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We have developed a method called ‘gene gorging’ to make precise mutations in the Escherichia coli genome at frequencies high enough (1–15%) to allow direct identification of mutants by PCR or other screen rather than by selection. Gene gorging begins by establishing a donor plasmid carrying the desired mutation in the target cell. This plasmid is linearized by in vivo expression of the meganuclease I-SceI, providing a DNA substrate for lambda Red mediated recombination. This results in efficient replacement of the wild type allele on the chromosome with the modified sequence. We demonstrate gene gorging by introducing amber stop codons into the genes xylA, melA, galK, fucI, citA, ybdO, and lacZ. To compliment this approach we developed an arabinose inducible amber suppressor tRNA. Controlled expression mediated by the suppressor was demonstrated for the lacZ and xylA amber mutants. [Copyright &y& Elsevier]

Brazzein is a small, heat-stable, intensely sweet protein consisting of 54 amino acid residues. Based on the wild-type brazzein, 25 brazzein mutants have been produced to identify critical regions important for sweetness. To assess their sweetness, psychophysical experiments were carried out with 14 human subjects. First, the results suggest that residues 29–33 and 39–43, plus residue 36 between these stretches, as well as the C-terminus are involved in the sweetness of brazzein. Second, charge plays an important role in the interaction between brazzein and the sweet taste receptor. [Copyright &y& Elsevier]

The forkhead box f1 (Foxf1) transcription factor is expressed in septum transversum mesenchyme and splanchnic (visceral) mesoderm, which give rise to mesenchymal cells of gut-derived organs such as liver, gall bladder, lung, stomach, and intestine. Foxf1 −/− embryos die in utero and haploinsufficiency of the Foxf1 gene resulted in a variety of developmental abnormalities of the lung, gall bladder, esophagus, and trachea and caused defects in liver and lung repair. In this study, we used Foxf1 +/− mouse embryos containing the β-Galactosidase (β-Gal) gene knocked into the Foxf1 locus to examine whether Foxf1 is expressed in the developing brain and head region. In addition to previously reported Foxf1 expression patterns, β-Gal staining was also found in the mesenchyme of developing pituitary gland, eye, pancreas, heart, notochord, genital tubercle, Meckel''s cartilage, and cartilage primordia of nasal septum and vertebral bodies. In adult Foxf1 +/− mice, β-Gal staining was detected in anterior and posterior pituitary gland, astrocytes of the cerebellum and cerebral cortex, lens and retina of the eye, tracheal cartilage, parathyroid, cortical layer of thymus, capsule of the spleen, coronary arteries, and pancreatic blood vessels. [Copyright &y& Elsevier]

A novel gap junction forming protein, mouse connexin-30.2 (Cx30.2) contains 278 amino acid residues, and is 79% identical to human Cx31.9 (GJA11). Northern analysis showed that Cx30.2 is ubiquitously expressed, most prominently in testis. Polyclonal antibodies against Cx30.2 detected a 30 kDa protein in cells overexpressing Cx30.2, and in mouse testis. Immunofluorescence showed that Cx30.2 was expressed in vascular smooth muscle, but also in cell types where Cx31.9 was not detected. These data demonstrate that Cx30.2 is a bona fide gene, and suggest that it is the orthologue of Cx31.9, but that it may have additional roles compared with Cx31.9. [Copyright &y& Elsevier]

Intron splicing is a prime example of the many types of RNA processing catalyzed by small nuclear ribonucleoprotein (snRNP) complexes. Sm proteins form the cores of most snRNPs, and thus to learn principles of snRNP assembly we characterized the oligomerization and ligand-binding properties of Sm-like archaeal proteins (SmAPs) from Pyrobaculum aerophilum ( Pae) and Methanobacterium thermautotrophicum ( Mth). Ultracentrifugation shows that Mth SmAP1 is exclusively heptameric in solution, whereas Pae SmAP1 forms either disulfide-bonded 14-mers or sub-heptameric states (depending on the redox potential). By electron microscopy, we show that Pae and Mth SmAP1 polymerize into bundles of well ordered fibers that probably form by head-to-tail stacking of heptamers. The crystallographic results reported here corroborate these findings by showing heptamers and 14-mers of both Mth and Pae SmAP1 in four new crystal forms. The 1.9 Å-resolution structure of Mth SmAP1 bound to uridine-5′-monophosphate (UMP) reveals conserved ligand-binding sites. The likely RNA binding site in Mth agrees with that determined for Archaeoglobus fulgidus ( Afu) SmAP1. Finally, we found that both Pae and Mth SmAP1 gel-shift negatively supercoiled DNA. These results distinguish SmAPs from eukaryotic Sm proteins and suggest that SmAPs have a generic single-stranded nucleic acid-binding activity. [ABSTRACT FROM AUTHOR]

Human host cell factor-1 (HCF-1) is a large, 2035-residue nuclear protein that interacts with cellular and viral transcription factors. It contains an N-terminal kelch domain, C-terminal fibronectin type III (FnIII) domain, and a central region including tandem repeats which act as cleavage sites. A second human HCF-1 related gene encodes a protein with a high degree of homology in both the N-terminal kelch domain and C-terminal FnIII domain, but lacks the central portion and as a result is considerably smaller at 792 residues. A unique HCF orthologue has been found in Caenorhabditis elegans which is structurally more related to HCF-2 than HCF-1. Here we report the cloning and expression of the single Drosophila melanogaster host cell factor orthologue (dHCF). The dHCF is 1500 residues in size, intermediate between HCF-1 and HCF-2 and contains an N-terminal kelch domain, and C-terminal FnIII domain both of which show a very high degree of identity, and a central region of some 700 residues with more limited homology. Despite containing a central region no repeat-related motifs were apparent. The dHCF is expressed as a single unprocessed polypeptide consistent with the lack of the internal HCF-1 processing sites, and exhibits a predominantly nuclear localization. We show that this nuclear localization is dependent on a bipartite nuclear localization signal at the C-terminus of the protein, which contains a long spacer of 20 amino acids between two basic clusters. Finally, we also show that dHCF is unable to rescue the tsBN67 cell cycle arrest phenotype. These results indicate that dHCF is an orthologue of HCF-1, although both proteins might not be functionally exchangeable. [Copyright &y& Elsevier]

In this study we have modified the neuroinvasiveness of pseudorabies virus strain Bartha, a commonly utilized trans-synaptic tract-tracer. In addition, we sought to facilitate detection of cellular mRNAs in neurons infected with the virus. In order to modify spreading characteristics, we inserted the lacZ or the GFP (green fluorescent protein) genes into the genomic loci containing the putative latency-associated transcript promoter (PLAT2), resulting in the disruption of the promoter function. Following rat kidney injection, mutant viruses labeled central autonomic neurons in a slower and much more restricted manner than the parent Bartha strain. Since both reporter genes were controlled by the human cytomegalovirus immediate early (IE) 1 promoter, they exhibited IE expression kinetics. This property proved to be important for the co-detection of reporter proteins with neuronal mRNAs, readily detected at early but not at late stage of infection, as shown in tyrosine-hydroxylase expressing A5 catecholaminergic neurons and in serotonin transporter expressing raphe magnus neurons. [Copyright &y& Elsevier]

Both the β-d-(+) and β-l-(−)-enantiomers of 2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine (D4FC) are clinically relevant compounds because of their potent anti-HIV and anti-HBV activities. Cross-resistance to l-D4FC with HBV containing a mutation in the conserved polymerase YMDD region has been observed. In order to better understand the effects of stereochemistry on planar 5-fluorinated cytidine analogs and to gain insight into resistance caused by YMDD mutations in HIV-1 reverse transcriptase (RT), a combination of transient kinetic studies and computer modeling were employed. In contrast to studies with the (+) and (−) isomers of 3TC-TP and FTC-TP, it was found that wild type RT had a high enantiomeric selectivity between the d-(+) and l-(−) isomers of D4FC-TP. While no resistance was conferred by the methionine 184 to valine mutation to d-D4FC-TP, l-D4FC-TP was incorporated 50- to 70-fold less efficiently. The kinetic parameters of incorporation in the presence of l-D4FC-TP by RTWT and the mechanism of resistance by RTM184V were found to be distinct from those seen with the corresponding l-isomers containing an oxathiolane ring: (−)-3TC-TP and (−)-FTC-TP. Molecular modeling suggests that l- and d-D4FC-TP are positioned in the active site favorably for incorporation by RTWT and that l-D4FC-TP, but not d-D4FC-TP, is sterically hindered by the addition of a β branched amino acid at position 184 of RTM184V. [Copyright &y& Elsevier]

Neuronal nicotinic acetylcholine receptors (nAChR), composed of 12 subunits (α2–α10, β2–β4), are expressed in autonomic ganglia, playing a central role in autonomic transmission. The repertoire of nicotinic subunits in autonomic ganglia includes α3, α5, α7, β2 and β4 subunits. In the last 10 years, heterologous expression studies have revealed much about the nature of neuronal nAChRs. However, there is only limited understanding of subunit actions in autonomic system. Functional deletions of subunit by gene knockout in animals could overcome these limitations. We review recent studies on nAChRs on autonomic ganglia for physiological and pharmacological properties and potential locations of the subunits. [Copyright &y& Elsevier]

Certain antibodies (Abs) elicited using the cardiac glycoside digoxin (digoxigenin tridigitoxoside) bind preferentially to analogs that differ from digoxin by substitutions on the cardenolide rings, the lactone, or by the presence or absence of attached sugars. Antibody 26-10 binds equally well to digoxin and digitoxin, which differ only by the presence in the former and the absence in the latter of an hydroxyl group at C12. Other antidigoxin Abs, however, can distinguish between these ligands by three orders of magnitude in binding. Inspection of the structure of Fab 26-10 complexed with digoxin shows a gap in complementarity in the region between the digoxin O12 and LCDR3. We proposed that insertions in LCDR3 might result in Abs that bind digitoxin preferentially. We produced libraries of mutants displayed on bacteriophage which were randomized at LCDR3 and contained LCDR3 insertions. Mutants were selected by panning against digoxin and analogs. The mutants bound digitoxin preferentially up to 47-fold greater than digoxin. The mutants that bound well to digitoxin demonstrated a consensus sequence including the substitution of Trp at position L:94. Using site-directed mutagenesis, the binding to digitoxin was shown to be maximized by the combination of an insertion and L:Trp94 mutation, moving the L 94 side chain closer to digoxin. We also selected mutants that bound preferentially to gitoxin, which, like digitoxin, lacks the 12-hydroxyl, increasing relative binding to gitoxin up to 600-fold compared to the unmutated Ab 26-10. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor (VEGF) is a potent angiogenic mediator in tissue repair. In non-healing human wounds plasmin cleaves and inactivates VEGF165. In the present study, we generated recombinant VEGF165 mutants resistant to plasmin proteolysis. Substitution of Arg110 with Ala110 or Gln110, and Ala111 with Pro111 yielded plasmin-resistant and biologically active VEGF165 mutants. In addition, substitution of Ala111 with Pro111 resulted in a substantial degree of stabilization when incubated in wound fluid obtained from non-healing wounds. These results suggest that the plasmin cleavage site Arg110/Ala111 and the carboxyl-terminal domain play an important role in the mitogenic activity of VEGF165. [Copyright &y& Elsevier]

Dystrophin, the protein which is absent or non-functional in Duchenne muscular dystrophy, consists of four main domains: an N-terminal actin binding domain, a rod shaped domain of spectrin-like repeats, a cysteine-rich domain and a unique C-terminal domain. In muscle, dystrophin forms a linkage between the cytoskeletal actin and a group of membrane proteins (dystrophin associated proteins). The N-terminal domain binds to the cytoskeletal actin and the association with the dystrophin associated proteins is mediated mainly by the cysteine-rich and C-terminal domains of dystrophin. The dystrophin gene also encodes two isoforms of non-muscle dystrophins and a number of smaller products consisting of the two C-terminal domains with different extensions into the spectrin-like repeat domain. Dp71, which consist of the C-terminal and the cysteine-rich domains of dystrophin, is the major product of the gene in all non-muscle tissues tested so far, but it is absent in differentiated skeletal muscle. In an attempt to understand the functions of Dp71, we produced transgenic mice over-expressing this protein in several tissues. The highest levels of exogeneous Dp71 were detected in skeletal muscle, in association with the sarcolemma. This resulted in muscle damage similar to that found in mice which lack dystrophin. The data indicates that Dp71 competes with dystrophin for the binding to the dystrophin associated proteins. Since Dp71 lacks the actin binding domain, it cannot form the essential linkage between the dystrophin associated proteins complex and the cytoskeleton. [Copyright &y& Elsevier]

The putative cell-lysis gene lys of Lactobacillus plantarum G1e phage ϕg1e encodes for a 442 amino-acids protein Lys. The N-terminal region (about 80 amino acids) of Lys consists of two discrete regions (the signal-peptide-like domain and the DE domain containing putative active sites of endolysin). To elucidate functions of the regions of Lys, mutational (random, site-directed, and/or fusion) analysis was performed. The plasmid pNdEHL, expressing the wild type Lys protein under promoter of lacZ′ gene in Escherichia coli, was constructed. Two molecular species (44 kDa; referred to as pre-Lys, and 42 kDa; mature-Lys) from the protein extract of XL1-Blue/pNdEHL were detected on a sodium dodecyl sulfate gel and zymogram with L. plantarum G1e cells. Based on the N-terminal amino acid sequences, the two molecules were determined as; pre-Lys (the amino acid position deduced from lys gene, 1–7) MKLKNKL, mature-Lys (27–33) QTLSSQS. The mature Lys was hardly detected in the cells treated with sodium azide.These results suggested that the N-terminal 26 amino acids region of Lys precursor form is possibly processed posttranslationally, by a SecA-dependent manner at least in E. coli.Analysis of the point mutants (pLD36A, pLE39A, pLE55A, pLE67A and pLD71A), indicated that the acidic residues (aspartic acids at position 36, 71 and glutamic acids at position 39, 55) of N-terminal region and the serine at the position 48 of ϕg1e Lys are essential for the lytic activity. [Copyright &y& Elsevier]

Thrombopoietin (Tpo) and its receptor, c-mpl, are expressed in murine embryonic stem (ES) cells. ES cells are maintained in a pluripotent state by leukemia inhibitory factor (LIF) via activation of the Janus kinase (Jak)–STAT3 signaling pathway. Tpo, like LIF, activates STAT3. We report that Tpo increases the number of undifferentiated colonies derived from wild type or Shp-2 mutant (Shp-2Δ46–110) ES cells. Tpo plus LIF acted synergistically on the Shp-2Δ46–110 ES cells to maintain undifferentiated colonies but no evidence of synergism via Jak–STAT3 activation was detected. Collectively, these data suggest that Tpo can play a role in preventing ES cell differentiation via Jak–STAT3 activation and perhaps via novel pathways that are enhanced in the absence of functional Shp-2. [Copyright &y& Elsevier]

The anti- hen egg- white lysozyme (HEWL) antibodies HyHEL-10 and F9.13.7 recognize a common epitope. The structures of the complexes differ, however, in the numbers of electrostatic and hydrogen-bond interactions and in the distributions of contacts between the light and heavy chains. The equilibria and kinetics characterizing the F9.13.7 complex formation were evaluated for both wild-type and mutant derivatives of HEWL to help to understand how the different contacts are effectively used in the complexes with the two antibodies. Three epitope hot spots, Y20, K96, and R73 (destabilization > 4 kcal/mole), were found by alanine scanning mutagenesis. The first two constitute two of the three hot spots in the HyHEL-10 complex. The hot spots of the HyHEL-10 paratope are centered on the HEWL epitope; whereas R73 (HEWL), the only important light-chain-contacting residue, is clearly separated from the other hot spots of the F9.13.7 complex. The larger number of epitope warm plus hot spots found in the F9.13.7 complex compared with that of HyHEL-10 shows that the specificity of the former is greater even though the KD value is 20-fold larger. Conservative mutations showed that the specificity enhancement is related to the greater number of functional polar and hydrogen bond interactions in the F9.13.7 complex. Alanine scanning mutagenesis would not have illuminated these distinctions. It is shown that the concept of antigen specificity, as defined by cross-reactivity with natural variant antigens, is flawed by phylogenetic bias, and that specificity can only be defined by the use of unbiased epitopes, which are conveniently accessed by site-directed mutagenesis. [ABSTRACT FROM AUTHOR]

Cultured murine F9 teratocarcinoma stem cells resemble pluripotent stem cells of the inner cell mass of the mouse blastocyst and, depending upon their treatment, can be induced to differentiate along the primitive endoderm, the parietal endoderm (PE), or the visceral endoderm (VE) pathway. The Rex-1 gene encodes a zinc finger family transcription factor which is expressed at high levels in undifferentiated F9 stem cells, embryonic stem cells, and in other types of stem cells. To examine whether the Rex-1 protein plays a role in F9 cell differentiation, homologous recombination was employed to generate F9 cell lines which lack both alleles of Rex-1. F9 wild type cells in monolayer culture require both retinoic acid and cyclic AMP analogs to differentiate into PE, whereas the F9 Rex-1−/− cells differentiate into PE, as assessed by several molecular markers, including thrombomodulin and laminin B1, in the presence of RA alone. The F9 Rex-1−/− cells do not completely differentiate into VE after RA treatment in aggregate culture; they do not express alpha-fetoprotein, a definitive marker of VE differentiation. These results indicate that the Rex-1 transcription factor regulates the differentiation of F9 stem cells along several distinct cell lineages found in the early embryo. [Copyright &y& Elsevier]

The dopamine D2 receptor (D2) is implicated in drug addiction, learning and memory. Two isoforms of the D2 receptor, termed D2L (long form) and D2S (short form), have been identified. We previously generated mice lacking D2L (D2L−/−), but expressing functional D2S. In this study, we investigated the role of D2L in the positive and negative reinforcing properties of abused drugs and electrical stimuli, using D2L−/− mice as a model system. Mice were trained in three associative learning tasks: conditioned place preference to morphine and cocaine, conditioned place aversion to naloxone-precipitated morphine withdrawal, and active avoidance. D2L−/− mice, like wild type mice, developed a place preference to cocaine. In contrast to wild type mice, D2L−/− mice did not develop a place preference to morphine, nor did they attain a place aversion to morphine withdrawal. D2L−/− mice also failed to acquire avoidance behavior in response to electrical stimuli. There were no significant differences between D2L−/− and wild type mice in μ-opioid receptor density, morphine-induced locomotor stimulation and morphine withdrawal symptoms.These results suggest that D2L may have a greater impact than D2S on the rewarding aspects of morphine, and the aversive properties of morphine withdrawal and electrical stimulus. These findings also suggest that the presence of D2L is critical in the acquisition (learning) and/or retention (memory) of context–stimulus associations in certain situations. On the other hand, D2L is not essential for the rewarding aspects of cocaine and for the development of morphine dependence. Thus, these studies reveal distinct functional roles of D2L and/or D2S in drug addiction and avoidance learning, which may lead to a better understanding of the neurobiological basis underlying these behaviors. [Copyright &y& Elsevier]

Apolipophorin III (ApoLp-III) from the Sphinx moth, Manduca sexta, is an 18 kDa protein that binds reversibly to hydrophobic surfaces generated on metabolizing lipoprotein particles. It is comprised of amphipathic α-helices (H1–H5) organized in an up-and-down topology forming a helix bundle in the lipid-free state. Upon interaction with lipids, apoLp-III has been proposed to undergo a dramatic conformational change, involving helix bundle opening about putative hinge loops such that H1, H2 and H5 move away from H3 and H4. In the present study, we examine the relative spatial disposition of H1 and H5 on discoidal phospholipid complexes and spherical lipoproteins. Cysteine residues were engineered at position 8 in H1 and/or at position 138 in H5 in apoLp-III (which otherwise lacks Cys) yielding A8C-, A138C- and A8C/A138C–apoLp-III. Tethering of H1 and H5 by a disulfide bond between A8C and A138C abolished the ability of apoLp-III to transform phospholipid vesicles to discoidal particles, or to interact with lipoproteins, demonstrating that these helices are required to reposition during lipid interaction. Site-specific labeling of A8C/A138C–apoLp-III with N-(1-pyrene)maleimide in the lipid-free state resulted in intramolecular pyrene “excimer” fluorescence emission indicative of spatial proximity between these sites. Upon association with dimyristoylphosphatidylcholine (DMPC) discoidal complexes, the intramolecular excimer was replaced by intermolecular excimer fluorescence due to proximity between pyrene moieties on A8C and A138C in neighboring apoLp-III molecules on the discoidal particle. No excimer emission was observed in the case of pyrene–A8C–apoLp-III/DMPC or pyrene–A138C–apoLp-III/DMPC complexes. However, equimolar mixing of the two labeled single-cysteine mutants prior to disc formation resulted in excimer emission. In addition, intramolecular pyrene excimer formation was diminished upon binding of pyrene–A8C/A138C–apoLp-III to spherical lipoproteins. The data are consistent with repositioning of H1 away from H5 upon encountering a lipid surface, resulting in an extended conformation of apoLp-III that circumscribes the discoidal bilayer particle. [Copyright &y& Elsevier]

Application of delivery systems in cancer therapy is restricted as a result of the lack of cell specificity of the respective vectors. Recently, a vector system based on virus-like particles (VLPs) of modified polyoma-VP1 was described which were able to bind specifically a tumor-specific antibody fragment, thus directing the vector system towards tumor cells. The functional gene transfer using the VP1 variant VP1-E8C, coupled with the antibody fragment of the tumor-specific antibody B3 is described in this paper. The specific targeting of the antigen expressing cells was highly efficient as determined by fluorescence microscopy. However, only a low percentage of these cells showed a functional gene transfer. This discrepancy could be accounted for by a rather low capacity of the virus like particles to transport DNA and the mechanism of their internalization by the target cells, which led to a lysosomal degradation of the particles. These limitations could be surmounted partially in cell culture experiments, and the principles suitable for applying this vector system in vivo are discussed. [Copyright &y& Elsevier]

The activity of the Alzheimer's amyloid β-peptide is a sensitive function of the peptide's sequence. Increased fibril elongation rate of the E22Q Dutch mutant of the Alzheimer's amyloid β-peptide relative to that of the wild-type peptide has been observed. The increased activity has been attributed to a larger propensity for the formation of β structure in the monomeric E22Q mutant peptide in solution relative to the WT peptide. That hypothesis is tested using four nanosecond timescale simulations of the WT and Dutch mutant forms of the Aβ(10-35)-peptide in aqueous solution. The simulation results indicate that the propensity for formation of β-structure is no greater in the E22Q mutant peptide than in the WT peptide. A significant measure of 'flickering' of helical structure in the central hydrophobic cluster region of both the WT and mutant peptides is observed. The simulation results argue against the hypothesis that the Dutch mutation leads to a higher probability of formation of β-structure in the monomeric peptide in aqueous solution. We propose that the greater stability of the solvated WT peptide relative to the E22Q mutant peptide leads to decreased fibril elongation rate in the former. Stability difference is due to the differing charge state of the two peptides. The other proposal leads to the prediction that the fibril elongation rates for the WT and the mutant E22Q should be similar under acid conditions. [ABSTRACT FROM AUTHOR]

The Factor for Inversion Stimulation (FIS) is a dimeric DNA binding protein found in enteric bacteria that is involved in various cellular processes, including stimulation of certain specialized DNA recombination events and transcription regulation of a large number of genes. The intracellular FIS concentration, when cells are grown in rich media, varies dramatically during the early logarithmic growth phase. Its broad range of concentrations could potentially affect the nature of its quaternary structure, which in turn, could affect its ability to function in vivo. Thus, we examined the stability of FIS homodimers under a wide range of concentrations relevant to in vivo expression levels. Its urea-induced equilibrium denaturation was monitored by far- and near-UV circular dichroism (CD), tyrosine fluorescence, and tyrosine fluorescence anisotropy. The denaturation transitions obtained were concentration-dependent and showed similar midpoints (Cm) and m values, suggesting a two-state denaturation process involving the native dimer and unfolded monomers (N2 ↔ 2U). The Δ G [ABSTRACT FROM AUTHOR]

A long-hypocotyl phytochrome B deficient mutant (lh) and its isogenic wild type (WT) of cucumber (Cucumis sativus L.) were used to study interacting effects between thermo- and photomorphogenetic stem elongation in long- and short-term experiments. The plants were grown under a 12 h high light intensity period and day/night temperature (DT/NT) of 25/19 °C (positive DIF, difference between DT and NT) or 19/25 °C (negative DIF) in combination with 30 min end-of-day (EOD)-red (R) or far red (FR) light. Thirty minutes EOD-FR, compared to EOD-R, significantly increased the length of stem, hypocotyl and internodes, and the number of visible internodes in WT. Positive DIF enhanced stem elongation compared to negative DIF under an EOD-R treatment, while the effect of negative DIF was strongly reduced by EOD-FR. For instance, an exposure to EOD-FR increased the plant height 68 and 41% under negative and positive DIF, respectively. In contrast, the lh mutant showed little difference in stem elongation in response to EOD-R and EOD-FR treatments, and small difference between positive and negative DIF treatments. These results indicate that DIF interacts significantly with EOD light quality on stem elongation, and that phytochrome B may play a key role in the interaction between thermo- and photomorphogenetic responses. A study on the kinetics of hypocotyl elongation showed that DIF interacted with EOD treatments in manipulating hypocotyl elongation rate (HER). In the photoperiod, positive DIF resulted in greater average HER than zero DIF and negative DIF, and the effect of DIF was stronger under EOD-R than EOD-FR. In the dark period, negative DIF showed larger HER compared to positive and zero DIF, and the effect was enhanced by EOD-FR compared to EOD-R. An EOD-FR increased HER both in photoperiod and dark period, in comparison to an EOD-R. Further, under EOD-R, negative DIF postponed a distinct peak of HER early in the dark period by approximately 4 h compared to positive DIF. A sharp peak was only found in the photoperiod under positive DIF. Under EOD-FR, a sharp peak of HER appeared at the end of the dark period or early in the photoperiod under negative DIF. This may indicate that the phytochrome is involved in thermoperiodic control of the kinetics of hypocotyl elongation. [Copyright &y& Elsevier]

Bisdioxopiperazine anti-cancer agents are catalytic inhibitors of topoisomerase II which by unknown means lock the enzyme in a closed clamp form and inhibit its ATPase activity. In order to demarcate a putative pharmacophore, we here describe a novel Tyr165Ser mutation in the enzyme’s Walker A ATP binding site leading to specific bisdioxopiperazine resistance when transformed into a temperature-conditional yeast system. The Tyr165Ser mutation differed from a previously described Arg162Gln by being heterozygous and by purified Tyr165Ser enzyme being drug-resistant in a kinetoplast DNA decatenation enzymatic assay. This suggested dominant nature of Tyr165Ser was supported by co-transformation studies in yeast of plasmids carrying wild type and mutant genes. These results enable a model of the bisdioxopiperazine pharmacophore using the proposed asymmetric ATP hydrolysis of the enzyme. [Copyright &y& Elsevier]

Mouse Emx2 homeobox gene is a very good dorsal marker for the developing cerebral cortex, as it is mainly expressed in this area from the very beginning of corticogenesis. Its cortical expression includes proliferating neuroblasts of the neuroepithelium, or ventricular zone, and the postmitotic Cajal-Retzius cells, known to control neuronal radial migration. Analysis of the phenotype of Emx2 null embryos has shown that this transcription factor plays important roles in neuroblast proliferation, migration and differentiation, as well as in the development of the diencephalon, where it has been shown to cooperate with Otx2. Moreover, the graded distribution of EMX2 homeoprotein along the antero-posterior and medial-lateral cortical axis, is responsible for the patterning of the forebrain, in particular for the specification process that defines cortical territories and area identity during neocortical development. Emx2 participates to this process as Pax6 and COUP-TF1. Finally Emx2 is very interesting from the evolutionary point of view, as it has been shown to share a high degree of homology in its sequence and function with Vax1, another homeobox gene regulating basal forebrain development. This homology traces back to Emx and Vax gene families, which are strongly related, as they are thought to derive from a common ancestor gene. [Copyright &y& Elsevier]

The role of the Lys68* :Glu265 intersubunit salt bridge that is conserved (Csb) in all known aspartate aminotransferases (AATases), except those of animal cytosolic, Ac (His68*:Glu265), and plant mitochondrial, Pm (Met68*:Gln265), origins, was evaluated in the Escherichia coli AATase. Two double-mutant cycles, to K68M/E265Q and the charge reversed K68E/E265K, were characterized with the context dependence ( C) and impact ( I) formalism, previously defined for functional chimeric analysis. Mutations of Lys68* with Glu265 fixed are generally more deleterious than the converse mutations of Glu265 with Lys68* fixed, showing that buried negative charges have greater effects than buried positive charges in this context. Replacement of the charged Lys68*:Glu265 with the K68M/E265Q neutral pair introduces relatively small effects on the kinetic parameters. The differential sensitivity of kcat/ KM, L-Asp and kcat/ KM, α-KG to salt bridge mutagenic replacements is shown by a linear-free energy relationship, in which the logarithms of the latter second order rate constants are generally decreased by a factor of two more than are those of the former. Thus, kcat/ KM, L-Asp and kcat/ KM, α-KG are 133 and 442 mM−1s−1 for the wild-type (WT) enzyme, respectively, but their relative order is reversed in the more severely compromised mutants (14.8 and 5.3 mM−1s−1 for K68E). A Venn diagram illustrates apparent forced covariances of groups of amino acids that accompany the naturally occurring salt bridge replacements in the Pm and Ac classes. The more deeply rooted tree indicates that the Csb variant was the ancestral specie. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus type-1 envelope glycoprotein (gp120env) binding to cell surface CD4 receptor triggers a broad range of intracellular effects leading to T cell activation and cell cycle entry. Among these effects we and others previously reported on the nuclear translocation of the nuclear factor-κB (NF-κB) transcription factor. The present work further investigates the signal transduction pathways involved in gp120env-induced NF-κB activation. We demonstrate that gp120env–CD4 interaction stimulates the hyperphosphorylation of IκB-α inhibitory protein. Conversely, overexpression of a dominant-negative IκB-α transgene mutated at S32 and S36 residues, abolishes gp120env-induced NF-κB activation. IκB kinases (IKKs) activity was found to be selectively enhanced following CD4 engagement with gp120env and to mediate the phosphorylation of IκB-α while co-transfection experiments using dominant-negative forms of IKKs inhibited gp120env-induced NF-κB activation. Taken together, these results confirm that IKKs complex play a key role in gp120env-induced NF-κB activation. [Copyright &y& Elsevier]

Cardiac troponin I, the inhibitory subunit of the heterotrimeric cardiac troponin (cTn) complex is phosphorylated by protein kinase A at two serine residues located in its heart-specific N-terminal extension. This flexible arm interacts at different sites within cTn dependent on its phosphorylation degree. Bisphosphorylation is known to induce conformational changes within cTnI which finally lead to a reduction of the calcium affinity of cTnC. However, as we show here, the bisphosphorylated cTnI arm does not interact with cTnC, but with cTnT and/or cTnI. [Copyright &y& Elsevier]

Xanthophylls (oxygen derivatives of carotenes) are essential components of the plant photosynthetic apparatus. Lutein, the most abundant xanthophyll, is attached primarily to the bulk antenna complex, light-harvesting complex (LHC) II. We have used mutations in Arabidopsis thaliana that selectively eliminate (and substitute) specific xanthophylls in order to study their function(s) in vivo. These include two lutein-deficient mutants, lut1 and lut2, the epoxy xanthophyll-deficient aba1 mutant and the lut2aba1 double mutant. Photosystem stoichiometry, antenna sizes and xanthophyll cycle activity have been related to alterations in nonphotochemical quenching of chlorophyll fluorescence (NPQ). Nondenaturing polyacrylamide gel electrophoresis indicates reduced stability of trimeric LHC II in the absence of lutein (and/or epoxy xanthophylls). Photosystem (antenna) size and stoichiometry is altered in all mutants relative to wild type (WT). Maximal ΔpH-dependent NPQ (qE) is reduced in the following order: WT>aba1>lut1≈lut2>lut2aba1, paralleling reduction in Photosystem (PS) II antenna size. Finally, light-activation of NPQ shows that zeaxanthin and antheraxanthin present constitutively in lut mutants are not qE active, and hence, the same can be inferred of the lutein they replace. Thus, a direct involvement of lutein in the mechanism of qE is unlikely. Rather, altered NPQ in xanthophyll biosynthetic mutants is explained by disturbed macro-organization of LHC II and reduced PS II-antenna size in the absence of the optimal, wild-type xanthophyll composition. These data suggest the evolutionary conservation of lutein content in plants was selected for due to its unique ability to optimize antenna structure, stability and macro-organization for efficient regulation of light-harvesting under natural environmental conditions. [Copyright &y& Elsevier]

We report that the Vps10p domain receptor sorLA binds the adaptor proteins GGA1 and -2, which take part in Golgi–endosome sorting. The GGAs bind with differential requirements via three critical residues in the C-terminal segment of the sorLA cytoplasmic tail. Unlike in sortilin and the mannose 6-phosphate receptors, the GGA-binding segment in sorLA contains neither an acidic cluster nor a dileucine. Our results support the concept of sorLA as a potential sorting receptor and suggest that key residues in sorLA and sortilin conform to a new type of motif (Ψ–Ψ–X–X–∅) defining minimum requirements for GGA binding to cytoplasmic receptor domains. [Copyright &y& Elsevier]

A novel polypeptide with an apparent molecular mass of 9 kDa was detected after sodium dodecyl sulphate–polyacrylamide gel electrophoresis of Arabidopsis photosystem I (PSI) and was N-terminally sequenced. Corresponding cDNA clones encode a precursor protein of 140 amino acid residues which was imported into isolated intact chloroplasts and processed to the mature protein, designated PSI-O. The mature protein has two transmembrane helices and a calculated mass of 10 104 Da. The PSI-O protein was also shown to be present in PSI isolated from barley and spinach, and was essentially absent in chloroplast grana. Expressed sequences encoding similar proteins are available from many species of plants and green algae. [Copyright &y& Elsevier]

The structures and dynamics of the native states of two mutational variants of human lysozyme, I56T and D67H, both associated with non-neuropathic systemic amyloidosis, have been investigated by NMR spectroscopy. The 1H and 15N main-chain amide chemical shifts of the I56T variant are very similar to those of the wild-type protein, but those of the D67H variant are greatly altered for 28 residues in the β-domain. This finding is consistent with the X-ray crystallographic analysis, which shows that the structure of this variant is significantly altered from that of the wild-type protein in this region. The 1H-15N heteronuclear NOE values show that, with the exception of V121, every residue in the wild-type and I56T proteins is located in tightly packed structures characteristic of the native states of most proteins. In contrast, D67H has a region of substantially increased mobility as shown by a dramatic decrease in heteronuclear NOE values of residues near the site of mutation. Despite this unusual flexibility, the D67H variant has no greater propensity to form amyloid fibrils in vivo or in vitro than has I56T. This finding indicates that it is the increased ability of the variants to access partially folded conformations, rather than intrinsic changes in their native state properties, that is the origin of their amyloidogenicity. [ABSTRACT FROM AUTHOR]

Background & Aims: Primary biliary cholangitis (PBC) is a chronic cholangiopathy characterised by immuno-mediated injury of interlobular bile ducts leading to intrahepatic cholestasis and progressive liver fibrosis. PBC histology is characterised by portal inflammation, progressive fibrosis, ductopenia, and the appearance of the so-called ductular reaction. The aim of the present study was to investigate the pathogenetic relevance of ductular reaction in PBC., Methods: Liver biopsies were collected from naïve people with PBC (N = 87). Clinical-serological parameters were obtained at diagnosis and after 1 year of ursodeoxycholic acid (UDCA) treatment. Histological staging was performed on all slides according to multiple scoring systems and criteria for PBC. Liver samples were obtained from Mdr2 -/- mice treated with or without UDCA. Samples were processed for histology, immunohistochemistry, and immunofluorescence., Results: Ductular reaction in people with PBC correlated with the disease stage and liver fibrosis, but not with disease activity; an extensive ductular reaction correlated with serum alkaline phosphatase levels at diagnosis, response to UDCA, and individuals' estimated survival, independently from other histological parameters, including disease stage. In people with PBC, reactive ductules were associated with the establishment of junctions with bile canaliculi and with fibrogenetic cell activation. Consistently, in a mouse model of intrahepatic cholestasis, UDCA treatment was effective in reducing ductular reaction and fibrosis and increasing ductular-canalicular junctions., Conclusions: Extensive ductular reaction outlines a severe histologic phenotype in PBC and is associated with an inadequate therapy response and a worse estimated prognosis., Lay Summary: In people affected by primary biliary cholangitis (PBC), the histological appearance of extensive ductular reaction identifies individuals at risk of progressive fibrosis. Ductular reaction at diagnosis correlates with the lack of response to first-line therapy with ursodeoxycholic acid and serves to restore ductular-canalicular junctions in people with PBC. Assessing ductular reaction extension at diagnosis may add valuable information for clinicians., Competing Interests: The authors declare that there is no conflict of interest. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2022 The Author(s).)

Intrinsic protein flexibility is of overwhelming relevance for intermolecular recognition and adaptability of highly dynamic ensemble of complexes, and the phenomenon is essential for the understanding of numerous biological processes. These conformational ensembles-encounter complexes-lack a unique organization, which prevents the determination of well-defined high resolution structures. This is the case for complexes involving the oncoprotein SET/template-activating factor-Iβ (SET/TAF-Iβ), a histone chaperone whose functions and interactions are significantly affected by its intrinsic structural plasticity. Besides its role in chromatin remodeling, SET/TAF-Iβ is an inhibitor of protein phosphatase 2A (PP2A), which is a key phosphatase counteracting transcription and signaling events controlling the activity of DNA damage response (DDR) mediators. During DDR, SET/TAF-Iβ is sequestered by cytochrome c (C c ) upon migration of the hemeprotein from mitochondria to the cell nucleus. Here, we report that the nuclear SET/TAF-Iβ:C c polyconformational ensemble is able to activate PP2A. In particular, the N-end folded, globular region of SET/TAF-Iβ (a.k.a. SET/TAF-Iβ ΔC)-which exhibits an unexpected, intrinsically highly dynamic behavior-is sufficient to be recognized by C c in a diffuse encounter manner. C c -mediated blocking of PP2A inhibition is deciphered using an integrated structural and computational approach, combining small-angle X-ray scattering, electron paramagnetic resonance, nuclear magnetic resonance, calorimetry and molecular dynamics simulations., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

CD4 + T cells turn pathological during heart failure (HF). We show that the expression of tumor necrosis factor (TNF)-α and tumor necrosis factor receptor (TNFR1) increases in HF-activated CD4 + T cells. However, the role of the TNF-α/TNFR1 axis in T-cell activation/proliferation is unknown. We show that TNFR1 neutralization during T-cell activation (ex vivo) or the loss of TNFR1 in adoptively transferred HF-activated CD4 + T cells (in vivo) augments their prosurvival and proliferative signaling. Importantly, TNFR1 neutralization does not affect CD69 expression or the pathological activity of HF-activated TNFR1 -/- CD4 + T cells. These results show that during HF TNFR1 plays an important role in quelling prosurvival and proliferative signals in CD4 + T cells without altering their pathological activity., Competing Interests: This work was supported by the National Institutes of Health grants to Dr Bansal (R00 HL132123 and R01 HL153164). Funding sources did not influence any part of this work. The authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2022 The Authors.)

Cardiovascular diseases are a major cause of mortality, and vascular injury, a common pathological basis of cardiovascular disease, is deeply correlated with macrophage apoptosis and inflammatory response. Genistein, a type of phytoestrogen, exerts cardiovascular protective activities, but the underlying mechanism has not been fully elucidated. In this study, RAW264.7 cells were treated with genistein, lipopolysaccharide (LPS), nuclear factor-kappa B (NF-κB) inhibitor, and/or protein kinase B (AKT) agonist to determine the role of genistein in apoptosis and inflammation in LPS-stimulated cells. Simultaneously, high fat diet-fed C57BL/6 mice were administered genistein to evaluate the function of genistein on LPS-induced cardiovascular injury mouse model. Here, we demonstrated that LPS obviously increased apoptosis resistance and inflammatory response of macrophages by promoting miR-21 expression, and miR-21 downregulated tumor necrosis factor-α-induced protein 8-like 2 (TIPE2) expression by targeting the coding region. Genistein reduced miR-21 expression by inhibiting NF-κB, then blocked toll-like receptor 4 (TLR4) pathway and AKT phosphorylation dependent on TIPE2, resulting in inhibition of LPS. Our research suggests that miR-21/TIPE2 pathway is involved in M1 macrophage apoptosis and inflammatory response, and genistein inhibits the progression of LPS-induced cardiovascular injury at the epigenetic level via regulating the promoter region of Vmp1 by NF-κB., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Osteosarcoma is one of the commonest metastatic tumor in children and teenagers, and has a hopeless, prognosis. Long non-coding RNA (lncRNA) acts momentous roles as a regulator on the proliferation and migration of cancer. Here, we performed GEO database analysis and qPCR to identify differentially expressed lncRNAs in osteosarcoma cells. Knockdown of lncRNA LINC01140 was used to detect the effect of LINC01140 on the proliferation, invasion, and epithelial-mesenchymal transition (EMT) of osteosarcoma cells. Bioinformatics analysis and qPCR identified the LINC01140/miR-139-5p/Homeobox A9 (HOXA9) regulatory axis. RNA immunoprecipitation assay, Dual-luciferase assay, and rescue experiments confirmed the interaction of LINC01140/miR-139-5p/HOXA9 in osteosarcoma. LINC01140 was overexpressed in osteosarcoma and knocking down LINC01140 restrained the proliferation and invasion of osteosarcoma cells and EMT. In Saos2 and MG63 cells, LINC01140 sponged miR-139-5p, and a miR-139-5p inhibitor overturned the suppression of LINC01140 knockdown on the proliferation and migration of osteosarcoma cells. Moreover, miR-139-5p depressed the invasion, proliferation, and EMT of osteosarcoma cells via targeting HOXA9. Our results indicate that LINC01140 downregulation inhibits the invasion, proliferation, and EMT in osteosarcoma cells through targeting the miR-139-5p/HOXA9 axis. Therefore, LINC01140 is a potential therapeutic target for osteosarcoma., Competing Interests: All authors disclosed no relevant relationship., (© 2022 Published by Elsevier B.V.)

Background & Aims: The chronicity of HBV (and resultant liver disease) is determined by intrahepatic persistence of the HBV covalently closed circular DNA (cccDNA), an episomal form that encodes all viral transcripts. Therefore, cccDNA is a key target for new treatments, with the ultimate therapeutic aim being its complete elimination. Although established cccDNA molecules are known to be stable in resting hepatocytes, we aimed to understand their fate in dividing cells using in vitro models., Methods: We infected HepG2-NTCP and HepaRG-NTCP cells with HBV and induced mitosis by passaging cells. We measured cccDNA copy number (by precise PCR assays) and HBV-expressing cells (by immunofluorescence) with wild-type HBV. We used reporter viruses expressing luciferase or RFP to track number of HBV-expressing cells over time after mitosis induction using luciferase assays and live imaging, respectively., Results: In all cases, we observed dramatic reductions in cccDNA levels, HBV-positive cell numbers, and cccDNA-dependent protein expression after each round of cell mitosis. The rates of reduction were highly consistent with mathematical models of a complete cccDNA loss in (as opposed to dilution into) daughter cells., Conclusions: Our results are concordant with previous animal models of HBV infection and show that HBV persistence can be efficiently overcome by inducing cell mitosis. These results support therapeutic approaches that induce liver turnover ( e.g. immune modulators) in addition to direct-acting antiviral therapies to achieve hepatitis B cure., Lay Summary: Chronic hepatitis B affects 300 million people (killing 884,000 per year) and is incurable. To cure it, we need to clear the HBV genome from the liver. In this study, we looked at how the virus behaves after a cell divides. We found that it completely clears the virus, making 2 new uninfected cells. Our work informs new approaches to develop cures for chronic hepatitis B infections., Competing Interests: SU is co-applicant and co-inventor on patents protecting HBV preS-derived lipopeptides (Myrcludex B) for the use of HBV/HDV entry inhibitors. UP is a co-founder and shareholder of SCG Cell Therapy. The other authors in this study declare no relevant competing interests. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2022 The Author(s).)

Since the beginning of the of SARS-CoV-2 (Covid-19) pandemic, variants of concern (VOC) have emerged taxing health systems worldwide. In October 2020, a new variant of SARS-CoV-2 (B.1.617+/Delta variant) emerged in India, triggering a deadly wave of Covid-19. Epidemiological data strongly suggests that B.1.617+ is more transmissible and previous reports have revealed that B.1.617+ has numerous mutations compared to wild type (WT), including several changes in the spike protein (SP). The main goal of this study was to use In Silico (computer simulation) techniques to examine mutations in the SP, specifically L452R and E484Q (part of the receptor binding domain (RBD) for human angiotensin-converting enzyme 2 (hACE2)) and P681R (upstream of the Furin cleavage motif), for effects in modulating the transmissibility of the B.1.617+ variant. Using computational models, the binding free energy (BFE) and H-bond lengths were calculated for SP-hACE2 and SP-Furin complexes. Comparison of the SP-hACE2 complex in the WT and B.1.617+ revealed both complexes have identical receptor-binding modes but the total BFE of B.1.617+ binding was more favorable for complex formation than WT, suggesting L452R and E484Q have a moderate impact on binding affinity. In contrast, the SP-Furin complex of B.1.617+ substantially lowered the BFE and revealed changes in molecular interactions compared to the WT complex, implying stronger complex formation between the variant and Furin. This study provides an insight into mutations that modulate transmissibility of the B.1.617+ variant, specifically the P681R mutation which appears to enhance transmissibility of the B.1.617+ variant by rendering it more receptive to Furin., Competing Interests: No conflict of interests to declare., (Crown Copyright © 2022 Published by Elsevier Inc. All rights reserved.)

Cardiovascular disease remains the leading cause of death globally, and heart failure (HF) represents its terminal stage. Asthma, one of the most common chronic diseases, has been reported to be associated with an increased risk of cardiovascular disease. However, the link between asthma and HF has rarely been studied, and the possible mechanisms by which asthma affects HF are unclear. This study aimed to explore the influence of asthma on HF and the possible mechanisms. We analyzed data from the National Health and Nutrition Examination Survey and found a higher prevalence of HF among asthmatic individuals, and identified an independent association between HF and asthma. Subsequently, we produced mice with concurrent ovalbumin (OVA) sensitization-induced allergic asthma and angiotensin Ⅱ infusion-induced cardiac remodeling to explore the effect of asthma on cardiac remodeling in vivo. The results showed that OVA-induced asthma impaired heart function and aggravated cardiac remodeling in mice. We also found that OVA sensitization increased the expression levels of immunoglobulin E (IgE) in serum and IgE receptor (FcεR1) in the heart, and enhanced the activation of downstream signaling molecules of IgE-FcεR1 in the heart. Importantly, blockage of IgE-FcεR1 using FcεR1-deficient mice or an anti-IgE antibody prevented asthma-induced decline of cardiac function, and alleviated cardiac remodeling. These findings demonstrate the adverse effects of allergic asthma on the heart, and suggest the potential application of anti-IgE therapy in the treatment of asthma complicated with heart conditions., (Copyright © 2022 Elsevier Inc. All rights reserved.)