Search

Sensitization to cockroach allergen is one of the strongest predictors of asthma morbidity, especially among African Americans.Our aims were to determine the genomic basis of cockroach sensitization and the specific response to cockroach antigen.We investigated the Th1/Th2 cytokine profile of co-cultured plasmacytoid dendritic cells (pDCs) and CD4+ T cells and the 'transcript signature' of the immune response to cockroach antigen using high-throughput expression profiling of co-cultured cells.We observed significantly elevated levels of IL-13, IL-10, and TNF-alpha, but undetectable levels of IL-12p70 and IFN-alpha, when cultures were exposed to crude cockroach antigen. A significant difference was observed for IL-13 between cockroach-allergic and non-allergic individuals (P=0.039). Microarray analyses demonstrated a greater response at 48 h compared with 4 h, with 50 genes being uniquely expressed in cockroach antigen-treated cells, including CD14, S100A8, CCL8, and IFI44L. The increased CD14 expression was further observed in purified pDCs, human monocytic THP-1 cells, and the supernatant of co-cultured pDCs and CD4+ T cells on exposure to cockroach extract. Furthermore, the most differential expression of CD14 between cockroach allergy and non-cockroach allergy was only observed among individuals with the CC 'high-risk' genotype of the CD14-260C/T. Ingenuity Pathways Analysis analyses suggested the IFN signalling as the most significant canonical pathway.Our results suggest that these differentially expressed genes, particularly CD14, and genes in the IFN signalling pathway may be important candidates for further investigation of their role in the immune response to cockroach allergen.

In higher eukaryotes, the 5S ribosomal DNA (5S rDNA) is organized in tandem arrays with repeat units composed of a coding region and a non-transcribed spacer sequence (NTS). These tandem arrays can be found on either one or more chromosome pairs. 5S rDNA copies from the tilapia fish, Oreochromis niloticus, were cloned and the nucleotide sequences of the coding region and of the non-transcribed spacer were determined. Moreover, the genomic organization of the 5S rDNA tandem repeats was investigated by fluorescence IN SITU hybridization (FISH) and Southern blot hybridization. Two 5S rDNA classes, one consisting of 1.4-kb repeats and another one with 0.5-kb repeats were identified and designated 5S rDNA type I and type II, respectively. An inverted 5S rRNA gene and a 5S rRNA putative pseudogene were also identified inside the tandem repeats of 5S rDNA type I. FISH permitted the visualization of the 5S rRNA genes at three chromosome loci, one of them consisting of arrays of the 5S rDNA type I, and the two others corresponding to arrays of the 5S rDNA type II. The two classes of the 5S rDNA, the presence of pseudogenes, and the inverted genes observed in the O. niloticus genome might be a consequence of the intense dynamics of the evolution of these tandem repeat elements.

There are few reports on the genomic organization of 5S rDNA in fish species. To characterize the 5S rDNA nucleotide sequence and chromosomal localization in the Neotropical fishes of the genus Brycon, 5S rDNA copies from seven species were generated by PCR. The nucleotide sequences of the coding region (5S rRNA gene) and the nontranscribed spacer (NTS) were determined, revealing that the 5S rRNA genes were highly conserved, while the NTSs were widely variable among the species analyzed. Moreover, two classes of NTS were detected in each species, characterized by base substitutions and insertions-deletions. Using fluorescence in situ hybridization (FISH), two 5S rDNA chromosome loci that could be related to the two 5S rDNA NTS classes were observed in at least one of the species studied. 5S rDNA sequencing and chromosomal localization permitted the characterization of Brycon spp. and suggest a higher similarity among some of them. The data obtained indicate that the 5S rDNA can be an useful genetic marker for species identification and evolutionary studies.

We demonstrate that the homozygous weaver granule neurons cultured on a laminin substratum fail to express inwardly rectifying potassium currents, including a functional G-protein coupled inwardly rectifying potassium (GIRK)2 potassium channel. By contrast, both normal and weaver Purkinje cells express inwardly rectifying potassium currents, and normal granule cells exhibit inwardly rectifying potassium currents inducible with GTP-gamma-S. In protein extracts of the vermal postnatal day (P)5-9 weaver cerebellum, the GIRK2 protein could not be detected by Western analysis, although the GIRK2 protein was detectable in extracts of the normal vermis. Northern analysis indicated that during early postnatal cerebellar development, the GIRK2 mRNA is expressed at extremely low levels being detectable at P18-23 in the normal but not yet in the homozygous weaver cerebellum. Using reverse transcriptase-polymerase chain reaction (RT-PCR), the GIRK2 mRNA was detected in both normal and weaver cerebella, but quantitative PCR confirmed that the weaver cerebellum expressed the GIRK2 gene at significantly lower levels as compared to the normal cerebellum (P = 0.01, paired t-test). Sequencing indicated that the weaver GIRK2 channel gene had the point mutation proposed to be responsible for the weaver phenotype. Rescue of both survival and neurite outgrowth of the cultured vermal weaver granule neurons by verapamil (Liesi and Wright, 1996; Liesi et al., 1999) induced expression of immunocytochemically detectable levels of the GIRK2 protein. Sequencing revealed that the GIRK2 mRNA of the rescued weaver granule neurons remained the mutated variant of the GIRK2 channel gene. Our results indicate that expression of the mutated GIRK2 protein and/or mRNA in the weaver granule neurons may be an indicator of rescue rather than death of the weaver granule neurons. That the weaver granule neurons expressed no functional GIRK2 receptors during a time period of neuronal death and migration failure suggests that the point mutation in the H5 membrane spanning region of the GIRK2 gene may associate with, but not be responsible for the weaver phenotype.

Isoniazid (INH), a widely used drug in the prophylaxis and treatment of tuberculosis, is associated with a 1 to 2% risk of severe and potentially fatal hepatotoxicity. There is evidence that the INH metabolite hydrazine plays an important role in the mechanism of this toxicity. Metabolism of INH leads to the production of hydrazine via both direct and indirect pathways. In both cases, the activity of an INH amidase is required to hydrolyze an amide bond. In the present study, using a model of INH-induced hepatotoxicity in rabbits, pretreatment of rabbits with the amidase inhibitor bis-p-nitrophenyl phosphate 30 min before injection of INH inhibited the formation of INH-derived hydrazine and decreased measures of hepatocellular damage, hepatic triglyceride accumulation, and hypertriglyceridemia. Bis-p-nitrophenyl phosphate also potently inhibited the production of hydrazine from INH in in vitro microsomal incubations (IC50 2 microM). Although hepatic glutathione stores are decreased, they are not depleted in animals with INH-induced hepatotoxicity. Significant effects on hepatic microsomal cytochrome P-450 1A1/2 and cytochrome P-450 2E1 activities suggest that these isozymes may be involved in the mechanism of the toxicity. In conclusion, this study demonstrates the importance of amidase activity in this rabbit model of hepatotoxicity and provides additional evidence in support of the role of hydrazine in the mechanism of INH-induced hepatotoxicity.

Biochemical, immunocytochemical, and molecular biological techniques were used to investigate the expression of N-methyl-D-aspartate (NMDA) receptor subunits in migration-deficient weaver mouse cerebellum in vivo and in primary cultures of the vermal weaver granule neurons with or without a rescue by verapamil. We found that both NMDAR1(zeta1) message and protein were expressed by the weaver granule neurons in situ. Immunocytochemical and biochemical analyses indicated that granule neurons of the weaver cerebellum expressed R1(zeta1) and R2A(epsilon1) subunits but showed little expression of the R2B(epsilon2) subunit. In weaver cerebellum, the R2B(epsilon2) subunit was primarily expressed in nerve fibers of the internal granule cell layer and white matter. Reverse-transcriptase-polymerase chain reaction followed by sequence analysis of the R1(zeta1) subunit indicated that the zeta1 subunit amplicons of both normal and weaver cerebella were identical, and that splice variants with exon 22 (1-2) and with or without exon 5 (a/b) or exon 21 (1-4) were detectable. The R2A(epsilon1), and R2B(epsilon2) subunits of the normal and weaver mouse cerebellum revealed no primary structural differences between the normal and weaver NMDA receptor subunits or the cloned mouse NMDA receptor subunits. In vermal cultures, normal granule neurons expressed all three NMDA receptor subunits (zeta1, epsilon1, and epsilon2), whereas the weaver neurons failed to express the epsilon2 subunit. Rescue of the weaver neurons by verapamil induced expression of the epsilon2 protein along the granule neuronal surfaces. The present results suggest that lack of the epsilon2 subunit in the weaver cerebellum may relate to the lack of functional NMDA receptors and/or to the migratory failure of the weaver granule neurons. Our data further suggest that NMDA receptor-mediated neurotoxicity is an unlikely mediator of neuronal death of the weaver granule neurons. In fact, down-regulation of the NMDA receptor expression and function may be a protective measure of the weaver granule neurons to reduce calcium entry via these receptors.

The structure of the heterochromatic bands in mitotic chromosomes of the important tropical aquaculture species of tilapia, Oreochromis niloticus, was investigated by the combination of the C-banding technique, chromosomal digestion with two restriction endonucleases and fluorescence in situ hybridization (FISH) of two satellite DNAs (SATA and SATB). The tilapia chromosomes presented heterochromatic bands in the centromeres and in the short arms of almost all chromosomes that were differentially digested by the restriction endonucleases HaeIII and EcoRI. FISH of SATA showed that this satellite sequence is distributed in the centromeric region of all chromosomes of tilapia. FISH also revealed an intense hybridization signal for SATB in only one chromosome pair, but less intense signals were also present in several other pairs. The digestion of tilapia chromosomes by HaeIII and EcoRI was positively correlated with the position of SATA and SATB in chromosomes as revealed by FISH. The results obtained may be useful in future molecular and genetic studies of tilapias.

A SINE-like repetitive element (ROn-1) has been cloned from the tilapiine cichlid fish Oreochromis niloticus. The element is 345 base pairs (bp) in length and consists of a transfer-RNA-like domain with putative RNA polymerase III recognition sequences, a tRNA-unrelated region, and a poly(A) tail. Approximately 6000 copies of ROn-1 occur in the haploid genome of O. niloticus. Southern blot analysis revealed that ROn-1 is an abundant element in the genomes of many African cichlid fishes, but absent from the genome of the Indian cichlid Etroplus.

Three species of redfish (Sebastes) share a common pattern of mitochondrial DNA tandem repeat polymorphism and heteroplasmy in the northwest Atlantic Ocean. All three species exhibit 9-17 copies of an approximately 275 base pair (bp) tandem repeat situated within the 3' domain of the control region. Sequence analysis of cloned mtDNA from S. mentella revealed that the tandem array is adjacent to the tRNA(phe) gene, and that the repeat shares 53% identity with the tRNA(phe) gene and part of the 12S rRNA gene. These features, as well as potential secondary structure assumed by the repeat, are consistent with previously proposed models explaining tandem duplications in the 3' end of the control region. In a sample comprising 36 S. fasciatus, 52 S. mentella, and 13 S. marinus taken near Newfoundland, neither the mean number of repeats per fish (12.2-12.7) nor the frequency of heteroplasmy varied significantly among species. A total of 42% of the redfishes were heteroplasmic, bearing either two or three repeat variants (33% and 9%, respectively). The similarity of the frequency distributions of tandem repeat variants in the three species suggests either a common balance between mutation and selection in the three species, or mitochondrial gene flow between them.

Isoniazid, a highly effective drug for the chemoprophylaxis and treatment of tuberculosis, is associated with severe hepatotoxicity in 1-2% of individuals. In a rabbit model of isoniazid-induced hepatotoxicity, we have measured hepatic necrosis (quantitated by elevation of plasma argininosuccinic acid lyase (ASAL) activity), hepatic steatosis (quantitated by elevation of hepatic triglyceride content), and elevation in plasma triglyceride concentration in 15 rabbits. Eight of 15 rabbits were male, and 14 of 15 were rapid acetylators of sulfamethazine. Administration of isoniazid to rabbits resulted in a 27-fold increase in plasma ASAL activities, a 7.5-fold increase in hepatic triglyceride content, and a 13-fold increase in plasma triglyceride levels. This study demonstrated no effect of gender on these three pathological changes that occur in this model of isoniazid-induced hepatotoxicity in rabbits.

The mechanisms by which the weaver gene (Reeves et al., 1989; Patil et al., 1995) inhibits neurite extension and/or induces death of the granule neurons in homozygous weaver mouse cerebellum are not presently understood. Here we show that BAPTA-AM and ethanol, which either reduce cytosolic levels of free calcium or prevent calcium entry, promote neurite outgrowth of the weaver neurons similar to the L-type calcium channel blocker verapamil (Liesi and Wright, 1996). Importantly, BAPTA-AM, ethanol, and verapamil not only restore neurite outgrowth of the weaver neurons but adjust their depolarized resting membrane potentials to the levels of normal neurons. These results indicate that calcium-dependent mechanisms mediate the action of the weaver gene and that the weaver neurons can be normalized by blocking this calcium effect. We further report that BAPTA-AM and verapamil also have a neuroprotective effect on normal neurons exposed to high concentrations of ethanol. We suggest that verapamil should be evaluated as a drug for treatment of alcohol-induced brain damage and neurodegenerative disorders.

To use lipopolysaccharide (LPS) to create synovitis in the midcarpal joint of ponies, and to assess the morphologic, histochemical, and immunohistochemical effects of synovitis on articular cartilage of the third carpal bone.2- to 3-year-old ponies, 6 control (group 1) and 6 treated (group 2).Synovitis was induced in 1 midcarpal joint of group-2 ponies by intra-articular injections of LPS (0.02 micrograms/kg of body weight), morphine (0.1 mg/kg), and saline solution (group 2a) and a morphine and saline solution alone in the contralateral midcarpal joint (group 2b). Articular cartilage sections and attached synovial membrane from the third carpal bones were examined by immunohistochemical distribution of interleukin 1 beta, tumor necrosis factor (TNF)-alpha, TNF receptors (P55, P75) and 3-B-3(-) epitopes, and by localization of proteoglycans (metachromatic staining). Proteoglycan extracts were assessed by metachromatic staining or western blotting and immunohistochemical staining, using anti-3-B- antibodies.Enhanced immunoreactivity for the cytokines and receptors was found in inflamed synovial membrane and noncalcified cartilage (group 2a more than 2b). Metachromasia of the noncalcified cartilage was greater in group-1 than in group-2a and group-2b specimens. In group 2a, chondrocyte hypertrophy and enhanced immunoreactivity for 3-B-3(-) epitope in areas of increased cytokine immunoreactivity suggested possible phenotypic change of the chondrocytes in response to synovitis. Immunohistochemical analysis by western blotting of proteoglycan extracts indicated strong 3-B-3(-) epitope immunolocalization in group-2a, weaker staining in group-2b, and barely detectable stain in group-1 specimens, which correlated with in situ immunolocalization.Intra-articular administration of LPS may be used to induce a synovial environment conductive to increased immunoreactivity of interleukin 1 beta, TNF-alpha, and its receptors in equine synovial membrane and articular cartilage. These cytokines may be involved in the early phenotypic change of chondrocytes that is believed to occur in osteoarthritis and is characterized in this study by enhanced 3-B-3(-) epitope immunoreactivity and chondrocyte hypertrophy.

We have amplified, by the polymerase chain reaction, and have sequenced the D-loop region of the mitochondrial DNA from the sperm whale (Physeter macrocephalus). The sperm whale D-loop was aligned with D-loop sequences from four other cetaceans (Commerson's dolphin, orca, fin whale, and minke whale) and an out-group (cow). This alignment showed the sperm whale sequence to be larger than that of other cetaceans. In addition, some sequence blocks were highly conserved among all six species, suggesting roles in the functioning of mitochondrial DNA. Other blocks that were previously reported to be well conserved among cetaceans showed little sequence conservation with the sperm whale D-loop, which argues against the functional importance of these sequence blocks in cetaceans.

The effects of ethanol on excitatory amino acid activated ion channels were investigated using patch-clamp recording methods. Intoxicating concentrations of ethanol (5-50 mM) inhibited ion current activated by the glutamate receptor agonist N-methyl-D-aspartate (NMDA) in a concentration-dependent manner (IC50 = 30 mM). The intoxicating potency of different alcohols was correlated with their potency for inhibiting NMDA-activated current, suggesting that alcohol-induced inhibition of NMDA channel function may contribute to the neural and cognitive impairments associated with intoxication. Analysis of mechanism suggests that ethanol inhibits NMDA-activated current by altering gating of the channel, rather than by affecting channel conductance, ion permeance or regulatory sites on the channel. Anesthetic concentrations of ethanol (50 mM) produced a concentration-dependent inhibition of kainate- and quisqualate-activated currents (IC50 = 220 mM), suggesting that this inhibition may contribute to the general anesthetic effects of ethanol. This hypothesis is supported by the observations that the general anesthetic agents, trichloroethanol (the active metabolite of chloral hydrate), pentobarbital and halothane, all inhibit kainate- and quisqualate-activated currents.

Bicuculline methiodide (BIC-Mel) (10-100 microM) altered the kinetics of N-methyl-D-aspartate (NMDA) responses in single-channel and whole-cell recordings. The principal effect of BIC-Mel (10-100 microM) on NMDA channels was a dose-dependent decrease in mean channel open time (tau o), accompanied by the introduction of a new closed time (tau B) of 14.0 +/- 3.5 msec (mean +/- standard deviation; n = 14) in closed time distributions, which was independent of BIC-Mel concentration. BIC-Mel (10-100 microM) increased the frequency of NMDA channel opening in a dose-dependent manner, offsetting the decrease in tau o, such that the total time spent in the open state per minute was unchanged, and thus the total charge/min through NMDA channels was unchanged. Similarly, the amplitudes of NMDA whole-cell current responses were not noticeably affected by 10-80 microM BIC-Mel, even though power spectra density analysis of the whole-cell NMDA-stimulated noise revealed changes in the underlying channel kinetics in the presence of BIC-Mel. Taken together, the effects of 10-80 microM BIC-Mel on NMDA responses were consistent with the predictions of the sequential block model; however, the effects of BIC-Mel exhibited no obvious voltage dependence. In addition to the low-dose effects of BIC-Mel, 100 and 200 microM BIC-Mel inhibited whole-cell NMDA responses. The inhibition by 100 microM BIC-Mel was not large, but it was augmented from 15% to 30% by increasing the NMDA concentration from 10 microM NMDA to 20 microM NMDA, indicating that channel activation was necessary for BIC-Mel-mediated inhibition. Preliminary single-channel experiments performed under conditions conducive to trapping of an open channel blocker at its binding site indicated that the effect of BIC-Mel on tau o persisted after the removal of the blocker, consistent with use dependence of the dissociation of BIC-Mel from the NMDA receptor-channel complex.

1. The mechanisms of tetraethylammonium (TEA) antagonism of N-methyl-D-aspartate (NMDA) responses were investigated in cultured mouse cortical neurons by analysing single-channel and whole-cell currents from patch clamp recordings. TEA (1-5 mM) decreased whole-cell NMDA responses. Kainate and quisqualate receptor-mediated responses were unaffected at these TEA concentrations. 2. In whole-cell recordings, increasing the NMDA concentration while keeping the TEA concentration constant resulted in greater inhibition by TEA. Thus, TEA-mediated inhibition of NMDA responses was not due to competitive antagonism, and the greater inhibition by a single dose of TEA as NMDA concentration was elevated indicated some form of non-competitive inhibition. In single-channel recordings, two inhibitory effects were seen in 1-5 mM-extracellular TEA: single-channel conductance (gamma) was decreased, and the frequency of channel events was decreased. These effects were not accompanied by any change in average channel open time. 3. Single-channel current-voltage (I-V) curves obtained in 2, 5, 10 and 30 mM-TEA indicated the decrease in NMDA channel conductance was voltage dependent with larger reduction occurring as patches were hyperpolarized. The data were well fitted by the Woodhull model with the dissociation constant (KD) showing an e-fold increase in inhibition for a 43-45 mV change in membrane potential. The 0 mV KD was 45 mM-TEA decreasing to about 11 mM at -60 mV. The TEA block site appeared to sense approximately 60% of the transmembrane potential field (delta = 0.6) for extracellular application of TEA. 4. The decrease in channel opening frequency seen in TEA was concentration dependent and generally more sensitive to extracellular TEA than the channel block effect. There was a 50% reduction in the number of NMDA channel openings observed in 5 mM-TEA. Increasing either NMDA or glycine concentrations in constant TEA concentration caused an additional decrease in the frequency of NMDA channel opening. In contrast to extracellular TEA, intracellular TEA had no noticeable effect on open-state probability. 5. NMDA single-channel currents were observed at positive potentials after completely replacing pipette Cs+ by 140 mM-TEA-Cl indicating TEA could serve as a current carrier through NMDA channels. Single channel I-V curves obtained with pipettes containing 70 or 140 nM-TEA in place of equivalent amounts of Cs+ were fitted by the Goldman-Hodgkin-Katz (GHK) equation over the range of -80 to +70 mV assuming a permeability of 0.45 compared with a Cs+ permeability of 1.0.(ABSTRACT TRUNCATED AT 400 WORDS)

The terminal changes following Borrelia duttoni infection in mice consisted of disseminated intravascular coagulation with associated depression in temperature, and in white cell and platelet counts with the development of a positive limulus lysate test. Apart from thrombosis and haemorrhage, similar changes were found in mice given cyclophosphamide prior to infection. The results suggest that neither intravascular coagulation nor immune complex formation are major factors in causing death in borreliosis.

The site of neuromuscular blockade induced by clindamycin and lincomycin was studied on isolated nerve and nerve–muscle preparations. Clindamycin (3.6 × 10−3 M) but not lincomycin (up to 1.5 × 10−2 M) had a local anaesthetic effect on a frog desheathed nerve preparation. Clindamycin (8 × 10−4 M) and lincomycin (4 × 10−3 M) depressed the response of the rat diaphragm to nerve stimulation and to direct muscle stimulation in parallel. This indicated that the predominant neuromuscular blocking effect of these antibiotics was due to an effect on the muscle. Clindamycin was fivefold more potent than lincomycin in this effect, and the unionized form of both drugs was the active form. Lincomycin (4 × 10−3 M) but not clindamycin (8 × 10−4 M) also had some depressant effect on nerve–muscle transmission as indicated by the interaction of the effects of the antibiotics and d-tubocurarine. The significance of these findings is discussed in relation to the acute clinical toxicity of these antibiotics.

Atropine is known to increase the release of acetylcholine (ACh) from cerebral cortex, and the present experiments tested the effect of this drug upon ACh release in the superior cervical ganglion of the cat. The release of ACh was measured by a radio-enzymic method, which was shown to provide an estimate of the ACh content of samples collected from perfused ganglia that was similar (102%) to that obtained by the method of bioassay more usually used. Atropine (3 × 10−6 M) increased (3.5- to 4-fold) the amount of ACh released by rat's sliced cerebral cortex incubated in a high (23 mM) potassium medium. However atropine (3 × 10−6–3 × 10−5 M) did not change the amount of ACh released by ganglia during preganglionic nerve stimulation (5–10 Hz). It is concluded that cholinergic nerve terminals in different tissues appear to have different pharmacological properties.

We examined the effects of simian virus 40 infection on the temporal and spatial organization of initiation sites for DNA replication in Muntjac cells by means of light microscopic DNA fiber autoradiography. Initiation at multiple sites along the DNA fiber in virus-infected confluent Muntjac cells was more nearly synchronous than in serum-deprived controls, although temporal control in the infected cells did not reach the level observed in cells incubated in serum-enriched medium. Initiation sites in virus-infected cells appeared to be spatially closer together than in either uninfected serum-deprived or uninfected serum-enriched cells. This change did not appear to be the result of the induction or repression by simian virus 40 of clusters of replication units with new and different organizations.

Guinea pigs with anteroventral third ventricle region (AV3V) lesions fail to develop fever and the associated rise in acute-phase plasma protein levels following systemic injections of lipopolysaccharide (LPS). Since endogenous pyrogen (EP) injected directly into the preoptic area of animals with AV3V lesions causes appropriate elevations in core temperature (Tco) and acute-phase plasma proteins levels, the blocked responses to LPS probably are not due to damage to the adjacent preoptic area. We proposed, therefore, that EP may pass from blood into brain in the AV3V, presumably through the organum vasculosum laminae terminalis. However, the possibility that a more generalized impairment due to damaged pathways within the AV3V could account for the observed effects was not examined. To investigate this possibility, guinea pigs were given AV3V lesions. Pending histological verification of the ablated sites, AV3V lesions were presumed to be placed correctly if the animals did not develop fever following LPS (Salmonella enteritidis, 2 μg/kg i.p., at ambient temperature (Ta) 22 °C); those failing to meet this criterion were designated as sham-operated. Two experiments were conducted. In the first, metabolic rates, Tco, and two skin temperatures (Tsk) were measured at Ta 12°, 22°, and 32 °C over an 8-month postlesion period during which failure to fever persisted; the data were collected during a 30-min period after thermal balance had been achieved at any given Ta. There were no differences in the variables measured between sham-operated and AV3V-lesioned animals at Ta 22 °C. At Ta 12° and 32 °C, the Tsk of both the sham-operated and the AV3V-lesioned guinea pigs were reduced and elevated, and metabolic rate increased and decreased, respectively. However, Tco at Ta 32 °C and metabolic rate at Ta 12° and 32 °C were significantly higher in the AV3V-lesioned than in the sham-operated animals. In the second experiment, only Tco was measured, but under more severe ambient conditions and over a briefer duration. Thus, 11 and 14 days postlesion, the animals were exposed to Ta 35.5 ± 1.8 °C (means ± SD) until their Tco stabilized between 40.5 and 41.5 °C, and to Ta 2.7 ± 1.5 °C for 2.5 h, respectively. In addition, as indices of the acute-phase plasma protein response, their plasma levels of copper (Cu) and N-acteylneuraminic acid (NANA) were assayed 28 h after the onset of these exposures. There were no significant differences in the Tco changes between sham-operated and AV3V-lesioned guinea pigs induced by each of these conditions. Plasma Cu and NANA levels also were not affected by the lesions or the environments. It would appear, therefore, that ablation of the AV3V causes enhanced metabolic responses to thermal stress; but since these changes lead to elevated rather than to depressed Tco and do not alter acute-phase plasma protein levels, they cannot account for the blocked febrile and acute-phase plasma protein responses to LPS.

Summary A hitherto unrecognised lipoprotein liver-cell inclusion is described in elderly mice. The significance of these inclusions is unknown. In an experiment to determine the effect of Treponema pallidum on the lifespan of mice (Wright & Wharton, ] 977) an inclusion which had not previously been described was detected in the liver. 30 were inoculated intra peritoneally with a formalinkilled suspension, JO with only the suspending medium, and 20 uninoculated mice were held over for the period of the experiment. From the infected group JO mice were sacrificed at intervals of 6 months, while 10 mice inoculated with dead spirochaetes were killed at 30, 36 and 42 months following the commencement of the experiment. Organs of all these mice were submitted to microscopical examination.

The interaction of isoniazid with its hepatotoxic metabolite acetylhydrazine has been studied. In vitro, isoniazid at a concentration of 0.5 mM was found to inhibit the microsomal metabolism of acetylhydrazine to a reactive acylating species. In vivo, however, coadministered isoniazid inhibited the acetylation to diacetylhydrazine and concomitantly increased the microsomal metabolism of 14C-acetylhydrazine to a reactive intermediate which covalently bound to liver macromolecules. Comparison of urinary metabolites of isoniazid at two dose levels indicated that acetylation of acetylhydrazine produced as a metabolite may be inhibited by the parent drug.

1. The cardiovascular effects of intravenous injections of interleukin-1 (IL-1) and tumour necrosis factor (TNF) have been investigated in the conscious rabbit. They have been compared with the effects of bacterial lipopolysaccharide (LPS) because both IL-1 and TNF are released from macrophages by LPS. 2. IL-1, TNF and Escherichia coli J5-LPS all caused hypotension when given intravenously in a dose with low mortality. The time course of the hypotension caused by IL-1 and LPS was similar, although the maximal fall in mean blood pressure occurred earlier after IL-1. TNF produced a more sustained fall in blood pressure. Hypotension was not accompanied by a compensatory tachycardia after any of the test substances. Hypotension was associated with a fever after TNF, hypothermia after LPS and no significant change in temperature after IL-1. 3. The packed cell volume did not change during hypotension in any of the study groups, implying that the hypotension was not due to fluid loss resulting from increased capillary permeability. 4. IL-1 and TNF are candidates for the role of effectors of LPS-induced hypotension.

The site of neuromuscular blockade induced by polymyxin B and rolitetracycline was studied on isolated nerve and nerve–muscle preparations. Polymyxin B (1.8 × 10−4 M) was equipotent to lidocaine as a local anaesthetic on a frog desheathed nerve preparation, while rolitetracycline (up to 3.6 × 10−3 M) had no local anaesthetic effect. Polymyxin B (6 × 10−5 M) and rolitetracycline (7 × 10−4 M) blocked by 50% the response of rat diaphragm induced by phrenic nerve stimulation, but did not decrease the amount of acetylcholine (ACh) released from this preparation during nerve stimulation. Both antibiotics depressed the response of the rat diaphragm to inject ACh, and this response was more sensitive to inhibition by the drugs than was the response to nerve stimulation. With rolitetracycline, a concentration that blocked the response to nerve stimulation by 50% inhibited the response to injected ACh by 85%, and this relationship was similar to that with d-tubocurarine; however, polymyxin B was relatively more effective than d-tubocurarine in inhibiting the effect of ACh. Polymyxin B (1–1.5 × 10−4 M) but not rolitetracycline (1 × 10−3 M) depressed the response of the diaphragm to direct muscle stimulation. It is concluded that polymyxin B and rolitetracycline block neuromuscular transmission predominantly by an effect to depress the muscle's sensitivity to ACh; polymyxin B probably acts by an effect similar to that of local anaesthetics, while rolitetracycline probably acts by an effect similar to that of d-tubocurarinc.

A water-borne Bacillus licheniformis infection was associated with depressed haemoglobin content, white cell and platelet count. The epidemic was resolved by changing from tanked to mains water supply.

SUMMARY Intraperitoneal infection with Treponema pallidum did not shorten the lives of inbred CBA mice. One hundred and fifty infected mice survived to a mean age of 41-24 months and 60 uninfected mice to a mean age of 41-38 months.

Some factors affecting the metabolism of the potent hepatotoxin, acetylhydrazine, were studied in rats. After ip administration of [14C]acetylhydrazine, 36% and 38% of the dose was recovered in the urine and as 14CO2, respectively. The major urinary metabolites were diacetylhydrazine and the pyruvic acid and alpha-oxoglutaric acid acetylhydrazones. The acetylation of acetylhydrazine to diacetylhydrazine was found to be dose-dependent and to be inhibited by coadministered isoniazid and p-aminosalicylic acid. Coadministered acetylisoniazid had no effect on acetylation. The proportion of acetylhydrazine recovered as 14CO2 presumably reflects the amount metabolized by the microsomal oxidation pathway, thought to be responsible for the toxicity, and also possibly by hydrolysis to acetate and hydrazine. This latter pathway could not be confirmed, as only a small proportion of hydrazine administered to rats was recovered. The inhibition of acetylation by p-aminosalicyclic acid, but not isoniazid, significantly increased the excretion of 14CO2, suggesting that isoniazid may also inhibit a pathway resulting in the production of 14CO2. These results indicate that the metabolism and hepatotoxicity of acetylhydrazine may be different when it is produced as a metabolite of isoniazid than when it is given alone.

The prolonged anti-rheumatic effects produced by some higher 21-esters of intra-articular corticosteroids have been ascribed to their low aqueous solubility or, alternatively, to their slow release of free (21-OH) steroid in the inflamed synovium. Experiments were designed to test this hypothesis. Twelve 21-carboxyl esters of dexamethasone and [3H]dexamethasone were prepared. Their side-chain structures were chosen to provide systematic steric hindrance of the scissile bond. Four dexamethasone/[3H]dexamethasone 21-carbamates were also prepared. When incubated with a 10% (w/v) homogenate of rabbit synovial tissue, esters providing steric hindrance, e.g. t-butylacetate, were more slowly hydrolysed than those which were linear, e.g. n-hexanoate, or cyclic e.g. cyclohexane acetate. Carbamate esters remained unhydrolysed during 24 hours' incubation. The partition coefficients of these compounds, derived using reversed-phase thin-layer chromatography and hydrophobic fragmental constants, were not correlated with their hydrolysis rates. Isomeric 21-substituents had similar partition coefficients. The affinity of the isomers, dexamethasone n-hexanoate and dexamethasone t-butylacetate, for the glucocorticoid receptor of mouse fibroblast cytosol, was determined by a competitive binding assay using [3H]triamcinolone acetonide. Dexamethasone t-butylacetate had 1/10 binding affinity relative to that of dexamethasone. Dexamethasone n-hexanoate was inactive. The therapeutic activities of dexamethasone n-hexanoate and dexamethasone t-butylacetate were compared at a single dose (2 mg), injected into experimentally-arthritic rabbit knee joints. These preparations reduced the swelling and histopathological changes in the treated joints by the same extent, indicating that the local anti-rheumatic activity of corticosteroid 21-esters is unrelated to their hydrolysis rates in vitro.

Endotoxins, which are lipopolysaccharide complexes derived from the cell walls of gram-negative bacteria, have been implicated in the pathogenesis of gram-negative septic shock. One possible mechanism of endotoxin-induced damage may involve an action at cell surface membranes resulting in cell injury and lysosomal enzyme release. In our experiments, the administration of purified E. coli endotoxin (2 mg/kg intravenously) to guinea pigs produced elevations in the plasma activity of the lysosomal hydrolases glucosaminidase, acid phosphatase and Cathepsin D of approx. 2-, 3- and 4-fold, respectively, at 5 h following endotoxin injection. Animals haemorrhaged to produce sustained hypotension that was greater than the reduction in blood pressure seen with endotoxin treatment, exhibited an elevation only in plasma Cathepsin D activity that was, however, significantly lower than the increase associated with endotoxemia. The three lysosomal hydrolases were also measured in man, including a control group, patients with gram-negative septic shock, other shock, gram-positive and gram-negative septicaemia without shock. Plasma Cathepsin D activity was significantly elevated (26-fold above control) in the group with gram-negative septic shock as compared to all other groups. Patients in the gram-negative septic shock group and the other shock group both had significantly greater glucosaminidase activity than controls. Our results suggest that plasma Cathepsin D measurements may be of diagnostic and prognostic value in the clinical management of gram-negative septic shock.

These experiments were designed to determine the site and mechanism of action of neomycin on cholinergic transmission. These agents depressed the response of rat diaphragm preparations to phrenic nerve stimulation and to injected acetylcholine (ACh); however, equi-effective neuromuscular blocking concentrations of neomycin (6 x 10(-4) M), streptomycin (1.2 x 10(-3) M) or d-tubocurarine (6.5 x 10(-7) M) reduced the muscle response to injected ACh to 54,27 and 15% of control, respectively, suggesting that neomycin and streptomycin have a presynaptic effect. This finding was confirmed by measuring ACh release from the diaphragm during phrenic nerve stimulation; neomycin (6x10(-4) M) and streptomycin (1.2 x 10(-4) M) depressed ACh release to 29 and 41% of control, respectively. In the cat superior cervical ganglion neomycin (2 x 10(-3) M) blocked ganglionic transmission, did not reduce the response of ganglion cells to injected nicotine and depressed ACh release during preganglionic nerve stimulation to 61% of control in normal Ca++ (2.5 mM) medium and to less than 10% of control in low Ca++ (0.5 mM) medium. The increased accululation of 45Ca induced in rat isolated ganglia by preganglionic nerve stimulation was not changed by d-tubocurarine (2 x 10(-4) M), but was abolished by neomycin (2 x10(-3) M). It is concluded that neomycin blocks ACh release by blocking the influx of Ca++ necessary for transmitter release. This conclusion suggested that neomycin should block noradrenaline release, and this was shown using the anococcygeus preparation from the rat.

Long chain esters of cortisol have shown prolonged anti-inflammatory activity in both clinical and animal studies. This effect has been ascribed to the decreased water-solubility of the higher esters, but an alternative explanation is that the higher esters are hydrolysed more slowly to free steroid by the synovial tissue enzymes. In order to investigate the influence of chain length on hydrolysis rate we synthesized a series of cortisol 21-esters. When incubated in a 0.1% (w/v) homogenate of inflamed rabbit synovial tissue the esters with chain lengths of 4, 6, 8 and 10 carbon atoms were hydrolysed much faster than those with 2, 12, 14 and 16 carbon atoms. At tissue concentrations of 10% (w/v), however, the breakdown of cortisol acetate was greatly accelerated, whereas cortisol palmitate remained quite stable. Although cortisol esters were hydrolysed in 50% (v/v) rheumatoid synovial fluid, the rates of hydrolysis were relatively slow. The chain length dependence was similar to that seen with the tissue homogenate.