Intact amino acid uptake by northern hardwood and conifer trees

Empirical and modeling studies of the N cycle in temperate forests of eastern North America have focused on the mechanisms regulating the production of inorganic N, and assumed that only inorganic forms of N are available for plant growth. Recent isotope studies in field conditions suggest that amino acid capture is a widespread ecological phenomenon, although northern temperate forests have yet to be studied. We quantified fine root biomass and applied tracer-level quantities of U–¹³C₂–¹⁵N-glycine, ¹⁵NH₄ ⁺ and ¹⁵NO₃ ⁻ in two stands, one dominated by sugar maple and white ash, the other dominated by red oak, beech, and hemlock, to assess the importance of amino acids to the N nutrition of northeastern US forests. Significant enrichment of ¹³C in fine roots 2 and 5 h following tracer application indicated intact glycine uptake in both stands. Glycine accounted for up to 77% of total N uptake in the oak–beech–hemlock stand, a stand that produces recalcitrant litter, cycles N slowly and has a thick, amino acid-rich organic horizon. By contrast, glycine accounted for only 20% of total N uptake in the sugar maple and white ash stand, a stand characterized by labile litter and rapid rates of amino acid production and turnover resulting in high rates of mineralization and nitrification. This study shows that amino acid uptake is an important process occurring in two widespread, northeastern US temperate forest types with widely differing rates of N cycling.     

Dissecting the Mechanisms of Tissue Transglutaminase-induced Cross-linking of α-Synuclein: IMPLICATIONS FOR THE PATHOGENESIS OF PARKINSON DISEASE*S?

Tissue transglutaminase (tTG) has been implicated in the pathogenesis of Parkinson disease (PD). However, exactly how tTG modulates the structural and functional properties of α-synuclein (α-syn) and contributes to the pathogenesis of PD remains unknown. Using site-directed mutagenesis combined with detailed biophysical and mass spectrometry analyses, we sought to identify the exact residues involved in tTG-catalyzed cross-linking of wild-type α-syn and α-syn mutants associated with PD. To better understand the structural consequences of each cross-linking reaction, we determined the effect of tTG-catalyzed cross-linking on the oligomerization, fibrillization, and membrane binding of α-syn in vitro. Our findings show that tTG-catalyzed cross-linking of monomeric α-syn involves multiple cross-links (specifically 2-3). We subjected tTG-catalyzed cross-linked monomeric α-syn composed of either wild-type or Gln → Asn mutants to sequential proteolysis by multiple enzymes and peptide mapping by mass spectrometry. Using this approach, we identified the glutamine and lysine residues involved in tTG-catalyzed intramolecular cross-linking of α-syn. These studies demonstrate for the first time that Gln⁷⁹ and Gln¹⁰⁹ serve as the primary tTG reactive sites. Mutating both residues to asparagine abolishes tTG-catalyzed cross-linking of α-syn and tTG-induced inhibition of α-syn fibrillization in vitro. To further elucidate the sequence and structural basis underlying these effects, we identified the lysine residues that form isopeptide bonds with Gln⁷⁹ and Gln¹⁰⁹. This study provides mechanistic insight into the sequence and structural basis of the inhibitory effects of tTG on α-syn fibrillogenesis in vivo, and it sheds light on the potential role of tTG cross-linking on modulating the physiological and pathogenic properties of α-syn.Parkinson disease (PD)² is a progressive movement disorder that is caused by the loss of dopaminergic neurons in the substantia nigra, the part of the brain responsible for controlling movement. Clinically, PD is manifested in symptoms that include tremors, rigidity, and difficulty in initiating movement (bradykinesia). Pathologically, PD is characterized by the presence of intraneuronal, cytoplasmic inclusions known as Lewy bodies (LB), which are composed primarily of the protein “α-synuclein” (α-syn) (1) and are seen in the post-mortem brains of PD patients with the sporadic or familial forms of the disease (2). α-Syn is a presynaptic protein of 140 residues with a “natively” unfolded structure (3). Three missense point mutations in α-syn (A30P, E46K, and A53T) are associated with the early-onset, dominant, inherited form of PD (4, 5). Moreover, duplication or triplication of the α-syn gene has been linked to the familial form of PD, suggesting that an increase in α-syn expression is sufficient to cause PD. Together, these findings suggest that α-syn plays a central role in the pathogenesis of PD.The molecular and cellular determinants that govern α-syn oligomerization and fibrillogenesis in vivo remain poorly understood. In vitro aggregation studies have shown that the mutations associated with PD (A30P, E46K, and A53T) accelerate α-syn oligomerization, but only E46K and A53T α-syn show higher propensity to fibrillize than wild-type (WT) α-syn (6-8). This suggests that oligomerization, rather than fibrillization, is linked to early-onset familial PD (9). Our understanding of the molecular composition and biochemical state of α-syn in LBs has provided important clues about protein-protein interactions and post-translational modifications that may play a role in modulating oligomerization, fibrillogenesis, and LB formation of the protein. In addition to ubiquitination (10), phosphorylation (11, 12), nitration (13, 14), and C-terminal truncation (15, 16), analysis of post-mortem brain tissues from PD and Lewy bodies in dementia patients has confirmed the colocalization of tissue transglutaminase (tTG)-catalyzed cross-linked α-syn monomers and higher molecular aggregates in LBs within dopaminergic neurons (17, 18). Tissue transglutaminase catalyzes a calcium-dependent transamidating reaction involving glutamine and lysine residues, which results in the formation of a covalent cross-link via ε-(γ-glutamyl) lysine bonds (Fig. 2F). To date, seven different isoforms of tTGs have been reported, of which only tTG2 seems to be expressed in the human brain (19), whereas tTG1 and tTG3 are more abundantly found in stratified squamous epithelia (20). Subsequent immuno-histochemical, colocalization, and immunoprecipitation studies have shown that the levels of tTG and cross-linked α-syn species are increased in the substantia nigra of PD brains (17). These findings, combined with the known role of tTG in cross-linking and stabilizing bimolecular assemblies, led to the hypothesis that tTG plays an important role in the initiation and propagation of α-syn fibril formation and that it contributes to fibril stability in LBs. This hypothesis was initially supported by in vitro studies demonstrating that tTG catalyzes the polymerization of the α-syn-derived non-amyloid component (NAC) peptide via intermolecular covalent cross-linking of residues Gln⁷⁹ and Lys⁸⁰ (21) and by other studies suggesting that tTG promotes the fibrillization of amyloidogenic proteins implicated in the pathogenesis of other neurodegenerative diseases such as Alzheimer disease, supranuclear palsy, Huntington disease, and other polyglutamine diseases (22-24). However, recent in vitro studies with full-length α-syn have shown that tTG catalyzes intramolecular cross-linking of monomeric α-syn and inhibits, rather than promotes, its fibrillization in vitro (25, 26). The structural basis of this inhibitory effect and the exact residues involved in tTG-mediated cross-linking of α-syn, as well as structural and functional consequences of these modifications, remain poorly understood.Open in a separate windowFIGURE 2.tTG-catalyzed cross-linking of α-syn involves one to three intramolecular cross-links. A-C, MALDI-TOF/TOF analysis of native (—) and cross-linked (- - -) α-syn, showing that most tTG-catalyzed cross-linking products of WT or disease-associated mutant forms of α-syn are intramolecularly linked (predominant peak with two cross-links), and up to three intramolecular cross-links can occur (left shoulder). The abbreviations M and m/cl are used to designate native and cross-linked α-synuclein, respectively. D and E, kinetic analysis of α-syn (A30P) cross-linking monitored by MALDI-TOF and SDS-PAGE. F, schematic depiction of the tTG-catalyzed chemical reaction (isodipeptide formation) between glutamine and lysine residues.In this study, we have identified the primary glutamine and lysine residues involved in tTG-catalyzed, intramolecularly cross-linked monomeric α-syn and investigated how cross-linking these residues affects the oligomerization, fibrillization, and membrane binding of α-syn in vitro. Using single-site mutagenesis and mass spectrometry applied to exhaustive proteolytic digests of native and cross-linked monomeric α-syn, we identified Gln¹⁰⁹ and Gln⁷⁹ as the major tTG substrates. We demonstrate that the altered electrophoretic mobility of the intramolecularly cross-linked α-syn in SDS-PAGE occurs as a result of tTG-catalyzed cross-linking of Gln¹⁰⁹ to lysine residues in the N terminus of α-syn, which leads to the formation of more compact monomers. Consistent with previous studies, we show that intramolecularly cross-linked α-syn forms off-pathway oligomers that are distinct from those formed by the wild-type protein and that do not convert to fibrils within the time scale of our experiments (3-5 days). We also show that membrane-bound α-syn is a substrate of tTG and that intramolecular cross-linking does not interfere with the ability of monomeric α-syn to adopt an α-helical conformation upon binding to synthetic membranes. These studies provide novel mechanistic insight into the sequence and structural basis of events that allow tTG to inhibit α-syn fibrillogenesis, and they shed light on the potential role of tTG-catalyzed cross-linking in modulating the physiological and pathogenic properties of α-syn.     

PTools: an opensource molecular docking library

Background  

Macromolecular docking is a challenging field of bioinformatics. Developing new algorithms is a slow process generally involving routine tasks that should be found in a robust library and not programmed from scratch for every new software application.     

Copy Number Variation of CCL3-like Genes Affects Rate of Progression to Simian-AIDS in Rhesus Macaques (Macaca mulatta)

Variation in genes underlying host immunity can lead to marked differences in susceptibility to HIV infection among humans. Despite heavy reliance on non-human primates as models for HIV/AIDS, little is known about which host factors are shared and which are unique to a given primate lineage. Here, we investigate whether copy number variation (CNV) at CCL3-like genes (CCL3L), a key genetic host factor for HIV/AIDS susceptibility and cell-mediated immune response in humans, is also a determinant of time until onset of simian-AIDS in rhesus macaques. Using a retrospective study of 57 rhesus macaques experimentally infected with SIVmac, we find that CCL3L CNV explains approximately 18% of the variance in time to simian-AIDS (p<0.001) with lower CCL3L copy number associating with more rapid disease course. We also find that CCL3L copy number varies significantly (p<10⁻⁶) among rhesus subpopulations, with Indian-origin macaques having, on average, half as many CCL3L gene copies as Chinese-origin macaques. Lastly, we confirm that CCL3L shows variable copy number in humans and chimpanzees and report on CCL3L CNV within and among three additional primate species. On the basis of our findings we suggest that (1) the difference in population level copy number may explain previously reported observations of longer post-infection survivorship of Chinese-origin rhesus macaques, (2) stratification by CCL3L copy number in rhesus SIV vaccine trials will increase power and reduce noise due to non-vaccine-related differences in survival, and (3) CCL3L CNV is an ancestral component of the primate immune response and, therefore, copy number variation has not been driven by HIV or SIV per se.     

Temporal habitat variability and the maintenance of sex in host populations of the pea aphid

The evolutionary maintenance of sex, despite competition from asexual reproduction, has long intrigued the evolutionary biologists owing to its numerous apparent short-term costs. In aphids, winter climate is expected to determine the maintenance of sexual lineages in the high latitude zones owing to their exclusive ability to produce frost-resistant eggs. However, diverse reproductive modes may coexist at a local scale where climatic influence is counteracted by microgeographical factors. In this study, we tested the influence of local habitat characteristics on regional coexistence of reproductive modes in the pea aphid, Acyrthosiphon pisum. In the laboratory, the induction of sexual morph production of many pea aphid genotypes from the local fields of annual (pea and faba bean) and perennial (alfalfa and red clover) crops in Western France indicated that A. pisum lineages from annual crops had a significantly higher investment in sexual reproduction than A. pisum lineages from the perennial hosts. We propose that temporal habitat variability exerts a selective pressure to maintain the sexual reproduction in A. pisum. The ecological and evolutionary consequences of the association between the mode of reproduction and the host population on gene flow restriction and on ecological specialization are discussed.     

Gender-dependent regulation of G-protein-gated inwardly rectifying potassium current in dorsal raphe neurons in knock-out mice devoid of the 5-hydroxytryptamine transporter

Agonists at G-protein-coupled receptors in neurons of the dorsal raphe nucleus (DRN) of knock-out mice devoid of the serotonin transporter (5-HTT(-/-)) exhibit lower efficacy to inhibit cellular discharge than in wild-type counterparts. Using patch-clamp whole-cell recordings, we found that a G-protein-gated inwardly rectifying potassium (GIRK) current is involved in the inhibition of spike discharge induced by 5-HT1A agonists (5-carboxamidotryptamine (5-CT) and (+/-)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (8-OH-DPAT); 50 nM-30 microM) in both wild-type and 5-HTT(-/-) female and male mice. These effects were mimicked by 5'-guanylyl-imido-diphosphate (Gpp(NH)p; 400 microM) dialysis into cells with differences between genders. The 5-HTT(-/-) knock-out mutation reduced the current density induced by Gpp(NH)p in females but not in males. These data suggest that the decreased response of 5-HT1A receptors to agonists in 5-HTT(-/-) mutants reflects notably alteration in the coupling between G-proteins and GIRK channels in females but not in males. Accordingly, gender differences in central 5-HT neurotransmission appear to depend-at least in part-on sex-related variations in corresponding receptor-G protein signaling mechanisms.     

How to Build a Laser Speckle Contrast Imaging (LSCI) System to Monitor Blood Flow

Laser Speckle Contrast Imaging (LSCI) is a simple yet powerful technique that is used for full-field imaging of blood flow. The technique analyzes fluctuations in a dynamic speckle pattern to detect the movement of particles similar to how laser Doppler analyzes frequency shifts to determine particle speed. Because it can be used to monitor the movement of red blood cells, LSCI has become a popular tool for measuring blood flow in tissues such as the retina, skin, and brain. It has become especially useful in neuroscience where blood flow changes during physiological events like functional activation, stroke, and spreading depolarization can be quantified. LSCI is also attractive because it provides excellent spatial and temporal resolution while using inexpensive instrumentation that can easily be combined with other imaging modalities. Here we show how to build a LSCI setup and demonstrate its ability to monitor blood flow changes in the brain during an animal experiment.Download video file.^{(31M, mov)}