Cryopreventive temperatures prior to chemotherapy.

The superiority of oral cryotherapy (OC) for prevention of chemotherapy-induced oral mucositis (OM) has been demonstrated in several trials. In clinical settings, cooling is usually initiated prior to the chemotherapy infusion. It then continues during the infusion, and for a period after the infusion has been completed. While the cooling period post-infusion depends on the half-life of the chemotherapeutic drug, there is no consensus on when cooling should be initiated prior to the infusion. The lowest achieved temperature in the oral mucosa is believed to provide the best condition for OM prevention. Given this, it was of interest to investigate when along the course of intraoral cooling this temperature is achieved. In total, 20 healthy volunteers participated in this randomized crossover trial. Each subject attended three separate cooling sessions of 30 min each, with ice chips (IC) and the intraoral cooling device (ICD) set to 8 and 15 °C, respectively. At baseline and following 5, 10, 15, 20 and 30 min of cooling, intraoral temperatures were registered using a thermographic camera. The greatest drop in intraoral temperature was seen after 5 min of cooling with IC, ICD8°C and ICD15°C, respectively. A statistically significant difference, corresponding to 1.4 °C, was seen between IC and the ICD15°C (p < 0.05). The intraoral temperature further declined throughout the 30 min of cooling, showing an additional temperature reduction of 3.1, 2.2, and 1.7 °C for IC, ICD8°C and ICD15°C, respectively.

Postnatal development of presynaptic receptors that modulate noradrenaline release in mice.

The objective of the study was to clarify the postnatal development of the following transmitter release-modulating receptors of noradrenergic neurons in mice: alpha2-adrenoceptors, muscarinic, opioid and cannabinoid receptors (inhibitory), beta-adrenoceptors and receptors for angiotensin II and bradykinin (facilitatory). Wildtype (NMRI) and in some cases alpha2A/D-adrenoceptor-deficient mice aged 1 day (P1) or 8-16 weeks (adults) were used. Hippocampal and occipito-parietal cortex slices and sympathetically innervated tissues (atria and vas deferens) were preincubated with [3H]-noradrenaline and then superfused and stimulated electrically. Stimulation led to distinct increases in tritium efflux which were abolished by tetrodotoxin or removal of calcium. Concentration-response curves of appropriate agonists and in the case of alpha2-autoreceptors antagonists were determined. For beta-adrenoceptors and angiotensin receptors, the interaction of agonists with antagonists was also examined. Results demonstrate that alpha2A/D-autoreceptors operate already at P1 whereas nonalpha2A/D-autoreceptors, presumably alpha2C, develop later. Of the various heteroreceptors, those of brain noradrenergic neurons (OP3 and ORL1) modulate the release of [3H]-noradrenaline at least as effectively at P1 as in adults. Those of peripheral sympathetic neurons (muscarinic, probably mainly M2, OP1, OP2, OP3, CB1, AT1 and B1), in contrast, operate less effectively or not at all at P1, with one exception: beta2-adrenoceptors increase the release of [3H]-noradrenaline (atria) to the same extent, irrespective of age. Overall, results indicate that brain and peripheral noradrenergic neurons release their transmitter already shortly after birth. Presynaptic receptor mechanisms mature differentially in the brain and the periphery. Moreover, the various presynaptic receptors differ in their postnatal development and may play differential roles at different ages.

Modulation of (3)H-noradrenaline release by presynaptic opioid, cannabinoid and bradykinin receptors and beta-adrenoceptors in mouse tissues.

Release-modulating opioid and cannabinoid (CB) receptors, beta-adrenoceptors and bradykinin receptors at noradrenergic axons were studied in mouse tissues (occipito-parietal cortex, heart atria, vas deferens and spleen) preincubated with (3)H-noradrenaline. Experiments using the OP(1) receptor-selective agonists DPDPE and DSLET, the OP(2)-selective agonists U50488H and U69593, the OP(3)-selective agonist DAMGO, the ORL(1) receptor-selective agonist nociceptin, and a number of selective antagonists showed that the noradrenergic axons innervating the occipito-parietal cortex possess release-inhibiting OP(3) and ORL(1) receptors, those innervating atria OP(1), ORL(1) and possibly OP(3) receptors, and those innervating the vas deferens all four opioid receptor types. Experiments using the non-selective CB agonists WIN 55,212-2 and CP 55,940 and the CB(1)-selective antagonist SR 141716A indicated that the noradrenergic axons of the vas deferens possess release-inhibiting CB(1) receptors. Presynaptic CB receptors were not found in the occipito-parietal cortex, in atria or in the spleen. Experiments using the non-selective beta-adrenoceptor agonist isoprenaline and the beta(2)-selective agonist salbutamol, as well as subtype-selective antagonists, demonstrated the occurrence of release-enhancing beta(2)-adrenoceptors at the sympathetic axons of atria and the spleen, but demonstrated their absence in the occipito-parietal cortex and the vas deferens. Experiments with bradykinin and the B(2)-selective antagonist Hoe 140 showed the operation of release-enhancing B(2) receptors at the sympathetic axons of atria, the vas deferens and the spleen, but showed their absence in the occipito-parietal cortex. The experiments document a number of new presynaptic receptor locations. They confirm and extend the existence of marked tissue and species differences in presynaptic receptors at noradrenergic neurons.

Methionine overcomes neural tube defects in rat embryos cultured on sera from laminin-immunized monkeys.

Sera from laminin-immunized monkeys were previously found to cause neural tube defects in cultures of whole rat embryos by unknown mechanisms. In the present study, adding L-methionine to either the culture media or to the diets of the monkeys overcame the toxicity of the serum from one of these monkeys (LAM3) but not the other (LAM4). The antilaminin antibody levels and avidities for isolated murine laminin of sera from the two monkeys were comparable. However, when yolk sac homogenates were tested on ELISA, antibodies from LAM4 had greater binding than LAM3, which was further supported by immunoelectron microscopy. These differences in antibody binding were explained by the findings that antibodies from LAM4 recognized more epitopes than LAM3 and that LAM4 recognized specific epitopes not recognized by LAM3. These antibodies caused reductions in the number of microvilli on the cells and the cell sizes of the yolk sac endoderm. In addition, uptake of [14C]methionine, [14C]sucrose and [14C]valine by yolk sacs from embryos cultured on serum from LAM4 was less than that for LAM3. We suggest that the neural tube defects caused by the antilaminin antibodies were a result of reduced nutrient flow caused by the reduction in the number of microvilli on the cells of the yolk sac endoderm.

Choroid tissue supports the survival of ciliary ganglion neurons in vitro.

It is well established that during in vivo development the neurons of the avian ciliary ganglion are dependent for their survival on structures in the eye. Separate neuron populations innervate intraocular smooth and striated muscle targets. All ciliary neurons survive when cocultured with striated muscle. We demonstrate that when ciliary ganglion neurons are plated on explants of the choroid coat (a smooth muscle-containing target tissue) using a defined medium (N2), the neurons survive and grow vigorously into the tissue, forming contacts between axons and target cells identified as smooth muscle. Conditioned medium from choroid explants also rescues all the neurons, as does coculturing ciliary ganglion neurons with dissociated choroid cells. However, the presence of horse serum and chick embryo extract in the medium inhibits the choroid's ability to support ciliary neurons. The effects of these additives on the phenotypic expression of the smooth muscle may explain the inability of previous investigators to demonstrate target-derived support from smooth muscle preparations. Because the choroid contains cell types other than smooth muscle (e.g., fibroblasts and endothelial cells), we could not identify smooth muscle as the only cell type responsible for the release of the soluble trophic factor present in the target tissue. However, indirect evidence using avian primary fibroblast cultures, a fibroblast cell line, and an anatomically simple smooth muscle preparation, the avian amnion, suggests that smooth muscle cells are sufficient to account for the observed trophic activity, and that similar target-derived molecules support the survival of both types of ciliary ganglion cells.

Importance of interferons in recovery from mousepox.

Gamma interferon is shown to be critical in recovery of C57BL/6 mice from mousepox. Anti-gamma interferon treatment of mice infected in the footpad with ectromelia virus resulted in enhanced spread to and efficient virus replication in the spleen, lungs, ovaries, and, especially, liver. All treated, infected mice died within a mean of 7 days, 2.5 days earlier than mice with severe combined immunodeficiency that were given a comparable infection. On the other hand, alpha interferon appeared not to have a major role in controlling virus replication in tissues examined, and beta interferon was important for virus clearance in the liver and ovaries but not the spleen. Either anti-alpha, beta interferon or anti-beta interferon antibody therapy resulted in only 25% mortality. Infected control mice survived but showed persistence of ectromelia virus at the site of infection (the footpad) and transient presence of the virus in the spleen, liver, lungs, and ovaries and in the fibroreticular but not lymphoid cells of the draining popliteal lymph node. Depletion of gamma interferon but not alpha and/or beta interferon resulted in a significant reduction in the numbers of splenic T (especially gamma delta-TCR+), B, and Mac-1+ cells, although the proportion of Mac-1+ cells in the spleen increased compared with control values. Depletion of alpha, beta, or gamma interferons did not severely affect the generation of virus-specific cytotoxic T-lymphocyte responses or natural killer cell cytolytic activity. This study, in which a natural virus disease model was used, underscores the crucial importance of gamma interferon in virus clearance at all stages of infection and in all tissues tested except the primary site of infection, where virus clearance appears to be delayed.

Acute inflammatory response to cowpox virus infection of the chorioallantoic membrane of the chick embryo.

The chick embryo chorioallantoic membrane was used to study the acute inflammatory response in the absence of contributions from the immune system. In preliminary experiments, lesions of wild-type cowpox virus strain Brighton (CPV-BR) and a 38K gene deletion mutant of CPV-BR (CPV-BR.D1) were compared with vaccinia virus (strains WR and Copenhagen), fowlpox virus, laryngotracheitis virus, and infectious tenosynovitis virus, and were ranked for degree of induced inflammation. The maximal and minimal inflammatory responses were observed with CPV-BR.D1 and CPV-BR viruses, respectively. CPV-BR.D1 lacks a 38K gene which encodes an anti-inflammatory 38-kDa protein that has homology to SERPINs. The kinetics and character of the inflammatory response were examined further in the wild-type CPV-BR and mutant CPV-BR.D1 infections using cell counts, electron microscopy, and assays for inflammatory cell activation. CPV-BR virus infection rapidly spread through the ectoderm, uniformly infecting all cells with the production of large amounts of virions and viral-induced cytopathic effect, but evoking little or no inflammatory response until 144 hr p.i. The CPV-BR.D1 infection, on the other hand, was rapidly contained by a dexamethasone-sensitive inflammatory response mainly of activated heterophils which was advanced by 36 hr p.i. Both infections resulted in disseminated disease with similar numbers of liver lesions and only a slight difference in the LD50, with the CPV-BR.D1 values being higher than that for CPV-BR virus. In this model, the acute inflammatory response alone is unable to prevent disseminated disease and associated mortality.

Isolation and structure of the lipid envelopes from the nitrogen-fixing vesicles of Frankia sp. strain CpI1.

Frankia vesicles are differentiated during nitrogen starvation; they contain nitrogenase whether produced by free-living frankiae or by frankiae in actinorhizal root nodules. Vesicles are surrounded by envelopes of several monolayers of uncharacterized lipid. It has been suggested that the envelope limits diffusion of O2 into the vesicle cytoplasm, thereby preventing inactivation of nitrogenase. Whole vesicles were prepared on sucrose gradients and sonicated, and vesicle envelopes were isolated on top of a cushion of 40% sucrose. Transmission electron microscopy of potassium permanganate-fixed envelopes confirmed the purity of these preparations. Only the outer and inner envelope layers were visible in permanganate-fixed intact vesicles; the laminae were not visible in aldehyde-osmium-fixed, lead citrate-uranyl acetate-stained whole vesicles. However, the laminated nature of the envelope was clearly evident in sonicated vesicles and in envelope fragments fixed with KMnO4. The observations indicate that partial disruption of the vesicle envelope enables its visualization with permanganate fixation, and these observations open the way for further studies on the relationship of the vesicle surface to environmental conditions.

Preparation and properties of recombinant DNA derived tobacco mosaic virus coat protein.

Recombinant DNA derived tobacco mosaic virus (vulgare strain) coat protein (r-TMVP) was obtained by cloning and expression in Escherichia coli and was purified by column chromatography, self-assembly polymerization, and precipitation. SDS-PAGE, amino terminal sequencing, and immunoblotting with polyclonal antibodies raised against TMVP confirmed the identify and purity of the recombinant protein. Isoelectric focusing in 8 M urea and fast atom bombardment mass spectrometry demonstrated that the r-TMVP is not acetylated at the amino terminus, unlike the wild-type protein isolated from the tobacco plant derived virus. The characterization of r-TMVP with regard to its self-assembly properties revealed reversible endothermic polymerization as studied by analytical ultracentrifugation, circular dichroism, and electron microscopy. However, the details of the assembly process differed from those of the wild-type protein. At neutral pH, low ionic strength, and 20 degrees C, TMVP forms a 20S two-turn helical rod that acts as a nucleus for further assembly with RNA and additional TMVP to form TMV. Under more acidic conditions, this 20S structure also acts as a nucleus for protein self-assembly to form viruslike RNA-free rods. The r-TMVP that is not acetylated carries an extra positive charge at the amino terminus and does not appear to form the 20S nucleus. Instead, it forms a 28S four-layer structure, which resembles in size and structure the dimer of the bilayer disk formed by the wild-type protein at pH 8.0, high ionic strength, and 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and function of disk aggregates of the coat protein of tobacco mosaic virus.

Experiments have been carried out on the coat protein of tobacco mosaic virus (TMVP) to test for the occurrence of the previously postulated RNA-induced direct switching, during in vitro assembly of tobacco mosaic virus (TMV), of the subunit packing from the cylindrical bilayer disk to the virus helical arrangement. No evidence was found for such RNA-induced switching and no evidence for the direct participation of the bilayer disk in either the nucleation or elongation phases of the in vitro virus assembly. Instead, virus assembly proceeds by an initiation step involving the binding of the RNA to the previously characterized two-plus turn helical aggregate that is formed from small oligomers of subunits. However, a bilayer disk, which has been characterized in high ionic strength crystals, has been observed in low ionic strength virus assembly solutions only as a transient species upon depolymerization of dimers of bilayer disks formed in solution at high ionic strength, and not as an equilibrium species of TMVP.

Elemental analysis of freeze-dried thin sections of Samanea motor organs: barriers to ion diffusion through the apoplast.

Leaflet movements in the legume Samanea saman are dependent upon massive redistribution of potassium (K), chloride (Cl), and other solutes between opposing (extensor and flexor) halves of the motor organ (pulvinus). Solutes are known to diffuse through the apoplast during redistribution. To test the possibility that solute diffusion might be restricted by apoplastic barriers, we analyzed elements in the apoplast in freeze-dried cryosections of pulvini using scanning electron microscopy/x-ray microanalysis. Large discontinuities in apoplastic K and Cl at the extensor-flexor interface provide evidence for a barrier to solute diffusion. The barrier extends from the epidermis on upper and lower sides of the pulvinus to cambial cells in the central vascular core. It is completed by hydrophobic regions between phloem and cambium, and between xylem rays and surrounding vascular tissue, as deduced by discontinuities in apoplastic solutes and by staining of fresh sections with lipid-soluble Sudan dyes. Thus, symplastic pathways are necessary for ion redistribution in the Samanea pulvinus during leaflet movement. In pulvini from leaflets in the closed state, all cells on the flexor side of the barrier have high internal as well as external K and Cl, whereas cells on the extensor side have barely detectable internal or external K or Cl. Approximately 60% of these ions are known to migrate to the extensor during opening; all return to the flexor during subsequent closure. We propose that solutes lost from shrinking cells in the outer cortex diffuse through the apoplast to plasmodesmata-rich cells of the inner cortex, collenchyma, and phloem; and that solutes cross the barrier by moving through plasmodesmata.