Short-lived alpha-helical intermediates in the folding of beta-sheet proteins.

Several lines of evidence point strongly toward the importance of highly alpha-helical intermediates in the folding of all globular proteins, regardless of their native structure. However, experimental refolding studies demonstrate no observable alpha-helical intermediate during refolding of some beta-sheet proteins and have dampened enthusiasm for this model of protein folding. In this study, beta-sheet proteins were hypothesized to have potential to form amphiphilic helices at a period of <3.6 residues/turn that matches or exceeds the potential at 3.6 residues/turn. Hypothetically, such potential is the basis for an effective and unidirectional mechanism by which highly alpha-helical intermediates might be rapidly disassembled during folding and potentially accounts for the difficulty in detecting highly alpha-helical intermediates during the folding of some proteins. The presence of this potential was confirmed, indicating that a model entailing ubiquitous formation of alpha-helical intermediates during the folding of globular proteins predicts previously unrecognized features of primary structure. Further, the folding of fatty acid binding protein, a predominantly beta-sheet protein that exhibits no apparent highly alpha-helical intermediate during folding, was dramatically accelerated by 2,2,2-trifluoroethanol, a solvent that stabilizes alpha-helical structure. This observation suggests that formation of an alpha-helix can be a rate-limiting step during folding of a predominantly beta-sheet protein and further supports the role of highly alpha-helical intermediates in the folding of all globular proteins.

Direct Carbon-Carbon Bond Formation via Reductive Soft Enolization: A Kinetically Controlled syn-Aldol Addition of alpha-Halo Thioesters and Enolizable Aldehydes

The direct addition of enolizable aldehydes and a-halo thioesters to produce beta-hydroxy thioesters enabled by reductive soft enolization is reported. The transformation is operationally simple and efficient and has the unusual feature of giving high syn-selectivity, which is the opposite of that produced for (thio)esters under conventional conditions. Moreover, excellent diastereoselectivity results when a chiral nonracemic alpha-hydroxy aldehyde derivative is used.

Organic nitrogen in PM2.5 aerosol at a forest site in the Southeast US

There is growing evidence that organo-nitrogen compounds may constitute a significant fraction of the aerosol nitrogen (N) budget. However, very little is known about the abundance and origin of this aerosol fraction. In this study, the concentration of organic nitrogen (ON) and major inorganic ions in PM2.5 aerosol were measured at the Duke Forest Research Facility near Chapel Hill, NC, during January and June of 2007. A novel on-line instrument was used, which is based on the Steam Jet Aerosol Collector (SJAC) coupled to an on-line total carbon/total nitrogen analyzer and two on-line ion chromatographs. The concentration of ON was determined by tracking the difference in concentrations of total nitrogen and of inorganic nitrogen (determined as the sum of N-ammonium and N-nitrate). The time resolution of the instrument was 30 min with a detection limit for major aerosol components of ∼0.1 mu;gm-3. Nitrogen in organic compounds contributed ∼33% on average to the total nitrogen concentration in PM2.5, illustrating the importance of this aerosol component. Absolute concentrations of ON, however, were relatively low (lt;1.0 mu;gm-3) with an average of 0.16 mu;gm-3. The absolute and relative contribution of ON to the total aerosol nitrogen budget was practically the same in January and June. In January, the concentration of ON tended to be higher during the night and early morning, while in June it tended to be higher during the late afternoon and evening. Back-trajectories and correlation with wind direction indicate that higher concentrations of ON occur in air masses originating over the continental US, while marine air masses are characterized by lower ON concentrations. The data presented in this study suggests that ON has a variety of sources, which are very difficult to quantify without information on chemical composition of this important aerosol fraction.

Global fire emissions and the contribution of deforestation, savanna, forest, agricultural, and peat fires (1997-2009)

New burned area datasets and top-down constraints from atmospheric concentration measurements of pyrogenic gases have decreased the large uncertainty in fire emissions estimates. However, significant gaps remain in our understanding of the contribution of deforestation, savanna, forest, agricultural waste, and peat fires to total global fire emissions. Here we used a revised version of the Carnegie-Ames-Stanford-Approach (CASA) biogeochemical model and improved satellite-derived estimates of area burned, fire activity, and plant productivity to calculate fire emissions for the 1997-2009 period on a 0.5° spatial resolution with a monthly time step. For November 2000 onwards, estimates were based on burned area, active fire detections, and plant productivity from the MODerate resolution Imaging Spectroradiometer (MODIS) sensor. For the partitioning we focused on the MODIS era. We used maps of burned area derived from the Tropical Rainfall Measuring Mission (TRMM) Visible and Infrared Scanner (VIRS) and Along-Track Scanning Radiometer (ATSR) active fire data prior to MODIS (1997-2000) and estimates of plant productivity derived from Advanced Very High Resolution Radiometer (AVHRR) observations during the same period. Average global fire carbon emissions according to this version 3 of the Global Fire Emissions Database (GFED3) were 2.0 PgC year-1 with significant interannual variability during 1997-2001 (2.8 Pg Cyear-1 in 1998 and 1.6 PgC year-1 in 2001). Globally, emissions during 2002-2007 were rela-tively constant (around 2.1 Pg C year-1) before declining in 2008 (1.7 Pg Cyear-1) and 2009 (1.5 PgC year-1) partly due to lower deforestation fire emissions in South America and tropical Asia. On a regional basis, emissions were highly variable during 2002-2007 (e.g., boreal Asia, South America, and Indonesia), but these regional differences canceled out at a global level. During the MODIS era (2001-2009), most carbon emissions were from fires in grasslands and savannas (44%) with smaller contributions from tropical deforestation and degradation fires (20%), woodland fires (mostly confined to the tropics, 16%), forest fires (mostly in the extratropics, 15%), agricultural waste burning (3%), and tropical peat fires (3%). The contribution from agricultural waste fires was likely a lower bound because our approach for measuring burned area could not detect all of these relatively small fires. Total carbon emissions were on average 13% lower than in our previous (GFED2) work. For reduced trace gases such as CO and CH4, deforestation, degradation, and peat fires were more important contributors because of higher emissions of reduced trace gases per unit carbon combusted compared to savanna fires. Carbon emissions from tropical deforestation, degradation, and peatland fires were on average 0.5 PgC year-1. The carbon emissions from these fires may not be balanced by regrowth following fire. Our results provide the first global assessment of the contribution of different sources to total global fire emissions for the past decade, and supply the community with an improved 13-year fire emissions time series. © 2010 Author(s).

Morphometrics and testicle size of rain forest lemur species from southeastern Madagascar

Six species of prosimians inhabiting the montane rain forest of the Ranomafana National Park located in southeastern Madagascar were captured, weighed, and measured during the months of May or June of 1987, 1988, and 1989. There were no significant differences in body weights and measurements between male and femaleEulemur rubriventer (red-bellied lemur) orEulemur fulvus rufus (red-fronted lemur). Adult femalePropithecus diadema edwardsi (Milne Edward's sifaka) were heavier than males but the difference was not significant. A fewAvahi laniger laniger (woolly lemur),Hapalemur aureus (golden bamboo lemur) andH. g. griseus (gentle bamboo lemur) also were captured and measured. Body weights of the same individual adultP. d. edwardsi changed over the three years, suggesting variation in food availability. Although there was no difference in body weight among adult males of two groups ofP. d. edwardsi, one male in each group had a testicular volume four times larger than that of other males, even though these measurements were taken five months after the breeding season. These data suggest that only one adult male mates in each group. Testicular size of the polygynousE. f. rufus males was significantly larger than that of the monogamousE. rubriventer. © 1992 Academic Press Limited.

Seeing the forest for the fuel

We demonstrate a new approach to understanding the role of fuelwood in the rural household economy by applying insights from travel cost modeling to author-compiled household survey data and meso-scale environmental statistics from Ruteng Park in Flores, Indonesia. We characterize Manggarai farming households' fuelwood collection trips as inputs into household production of the utility yielding service of cooking and heating. The number of trips taken by households depends on the shadow price of fuelwood collection or the travel cost, which is endogenous. Econometric analyses using truncated negative binomial regression models and correcting for endogeneity show that the Manggarai are 'economically rational' about fuelwood collection and access to the forests for fuelwood makes substantial contributions to household welfare. Increasing cost of forest access, wealth, use of alternative fuels, ownership of kerosene stoves, trees on farm, park staff activity, primary schools and roads, and overall development could all reduce dependence on collecting fuelwood from forests. © 2004 Cambridge University Press.

Four-decade responses of soil trace elements to an aggrading old-field forest: B, Mn, Zn, Cu, and Fe.

In the ancient and acidic Ultisol soils of the Southern Piedmont, USA, we studied changes in trace element biogeochemistry over four decades, a period during which formerly cultivated cotton fields were planted with pine seedlings that grew into mature forest stands. In 16 permanent plots, we estimated 40-year accumulations of trace elements in forest biomass and O horizons (between 1957 and 1997), and changes in bioavailable soil fractions indexed by extractions of 0.05 mol/L HCl and 0.2 mol/L acid ammonium oxalate (AAO). Element accumulations in 40-year tree biomass plus O horizons totaled 0.9, 2.9, 4.8, 49.6, and 501.3 kg/ha for Cu, B, Zn, Mn, and Fe, respectively. In response to this forest development, samples of the upper 0.6-m of mineral soil archived in 1962 and 1997 followed one of three patterns. (1) Extractable B and Mn were significantly depleted, by -4.1 and -57.7 kg/ha with AAO, depletions comparable to accumulations in biomass plus O horizons, 2.9 and 49.6 kg/ha, respectively. Tree uptake of B and Mn from mineral soil greatly outpaced resupplies from atmospheric deposition, mineral weathering, and deep-root uptake. (2) Extractable Zn and Cu changed little during forest growth, indicating that nutrient resupplies kept pace with accumulations by the aggrading forest. (3) Oxalate-extractable Fe increased substantially during forest growth, by 275.8 kg/ha, about 10-fold more than accumulations in tree biomass (28.7 kg/ha). The large increases in AAO-extractable Fe in surficial 0.35-m mineral soils were accompanied by substantial accretions of Fe in the forest's O horizon, by 473 kg/ha, amounts that dwarfed inputs via litterfall and canopy throughfall, indicating that forest Fe cycling is qualitatively different from that of other macro- and micronutrients. Bioturbation of surficial forest soil layers cannot account for these fractions and transformations of Fe, and we hypothesize that the secondary forest's large inputs of organic additions over four decades has fundamentally altered soil Fe oxides, potentially altering the bioavailability and retention of macro- and micronutrients, contaminants, and organic matter itself. The wide range of responses among the ecosystem's trace elements illustrates the great dynamics of the soil system over time scales of decades.

Myocardial expression of a constitutively active alpha 1B-adrenergic receptor in transgenic mice induces cardiac hypertrophy.

Transgenic mice were generated by using the alpha-myosin heavy chain promoter coupled to the coding sequence of a constitutively active mutant alpha 1B-adrenergic receptor (AR). These transgenic animals demonstrated cardiac-specific expression of this alpha 1-AR with resultant activation of phospholipase C as shown by increased myocardial diacylglycerol content. A phenotype consistent with cardiac hypertrophy developed in adult transgenic mice with increased heart/body weight ratios, myocyte cross-sectional areas, and ventricular atrial natriuretic factor mRNA levels relative to nontransgenic controls. These transgenic animals may provide insight into the biochemical triggers that induce hypertrophy in cardiac disease and serve as a convenient experimental model for studies of this condition.

Tyrosine phosphorylation of G protein alpha subunits by pp60c-src.

A number of lines of evidence suggest that cross-talk exists between the cellular signal transduction pathways involving tyrosine phosphorylation catalyzed by members of the pp60c-src kinase family and those mediated by guanine nucleotide regulatory proteins (G proteins). In this study, we explore the possibility that direct interactions between pp60c-src and G proteins may occur with functional consequences. Preparations of pp60c-src isolated by immunoprecipitation phosphorylate on tyrosine residues the purified G-protein alpha subunits (G alpha) of several heterotrimeric G proteins. Phosphorylation is highly dependent on G-protein conformation, and G alpha(GDP) uncomplexed by beta gamma subunits appears to be the preferred substrate. In functional studies, phosphorylation of stimulatory G alpha (G alpha s) modestly increases the rate of binding of guanosine 5'-[gamma-[35S]thio]triphosphate to Gs as well as the receptor-stimulated steady-state rate of GTP hydrolysis by Gs. Heterotrimeric G proteins may represent a previously unappreciated class of potential substrates for pp60c-src.

G-protein-coupled receptor genes as protooncogenes: constitutively activating mutation of the alpha 1B-adrenergic receptor enhances mitogenesis and tumorigenicity.

The alpha 1B-adrenergic receptor (alpha 1B-ADR) is a member of the G-protein-coupled family of transmembrane receptors. When transfected into Rat-1 and NIH 3T3 fibroblasts, this receptor induces focus formation in an agonist-dependent manner. Focus-derived, transformed fibroblasts exhibit high levels of functional alpha 1B-ADR expression, demonstrate a catecholamine-induced enhancement in the rate of cellular proliferation, and are tumorigenic when injected into nude mice. Induction of neoplastic transformation by the alpha 1B-ADR, therefore, identifies this normal cellular gene as a protooncogene. Mutational alteration of this receptor can lead to activation of this protooncogene, resulting in an enhanced ability of agonist to induce focus formation with a decreased latency and quantitative increase in transformed foci. In contrast to cells expressing the wild-type alpha 1B-ADR, focus formation in "oncomutant"-expressing cell lines appears constitutively activated with the generation of foci in unstimulated cells. Further, these cell lines exhibit near-maximal rates of proliferation even in the absence of catecholamine supplementation. They also demonstrate an enhanced ability for tumor generation in nude mice with a decreased period of latency compared with cells expressing the wild-type receptor. Thus, the alpha 1B-ADR gene can, when overexpressed and activated, function as an oncogene inducing neoplastic transformation. Mutational alteration of this receptor gene can result in the activation of this protooncogene, enhancing its oncogenic potential. These findings suggest that analogous spontaneously occurring mutations in this class of receptor proteins could play a key role in the induction or progression of neoplastic transformation and atherosclerosis.

Expansion of the alpha 2-adrenergic receptor family: cloning and characterization of a human alpha 2-adrenergic receptor subtype, the gene for which is located on chromosome 2.

Pharmacologic, biochemical, and genetic analyses have demonstrated the existence of multiple alpha 2-adrenergic receptor (alpha 2AR) subtypes. We have cloned a human alpha 2AR by using the polymerase chain reaction with oligonucleotide primers homologous to conserved regions of the previously cloned alpha 2ARs, the genes for which are located on human chromosomes 4 (C4) and 10 (C10). The deduced amino acid sequence encodes a protein of 450 amino acids whose putative topology is similar to that of the family of guanine nucleotide-binding protein-coupled receptors, but whose structure most closely resembles that of the alpha 2ARs. Competition curve analysis of the binding properties of the receptor expressed in COS-7 cells with a variety of adrenergic ligands demonstrates a unique alpha 2AR pharmacology. Hybridization with somatic cell hybrids shows that the gene for this receptor is located on chromosome 2. Northern blot analysis of various rat tissues shows expression in liver and kidney. The unique pharmacology and tissue localization of this receptor suggest that this is an alpha 2AR subtype not previously identified by classical pharmacological or ligand binding approaches.

Regions of the alpha 1-adrenergic receptor involved in coupling to phosphatidylinositol hydrolysis and enhanced sensitivity of biological function.

Regions of the hamster alpha 1-adrenergic receptor (alpha 1 AR) that are important in GTP-binding protein (G protein)-mediated activation of phospholipase C were determined by studying the biological functions of mutant receptors constructed by recombinant DNA techniques. A chimeric receptor consisting of the beta 2-adrenergic receptor (beta 2AR) into which the putative third cytoplasmic loop of the alpha 1AR had been placed activated phosphatidylinositol metabolism as effectively as the native alpha 1AR, as did a truncated alpha 1AR lacking the last 47 residues in its cytoplasmic tail. Substitutions of beta 2AR amino acid sequence in the intermediate portions of the third cytoplasmic loop of the alpha 1AR or at the N-terminal portion of the cytoplasmic tail caused marked decreases in receptor coupling to phospholipase C. Conservative substitutions of two residues in the C terminus of the third cytoplasmic loop (Ala293----Leu, Lys290----His) increased the potency of agonists for stimulating phosphatidylinositol metabolism by up to 2 orders of magnitude. These data indicate (i) that the regions of the alpha 1AR that determine coupling to phosphatidylinositol metabolism are similar to those previously shown to be involved in coupling of beta 2AR to adenylate cyclase stimulation and (ii) that point mutations of a G-protein-coupled receptor can cause remarkable increases in sensitivity of biological response.

Molecular cloning and expression of the cDNA for the hamster alpha 1-adrenergic receptor.

The cDNA for the Syrian hamster alpha 1-adrenergic receptor has been cloned with oligonucleotides corresponding to the partial amino acid sequence of the receptor protein purified from DDT1MF-2 smooth muscle cells. The deduced amino acid sequence encodes a 515-residue polypeptide that shows the most sequence identity with the other adrenergic receptors and the putative protein product of the related clone G-21. Similarities with the muscarinic cholinergic receptors are also evident. Expression studies in COS-7 cells confirm that we have cloned the alpha 1-adrenergic receptor that couples to inositol phospholipid metabolism.

Cloning and expression of a human kidney cDNA for an alpha 2-adrenergic receptor subtype.

An alpha 2-adrenergic receptor subtype has been cloned from a human kidney cDNA library using the gene for the human platelet alpha 2-adrenergic receptor as a probe. The deduced amino acid sequence resembles the human platelet alpha 2-adrenergic receptor and is consistent with the structure of other members of the family of guanine nucleotide-binding protein-coupled receptors. The cDNA was expressed in a mammalian cell line (COS-7), and the alpha 2-adrenergic ligand [3H]rauwolscine was bound. Competition curve analysis with a variety of adrenergic ligands suggests that this cDNA clone represents the alpha 2B-adrenergic receptor. The gene for this receptor is on human chromosome 4, whereas the gene for the human platelet alpha 2-adrenergic receptor (alpha 2A) lies on chromosome 10. This ability to express the receptor in mammalian cells, free of other adrenergic receptor subtypes, should help in developing more selective alpha-adrenergic ligands.

Phorbol esters promote alpha 1-adrenergic receptor phosphorylation and receptor uncoupling from inositol phospholipid metabolism.

DDT1 MF-2 cells, which are derived from hamster vas deferens smooth muscle, contain alpha 1-adrenergic receptors (54,800 +/- 2700 sites per cell) that are coupled to stimulation of inositol phospholipid metabolism. Incubation of these cells with tumor-promoting phorbol esters, which stimulate calcium- and phospholipid-dependent protein kinase, leads to a marked attenuation of the ability of alpha 1-receptor agonists such as norepinephrine to stimulate the turnover of inositol phospholipids. This turnover was measured by determining the 32P content of phosphatidylinositol and phosphatidic acid after prelabeling of the cellular ATP pool with 32Pi. These phorbol ester-treated cells also displayed a decrease in binding affinity of cellular alpha 1 receptors for agonists with no change in antagonist affinity. By using affinity chromatography on the affinity resin Affi-Gel-A55414, the alpha 1 receptors were purified approximately equal to 300-fold from control and phorbol ester-treated 32Pi-prelabeled cells. As assessed by NaDodSO4/polyacrylamide gel electrophoresis, the Mr 80,000 alpha 1-receptor ligand-binding subunit is a phosphopeptide containing 1.2 mol of phosphate per mol of alpha 1 receptor. After phorbol ester treatment this increased to 3.6 mol of phosphate per mol of alpha 1 receptor. The effect of phorbol esters on norepinephrine-stimulated inositol phospholipid turnover and alpha 1-receptor phosphorylation showed the same rapid time course with a t1/2 less than 2 min. These results indicate that calcium- and phospholipid-dependent protein kinase may play an important role in regulating the function of receptors that are coupled to the inositol phospholipid cycle by phosphorylating and deactivating them.