Highly conserved piggyBac elements in noctuid species of Lepidoptera

The piggyBac transposable element was originally discovered in a Trichoplusia ni cell line and nearly identical elements were subsequently discovered in the tephritid fly, Bactrocera dorsalis. This suggested the existence of piggyBac in additional insects and this study shows highly conserved, though not identical, piggyBac sequences in the noctuid species Heliocoverpa armigera, H. zea, and Spodoptera frugiperda, as well as new piggyBac sequences from the T. ni organismal genome. Genomic piggyBac elements could not be unambiguously identified in several other moth species indicating a discontinuous presence of piggyBac in the Lepidoptera. Most sequences have greater than 95% nucleotide identity to the original IFP2 piggyBac, except for a more diverged sequence in S. frugiperda, having approximately 78% identity. Variants of 1.3 and 0.8kb sequences found in both H. armigera and H. zea most likely became established by interbreeding, supporting the notion that the species are conspecific. None of the independent piggyBac sequences isolated from T. ni larval genomes are identical to IFP2, though all have an uninterrupted reading frame with the potential for encoding a functional transposase. The piggyBac sequences from T. ni and the Helicoverpa species, as well as those previously reported from B. dorsalis, all share three common nucleotide substitutions resulting in a single amino acid substitution in the transposase. This suggests that the original IFP2 piggyBac is a related variant of a predecessor element that became widespread. The existence of conserved piggyBac elements, some of which may have been transmitted horizontally between lepidopteran species, raises important considerations for the stability and practical use of piggyBac transformation vectors.     

Genetic similarity of vancomycin resistant strains of Enterococcus faecium isolated from clinical specimens

Twenty vancomycin resistant E. faecium strains (VRE) isolated from patients of three different hospital wards in 2005-2008 were examined. The strains originated from patients of intensive therapy, urological and internistic wards. The chosen wards differ significantly in their specificity. In all cases the presence of o vanA and lack of vanB, vanD, vanE and vanG genes and were found. Strains were compared by using RFLP-PFGE, the reference method for molecular typing of VRE. One group including fourteen strains showing similarity higher than 79.5% was distinguished. This group was divided into subgroups. The greatest similarity was found among strains from patients of intensive therapy ward. Two subgroups of strains showing similarity more than 93.3%, of four strains each were identified. The similarity between these two subgroups was 79.5%. Most strains from other two wards showed less than 79.5% similarity and they could be recognised as not related. Only one strain from internal ward and two strains from urologic ward were similar in 82.1 - 86.4% to one of subgroups of strains originated from intensive therapy.     

Comparative structural studies of two natural isoforms of ammodytoxin,phospholipases A2 from Vipera ammodytes ammodytes which differ in neurotoxicity and anticoagulant activity

Ammodytoxin A (AtxA) and its natural isoform AtxC from the venom of Vipera ammodytes ammodytes belong to group IIA-secreted phospholipases A₂ which catalyze the hydrolysis of glycerophospholipids and exhibit strong neurotoxic and anticoagulant effects. The two isoforms, which differ in sequence by only two amino acid residues (Phe124 > Ile and Lys128 > Glu), display significant differences in toxicity and anticoagulant properties and act on protein targets including neurotoxic proteic receptors and coagulation factor Xa with significantly different strengths of binding.In order to characterize the structural basis of these functional differences, we have determined the crystal structures of the two isoforms. Comparison of the structures shows that the mutation Lys128 > Glu in AtxC could perturb interactions with FXa, resulting in lower anticoagulant activity, since the side chain of Glu128 is partly buried, making a stabilizing hydrogen bond with the main-chain nitrogen atom of residue Thr35. This interaction leads to a displacement of the main polypeptide chain at positions 127 and 128 (identified by mutagenesis as important for interaction with FXa), and a different orientation of the side chain of unmutated Lys127. The mutation Phe124 > Ile in AtxC induces no significant conformational changes, suggesting that the differences in toxicity of the two isoforms are due essentially to differences in surface complementarity in the interaction of the toxin with the neurotoxic protein receptor. The crystal structures also reveal a novel dimeric quaternary association involving significant hydrophobic interactions between the N-terminal α-helices of two molecules of ammodytoxin related by crystallographic symmetry. Interactions at the dimer interface include important contributions from Met7, which is unique to ammodytoxin. Equilibrium sedimentation experiments are consistent with the crystallographic model.Competition experiments using SPR technology show complete inhibition of AtxA binding to FXa by calmodulin (CaM). The crystal structure shows that the C-terminal region, important for binding to FXa and CaM, is fully exposed and accessible for interaction with proteic receptors in both the monomeric and dimeric forms of ammodytoxin described here.     

Comparative binding of 125I-and 99mTc-d-labeled native and glycated low-density lipoprotein to human microvascular endothelial cells-potential for atherosclerosis imaging?

Native (n), glycated (g), and glycoxidated (go) low-density lipoproteins (LDL) were labeled with 125I or 99mTc, and the labeling efficiency and binding were assessed for potential use of these LDL compounds in imaging analysis of atherosclerotic lesions (PPAR-gamma receptors) by determining the number of specific receptors for nLDL, gLDL or goLDL on human microvascular endothelial cells as well as the KDs using either 125I-or 99mTc-labeled LDLs. The specific activity of labeled gLDL and goLDL was much higher (for goLDL 20 times higher) than that of nLDL. Gel filtration of labeled LDLs revealed, however, that 99mTc-g/goLDL is significantly degraded by the labeling reaction. No fragmentation was observed for 99mTc-nLDL and all the 125I-labeled LDL forms. Binding studies using both 125I-and 99mTc-nLDL indicated a weak binding affinity (KD 10- 7mol/L) to human microvascular endothelial cells. The binding affinity of 125I-g/goLDL to these cells was significantly higher (KD 10- 9mol/L) and could be increased further by preactivation of the endothelial cells using TNFalpha. Incubation with 99mTc-goLDL, however, did not result in specific binding of the ligand, possibly as a consequence of the fragmentation of the lipoprotein during the labeling. Scatchard transformation of the binding data with 99mTc-gLDL revealed the presence of only a few binding sites. This was in contrast to the results obtained with 125I-labeled gLDL, which revealed a much higher membrane density of scavenger receptors for this ligand. We conclude that for in vitro binding studies as well as for potential in vivo imaging, only 125I-labeled goLDL should be used, whereas nLDL may be applied as 125I-or 99mTc-labeled ligand.     

Identification of proteins that associate with protein kinase CK2

In order to aid in an understanding of the cellular functions of protein kinase CK2, a search for interacting proteins was carried out using a 32P-labeled CK2 overlay method. Several proteins were found to associate with CK2 by this assay; among them, one protein of 110 kDa appeared to be the most prominent one. The possible association of CK2 with p110 was suggested by experiments involving the co-immunoprecipitation using anti-CK2 antibodies. Further analysis using GST-CK2 fusion proteins demonstrated that the CK2-p110 interaction occurred through the CK2/ subunits. To identify p110, it was purified using a GST-CK2 affinity column, and internal amino acid sequencing was then performed. p110 was found to be nucleolin, a nucleolar protein that may be important for rRNA synthesis; a possible role of CK2 in the control of this process is suggested. Using the same CK2 overlay technique, another interacting protein, insulin receptor substrate 1 (IRS-1), was also identified. By applying a modified overlay method using individual 35S-labeled CK2 subunits, obtained by in vitro translation in rabbit reticulate lysates, it was determined that CK2 associates with IRS-1 through its / subunits; i.e. in keeping with the fact that IRS-1 is a known substrate for CK2. However, further work is needed to examine the association of CK2 with IRS-1 in vivo in order to fully understand the significance of the interaction.     

Is there a signal transduction pathway that links events at the plasma membrane to the phosphorylation state of the mitochondrial pyruvate dehydrogenase complex?

Monoclonal antibodies against the E1α subunit of pyruvate dehydrogenase (PDH) were used to quantify the mitochondrial pyruvate dehydrogenase complex (mtPDC) by enzyme-linked immunosorbent assay (ELISA). Recombinant Arabidopsis thaliana PDH (E1) was used to calibrate the ELISA. Antibodies against a synthetic phosphopeptide corresponding to phosphorylation site one of E1α were used in an ELISA to quantify phospho-PDC (P-PDC). For calibration of the second ELISA, recombinant E1 was phosphorylated in vitro with recombinant A. thaliana E1-kinase. The two ELISA were used to quantify mitochondrial total- and P-PDC in clarified homogenates from Nicotiana tabacum BY-2 suspension cells. The level of mtPDC remained constant throughout the 7-day growth cycle at 25.1 g⁻¹ FW. During the lag (days 0–2) and stationary (day 7) stages of the growth cycle, the mtPDC was completely phosphorylated (inactive), whereas during the log-growth stage it was completely dephosphorylated (active). Exposure of 3- or 7-day posttransfer suspension cells to osmotic stress significantly decreased proportion of P-PDC. A series of pharmacological studies were undertaken to gain insight into the signal transduction pathways coupling osmotic stress perception with control of mitochondrial respiration. Results from these studies indicate a signal transduction pathway linking stress perception to control of mitochondrial respiration that includes protein kinases and phosphoprotein phosphatases.     

Bidirectional regulation of renal cortical Na+,K+-ATPase by protein kinase C

We examined the role of protein kinase C (PKC) in the regulation of Na+,K+- ATPase activity in the renal cortex. Male Wistar rats were anaesthetized and the investigated reagents were infused into the abdominal aorta proximally to the renal arteries. A PKC-activating phorbol ester, phorbol 12,13-dibutyrate (PDBu), had a dose-dependent effect on cortical Na+,K+-ATPase activity. Low dose of PDBu (10(-11) mol/kg per min) increased cortical Na+,K+-ATPase activity by 34.2%, whereas high doses (10(-9) and 10(-8) mol/kg per min) reduced this activity by 22.7% and 35.0%, respectively. PDBu administration caused changes in Na+,K+-ATPase Vmax without affecting K(0.5) for Na+, K+ and ATP as well as Ki for ouabain. The effects of PDBu were abolished by PKC inhibitors, staurosporine, GF109203X, and G? 6976. The inhibitory effect of PDBu was reversed by pretreatment with inhibitors of cytochrome P450-dependent arachidonate metabolism, ethoxyresorufin and 17-octadecynoic acid, inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002, and by actin depolymerizing agents, cytochalasin D and latrunculin B. These results suggest that PKC may either stimulate or inhibit renal cortical Na+,K+-ATPase. The inhibitory effect is mediated by cytochrome P450-dependent arachidonate metabolites and PI3K, and is caused by redistribution of the sodium pump from the plasma membrane to the inactive intracellular pool.     

Plasma levels of total, free and protein bound thiols as well as sulfane sulfur in different age groups of rats

The redox status of plasma thiols can be a diagnostic indicator of different pathological states. The aim of this study was to identify the age dependent changes in the plasma levels of total, free and protein bound glutathione, cysteine and homocysteine. The determination was conducted in plasma of three groups of rats: 1) young (3-month-old), 2) middle aged (19-month-old), and 3) old (31-month-old). Total levels of glutathione, cysteine and homocysteine and their respective free and protein-bound fractions decreased with age. The only exception was a rise in free homocysteine concentration in the middle group, which indicates a different pattern of transformations of this thiol in plasma. The drop in the level of protein-bound thiols suggests that the antioxidant capacity of plasma diminishes with age, which, consequently, leads to impaired protection of -SH groups through irreversible oxidation. The plasma sulfane sulfur level also declines with age, which means that aging is accompanied by inhibition of anaerobic sulfur metabolism.     

Fcgamma receptor-like activity of hepatitis C virus core protein

We have previously demonstrated that viral particles with the properties of nonenveloped hepatitis C virus (HCV) nucleocapsids occur in the serum of HCV-infected individuals (1). We show here that nucleocapsids purified directly from serum or isolated from HCV virions have FcgammaR-like activity and bind "nonimmune" IgG via its Fcgamma domain. HCV core proteins produced in Escherichia coli and in the baculovirus expression system also bound "nonimmune" IgG and their Fcgamma fragments. Folded conformation was required for IgG binding because the FcgammaR-like site of the core protein was inactive in denaturing conditions. Studies with synthetic core peptides showed that the region spanning amino acids 3-75 was essential for formation of the IgG-binding site. The interaction between the HCV core and human IgG is more efficient in acidic (pH 6.0) than in neutral conditions. The core protein-binding site on the IgG molecule differs from those for C1q, FcgammaRII (CD32), and FcgammaRIII (CD16) but overlaps with that for soluble protein A from Staphylococcus aureus (SpA), which is located in the CH2-CH3 interface of IgG. These characteristics of the core-IgG interaction are very similar to those of the neonatal FcRn. Surface plasmon resonance studies suggested that the binding of an anti-core antibody to HCV core protein might be "bipolar" through its paratope to the corresponding epitope and by its Fcgamma region to the FcgammaR-like motif on this protein. These features of HCV nucleocapsids and HCV core protein may confer an advantage for HCV in terms of survival by interfering with host defense mechanisms mediated by the Fcgamma part of IgG.     

Ultrastructural studies on the formation of egg envelopes in fulgoromorphans (Insecta, Hemiptera, Fulgoromorpha: Dictyopharidae)

Follicular cells in ecuadorian dictyopharids diversify into two subpopulations: the main body cells (MFs) and cells surrounding the anterior pole of the oocyte (AFs). The synthetic activity of both categories of follicular cells is manifested by the presence of numerous cisternae of rough endoplasmic reticulum, Golgi complexes and vacuoles containing electron-dense material in their cytoplasm. The MFs synthesize precursors of the main body chorion, whereas the AFs are responsible for the formation of micropylar apparatus and respiratory tubules. The main body chorion is composed of thin endochorion and exochorion which forms pillar-like projections.