The JNK binding domain of islet-brain 1 inhibits IL-1 induced JNK activity and apoptosis but not the transcription of key proapoptotic or protective genes in insulin-secreting cell lines.

The stress-activated protein kinase c-Jun NH2-terminal kinase (JNK) is a central signal for interleukin-1beta (IL-1beta)-induced apoptosis in insulin-producing beta-cells. The cell-permeable peptide inhibitor of JNK (JNKI1), that introduces the JNK binding domain (JBD) of the scaffold protein islet-brain 1 (IB1) inside cells, effectively prevents beta-cell death caused by this cytokine. To define the molecular targets of JNK involved in cytokine-induced beta-cell apoptosis we investigated whether JNKI1 or stable expression of JBD affected the expression of selected pro- and anti-apoptotic genes induced in rat (RIN-5AH-T2B) and mouse (betaTC3) insulinoma cells exposed to IL-1beta. Inhibition of JNK significantly reduced phosphorylation of the specific JNK substrate c-Jun (p<0.05), IL-1beta-induced apoptosis (p<0.001), and IL-1beta-mediated c-fos gene expression. However, neither JNKI1 nor JBD did influence IL-1beta-induced NO synthesis or iNOS expression or the transcription of the genes encoding mitochondrial manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase rho (GSTrho), heat shock protein (HSP) 70, IL-1beta-converting enzyme (ICE), caspase-3, apoptosis-inducing factor (AIF), Bcl-2 or Bcl-xL. We suggest that the anti-apoptotic effect of JNK inhibition by JBD is independent of the transcription of major pro- and anti-apoptotic genes, but may be exerted at the translational or posttranslational level.

Monotreme IGF2 expression and ancestral origin of genomic imprinting.

IGF2 (insulin-like growth factor 2) and M6P/IGF2R (mannose 6-phosphate/insulin-like growth factor 2 receptor) are imprinted in marsupials and eutherians but not in birds. These results along with the absence of M6P/IGF2R imprinting in the egg-laying monotremes indicate that the parental imprinting of fetal growth-regulatory genes may be unique to viviparous mammals. In this investigation, we have cloned IGF2 from two monotreme mammals, the platypus and echidna, to further investigate the origin of imprinting. We report herein that like M6P/IGF2R, IGF2 is not imprinted in monotremes. Thus, although IGF2 encodes for a highly conserved growth factor in chordates, it is only imprinted in therian mammals. These findings support a concurrent origin of IGF2 and M6P/IGF2R imprinting in the late Jurassic/early Cretaceous period. The absence of imprinting in monotremes, despite apparent interparental conflicts over maternal-offspring exchange, argues that a fortuitous congruency of genetic and epigenetic events may have limited the phylogenetic breadth of genomic imprinting to therian mammals. J. Exp. Zool. (Mol. Dev. Evol.) 291:205-212, 2001.

Glucose potentiates interleukin-1 beta (IL-1 beta)-induced p38 mitogen-activated protein kinase activity in rat pancreatic islets of Langerhans.

The cytokine interleukin-1 beta (IL-1 beta) is cytotoxic to rat pancreatic beta-cells and has been implicated in the pathogenesis of insulin-dependent diabetes mellitus. IL-1 beta causes expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO). NO may be the mediator of the cytotoxic effect of IL-1 beta in rat islets and beta-cell lines. Glucose has been shown to modulate the effects of IL-1 beta on accumulated insulin release and potentiate NO production in rat islets, but the biochemical mechanism is unknown. IL-1 beta activates the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38 and c-jun NH2-terminal kinase (JNK) in rat islets and beta-cells. Glucose may modulate MAPK activity although contrasting data have been published. The aim of this study was to investigate whether glucose potentiated IL-1 beta-induced p38 and ERK1/2 activity in rat islets. It was shown that glucose alone increased the phosphorylation of the MAPK substrates Elk-1 and activating transcription factor 2 (ATF2). D-glucose potentiated the p38 activity induced by a low concentration of IL-1 beta, whereas no effect was seen at high concentrations of IL-1 beta. Inhibition of p38 activity prevented IL-1 beta-induced nitrite production in the presence of D-glucose. We conclude that IL-1 beta-induced NO production in the presence of glucose is signalled by the p38 pathway.

TNFalpha and IFNgamma potentiate IL-1beta induced mitogen activated protein kinase activity in rat pancreatic islets of Langerhans.

AIMS/HYPOTHESIS: Interleukin-1 beta (IL-1beta) in synergy with tumour necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) is cytotoxic to pancreatic beta cells. Mitogen-activated protein kinase (MAPK) activity that is induced by interleukin-1 beta has been suggested to signal nitric oxide-dependent as well as nitric oxide-independent beta-cell destructive pathways. The aim of this study was to investigate if TNFalpha and IFNgamma signal through mitogen-activated protein kinases in isolated rat islets of Langerhans and if they potentiate mitogen-activated protein kinase activity induced by IL-1beta. METHODS: Islets of Langerhans were isolated from 5- to 7-day-old Wistar rats and precultured for 7 days before stimulation with IL-1beta, TNFalpha and/or IFNgamma for 20 min followed by lysis. Kinase activity was measured with a whole cell lysate kinase assay and after immunoprecipitation of the kinase using immunocomplex kinase assay. RESULTS: Exposure to IL-1beta or TNFalpha significantly increased mitogen-activated protein kinase activity, whereas IFNgamma tended to decrease extracellular-signal-regulated kinase activity. Further, TNFalpha and IFNgamma were found to synergistically increase mitogen-activated protein kinase activity induced by IL-1beta. CONCLUSION/INTERPRETATION: We hypothesise that the synergistic effect of IL-1beta, TNFalpha and IFNgamma in the functional inhibition and induction of cell death in pancreatic beta cells is signalled through a synergistic activation of mitogen-activated protein kinase activity.

Interleukin-18 mRNA, but not interleukin-18 receptor mRNA, is constitutively expressed in islet beta-cells and up-regulated by interferon-gamma.

Interleukin-18 (IL-18) mRNA is expressed in islets of NOD mice during the early stages of insulitis and IL-18 has therefore been implicated as a contributing factor in immune-mediated beta-cell destruction. However, a recent study failed to show any effect of human IL-18 on the function of isolated rat islets. Since species differences have been shown between human and murine IL-18, the aims of this study were to investigate 1) if species homologous IL-18 alone or following IL-12 pre-exposure affected rat islet function, 2) if IL-18 dose-dependently modulated IL-1 beta or interferon-gamma (IFN-gamma) + tumor necrosis factor-alpha (TNF-alpha) actions on islet function, and 3) if IL-18 and IL-18 receptor (IL-18R) were expressed in rat islet beta-cells. Insulin release and nitric oxide (NO) production from isolated rat islets were measured after incubation with or without cytokines. RT-PCR was used to quantitate mRNA expression of IL-18 and the IL-18R signaling chain (IL-18R beta). There were no significant effects of 0.625-10 nM recombinant murine (rm) IL-18 alone on accumulated or glucose-challenged insulin release or NO production after 24 hours. Fifteen pg/ml of recombinant human (rh) IL-1 beta as well as 200 U/ml recombinant rat (rr) IFN-gamma + 250 U/ml rhTNF-alpha significantly increased islet NO production and inhibited both accumulated and glucose-challenged islet insulin release. However, rmIL-18 failed to modulate these effects of IL-1 beta or IFN-gamma + TNF-alpha. Although IL-12 induces IL-18R expression in Th1 and B lymphocytes, 24-hours rmIL-12 preincubation neither sensitized islets to effects of 10 nM of rm or rrIL-18 alone nor primed the islets to IL-1 beta actions on insulin release and NO production. IL-18R beta mRNA, which was expressed in human peripheral blood mononuclear cells (PBMC), was not expressed in rat insulinoma (RIN) cells or in isolated rat islets, even after exposure to IL-1 beta and/or IFN-gamma + TNF-alpha or IL-12. IL-18 mRNA was constitutively expressed in RIN cells, in FACS-purified rat beta-cells and in intact rat and mouse islets, and was up-regulated by IFN-gamma in an interferon regulatory factor-1- IRF-1) and NO - independent manner. However, IL-18 protein was undetectable in lysates and supernates of RIN cells by ECL, Western blotting and immunoprecipitation. In conclusion, we show for the first time that IL-18 but not IL-18R is expressed in rodent islet beta-cells. The physiological importance and pathological role of IL-18 originating from islet beta-cells deserve further investigation.

Activation of extracellular signal-regulated kinase (ERK)1/2 contributes to cytokine-induced apoptosis in purified rat pancreatic beta-cells.

Cytokines may contribute to beta-cell apoptosis in the early stages of type 1 diabetes mellitus. It has been reported recently that interleukin-1 beta (IL-1 beta) induces activation of the mitogen-activated protein kinases (MAPK) p38 and ERK1/2 in neonatal rat islets. Since these kinases may participate in cytokine-induced apoptosis, we evaluated whether cytokines induce activation of MAPKs in FACS-purified primary rat beta-cells, and whether blockers of p38 and/or ERK1/2 prevent beta-cell death. IL-1 beta, but not interferon-gamma (IFN-gamma), caused phosphorylation of the substrates Elk-1, ATF-2 and hsp25, and the phosphorylation of both Elk-1 and hsp25 were decreased by the p38 blocker SB203580 (p38i) and the MAPK/ERK blocker PD 098059 (MEKi). When added together, p38i and MEKi decreased IL-1 beta-induced nitrite production over 24 hours by 60%, but did not affect IL-1 beta-induced manganese superoxide dismutase (MnSOD) mRNA expression. To test the effects of MAPK inhibitors on beta-cell death by necrosis or apoptosis, these cells were exposed for 6 or 9 days to IL-1 beta + IFN-gamma. This treatment induced cell death, mostly by apoptosis. The MEKi, but not the p38i, significantly decreased cytokine-induced apoptosis, thus decreasing the total number of dead cells. This protection was only partial, suggesting that ERK1/2 activation is not the only mechanism by which cytokines induce beta-cell apoptosis. We conclude that IL-1 beta induces activation of both p38 and ERK1/2, and that ERK1/2 contributes to the pro-apoptotic effects of the cytokine in primary beta-cells.

The echidna manifests typical characteristics of rapid eye movement sleep.

The failure to identify rapid eye movement sleep (REMS) in an early study of the echidna at an unmeasured ambient temperature (T(a)) was unexpected, as its brain stem structures resemble those that generate REMS in other mammals. However, typical mammalian REMS was evident in echidnas exposed to several T(a)s. The parallel presence of REMS in birds points to its reptilian origin.

Descriptive study of the diaphragm and lungs in the short-nosed echidna, Tachyglossus aculeatus (Mammalia: monotremata).

In this descriptive study, we characterize the diaphragm and lungs of the short-nosed echidna, Tachyglossus aculeatus, using a combination of gross anatomical, light-microscopic, electron microscopic, and morphometric techniques, including airway casting. The diaphragm is inclined from ventro-cranial to dorso-caudal and possesses a large central tendon (centrum tendineum). The crural and costal muscle groups and the associated trigoni are located in the same positions as in other mammals. The bronchial branching pattern reveals cranially broad, tapering stem bronchi and an unusually small number of first order bronchi. The asymmetrical primary branching pattern and possibly also the asymmetry of right and left lungs are plesiomorphic within the Mammalia. The histology and ultrastructure of the airways and lung parenchyma reveal no unusual features: alveolar type 1 and type 2 cells in the parenchyma; type 2 cells, exocrine bronchiolar cells (Clara cells), ciliated cells, and goblet cells in the terminal airways and the latter two cell types in the bronchi. Both a double and a single capillary net are found on the interalveolar septa. The high capillary loading of the double net may be of selective advantage because of long apneas and low metabolic rate in the echidna.

Seasonal variations in thyroid hormone levels in free-living echidnas (Tachyglossus aculeatus).

Hulbert and Augee (1982) have suggested that the thyroid has little effect on energy metabolism in the echidna. In order to investigate whether thyroid status changes during hibernation, when metabolism drops dramatically, we measured levels of thyroid hormones in 31 free-living echidnas at various times during the year. Unlike eutherian hibernators, in which thyroid hormone levels may rise to seasonally high values in late hibernation, total and free T(4) and T(3) were all significantly depressed throughout hibernation. TT(4) from nonhibernating echidnas was 11.8 +/- 0.9 ng/ml (n = 23), confirming previously published values, but fell to half this level (5.9 +/- 0.7 ng/ml, n = 8) during hibernation. By contrast to the low TT(4) values, nonhibernating FT(4), TT(3), and FT(3) values were similar to normal values for eutherian mammals. Differences in the seasonal pattern of variation in thyroid hormones between echidnas and hibernating eutherians may be due to differences in thyroid hormone transporting proteins.

Characterization of new polymorphisms in the 5' UTR of the human interleukin-1 receptor type 1 (IL1R1) gene: linkage to type 1 diabetes and correlation to IL-1RI plasma level.

The human interleukin-1 type I receptor (IL-1RI) is the signal transducing receptor for IL-1, a principal proinflammatory cytokine, which is cytotoxic to pancreatic islet beta cells. The IL-1RI gene, IL1R1, maps to chromosome 2q12. We have previously examined part of the IL1R1 promoter region and in the present study we further characterized the promoter region demarcating exon 1B and 1C by sequencing and mutation scanning. New sequence was obtained 1762 bp upstream and 1609 bp downstream the known region. Within this sequence, we identified four frequent single nucleotide polymorphisms (SNPs). PCR-based RFLP assays were established and three of the polymorphisms were typed in a Danish Type 1 (insulin-dependent) diabetes mellitus (T1DM) family collection comprising 103 simplex and 150 sib-pair affected families. Linkage was evaluated by the sib-TDT (transmission disequilibrium test). One of the polymorphisms, defined by a Hinfl RFLP assay, demonstrated linkage to T1DM, P(sTDT) = 0.026. Random transmission was observed to unaffected offspring from heterozygous parents, P = 0.87. No evidence for positive linkage was seen for the other tested polymorphisms, P = 0.14 and P = 0.21, respectively. To evaluate the possible functional significance of the Hinfl polymorphism, we measured circulating IL-1RI plasma level in 30 T1DM patients and in 30 control subjects--10 with each genotype in both groups. Significant differences in plasma levels in relation to genotype--independent of disease status--were found (P = 0.04). In both diabetic and non-diabetic subjects, the wt/wt genotype correlated with the highest IL-1RI plasma level, whereas the plasma levels were lowest for the mt/mt genotype.

The fetal glycoprotein, fetuin, counteracts ill-effects of the bacterial endotoxin, lipopolysaccharide, in pregnancy.

We show that fetuin, a fetal glycoprotein present in fetal plasma in concentrations substantially higher than in the adult, can exert a protective function against ill-effects of the bacterial endotoxin, lipopolysaccharide (LPS), in pregnancy. The concentration of fetuin in plasma of pregnant rats (E17 gestation) was artificially increased by repeated intraperitoneal administration of this protein. This was followed by an injection of a standard dose of LPS, a dose that produced in control animals (no additional fetuin) nearly 50% abortions and 40% maternal deaths. None of the females exposed to increased fetuin died following LPS injection. Nine out of 10 multigravida animals carried their pregnancy to full term but all primagravidas aborted.

Modification of macrophage response to lipopolysaccharide by fetuin.

The physiological role(s) of fetuin, a protein present in plasma and many tissues of developing animals at levels much higher than in the adult, is unknown. Here we show that fetuin can modify the responses of macrophages to lipopolysaccharide (LPS) stimulation. At concentrations of fetuin in the medium, corresponding to fetal levels of this protein in plasma, the production and secretion of interleukin 1beta (IL-1beta) and nitric oxide (NO) is almost abolished, tumour necrosis factor-alpha (TNF-alpha) reduced, while that of IL-6 is not affected. On the other hand, concentrations of fetuin corresponding to adult plasma levels (i.e. 40-60 mg/100 ml) were without much effect on macrophage synthesis and secretion of these cytokines.

The acute phase response of plasma proteins in the polyprotodont marsupial Monodelphis domestica.

In eutherians, patterns of plasma protein levels in blood change during the acute phase response to trauma and inflammation. Until now, such an acute phase response has not been characterised in a noneutherian species. Here we describe the acute phase response in a marsupial species, the South American polyprotodont marsupial Monodelphis domestica, after brain surgery or injection of lipopolysaccharide. Several days after brain surgery, transthyretin was not detected in plasma. For 48 hr following injection of lipopolysaccharide, the concentration of haptoglobin in plasma increased, that of transthyretin decreased, and the concentration of albumin in plasma did not change significantly. The American polyprotodont marsupials are probably more closely related to the common ancestor marsupial than the Australian marsupials are. It is most likely that the transthyretin gene was not expressed in the liver of this common ancestor. As the transthyretin gene is expressed in the liver of M. domestica, it seems that as soon as transthyretin is synthesised by the liver, it is under negative acute phase control.

Nonshivering thermogenesis in marsupials: absence of thermogenic response to beta 3-adrenergic agonists.

The status of nonshivering thermogenesis (NST) in marsupials remains controversial. Although morphological studies have failed to find evidence for the presence of brown adipose tissue (BAT) in adults or juveniles of species from all extant families of marsupial, a number of studies have investigated the metabolic response of marsupials to noradrenaline (NA) and yielded conflicting results. In eutherian mammals, NA stimulates NST in BAT by acting on beta 3-receptors, and in the experiments reported here we investigated the response of adult and juvenile brush tail possums (Trichosurus vulpecula), a Brazilian opossum (Monodelphis domestica), adult and juvenile red-necked (Bennett's) wallabies (Macropus rufogriseus) and the laboratory rat to selective beta 3-agonists (ICI D7114 and BRL 35135) and to NA. Wallabies were tested with the beta 3-agonists only. Although NA and both beta 3-agonists caused an 85% increase in oxygen consumption in rats, there was no significant effect on any of the marsupials. These results clearly indicate no beta 3-stimulated NST in these marsupials. All reports of metabolic responses to NA are from macropods, and a recent study demonstrates that NA and other alpha-adrenergic agonists stimulate thermogenesis in a small macropod, the bettong (Bettongia gaimardi), by acting on alpha 1-receptors. Thermogenic responses to NA seems to be restricted to macropods, showing the danger of characterising the response of any one marsupial species as being representative of marsupials as a group.

Phrenicotomy in the rat: acute changes in blood gases, pH and body temperature.

Adult male rats were used to compare blood gases, pH and body temperature (Tb) before and after acute bilateral phrenicotomy. Under anaesthesia a femoral artery was catheterised and ties were placed round the phrenic nerves of seven rats (PNX group), while in five rats the ties were placed in the vicinity of the phrenic nerves (SHAM group). Twenty-four hours after surgery arterial blood samples were collected during quiet wakefulness (QW) and grooming (G), before and 1 h after the ties were pulled, and analysed for PO2, PCO2 and pH. No changes were detected in the SHAM samples taken before and after the ties were pulled. In the PNX group a significant decrease in Tb occurred (QW, 0.6 degrees C; G, 1.5 degrees C). Following PNX PaO2 decreased by 11.2 mmHg (QW) and 10.0 mmHg (G); PaCO2 increased by 2.6 mmHg (QW) and 2.4 mmHg (G) and pH fell by 0.04 (QW) and 0.03 (G). All changes except in PaCO2 (QW) were significant. It is concluded that the changes in Tb, blood gases and pH which follow phrenicotomy in the rat are due to an increase in dead space ventilation (VD) and a small reduction in alveolar ventilation (VA) associated with a faster, shallower pattern of breathing.

Interrelationships between red cell nucleoside triphosphate content, and blood pH, O2-tension and haemoglobin concentration in the carp, Cyprinus carpio.

As a starting point for investigations of possible control factors for the nucleoside triphosphate content in carp red cells, we utilized the natural variability in blood physico-chemical parameters to test for interrelationships. By application of two-variable regression analysis, the red cell NTP content was found to be significantly correlated with arterial PO 2 and pH as well as with the blood haemoglobin (Hb) concentration. These correlations show a rise in red cell NTP content with falling pH as well as with falling Hb concentration, whereas a decrease in PO 2 was associated with a decrease in the content of NTP, particularly at low PO 2 values. Evaluation based on multiple regression analysis suggested that only PO 2 and pH significantly affected the red cell NTP content, and that the influence of changes in Hb concentration could be accounted for by naturally occurring simultaneous changes in PO 2 and pH. The multifactorial control of red cell nucleoside triphosphates is discussed in relation to the role of still unknown factors.

Are general practitioners in favour of quality assurance?

A survey of established general practitioners in New South Wales and South Australia and of Family Medicine Programme trainees in South Australia was undertaken to investigate their attitudes towards a general practice based Quality Assurance Programme. Broad support for such a programme was found and was strongest among younger practitioners; those who had, or were undertaking, FMP training, those belonging to the RACGP; and those who hold the DipObstRACOG.

Effects of nitrite exposure on blood respiratory properties, acid-base and electrolyte regulation in the carp (Cyprinus carpio).

Adult carp were subjected to 1 mM environmental nitrite for 48 h and nitrite uptake and changes in blood respiratory properties, extracellular electrolyte composition and acid-base status were examined. A constant influx of nitrite caused an accumulation of NO2- in plasma to 5.4 mM in 48 h. The fraction of methaemoglobin rose with plasma [NO2-] to 83%, and the arterial oxygen content decreased to extremely low values. Arterial PO2 increased as a compensation to this O2-shortage, whereas the O2 saturation of the functional (unoxidized) haemoglobin decreased, revealing a reduction in its O2 affinity. Blood haematocrit decreased as a result of red cell shrinkage, which caused very high red cell haemoglobin (Hb) concentrations. The erythrocytic nucleoside triphosphate (NTP) concentration showed a parallel increase whereby NTP/Hb, as well as the relative contributions of ATP and GTP to NTP, remained unchanged. Plasma [Cl-] declined by 15 mM in 48 h, offsetting the plasma [NO2-] increase, minor changes in plasma [HCO3-] and a considerable increase in plasma [lactate]. Arterial pH and [HCO3-] rose slightly during the first 24 h of nitrite exposure, but returned to control values at 48 h. The rise in plasma [lactate] was not reflected in an extracellular metabolic acidosis. Plasma [K+] increased by 94% in 48 h, revealing an uncompensated extracellular hyperkalemia, whereas plasma [Na+] decreased, and plasma [Ca++] was unchanged. Plasma osmolality remained essentially constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Respiratory properties of blood and arterial blood gases in the tegu lizard: effects of temperature and hypercapnia.

The effects of body temperature and hypercapnia (7% inspired CO2) on arterial blood gases, plasma pH, and the characteristics of the blood oxygen dissociation curve were determined in Tegu lizards (Tupinambis nigropunctatus). Arterial pH fell from 7.59 to 7.50 when body temperature was increased from 25 to 35 degrees C. The pH/temperature coefficient (delta pH/delta t = -0.009 U/degrees C) was half of that predicted on the basis of 'constant relative alkalinity' and the alphastat hypothesis. The fall in plasma pH resulted from a decrease in plasma [HCO3-], and a rise in plasma Pco2. The O2 affinity of Tegu blood, expressed by the partial pressure at half saturation (P50), decreased with temperature in vitro from 42.3 to 49.6 torr at pH 7.4. The apparent enthalpy (delta H = -3.1 kcal/mol) is about 1/4 of that of human blood. In vivo, the arterial blood oxygen saturation decreased from 89% at 25 degrees to 82% at 35 degrees C. Arterial Po2 increased from 61 to 71 torr as expected from the right-shift of the oxygen dissociation curve. During environmental hypercapnia (7% CO2, 21% O2, 72% N2 inspired concentrations), arterial pH decreased to 7.28. Arterial O2 saturation remained constant and arterial Po2 increased from 61 to 85 torr due to the right-shift of the oxygen dissociation curve. The comparatively small effect of changes in temperature on the oxygen affinity of Tegu blood (directly according to the delta H value, and indirectly via changes in blood pH) results in a relatively small right shift of the oxygen dissociation curve, and accordingly in relatively high arterial and tissue Po2 values also at higher temperatures.