The intact retroviral Env glycoprotein of human foamy virus is a trimer

Electron microscopy of negatively stained human foamy virus particles provides direct evidence for the trimeric nature of intact Env surface glycoproteins. Three-dimensional image reconstruction reveals that the Env trimer is a tapering spike 14 nm in length. The spikes were often arranged in hexagonal rings which shared adjacent Env trimers.     

Small, stable shuttle vectors based on the minimal pVS1 replicon for use in gram-negative, plant-associated bacteria

The minimal replicon of the Pseudomonas plasmid pVS1 was genetically defined and combined with the Escherichia coli p15A replicon, to provide a series of new, oligocopy cloning vectors (5.3 to 8.3 kb). Recombinant plasmids derived from these vectors were stable in growing and nongrowing cells of root-colonizing P. fluorescens strains incubated under different environmental conditions for more than 1 month.     

Chemical and posttranslational modification of Escherichia coli acyl carrier protein for preparation of dansyl-acyl carrier proteins

Escherichia coli acyl carrier protein (ACP) contains a single tyrosine residue at position 71. The combined o-nitration of apo-ACP Y71 by tetranitromethane and reduction to 3-aminotyrosyl-apo-ACP were performed to introduce a specific site for attachment of a dansyl fluorescent label. Conditions for purification and characterization of dansylaminotyrosyl-apo-ACP are reported. Dansylaminotyrosyl-apo-ACP was enzymatically phosphopantetheinylated and acylated in vitro with an overall approximately 30% yield of purified stearoyl-dansylaminotyrosyl-ACP starting from unmodified apo-ACP. The steady-state kinetic parameters k(cat) = 22 min(-1) and K(M) = 2.7 microM were determined for reaction of stearoyl-dansylaminotyrosyl-ACP with stearoyl-ACP Delta(9)-desaturase. These results show that dansylaminotyrosyl-ACP will function well for studying binding interactions with the Delta(9)-desaturase and suggest similar possibilities for other ACP-dependent enzymes. The efficient in vivo phosphopantetheinylation of E. coli apo-ACP by coexpression with holo-ACP synthase in E. coli BL21(DE3) using fructose as the carbon source is also reported.     

Inhibition of death receptor-mediated gene induction by a cycloheximide-sensitive factor occurs at the level of or upstream of Fas-associated death domain protein (FADD)

In HeLa cells, induction of apoptosis and nuclear factor kappaB (NF-kappaB) activation initiated by TRAIL/Apo2L or the agonistic Apo1/Fas-specific monoclonal antibody anti-APO-1 require the presence of cycloheximide (CHX). Inhibition of caspases prevented TRAIL/anti-APO-1-induced apoptosis, but not NF-kappaB activation, indicating that both pathways bifurcate upstream of the receptor-proximal caspase-8. Under these conditions, TRAIL and anti-APO-1 up-regulated the expression of the known NF-kappaB targets interleukin-6, cellular inhibitor of apoptosis 2 (cIAP2), and TRAF1 (TRAF, tumor necrosis factor receptor-associate factor). In the presence of CHX, the stable overexpression of a deletion mutant of the Fas-associated death domain molecule FADD comprising solely the death domain of the molecule but lacking its death effector domain (FADD-(80-208)) led to the same response pattern as TRAIL or anti-APO-1 treatment. Moreover, the ability of death receptors to induce NF-kappaB activation was drastically reduced in a FADD-deficient Jurkat cell line. TRAIL-, anti-APO-1-, and FADD-(80-208)-initiated gene induction was blocked by a dominant-negative mutant of TRAF2 or the p38 kinase inhibitor SB203580, similar to tumor necrosis factor receptor-1-induced NF-kappaB activation. CHX treatment rapidly down-regulated endogenous cFLIP protein levels, and overexpression of cellular FLICE inhibitory protein (cFLIP) inhibited death receptor-induced NF-kappaB activation. Thus, a novel functional role of cFLIP as a negative regulator of gene induction by death receptors became apparent.     

Dissociation from BiP and retrotranslocation of unassembled immunoglobulin light chains are tightly coupled to proteasome activity

Unassembled immunoglobulin light chains expressed by the mouse plasmacytoma cell line NS1 (kappa(NS1)) are degraded in vivo with a half-life of 50-60 min in a way that closely resembles endoplasmic reticulum (ER)-associated degradation (). Here we show that the peptide aldehydes MG132 and PS1 and the specific proteasome inhibitor lactacystin effectively increased the half-life of kappa(NS1), arguing for a proteasome-mediated degradation pathway. Subcellular fractionation and protease protection assays have indicated an ER localization of kappa(NS1) upon proteasome inhibition. This was independently confirmed by the analysis of the folding state of kappa(NS1) and size fractionation experiments showing that the immunoglobulin light chain remained bound to the ER chaperone BiP when the activity of the proteasome was blocked. Moreover, kinetic studies performed in lactacystin-treated cells revealed a time-dependent increase in the physical stability of the BiP-kappa(NS1) complex, suggesting that additional proteins are present in the older complex. Together, our data support a model for ER-associated degradation in which both the release of a soluble nonglycosylated protein from BiP and its retrotranslocation out of the ER are tightly coupled with proteasome activity.     

Epicuticular waxes and flavonol aglycones of the European mistletoe, Viscum album L

Cuticular waxes of Viscum album ssp. album contain oleanolic acid as main constituent, accompanied by aliphatic compounds like alkanes, esters and primary alcohols. A number of flavonol aglycones (methyl ethers of quercetin and kaempferol) have also been identified. Seasonal changes in amount and composition of cuticular waxes and the presence of flavonol aglycones are described and the ecophysiological significance of flavonoids on the surface of the mistletoe is briefly discussed.     

Troglitazone stimulates acetyl-CoA carboxylase activity through a post-translational mechanism

Troglitazone, a thiazolidinedione, is known to act as an insulin sensitizer. The various effects of the drug include stimulation of glucose utilization and inhibition of gluconeogenesis and fatty acid oxidation. We studied the effect of troglitazone treatment on rat liver acetyl-CoA carboxylase (ACC), the key enzyme that catalyzes the formation of malonyl-CoA, the rate-limiting step in the synthesis of long chain fatty acids. Treatment of rats with troglitazone for 18 days resulted in more than 200% increase in the activity of hepatic acetyl-CoA carboxylase (1.01+/-0.14 and 2.33+/-0.28 mU/mg supernatant protein for control and troglitazone-treated rats, respectively) (p<0.001). The expression of acetyl-CoA carboxylase mRNA, as studied by RNAse protection assay, was not significantly different between the two groups of animals. The ACC from control and troglitazone-treated groups was purified by avidin-affinity chromatography. The purified enzyme migrated as a major protein band (Mr 262,000) on SDS-polyacrylamide gels. Troglitazone treatment was associated with increased citrate sensitivity of ACC. The specific activity of the purified preparation in troglitazone-treated rats was increased by 67% (2.5 vs. 1.5 U/mg). Quantitation of alkali-labile phosphate content of the purified preparation revealed 5.66+/-0.17 and 6.29+/-0.13 mol Pi/mol subunit of 262 Kda for control and troglitazone-treated rats, respectively (P<0.01). The subtle increase in phosphate content does not explain the observed activation of the enzyme. It is possible that additional mechanisms such as troglitazone related rearrangement of the occupancy of select phosphate binding sites or altered binding of the biotin cofactor may also contribute to the observed activation of ACC.     

Cutting edge: IPSE/alpha-1, a glycoprotein from Schistosoma mansoni eggs, induces IgE-dependent, antigen-independent IL-4 production by murine basophils in vivo

During infection with the helminth parasite Schistosoma mansoni, the deposition of eggs coincides with the onset of IL-4 production and Th2 development. Although IL-4 is known as a potent inducer of Th2 differentiation, the mechanism by which schistosome eggs induce IL-4 production is not clear. In this study, we demonstrate that the S. mansoni egg Ag (SmEA) induces IgE-dependent IL-4 production by basophils derived from Heligmosomoides polygyrus-infected or OVA/alum-immunized mice in the absence of pathogen-specific IgE. The effect is mediated by the secretory glycoprotein IPSE/alpha-1, because IPSE/alpha-1-depleted SmEA no longer induces cytokine production. Conversely, recombinant IPSE/alpha-1 is sufficient to induce IL-4 production. Importantly, the injection of SmEA or recombinant IPSE/alpha-1 into H. polygyrus-infected 4get/KN2 IL-4 reporter mice rapidly induces the dose-dependent IL-4 production by basophils in the liver, a major site of egg deposition. Thus, IPSE/alpha-1 induces basophils to produce IL-4 even in the absence of Ag-specific IgE.     

Isolation and characterization of microsatellites in Neogobius kessleri (Perciformes,Gobiidae) and cross‐species amplification within the family Gobiidae

Fourteen polymorphic microsatellites were isolated from Neogobius kessleri, a benthic fish of Ponto‐Caspian origin which has been recently introduced into the Middle and Upper Danube River. Number of alleles and heterozygosity per locus in a sample of 32 fish individuals ranged from two to four and from 0.13 to 0.75, respectively. These primers will be useful in determining the population structure of N. kessleri. In addition, successful cross‐amplification was obtained for four related species, N. melanostomus, N. fluviatilis, N. gymnotrachelus and Proterorhinus marmoratus. These microsatellite loci may be useful for the evaluation of the origin of non‐native goby populations.