STUDIES OF AMINES IN THE STRIATUM IN MONKEYS WITH NIGRAL LESIONS

The effects of ventromedial tegmental lesions on the biosynthesis and disposition of biogenic amines in the striatum of monkeys were investigated. The concentrations of endogenous dopamine and of the intraventricularly injected [³H]dopamine were distinctly lower in the striatum on the lesion side than on the intact side. The storage of [³H]dopamine in the caudate nucleus was impaired to a much greater extent than the storage of the newly synthesized [³H]norepinephrine. The concentrations of endogenous serotonin and of the intraventricularly injected [¹⁴C]serotonin were lower in the striatum on the lesion side than on the intact side. However following MAO inhibition, the concentration of [¹⁴C]serotonin did not differ significantly on the two sides of the caudate nucleus. The in vivo biosynthesis of dopamine from tyrosine was significantly reduced in the striatum on the lesion side. Tyrosine hydroxylase and DOPA decarboxylase activities were decreased on the lesion side of the striatum as compared with the intact side. Thus, the ventromedial tegmental lesions affect the storage and the synthesis of dopamine and serotonin in the ipsilateral striatum.     

Specificity and Sensitivity of Insulin Staining by Aldehyde Fuchsin, Pseudoisocyanin and Toluidine Blue

Aldehyde fuchsin, pseudoisocyanin and toluidine blue, histochemical dyes reported to be specific for insulin-containing granules of the pancreatic beta cell, were applied to insulin fixed in polyacrylamide gel by disc electrophoresis. Two major and four minor bands were resolved as demonstrated by staining with amidoschwarz; only the two major bands, were stained by aldehyde fuchsin. The addition of serum did not affect this reaction. Serum or insulin components gave no metachromatic reactions to the other stains. Under the conditions applied, aldehyde fuchsin is the only one of these dyes specific for insulin in this, system, but this stain is not sufficiently sensitive to detect normal serum levels of the hormone.     

Factors that promote progressive development of the osteoblast phenotype in cultured fetal rat calvaria cells

Rat calvaria osteoblasts derived from 21-day-old fetal rat pups undergo a temporal expression of markers of the osteoblast phenotype during a 5 week culture period. Alkaline phosphatase and osteocalcin are sequentially expressed in relation to collagen accumulation and mineralization. This pattern of expression of these osteoblast parameters in cultured rat osteoblasts (ROB) is analogous to that seen in vivo in developing fetal rat calvaria tissue (Yoon et. al: Biochem. Biophis. Res. Commun. 148:1129, 1987) and is similar to that observed in cultures of subcultivated 16-day-old embryonic chick calvaria-derived osteoblasts (COB) (Gerstenfeld, et.al: Dev. Biol. 122:46, 1987). While the cellular organization of subcultivated COB and primary ROB cultures are somewhat different, the temporal expression of the parameters remains. Both the rat and chick culture systems support formation of matrix mineralization even in the absence of beta-glycerol-phosphate. A systematic examination of factors which constitute conditions supporting complete expression of the osteoblast phenotype in ROB cultures indicate requirements for specific serum lots, ascorbic acid and the ordered deposition of mineral in the extracellular matrix. The present studies suggest that formation of a collagenous matrix, dependent on ascorbic acid, is requisite for expression of the osteoblast phenotype. In ROB cultures, expression of osteocalcin synthesis occurs subsequent to initiation of alkaline phosphatase activity and accompanies the formation of mineralized nodules. Thus, extracellular matrix mineralization (deposition of hydroxyapatite) is required for complete development of the osteoblast phenotype, as reflected by a 200-fold increase in osteocalcin synthesis. These data show the temporal expression of the various osteoblast parameters during the formation and mineralization of an extracellular matrix can provide markers reflective of various stages of osteoblast differentiation/maturation in vitro.     

Characterization of envelope proteins from Pasteurella haemolytica and Pasteurella multocida

A method was devised for the reproducible isolation of envelopes from Pasteurella haemolytica serotype A2. It was also possible to prepare envelopes from other serotypes of P. haemolytica and Pasteurella multocida using this methodology. Examination of these preparations by SDS-PAGE showed major differences between strains of P. haemolytica and strains of P. multocida which allowed the clear distinction of isolates of these species. Amongst the P. haemolytica serotypes it was possible to distinguish envelope preparations made from A biotype and T biotype organisms easily, but it was not possible to identify individual serotypes from each other. Envelope profiles were sufficiently different between the individual P. multocida serotypes examined to allow each to be identified by its polypeptide profile. Experiments using radiolabelling, antibody absorption, and susceptibility to protease digestion, together with heat modifiability and detergent solubility characteristics indicated that 13 of the envelope proteins were probably surface-located. A high molecular mass immunogenic envelope protein was shown, by immunoblotting, to be present in all strains of P. haemolytica and P. multocida examined.     

Binding of apoE-rich high density lipoprotein particles by saturable sites on human blood platelets inhibits agonist-induced platelet aggregation

High density lipoproteins (HDL, d 1.063-1.21 g/ml) are reported to stimulate, to have no effect on, or to inhibit agonist-induced platelet aggregation. We have hypothesized that these conflicting reports might be explained by opposing effects of individual HDL subclasses on platelet aggregability. Physiologic concentrations of HDL3 had little effect on ADP-induced aggregation of washed platelet suspensions, although higher levels were stimulatory. Normal concentrations of HDL2 (0.2-0.4 mg of protein/ml) inhibited aggregation; further fractionation by heparin-Sepharose chromatography identified the particles rich in apolipoprotein E, termed HDL-E, as the major anti-aggregatory subclass. Washed platelets bound radioiodinated HDL-E to a uniform class of saturable sites; they numbered 4,200 per platelet and the KD was 7.9 x 10(-7) M. Binding of HDL-E by platelets, and its anti-aggregatory action, showed a similar rapidity and both occurred within the physiologic concentration range. Moreover, the two processes were independent of the presence of divalent ions and were impaired by chemical modification of the apolipoprotein constituents of HDL-E. We conclude that occupation of cell-surface receptors by HDL-E particles impairs platelet responsiveness to exogenous agonists and that platelet aggregability in the presence of whole HDL may reflect the relative concentrations of the individual subclasses in the particular sample.     

The identification of a distinct export step following the biosynthesis of leukotriene C4 by human eosinophils

We have examined the requirements for the export of leukotriene C4 (LTC4) from cultured human eosinophils. To define saturability and kinetics of LTC4 export, eosinophils were interacted with leukotriene A4 (LTA4) at 37 degrees C, and the methanolic extracts of the cell-associated and extracellular compartments were then analyzed for LTC4 content by reverse phase high performance liquid chromatography with on-line monitoring of absorbance at 280 nm. When LTA4 was added at concentrations from 0 to 100 microM for 10 min at 37 degrees C, the amount of LTC4 released extracellularly became constant at an LTA4 concentration of 7.5 microM or greater even though the amount of intracellular LTC4 continued to increase. When eosinophils were incubated with 50 microM LTA4 for 0-60 min at 37 degrees C and then held at 0 degrees C for the remainder of the 60-min interval, 54.2 and 77.3% (n = 3), respectively, of the total LTC4 was released extracellularly after 15 and 30 min of incubation at 37 degrees C. Eosinophils incubated with 50 microM LTA4 at 0 degrees C for 1 h synthesized 290 pmol of LTC4 (n = 3) which was approximately half-maximal, all of which was retained intracellularly. We utilized the time and temperature dependence of LTC4 export to preload eosinophils with both LTC4 and leukotriene C5 (LTC5) by sequentially supplying them with specific substrates. With increasing concentrations of intracellular LTC5, there was dose-dependent inhibition of the subsequent release of LTC4 at 37 degrees C, with the sum of the released glutathionyl leukotrienes remaining constant. In addition, only minimal competition for LTC4 release occurred when cells were preloaded with both LTC4 and the conjugate of 1-chloro-2,4-dinitrobenzene and reduced glutathione, S-(dinitrophenyl)glutathione. The criteria of saturability, time dependence of LTC4 release at 37 degrees C, competition of LTC4 with LTC5 for release, and the inhibition of LTC4 release at 0 degrees C establish the export of LTC4 from cells as a novel and specific biochemical step distinct from both LTA4 uptake and the conjugation of LTA4 with reduced glutathione by LTC4 synthase to form LTC4.