Policy implications of human-accelerated nitrogen cycling

The human induced input of reactive N into the globalbiosphere has increased to approximately 150 Tg N eachyear and is expected to continue to increase for theforeseeable future. The need to feed (125 Tg N) andto provide energy (25 Tg N) for the growing worldpopulation drives this trend. This increase inreactive N comes at, in some instances, significantcosts to society through increased emissions of NO_x,NH₃, N₂O and NO₃ ^– and deposition of NO_y and NH_x.In the atmosphere, increases in tropospheric ozone andacid deposition (NO_y and NH_x) have led toacidification of aquatic and soil systems and toreductions in forest and crop system production. Changes in aquatic systems as a result of nitrateleaching have led to decreased drinking water quality,eutrophication, hypoxia and decreases in aquatic plantdiversity, for example. On the other hand, increaseddeposition of biologically available N may haveincreased forest biomass production and may havecontributed to increased storage of atmospheric CO₂ inplant and soils. Most importantly, syntheticproduction of fertilizer N has contributed greatly tothe remarkable increase in food production that hastaken place during the past 50 years.The development of policy to control unwanted reactiveN release is difficult because much of the reactive Nrelease is related to food and energy production andreactive N species can be transported great distancesin the atmosphere and in aquatic systems. There aremany possibilities for limiting reactive N emissionsfrom fuel combustion, and in fact, great strides havebeen made during the past decades. Reducing theintroduction of new reactive N and in curtailing themovement of this N in food production is even moredifficult. The particular problem comes from the factthat most of the N that is introduced into the globalfood production system is not converted into usableproduct, but rather reenters the biosphere as asurplus. Global policy on N in agriculture isdifficult because many countries need to increase foodproduction to raise nutritional levels or to keep upwith population growth, which may require increaseduse of N fertilizers. Although N cycling occurs atregional and global scales, policies are implementedand enforced at the national or provincial/statelevels. Multinational efforts to control N loss tothe environment are surely needed, but these effortswill require commitments from individual countries andthe policy-makers within those countries.     

Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase

We report a highly specific, robust and rapid new method for labeling cell surface proteins with biophysical probes. The method uses the Escherichia coli enzyme biotin ligase (BirA), which sequence-specifically ligates biotin to a 15-amino-acid acceptor peptide (AP). We report that BirA also accepts a ketone isostere of biotin as a cofactor, ligating this probe to the AP with similar kinetics and retaining the high substrate specificity of the native reaction. Because ketones are absent from native cell surfaces, AP-fused recombinant cell surface proteins can be tagged with the ketone probe and then specifically conjugated to hydrazide- or hydroxylamine-functionalized molecules. We demonstrate this two-stage protein labeling methodology on purified protein, in the context of mammalian cell lysate, and on epidermal growth factor receptor (EGFR) expressed on the surface of live HeLa cells. Both fluorescein and a benzophenone photoaffinity probe are incorporated, with total labeling times as short as 20 min.     

Effects of halothane, isoflurane, sevoflurane and desflurane on contraction of ventricular myocytes from streptozotocin-induced diabetic rats

Various clinically used volatile general anaesthetics (e.g. sevoflurane, halothane, isoflurane and desflurane) have been shown to have significant negative inotropic effects on normal ventricular muscle. However, little is known about their effects in ventricular tissue from diabetic animals. Streptozotocin (STZ)-induced diabetes is known to induce changes in the amplitude and time course of shortening and one report suggests that the inotropic effects of anaesthetics are ameliorated in papillary muscles from diabetic animals. The aim of these studies was to investigate this further in electrically stimulated (1 Hz) ventricular myocytes. Cells were superfused with either normal Tyrode (NT) solution or NT containing anaesthetic (1 mM) for a period of 2 min (at 30-32 degrees C). Myocytes from STZ rats were shown to have a significantly longer time to peak shortening (p > 0.001, n = 50) and the amplitude of shortening tended to be greater but this was not significant (p = 0.13, n = 50). Halothane, isoflurane, desflurane and sevoflurane significantly (p < 0.05) reduced the magnitude of shortening of control cells by 72.5 +/- 3.2%, 46.5 +/- 9.7%, 28.9 +/- 4.3% and 22.8 +/- 5.6%, respectively (n > 11 per group) but their steady-state negative inotropic effect was found to be no different in cells from STZ-treated rats (73.0 +/- 4.8%, 40.7 +/- 4.7%, 25.0 +/- 5.2% and 19.8 +/- 5.2%, respectively, n > 10 per group). Therefore, we conclude that the inotropic effects of volatile anaesthetics were not altered by STZ treatment.     

Halothane alters contractility and Ca2+ transport in ventricular myocytes from streptozotocin-induced diabetic rats

General anaesthetics have previously been shown to have profound effects on myocardial function. Moreover, many patients suffering from diabetes mellitus are anaesthetised during surgery. This study investigated compromised functioning of cardiac myocytes from streptozotocin (STZ)-induced diabetic rats and the additive effects of halothane on these dysfunctions. Ventricular myocytes were isolated from 8 to 12 weeks STZ-treated rats. Contraction and intracellular free calcium concentration ([Ca2+]i) were measured in electrically field-stimulated (1 Hz) fura-2-AM-loaded cells using a video-edge detection system and a fluorescence photometry system, respectively. L-type Ca2+ current was measured in whole cell, voltage-clamp mode. Halothane significantly (p < 0.01) depressed the amplitude and the time course of the Ca2+ transients in a similar manner in myocytes from control and STZ-treated rats. However, the effect of halothane on the amplitude of shortening and L-type Ca2+ current was more pronounced in myocytes from STZ-treated animals compared to age-matched controls. Myofilament sensitivity to Ca2+ was significantly (p < 0.01) increased in myocytes from STZ-treated rats compared to control. However, in the presence of halothane the myofilament sensitivity to Ca2+ was significantly (p < 0.05) reduced to a greater extent in myocytes from STZ-treated rats compared to controls. In conclusion, these results show that contractility, Ca2+ transport and myofilament sensitivity were all altered in myocytes from STZ-treated rats and these processes were further altered in the presence of halothane suggesting that hearts from STZ-induced diabetic rats are sensitive to halothane.     

Estimation of breeding values using selected pedigree records

Fish bred in tanks or ponds cannot be easily tagged individually. The parentage of any individual may be determined by DNA fingerprinting, but is sufficiently expensive that large numbers cannot be so finger-printed. The measurement of the objective trait can be made on a much larger sample relatively cheaply. This article deals with experimental designs for selecting individuals to be finger-printed and for the estimation of the individual and family breeding values. The general setup provides estimates for both genetic effects regarded as fixed or random and for fixed effects due to known regressors. The family effects can be well estimated when even very small numbers are finger-printed, provided that they are the individuals with the most extreme phenotypes.     

Use of combined microautoradiography and fluorescence in situ hybridization to determine carbon metabolism in mixed natural communities of uncultured bacteria from the genus Achromatium

Combined microautoradiography and fluorescence in situ hybridization (FISH) was used to investigate carbon metabolism in uncultured bacteria from the genus Achromatium. All of the Achromatium species identified in a freshwater sediment from Rydal Water, Cumbria, United Kingdom, which were distinguishable only by FISH, assimilated both [(14)C]bicarbonate and [(14)C]acetate. This extends previous findings that Achromatium spp. present at another location could only utilize organic carbon sources. Achromatium spp., therefore, probably exhibit a range of physiologies, i.e., facultative chemolithoautotrophy, mixotrophy, and chemoorganoheterotrophy, similar to other large sulfur bacteria (e.g., Beggiatoa spp.).     

Protein global fold determination using site-directed spin and isotope labeling

We describe a simple experimental approach for the rapid determination of protein global folds. This strategy utilizes site-directed spin labeling (SDSL) in combination with isotope enrichment to determine long-range distance restraints between amide protons and the unpaired electron of a nitroxide spin label using the paramagnetic effect on relaxation rates. The precision and accuracy of calculating a protein global fold from only paramagnetic effects have been demonstrated on barnase, a well-characterized protein. Two monocysteine derivatives of barnase, (H102C) and (H102A/Q15C), were 15N enriched, and the paramagnetic nitroxide spin label, MTSSL, attached to the single Cys residue of each. Measurement of amide 1H longitudinal relaxation times, in both the oxidized and reduced states, allowed the determination of the paramagnetic contribution to the relaxation processes. Correlation times were obtained from the frequency dependence of these relaxation processes at 800, 600, and 500 MHz. Distances in the range of 8 to 35 A were calculated from the magnitude of the paramagnetic contribution to the relaxation processes and individual amide 1H correlation times. Distance restraints from the nitroxide spin to amide protons were used as restraints in structure calculations. Using nitroxide to amide 1H distances as long-range restraints and known secondary structure restraints, barnase global folds were calculated having backbone RMSDs <3 A from the crystal structure. This approach makes it possible to rapidly obtain the overall topology of a protein using a limited number of paramagnetic distance restraints.     

Endothelial dysfunction in Type 2 diabetes correlates with deregulated expression of the tail-anchored membrane protein SLMAP

The Type 2 diabetic db/db mouse experiences vascular dysfunction typified by changes in the contraction and relaxation profiles of small mesenteric arteries (SMAs). Contractions of SMAs from the db/db mouse to the alpha1-adrenoceptor agonist phenylephrine (PE) were significantly enhanced, and acetylcholine (ACh)-induced relaxations were significantly depressed. Drug treatment of db/db mice with a nonthiazolidinedione peroxisome prolifetor-activated receptor-gamma agonist and insulin sensitizing agent 2-[2-(4-phenoxy-2-propylphenoxy)ethyl]indole-5-acetic acid (COOH) completely prevented the changes in endothelium-dependent relaxation, but, with the discontinuation of therapy, endothelial dysfunction returned. Dysfunctional SMAs were found to specifically upregulate the expression of a 35-kDa isoform of sarcolemmal membrane-associated protein (SLMAP), which is a component of the excitation-contraction coupling apparatus and implicated in the regulation of membrane function in muscle cells. Real-time PCR revealed high SLMAP mRNA levels in the db/db microvasculature, which were markedly downregulated during COOH treatment but elevated again when drug therapy was discontinued. These data reveal that the microvasculature in db/db mice undergoes significant changes in vascular function with the endothelial component of vascular dysfunction specifically correlating with the overexpression of SLMAP. Thus changes in SLMAP expression may be an important indicator for microvascular disease associated with Type 2 diabetes.     

Genetic analysis of natural hybrids between endemic and alien Rubus (Rosaceae) species in Hawai`i

A population of putative hybrids between theendemic Rubus hawaiensis and naturalizedR. rosifolius was discovered inKpahulu Valley, on the island of Maui inthe Hawaiian archipelago. The goal of thisstudy was to molecularly characterize thisnatural hybridization event, investigate themode of hybridization, and determine the malefertility of the hybrid individuals. Bothmorphological and RAPD marker data indicatethat the putative hybrid individuals are theprogeny of R. rosifolius and R.hawaiensis. All 39 hybrid individuals sampledhad the chloroplast DNA haplotype of R.rosifolius. Thus hybridization appears to beasymmetric, with R. rosifolius acting asthe maternal parent. All hybrid individualsassessed for pollen stainability were sterile,and there was no evidence of backcrossing toeither parent. This result suggests thathybrids are of the first filial generation andthat variation among hybrids reflectsdifferences within the parental populations.Sympatric populations of R. hawaiensisand R. rosifolius occur on four islandsand six additional alien species of Rubusare naturalized and sympatric with R. hawaiensisin Hawai`i. Further investigationis merited to assess whether hybridization maypose a threat to the long term viability ofR. hawaiensis. This study highlights theincreasing frequency and negative consequencesof native-alien hybridization and theimportance of maintaining active alien speciescontrol programs in the Hawaiian Islands.