Modelling the influence of land-use changes on biophysical and biochemical interactions at regional and global scales.

Land-use changes since the start of the industrial era account for nearly one-third of the cumulative anthropogenic CO2 emissions. In addition to the greenhouse effect of CO2 emissions, changes in land use also affect climate via changes in surface physical properties such as albedo, evapotranspiration and roughness length. Recent modelling studies suggest that these biophysical components may be comparable with biochemical effects. In regard to climate change, the effects of these two distinct processes may counterbalance one another both regionally and, possibly, globally. In this article, through hypothetical large-scale deforestation simulations using a global climate model, we contrast the implications of afforestation on ameliorating or enhancing anthropogenic contributions from previously converted (agricultural) land surfaces. Based on our review of past studies on this subject, we conclude that the sum of both biophysical and biochemical effects should be assessed when large-scale afforestation is used for countering global warming, and the net effect on global mean temperature change depends on the location of deforestation/afforestation. Further, although biochemical effects trigger global climate change, biophysical effects often cause strong local and regional climate change. The implication of the biophysical effects for adaptation and mitigation of climate change in agriculture and agroforestry sectors is discussed.

Simulating the effects of climate change on the carbon balance of North American high-latitude forests.

The large magnitude of predicted warming at high latitudes and the potential feedback of ecosystems to atmospheric CO2 concentrations make it important to quantify both warming and its effects on high-latitude carbon balance. We analysed long-term, daily surface meteorological records for 13 sites in Alaska and north-western Canada and an 82-y record of river ice breakup date for the Tanana River in interior Alaska. We found increases in winter and spring temperature extrema for all sites, with the greatest increases in spring minimum temperature, average 0.47 °C per 10 y, and a 0.7-day per 10 y advance in ice breakup on the Tanana River. We used the climate records to drive an ecosystem process model, BIOME_BGC, to simulate the effects of climate change on the carbon and water balances of boreal forest ecosystems. The growing season has lengthened by an average of 2.6 days per 10 y with an advance in average leaf onset date of 1.10 days per 10 y. This advance in the start of the active growing season correlates positively with progressively earlier ice breakup on the Tanana River in interior Alaska. The advance in the start of the growing season resulted in a 20% increase in net primary production for both aspen (Populus tremuloides) and white spruce (Picea glauca) stands. Aspen had a greater mean increase in maintenance respiration than spruce, whereas spruce had a greater mean increase in evapotranspiration. Average decomposition rates also increased for both species. Both net primary production and decomposition are enhanced in our simulations, suggesting that productive forest types may not experience a significant shift in net carbon flux as a result of climate warming.

Reactivity of sulfhydryl groups of the catalytic subunits of rabbit skeletal muscle protein phosphatases 1 and 2A.

Chemical modification of the sulfhydryl residues of the catalytic subunits of protein phosphatases 1 and 2A was studied. Both enzymes were inactivated by a variety of thiol group reagents. Mercurial compounds were the most effective inactivators. Of the alkylating agents the maleimides were more effective than iodoacetate or iodoacetamide which were relatively slow reacting. Both enzymes were also inactivated by disulfides, including glutathione disulfide, 5,5'-dithiobis(2-nitrobenzoic acid), and 4,4'-dipyridyl disulfide. The latter two were much more reactive than glutathione disulfide and, in addition, displayed a significant differential reactivity toward phosphatase 1. The apparent second-order rate constants for the inactivation of phosphatase 1 were 20- to 70-fold higher than for phosphatase 2A with 5,5'-dithiobis(2-nitrobenzoic acid) and 4,4'-dipyridyl disulfide. Kinetic analysis indicated that inactivation of both enzymes could be correlated with the modification of one cysteine per one mole of enzyme.

Measurement of protein phosphatase activity in biological samples using synthetic phosphopeptides.

A method has been developed for measuring specific protein phosphatase activity in biological samples using synthetic, phospho-Kemptide and phospho-GS-peptide. This method uses ion-exchange chromatography to determine phosphatase activity by quantifying the release of [32P]phosphate directly. The method was used to measure phosphatase activity of rat kidney, adrenals, heart, and liver cytosol and the activity of purified alkaline phosphatases, protein phosphatase 1, and protein phosphatase 2A. Ion-exchange chromatography was also used for the preparation of the radiolabeled phosphopeptide substrates. This method results in high recovery and specific activity of the labeled peptides. These techniques should be useful in isolating and characterizing specific protein phosphatases found in cells.

Effect of parathyroid hormone on rat renal calcium/calmodulin-dependent protein kinase II.

Rat parathyroid hormone (PTH) stimulates cAMP-dependent protein kinase and protein kinase C activity in the kidney. However, PTH increases intracellular Calcium in primary cultures of proximal tubular cells. We have investigated the possibility that PTH also stimulates Calcium/calmodulin-dependent protein kinase II (CaM kinase II). We have employed the tandem chromatographic column method, using synthetic peptide as a substrate, to measure the renal CaM kinase II activity. PTH (250 nM) stimulated CaM kinase II activity by about 50% after 15 sec., and activity returned to baseline by 2 min. Calmodulin antagonists significantly impaired the stimulatory action of PTH whereas basal levels of CaM kinase II activity were relatively unaffected. This study demonstrates that PTH does activate CaM kinase II in renal tissue, and suggests another pathway for the actions of PTH in the kidney.

Characterization and regulation of the vitamin D hydroxylases.

The metabolism of vitamin D is regulated by three major cytochrome P450-containing h hydroxylases-the hepatic 25-hydroxylase, the renal 1α-hydroxylase, and the renal and intestinal 24-hydroxylase. In the liver, the 25-hydroxylation reaction is catalyzed by microsomal and mitochondrial cytochrome P450cc25. The microsomal P450 accepts electrons from the NADPH-cytochrome P450 reductase, and the mitochondrial P450 accepts electrons from NADPH-ferredoxin reductase and ferredoxin. In the kidney, the 1α- and 24-hydroxylation reactions are catalyzed by mitochondrial cytochromes P450cc1α and P450cc24, respectively. The 24-hydroxylase is also found in vitamin D target tissues such as the intestine. The rat hepatic mitochondrial P450cc25 and the rat renal mitochondrial P450cc24 have been purified, and their cDNAs have been cloned and sequenced. 1,25-Dihydroxyvitamin D, the active metabolite of vitamin D, markedly stimulates renal P450cc24 mRNA and 24-hydroxylase activity in the intact animal and in renal cell lines. This stimulation occurs via a receptor-mediated mechanism requiring new protein synthesis. Despite the availability of a clone, no studies have yet been reported of the regulation of hepatic P450cc25 at the mRNA level. The study of one of the most important enzymes in vitamin D metabolism, the renal 1α-hydroxylase which produces the active metabolite, awaits the definitive cloning of the cDNA for the P450cc1α.

Effect of parathyroid hormone on rat renal cAMP-dependent protein kinase and protein kinase C activity measured using synthetic peptide substrates.

The actions of parathyroid hormone (PTH) on the renal cortex are thought to be mediated primarily by cAMP-dependent protein kinase (PKA) with some suggestion of a role for protein kinase C (PKC). However, present methods for assaying PKA and PKC in subcellular fractions are insensitive and require large amounts of protein. Recently, a sensitive method for measuring the activity of protein kinases has been reported. This method uses synthetic peptides as substrates and a tandem chromatographic procedure for isolating the phosphorylated peptides. We have adapted this method to study the effect of PTH on PKA and PKC activity using thin slices of rat renal cortex. PTH (250 nM) stimulated cytosolic PKA activity four- to fivefold within 30 s, and PKA activity was sustained for at least 5 min. PTH also rapidly stimulated PKC activity in the membrane fraction and decreased PKC activity in the cytosol. These changes were maximal at 30 s, but unlike changes in PKA, they declined rapidly thereafter. PTH significantly activated PKC only at concentrations of 10 nM or greater. This study demonstrates that PTH does activate PKC in renal tissue, although the duration of activation is much less than for PKA. It also demonstrates that a combination of synthetic peptides with tandem chromatography can be used as a sensitive assay procedure for protein kinase activity in biological samples.

Phosphorylation of ferredoxin and regulation of renal mitochondrial 25-hydroxyvitamin D-1 alpha-hydroxylase activity in vitro.

The kidney is the principal physiologic site of production of biologically active 1,25-dihydroxyvitamin D. The 25-hydroxyvitamin D-1 alpha-hydroxylase (1-OHase) activity found in renal mitochondria is under tight hormonal control. Parathyroid hormone stimulates the renal conversion of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D in young animals, which is accompanied by dephosphorylation of ferredoxin (Fx), a component of the mitochondrial 1-OHase enzyme complex (Siegel, N., Wongsurawat, N., and Armbrecht, H. J. (1986) J. Biol. Chem. 261, 16998-17003). The present study investigates the capacity of Fx to be phosphorylated in vitro and to modulate the 1-OHase activity of a reconstituted system. Fx was phosphorylated by renal mitochondrial type II protein kinase. Phosphorylation did not alter Fx mobility on sodium dodecyl sulfate gels but did decrease the pI as measured by isoelectric focusing. Amino acid analysis demonstrated that 1 mol of serine and 1 mol of threonine were phosphorylated per mol of Fx. Peptide mapping of phosphorylated Fx was consistent with phosphorylation of serine 88 and threonine 85 or 97. Fx was selectively dephosphorylated by rabbit skeletal muscle protein phosphatase C2 but not C1. Phosphorylation of Fx significantly inhibited the 1-OHase activity of a reconstituted system consisting of Fx reductase, Fx, and renal mitochondrial cytochrome P-450. These findings suggest that phosphorylation/dephosphorylation of Fx may play a role in modulating renal 1,25-dihydroxyvitamin D production.

Isolation and characterization of rabbit skeletal muscle protein phosphatases C-I and C-II.

Previous studies have shown that phosphorylase phosphatase can be isolated from rabbit liver and bovine heart as a form of Mr approximately 35,000 after an ethanol treatment of tissue extracts. This enzyme form was designated as protein phosphatase C. In the present study, reproducible methods for the isolation of two forms of protein phosphatase C from rabbit skeletal muscle to apparent homogeneity are described. Protein phosphatase C-I was obtained in yields of up to 20%, with specific activities toward phosphorylase a of 8,000-16,000 units/mg of protein. This enzyme represents the major phosphorylase phosphatase activity present in the ethanol-treated muscle extracts. The second enzyme, protein phosphatase C-II, had a much lower specific activity toward phosphorylase a (250-900 units/mg). Phosphatase C-I and phosphatase C-II had Mr = 32,000 and 33,500, respectively, as determined by sodium dodecyl sulfate disc gel electrophoresis. The two enzymes displayed distinct enzymatic properties. Phosphatase C-II was associated with a more active alkaline phosphatase activity toward p-nitrophenyl phosphate than was phosphatase C-I. Phosphatase C-II activities were activated by Mn2+, whereas phosphatase C-I was inhibited. Phosphatase C-I was inhibited by rabbit skeletal muscle inhibitor 2 while phosphatase C-II was not inhibited. Both enzymes dephosphorylated glycogen synthase and phosphorylase kinase, but displayed different specificities toward the alpha- and beta-subunit phosphates of phosphorylase kinase (Ganapathi, M. K., Silberman, S. R., Paris, H., and Lee, E. Y. C. (1980) J. Biol. Chem. 246, 3213-3217). The amino acid compositions of the two proteins were similar. Peptide mapping of the two proteins showed that they are distinct proteins and do not have a precursor-proteolytic product relationship.