Glutamate receptor antagonists inhibit calpain-mediated cytoskeletal proteolysis in focal cerebral ischemia

Excitatory amino acids may promote microtubular proteolysis observed in ischemic neuronal degeneration by calcium-mediated activation of calpain, a neutral protease. We tested this hypothesis in an animal model of focal cerebral ischemia without reperfusion. Spontaneously hypertensive rats were treated with 2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo-(F)quinoxaline (NBQX), a competitive antagonist of the neuronal receptor for alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or cis-4-[phosphono-methyl]-2-piperidine carboxylic acid (CGS 19755), a competitive antagonist of the N-methyl-d-aspartate (NMDA) receptor. After treatment, all animals were subjected to permanent occlusion of the middle cerebral artery for 6 or 24 h. Infarct volumes measured in animals pretreated with CGS 19755 after 24 h of ischemia were significantly smaller than those quantified in ischemic controls. Rats pretreated with NBQX showed partial amelioration of cytoskeletal injury with preserved immunolabeling of microtubule-associated protein 2 (MAP 2) at 6 and 24 h and reduced accumulation of calpain-cleaved spectrin byproducts only at 6 h. Prevention of cytoskeletal damage was more effective after pretreatment with CGS 19755, as shown by retention of MAP 2 immunolabeling and significant restriction of calpain activity at both 6 and 24 h. Preserved immunolabeling of tau protein was observed at 6 and 24 h only in animals pretreated with CGS 19755. Western analysis performed on ischemic cortex taken from controls or rats pretreated with either NBQX or CGS 19755 suggested that loss of tau protein immunoreactivity was caused by dephosphorylation, rather than proteolysis. These results demonstrate a crucial link between excitotoxic neurotransmission, microtubular proteolysis, and neuronal degeneration in focal cerebral ischemia.     

Ontogeny of alpha-crystallin subunits in the lens of human and rat embryos

The distribution of alpha A- and alpha B-crystallin in the developing lens of human (Carnegie stages 13 to 23) and rat embryos (embryonic days E11 to 18) was examined immunohistochemically. In a human embryo at stage 13, the lens placode was already immunoreactive to alpha B-crystallin, but not to alpha A-crystallin. At stage 15, the lens vesicle was intensely immunoreactive both to alpha A- and alpha B-crystallin. From stages 16 to 23, the lens epithelial cells and fiber cells were immunoreactive to alpha A- and alpha B-crystallin. In rat embryos, alpha A-crystallin appeared in the lens pit at E12, and alpha B-crystallin appeared in the elongating lens fiber cells at E14. From E15 to E18, the lens epithelial cells and fiber cells were immunoreactive to alpha A-crystallin. The lens fiber cells were also immunoreactive to alpha B-crystallin, but the epithelial cells were not. These findings suggest that alpha B-crystallin appears earlier than alpha A-crystallin in the human lens, but at a later period than alpha A-crystallin in the rat lens. alpha B-Crystallin was not detected in the epithelial cells of the rat lens, but was persistently present in the epithelial cells of the human lens.     

Modelling cortical cataractogenesis. XXIX. Calpain proteolysis of lens fodrin in cataract

The relation between cataract and calpain proteolysis of lens fodrin was studied in two systems: elevated glucose (55.6 mM, diabetic model), and cytochalasin D (CD, 10(-2) mM, actin depolymerization-induced opacity model). Glucose treatment (48 h) caused a visible opaque layer and enzyme leakage, with a concomitant accumulation of ([Ca2+]i) around the lens equatorial cortex. CD caused both earlier and greater opacity and enzyme leakage than glucose. Lens fodrin digestion occurred in parallel with the timing and extent of calcium elevation. A calpain inhibitor peptide (CIP, 10(-2) mM) reduced the proteolysis of fodrin, opacity, and enzyme leakage in glucose-treated lenses but only partially retarded them in CD-treated lenses. These results suggest a mechanism in which calpain proteolysis of fodrin is a critical event in lens damage during opacification of cortical cataract.     

Immunohistochemical study on the development of the rat heart conduction system using anti-Leu-7 antibody

Morphological aspects of the heart conduction system have been studied by various histochemical markers. However, the actual presence of markers in the conduction system and its developmental mode remain controversial. We have shown the anti-Leu-7 antibody to cross-react with cells of the rat embryonic heart conduction system by an immuno-electron microscopic study. A comparison was thus made between the results of Leu-7 immunohistochemistry and those by other markers previously used for studying the development of the conduction system. As a result, Leu-7 immunoreactivity proved the most reliable marker for studying the conduction system of the developing rat heart. Examination of the developmental mode of a rat conduction system was facilitated with the use of this marker. The immunoreactivity for Leu-7 initially appeared in the anterior wall of the bulboventricular region at 10 days of development. The development of three internodal tracts and two atrioventricular nodes was demonstrated in the following stage. These primordial atrioventricular nodes, one of which connects to a bundle of His, fused to form a single distinctive node followed by completion of the conduction system in the 18 day fetus.     

Immunohistochemical evidence for the existence of rat cytosolic sialidase in rat skeletal muscles

Six male rowers rowed maximally for 2500 m in ergometer tests during normoxia (fractional concentration of oxygen in inspired air, F(I)O2 0.209), in hyperoxia (F(I)O2 0.622) and in hypoxia (F(I)O2 0.158) in a randomized single-blind fashion. Oxygen consumption (VO2), force production of strokes as well as integrated electromyographs (iEMG) and mean power frequency (MPF) from seven muscles were measured in 500-m intervals. The iEMG signals from individual muscles were summed to represent overall electrical activity of these muscles (sum-iEMG). Maximal force of a stroke (Fmax) decreased from the 100% pre-exercise maximal value to 67 (SD 12)%, 63 (SD 15)% and 76 (SD 13)% (P < 0.05 to normoxia, ANOVA) and impulse to 78 (SD 4)%, 75 (SD 14)% and 84 (SD 7)% (P < 0.05) in normoxia, hypoxia and hyperoxia, respectively. A strong correlation between Fmax and VO2 was found in normoxia but not in hypoxia and hyperoxia. The mean sum-iEMG tended to be lower (P < 0.05) in hypoxia than in normoxia but hyperoxia had no significant effect on it. In general, F(I)O2 did not affect MPF of individual muscles. In conclusion, it was found that force output during ergometer rowing was impaired during hypoxia and improved during hyperoxia when compared with normoxia. Moreover, the changes in force output were only partly accompanied by changes in muscle electrical activity as sum-iEMG was affected by hypoxic but not by hyperoxic gas. The lack of a significant correlation between Fmax and VO2 during hypoxia and hyperoxia may suggest a partial uncoupling of these processes and the existence of other limiting factors in addition to VO2.     

Immunohistochemical localization of the neural cannabinoid receptor in rat brain

A sample of UK consumers (N = 311) was interviewed in order to identify the attitudinal, cognitive and involvement characteristics of probable early adopters of polyunsaturated fatty acid (PUFA) fed fish. Attitude to fish significantly influenced PUFA fish, premium price PUFA fish, PUFA salmon, PUFA eel and PUFA sturgeon purchase. Involvement in healthy eating influenced PUFA fish, premium price PUFA fish and PUFA salmon purchase. Cognitive style did not influence PUFA fish and premium price PUFA fish purchase; nor, contrary to earlier research, did cognitive style and involvement interact to influence intended PUFA fish purchases.     

Immunohistochemical localization of enkephalin in peripheral sensory axons in the rat

Impaired energy metabolism plays an important role in neuronal cell death after brain ischemia, and apoptosis has been implicated in cell death induced by metabolic impairment. In the present study, metabolic impairment was induced by 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase. In order to clarify the involvement of poly(ADP-ribosyl)ation and apoptotic pathway in 3-NP induced cell death, we examined poly(ADP-ribosyl)ation and the apoptosis related gene protein expression after systemic administration of 3-NP by immunohistochemistry. Poly(ADP-ribosyl)ation was evidently detected in the striatal lesion but not in any other region. Immunoreactive ratio of Bcl-2 to Bax significantly increased both in the striatum and cortex. The data suggest that striatal cell death involves poly(ADP-ribosyl)ation and also apoptotic pathway in part following administration of 3-NP.     

Immunohistochemical localization of caffeine-induced c-Fos protein expression in the rat brain

Although caffeine is the most widely used central nervous system stimulant, the neuronal populations and pathways mediating its stimulant effects are not well understood. Using c-Fos protein as a marker for neuronal activation, the present study investigated the pattern of c-Fos induction at 2 hours after low locomotor-stimulant doses (1, 5, 10, and 30 mg/kg, i.p.) of caffeine and compared them with those after a higher dose (75 mg/kg, i.p.) or saline injection in adult male rats. Fos-immunoreactive neurons were counted in selected nuclei across the entire brain. Caffeine induced an increase in locomotor activity in a dose-dependent manner up to doses of 30 mg/kg and a decline at 75 mg/kg. Quantitative analysis of Fos-immunoreactive neurons indicated that no structures showed significant Fos expression at doses below 75 mg/kg or a biphasic pattern of Fos expression, as in locomotion. In contrast, caffeine at 75 mg/kg induced a significant increase compared with the saline condition in the number of Fos-immunoreactive neurons in the majority of structures examined. The structures included the striatum, nucleus accumbens, globus pallidus, and substantia nigra pars reticulata and autonomic and limbic structures including the basolateral and central nuclei of the amygdala, paraventricular and supraoptic hypothalamic nuclei, periventricular hypothalamus, paraventricular thalamic nuclei, parabrachial nuclei, locus coeruleus, and nucleus of the solitary tract. The locomotor-enhancing effects of low doses of caffeine did not appear to be associated with significant Fos expression in the rat brain.     

Immunohistochemical study of microglia in the Creutzfeldt-Jakob diseased brain

Immunohistochemical techniques have been used to investigate microglial reaction in Creutzfeldt-Jakob diseased (CJD) brains. Autopsy cases of six patients with CJD and age-matched controls were studied. Formalin-fixed, paraffin-embedded brain tissue samples were stained with antibodies against major histocompatibility complex (MHC) class II antigen (Ag), leukocyte common antigen (LCA), CDw75, CD68 and glial fibrillary acidic protein. Of the patients with CJD, two with a subacute spongiform encephalopathic type and short-survival periods after onset of the disease showed an increased number of reactive microglia labeled with anti-MHC class II Ag or LCA in the affected cerebral cortex. In advanced cases of the panencephalopathic type of CJD, in which both cerebral atrophy and astrocytosis were marked, the increase of reactive microglia was small. Some vacuoles developing in the neuropil of the CJD patients were surrounded by MHC class II Ag- or LCA-immunoreactive microglial cells. The number of ramified microglia in the affected lesions was decreased, although their number in the hippocampus was not affected. These results indicate that microglia can frequently be involved in the process of CJD and may be activated at the early stage of the disease.     

Immunohistochemical localisation of alpha1B-adrenergic receptors in the rat iris

This article reports results from a meta-analysis on adult age differences in the negative priming effect (21 studies on identity negative priming and 8 on location negative priming). Both younger and older adults were found to be susceptible to the negative priming effect in identity and location tasks. Effect sizes were homogeneous for both tasks, indicating that the data are adequately described without reference to moderator variables. State trace analysis on identity tasks, in which mean latencies in negative priming conditions were regressed onto mean latencies in baseline conditions, showed (a) that in both age groups the negative priming effect is proportional rather than additive and (b) that the negative priming effect is smaller in older adults as compared with younger adults.     

Immunohistochemical localization of ORL-1 in the central nervous system of the rat

A novel member of the opioid receptor family (ORL-1) has been cloned from a variety of vertebrates. ORL-1 does not bind any of the classical opioids, although a high affinity endogenous agonist with close homology to dynorphin has recently been identified. We have generated a monoclonal antibody to the N-terminus of ORL-1 to map areas of receptor expression in rat central nervous system (CNS). Intense and specific immunolabeling was observed in multiple areas in the diencephalon, mesencephalon, pons/medulla, and spinal cord. In the telencephalon, intense labeling was observed in the neuropil throughout layers II-V in the neocortex, the anterior olfactory nuclear complex, the pyriform cortex, the CA1-CA4 fields and dentate gyrus of the hippocampus, and in many of the septal and basal forebrain areas. In contrast to other members of the opioid receptor family, light labeling for ORL-1 was observed in telencephalic areas such as caudate-putamen. In the cerebellum, ORL-1 immunoreactivity was only observed in the deep nuclei. Throughout the CNS the majority of labelling was localized to fiber processes and fine puncta, although labeled scattered perikarya were observed in a few brain areas such as the hilus dentate in the hippocampus and some nuclei in the brainstem and spinal cord. The present mapping study is consistent with the reported distribution of ORL-1 mRNA and provides the first immunohistochemical report on anatomical and cellular distribution of ORL-1 receptor in the rat CNS.     

Protein turnover in skeletal muscle of the diabetic rat: activation of ubiquitin-dependent proteolysis

The JCR:LA-cp rat is a unique strain that, if homozygous for the autosomal recessive cp gene, is obese and exhibits the metabolic syndrome of insulin resistance, hyperinsulinemia, and hypertriglyceridemia. Obese male rats spontaneously develop advanced atherosclerosis and ischemic myocardial lesions. The angiotensin-converting enzyme inhibitor, captopril, was administered to obese rats at 30 mg/kg body weight from 6 to 39 weeks of age. There were no significant changes in food consumption or body weights of the treated animals. Insulin sensitivity was not improved. Plasma insulin levels were unaltered, but the volume density of the islets of Langerhans was halved, reflecting both reduced hyperplasia and a more normal islet structure. Triglyceride concentrations were not reduced, but unesterified cholesterol and cholesteryl esters decreased by 50% and 34%, respectively (p < 0.01). The impaired nitric oxide-mediated vascular relaxation of the obese rats was not improved, and the relaxant sensitivity to acetylcholine as indicated by the median effective concentration (EC50) was reduced. In vitro, captopril significantly reduced the basal tension of aortic rings from untreated rats, antagonized the contractile effects of norepinephrine, and induced complete relaxation of the contraction in response to 10(-7) M norepinephrine. The severity of spontaneous, raised atherosclerotic lesions of the aortic arch at age 39 weeks was not significantly decreased by captopril treatment. In contrast, the frequency of ischemic myocardial lesions was reduced by 78% (p < 0.01). The protective effects of captopril on the heart and pancreas in this animal model of type II diabetes and atherosclerosis are probably the result of its bradykinin-enhancing effects.     

Effect of proteolysis on the state of lipid phase in rat brain synaptosomal membranes

Polymethylmethacrylate (PMMA) contact lenses can alter corneal shape and induce corneal warpage or distortion. The purpose of our study was to determine the effects on the corneal topography after immediate refitting of long-term PMMA contact lens wearers into rigid gas permeable (RGP) materials. Six eyes with contact lens induced corneal warpage from PMMA contact lenses were assessed using the Topographical Mapping System-1. Statistical analysis was performed for the following variables prior to and approximately 6 months after contact lens refitting: best spectacle visual acuity, manifest refraction, surface regularity index, surface asymmetry index, keratometry, and simulated keratometry. Best spectacle visual acuity improved an average of 1.8 +/- 1.0 (mean +/- SD, P < 0.05) lines of Snellen visual acuity, while refraction did not change appreciably. The surface regularity index diminished by 0.51 +/- 0.32 (P = 0.01). The surface asymmetry index improved by 0.32 +/- 0.26 (P < 0.05). There was a good correlation between keratometry and simulated keratometry, and neither changed significantly after refitting with RGP contact lenses. All general topographic patterns remained unchanged throughout the study. Immediate refitting of long-term PMMA contact lens wearers into RGP materials of similar fit allows a slightly more regular and symmetric central corneal shape, which can result in improved spectacle visual acuity. The general corneal topographic patterns of contact lens induced corneal warpage did not change or improve after refitting to RGP material.     

Identification of photooxidation sites in bovine alpha-crystallin

Because UV irradiation of proteins can produce reactive oxygen species and exposure to UV light has been implicated in cataractogenesis, the sites of photooxidation of bovine alpha-crystallin, a major lens protein with molecular chaperone activity, were identified using tandem mass spectrometry (MS/MS). Bovine alpha-crystallin was irradiated with UV light (> 293 nm) for 1, 4 and 8 h, digested with trypsin and analyzed by matrix-assisted laser desorption ionization, time-of-flight mass spectrometry (MALDI) to identify the oxidized sequences. Tryptic peptides were purified by reverse-phase HPLC and oxidized peptides were sequenced by MS/MS to determine the sites of oxidation. Tryptophan fluorescence decreased exponentially with increasing time of UV exposure and peptides containing residues 1-11 of alpha A-crystallin and 1-11, 12-22 and 57-69 of alpha B-crystallin were determined to be oxidized by shifts of 16 D or multiples of 16 Da above the mass of the unmodified peptide. The MALDI analysis revealed single oxidation of all four sequences, which increased with increasing time of UV exposure and possible double oxidation of alpha B 12-22. The specific sites of photooxidation indicate that the N-terminal regions of alpha A- and alpha B-crystallin are exposed to an aqueous environment and are in the vicinity of tryptophan residues from neighboring subunits.     

Immunohistochemical localization of somatostatin receptor SST2A in the rat pancreas

Somatostatin (SRIF) acts on specific membrane receptors to inhibit exocrine and endocrine pancreatic functions. Five SRIF receptor genes have been cloned, producing six receptor proteins (sst-s). We used a recently developed antibody to localize the sst2A splice variant in the rat pancreas. Western blots identified the sst2A receptor as an 90 kDa glycosylated protein in pancreatic tissue. In tyramide-amplified immunostainings all acinar cells, and the glucagon and pancreatic polypeptide immunoreactive cells (A and PP, respectively) were intensely labeled for sst2A, while no signal was detected in SRIF producing (D) cells. A very few insulin immunoreactive (B) cells were also labeled for sst2A, but the signal in these cells was lower than in exocrine, A or PP cells. Absorption of the sst2A antibody with the receptor peptide abolished specific staining in both immunoblots and tissue sections (negative control). These studies are the first to localize any SRIF receptor subtype in the rat pancreas. The specific localization of sst2A receptor in acinar, A and PP cells if confirmed in humans, would suggest that subtype specific analogs will be useful for the therapeutic regulation of exocrine and/or endocrine pancreatic secretion.     

Immunohistochemical analysis of rat S-adenosylmethionine synthetase isozymes in developmental liver

Ins(1,4,5)P3 receptors in adrenal cortical and cerebellar membranes can be distinguished by their affinities for Ins(1,4,5)P3 as well as the potencies with which heparin and Mg2+ inhibit binding. We have found that the differences in Ins(1,4,5)P3 affinity and heparin inhibition are maintained upon receptor solubilization and purification. In contrast to this, heparin-agarose affinity purification of solubilized cerebellar receptors reduces the potency of Mg2+ inhibition to that in adrenal cortex. These results suggest that Ins(1,4,5)P3 receptors in adrenal cortex are structurally distinct from those in cerebellum. Monoclonal antibodies raised against C- and N-terminal regions of mouse cerebellar Ins(1,4,5)P3 receptors recognize 250-300-kDa proteins in both rat cerebellum and bovine adrenal cortex.     

Immunohistochemical study on myoglobin in electrocution]

We have studied the changes of myoglobin (MB) IN heart muscle cells from eight corpses died of electrocution. The changes were compared with that of the heart muscle cells died of fall. The results indicated that the immunohistochemical changes of Mb were as follows: (1) blocky separate from heart muscle cell; (2) drift away into the interval of the heart muscle. The Mb changes were small parts of an area separate from heart muscle cells in control. The authors emphasized these changes were the characteristics of oxygen deficiency of the heart by electrocution.