqueous solution when delta H and delta S are normalized with respect to number of residues. It has been previously hypothesized that these convergence temperatures correspond to the temperatures at which the hydrophobic contributions to delta H and delta S are zero; the results presented here agree with this viewpoint.


Expression cloning of a cDNA encoding UDP-GlcNAc:Gal beta 1-3-GalNAc-R (GlcNAc to GalNAc) beta 1-6GlcNAc transferase by gene transfer into CHO cells expressing polyoma large tumor antigen. A cDNA encoding UDP-GlcNAc:Gal beta 1-3GalNAc-R (GlcNAc to GalNAc) beta 1-6GlcNAc transferase (EC 2.4.1.102), which forms critical branches in O-glycans, has been isolated by an expression cloning approach using Chinese hamster ovary (CHO) cells. Increased activity of this enzyme and the concomitant occurrence of the O-glycan core 2 structure [Gal beta 1-3(GlcNAc beta 1-6)GalNAc] has been observed in a variety of biological processes, such as T-cell activation and immunodeficiency due to the Wiskott-Aldrich syndrome and AIDS. Since CHO cells do not express this enzyme, CHO cell lines were established to stably express polyoma large tumor (T) antigen, which enables transient expression cloning. Because the antibody used was found to detect most efficiently the oligosaccharide products attached to leukosialin, the CHO cells were also stably transfected with leukosialin cDNA. By using this particular CHO cell line, a cDNA that encodes a protein determining the formation of the core 2 structure was isolated from an HL-60 cDNA library. The cDNA sequence predicts a protein with type II membrane topology, as has been found for all other mammalian glycosyltransferases cloned to date. The expression of the presumed catalytic domain as a fusion protein with the IgG binding domain of protein A enabled us to demonstrate unequivocally that the cDNA encodes the core 2 beta-1,6-N-acetylglucosaminyltransferase, the enzyme responsible for the formation of Gal beta 1-3(GlcNAc beta 1-6)GalNAc structures. No activity with this enzyme was detected toward the acceptors for other beta 1-6GlcNAc transferases.


A 5' to 3' exonuclease functionally interacts with calf DNA polymerase epsilon. Analysis of fractions containing purified DNA polymerase epsilon from calf thymus has revealed the presence of a 5' to 3' exonuclease activity that is specific for a single strand of duplex DNA. This activity is capable of degrading a 3'-labeled oligonucleotide hybridized to M13mp18 DNA. When a second oligonucleotide primer is annealed 3 bases upstream, degradation of the downstream primer is strictly dependent on DNA synthesis from the upstream primer. Replacement of the downstream primer by an oligoribonucleotide of identical sequence results in a similar pattern of exonucleolytic activity. The activity has been highly purified and found to cosediment in glycerol gradients with a peptide of 56 kDa as judged by SDS/PAGE analysis. Effects of calf DNA polymerase alpha and delta on exonuclease activity are also observed but with differences in the pattern of products.


Hypoxia and N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate, but not nerve growth factor, induce Na+ channels and hypertrophy in chromaffin-like arterial chemoreceptors. Chronic hypoxia sensitizes the ventilatory reflex in mammals and causes enlargement of the carotid body, a peripheral arterial chemosensory organ. To investigate possible underlying mechanisms, in the absence of circulatory changes, we exposed cultures of dissociated rat carotid body containing the oxygen sensors (i.e., chromaffin-like glomus cells) to chronic hypoxia (6% O2) over a period of 2 weeks. After a delay of a few days, the Na+ current density in hypoxia-treated glomus cells increased significantly, reaching values up to 6 times that seen in normoxic (20% O2) controls. In addition the whole-cell capacitance, an indicator of cell size, was also significantly larger (3-4 times control) in glomus cells exposed to chronic hypoxia. Both effects were mimicked qualitatively by chronic treatment of normoxic cultures with N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate, but not nerve growth factor, which is known to induce similar changes in the chromaffin cell line PC12. Thus, the physiological and morphological effects of chronic hypoxia on the carotid body in vivo may be due in part to a cAMP-mediated stimulation of Na+ channel expression and hypertrophy in the chemosensory glomus cells.


Cyclic amplification and selection of targets for multicomponent complexes: myogenin interacts with factors recognizing binding sites for basic helix-loop-helix, nuclear factor 1, myocyte-specific enhancer-binding factor 2, and COMP1 factor. Myogenin is one of four muscle-specific basic helix-loop-helix regulatory factors involved in controlling myogenesis. We here describe various protein complexes that increase the affinity of myogenin for DNA. We mixed an oligonucleotide containing a degenerate center large enough to accommodate multiple binding sites with crude myotube nuclear extracts and used cyclic amplification and selection of targets with an antimyogenin monoclonal antibody to isolate protein-DNA complexes. Since each cycle of selection results in the enrichment for the sequences with the highest affinity, we isolated multicomponent sites in which myogenin binding was increased by its interaction with other DNA binding proteins. Myogenin interacts with members of the nuclear factor 1 family, the muscle-specific factor myocyte-specific enhancer-binding factor 2, and another factor, COMP1 (cooperates with myogenic proteins 1), that binds to the sequence TGATTGAC. Myogenin also exhibits cooperative binding with other proteins that recognize CANNTG motifs, and various constraints on spacing and orientation were observed. The application of this approach to other transcription factors should not only help identify the different functions of myogenin versus other members of the muscle basic helix-loop-helix regulatory family but also help define the general combinatorial mechanisms involved in eukaryotic gene regulation.


Origins of genes: "big bang" or continuous creation? Many protein families are common to all cellular organisms, indicating that many genes have ancient origins. Genetic variation is mostly attributed to processes such as mutation, duplication, and rearrangement of ancient modules. Thus it is widely assumed that much of present-day genetic diversity can be traced by common ancestry to a molecular "big bang." A rarely considered alternative is that proteins may arise continuously de novo. One mechanism of generating different coding sequences is by "overprinting," in which an existing nucleotide sequence is translated de novo in a different reading frame or from noncoding open reading frames. The clearest evidence for overprinting is provided when the original gene function is retained, as in overlapping genes. Analysis of their phylogenies indicates which are the original genes and which are their informationally novel partners. We report here the phylogenetic relationships of overlapping coding sequences from steroid-related receptor genes and from tymovirus, luteovirus, and lentivirus genomes. For each pair of overlapping coding sequences, one is confined to a single lineage, whereas the other is more widespread. This suggests that the phylogenetically restricted coding sequence arose only in the progenitor of that lineage by translating an out-of-frame sequence to yield the new polypeptide. The production of novel exons by alternative splicing in thyroid receptor and lentivirus genes suggests that introns can be a valuable evolutionary source for overprinting. New genes and their products may drive major evolutionary changes.


Engagement of the T-cell receptor during positive selection in the thymus down-regulates RAG-1 expression. We have examined the expression of the recombination activating gene RAG-1 by in situ hybridization to thymi from mice bearing transgenes for the T-cell receptor (TCR) alpha chain, TCR beta chain, or both TCR alpha and beta chains. RAG-1 transcription was found in the thymic cortex of transgenic mice carrying a single TCR alpha- or TCR beta-chain transgene, comparable to normal mice. However, RAG-1 transcription was strikingly reduced in the thymic cortex from transgenic mice carrying both TCR alpha- and beta-chain genes and expressing major histocompatibility complex (MHC) class I (H-2b) molecules necessary for positive selection of the transgenic TCR. In contrast, thymi of transgenic mice also carrying both TCR alpha- and beta-chain genes but expressing MHC molecules (H-2d) that did not positively select the transgenic TCR displayed high levels of RAG-1 transcription. The low thymic RAG-1 expression coincided with high transgenic TCR alpha-chain surface expression and with inhibition of endogenous TCR alpha-chain rearrangement. Our findings suggest that binding of the TCR to self MHC molecules during positive selection down-regulates RAG-1 transcription in cortical thymocytes and thereby prevents further TCR alpha-chain rearrangements.


Genetic predisposition of transgenic mouse melanocytes to melanoma results in malignant melanoma after exposure to a low ultraviolet B intensity nontumorigenic for normal melanocytes. Tyr-SV40E transgenic mice are susceptible to melanoma due to simian virus 40 oncogenic sequences specifically expressed in pigment cells. Skin melanomas form relatively late. Therefore, melanocyte cell lines have been established from very young transgenic animals, when they showed no skin lesions, so that the spontaneous and gradual progress of the cells toward tumorigenesis could be characterized under culture conditions in which wild-type cells of the same inbred strain remain untransformed. Melanocytes of an in vitro transgenic line were irradiated with very low intensities of ultraviolet B (UVB) (280- to 320-nm wavelength) light at culture passages when the cells had not achieved anchorage independence. After a single exposure to 0.7 mJ/cm2 of UVB radiation, the cells became anchorage independent and formed foci at confluence; however, cells propagated from the foci were not tumorigenic. After one exposure to 1.75 mJ/cm2, more numerous and larger foci resulted, and the cells grown from them yielded malignant melanomas in graft hosts. Wild-type melanocytes were not transformed at these UVB doses. At least two genetic changes contributing to malignant conversion--in addition to the initiating effect of the transgene--are likely to have occurred, one change leading to anchorage independence and another to further progress toward malignancy. Cells at these stages provide an opportunity to isolate the relevant genes and identify any molecular defects attributable to UVB. Tumorigenesis after a very low UVB dose in cells where an initiating stimulus is already present suggests that some other stimulus, such as a gene or a carcinogen, might lead to melanoma in conjunction with exposure to relatively little UVB.


Characterization of muscarinic receptor subtypes inhibiting Ca2+ current and M current in rat sympathetic neurons. Muscarinic receptors mediating suppression of Ca2+ current and of M-type K+ current in rat superior cervical ganglion neurons were subclassified pharmacologically by using the muscarinic receptor antagonists pirenzepine and himbacine. Our voltage clamp experiments previously distinguished fast and slow intracellular signaling pathways coupling muscarinic receptors to calcium channels. We now establish that the fast, pertussis toxin-sensitive suppression of Ca2+ current is mediated primarily by muscarinic receptors of the M4 subtype, whereas the slow, bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetate (BAPTA)-sensitive suppression of Ca2+ current is mediated primarily by muscarinic receptors of the M1 subtype. Both actions on Ca2+ current are blocked by guanosine 5'-[beta-thio]diphosphate. Muscarinic suppression of M current is slow, BAPTA-sensitive, and mediated by receptors of the M1 subtype. Hence the two muscarinic pathways use different receptors and different guanine nucleotide binding proteins to produce different actions on channels.


Large and small vertebrate sensory neurons express different Na and K channel subtypes. Sensory neurons of frog dorsal root ganglia (DRG) express at least two subtypes of voltage-gated Na channel and at least two subtypes of voltage-gated K channel. The Na channel subtypes have different sensitivities to tetrodotoxin (TTX) and different kinetics. The TTX-sensitive (TTX-s) Na channel inactivates rapidly and is blocked by nanomolar TTX. The TTX-insensitive (TTX-i) Na channel resists blockage by up to 100 microM TTX (it is blocked by saxitoxin) and inactivates 2-6 times more slowly. The two subtypes of voltage-gated K channel differ in activation kinetics: the fast subtype activates 2-8 times faster than the slow subtype. These Na and K channel subtypes are distributed differentially by cell size, falling into three major groups: (i) small cells with slowly activating K channels and a mixture of TTX-s and TTX-i Na channels; (ii) small cells with slowly activating K channels and TTX-s Na channels; and (iii) large cells with rapidly activating K channels and TTX-s Na channels. The contributions of these channel subtypes to the electrical properties of sensory neurons were investigated under conditions that minimized the contribution of Ca current. Under these conditions, action potential duration is correlated with the channel subtypes expressed: cells with both TTX-i and TTX-s Na channels and slowly activating K channels exhibit long-duration action potentials, cells with TTX-s Na channels and slowly activating K channels exhibit intermediate-duration action potentials, and cells with TTX-s Na channels and rapidly activating K channels exhibit short-duration action potentials.


Low frequency of p53 mutations observed in a diverse collection of primary hepatocellular carcinomas. Recent studies of the p53 tumor suppressor locus (designated TP53) in primary hepatocellular carcinoma (PHC) have identified a high frequency of codon 249 mutations. Due to the geographic location from which the samples were obtained and the substitution observed, the mutation was suggested to be attributable to aflatoxin B1 (AFB1) exposure. To determine the generality of this phenomenon, we have examined PHC tissues from 107 geographically and ethnically diverse sources. The frequency of p53 gene mutations was evaluated by using PCR/restriction-digest methods, GC-clamp (G+C-rich sequence) denaturing gradient gel electrophoresis, and DNA sequencing. The mutation rate observed in tumors from high-AFB1-exposure regions (25%) was more than double the rate observed in low-exposure regions (12%) but lower than the 50% frequency previously reported. Codon 249 mutations occurred at a much lower frequency than previously reported (2 of 107 samples examined). These results suggest that changes in DNA encoding p53 may not represent primary oncogenic effects but instead represent genetic changes related to tumor progression. High AFB1 levels may facilitate the generation of these progressional changes, but not by inducing a specific p53 gene mutation at codon 249 as previously reported.


Evidence for the role of dendritic spines in the temporal filtering properties of neurons: the decoding problem and beyond. The question of relations between structure and function acutely applies to the search for the functional role of dendritic spines. While dendritic spines are a prominent and widespread structural feature of neurons in the central nervous system, their function is poorly understood. Because the conducting core of a spine stem can be of extremely small dimensions, a large axial resistance to current flow and "low-pass" filtering of inputs have been hypothesized. Here we show that neurons in the dorsal torus semicircularis of the electric fish Eigenmannia show real-time fluctuations in their transmembrane potential that reflect modulations in the amplitude of a high-frequency sinusoidal carrier signal. In 18 neurons recorded intracellularly and labeled with Lucifer yellow, the decrease in the magnitude of these potentials with increasing rate of amplitude modulation (i.e., low-pass temporal filtering) was positively correlated (r = 0.79, P < 0.001, over a range of one to two octaves in modulation rate) with the mean dendritic spine density (range, 0-0.38 spine per micron of dendritic length) of the cell. The acquisition of synaptic input through dendritic spines may be a general mechanism for achieving the temporal filtering that underlies real-time signal processing in the central nervous system.


Extracellular superoxide dismutase, nitric oxide, and central nervous system O2 toxicity. Although reactive O2 species appear to participate in central nervous system (CNS) O2 toxicity, the exact roles of different reactive O2 species are undetermined. To study the contribution of extracellular superoxide anion (O2-) to CNS O2 toxicity we constructed transgenic mice overexpressing human extracellular superoxide dismutase (ECSOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) in the brain. Remarkably, when exposed to 6 atm (1 atm = 101.3 kPA) of hyperbaric oxygen for 25 min, transgenic mice demonstrated higher mortality (83%) than nontransgenic litter-mates (33%; P < 0.017). Pretreatment with diethyldithiocarbamate, which inhibits both ECSOD and Cu/Zn superoxide dismutase (Cu/Zn SOD) activity, increased resistance to CNS O2 toxicity, in terms of both survival (100% in transgenics and 93% in nontransgenics) and resistance to seizures (4-fold increase in seizure latency in both transgenic and nontransgenic mice; P < 0.05). Thus, O2- apparently protects against CNS O2 toxicity. We hypothesized that O2- decreased toxicity by inactivating nitric oxide (NO.). To test this, we inhibited NO. synthase (EC 1.14.23) with N omega-nitro-L-arginine to determine whether NO. contributes to enhanced CNS O2 toxicity in transgenic mice. N omega-nitro-L-arginine protected both transgenic and nontransgenic mice against CNS O2 toxicity (100% survival and a 4-fold delay in time to first seizure; P < 0.05), as well as abolishing the difference in sensitivity to CNS O2 toxicity between transgenic and nontransgenic mice. These results implicate NO. as an important mediator in CNS O2 toxicity and suggest that ECSOD increases CNS O2 toxicity by inhibiting O2(-)-mediated inactivation of NO.


Sequence-specific recognition of DNA by zinc-finger peptides derived from the transcription factor Sp1. We have overexpressed and purified two peptide fragments of Sp1 that contain the three "zinc-finger" domains necessary for specific Sp1 DNA binding. These peptides assume a stable, folded conformation in solution in the presence of Zn2+ as shown by DNA binding assays and NMR spectroscopy. Mobility-shift assays demonstrate that the Sp1 peptides recognize a number of different Sp1 DNA binding sites (GC boxes, with the core sequence GGGCGG). The dissociation constant for a 92-amino acid peptide binding to the GGGGCGGGGC sequence (Kd approximately 10 nM) and the relative affinities for several other DNA sequences definitively demonstrate Sp1-like binding properties. The thermodynamic binding site for Sp1-Zn92 has been mapped using the primer-extension/mobility-shift assay revealing that the 5' portion of the GC box DNA sequence (GGG GCG) contributes more strongly to the total binding energy than the 3' portion (GGGC). These findings are interpreted in the context of the Sp1 amino acid sequence in comparison with the structurally characterized Zif-268/DNA complex. A model is proposed that offers a structural explanation for the ability of Sp1 to recognize a diverse array of DNA sequences in terms of the individual (and different) DNA binding properties of each of the three zinc-finger domains.


Competitive adsorption of human immunoglobulin G and albumin: consequences for structure and reactivity of the adsorbed layer. The affinity of polyclonal anti-IgG for human IgG adsorbed on silica surfaces was investigated by two complementary techniques, scanning angle reflectometry and 125I radiotracing. Special attention was paid to compare the reactivity of IgG adsorbed directly or by exchange with already adsorbed albumin. In particular it was shown that (i) in the first case (direct adsorption) the reaction between anti-IgG and adsorbed IgG was in the ratio 1:1 and (ii) in the second case (adsorption by exchange) there was no reaction.


A single-pool inositol 1,4,5-trisphosphate-receptor-based model for agonist-stimulated oscillations in Ca2+ concentration. Relying on quantitative measurements of Ca2+ activation and inhibition of the inositol 1,4,5-trisphosphate (IP3) receptor in the endoplasmic reticulum, we construct a simplified kinetic model to describe the properties of this channel. Selecting rate constants to fit key kinetic and equilibrium data, we find that the model reproduces a variety of in vivo and in vitro experiments. In combination with Ca(2+)-ATPase activity for Ca2+ uptake into the endoplasmic reticulum, the model leads to cytoplasmic oscillations in Ca2+ concentration at fixed IP3 concentration and only a single pool of releasable Ca2+, the endoplasmic reticulum. Incorporation of a positive-feedback mechanism of Ca2+ on IP3 production by phospholipase C enriches the properties of the oscillations and leads to oscillations in Ca2+ concentration accompanied by oscillations in IP3 concentration. We discuss the possible significance of these results for the interpretation of experiments.


Modulation of interleukin 2 activity by delta 9-tetrahydrocannabinol after stimulation with concanavalin A, phytohemagglutinin, or anti-CD3 antibody. The effects of delta 9-tetrahydrocannabinol (THC) on lymphocyte proliferation and interleukin (IL) 2 activity was investigated using adult murine spleen cells stimulated with either the mitogens concanavalin A, phytohemagglutinin, or anti-CD3 antibody. THC was found to suppress mitogen-induced proliferation, but to enhance anti-CD3-antibody-induced proliferation. These results reflected THC-induced suppression of Ly2 cells following concanavalin A or phytohemagglutinin stimulation and THC-induced enhancement of Ly2 cells following CD3 stimulation. The combination of THC and concanavalin A or phytohemagglutinin resulted in suppressed IL-2 activity, whereas the combination of THC and anti-CD3 antibody resulted in enhanced IL-2 activity. This drug-related modulation of IL-2 activity corresponded to the changes in blastogenic activity as well as to variations in numbers of Tac positive cells. These results suggest that the dysregulation in immune responses following THC treatment, either suppression or enhancement, may relate to the effects of THC on IL-2 production.


RNA binding determinant in some class I tRNA synthetases identified by alignment-guided mutagenesis. The N-terminal nucleotide binding folds of all 10 class I tRNA synthetases (RSs) contain characteristic conserved sequence motifs that define this class of synthetases. Sequences of C-terminal domains, which in some cases are known to interact with anticodons, are divergent. In the 676-amino acid Escherichia coli methionyl-tRNA synthetase (MetRS), interactions with the methionine tRNA anticodon are sensitive to substitutions at a specific location on the surface of the C-terminal domain of this protein of known three-dimensional structure. Although four class I synthetases of heterogeneous lengths and unknown structures are believed to be historically related to MetRS, pair-wise sequence similarities in the region of this RNA binding determinant are obscure. A multiple alignment of all sequences of three of these synthetases with all MetRS sequences suggested a location for the functional analog of the anticodon-binding site in these enzymes. We chose a member of this set for alignment-guided mutagenesis, combined with a functional analysis of mutant proteins. Substitutions within two amino acids of the site fixed by the multiple sequence alignment severely affected interactions with tRNA but not with ATP or amino acid. Multiple individual replacements at this location do not disrupt enzyme stability, indicating this segment is on the surface, as in the MetRS structure. The results suggest the location of an RNA binding determinant in each of these three synthetases of unknown structure.


Histopathologic effects of soluble glucan and WR-2721, independently and combined in C3H/HeN mice. Soluble glucan, an immunomodulator, and Walter Reed (WR)-2721, a radioprotectant, increase postirradiation survival when administered before and after exposure, respectively. Combined, these agents act synergistically through WR-2721's ability to spare hematopoietic stem/progenitor cells from radiation injury and glucan's ability to subsequently stimulate spared cells to proliferate. In this study, the histopathologic effects of WR-2721 (200 mg/kg, ip) and glucan (250 mg/kg, iv), at doses capable of increasing survival in lethally irradiated mice, were evaluated in unirradiated and irradiated female C3H/HeN mice. After treatment, whole body weights and wet organ weights of liver, spleen, and kidney, as well as gross and histologic changes in these and other tissues, were monitored on Days 1, 4, 7, 11, 15, 21, and 28. Morphometric studies of splenic white and red pulps were also performed. Soluble glucan, with or without WR-2721, in unirradiated groups, was associated with splenomegaly, transient morphometrically determined perturbations of white and red pulp areas, and histologic alterations of white pulp. In irradiated mice, splenic weight loss was initially dampened in glucan groups and accompanied by morphologic and histologic changes similar to those seen in unirradiated counterparts. The subsequent rebound of splenic parameters in irradiated mice was limited to WR-2721-treated mice and was associated with hematopoietic reconstitution. Glucan, with or without WR-2721, in unirradiated groups was associated with transient hepatomegaly and associated histologic changes. Similar changes in irradiated animals were seen only in the combined treatment group.


Flow cytometric evaluation of the effects of leukotriene B4 receptor antagonists (LY255283 and SC-41930) on calcium mobilization and integrin expression of activated human neutrophils. Leukotriene B4 (LTB4) is a naturally occurring eicosanoid mediator which chemoattracts and stimulates human neutrophils to an activated state. In an attempt to identify novel antiinflammatory drugs, synthetic LTB4 receptor antagonists have been developed in several laboratories. In this study, the effects of two such LTB4 receptor antagonists were examined for their influences on two elements of human neutrophil activation using flow cytometric techniques. Quantitative flow cytometric assays of human neutrophil intracellular calcium mobilization and up-regulation of integrin (CD11b/CD18) cell surface expression were developed and used to determine the potency and selectivity of compounds LY255283 and SC-41930 on these activities. Our results indicate that both compounds preferentially block these functions of LTB4-induced human neutrophil activation in a concentration dependent manner and fall in the 1-2 microM range of antagonist activity. Compound SC-41930 was approximately twice as potent as LY255283 in blocking the targeted agonist effects. Both compounds were approximately 100-fold less potent in blocking the same functions of interleukin-8-induced human neutrophil activation.


Mechanism of prostaglandin E2-induced arachidonic acid release in osteoblast-like cells: independence from phosphoinositide hydrolysis. We previously reported that pertussis toxin (PTX)-sensitive GTP-binding protein is involved in the coupling of prostaglandin E2 (PGE2) receptor to phospholipase C in osteoblast-like MC3T3-E1 cells (1). In the present study, we analyzed the mechanism of PGE2-induced arachidonic acid (AA) release in MC3T3-E1 cells. PGE2 stimulated the release of AA and the formation of inositol trisphosphate (IP3) dose dependently in the range between 1 nM and 10 microM. The effect of PGE2 on AA release (ED50 was 80 nM) was more potent than that on IP3 formation (ED50 was 0.8 microM). Quinacrine, a phospholipase A2 inhibitor, suppressed the PGE2-induced AA release but had little effect on the IP3 formation. NaF, a GTP-binding protein activator, mimicked PGE2 by stimulating the AA release. The AA release stimulated by a combination of PGE2 and NaF was not additive. PTX had little effect on the PGE2-induced AA release. These results strongly suggest that the AA release and the phosphoinositide hydrolysis are separately stimulated by PGE2 in osteoblast-like cells, and the PGE2-induced AA release is mediated by PTX-insensitive GTP-binding protein.


Impact of psychological dynamics of stress on the peripheral benzodiazepine receptor. In an attempt to dissociate the relative impact of psychological vs. physiological concomitants of stress on the peripheral benzodiazepine receptor (PBR), the influence of stressor controllability and predictability was investigated in rats. In addition, the effect of a purely psychological stressor, contextually conditioned fear, was examined. The response of the PBR in rats confronted with a naturalistic threat, a cat, was also tested. Various peripheral and CNS tissues were analyzed. Specific binding of [3H]Ro 5-4864 was significantly reduced in the kidneys of subjects receiving either controllable or uncontrollable shock. Similar changes were seen in the kidneys of subjects receiving either predictable or unpredictable shock. Mean [3H]Ro 5-4864 binding in lung was reduced following both predictable and unpredictable shock, but only the reduction in the predictable shock group reached significance. Controllability appeared to protect against the stress-induced reduction in [3H]Ro 5-4864 binding in lung. Contextually conditioned fear only affected PBR in the olfactory bulb, and exposure to a cat was without effect. These data suggest that the PBR responds only to potent stressors, and psychological influences on the PBR are tissue specific.


General activity in baboons measured with a computerized, lightweight piezoelectric motion sensor: effects of drugs. A small, 1-oz activity-monitoring device is described for measuring motor activity continuously for periods of up to 42 days. The monitor employs a piezoelectric sensor that detects extremely small accelerations induced by movements. The monitor can be placed on collars or harnesses (e.g., for rabbits, cats, dogs, nonhuman primates, etc.). The use of the monitor is described within numerous laboratories studying the behavioral pharmacology of drugs in individually caged laboratory baboons. Patterns of daily activity were reliably recorded over periods of several months, and reflected the normal activity patterns of animals. The activity monitor recorded reliable, drug-induced changes in general activity that paralleled the known effects of the same drugs on learned behaviors. Low doses of the stimulants cocaine and d-amphetamine both increased general activity. Marked reductions in general activity were observed following both the administration of delta-9-tetrahydrocannabinol and an antihypertensive drug combination of diuretic and verapamil.


Opioid receptor subtypes mediating the noise-induced decreases in high-affinity choline uptake in the rat brain. Acute (20 min) exposure to 100-dB white noise elicits a naltrexone-sensitive decrease in sodium-dependent high-affinity choline uptake in the frontal cortex and hippocampus of the rat. In the present study, the subtypes of opioid receptors involved were investigated by pretreating rats with microinjection of specific opioid-receptor antagonists into the lateral cerebroventricle before noise exposure. We found that the noise-induced decrease in high-affinity choline uptake in the hippocampus was blocked by pretreatment with either mu-, delta-, or kappa-opioid-receptor antagonists, whereas the effect of noise on frontal cortical high-affinity choline uptake was blocked by a mu- and delta- but not by a kappa-antagonist. These data further confirm the role of endogenous opioids in mediating the effects of noise on central cholinergic activity and indicate that different neural mechanisms are involved in the effects of noise on the frontal cortical and hippocampal cholinergic systems.


Transient suppression of a secondary humoral response in rats is evoked by lithium-pilocarpine-induced limbic seizures. Several experiments were designed to evaluate a secondary humoral response following limbic seizures. After baseline antigen binding capacity (ABC) had been determined for the primary response, a second subcutaneous injection of the antigen (human serum albumin) was accompanied by an injection of either lithium (3 mEq/kg)-pilocarpine (30 mg/kg) or one of two comparator treatments: metrazol (30 mg/kg) or cyclophosphamide (50 mg/kg); other rats served as drug controls. Only the groups that received the lithium-pilocarpine (status epilepticus) or cyclophosphamide (no seizure) displayed significant immunosuppression after 5 but not 10 days. The results support the hypothesis that seizure activity within the amygdaloid-hippocampal complex modulates immunocompetence through corticotropin mechanisms.


Delta 9-tetrahydrocannabinol interactions with phencyclidine and ethanol: effects on accuracy and rate of responding. The effects of delta 9-tetrahydrocannabinol (delta 9-THC) in combination with phencyclidine (PCP) or ethanol were examined in rats responding under a fixed-consecutive-number schedule of food presentation. Under this schedule, a minimum of 13 consecutive responses on one lever followed by one response on another lever produced food. When administered alone, PCP (0.1-10.0 mg/kg) and delta 9-THC (0.1-5.6 mg/kg), but not ethanol (0.3-1.7 g/kg), decreased accuracy. PCP, delta 9-THC, and ethanol alone all produced dose-dependent decreases in rate of responding. A dose-effect curve for PCP or ethanol was then redetermined in combination with selected doses of delta 9-THC (0.125-1.75 mg/kg) and the data were analyzed according to the effect-addition and dose-addition models of additivity. When administered in combination, delta 9-THC produced dose-dependent leftward shifts in the PCP dose-effect curves for both accuracy and rate of responding. The interactions for PCP + delta 9-THC combinations were effect-additive for accuracy. In contrast, the type of interaction obtained for PCP + delta 9-THC combinations on rate of responding depended upon the particular doses combined, as well as on the model used to analyze the interactions. According to the effect-addition model, these interactions were additive at low doses of delta 9-THC and supraadditive at the highest dose. However, according to the dose-addition model the interactions at the higher doses of delta 9-THC were infraadditive. Delta 9-THC also shifted the ethanol dose-effect curve for rate of responding to the left but did not alter the ethanol dose-effect curve for accuracy. The interactions for ethanol + delta 9-THC combinations were effect-additive for accuracy and both effect- and dose-additive for rate of responding. The present investigation clearly illustrates the importance of examining an extensive range of dose combinations on different behavioral measures, as well as the use of appropriate analyses in studies designed to evaluate the interactions between drugs.


Effects of midazolam on T-cell immunosuppressive response to surgical stress in mice. Mice submitted to surgical stress induced by laparotomy and treated with chronic midazolam (1 mg/kg) showed a reduction in stress-induced suppression of thymus and spleen cellularity and in peripheral lymphocyte population. The blastogenic response of spleen lymphoid cells was also assessed and midazolam was found to partially attenuate the suppressive effect of surgery.


Biphasic effect of local anesthetic carbisocaine on fluidity of phosphatidylcholine bilayer. The local anesthetic carbisocaine (1), the monohydrochloride of [2-(heptyloxy)phenyl]-2-(diethylamino)-1-methylethyl ester of carbamic acid, exerts a biphasic effect on the fluidity of egg yolk phosphatidylcholine model membranes as detected by the stearic acid spin probes with the paramagnetic doxyl group bound to C-5 or C-16. The fluidity initially increases with an increase in the concentration of 1, but at concentrations above 25 mmol/l a decrease of fluidity has been observed. 31P NMR spectroscopy indicates changes in the phosphocholine head-group conformation in the presence of 1. The decrease of fluidity is caused by the increased packing density in the bilayer hydrophobic region at high concentrations of 1, probably due to interdigitation of hydrocarbon chains from apposing monolayers in the hydrophobic center of the bilayer.


Free radicals and excited species in the metabolism of indole-3-acetic acid and its ethyl ester by horseradish peroxidase and by neutrophils. The peroxidative metabolization of indole-3-acetic acid, a biologically important process, has been followed by EPR spectroscopy with the aim of obtaining information on the mechanism of generation of electronically excited species. The skatole-3-methylene radical detected during oxidation by horseradish peroxidase, does not appear to be involved in a major oxygen consuming process or in the generation of singlet oxygen. The chemiluminescence spectrum exhibits several maxima, which are also observed when the ethyl ester of indole-3-acetic acid is metabolized by horseradish peroxidase or by myeloperoxidase in neutrophils. When the ester is metabolically activated in either of these systems, the EPR spectrum indicates a tertiary carbon-centered radical. This radical centered on the carbon in the 3-position participates in a chemiexcitation/emissive route. Within the cell, this emissive process is responsible for a large part of the oxygen consumed. Some of the emitters originate in the cleavage of the 2,3 double bond. The ester, which is capable of penetrating into the cells, also emits with other myeloperoxidase-containing cells. This compound may have useful applications as an intracellular chemiluminescent probe for the presence of myeloperoxidase.


Absence of maternal behavior in rats with lithium/pilocarpine seizure-induced brain damage: support of MacLean's triune brain theory. Female rats, with and without maternal experience, received limbic seizure-inducing (SC) injections of lithium and pilocarpine. Following the subsequent parturitions, these rats displayed a complete absence of maternal behavior. Rats that did not display seizures after receiving the lithium/pilocarpine injections displayed behaviors that were comparable to normal controls. Although the multifocal limbic, thalamic, and cingulate damage abolished maternal care, there was no evidence of aberrant effects upon fecundity, litter size, or mammary function; infanticide was negligible. The pattern of brain damage involves the evolutionarily more recent thalamocingulate system of mammals and supports MacLean's theory that these pathways are required for normal mother-offspring interaction.


Hormonal milieu affects tailflick latency in female rats and may be attenuated by access to sucrose. The steroid hormones estrogen (E) and progesterone (P) are known to modify pain sensitivity; however, the relative role of each of these hormones in this process is not well understood. To systematically investigate the effects of E and P on nociception, pain sensitivity was assessed under several hormone conditions. Tailflick (TF) latencies were measured every other day in 10 cycling female rats and 10 female rats during luteal functioning (pseudopregnancy). Thirty ovariectomized (OVX) rats were tested for TF latency following administration of 10 micrograms estradiol benzoate (EB) and either 0.0, 0.5, or 1.0 mg of P. Significant differences in TF latency were seen across days of the estrous cycle but not during luteal functioning. Tailflick latencies during luteal functioning were elevated relative to latencies in normally cycling animals. Among OVX rats, those administered EB and P (1.0 mg) displayed significant reductions in TF latency compared to vehicle controls. As a separate line of research indicated that consumption of highly palatable foods modified pain sensitivity, whether chronic sucrose consumption might overide the influence of hormones on nociception was examined. Ovariectomized rats given EB and P (0.0, 0.5, or 1.0 mg) were allowed chronic exposure to a 32% sucrose solution. Our preliminary findings suggest that chronic sucrose consumption attenuates hormonally induced differences in nociception.


Obesity is the major cause of alterations in insulin secretion and calcium fluxes by isolated islets from aged rats. To investigate the alterations in insulin secretion induced by aging, 2-month-old, 12-month-old, and 12-month old lean rats (submitted to a caloric restriction during the last month that causes a weight loss of approximately 20%) were studied. As expected, glucose intolerance and increased insulin response were observed during IV-GTT in 12-month-old rats. These effects were, however, reversed by weight loss. Insulin secretion was investigated in isolated islets both during static incubation and perifusion. In 12-month-old rats insulin secretion and 45Ca2+ efflux were lower only in the second phase of the hormonal secretion, suggesting an involvement of voltage-sensitive calcium channels in these phenomena. Considering that in vivo and in vitro alterations were reversed after weight loss, it is possible to conclude that obesity is probably a major cause of impaired insulin secretion in 12-month-old albino rats. Since 14C-glucose metabolism was not changed in islets from aged rats, the effect of obesity on insulin secretion is not due to altered glucose metabolism in pancreatic B-cells.


Cloning and transposon vectors derived from satellite bacteriophage P4 for genetic manipulation of Pseudomonas and other gram-negative bacteria. We developed transposon and cloning shuttle vectors for genetic manipulation of Pseudomonas and other gram-negative bacteria, exploiting the unique properties and the broad host range of the satellite bacteriophage P4. P4::Tn5 AP-1 and P4::Tn5 AP-2 are suicide transposon vectors which have been used for efficient Tn5 mutagenesis in Pseudomonas putida. pKGB2 is a phasmid vector with a cloning capacity of about 7.5 kb; useful unique cloning sites are SacI and SacII in the streptomycin resistance determinant and PvuI and XhoI in the kanamycin resistance determinant. pKGB4 is a cosmid derived from pKGB2 and carries the additional cloning site SmaI in the kanamycin resistance determinant; its cloning capacity is about 18 kb. These vectors and their recombined derivatives were transferred from Escherichia coli to P. putida by transduction and may be used for other bacterial species susceptible to P4 infection.


Multiple glomus tumors associated with arteriovenous fistulas and with nodular lesions of the finger joints. A patient is reported with an extremely unusual clinical presentation comprising painful subungual glomus tumors, hemangioma-like skin lesions of the trunk, and nodular lesions of the finger joints. Prominent arteriovenous shunting also was present. The etiology of all these abnormalities was thought to be extensive proliferation of glomus cells.


A study on bone induction in hydroxyapatite combined with bone morphogenetic protein. Our present study consisted of an implantation of artificially made hydroxyapatite (HAP) ceramic pellets under the periosteum of the rabbit skull with subsequent inspection of further progress of bone formation and also of an evaluation of the effects of bone morphogenetic protein (BMP). The results revealed that the alkali phosphatase (AL-P) activity of the pellets was elevated only in those of the bone morphogenetic protein group. The results of determination of bone mineral density at the site of the pellets revealed that the increase in bone mineral density was the most remarkable in the bone morphogenetic protein group rather than the control group. The results of the histopathologic examinations revealed that marginal bone formation was found in the pores on the surface between the pellets and the skull in the control group and in the collagen group, whereas in the bone morphogenetic protein group very active bone formation was found not only on the interface in contact with the skull but also surrounding the whole pellet. It also was noted in the animals in the bone morphogenetic protein group that the pellets were corrupted from the peripheries and then absorbed into the newly formed bone. From these results, the efficacy of the hydroxyapatite-collagen-bone morphogenetic protein complex was made clear, and applications in clinical practice are expected in the near future.


Nutrition and cancer prevention.

Nutrition and cancer prevention. Of all cancers in the United States, 35% are estimated to be caused by dietary factors and may be preventable. Diets high in fat or calories, for example, are said to be associated with five of the six most common cancers: breast, colorectal, pancreatic, prostatic, and uterine. Conversely, some dietary components such as vitamin A, in fruits and vegetables, and fiber may help protect against certain cancers. Obesity may confer a small risk of breast cancer on a woman, but women with upper body fat localization are at significantly higher risk of developing breast cancer and endometrial cancer.


Thyroid stimulating hormone and prolactin responses to thyrotropin releasing hormone in nondepressed alcoholic inpatients. Thyroid stimulating hormone (TSH) and prolactin (PRL) responses to thyrotropin releasing hormone (TRH) stimulation are sometimes blunted in alcoholic subjects; however, the mechanisms involved in these phenomena have not been established. We hypothesized that elevations in free thyroid concentrations might be related to these abnormal responses and then tested that hypothesis in a sample of nondepressed alcoholic inpatients (n = 21). Four alcoholic patients had delta max TSH responses that were < 7 mIU/l; three had PRL responses at or below 8 micrograms/l. Baseline TSH was the only significant predictor of peak TSH; however, free thyroxine (FT4) and baseline TSH both were significant predictors of peak PRL. The average baseline FT4 concentration in alcoholic patients was significantly higher than that in healthy control subjects (n = 10). Our data, thus, suggest that small elevations of FT4 play a role in the inhibition of TSH and PRL responses to TRH among nondepressed, abstinent alcoholic patients.


[11C]Ro 15-4513, a ligand for visualization of benzodiazepine receptor binding. Preparation, autoradiography and positron emission tomography. Ro 15-4513, a partial inverse agonist at the benzodiazepine (BZ) receptor site was labelled with 11C and used for in vitro autoradiography on human post mortem brain sections and for positron emission tomography (PET) on Cynomolgus monkeys. The total radiochemical yield of [11C]Ro 15-4513 was 30-40% with an overall synthesis time of 40 min. The specific radioactivity was about 1000 Ci/mmol at end of synthesis. In vitro autoradiography showed that [11C]Ro 15-4513 bound specifically predominantly in the neocortex of the human brain. Specific binding was also demonstrated in the basal ganglia and the cerebellar cortex. Flumazenil (Ro 15-1788) and clonazepam inhibited the binding in cerebral regions, but a significant proportion in the cerebellum was not inhibited by these agents. This proportion may represent alpha 6-containing BZ receptors. PET examination of [11C]Ro 15-4513 binding in Cynomolgus monkeys demonstrated high uptake of radioactivity in neocortex. The uptake of radioactivity was markedly displaced by high doses of Ro 15-4513 or clonazepam. [11C]Ro 15-4513 should be a useful ligand to examine BZ receptor characteristics in the living human brain by PET.


FG 7142 selectively decreases nonpunished responding, but has no anxiogenic effects on time allocation in a conflict schedule. Previous work (Thomas et al. 1990) showed that an anxiolytic benzodiazepine increased the time allocated to responding in a conflict situation (where responses were both food-reinforced and shock-punished) versus a nonpunishment situation. The present experiment tested whether a benzodiazepine-receptor inverse agonist (FG 7142, 1-30 mg/kg) would have the opposite effect (i.e., decrease time spent responding in a punishment situation). Chain pulls determined whether a rat's lever presses were reinforced on 1) a lean variable-interval schedule, or 2) a richer variable-interval schedule in which responding also produced shock intermittently. FG 7142 dose-dependently decreased nonpunished lever responding, but did not affect punished responding. The drug nonselectively decreased chain pulling (the schedule-switching response). Like chlordiazepoxide, FG 7142 increased the time spent in the punishment component, showing that not all effects of benzodiazepine-receptor agonists and inverse agonists are opposite. These results are inconsistent with expectations that anxiogenic actions of FG 7142 should 1) decrease punished responding; 2) increase the rate of responses that terminate the punishment condition; and 3) decrease time spent in the punishment component. Rather, nonsuppressed responding seems most sensitive to decreases by FG 7142.


Increased GABAA-dependent chloride uptake in mice selectively bred for low aggressive behavior. Selective breeding for aggressive behavior alters GABA-dependent chloride uptake and behavioral response to benzodiazepine treatment. Pharmacological and biochemical studies examined subjects from three lines of adult male ICR mice selectively bred for either high levels or low levels of aggressive behavior, as well as unselected controls. Selective breeding produced two lines of behaviorally distinct males. During 5-min dyadic confrontations with an outbred stimulus animal, untreated low-aggressive mice spent more time in walking, rearing, and social interaction than untreated high-aggressive mice. The three lines also showed different responsiveness to the aggression increasing and decreasing effects as well as the sedative effects of benzodiazepine treatment. High doses of chlordiazepoxide (17, 30 mg/kg) reduced motor behaviors (walk, rear and groom) in the low-aggressive line without altering these behaviors in the high aggressive line. In the high-aggressive line, the same doses of chlordiazepoxide (17, 30 mg/kg) produced a behavioral shift; aggressive behaviors were reduced while social behaviors increased to levels similar to the untreated low-aggressive line. In contrast, only the unselected line pursued and threatened more after a low dose of chlordiazepoxide (3 mg/kg). The three lines also showed alterations at the GABAA-benzodiazepine receptor complex. Specific uptake of [3H]Ro-15-1788 was increased in cerebral cortex, hypothalamus and hippocampus in the low-aggressive line, and was reduced in these areas in high-aggressive line when compared with unselected controls. Similarly, GABA-dependent chloride uptake in cortical synaptoneurosomes was augmented in low-aggressive mice and decreased in high-aggressive mice when compared to unselected controls.(ABSTRACT TRUNCATED AT 250 WORDS)


Beta--CCE and FG 7142 increase defensiveness during intraspecies encounters in mice. The effects were ascertained of two partial inverse agonists at benzodiazepine receptors (beta-CCE and FG 7142) on the incidence of timid (defensive-escape), aggressive, sociable and locomotor activities in both timid and aggressive singly-housed male mice, treated with drugs in paired interactions with untreated non-aggressive males. FG 7142 (5 mg/kg) and beta-CCE (8 mg/kg) increased defenses and escapes without producing other behavioral changes in timid mice. FG 7142 (20 mg/kg) and beta-CCE (1-8 mg/kg) moderately increased defenses and alert postures in aggressive mice and this effect was associated with marked reduction of aggressive behavior. FG 7142 (20 and 80 mg/kg) also decreased walking across cage in aggressive mice. It is concluded that beta-CCE and FG 7142 produced behavioral changes which could be interpreted as "anxiogenic".


Evaluation of the apical sealing ability of apatite root canal sealer. The apical sealing ability of a tricalcium phosphate sealer was compared with that of three other sealers. Seventy roots of human incisors were cleansed and shaped and randomly assigned to one of seven groups of ten roots each. The root canal systems were obturated with gutta-percha and one sealer using the lateral-vertical condensation technique. The canal was sealed with Roth's sealer, Sealapex, Kerr root canal sealer, or Sankin apatite root sealer (Type I, II, or III). One group was filled with gutta-percha without sealer to serve as a control. After the roots were immersed in silver nitrate, the degree of dye penetration was measured under a dissecting microscope. Results indicated that Sealapex had the best sealing ability, followed by Sankin apatite root sealer, Type II. Roth's cement showed the most dye penetration. Canals that were obturated without sealer showed significantly greater apical leakage.


Paramagnetic molecular centers in gamma-irradiated precipitates in the system AlCl3-DL-alpha-valine-NaOH. Formation and stability of paramagnetic molecular centers were studied in AlCl3-NaOH-DL-alpha-valine by ESR spectroscopy. In Al3(OH)9(valine)1 x 3H2O gamma-irradiated at room temperature the valine radical [formula: see text] was detected. The radical was formed by abstraction of a hydrogen atom from the valine molecule coupled to the aluminum hydroxide matrix. Stability of the radical depended critically on structural properties of the aluminum hydroxide matrix. In aluminum hydroxide with the ratio (Al)/(Valine) = 20, either no paramagnetic species were detected (irradiation in air) or a singlet at g = 2.008 of 1.8 mT linewidth was detected (irradiation in vacuum) at room temperature. Primary paramagnetic species (gamma irradiation at 77 K) in Al3(OH)9(Val)1 x 3H2O are chloride paramagnetic centers and the primary neutral valine radical [formula: see text] It was formed by abstraction of the NH2 group from the valine molecule. With warming, this radical was not transformed to the radical (I).


Thin-walled cystic lesions in bronchioalveolar carcinoma. In a retrospective study, thin-walled cystic lesions were identified in four patients with proved bronchioalveolar carcinoma (BAC). The radiographic appearances are described, and possible mechanisms of formation of the lesions are discussed. Although such appearances due to BAC have rarely been described in the literature, the authors believe that the differential diagnosis of thin-walled cystic lesions associated with consolidation should include BAC.


[The effect of the dose rate on the development of rat pups under chronic gamma irradiation]. Chronic exposure of rat pups to gamma radiation, during their intensive growth, at a dose rate of 0.01, 0.11 and 0.5 cGy/day did not affect their development throughout a two-month period of observation. At a dose rate of 12.9 cGy/day rat growth was inhibited during the first 15 days. With further exposure at the same dose rate (over a period of up to five months) the rate of rat pup growth was restored, which indicated the presence of adaptation processes.


Estimating the prevalence of chronic fatigue syndrome and associated symptoms in the community. Chronic fatigue syndrome is a poorly understood disease characterized by debilitating fatigue and neuromuscular and neuropsychological symptoms. Despite numerous studies on the subject, the epidemiology of the syndrome in the community remains largely unexplored. An estimate of the prevalence in the population is presented, approximating the Centers for Disease Control criteria as well as the prevalence estimates of the fatigue symptom complex that include fatigue, disability, and neuromuscular and neuropsychological symptoms. The study population consisted of a very large, multicenter, stratified, and random sample of a general population health survey known as the Epidemiologic Catchment Area Program. Data used for this study were gathered between 1981 and 1984. The Diagnostic Interview Schedule, a highly structured mental health interview, was used to assess the lifetime prevalence of medical and psychological symptoms. Chronic fatigue was common. A total of 23 percent of the subjects reported having experienced the symptom of persistent fatigue sometime during their lives. Chronic fatigue syndrome, however, as defined by the Centers for Disease Control, appeared to be quite rare in the general population. Only 1 of 13,538 people examined was found to meet a diagnosis of the syndrome with an approximation of the CDC criteria. Fatigue symptom complex was frequently related to medical or psychiatric illness or substance abuse; thus, persons meeting partial criteria of chronic fatigue syndrome were also found to be rare when psychiatric or medical exclusions were applied.


Flow cytometric DNA analysis of excised breast lesions: use of fresh tissue needle aspirates obtained under guidance with mammography of the specimen. Eighty consecutive biopsy specimens were studied to determine whether DNA analysis could be performed on fine-needle aspirates of excised clinically occult breast lesions obtained under guidance with mammography of the specimen before fixation to offer the advantages of fresh-tissue analysis. With use of single aspirates, cytologic analysis was possible in 50 cases (62%); DNA analysis was possible in 75 cases (94%). These methods combined offered no statistically significant increase in sensitivity for detection of malignancy compared with cytologic analysis alone. Forty-one percent of the analyzable invasive carcinomas showed aneuploidy. Aneuploidy and high S phase fractions of the invasive carcinomas showed no substantial correlation with patient age, nodal status, and size or appearance of the mammographic lesion. Aneuploidy was also seen in zero of four analyzable lesions showing ductal carcinoma in situ, two of 13 showing atypical hyperplasia (15%), and one of the 28 remaining benign lesions (4%). The authors conclude that this mammographic intervention is an effective means of obtaining fresh tissue samples of clinically occult lesions for DNA analysis.


[The role of membrane receptors in stimulating cell multiplication under the influence of low doses of atomic radiation]. In experiments with Raji cells, it has been shown that the serum growth factors activate the growth of gamma-irradiated (0.36 mGy/day) culture. The authors discuss the role of radiation stimulation of membrane receptors in recepting natural effectors.


Breast conservation therapy in patients with mammographically undetected breast cancer. The authors reviewed their experience with 542 patients with breast cancer who were treated with conservative surgery and radiation therapy (CSRT) and analyzed the outcome in those patients whose tumors could not be detected with mammography. Fifty-five of the patients (10.1%) had a palpable, pathologically confirmed breast carcinoma and a negative preoperative mammogram. Routine follow-up included annual mammography and physical examination. The local recurrence, 5-year actuarial survival, and 5-year disease-free survival rates for these 55 patients did not differ significantly from those for patients with positive mammograms. There were six cases of local breast recurrence in this subgroup. Four of five cases were visible on mammograms (one patient did not undergo mammography at the time of recurrence); two of the cases were detected with mammography alone following physical examination with negative results. The authors conclude that patients with palpable but mammographically occult early-stage breast cancer are suitable candidates for CSRT and that mammography is a mandatory part of follow-up of conservatively treated patients.


[The postradiation effect of acetylcholine and GABA on the active potassium uptake by slices of rat cerebral cortex]. Whole-body X irradiation (0.155 and 0.310 C/kg) was shown to modify the biphase effect of acetylcholine and GABA on antigradient K+ uptake by rat brain sections. Radiation made the effects of neuromediators on active K+ transport be differently directed: acetylcholine enhanced the inhibitory effect of radiation and GABA restored the Na-K-pump function.


Hepatocellular carcinoma: treatment with percutaneous ethanol injection and transcatheter arterial embolization. The therapeutic effectiveness of a new combination therapy--pretreatment with transcatheter arterial embolization (TAE) and subsequent percutaneous ethanol injection (PEI)--for solitary large (> 3.0 cm in diameter) primary hepatocellular carcinoma lesions was compared with that of TAE alone. With TAE alone, a partial response of the tumor was seen in only 10% of the patients, and the 1-, 2-, and 3-year survival rates were calculated to be 68%, 37%, and 0%, respectively. Histologic examination of specimens obtained at hepatectomy showed that TAE alone caused complete necrosis in only 20% of the tumors. In contrast, PEI combined with TAE significantly (P < .05) increased the partial response rate (45%) and significantly (P < .01) prolonged the 1-, 2-, and 3-year survival rates (100%, 85%, and 85%, respectively). Combination therapy caused complete histologic necrosis in 83% of the tumors. It also was significantly (P < .05) better than TAE alone in terms of rate of primary tumor recurrence during follow-up.


Hepatocellular carcinoma not detected with plain US: treatment with percutaneous ethanol injection under guidance with enhanced US. To evaluate the usefulness of contrast material-enhanced ultrasound (US) in detection and treatment of hepatocellular carcinoma (HCC), carbon dioxide was injected as a contrast agent into the hepatic artery in 22 patients with HCC. Plain US had enabled detection of 24 HCC nodules in these patients. Contrast material-enhanced US enabled detection of seven additional nodules, which were confirmed as HCC by means of fine-needle aspiration biopsy performed under guidance with contrast-enhanced US. Six of these seven nodules were detected incidentally during examination of other suspected HCC nodules. Five of the seven nodules were treated with percutaneous ethanol injection (PEI) performed under guidance with contrast-enhanced US; the two other nodules were resected. Contrast-enhanced US made the HCC lesions visible for 15-60 minutes, sufficient time to mark the nodule with an iodized oil-ethanol solution for PEI. Because contrast-enhanced US enabled detection of additional nodules and performance of PEI in lesions not detected with plain US, it may help improve the treatment of HCC.


Choroid plexus, ependyma and arachnoidea express receptors for vitamin D: differences between "seasonal" and "non-seasonal" breeders. Autoradiographic studies with [3H]1,25-dihydroxyvitamin D3 demonstrated nuclear high affinity binding sites (receptors) in epithelial cells of choroid plexus, ependyma and arachnoid in "seasonal" breeders of various vertebrate phyla, namely Xiphophorus helleri, Hyla crucifer, Xenopus laevis, Bufo woodhousei, Chrysemys scripta, Anolis carolinensis, Nerodia sipedon and Phodopus sungorus, but vitamin D receptors were undetectable in these tissues in non-seasonal breeders, such as zebra finch, laboratory mice and rats. Both "seasonal" and "non-seasonal" species, however, exhibited nuclear binding in distinct nuclei of CNS. The data suggest that vitamin D is involved in the regulation of certain functions at the level of the blood-brain barrier in species strongly affected by seasonal changes.


Central effects of tricyclic compounds on the endocrine system--an in vitro study. The present study involves the effects on corticotropin (ACTH) release of neuro- and thymoleptic tricyclic antidepressant compounds (TrcACs: chlorpromazine, promethazine, haloperidol, imipramine, amitriptyline) and their interactions with lysine-8-vasopressin (LVP) and corticosterone (B). As an in vitro model, 14-day monolayer pituitary cell cultures of Wistar rats were employed. The ACTH concentrations of the supernatant media were measured by radioimmunoassay. TrcACs augmented ACTH release; their combination with LVP, however, did not result in further stimulation; moreover, when combined with TrcACs + LVP, B did not inhibit, but rather paradoxically increased their ACTH-releasing action. As none of these phenomena were followed by relevant changes in intracellular cyclic adenosine monophosphate content, the mechanism of action may be proposed to involve a protein kinase C route.


N-acetyl[3H]leukotriene D4: a stable internal standard for automated BIO-FAST HPLC extraction and separation of LTE4 from human urine. The BIO-FAST (Fully Automated Sample Treatment) HPLC can be used for the isolation and separation of leukotriene E4 (LTE4) from the urine of asthmatic patients. A chemically related leukotriene, N-acetyl[14,15-3H]leukotriene D4 (NAc[3H]LTD4), has been evaluated as an internal standard to allow full automation of the BIO-FAST method. NAcLTD4 is not a human metabolite, does not co-elute with endogenously produced LTs and is stable in native urine at 37 degrees C for at least 18 h. Recovery and stability studies were conducted by adding NAc[3H]LTD4 and [3H]LTE4 to the baseline urine of four asthmatic patients. Automated extraction of these four samples over 22 hours, using the BIO-FAST system, yielded recoveries of 80.5% (6.6 %CV, n = 12) and 72.4% (10.0 %CV, n = 12) for the NAc[3H]LTD4 and [3H]LTE4, respectively. The ratio of NAc[3H]LTD4 to [3H]LTE4 was 1.12 (6.3 %CV, n = 12) demonstrating the consistent relative extraction of these two leukotrienes.


Prostaglandin E2 binding site distribution and subtype classification in the rabbit iris-ciliary body. The distribution and characteristics of specific binding sites for tritium labeled prostaglandin E2 (3H-PGE2) were examined in membrane preparations from rabbit iris-sphincter, iris and ciliary body. The majority of 3H-PGE2 specific binding sites were found in the ciliary body (46%) followed by the iris (37%) and the iris-sphincter muscle (5%). Scatchard analysis of saturable 3H-PGE2 binding sites in the ciliary body indicated a single binding site with a Kd of 2.81 nM and Bmax value of 84 fmoles bound/mg protein. Competition by agonists selective for the EP1, EP2 and EP3 receptor subtypes of the EP (PGE2) prostanoid receptor indicated that the majority of rabbit ciliary body 3H-PGE2 binding sites are of the EP2 subtype. Incomplete displacement of labeled 3H-PGE2 from its binding sites by the EP2 selective agonist 11-deoxy PGE1 suggests the presence of additional EP or non-EP binding sites. There was essentially no binding to EP1 receptor sites as defined by the displacement of 3H-PGE2 by 17-phenyl-trinor PGE2. A weak displacement of 3H-PGE2 by the EP3/EP1 specific agonist, sulprostone, may account for the presence of a small number of EP3 specific binding sites in this tissue. The predominant distribution of PGE2 binding sites in the ciliary body and their identification as EP2 selective, supports recent functional studies where topical application of prostanoids with EP2 but not EP1 or EP3 agonist activity resulted in breakdown of the blood-aqueous barrier.


Different early effect of irradiation in brain and small cell lung cancer examined by in vivo 31P-magnetic resonance spectroscopy. Early effects of irradiation were evaluated by non-invasive in vivo 31P-magnetic resonance spectroscopy (31P-MRS) of two small cell lung cancer (SCLC) tumor lines CPH SCCL 54A and 54B, in nude mice. The tumors were originally derived from the same patient and have similar morphology and growth characteristics, but a different radiosensitivity. The 54A tumors are twice as radiosensitive as the 54B's. In the present study the tumors were treated with 2.5, 10, and 40 Gy. For comparison, nude mice were given cranial irradiation at the same three doses, and the effect was evaluated by in vivo 31P-MRS. No effect was observed in brain at any dose level. In contrast, 40 Gy induced a statistically significant reduction in ATP/Pi ratio during the 12-h post-irradiation period. This effect was more pronounced in 54A than in 54B. Some reduction was observed following 10 Gy, whereas 2.5 Gy induced no changes in ATP/Pi. The differential effect on tumors and brain might be relevant for monitoring irradiation effects by in vivo 31P-MRS in patients with brain metastases.


[Mucopolysaccharidoses: evaluation of the cranium by computed tomography and magnetic resonance]. Cranial CT and/or MRI examinations were performed in 8 patients affected with mucopolysaccharidosis. Two patients were affected with mucopolysaccharidosis IH,1 had mucopolysaccharidosis IS, 1 had mucopolysaccharidosis IV A and 4 presented mucopolysaccharidosis VI. CT and MRI showed white matter changes consisting of symmetric and diffuse hypodense areas on CT, large areas and multiple spots of prolonged T1 and T2 value on MRI. Reduced gray/white matter contrast was demonstrated on T2-weighted sequences. Furthermore, CT and MRI showed thickening of the dura mater at the cranio-cervical junction, which caused subarachnoid space narrowing in all patients. Spinal cord compression was detected in 4 patients. Additional findings were mild to severe hydrocephalus, skull dysplasia and odontoid dysplasia. Owing to the progressive clinical course of all mucopolysaccharidosis, the authors stress the importance of a careful study of the cranio-cervical junction in all the patients with mucopolysaccharidosis examined by CT or MRI in order to demonstrate essential thickening of the dura mater. In the patients with symptomatic cord compression, surgery should be considered.


Effect of diets containing different levels of calcium, phosphate, and vitamin D3 on tooth uptake of 47Ca-chloride in rat models: comparison with bone uptake. The effect of dietary manipulation on global tooth uptake of 47Ca-chloride was evaluated in three rat models of metabolic bone diseases verified by histologic bone study. The 24-hr tooth uptake of 47Ca-chloride was significantly decreased in the osteomalacic group, and was increased in the osteoporotic group at the fourth week. Tooth uptake closely paralleled femoral uptake at a correlation coefficient of 0.73 (p less than 0.01). The uptake of 47Ca-chloride in tooth was found to be more affected by dietary manipulation than that in femur in our rat models.


[Bronchiolo-alveolar carcinoma in a solitary site. The computed tomographic characteristics]. Bronchioloalveolar carcinoma is a peripheral, well-differentiated neoplasm arising beyond a recognizable bronchus, with a tendency to spread to the peripheral air spaces using the lung structure as stroma. This tumor has better prognosis than other lung neoplasms if it is diagnosed early (stage I). For this reason, the CT scans of 16 patients (all of them asymptomatic, only 2 smokers) were reviewed to assess CT capabilities. The cytologic diagnosis was made with CT-guided fine-needle aspiration biopsy in 4 patients (surgical confirmation followed); the histologic diagnosis was made in 12 patients after surgery. CT findings included the peripheral (14 cases) or subpleural (2 cases; 12.5%) location, irregular margins forming a star-like pattern (2 cases; 12.5%), pseudocavitation (13 cases; 81%), heterogeneous attenuation (11 cases; 69%), pleural tags (14 cases; 87.5%) and air bronchogram (2 cases; 12.5%). These CT findings, if compared with the same number of CT scans in patients with adenocarcinoma and squamous cell carcinoma, are present in 70% of patients (11/16) and, although not highly specific (specificity: 36%), they are typical enough to suggest the diagnosis.


Characterization of proton extrusion in sunflower cell cultures. Cell suspensions derived from callus root tips of sunflower (Helianthus annuus L., cv. enano) were obtained in order to assess the effects of different chemical and physical agents on cell H+ extrusion. Cell H+ efflux was sensitive to temperature, pH, inhibitors of plasmalemma H(+)-ATPase and Ca2+ and K+ concentrations in the assay medium, as well as to the light intensity at which cells were cultivated. Thus, in the darkness and at 60 mumol/m2/s of illumination, a strong inhibition of H+ extrusion was detected as compared to cells grown at 30 mumol/m2/s. H+ extrusion by cells grown at 30 mumol/m2/s was unaffected by the presence of calcium in the assay medium, while at 60 mumol/m2/s such an activity increased when calcium was removed. These results provide the basis for the use of cell suspensions as an appropriate model to investigate the involvement of membrane-associated processes in plant tolerance mechanisms to different environmental stresses.


[Specific inhibition by ATP of histamine-stimulated acid secretion in the gastric glands of the rabbit]. The influence of adenosine 5'-triphosphate on gastric acid secretion stimulated by histamine, carbachol, dibutyryl-cAMP and the phosphodiesterase inhibitors 8-phenyl-theophylline and rolipram in isolated rabbit gastric glands was studied. Changes oi gastric acid secretion were measured by the aminopyrine accumulation method. Histamine-stimulated acid secretion was significantly inhibited by ATP 1 mM, whereas the secretory responses elicited by carbachol, dibutyryl-cAMP, 8-phenyl-theophylline or rolipram were not. Assays with indomethacin, a well known prostaglandin synthesis inhibitor, showed that this agent significantly reduced the inhibitory effect of ATP on histamine responses. The results indicate that the antisecretory effect of ATP was specific for histamine and that it was mediated, at least in part, via stimulation of endogenous prostaglandin production.


Age-related alterations in second-messenger systems in the gerbil brain: autoradiographic analysis and effect of vinconate. Age-related alterations in binding sites of major second-messenger systems in the gerbil brain were analyzed by receptor autoradiography using [3H]phorbol 12,13-dibutyrate (PDBu), and [3H]inositol 1,4,5-trisphosphate (IP3). In middle-aged gerbils (16 months old), the reduction of PDBu-binding sites was noticed in various brain regions compared with young animals (4 weeks old). By contrast, the elevation of IP3-binding sites was seen in most of brain regions except for the reduction in the cerebellum in the middle-aged animals. Chronic treatment with vinconate partly regulated the age-related changes in the binding sites of these second messengers. The result demonstrates the age-related alterations in the binding sites of an intracellular second-messenger system. Furthermore, they suggest that vinconate partly modulates the age-related alterations in the bindings of second messengers.


Myoelectrical responses of pyloric antrum to GABAergic agents in conscious sheep. The role of GABA receptors in the control of abomasal motility was examined electromyographically in four conscious ewes by injecting intracerebroventricularly and intravenously selective GABA(A)- and GABA(B)-receptor agonists and antagonists. The study indicated that the stimulation of the GABA(B) receptors results in an inhibition of the antral motility via central mechanisms.


Hypothalamic-pituitary-adrenal axis responsiveness to insulin-induced hypoglycaemia is modified by trypanosome infection in Boran (Bos indicus) cattle. Ten Boran (Bos indicus) cattle were used to study the stress responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis during trypanosome infection. Five cattle were infected with Trypanosoma congolense IL 1180 by tsetse challenge and five cattle served as controls. All infected animals developed acute trypanosomiasis. Insulin-induced hypoglycaemia (50 per cent of pre-insulin glucose concentration) was used as a stress factor. Acute hypoglycaemia was observed in three infected and three control animals after insulin challenge. Two animals from each group either did not respond or responded slowly. Hypoglycaemia in infected animals completely failed to induce an HPA axis response, while in control animals an HPA axis response was indicated by a significant increase in plasma adrenocorticotrophic hormone (ACTH) and cortisol concentrations (P less than 0.01). The results show that trypanosomiasis in Boran cattle can cause a decrease in the stress responsiveness of the HPA axis as indicated by a blunted ACTH/cortisol response to insulin-induced hypoglycaemia.


[Changes in plasma cortisol and ACTH caused by diazepam, bromazepam, triazolam, and alprazolam in oral premedication]. Benzodiazepines relieve anxiety and modify the endocrine response to surgical-anesthetic stress. The aim of this study was to evaluate the effects of four benzodiazepines on preoperative secretion of cortisol and ACTH. We studied 60 patients programmed for abdominal surgery. Patients were randomly allocated into six groups of treatment: control group (placebo), diazepam 10 mg, triazolam 0.5 mg, alprazolam 0.5 mg, bromazepam 6 mg, and bromazepam 12 mg. Blood samples for cortisol and ACTH measurements were obtained at five different stages of the anesthetic-surgical procedure: baseline, preanesthesia, anesthesia, surgery, and postoperative phase. Diazepam and bromazepam (6 and 12 mg) lessened the increase in plasmatic cortisol induced by preanesthesia and anesthesia in control patients. Triazolam attenuated the cortisol response only during preanesthesia. Administration of 12 mg of bromazepam decreased ACTH levels during preanesthesia and anesthesia with respect to the control group. Benzodiazepines failed to modify cortisol and ACTH levels during surgery and during the immediate postoperative period. The most marked attenuation of adrenocortical response was achieved with 12 mg of bromazepam and the less marked lessening was induced by 0.5 mg of alprazolam.


Acute frosted retinal periphlebitis associated with cytomegalovirus retinitis. The clinical picture of an acute frosted periphlebitis has been reported in a limited number of otherwise healthy patients, and in one case of human immunodeficiency virus (HIV) infection. The disease is usually bilateral and highly sensitive to steroids. The case of a 26-year-old, HIV-seropositive woman who developed unilateral diffuse perivenous sheathing in the course of recurrent cytomegalovirus retinitis is reported. The appearance of the retinovasculitis was identical to that described as an acute frosted periphlebitis, and the disease proved to be highly sensitive to a combined treatment of steroids and ganciclovir. The clinical findings in this case support the possibility that the appearance of an acute frosted periphlebitis can occur in response to several different stimuli, including cytomegalovirus.


Retinal toxicity of 6-methoxypurine arabinoside (ara-M). A new selective and potent inhibitor of varicella-zoster virus. The toxicity of a new antiviral agent, 6-methoxypurine arabinoside (ara-M), as a selective and potent inhibitor against varicella-zoster virus, was investigated after intravitreal injection in rabbit eyes. Intravitreal doses of ara-M ranging from 20 micrograms to 400 micrograms produced no retinal toxicity, as assessed by electroretinography and light and transmission electron microscopy. These data suggest a potential role for intravitreal ara-M in the antiviral treatment of varicella-zoster viral retinitis.


The pattern of proteins synthesized in the liver is profoundly modified upon infection of susceptible mice with mouse hepatitis virus 3. Susceptible BALB/c mice, after experimental infection with mouse hepatitis virus 3 (MHV3), revealed virus titres in the liver that increased gradually to a peak of 8 x 10(5) PFU/g of tissue after 3 days' infection, when the mice died of acute hepatitis. BALB/c mice were infected with MHV3, subsequently labelled in vivo with 35S-methionine, and then the liver preparations from both infected and non-infected animals were subjected to two-dimensional gel electrophoresis. Comparisons of the patterns by computer image analysis revealed 17 gene products which increased, and 8 gene products which decreased, upon virus infection in their two-dimensional gel spot intensity. We conclude that during MHV3 infection of a susceptible strain of mice, a major modification in protein synthesis occurs. The pattern alterations were not related to the virus gene products but were mostly endogenous mouse proteins. Whether these proteins are a result of a defence attempt by the animal, or are dictated by the virus in order to prevent a protective response from happening, remains to be shown.


Transmissible gastroenteritis coronavirus: surface antigens induced by virulent and attenuated strains. Three strains of the transmissible gastroenteritis virus (TGEV) possessing different degrees of pathogenicity for piglets were examined for their capacity to express M and S glycoproteins on the infected cell surface using a microwell immunoperoxidase test. These two viral glycoproteins were easily detected on the plasma membrane of 0.1% paraformaldehyde-fixed swine testis (ST) or pig kidney (RP.D) cells which were infected with high-passaged Purdue-115 and low-passaged D-52 strains and a high-passaged attenuated (188-SG) mutant of TGEV. No significant differences were found between attenuated and virulent strains with regard to the viral antigen expression on the membrane of infected cells over a 14-h period.


Study duration for group sequential clinical trials with censored survival data adjusting for stratification. The study duration in a clinical trial with censored survival data is the sum of the accrual duration, which determines the sample size in a traditional sense, and the follow-up duration, which more or less controls the number of events to be observed. We propose a design procedure for determining the study duration or for calculating the power in a group sequential clinical trial with censored survival data and possibly unequal patient allocation between treatments, adjusting for stratified randomization. The group sequential method is based on the use function approach. We describe a clinical trial recently activated by the Eastern Cooperative Oncology Group for an illustration of the proposed procedure.


Mucosal delivery of herpes simplex virus vaccine. The mucosal route for the production of mucosal and systemic herpes simplex virus (HSV) antibodies was investigated using HSV1 subunit vaccine administered to guinea pigs. Groups of test animals (n = 13) were dosed, nasally or vaginally and compared with those injected subcutaneously (s.c.). The vaccines, in aqueous or gel form, were administered 5 and 3 weeks prior to vaginal challenge with HSV2 suspension. Control infected and non-infected animals were included for comparison. Animals which were vaccinated s.c. were shown to respond to subsequent infection with HSV by the production of serum HSV-specific IgG (and IgA) but negligible amounts of vaginal IgG and IgA. Control non-infected and infected-only groups produced none and only a small amount of vaginal HSV-specific antibodies, respectively. Substantial protection against HSV2 infection of the female guinea pig genital tract was provided by s.c. immunization with HSV vaccine. Protection was evaluated in terms of the reduction of histopathological lesions and clinical signs in vaccinated and control animals. The serum humoral response to nasal delivery in phosphate-buffered saline was comparable, and was superior for vaginal washes to that of parenteral vaccination. The nasally delivered free antigen gave significant (p < or = 0.05) reduction in the severity of the disease and higher levels of specific serum and vaginal immunoglobulin antibodies to HSV when compared with non-immunized infected-only controls, probably due to uptake of antigenically intact protein. Vaginal gel treatment slightly reduced the severity of the illness and gave higher humoral responses than those induced by vaginally delivered free antigen. Findings also indicate that these mucosal immune responses were produced at a site distant from the site of vaccination, suggesting a common immunological system.


Experimental neuroinvasiveness of wild and laboratory Junin virus strains. The neuroinvasiveness of Candid 1 and XJCL3 laboratory strains and CbalV4454 and CbaFHA5069 wild strains of Junin virus was studied in albino mice, guinea pigs, and a South American wild rodent, Calomys musculinus (Cm), of different ages inoculated by a non-neural route. Infectivity in brain, blood and organs, as well as lethality, were determined. The results with the 3 hosts indicate that Junin virus neuroinvasiveness is virus-strain-dependent, host species- and age-dependent, with the Candid 1 strain proving to be the least neuroinvasive of the strains studied. The lethal efficiency index (log PFU/LD50) in 2-day old albino mice and the neuroinvasiveness index (Log PFU/ND50) in 6 +/- 1 day-old Cm of the various strains using the intraperitoneal (ip) route could therefore be useful markers of Junin virus neuroinvasiveness. Moreover, different patterns of infection were established using the results of the presence of infectious virus in brain and viraemia in the 3 hosts. In nearly all cases, virus neuroinvasion was present without detectable viraemia (virus in plasma). Current evidence leads to the assumption that virus might reach the brain associated with the white cells in blood (undetectable by conventional isolation methods) or by another possible mechanism of neuroinvasion which is not haematogenous.


[Tumor necrosis factor alpha].

[Tumor necrosis factor alpha]. TNF-alpha (Cachectin) is a cytokine (tissue hormone) synthesized and secreted as a trimeric polypeptide; major producers are the cells of the RES after stimulation, e.g. by endotoxin. Its synthesis is strictly regulated. TNF elicits a great number of cell-specific responses. IL-1 and probably additional mediators cooperate in these effects. TNF is instrumental not only in the pathogenesis of inflammation, infections and some cell injuries, but also in the unspecific immune response, tumor toxicity and tissue homeostasis. TNF binding to specific receptors on cell surfaces initiates of secondary intracellular signals that, in turn, mediate the metabolic responses of the target cells. Pharmacological intervention may be envisaged at the level of synthesis (glucocorticoids, anti-oxidants) or interaction of the cytokine with its receptors.


[Peripheral nerve lesions in general surgery]. Surgical interventions can give rise to peripheral nerve lesions by various mechanisms. The neurologist is asked to define which nerve has been lesioned at which site. We also have to define the type of lesion and its severity. The electrophysiological investigation is a very important diagnostic tool in this particular situation. Together with the clinical examination it will allow to plan an optimal treatment for the patient.


[Polyneuropathies with an abnormal tendency for pressure-induced paralysis]. A 52-year old man is discussed. At age of 14 he was noticed to have pes cavus and thin shanks on both sides und drop foot on the right side. Since that time he has been suffering from episodic sensory loss in the distribution of the ulnar, median, peroneal and trigeminal nerves. An extended sensory loss around the pelvis and the posterior aspect of the thighs was a rather unique feature in this patient. As the patient refused nerve biopsy it cannot be decided whether peroneal muscular atrophy with multiple nerve compressions or if the entity of polyneuropathy with liability to pressure palsies was present.


[Value of interferon alpha determination in the diagnosis of meningoencephalitis presumed to be of viral origin]. Quantitative determination of alpha interferon (IFN) is used as an early marker in viral encephalitis. IFN is detected during 10 days following the onset of clinical symptoms. In 26 patients (11 children from 1 day to 6 year old and 15 adults from 17 to 70 year old) with central nervous system disorders (15 meningo-encephalitis, 5 meningitis, 1 myelitis, 1 polyradiculoneuritis, 1 dementia, 1 epilepsy and 2 other), alpha IFN is quantified using a cytopathic effect inhibition assay of VSV on MDBK cells. The mean value of alpha IFN is 80 UI/ml (range from 0 to 512 UI/ml). Virus involved are herpes virus in 38.5% of cases (10/26) and 66% of viral meningoencephalitis (8/12), H.I.V. in 3 cases, VZV in 2 and measles virus in 1 case. Viral aetiology is suspected in six other patients. The results show the importance of early determination of alpha IFN (immediately after the first symptoms and on the first admission to the hospital) in sera and cerebrospinal fluids (CSF) simultaneously with viral culture and antibody research. The presence of alpha IFN only in CSF and a higher titre of alpha IFN in CSF than in serum are important data to distinguish primitive acute necrotizing encephalitis from post eruptive or post infectious perivenous encephalitis. In herpes virus infections with specific treatment all the patients recover. However to prevent brain damage in survivors the treatment should be established as soon as possible.


Intranuclear inclusions in the pituitary gland of cynomolgus monkeys. In a 3-month oral toxicity study of a pharmaceutical agent, intranuclear inclusions were seen in the secretory cells of the pars anterior of the pituitary gland of 22 of 40 cynomolgus monkeys, with similar incidences in control and dosed groups. All monkeys were clinically healthy. Electron microscopic examination revealed that the intranuclear inclusions were cytoplasmic invaginations.


A simplified high-performance liquid chromatographic method for determination of vitamin D3, 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in human serum. Measurement of the serum level of 25-hydroxyvitamin D is the most useful parameter in evaluating vitamin D status. The serum level of vitamin D is a useful parameter in studying short time effects after exposure to ultraviolet light and absorption of the vitamin after oral administration. A method for simultaneous determinations of vitamin D3 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 is described. Serum or plasma was extracted by methanol-isopropanol (90:10, v/v) and hexane. The hexane layer was injected in to a reversed-phase (C18) high-performance liquid chromatographic (HPLC) system. 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were eluted by methanol-water (85:15, v/v), and vitamin D3 by a linear gradient of methanol-water (85:15) and methanol-isopropanol-water (87.5:10:2.5), and detected by u.v. absorption. This method gave separate determinations of the D2 and D3 forms of 25-hydroxyvitamin D, but owing to an interfering peak the method does not measure vitamin D2. The assay was very sensitive with a detection limit of 5 nmol l-1 for 25-hydroxyvitamin D2 and D3 and vitamin D3 by using 0.5 ml serum or plasma for analysis, so that for low vitamin D3 levels more than 1 ml of serum is desirable.


Acute development of low T3 syndrome and changes in pituitary-adrenocortical function after elective cholecystectomy in women: some differences between young and elderly patients. From two groups of female patients (young group: 15 patients aged less than 25 years; elderly group: 15 patients aged more than 65 years) who were subjected to cholecystectomy, blood samples were obtained on the day before surgery, on the day of surgery before premedication and after waking up from anaesthesia and also on days 1, 3 and 7 after surgery. In aliquots of sera the levels of TSH, ACTH, thyroxine (T4), triiodothyronine (T3), reverse triiodothyronine (rT3) and cortisol were estimated with the aid of radio-immunoassay (RIA). The differences between the young and elderly subjects were evaluated with the aid of four different statistical tests. Though no changes in the level of T4 were found, the level of T3 was significantly decreased and that of rT3 was significantly increased on Day 1 after surgery. However, the decrease of T3 was expressed more significantly in the young group and, in addition, an increase of rT3 in the same group was found even before premedication. The level of TSH showed a sharp increase at the end of surgery, which was expressed more in the young group. Though no differences between groups were observed in a sharp peak of the ACTH level during surgery, the increase of cortisol level in the elderly group was significantly higher and remained so during the post-operative period.


Interaction of prostaglandins with adenosine diphosphate induced increase in cytosolic free calcium in human platelets. The interaction of prostaglandins with changes in cytosolic Ca2+ concentration ([Ca2+]) and aggregation of human platelets induced by adenosine diphosphate (ADP) were investigated. Cytosolic [Ca2+] was measured with the fluorescent dye Quin2. Addition of ADP (0.25-2.5 mumol l-1) to platelet suspensions produced a dose dependent increase in cytosolic [Ca2+] from a basal level of 51 +/- 1 nmol l-1 to maximum levels exceeding 1 mumol l-1 and induced platelet aggregation. Chelation of extracellular calcium with 100 mumol l-1 EGTA markedly reduced the increase in cytosolic [Ca2+] induced by 0.25 mumol l-1 ADP, while pretreatment with the calcium entry blocker verapamil was without effect. Stimulation of cyclic AMP with prostaglandins (PGD2, PGE1, PGE2, PGI2, but not PGF2 alpha) and forskolin, or incubation with dibutyryl-cAMP, inhibited the rise in cytosolic [Ca2+] and platelet aggregation following ADP. We conclude that prostaglandins inhibit the increase in cytosolic [Ca2+] and aggregation of human platelets induced by ADP, probably by stimulation of cyclic AMP generation, thereby opposing the mechanism by which ADP increases cytosolic [Ca2+] and subsequently induces platelet aggregation.