A Chimera Carrying the Functional Domain of the Orphan Protein SLC7A14 in the Backbone of SLC7A2 Mediates Trans-stimulated Arginine Transport

In human skin fibroblasts, a lysosomal transport system specific for cationic amino acids has been described and named system c. We asked if SLC7A14 (solute carrier family 7 member A14), an orphan protein assigned to the SLC7 subfamily of cationic amino acid transporters (CATs) due to sequence homology, may represent system c. Fusion proteins between SLC7A14 and enhanced GFP localized to intracellular vesicles, co-staining with the lysosomal marker LysoTracker®. To perform transport studies, we first tried to redirect SLC7A14 to the plasma membrane (by mutating putative lysosomal targeting motifs) but without success. We then created a chimera carrying the backbone of human (h) CAT-2 and the protein domain of SLC7A14 corresponding to the so-called “functional domain” of the hCAT proteins, a protein stretch of 81 amino acids that determines the apparent substrate affinity, sensitivity to trans-stimulation, and (as revealed in this study) pH dependence. The chimera mediated arginine transport and exhibited characteristics similar but not identical to hCAT-2A (the low affinity hCAT-2 isoform). Western blot and microscopic analyses confirmed localization of the chimera in the plasma membrane of Xenopus laevis oocytes. Noticeably, arginine transport by the hCAT-2/SLC7A14 chimera was pH-dependent, trans-stimulated, and inhibited by α-trimethyl-l-lysine, properties assigned to lysosomal transport system c in human skin fibroblasts. Expression analysis showed strong expression of SLC7A14 mRNA in these cells. Taken together, these data strongly suggest that SLC7A14 is a lysosomal transporter for cationic amino acids.     

Evaluating the potential of night spotlighting as a method for assessing species composition and brown trout abundance: a comparison with electrofishing in small streams

Direct counts of fish obtained by night spotlighting were compared with species composition and population estimates obtained from three-pass electrofishing obtained across 29 sites along small clear streams in the Otago region of New Zealand. The influence of habitat variables on the relative efficiency of each method was also examined. The same seven species of fishes were identified by both methods. Juvenile brown trout Salmo trutta were the only species present in sufficient numbers to allow comparison of abundance estimates using the two methods. A total of 777 brown trout were counted by spotlight and 803 brown trout were caught using electrofishing. Estimates of abundance obtained by spotlighting reflected population estimates obtained by three-pass electrofishing across most habitats. Electrofishing produced higher population estimates relative to spotlighting in fast-flowing turbulent riffle habitats, whereas counts obtained by spotlighting tended to be higher relative to electrofishing in slow-flowing pool habitats. The results suggest that spotlighting is an effective method for assessing fish composition and brown trout abundance in small clear water streams, although the extremes of water velocity may influence efficiency of both spotlighting and electrofishing.     

Decoding the Substrate Supply to Human Neuronal Nitric Oxide Synthase

Nitric oxide, produced by the neuronal nitric oxide synthase (nNOS) from L-arginine is an important second messenger molecule in the central nervous system: It influences the synthesis and release of neurotransmitters and plays an important role in long-term potentiation, long-term depression and neuroendocrine secretion. However, under certain pathological conditions such as Alzheimer’s or Parkinson’s disease, stroke and multiple sclerosis, excessive NO production can lead to tissue damage. It is thus desirable to control NO production in these situations. So far, little is known about the substrate supply to human nNOS as a determinant of its activity. Measuring bioactive NO via cGMP formation in reporter cells, we demonstrate here that nNOS in both, human A673 neuroepithelioma and TGW-nu-I neuroblastoma cells can be fast and efficiently nourished by extracellular arginine that enters the cells via membrane transporters (pool I that is freely exchangeable with the extracellular space). When this pool was depleted, NO synthesis was partially sustained by intracellular arginine sources not freely exchangeable with the extracellular space (pool II). Protein breakdown made up by far the largest part of pool II in both cell types. In contrast, citrulline to arginine conversion maintained NO synthesis only in TGW-nu-I neuroblastoma, but not A673 neuroepithelioma cells. Histidine mimicked the effect of protease inhibitors causing an almost complete nNOS inhibition in cells incubated additionally in lysine that depletes the exchangeable arginine pool. Our results identify new ways to modulate nNOS activity by modifying its substrate supply.     

Protein kinase C-dependent ubiquitination and clathrin-mediated endocytosis of the cationic amino acid transporter CAT-1

Cationic amino acid transporter 1 (CAT-1) is responsible for the bulk of the uptake of cationic amino acids in most mammalian cells. Activation of protein kinase C (PKC) leads to down-regulation of the cell surface CAT-1. To examine the mechanisms of PKC-induced down-regulation of CAT-1, a functional mutant of CAT-1 (CAT-1-HA-GFP) was generated in which a hemagglutinin antigen (HA) epitope tag was introduced into the second extracellular loop and GFP was attached to the carboxyl terminus. CAT-1-HA-GFP was stably expressed in porcine aorthic endothelial and human epithelial kidney (HEK) 293 cells. Using the HA antibody internalization assay we have demonstrated that PKC-dependent endocytosis was strongly inhibited by siRNA depletion of clathrin heavy chain, indicating that CAT-1-HA-GFP internalization requires clathrin-coated pits. Internalized CAT-1-HA-GFP was accumulated in early, recycling, and late endosomes. PKC activation also resulted in ubiquitination of CAT-1. CAT-1 ubiquitination and endocytosis in phorbol ester-stimulated porcine aorthic endothelial and HEK293 cells were inhibited by siRNA knockdown of NEDD4-2 and NEDD4-1 E3 ubiquitin ligases, respectively. In contrast, ubiquitination and endocytosis of the dopamine transporter was dependent on NEDD4-2 in all cell types tested. Altogether, our data suggest that ubiquitination mediated by NEDD4-2 or NEDD4-1 leading to clathrin-mediated endocytosis is the common mode of regulation of various transporter proteins by PKC.     

Arginase Activity - A Marker of Disease Status in Patients with Visceral Leishmaniasis in Ethiopia

The underlying mechanisms resulting in the profound immune suppression characteristic of human visceral leishmaniasis (VL) are not fully understood.Here, we tested the hypothesis that arginase, an enzyme associated with immunosuppression, is higher in patients with VL and contributes to impaired T cell responses. We recruited patients with VL before and after treatment and healthy controls and measured the arginase metabolism in the blood of these individuals. Our results show that arginase activity is significantly higher in the blood of patients with active VL as compared to controls. These high levels of arginase decline considerably once the patients are successfully treated. We identified the phenotype of arginase-expressing cells among PBMCs as neutrophils and show that their frequency was increased in PBMCs of patients before treatment; this coincides with reduced levels of L-arginine in the plasma and decreased expression levels of CD3ζ in T cells.     

Arboreal arthropod sampling methods for urban trees

Large, isolated trees are a prominent part of town planning, and the nature of the urban environment poses unique challenges in sampling the arthropod assemblages found on them. As part of a larger study on the role of these trees in supporting wider biodiversity, we undertook a trial of arthropod sampling methods to compare the relative impact of sampling method versus the species of tree sampled. Using four species of study trees, three sampling methods were trialed: an inverted cone based bole trap, a compact flight intercept trap, and branch bagging and clipping. Total arthropod abundance, richness, and diversity were modeled using generalised linear mixed models, which showed that in many cases the variation between sampling method was larger than that found between tree species. All three of the sampling methods proved to be practical in the field, and overcame the challenges faced in sampling urban trees, thereby providing the opportunity for more detailed study. Our findings also highlight the case for a multifaceted approach to sampling arthropods if a complete picture of the community assemblage is to be gained.     

Continuous remote monitoring of fish activity with restricted home ranges using radiotelemetry

Signal strength received by a fixed antenna increased as a transmitter was positioned closer to it and decreased as the transmitter was positioned further away. Depth and transmitter orientation also influenced signal strength, but these were less pronounced than the effect of distance. The variations in signal strength recorded from fixed distances, depths or orientations were low, suggesting that variation in signal strength only occurs if the transmitter is moved. The use of signal strength variation as a measure of fish activity over a 24 h period was compared with observed patterns of point habitat use of a live fish. Close correspondence of assessments of activity using signal strength variation and habitat use was observed. This study demonstrated the potential utility of radiotelemetry in association with point-in-time habitat use data to determine home-range and diel and seasonal patterns of fish activity over 24 h periods.     

Observations on psychoneurophysiologically significant mushrooms

Summary The extracts ofP. venenosus, P. sphrinctrinus, Psil. mexicana, Psil. cubensis, andPsil. caerulescens have been compared by paper chromatography. It was found that neither Panaeoli contain psilocybin, the main constituent of the threePsilocybe species. The extract fromP. venenosus has been chromatographed, and two of the three major constituents were purified and obtained crystalline. One of these compounds could be shown to possess the same chromophore as psilocybin, a 4-oxygenated indole system, and seems most likely to be the active compound of the mushroom.Coordinator of Research and Clinical Investigator, The Bertram and Roberta Stein Neuropsychiatric Research Program, Inc.