Impact of hyperthermia on inflammation-related perinatal brain injury.

In a rat model of perinatal inflammation and hypoxia, we investigated the impact of hyperthermia on the deleterious events which are commonly associated with chorioamnionitis. Late-pregnancy gestational day 20 rats received a single injection of either lipopolysaccharide (LPS) Escherichia coli endotoxin or saline. The offspring were born 24-36 h later at full term. The pups underwent hypoxia on the first postnatal day (PND1) immediately after which they were maintained at a planned target temperature for 2 h, before being returned to the dams. The pups were sacrificed on PND5 and the brain tissue was examined. Results showed that LPS alone or in combination with hypoxia was well tolerated. The additional stress of moderate hyperthermia (39°C for 2 h) on PND1 resulted in (a) a significant increase in brain reactive nitrogen species (RNS), (b) a significant increase in caspase-3 activity, (c) a significant increase in c-jun, bax and bcl-2 gene expression and (d) a significant increase in apoptotic cells in the CA1 region of the hippocampus. Hyperthermia was also associated with reduced growth over the ensuing 4 days in a small number of pups. In this model of perinatal inflammation, we demonstrated that brief hyperthermia when superimposed on a perinatal inflammation stimulus and hypoxia led to brain injury while either inflammation alone, or combined inflammatory stimulus and hypoxia did not cause significant damage.

Modification of tubulin by tyrosylation in cells and extracts and its effect on assembly in vitro.

A post-translational modification of tubulin with potential regulatory significance has been revealed by the discovery of an enzyme (tubulin-tyrosine ligase) in brain extracts which can add a tyrosine residue to the alpha chain, apparently through peptide bond linkage to a C-terminal glutamate. We have investigated whether this modification also occurs in vivo, and whether it alters the extent to which tubulin can assemble in vitro. Cytoplasmic tubulin purified from bovine brain by cycles of assembly was shown to be partially tyrosylated. Carboxypeptidase A digestion of isolated alpha chains liberated about 0.3 equivalent of tyrosine. Brief digestion of native tubulin increased the proportion of alpha chains which could be tyrosylated by ligase, from 25 to 45%. The tubulin assembled to the same extent before and after carboxypeptidase treatment. When tubulin was purified after introducing labeled tyrosine with ligase, the labeled species assembled in the same proportion as unlabeled. Thus tubulin can be incorporated into microbubules in vitro with or without C-terminal tyrosine. An apparent resolution of alpha chain into two components by hydroxylapatite chromatography was shown not to be due to the presence or absence of C-terminal tyrosine. Tubulin-tyrosine ligase was found in extracts of every rat tissue examined, but was not detected in sea urchin eggs before or after fertilization, in Tetrahymena cells or cilia, or in yeast. Cultured neuroblastoma cells fixed tyrosine into tubulin alpha chains under conditions where protein synthesis was inhibited; this in vivo fixation appeared to be into an insoluble moiety of tubulin. Incidental to these studies, a new assay utilizing an enamine substrate for carboxypeptidase was investigated.

Stimulation of tubulin tyrosinolation in rabbit leukocytes evoked by the chemoattractant formyl-methionyl-leucyl-phenylalanine.

Cellular tubulin is subject to a posttranslational modification involving the reversible addition to tyrosine through peptide linkage to the C-terminal glutamate of the alpha-chain. The synthetic peptide chemoattractant, N-formyl-methionyl-leucyl-phenylalanine, causes a specific, dose-dependent stimulation of tubulin tyrosinolation in rabbit leukocytes. This stimulation is prevented by carbobenzoxy-phenylalanyl-methionine, benzoyl-tyrosine ethylester, and nordihydroguaiaretic acid, which are all inhibitors of chemotaxis presumed to act via membrane-associated events. The combination of 3-deazaadenosine and homocysteine thiolactone, which inhibits phospholipid methylation, and quinacrine, an inhibitor of phospholipase A2, also abolishes the response to the peptide. Colchicine, however, which causes a marked disassembly of cellular microtubules in these cells and also inhibits chemotaxis, does not have any inhibitory effect on the basal or peptide-stimulated rate of tubulin tyrosinolation. In contrast, taxol, a microtubule-stabilizing agent, has an inhibitory effect on both the basal and peptide-stimulated tyrosine incorporation. Taxol also inhibits chemotaxis in rabbit leukocytes. The results strongly suggest the role of closely linked membrane-cytoskeleton interactions in leukocyte chemotaxis, in which tyrosinolation of tubulin may be functionally involved.

Tubulin tyrosinolation in human polymorphonuclear leukocytes: studies in normal subjects and in patients with the Chediak-Higashi syndrome.

We have recently reported a specific dose-dependent stimulation of posttranslational incorporation of tyrosine into tubulin alpha-chains of rabbit peritoneal leukocytes as induced by the synthetic peptide chemoattractant formyl-methionyl-leucyl-phenylalanine (FMLP). The present study reports a similar, specific stimulation of tubulin tyrosinolation in human polymorphonuclear leukocytes (PMN). When compared to normal PMN, both the resting and FMLP-stimulated levels of posttranslational tyrosine incorporation were two- to threefold higher in PMN of three patients with the Chediak-Higashi syndrome (CHS). The concentration of cellular tubulin and the specific activity of tubulin tyrosine ligase were similar in PMN of CHS patients and normal donors and resembled that of other non-neuronal cells. The high levels of tyrosine incorporation in PMN of CHS patients were normalized by the administration of ascorbate, both in vitro and in in vivo experiments. In vitro addition of ascorbate also inhibited the FMLP-induced stimulation of tyrosine incorporation in both normal and CHS cells. Normalization of higher levels of tyrosine incorporation in PMN of CHS patients and the inhibition of FMLP-induced stimulation of tubulin tyrosinolation in normal and CHS cells as observed with ascorbate could also be affected by other reducing agents such as reduced glutathione, cysteine, or dithiothreitol. These results suggest a possible relationship between cellular redox and tubulin tyrosinolation in PMN.

In vivo analysis of the model tyrosine aminotransferase gene reveals multiple sequential steps in glucocorticoid receptor action.

We are studying the mechanisms of transcriptional activation by nuclear receptors and we focus our studies on the glucocorticoid regulation of the model tyrosine aminotransferase gene. Rather than using in vitro biochemical approaches, we determine the actual events occurring in the cells. Our experimental approaches include genomic footprinting, chromatin immunoprecipitation, in situ hybridization and transgenic mice. Our results show that the glucocorticoid receptor uses a dynamic multistep mechanism to recruit successively accessory DNA binding proteins that assist in the activation process. Chromatin is first remodelled, DNA is then demethylated, and the synthesis of an accessory factor is induced. Efficient transcription induction is finally achieved upon the formation of a 'stable' multiprotein complex interacting with the regulatory element. We discuss: the relative contribution of histone acetyltransferases and ATP-dependent remodelling machines to the chromatin remodelling event; the nature of the remodelled state; the contribution of regulated DNA demethylation to gene memory during development; the mechanisms of regulated DNA demethylation; the dynamics of protein recruitment at regulatory elements; the control of the frequency of transcription pulses and the control levels of the cell-type specificity of the glucocorticoid response.

A dynein ATPase inhibitor isolated from a commercial ATP preparation.

Preparations of ATP from equine muscle contained an inhibitor of dynein Mg2+-activated ATPase. The inhibitory material was separated from the ATP by molecular sieve filtration. The several molecular species of dynein extracted from three different axonemal sources were all inhibited; myosin ATPase was not. With increasing amounts of inhibitor the inhibition did not go to completion but reached a plateau when the rate had been reduced to 1/5 the uninhibited rate. A plot of 1/[S] against 1/v at several inhibitor concentrations yielded parallel lines. There was little inhibition of dynein ATPase when Mg2+ was replaced by Ca2+. The inhibitor appeared slightly smaller in molecular size than ATP, had anionic character, and was not adsorbed to charcoal.

Tubulin tyrosinolated in vivo can be different from that tyrosinolated in vitro.

Tubulin can be tyrosinolated, in the presence of ATP, by tubulin-tyrosine ligase, and tyrosine can be released by the same enzyme in the presence of ADP plus inorganic phosphate. There is however a 'non-substrate' component of tubulin which can not be tyrosinolated or detyrosinolated by this enzyme. Tubulin tyrosinolated in vivo was found to be the non-substrate species in HeLa cells, and the substrate species in cultured neuronal cells. In this respect HeLa tubulin resembled membrane-associated tubulin from brain, and neuronal cell tubulin resembled brain cytosolic tubulin.

115 kDa protein from Xenopus laevis embryos recognized by antibodies directed against the Xenopus homeoprotein XIHbox 1.

Using antibodies against homeoprotein XIHbox 1 from Xenopus laevis, we have detected a new embryonic protein with a much larger molecular weight, 115 kDa. Antibodies fractionated according to their affinity for 3 different domains of the XIHbox 1 protein were used to show that this new protein is related to the C-terminal region of XIHbox 1 protein, downstream from the homeodomain. By immunohistochemistry, the protein was shown to be localized in nuclei of embryonic cells. On SDS-polyacrylamide gels, the 115 kDa protein appears as a set of closely spaced bands whose pattern varies with the stage of development and with the parental origin of the embryos. The protein could be extracted from embryos in a multiprotein complex of approximately 600 kDa. In contrast, the 18 and 27 kDa proteins predicted from the sequence of cloned cDNA to be transcribed and translated from the XIHbox 1 gene could not be detected, suggesting that they are rare or unstable in embryos. These data suggest that the new protein is involved in the development of Xenopus embryos, with a function possibly related to that of the homeoprotein XIHbox 1.

Cloning and sequencing of mRNAs coding for two adult alpha globin chains of the salamander Pleurodeles waltlii.

A cDNA library of erythrocyte mRNAs was established from immature red blood cells of the adult amphibian, Pleurodeles waltlii (urodele; salamander). The cDNA clones corresponding to the four adult globin chains were first identified and characterized by positive selection and the cDNAs derived from the two (major and minor) alpha-globin chains sequenced. The sequences presented contain both the complete 3'-noncoding region and the coding region of both chains, with the exception of the first nine codons of the minor alpha-chain, and a portion of the 5'-noncoding region of the major chain. The amino acid sequences of the encoded alpha-globin polypeptides have been deduced and compared with those of Xenopus laevis and of man. These comparative studies suggest that the alpha-globins of Pleurodeles waltlii and Xenopus laevis may have diverged from a common ancestral gene at the time when mammalian and amphibian lines diverged, and that they then evolved separately. Duplication of the alpha-gene, which is responsible for the polypeptide heterogeneity, appears to have occurred earlier in Pleurodeles waltlii than in Xenopus laevis.

Sensitivity and resistance to (+)-calanolide A of wild-type and mutated forms of HIV-1 reverse transcriptase.

We have tested both wild-type and drug-resistant mutated, recombinant human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) molecules for sensitivity to each of two non-nucleoside RT inhibitors (NNRTI), (+)-calanolide A and nevirapine, in primer extension assays. We found that RT containing either the V106A or Y181C substitutions, associated with NNRTI resistance, displayed approximately 90-fold resistance to nevirapine but remained fully sensitive to (+)-calanolide A and that the Y181C mutation marginally enhanced susceptibility to the latter drug. In contrast, the Y188H substitution in RT resulted in about 30-fold resistance to (+)-calanolide A in these assays but did not result in diminished sensitivity to nevirapine. Tissue culture results indicated that the combination of (+)-calanolide A and nevirapine possessed an additive to weakly synergistic effect in blocking replication of HIV-1 in tissue culture. These results suggest that (+)-calanolide A and nevirapine might have rationale as a combination therapy for HIV disease.

Molecular modification of anticholinergics as probes for muscarinic receptors. 3. Conformationally restricted analogues of benactyzine.

The synthesis and pharmacological evaluation of conformationally restricted analogues of certain anticholinergic agents is a powerful method for probing the topography of the muscarinic receptor. In the present study, clues as to the binding conformation of structurally flexible anticholinergics are provided by approximating certain conformations of benactyzine by synthetic analogues 1-6, which are structurally locked into desired conformations. The pharmacological activity of each analogue is an indication of how well particular conformational models are accommodated by the receptor. The conformation of benactyzine in which an intramolecular hydrogen bond may exist between the hydroxyl group and the carbonyl oxygen of the ester group (conformation I) is approximated by the synthetic analogue 2,2-diphenyl-3-tetrahydrofuranyl (diethylamino)ethyl ether (1) and related analogues. Pharmacological evaluation using dose-response experiments on isolated rat ileum tissue demonstrated that these compounds noncompetitively inhibited acetylcholine-induced ileum contractions. Restriction of the conformational freedom of the amino side chain of 1 by synthesis of the hexahydro[3,4-b]furan derivative 3 provided a weak but competitive inhibitor at low concentration. The conformation of benactyzine in which an intramolecular hydrogen bond may exist between the hydroxyl group and the ether oxygen of the ester group (conformation II) is approximated by 2,2-diphenyl-4-[2-(diethylamino)ethyl]-3-tetrahydrofuranone (4). Pharmacological studies showed that this compound competitively inhibited acetylcholine-induced ileum contractions. These experiments provide evidence that receptor-bound conformation II for benactyzine is preferred over conformation I in providing competitive binding with the muscarinic receptor.

Structural analogues of the calanolide anti-HIV agents. Modification of the trans-10,11-dimethyldihydropyran-12-ol ring (ring C).

(+)-Calanolide A is a potent inhibitor of reverse transcriptase from human immunodeficiency virus type 1 (HIV-1), which was isolated from an extract of Calophyllum lanigerum, along with seven related compounds. In order to examine the structure-activity relationships of the trans-10,11-dimethyldihydropyran-12-ol ring (designated ring C), a series of structural analogues were prepared and evaluated using a whole cell cytopathicity assay (XTT). Removal of the 10-methyl group resulted in decreased activity, with only one epimer exhibiting anti-HIV activity. Substituting the 10-methyl group with an ethyl chain maintained anti-HIV activity, with only a 4-fold reduction in potency relative to racemic calanolide A. Substitution of the 10-methyl group with an isopropyl moiety completely eliminated the anti-HIV activity. Addition of an extra methyl group at either the 10- or 11-position maintained the basic stereochemical features of the parent calanolide system while removing the chirality at the respective carbon, but resulted in decreased activity relative to calanolide A. In all the above examples, analogues containing a cis relationship between the 10- and 11-alkyl moieties were completely devoid of activity. Synthetic intermediates in which the 12-hydroxyl group was in the ketone oxidation state exhibited suppressing anti-HIV activity, with EC50 values only 5-fold less potent than that of calanolide A for both the 10,11-cis (6) and -trans (5) series. These ketones represent the first derivatives in the calanolide series to exhibit anti-HIV activity while not containing a 12-hydroxyl group. Likewise, ketone derivative 6 was the first example of a compound in the calanolide series having a cis relationship between the 10- and 11-methyl groups found to exhibit anti-HIV activity. Analogues which showed anti-HIV activity in the CEM-SS cytoprotection assay were further confirmed to be inhibitors of HIV-1 reverse transcriptase.

Synthesis, chromatographic resolution, and anti-human immunodeficiency virus activity of (+/-)-calanolide A and its enantiomers.

The anti-HIV agent (+/-)-calanolide A (1) has been synthesized in a five-step approach starting with phloroglucinol [-->5-->6-->11-->18-->(+/-)-1], which includes Pechmann reaction, Friedel-Crafts acylation, chromenylation with 4,4-dimethoxy-2-methylbutan-2-ol, cyclization, and Luche reduction. Cyclization of chromene 11 to chromanone 18 was achieved by employing either acetaldehyde diethyl acetal or paraldehyde in the presence of trifluoroacetic acid and pyridine or PPTS. Luche reduction of chromanone 18 at lower temperature preferably yielded (+/-)-1. Reduction of chromone 12, synthesized by Kostanecki-Robinson reaction from chromene 11, failed to afford (+/-)-1. The synthetic (+/-)-1 has been chromatographically resolved into its optically active forms, (+)- and (-)-1. The anti-HIV activities for synthetic (+/-)-1, as well as resultant (+)- and (-)-1, have been determined. Only (+)-1 accounted for anti-HIV activity, which was similar to the data reported for the natural product, and (-)-1 was inactive.

Soluble macrophage factors trigger apoptosis in cultured hippocampal neurons.

It is not clear whether macrophages which can phagocytose dead cells, may also contribute to death of potentially viable neurons when they enter brain lesion sites after insult. We have initially examined the effects of macrophage-conditioned medium on the integrity of hippocampal neurons in culture. We assessed qualitative and quantitative changes in neuronal status in terms of nuclear morphology, internucleosomal cleavage, cell membrane integrity and process density. Cell morphology with manual counts to quantitate findings showed that macrophage conditioned medium significantly increased the percentage of neurons with abnormal nuclei. Aurintricarboxylic acid attenuated this effect. Demonstration of laddering of DNA on agarose gels suggested an apoptosis-like event. A commercially available kit used to detect high concentrations of 3'-OH DNA ends showed marked increase in labelled cells. These combined findings confirmed that apoptosis was the main event triggered by conditioned medium. Although the number of cells with incompetent membranes also increased with conditioned medium application the majority of cells with apoptotic nuclei maintained membrane integrity. Conditioned medium also resulted in significant loss of cell processes. Conditioned medium from stimulated microglia showed a similar pattern of injury. The response of stressed neurons to conditioned medium was also tested. Exposure of cultures to mild hypoxia resulted in injury but did not significantly alter their subsequent vulnerability to macrophage-conditioned medium. Early experiments suggest that the documented changes in neuronal status are caused by relatively large and stable secreted macrophage proteins.

Robustaflavone, a potential non-nucleoside anti-hepatitis B agent.

Robustaflavone, a naturally occurring biflavanoid isolated from Rhus succedanea, was found to be a potent inhibitor of hepatitis B virus (HBV) replication in 2.2.15 cells, with an effective concentration (EC50) of 0.25 microM, and a selectivity index (SI, IC50/EC90) of 153. Robustaflavone hexaacetate inhibited HBV replication with an EC50 of 0.73 microM, but exhibited no cytotoxicity at concentrations up to 1000 microM. Combinations of robustaflavone with penciclovir and lamivudine displayed synergistic anti-HBV activity, having the most pronounced effects when the combination ratios were similar to the ratio of EC50 potencies. Thus, a 1:1 combination of robustaflavone and penciclovir exhibited an EC50 of 0.11 microM and an SI of 684, while a 10:1 combination of robustaflavone and lamivudine exhibited an EC50 of 0.054 microM and an SI of 894. Statistical analyses of the combination data using the Combostat program confirmed that robustaflavone exhibited synergism with both penciclovir and lamivudine.

Amyloid fibril formation in microwell plates for screening of inhibitors.

Fibril formation is the basis of amyloid production in a number of disease states, such as Alzheimer's disease, diabetes and immunocytic dyscrasias. Compounds that inhibit fibril formation could be directly relevant to the treatment of amyloid diseases, and may also provide a foundation for the development of interventions in other molecular condensation diseases ranging from sickle cell anemia to atherosclerosis. We developed an economical and convenient high-throughput method for screening compounds against fibril formation in microwell plates. Chalcones, flavonoids and biflavonoids were screened against fibril formation by a recombinant antibody variable domain (V1). Chalcones 6 and 14 were found to demonstrate inhibition at 0.1 microM in 79 microM of protein solution in both test tube and microwell plate assays. The concentration of protein in the microwell plate assay could be as low as 5 microM using ThT as a monitoring agent. Molecular modeling studies indicated that both compounds could be individually docked into a binding site at the monomer-monomer interface of the V(L) protein dimer. These studies suggested that these compounds could potentially stabilize the VL dimer and therefore reduce its tendency to form fibrils. These findings may provide the basis for a new therapeutic approach to prevent or treat amyloid diseases.

Multiple sequence-specific single-strand-binding proteins for the promoter region of the rat albumin gene.

Previous work from our laboratory described a protein that binds to single-stranded DNA in the early promoter of simian virus 40 in a sequence specific fashion. We have now used the gel retardation assay to search for similar sequence-specific single-strand-binding proteins for the promoter region of the rat albumin gene in nuclear extracts of rat hepatoma cells. Several proteins of this kind were detected, three of which are described in the present paper. Two of them bind specifically to the noncoding strand and the third one binds to the coding strand. The most abundant of these proteins binds to a pyrimidine stretch inside the coding region of the gene and appears to be homologous to the previously observed SV40-binding protein. Possible functions for sequence-specific single-strand-binding proteins in transcription are discussed.

The effect of hypoxia on neurotransmitter phenotype of forebrain cholinergic neurons.

The effect of hypoxia on the neurotransmitter phenotype of rat forebrain cholinergic neurons was analyzed using a dissociated fetal rat culture system. The aims of this study were to examine the feasibility of using choline acetyltransferase (ChAT) activity as a measure of cell injury and/or recovery, to measure the time course of hypoxic effects on ChAT activity, to determine how changes in ChAT activity at 48 h post-injury relate to microscopic changes and LDH release into the medium during that time, and finally to explore the possible mechanisms of hypoxic injury in this model. At exposure to 0.5-1.5% O2 there was a time-dependent decrease in ChAT activity when cells were harvested 48 h after exposure. Forty-eight hours after 8-9 h hypoxic exposure ChAT activity was 50-60% that of controls without any alteration in morphology of neurons. An 8 h exposure to hypoxic conditions caused a post-exposure time-dependent decrease in ChAT activity to 20% of control level at 72 h. Thereafter there was spontaneous recovery of phenotype to 60% of control which remained stable between 5 and 7 days post-exposure. Loss of neurotransmitter phenotype was not well correlated with other measures of cytotoxicity including morphological changes and LDH release. The loss of phenotype observed with hypoxia was mimicked by glutamate and kainate but not by NMDA. Consistent with these observations, neither APV nor AP3 significantly altered the effect of hypoxia on forebrain cholinergic neurons, while the addition of APV and CNQX in combination protected the phenotype of these neurons only if there was 50% or less loss of phenotype following hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential sensitivity of rat hippocampal and cortical astrocytes to oxygen-glucose deprivation injury.

Selective vulnerability of hippocampal neurons to ischemia is felt to relate to intense glutamatergic input and glutamate receptor expression. Since astrocytes are thought to have a neuroprotective role we speculated that hippocampal astrocyte sensitivity to insult could also contribute to this regional vulnerability. The purpose of the study was to determine if there is a differential sensitivity of cultured hippocampal and cortical astrocytes to oxygen-glucose deprivation (OGD). Hippocampal and cortical astrocytes were grown to confluence at matching cell density. Cultures were exposed to OGD for 2, 4, 6, 8 and 10 h. Progressive reduction in viability occurred in hippocampal astrocytes beginning at 2 h OGD. Reduction in cortical astrocyte viability was not observed until 4 h OGD. Death of hippocampal astrocytes was significantly greater than that of cortical astrocytes at each period of OGD. Based on acid phosphatase activity data the LD(50) for OGD duration in hippocampal astrocytes was 2 h compared to 8 h in cortical astrocytes. Regional differences in sensitivity of astrocytes to OGD implies that this may contribute to regional differences in neuronal vulnerability to ischemia.

Hypoxic forebrain cholinergic neuron injury: role of glucose, excitatory amino acid receptors and nitric oxide.

Glucose depletion increased sensitivity to hypoxic insult in basal forebrain forebrain cultures in a dose-dependent manner as indicated by reduction of choline acetyltransferase (ChAT) activity, increased lactate dehydrogenase (LDH) release and disrupted morphology. The glutamate receptor antagonists 2-amino-5-phosphonovaleric acid (APV) and 6-cyano-2,3-nitroquinoxoline (CNQX) limited the degree of injury in combination and individually. The nitric oxide synthase (NOS) inhibitor N-nitro-L-arginine (NNLA) also either completely protected against mild injury or attenuated severe injury.