Expression of Glial Fibrillary Acidic Protein and Neurofilament mRNA in Gliosis Induced by Experimental Autoimmune Encephalomyelitis

We have previously shown that the content of glial fibrillary acidic protein (GFAP) gradually increases in the spinal cord of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE), reaching a level 1.5-2 times greater than that in controls by 35 days postimmunization (dpi). We report here that the increase in GFAP mRNA level followed a completely different time course and reached higher levels relative to controls than did that of the protein. Total RNA was isolated using a modified version of current methods using phenol/chloroform extractions to ensure optimal recovery from spinal cord. Control animals yielded 323 +/- 35 micrograms (mean +/- SD; n = 34) of total RNA/spinal cord throughout the experimental period. EAE animals contained up to three times as much total RNA during 11-14 dpi, a finding largely reflecting the infiltration of inflammatory cells. By 65 dpi, total RNA levels closely approached control values. As early as 10 dpi, increased amounts of GFAP mRNA were detected in EAE animals relative to controls. During 11-14 dpi, GFAP mRNA levels reached six- to eightfold greater than values in controls and then slowly declined throughout the remainder of the time course, with a fourfold increase still detected at 65 dpi. However, coinciding with the height of inflammation and clinical signs at 12 dpi, the GFAP mRNA content dropped to approximately 50% of the level at 11 dpi but rose again at 13 dpi. This dip was mirrored by a similar decrease in neurofilament mRNA content, but otherwise the level of this message remained relatively constant and equal to that in controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of hepatic glycine catabolism by glucagon

Glucagon stimulates 14CO2 production from [1-14C] glycine by isolated rat hepatocytes. Maximal stimulation (70%) of decarboxylation of glycine by hepatocytes was achieved when the concentration of glucagon in the medium reached 10 nM; half-maximal stimulation occurred at a concentration of about 2 nM. A lag period of 10 min was observed before the stimulation could be measured. Inclusion of beta-hydroxybutyrate (10 mM) or acetoacetate (10 mM) did not affect the magnitude of stimulation suggesting that the effects of glucagon were independent of mitochondrial redox state. Glucagon did not affect either the concentration or specific activity of intracellular glycine, thus excluding the possibilities that altered concentration or specific activity of intracellular glycine contributes to the observed stimulation. The stimulation of decarboxylation of glycine by glucagon was further studied by monitoring 14CO2 production from [1-14C]glycine by mitochondria isolated from rats previously injected with glucagon. Glycine decarboxylation was significantly stimulated in the mitochondria isolated from the glucagon-injected rats. We suggest that glucagon is a major regulator of hepatic glycine metabolism through the glycine cleavage enzyme system and may be responsible for the increased hepatic glycine removal observed in animals fed high-protein diets.     

Effects of body position on intracranial and cerebral perfusion pressures in isoflurane-anesthetized horses.

Inhalant anesthetics may interfere with normal cerebrovascular autoregulation. It was, therefore, hypothesized that intracranial pressure (ICP) and cerebral perfusion pressure (CPP) in isoflurane-anesthetized horses would be especially sensitive to body and head position because of the potential for large hydrostatic gradients between the brain and heart in this species. Anesthesia was induced and maintained in six clinically healthy, unmedicated geldings with 1.57% isoflurane in O(2); mechanical ventilation was used to maintain normocapnia. ICP was measured by using a subarachnoid strain-gauge transducer. Blood gases and carotid arterial, right atrial, and airway pressures were also measured. Five body positions were studied in semirandomized order: dorsal recumbency (DR) with head down (HD), DR with head level (HL), lateral recumbency (LR), sternal recumbency (SR) with HL, and SR with head up (HU). Data were analyzed by using paired t-tests. ICP and CPP values, respectively, are as follows (means +/- SD): 36 +/- 4 and 55 +/- 18 mmHg (DR-HD); 34 +/- 6 and 51 +/- 32 mmHg (DR-HL); 24 +/- 5 and 48 +/- 4 mmHg (LR); 19 +/- 11 and 87 +/- 12 mmHg (SR-HL); and -14 +/- 7 and 71 +/- 10 mmHg (SR-HU). Significant differences were found among all positions, except for SR-HL vs. LR. Significant increases in CPP were observed only in sternal positions. In conclusion, ICP in isoflurane-anesthetized horses changes inversely with the brain-to-heart hydrostatic gradient. DR may also cause increases in ICP, irrespective of head position.     

EAE tolerance induction with Hsp70-peptide complexes depends on H60 and NKG2D activity

Inflammation leads to induction of tissue stress conditions that might contribute to the generation of mechanisms limiting ongoing immune responses. We have shown previously that peptides derived from brain tissue of mice with experimental autoimmune encephalomyelitis (EAE) complexed with the chaperone heat shock protein 70 (Hsp70-pc) induce an NK-cell-dependent tolerance for subsequent EAE sensitization. We now present data that showed that the MHC class I-related glycoprotein H60 determines Hsp70-pc-induced EAE inhibition. Hsp70-pc led to significant and selective up-regulation of H60 expression in SJL/J mice, and Ab-blocking of H60 expression led to loss of EAE tolerance. Similarly, blocking of the NK cell receptor for H60, NKG2D, also reversed the Hsp70-pc-induced EAE inhibition. In contrast, in C57BL/6 mice H60 was not expressed, and Hsp70-pc-induced tolerance was not detected. The NK cell mediated Hsp70-pc-induced tolerance to EAE was dependent on modulation of dendritic cells function leading to diminished T cell reactivity to PLP. As, no increase of H60 expression on T cells from EAE mice immunized with PLP was detected, and no enhanced loss of CD3+ H60+ over CD3+ H60- cells in Hsp70-pc-induced EAE tolerance was found direct killing of H60+ PLP-reactive cells seems not to be involved in the Hsp70-pc-induced tolerance induction. We have provided evidence that Hsp70-pc-induced tolerance for EAE, mediated by NK cells, involves induction of H60 ligand and its interaction with NKG2D receptor. NK cells tolerization of EAE depends on altered dendritic cells activity leading to enhanced death of Ag reactive cells.     

Investigation of the mechanisms of irreversible thermoinactivation of Bacillus stearothermophilus alpha-amylase.

Bacillus stearothermophilus NCIB 11412 produces a highly thermostable alpha-amylase. The enzyme displayed half-lives of irreversible thermoinactivation at 90 degrees C of 1.9 min and 12.5 min at pH 5.0 and pH 8.0, respectively. Molecular mechanisms of irreversible thermoinactivation were investigated. At both pH 5.0 and pH 8.0 irreversible thermoinactivation was due to heat-induced breakdown of non-covalent interaction within the protein molecule, resulting in unfolding and consequent formation of altered structures. Hydrophobic interactions were shown to be the most important non-covalent mechanisms involved in this phenomenon. Although not dramatically effecting the rates of irreversible thermoinactivation, electrostatic interactions, including hydrogen bonding, were also shown to have a contributory role in this process. At pH 8.0 a covalent mechanism, that of oxidation of thiols was also shown to be of secondary importance to hydrophobic interactions in the irreversible thermoinactivation of this enzyme.     

Capuchin monkeys (Sapajus [Cebus] apella) play Nash equilibria in dynamic games,but their decisions are likely not influenced by oxytocin

Comparative approaches to experimental economics have shed light on the evolution of social decision‐making across a range of primate species, including humans. Here we replicate our previous work looking at six pairs of capuchin monkeys' (Sapajus [Cebus] apella) responses to scenarios requiring both coordination (Assurance Game) and anti‐coordination (Hawk‐Dove Game). This then provides a foundation for assessing their responses to two additional games, one with a scenario of beneficial cooperation with a temptation to defect (Prisoner's Dilemma) and one with an environment requiring changing strategies within short temporal proximity (Alternating Economic Game). We additionally explored the effects of exogenous oxytocin on decision‐making. Oxytocin did not affect decisions in any of our games. Results from the first two games largely replicated our previous findings. Responses to the Prisoner's Dilemma were more varied than was seen in previous games, with pairs respectively cooperating, defecting, and failing to establish stable strategies. Such variability indicates that this game may be a good assay for individual differences in social decision‐making. Finally, capuchins were able to flexibly switch between their previously established strategies within each of the different games, even when the games were presented within the same session, requiring strategy adjustments within short temporal proximity. These results build on earlier findings showing that capuchins can alter decision‐making strategies as the context demands, which is likely essential for decision‐making in naturally occurring contexts.