Big Moose Basin: simulation of response to acidic deposition

The ILWAS model has been enhanced for application to multiple-lake hydrologic basins. This version of the model has been applied to the Big Moose basin, which includes Big Moose Lake and its tributary streams, lakes, and watersheds. The basin, as defined, includes an area of 96 km², with over 20 lakes and ponds, and 70 km of streams. Hydrologic and chemical calibrations have been made using data from seven sampling stations. When total atmospheric sulfur loading to the basin is halved, the model predicts, after four years of simulation, a decreasing sulfate concentration and to a lesser extent a rising alkalinity at Big Moose Lake outlet. At the end of four years, the results show an increase in pH of 0.1 to 0.5 pH units depending upon season.     

Renal handling of taurine,l-alanine,l-glutamate andd-glucose inOpsanus tau: studies on isolated brush border membrane vesicles

Summary Renal brush border membrane vesicles (bbmv) from the aglomerular toadfish (Opsanus tau), isolated by differential precipitation, were tested for their ability to actively translocate (i) taurine, known to be secreted by the kidney of several marine teleosts, and (ii)l-alanine,l-glutamic acid, andd-glucose, solutes that are normally reabsorbed in the filtering nephron. Vesicular taurine uptake displayed a Na⁺ dependence. Transport was greatest under conditions of an inward-directed Na⁺ gradient, but a significant stimulation by Na⁺ over K⁺ could also be observed in the absence of a salt gradient. At high extravesicular K⁺, the addition of valinomycin reduced taurine uptake. Na⁺-dependent³H-taurine flux was almost completely inhibited by non-labeled taurine (tracer replacement) or -alanine, but was unaffected byl-alanine. Replacement of medium chloride by SCN^– or NO ₃ ^– in the presence of Na⁺ resulted in significantly lower uptake rates under both anion gradient and anion equilibrium conditions, whereas Br^– could almost fully substitute for the stimulatory Cl^– action. These results indicate the presence of an electrogenic Na⁺-cotransport mechanism with specificity for -amino acids in the toadfish renal brush border. Whether the system under physiological conditions mediates reabsorption or secretion of taurine remains to be determined. Toadfish bbmv also translocatedl-alanine andl-glutamic acid in a Na⁺-dependent manner. Possible roles for these most likely reabsorptive transport systems in a non-filtering kidney are discussed.d-glucose uptake, however, appeared to occur via Na⁺-independent pathways, since it was not affected by phlorizin in the presence of Na⁺, or by Na⁺ replacement.Abbreviation bbmv brush border membrane vesicles     

Formation of a barrier by brain microvessel endothelial cells in culture

Endothelial cells (EC) isolated from bovine brain microvessels produce a continuous monolayer when grown in primary culture. The EC are joined together by tight junctions and contain few pinocytotic vesicles. Horseradish peroxidase (HRP) is unable to penetrate this in vitro barrier system. Exposure of the cells to 1.6 M arabinose produces a reversible separation of the tight junctions with penetration of HRP across the monolayer in a pattern similar to that observed in animals after infusion of hyperosmotic solutions into the carotid artery. The behavior of brain microvascular cells in culture suggest that they retain properties important to the formation of the blood-brain barrier.     

Age-related changes in oxidized proteins

We have previously described the oxidative inactivation of several key metabolic enzymes by a variety of mixed function oxidation systems. Because many of the enzymes which are inactivated have been shown by others to accumulate as inactive or less active forms during cellular aging, we have examined the levels of oxidatively modified proteins in two model systems used for studies on aging. The results show that levels of oxidatively modified proteins increase with age in circulating erythrocytes, and this change is correlated with the loss of marker enzyme activity. Our studies also show that in cultured fibroblasts from normal donors the levels of oxidatively modified proteins increase only after the age of 60. However, the levels of oxidatively modified proteins in fibroblasts from individuals with progeria or Werner's syndrome are significantly higher than age-matched controls. Moreover, treatment of glucose-6-phosphate dehydrogenase with a mixed function oxidation system leads to oxidative modification and increased heat lability of the enzyme. Taken together these results suggest that loss of functional enzyme activity and increased heat lability of enzymes during aging may be due in part to oxidative modification by mixed function oxidation systems.     

Cell-free detection and characterization of a novel nerve growth factor-activated protein kinase in PC12 cells

We have developed a cell-free assay to detect and characterize nerve growth factor (NGF)-activated protein kinase activity. Cultured PC12 cells were briefly exposed to NGF, and extracts of these were assayed for phosphorylating activity using exogenously added tyrosine hydroxylase as substrate. Tyrosine hydroxylase was employed since it is an endogenous substrate of NGF-regulated kinase activity and is activated by phosphorylation. In the cell-free assay, extracts prepared from NGF-treated cells yielded a 2-3-fold greater incorporation of phosphate into tyrosine hydroxylase as compared with extracts of control, NGF-untreated cells. Activation did not occur, however, if NGF was added directly to cell extracts. The NGF-stimulated phosphorylating activity appeared to be due to regulation of a protein kinase rather than of a phosphoprotein phosphatase. Characterization of the kinase (designated as kinase N) showed that it is soluble, is detectably activated within 1-3 min after cells are exposed to NGF and maximally activated by 10 min, is half-maximally activated with 0.5 nM NGF and maximally activated with 1 nM NGF, is detectable in the presence of either Mg2+ or Mn2+ but does not require Ca2+, does not require nonmacromolecular cofactors, can use histone H1 as a substrate, and exhibits a 2-fold increase in apparent Vmax in response to NGF but does not undergo a significant change in apparent Km for either ATP or GTP. A number of characteristics of kinase N were assessed including susceptibility to inhibitors, substrate specificity, cofactor requirements, ATP dependence, and lack of down-regulation by prolonged expose to a phorbol ester. These studies indicated that it lacks tyrosine kinase activity and is distinct from a variety of well-characterized protein kinases including cAMP-dependent protein kinase, protein kinase C (Ca2+/phospholipid-dependent enzyme), Ca2+/calmodulin-dependent kinase, and casein kinase II. Preliminary purification data show that the kinase has a basic pI and that it has an apparent Mr of 22,000-25,000. The only amino acid in tyrosine hydroxylase found to be phosphorylated by the semipurified kinase is serine.     

Detection and partial characterization of a human mast cell carboxypeptidase

Using a high performance liquid chromatography assay that detects the cleavage of the C-terminal leucine from angiotensin I, we have identified a carboxypeptidase activity in mast cells from human lung and in dispersed mast cell preparations from human skin. The enzyme activity was detected in a preparation of dispersed human mast cells from lung of greater than 99% purity and was released with histamine after stimulation with goat anti-human IgE. In nine preparations of dispersed human mast cells from lung of 10 to 99% purity, net percentage of release of carboxypeptidase correlated with the release of histamine, localizing carboxypeptidase to mast cell secretory granules. The enzyme activity was also detected in preparations of dispersed human mast cells from skin and in extracts of whole skin. The inhibitor profile and m.w. of carboxypeptidase activity from preparations of dispersed mast cells from skin was similar to that from dispersed mast cells from lung. Mast cell carboxypeptidase had a m.w. on gel filtration of 30,000 to 35,000. The enzyme in crude lysates of dispersed mast cell preparations had optimal activity between pH 8.5 and 9.5 and was inhibited by potato inhibitor, which distinguished it from carboxypeptidase in cultured human foreskin keratinocytes and adult fibroblasts, and from other proteolytic mast cell enzymes. The enzyme activity was also inhibited by EDTA, o-phenanthroline, and, to a small extent, by 8-OH quinoline, but not by Captopril, soybean trypsin inhibitor, or pepstatin. These findings demonstrate that human mast cell secretory granules contain carboxypeptidase in addition to tryptase and chymase. It appears that mast cells from skin may have a higher content of carboxypeptidase than do mast cells from lung.     

Variation in insulin receptor messenger ribonucleic acid expression in human and rodent tissues

The expression of insulin receptor mRNA was studied in human and rodent tissues by Northern analysis. Human EBV-transformed lymphocytes contained four receptor mRNA species of sufficient length to encode the entire proreceptor: 9.5, 7.9, 7.1, and 5.7 kb. In human fibroblasts, the same four species were observed; however, the 7.9 and 5.7 kb mRNAs were markedly decreased. In mouse liver, rat hepatoma cells, and normal rat brain, kidney, liver, and muscle only two mRNA species (7.4 and 9.6 kb) were detected. Each of these human and rodent mRNAs hybridized equally well with cDNA sequences encoding the binding and kinase domains of the insulin receptor. Several smaller polyadenylated mRNAs (approximately 1.8 to 3.3 kb) were also identified in human cell lines that appeared to separately encode either alpha- or beta-subunit sequences of the receptor. In rats, liver had the highest content of insulin receptor mRNA, followed by kidney, brain, and muscle. The relative amount of the two mRNA species also varied among the rat tissues. The ratio of the 9.6-7.4 kb species was 2.7 in brain but only 1.0 to 1.6 in the other tissues (P less than 0.025). Dexamethasone treatment increased the content of the two insulin receptor mRNAs in rat liver by 2-fold. The half-life of both mRNA species was 70 min in rat hepatoma cells. These findings indicate that insulin receptor gene expression is complex and regulated with differential expression of insulin receptor mRNA and/or alterations in mRNA processing among various tissues.     

Phenotypic differences in expression of cytochrome P-450g but not its mRNA in outbred male Sprague-Dawley rats

Cytochrome P-450g was isolated from livers of adult male Sprague-Dawley (CD) rats. Antibody to P-450g cross-reacted with several proteins in Western blots of liver microsomes from male CD rats. An immunospecific antibody was prepared by adsorption over immunoaffinity columns of Sepharose-bound solubilized rat liver microsomes from female CD and male Fischer 344 rats containing little or no P-450g. The immunopurified antibody recognized a single protein on Western blots of liver microsomes from male CD rats with an electrophoretic mobility identical to that of P-450g. Using this antibody, P-450g was shown to be male specific in the CD rat and expressed at maturity. Adult male CD rats were shown to fall into two distinct populations, those expressing high levels of P-450g (+g) and those expressing low levels of P-450g (-g). The P-450g content of the two populations differed 10- to 20-fold. P-450g was low or absent in liver microsomes of both sexes of adult Fischer rats. Purified P-450g catalyzed the hydroxylation of testosterone and androstenedione principally at the 6 beta-position and progesterone at the 16 alpha- and 6 beta-positions in reconstituted systems. However, the hydroxylation of these steroids by liver microsomes from the (+g) phenotype did not differ from that of the (-g) phenotype. Translatable mRNA for P-450g could be detected in livers of adult male CD rats but not female rats. However, the level of P-450g mRNA in livers of adult male CD rats with the (+g) phenotype did not differ from that of (-g) phenotype. These data suggest that phenotypic differences in the expression of P-450g do not depend on differences in mRNA content. This study provides a clear example of a P-450 isozyme which is markedly variable in an outbred strain of rat and absent in an inbred strain. Such a marked variability in an enzyme involved in metabolism of endogenous and exogenous substrates could account for some of the strain differences in susceptibility to toxic chemicals.     

Immunological detection of low-density lipoproteins modified by malondialdehyde in vitro or in vivo

We describe an ELISA technique able to recognize malondialdehyde-modified low-density lipoproteins (LDL). For this purpose we produced antibodies to malondialdehyde-LDL, specific for the malondialdehyde modification of LDL; these antibodies recognized essentially malondialdehyde-LDL. Coating ELISA plates with the antibodies to malondialdehyde-LDL and using peroxidase-labelled antibodies to LDL, which reveal only apolipoprotein B, we obtained an accurate method of detecting malondialdehyde-modified apolipoprotein B. Preliminary studies demonstrated that this method allows the detection of lipoproteins containing malondialdehyde-modified apolipoprotein B in the serum of patients with cardiovascular diseases.     

The effect of receptor density on the forward rate constant for binding of ligands to cell surface receptors.

For monovalent ligands interacting with cell surface receptors we have directly observed the functional dependence of the forward rate constant on the number of receptors per cell (N). The experimental system we studied consisted of monovalent ligand, 2,4-dinitrophenyl (DNP)-aminocaproyl-L-tyrosine (DCT), binding to bivalent, monoclonal anti-DNP immunoglobulin E (IgE) anchored to its high affinity receptor on rat basophilic leukemia (RBL) cells. To measure the fractional occupation of antibody combining sites by DNP we employed a recently developed fluorescence technique (Erickson, J., Kane, B. Goldstein, D. Holowka, and B. Baird, 1986, Mol. Immunol., 72:769-781). Our results are well fitted by the equation (Berg and Purcell, 1977, Biophys. J., 20:193-219) konc = 4 pi DaN kappa on/[4 pi Da + N kappa on] where konc is the forward rate constant for binding to the cell, D is the diffusion constant of the ligand, a is the radius of the cell, and kappa on is the intrinsic forward rate constant describing a single IgE combining site-DNP interaction. If D is fixed at 10(-5) cm2/s, the best fit of accumulated data predicts an average cell radius of approximately 4 microns and kappa on of approximately 1.8 x 10(-13) cm3/s [1.1 x 10(8)(M . s)-1]; both in excellent agreement with RBL cell size and the single-site forward rate constant for the binding of DCT to IgE in solution, respectively. We believe this is the first report of experimental evidence that directly illustrates the effect of surface density in determining the rates of binding for small molecules to membrane receptors.