Ring cleavage and degradative pathway of cyanuric acid in bacteria.

The degradative pathway of cyanuric acid [1,3,5-triazine-2,4,6(1H,3H,5H)-trione] was examined in Pseudomonas sp. strain D. The bacterium grew with cyanuric acid, biuret, urea or NH4+ as sole source of nitrogen, and each substrate was entirely metabolized concomitantly with growth. Enzymes from strain D were separated by chromatography on DEAE-cellulose and three reactions were examined. Cyanuric acid (1 mol) was converted stoichiometrically into 1.0 mol of CO2 and 1.1 mol of biuret, which was conclusively identified. Biuret (1 mol) was converted stoichiometrically into 1.1 mol of NH4+, about 1 mol of CO2 and 1.0 mol of urea, which was conclusively identified. Urea (1 mol) was converted into 1.9 mol of NH4+ and 1.0 mol of CO2. The reactions proceeded under aerobic or anoxic conditions and were presumed to be hydrolytic. Data indicate that the same pathway occurred in another pseudomonad and a strain of Klebsiella pneumoniae.     

Glutathione peroxidase activity in insects: A reassessment

In vertebrate species, cytotoxic H₂O₂ and other lipid or organic hydroperoxides (ROOH) formed in aerobic metabolism are removed by a selenoprotein, glutathione peroxidase (GPOX). The GPOX activity in most rat tissues ranges from 100 to 1,000 units (1 unit = 1 nmol NADPH oxidized·mg protein^?1·min^?1), except for muscles (20–30 units). In contrast, GPOX activities of two strains of the housefly (Musca domestica), cabbage looper (Trichoplusia ni), southern armyworm (Spodoptera eridania), and black swallowtail butterfly (Papilio polyxenes), were found to be in the range 2–12 units. Trivial GPOX activity was detected in the confused flour beetle (Tribolium confusum). In the earthworm (Lumbricus terrestris), banana slug (Ariolimax columbianus), and market squid (Loligo opalescens), the GPOX activity ranged from 1 to 5 units. Tissue selenium concentrations were about 500–1,000 ppb for adult M. domestica, 600 ppb in T. confusum, 32 ppb in T. ni, 17 ppb in S. eridania, and 31 ppb in P. polyxenes larvae. The form of selenium incorporated at such high levels in tissues of invertebrates such as M. domestica remains an unresolved issue. Peroxidase activity of non-selenium glutathione-S-transferase (GT) against ROOH may compensate for the low GPOX activity. Catalase (CAT) has high activity and wide subcellular distribution in insects. This may be an evolutionary adaptation to GT's inability to catalyze the reduction of H₂O₂. The GT's peroxidase and CAT activities were not assessed for other invertebrate species, and warrants an investigation due to their reported low GPOX levels.     

Evidence for selenocysteine in ovine tissue organelles

Subcellular fractions were prepared by differential centrifugation from heart and liver of a lamb labeled with 75Se-selenite. Crude fractions of nuclei, mitochondria, and microsomes from both tissues were solubilized with sodium dodecyl sulfate and chromatographed on columns of sephacryl S-200. A low molecular weight (MW) 75Se labeled cardiac cytosol protein (approximately 10,000 daltons) was partially purified by gel filtration chromatography. The major 75Se peaks from the sephacryl columns and the low MW cardiac protein were hydrolyzed in HCl under an inert atmosphere. When chromatographed on an amino acid analysis column, 75Se from each hydrolysate chromatographed in the identical position of 2,7-diamino-4-thia-5-selenaoctanedioic acid, the mixed oxidized dimer of cysteine and selenocysteine. The low MW cardiac protein was reacted with chloroacetate after reduction with borohydride. 75Se from a hydrolysate of this derivatized protein eluted in the same position as Se-carboxymethylselenocysteine on the amino acid analysis column. Thus, selenocysteine appears to be the predominant form of selenium in ovine heart and liver.     

Dystonin/BPAG1 Promotes Plus-End-Directed Transport of Herpes Simplex Virus 1 Capsids on Microtubules during Entry

During infection by herpes simplex virus 1 (HSV-1), the viral capsid is transported around the cytoplasm along the microtubule (MT) network. Although molecular motors have been implicated in this process, the composition of the molecular machinery required for efficient directional transport is unknown. We previously showed that dystonin (BPAG1) is recruited to HSV-1 capsids by the capsid-bound tegument protein pUL37 to promote efficient cytoplasmic transport of capsids during egress. Dystonin is a cytoskeleton cross-linker which localizes at MT plus ends and has roles in retrograde and anterograde transport in neurons. In this study, we investigated the role of dystonin during the entry stages of HSV-1 infection. Because of the way in which the MT network is organized, capsids are required to change their direction of motion along the MTs as they travel from the point of entry to the nucleus, where replication takes place. Thus, capsids first travel to the centrosome (the principal microtubule organizing center) by minus-end-directed transport and then switch polarity and travel to the nucleus by plus-end-directed transport. We observed that transport of capsids toward the centrosome was slowed, but not blocked, by dystonin depletion. However, transport of capsids away from the centrosome was significantly impaired, causing them to accumulate in the vicinity of the centrosome and reducing the numbers reaching the nucleus. We conclude that, during entry of HSV-1, dystonin has a specific role in plus-ended transport of capsids from the centrosome to the nucleus.     

The proteome of cytosolic lipid droplets isolated from differentiated Caco-2/TC7 enterocytes reveals cell-specific characteristics

Background information. Intestinal absorption of alimentary lipids is a complex process ensured by enterocytes and leading to TRL [TAG (triacylglycerol)‐rich lipoprotein] assembly and secretion. The accumulation of circulating intestine‐derived TRL is associated with atherosclerosis, stressing the importance of the control of postprandial hypertriglyceridaemia. During the postprandial period, TAGs are also transiently stored as CLDs (cytosolic lipid droplets) in enterocytes. As a first step for determining whether CLDs could play a role in the control of enterocyte TRL secretion, we analysed the protein endowment of CLDs isolated by sucrose‐gradient centrifugation from differentiated Caco‐2/TC7 enterocytes, the only human model able to secrete TRL in culture and to store transiently TAGs as CLDs when supplied with lipids. Cells were analysed after a 24 h incubation with lipid micelles and thus in a state of CLD‐associated TAG mobilization. Results. Among the 105 proteins identified in the CLD fraction by LC‐MS/MS (liquid chromatography coupled with tandem MS), 27 were directly involved in lipid metabolism pathways potentially relevant to enterocyte‐specific functions. The transient feature of CLDs was consistent with the presence of proteins necessary for fatty acid activation (acyl‐CoA synthetases) and for TAG hydrolysis. In differentiated Caco‐2/TC7 enterocytes, we identified for the first time LPCAT2 (lysophosphatidylcholine acyltransferase 2), involved in PC (phosphatidylcholine) synthesis, and 3BHS1 (3‐β‐hydroxysteroid dehydrogenase 1), involved in steroid metabolism, and confirmed their partial CLD localization by immunofluorescence. In enterocytes, LPCAT2 may provide an economical source of PC, necessary for membrane synthesis and lipoprotein assembly, from the lysoPC present in the intestinal lumen. We also identified proteins involved in lipoprotein metabolism, such as ApoA‐IV (apolipoprotein A‐IV), which is specifically expressed by enterocytes and has been proposed to play many functions in vivo, including the formation of lipoproteins and the control of their size. The association of ApoA‐IV with CLD was confirmed by confocal and immunoelectron microscopy and validated in vivo in the jejunum of mice fed with a high‐fat diet. Conclusions. We report for the first time the protein endowment of Caco‐2/TC7 enterocyte CLDs. Our results suggest that their formation and mobilization may participate in the control of enterocyte TRL secretion in a cell‐specific manner.     

Development of a ponasterone A-inducible gene expression system for application in cultured skeletal muscle cells

The goal of this study was to develop an inducible gene expression system to assess functions of specific proteins in differentiated cultured skeletal muscle. We utilized and modified the ecdysone inducible system because others have used this system to express exogenous genes in vitro and in transgenic animals. A limitation of the commercially-available ecdysone system is its constitutive expression in all tissues. Hence, its application in vivo would result in expression of a cloned gene in undifferentiated and differentiated tissues. To target its expression to muscle, we removed the constitutively-active CMV promoter of pVgRXR and replaced it with a skeletal muscle alpha-actin promoter so that the regulatory features of the system would be expressed in differentiated muscle cells. We transfected our newly designed expression system into L8 muscle myoblasts and established stable cell lines via antibiotic selection. We determined that reporter gene activity was induced by ponasterone A in myotubes, a differentiated muscle phenotype, but not in myoblasts (undifferentiated cells). This proved the validity of the concept of an inducible muscle-specific expression system. We then determined that beta-galactosidase expression was dependent upon the dose of ponasterone A and duration of exposure to inducer. This creates potential to regulate both the level of expression and duration of expression of a cloned gene in differentiated muscle.     

Brassicaceae phylogeny inferred from phytochrome A and ndhF sequence data: tribes and trichomes revisited

The family Brassicaceae comprises 3710 species in 338 genera, 25 recently delimited tribes, and three major lineages based on phylogenetic results from the chloroplast gene ndhF. To assess the credibility of the lineages and newly delimited tribes, we sequenced an approximately 1.8-kb region of the nuclear phytochrome A (PHYA) gene for taxa previously sampled for the chloroplast gene ndhF. Using parsimony, likelihood, and Bayesian methods, we reconstructed the phylogeny of the gene and used the approximately unbiased (AU) test to compare phylogenetic results from PHYA with findings from ndhF. We also combined ndhF and PHYA data and used a Bayesian mixed model approach to infer phylogeny. PHYA and combined analyses recovered the same three large lineages as those recovered in ndhF trees, increasing confidence in these lineages. The combined tree confirms the monophyly of most of the recently delimited tribes (only Alysseae, Anchonieae, and Descurainieae are not monophyletic), while 13 of the 23 sampled tribes are monophyletic in PHYA trees. In addition to phylogenetic results, we documented the trichome branching morphology of species across the phylogeny and explored the evolution of different trichome morphologies using the AU test. Our results indicate that dendritic, medifixed, and stellate trichomes likely evolved independently several times in the Brassicaceae.     

Glutathione peroxidase activity and chemical forms of selenium in tissues of rats given selenite or selenomethionine

Glutathione peroxidase (GPx) activity and deposition of selenium (Se) were examined in tissues of rats given dietary Se for 7 wk as either selenite or selenomethionine (SeMet) with 75Se radiotracer of the same chemical form. On the basis of Se:75Se ratio, all tissues of the rats fed selenite were equilibrated with the dietary source, but tissues of the SeMet fed animals maintained a ratio of Se:75Se greater than the dietary ratio. Deposition of dietary Se and 75Se was higher in most tissues of rats fed SeMet. Muscle 75Se was the largest single tissue pool of 75Se in both groups accounting for one-third of recovered 75Se in the rats fed selenite, and one-half of recovered 75Se in the rats fed SeMet. Tissue GPx activities were not different between the two dietary groups. The proportion of Se as GPx in tissues was highest in erythrocytes of the rats fed selenite (.81) and lowest in testes and epididymides of the rats fed SeMet (.009). The proportion of Se present in cytosolic GPx was consistently higher in tissues of rats fed selenite. Erythrocytes of the rats fed SeMet had more 75Se associated with hemoglobin, and muscle cytosols of the rats fed selenite had more 75Se associated with the G-protein. The proportion of 75Se as SeMet determined by ion exchange chromatography of tissue hydrolysates was higher in tissues of rats fed SeMet (highest in muscle and hemoglobin, 70%, and lowest in testes, 16%). In contrast, selenocysteine was the predominant form of Se present in tissues of rats given selenite. These results indicate that the form of Se administered will influence the form in the tissues, the percentage of Se with GPx and the body burden of Se.