Is the IS1 transposase, InsAB', the only IS1-encoded protein required for efficient transposition?

The transposase of the bacterial insertion sequence IS1 is normally expressed by inefficient translational frameshifting between an upstream reading frame which itself specifies a transposition inhibitor, InsA, and a second consecutive reading frame located immediately downstream. A fused-frame mutant which carries an additional base pair inserted at the point of frameshifting was constructed. This mutant exhibits high transposition activity and should express the transposase, InsAB', constitutively without frameshifting. Unexpectedly, a second protein species was observed to be expressed from this mutant. We demonstrate here that this protein, InsA*, results from continued frameshifting on the modified frameshift motif. The protein retains the activities of the repressor InsA. Its elimination, by further modification of the frameshift motif, results in a further increase in various transposition activities of IS1. These results support the hypothesis that a single IS1-encoded protein, InsAB', is necessary for transposition.     

Chromosome localization of the genes for ENO1, HK1, ADK,ACP2, MPI,ITPA, ACON1 and α-GAL in the American mink (Mustela vison)

Summary Twenty-eight American mink × Chinese hamster somatic cell hybrids were analysed for the expression of mink enzymes and the segregation of mink chromosomes. The results demonstrated that the gene for enolase-1 is located on the long arm of mink chromosome 2, and those for hexokinase-1 and adenosine kinase, on its short arm. Segregation analysis of mink chromosomes and mink acid phosphatase-2, mannose phosphate isomerase, inosine triphosphatase and aconitase-1 provided data allowing us to assign the genes for these markers to mink chromosomes 7, 10, 11 and 12, respectively. The expression of mink -galactosidase was highly coincidental with mink × chromosome as well as with its markers: hypoxanthine-phosphoribosyltransferase, glucose-6-phosphate dehydrogenase and phosphoglycerate kinase-1. This result confirms the assignment of the gene for -galactosidase to the mink × chromosome.     

Structural Insights into the A1 ATPase from the archaeon, Methanosarcina mazei Gö1

The low-resolution structure and overall dimensions of the A(3)B(3)CDF complex of the A(1) ATPase from Methanosarcina mazei G?1 in solution is analyzed by synchrotron X-ray small-angle scattering. The radius of gyration and the maximum size of the complex are 5.03 +/- 0.1 and 18.0 +/- 0.1 nm, respectively. The low-resolution shape of the protein determined by two independent ab initio approaches has a knob-and-stalk-like feature. Its headpiece is approximately 9.4 nm long and 9.2 nm wide. The stalk, which is known to connect the headpiece to its membrane-bound A(O) part, is approximately 8.4 nm long. Limited tryptic digestion of the A(3)B(3)CDF complex was used to probe the topology of the smaller subunits (C-F). Trypsin was found to cleave subunit C most rapidly at three sites, Lys(20), Lys(21), and Arg(209), followed by subunit F. In the A(3)B(3)CDF complex, subunit D remained protected from proteolysis.     

Adipophilin affects the expression of TNF-α, MCP-1, and IL-6 in THP-1 macrophages

Macrophages accumulated in the arterial intima play an important role in the development of atherosclerosis by producing a large number of proinflammatory cytokines which accelerate the disease. Recent studies show that adipophilin might be involved in inflammatory processes in macrophages. In this study, we observe the effect of adipophilin on proinflammatory cytokine expression and secretion in THP-1 macrophages. SiRNA and adipophilin gene overexpression mediated by an pEGFP-C3 vector were used to observe the effect of adipophilin on proinflammatory cytokines in THP-1 macrophages in vitro. Realtime PCR and enzyme-linked immunosorbent assay (ELISA) were applied to detect the production of tumor necrosis factor α (TNF-α), monocyte chemoattractant protein 1 (MCP-1), and interleukin-6 (IL-6). It was found that acetylated low-density lipoprotein (AcLDL), pioglitazone [a peroxisome proliferator-activated receptor γ (PPARγ) agonist] increased adipophilin expression in macrophages, while glucose had no such affect. It was also shown that adipophilin augments TNF-α, MCP-1, and IL-6 expression in AcLDL induced macrophages. Our results suggest that adipophilin augment inflammation in macrophages, which might be one role of adipophilin in atherosclerosis.     

How Does KCNE1 Regulate the Kv7.1 Potassium Channel? Model-Structure,Mutations, and Dynamics of the Kv7.1-KCNE1 Complex

The voltage-gated potassium channel Kv7.1 and its auxiliary subunit KCNE1 are expressed in the heart and give rise to the major repolarization current. The interaction of Kv7.1 with the single transmembrane helix of KCNE1 considerably slows channel activation and deactivation, raises single-channel conductance, and prevents slow voltage-dependent inactivation. We built a Kv7.1-KCNE1 model-structure. The model-structure agrees with previous disulfide mapping studies and enables us to derive molecular interpretations of electrophysiological recordings that we obtained for two KCNE1 mutations. An elastic network analysis of Kv7.1 fluctuations in the presence and absence of KCNE1 suggests a mechanistic perspective on the known effects of KCNE1 on Kv7.1 function: slow deactivation is attributed to the low mobility of the voltage-sensor domains upon KCNE1 binding, abolishment of voltage-dependent inactivation could result from decreased fluctuations in the external vestibule, and amalgamation of the fluctuations in the pore region is associated with enhanced ion conductivity.     

Involvement of the Akt/NF-κB Pathways in the HTNV-Mediated Increase of IL-6, CCL5, ICAM-1, and VCAM-1 in HUVECs

Background

Hantaan virus (HTNV) infection causes a severe form of HFRS(hemorrhagic fever with renal syndrome)in Asia. Although HTNV has been isolated for nearly forty years, the pathogenesis of HFRS is still unknown, and little is known regarding the signaling pathway that is activated by the virus.

Methodology/Principal Findings

Cardamonin was selected as a NF-κB inhibitor, and indirect immunofluorescence assays were used to detect the effect of cardamonin on HTNV-infected HUVECs. The effect of cardamonin on the HTNV-induced phosphorylation of Akt and DNA-binding activity of NF-κB were determined using Western blot analysis and electrophoretic mobility shift assays (EMSAs), respectively. Then, flow cytometric and quantitative real-time PCR analyses were performed to quantify the expression levels of the adhesion molecules ICAM-1 and VCAM-1, and the concentrations of IL-6, IL-8, and CCL5 in HUVEC supernatants were examined using ELISA. The results showed that cardamonin did not effect the proliferation of HUVECs or the replication of HTNV in HUVECs. Instead, cardamonin inhibited the phosphorylation of Akt and nuclear transduction of NF-κB and further reduced the expression of the adhesion molecules ICAM-1 and VCAM-1 in HTNV-infected HUVECs. Cardamonin also inhibited the secretion of IL-6 and CCL5, but not IL-8.

Conclusion/Significance

HTNV replication may not be dependent upon the ability of the virus to activate NF-κB in HUVECs. The Akt/NF-κB pathways may be involved in the pathogenesis of HFRS; therefore, cardamonin may serve as a potential beneficial agent for HFRS therapy.     

Cloning and analysis of the cDNA encoding the rod G-protein transducin α, β1 and γ1 subunits from the canine retina

The canine (Canis familiaris) retinal rod transducin (G_T) α, β1 and γ1 subunits were sequenced. Cloning of the cDNAs was accomplished by polymerase chain reaction (PCR) using degenerate and wild type retinal cDNA libraries as templates. The deduced amino acid sequences were highly similar to rod transducins from other species: G_Tα differed by 5 amino acids from the corresponding human sequence, whereas β1 and γ1 were identical to human sequences. The coding sequence of rod transducin was evaluated as a possible cause for the recessively inherited retinal rod-cone degeneration: there were no nucleotide differences between the wild type and retinal degenerate strains.     

UCP1: The Original Uncoupling Protein—and Perhaps the Only One? New Perspectives on UCP1, UCP2, and UCP3 in the Light of the Bioenergetics of the UCP1-Ablated Mice

The availability of a UCP1-ablated mouse has enabled critical studies of the function of UCP1,UCP2, and UCP3. Concerning UCP1, its presence in brown-fat mitochondria is associatedwith innate uncoupling, high GDP-binding capacity, and GDP-inhibitable Cl^- permeabilityand uncoupling—but the high fatty acid sensitivity found in these mitochondria is observedeven in the absence of UCP1. The absence of UCP1 leads to low cold tolerance but not toobesity. UCP1 ablation also leads to an augmented expression of UCP2 and UCP3 in brownadipose tissue, making this tissue probably the one that boasts the highest expression ofthese UCPs. However, these very high expression levels are not associated with any inherentuncoupling, or with a specific GDP-binding capacity, or with a GDP-sensitive Cl^- permeability,or with any effect of GDP on mitochondrial membrane potential, or with an increased basalmetabolism of cells, or with the presence of norepinephrine- or fatty acid-induced thermogenesisin cells, and not with a cold-acclimation recruited, norepinephrine-induced thermogenicresponse in the intact animal. Therefore, it can be discussed whether any uncoupling effect isassociated with UCP2 or UCP3 when they are endogenously expressed and, consequently,whether (loss of) uncoupling (thermogenic) effects of UCP2 or UCP3 can be invoked toexplain metabolic phenomena, such as obesity.     

Phospholemman (FXYD1) raises the affinity of the human α1β1 isoform of Na,K-ATPase for Na ions

The human α(1)/His(10)-β(1) isoform of the Na,K-ATPase has been expressed in Pichia pastoris, solubilized in n-dodecyl-β-maltoside, and purified by metal chelate chromatography. The α(1)β(1) complex spontaneously associates in vitro with the detergent-solubilized purified human FXYD1 (phospholemman) expressed in Escherichia coli. It has been confirmed that FXYD1 spontaneously associates in vitro with the α(1)/His(10)-β(1) complex and stabilizes it in an active mode. The functional properties of the α(1)/His(10)-β(1) and α(1)/His(10)-β(1)/FXYD1 complexes have been investigated by fluorescence methods. The electrochromic dye RH421 which monitors binding to and release of ions from the binding sites has been applied in equilibrium titration experiments to determine ion binding affinities and revealed that FXYD1 induces an ～30% increase of the Na(+)-binding affinity in both the E(1) and P-E(2) conformations. By contrast, it does not affect the affinities for K(+) and Rb(+) ions. Phosphorylation induced partial reactions of the enzyme have been studied as backdoor phosphorylation by inorganic phosphate and in kinetic experiments with caged ATP in order to evaluate the ATP-binding affinity and the time constant of the conformational transition, Na(3)E(1)-P → P-E(2)Na(3). No significant differences with or without FXYD1 could be detected. Rate constants of the conformational transitions Rb(2)E(1) → E(2)(Rb(2)) and E(2)(Rb(2)) → Na(3)E(1), investigated with fluorescein-labeled Na,K-ATPase, showed only minor or no effects of FXYD1, respectively. The conclusion from all these experiments is that FXYD1 raises the binding affinity of α(1)β(1) for Na ions, presumably at the third Na-selective binding site. In whole cell expression studies FXYD1 reduces the apparent affinity for Na ions. Possible reasons for the difference from this study using the purified recombinant Na,K-ATPase are discussed.     

Effect of the association of IGF-I, IGF-II, bFGF, TGF-β1, GM-CSF, and LIF on the development of bovine embryos produced in vitro

This study examined the influence of the following growth factors and cytokines on early embryonic development: insulin-like growth factors I and II (IGF-I, IGF-II), basic fibroblast growth factor (bFGF), transforming growth factor (TGF-β), granulocyte-macrophage colony-stimulating factor (GM-CSF), and leukemia inhibitory factor (LIF). Synthetic oviduct fluid (SOF) was used as the culture medium. We studied the development of bovine embryos produced in vitro and cultured until Day 9 after fertilization. TGF-β1, bFGF, GM-CSF, and LIF used on their own significantly improved the yield of hatched blastocysts. IGF-I, bFGF, TGF-β1, GM-CSF, and LIF significantly accelerated embryonic development, especially the change from the expanded blastocyst to hatched blastocyst stages. Use of a combination of these growth factors and cytokines (GF-CYK) in SOF medium produced higher percentages of blastocysts and hatched blastocysts than did use of SOF alone (45% and 22% vs. 24% and 12%; P < 0.05) on Day 8 after in vitro fertilization and similar results to use of SOF + 10% fetal calf serum (38% and 16%, at the same stages, respectively). The averages of total cells, inner cell mass cells, and trophectoderm cells of exclusively in vitro Day-8 blastocysts for pooled GF-CYK treatments were higher than those for SOF and similar to those for fetal calf serum. The presence of these growth factors and cytokines in the embryo culture medium therefore has a combined stimulatory action on embryonic development; in particular through an increase in hatching rate and in the number of cells of both the inner cell mass and trophoblast. These results are the first to demonstrate that use of a combination of recombinant growth factors and cytokine, as IGF-I, IGF-II, bFGF, TGF-β1, LIF, and GM-CSF, produces similar results to 10% fetal calf serum for the development of in vitro-produced bovine embryos. This entirely synthetic method of embryo culture has undeniable advantages for the biosecurity of embryo transfer.     

Plumieribetin, a Fish Lectin Homologous to Mannose-binding B-type Lectins, Inhibits the Collagen-binding ��1��1 Integrin

Recently, a few fish proteins have been described with a high homology to B-type lectins of monocotyledonous plants. Because of their mannose binding activity, they have been ascribed a role in innate immunity. By screening various fish venoms for their integrin inhibitory activity, we isolated a homologous protein from the fin stings and skin mucus of the scorpionfish (Scorpaena plumieri). This protein inhibits α1β1 integrin binding to basement membrane collagen IV. By protein chemical and spectroscopic means, we demonstrated that this fish protein, called plumieribetin, is a homotetramer and contains a high content of anti-parallel β strands, similar to the mannose-binding monocot B-lectins. It lacks both N-linked glycoconjugates and common O-glycan motifs. Despite its B-lectin-like structure, plumieribetin binds to α1β1 integrin irrespective of N-glycosylation, suggesting a direct protein-protein interaction. This interaction is independent of divalent cations. On the cellular level, plumieribetin failed to completely detach hepatocarcinoma HepG2 cells and primary arterial smooth muscle cells from the collagen IV fragment CB3. However, plumieribetin weakened the cell-collagen contacts, reduced cell spreading, and altered the actin cytoskeleton, after the compensating α2β1 integrin was blocked. The integrin inhibiting effect of plumieribetin adds a new function to the B-lectin family, which is known for pathogen defense.     

Genomic organization, chromosomal mapping, and analysis of the 5’ promoter region of the human MAdCAM-1 gene

 MAdCAM-1, the endothelial addressin cell adhesion molecule-1, interacts preferentially with the leukocyte β7 integrin LPAM-1 (α4β7), but also with L-selectin, and with VLA-4 (α4β1) on myeloid cells, and serves to direct leukocytes into mucosal and inflamed tissues. Overlapping cosmid and phage λ genomic clones were isolated, revealing that the human MAdCAM-1 gene contains five exons where the signal peptide, two Ig domains, and mucin domain are each encoded by separate exons. The transmembrane domain, cytoplasmic domain, and 3′ untranslated region are encoded together on exon 5. The mucin domain contains eight repeats in total that are subject to alternative splicing. Despite the absence of a human counterpart of the third IgA-homologous domain and lack of sequence conservation of the mucin domain, the genomic organizations of the human and mouse MAdCAM-1 genes are similar. An alternatively spliced MAdCAM-1 variant was identified that lacks exon 4 encoding the mucin domain, and may mediate leukocyte adhesion to LPAM-1 without adhesion to the alternate receptor, L-selectin. The MAdCAM-1 gene was located at p13.3 on chromosome 19, in close proximity to the ICAM-1 and ICAM-3 genes (p13.2-p13.3). PMA-inducible promotor activity was contained in a 700 base pair 5’ flanking fragment conserved with the mouse MAdCAM-1 gene including tandem NF-kB sites, and an Sp1 site; and in addition multiple potential AP2, Adh1 (ETF), PEA3, and Sp1 sites. In summary, the data establish that the previously reported human MAdCAM-1 cDNA does indeed encode the human homologue of mouse MAdCAM-1, despite gross dissimilarities in the MAdCAM-1 C-terminal structures. Received: 5 December 1996 / Revised: 2 January 1997     

The effect of illumination on the pigment composition of the ζ-carotenic mutant,PG1, of Scenedesmus obliquus

Dark grown Scenedesmus obliquus strain PG1 accumulated -carotene and phytoene as its major carotenoids. On illumination of dark-grown cultures in air/CO₂, cyclic carotenes and xanthophylls were formed, apparently at the expense of the accumulated phytoene and -carotene. This interconversion of carotenoids was accompanied by chlorophyll synthesis. In an atmosphere of nitrogen/CO₂ the light-induced changes occurred more slowly and in nitrogen alone the changes were incomplete. No massive production of cyclic carotenes from the accumulated -carotene was observed in cultures illuminated under anaerobic conditions.     

Backbone 1H, 15N, and 13C resonance assignments for the NOXO1β PX domain

NOXO1 (Nox Organizer 1) is a homolog of the NAPDH oxidase protein p47 ^phox . NADPH oxidases transfer electrons from NADPH to molecular oxygen, generating the superoxide anion. NOXO1 contains an N-terminal PX (phox homology) domain and is one of several PX domain-containing proteins found in the cytosolic subunits of the NADPH oxidase complex. These PX domains bind to membrane lipids and target the protein to membranes, recruiting other cytosolic components to the membrane bound components and aiding formation of a active enzyme complex. This recruitment represents a level of regulation of these oxidases. Here we report the backbone assignments of NOXO1β PX.