Catecholaminergic contributions to the neuronal machinery of the olfactory bulb: aftoradiographic, immunohistochemical and evoked feild potential studies

aftographic exeperiments on the localization of radiolabelednoradrenaline, dopamine and dopa, as well as immunohistochemicalstudies on hydroxylase-like activity, are summarized and comparedin both rat and turtle olfactory bulbs. Evoked field potentialstudies on effects of dopamine are also discussed. The histochemicalstudies suggest that dopaminergic periglomerular neurons arethe most significant cellular component of the catecholaminergicsystem in the olfactory bulb of both species. Scattered fluorescentcell group was also present in the internal plexiform layerand superficial granule cell layer of the turtle olfactory bulb.Other fibres, not related to intrinsic bulbar neuronal cellbodies, were also labeled, mostly in the granule cell layerbut also in the external plexiform layer. These might belongto a centrifugal catecholaminergic system from brain stem neurons.In the in vitro turtle olfactory bulb, dopamine and apomorphinedepressed the amplitude of field potentials evoked by a singlevolley in the olfactory nerve or lateral olfactory tract, andreduced the depression and latency of reponses when paired volleywere delivered. It is suggested that catecholaminergic systemsplay a key role in modulating mitral cell activity through actionsin both superficial (glomerular) and deep (granule) layers.This may involve direct actions, or other, non-catecholaminergicinterneurons.     

Inducible (class 3) aldehyde dehydrogenase from rat hepatocellular carcinoma and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated liver: distant relationship to the class 1 and 2 enzymes from mammalian liver cytosol/mitochondria

Peptides from rat liver aldehyde dehydrogenase (AIDH) induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment match the AIDH structure from HTC rat hepatoma cells (HTC-AIDH) at all positions examined, indicating induction of the same gene product by two independent routes. This 452 amino acid residue, class 3 AIDH structure differs substantially from the 500-residue AIDH structures isolated from normal liver cytosol (class 1) and mitochondria (class 2). Despite a 29.8% identity in 429 overlapping amino acids vs the human class 1 enzyme (27.7% vs class 2), neither the N- nor C-termini coincide, and gaps are introduced to optimize the alignment. Two residues placed in the active site of human liver AIDH by chemical modification, Cys-302 and Glu-268, are conserved in class 3 AIDH as Cys-243 and Glu-209. Cys-243/302 is the only cysteine residue conserved in all known AIDH structures. Gly-245 and Gly-250 of class 1/2 AIDHs, fitting the patterns of glycine residues in coenzyme binding fold of other dehydrogenases, are also conserved. Otherwise, Cys-49, Cys-162, and Glu-487, to which functional importance has also been ascribed, are not retained in the class 3 structure. Overall, a high conservation of Gly, Pro, and Trp and similar patterns of predicted secondary structure indicate general conservation of tertiary structure, as noted with other distantly related proteins. Three exon boundaries from the human liver mitochondria AIDH gene directly correspond to the N-terminus of the rat class 3 protein and to two of the gaps in the alignment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ntau-methylhistidine excretion is a valid index of myofibrillar protein breakdown in the guineapig (Cavia porcellus).

The recovery of radioactivity in the urine of guineapigs following a bolus intravenous dose of chromatographically pure 14C-Ntau-methylhistidine was measured in order to test whether the excretion of Ntau-methylhistidine (Ntau-MH) is a valid index of myofibrillar protein breakdown in these animals. Four male and four female guineapigs were dosed and after 7 days, 91.65+/-2.82% and 3.58+/-0.91% of injected radioactivity was recovered in the excreta and tissues, respectively. The average total recovery of 95.2+/-3.0% was not significantly different from 100%. Male guineapigs excreted the radioactivity more slowly than females (70% of the dose excreted within 74 h vs 39 h, respectively) but cumulative excretion at 7 days was the same for each sex. Chromatographic analysis of the urine showed almost all of the radioactivity to be associated with a single peak corresponding to Ntau-MH, indicating a lack of significant metabolism. These data show that although the clearance of 14C-Ntau-MH is slower than in rats or humans the urinary excretion of Ntau-MH is a valid index for myofibrillar protein degradation in the guineapig.     

Increased Concentrations of 3-Hydroxykynurenine in Vitamin B6 Deficient Neonatal Rat Brain

Increased concentrations of the endogenous tryptophan metabolite 3-hydroxykynurenine (3-HK) were measured in the brains of vitamin B6 deficient neonatal rats. Mean concentrations of 3-HK in B6 deficient cerebellum, corpus striatum, frontal cortex, and pons/medulla ranged from 9.7 to 18.6 and 102 to 142 nmol/g of wet tissue at 14 and 18 days of age, respectively. 3-HK was not significantly increased in control neonatal or adult rat brain, vitamin B6 deficient rat brain at 7 days of age, or in brains from adult rats deprived of vitamin B6 for 58 days. The administration of daily intraperitoneal injections of vitamin B6 from the 14th to the 18th day of age decreased the concentration of 3-HK to control levels. 3-HK has been shown by other investigators to produce seizures when injected into the cerebral ventricles of adult rodents. Thus, our studies show the accumulation in brain of a putative endogenous convulsant as the result of a nutritional deficiency.     

Degradation of heparin proteoglycan in cultured mouse mastocytoma cells.

Pulse-labelling of mouse mastocytoma cell cultures, established from ascites fluid, with inorganic [35S]sulphate for 1 h yielded labelled heparin proteoglycan containing polysaccharide chains of Mr 60,000-100,000. After chase incubation for 24 h most of the 35S appeared in intracellular polysaccharide fragments similar in size to commercially available heparin, Mr 5000-25,000, as indicated by gel chromatography. Products isolated from cultures after 6 h of chase incubation consisted of partially degraded free polysaccharide chains and, in addition, residual proteoglycans that were of smaller size than the proteoglycans initially pulse-labelled. The polysaccharide chains released by alkali treatment from the residual chase-incubated proteoglycans were of the same size as the chains derived from proteoglycans after 1 h of pulse labelling. These results suggest that the intracellular degradation of heparin proteoglycan to polysaccharide fragments is initiated by release of intact polysaccharide chains, probably by action of a peptidase, and is pursued through cleavage of these chains by an endoglycosidase. An endoglucuronidase with stringent substrate specificity [Thunberg, Bäckström, Wasteson, Ogren & Lindahl (1982) J. Biol. Chem. 257, 10278-10282] has previously been implicated in the latter step. Cultures of more purified mastocytoma cells (essentially devoid of macrophages) did not metabolize [35S]heparin proteoglycan to polysaccharide fragments, but instead accumulated free intact polysaccharide chains, i.e. the postulated intermediate of the complete degradation pathway. When such purified cells were co-cultured with adherent mouse peritoneal cells, presumably macrophages, formation of polysaccharide fragments was observed. It is tentatively proposed that the expression of endoglucuronidase activity by the mast cells depends on collaboration between these cells and macrophages.     

M?ssbauer, EPR, and optical studies of the corrinoid/iron-sulfur protein involved in the synthesis of acetyl coenzyme A by Clostridium thermoaceticum

We have purified to homogeneity the 88-kDa corrinoid protein from Clostridium thermoaceticum which acts as a methyl carrier in the synthesis of acetyl-CoA. As shown here, this protein contains a [4Fe-4S]1+/2+ cluster in addition to a corrinoid. The corrinoid is 5-methoxybenzimidazolylcobamide, with an OH- group probably present as the upper axial ligand. Co+ is present in the reduced form, Co2+ in the as-isolated form, and Co3+ in the methylated form of the protein. The as-isolated corrinoid/Fe-S protein exhibits a Co2+ EPR signal lacking nitrogen superhyperfine splittings, indicating that the benzimidazole base is uncoordinated ("base-off") in the Co2+ state. Optical studies suggest that the Co3+-CH3 corrinoid is also base-off. In the as-isolated and methylated forms, the iron-sulfur cluster is diamagnetic, with quadrupole splittings and isomer shifts characteristic of [4Fe-4S]2+ clusters. The protein can be reduced by CO and CO dehydrogenase in the absence of ferredoxin. The EPR spectra of the reduced cluster exhibit two components: one with principal g-values at 2.07, 1.93, and 1.82 and the other at 2.02, 1.94, and 1.86. The M?ssbauer data show that these signals result from [4Fe-4S]1+ clusters. Chemical analysis shows that the iron:cobalt atomic ratio is close to 4:1, suggesting that a single [4Fe-4S]1+ cluster occurs in two distinct S = 1/2 spin states in the reduced state. Treatment with 1-2.5 M urea converts the two cluster forms into a single one, with EPR and M?ssbauer spectra of typical [4Fe-4S]1+ clusters. A 27-kDa corrinoid protein (Ljungdahl, L.G., LeGall, J., and Lee, J.P. (1973) Biochemistry 12, 1802-1808) also was purified and found to be inactive in the synthesis of acetyl-CoA, contrary to the suggestion of Ljungdahl et al. (1973).     

Rat hepatocytes in serum-free primary culture elaborate an extensive extracellular matrix containing fibrin and fibronectin

Adult rat hepatocytes cultured on type IV collagen, fibronectin, or laminin and maintained in serum-free medium were examined by indirect immunofluorescence using polyclonal antibodies against extracellular matrix proteins. An extensive fibrillar matrix containing fibronectin and fibrin was detected in all hepatocyte cultures irrespective of the exogenous matrix substratum used to support cell adhesion. Fibrils radiated from the cell periphery and covered the entire culture substratum. In addition, thicker fibers or bundles of fibers were localized on top of hepatocytes. This matrix did not contain laminin or the major types of collagen found in the liver biomatrix (types I, III, and IV). Isolation of the fibrillar matrix and analysis on polyacrylamide gels under reducing conditions demonstrated a major 58-kD polypeptide, derived from beta-fibrinogen as indicated by immunoblotting and two-dimensional peptide mapping. Plasmin rapidly dissolved the matrix. Deposition of the fibrin matrix in hepatocyte cultures was arrested by hirudin, by specific heparin oligosaccharides that potentiate thrombin inhibition by antithrombin III, and by dermatan sulfate, an activator of heparin cofactor II-mediated inhibition of thrombin. The results indicate that hepatocytes in culture synthesize and activate coagulation zymogens. In the absence of inhibitory and fibrinolytic mechanisms, a fibrin clot is formed by the action of thrombin on fibrinogen. Fibronectin attaches to this fibrin clot but fails to elaborate a fibrillar matrix on its own in the presence of coagulation inhibitors.     

Carbohydrate receptor specificity of K99 fimbriae of enterotoxigenicEscherichia coli

K99 Fimbriae from enterotoxigenicEscherichia coli (ETEC) were found to bind specifically to sialic acid, as measured in a haemagglutination inhibition assay using the intact bacteria and human erythrocytes. The affinity forN-glycolylneuraminic acid was about twice that ofN-acetylneuraminic acid (NeuAc), and other monosaccharides were found to be at least ten-fold less effective as inhibitors. The specificity was found to depend on electrostatic interaction where the carboxyl group and its orientation plays an important role. 2--Benzyl-NeuAc was a better inhibitor than 2--methyl-NeuAc suggesting a hydrophobic patch near the binding site on the protein. Axially oriented hydroxyl groups as in 4-epi-NeuAc and 3-hydroxy-NeuAc seemed to participate in binding since these derivatives were better inhibitors thanN-acetylneuraminic acid. K99 was found to have a higher affinity for 4-O-acetyl-NeuAc and lower affinity forN-acetylneuraminic acid withO-substituents at C7-C9 as compared toN-acetylneuraminic acid. Hence, the degree ofO-acetylation of sialic acid in the mucosa of the small intestine may influence colonization and determine susceptibility to infection.