Dissociation of polysome aggregates by protease k

Apparent large size-classes of zein-synthesizing polysomes from developing kernels of Zea mays L. were converted to smaller polysomes after treatment with Protease K. The reduction in polysome size was not a result of ribonuclease activity, inasmuch as the enzyme did not affect the free polysomes or the size of the mRNA from the membrane-bound polysomes. High concentrations of MgCl(2) in polysome buffer inhibited ribonuclease activity and appeared to cause protein interaction between nascent zein polypeptides. Although Protease K inhibited the polysome's capacity for protein synthesis, it was a useful reagent for determining if polysomes were aggregated by protein.     

Multifunctionality of lipoamide dehydrogenase: Role of histidine residues in reductase reaction

Rose bengal sensitizes photoinactivation of lipoamide dehydrogenase from pig heart to a constant residual reductase activity resulting from specific destruction of histidine residues. The rate of sensitized photoinactivation is pH dependent and is associated with an ionizable group with pK 6.6 ± 0.2. All steady-state kinetic parameters are markedly reduced by photooxidation. Spectroscopic studies indicate the contribution of oxidized flavin/dithiol to the half-reduced form of the photooxidized enzyme. The proton magnetic resonance spectrum of lipoamide dehydrogenase shows resolved histidine C₂ proton peak at δ9.18 ppm and a shoulder at δ9.23 ppm. The shoulder protons are eliminated by the sensitized photooxidation and shifted upfield on deprotonation. At high pH, the characteristic Faraday A term also disappears. These observations suggest that the essential histidine stabilizes the nascent thiolate via the ion pair formation to facilitate the reductase reaction catalyzed by lipoamide dehydrogenase.     

Five structural classes of major outer membrane proteins in Neisseria meningitidis

Group B Neisseria meningitidis is thus far subdivided into 15 protein serotypes based on antigenically different major outer membrane proteins. Most serotypes have three or four major proteins in their outer membranes. Comparative structural analysis by chymotryptic 125I-peptide mapping was performed on these major proteins from the prototype strains as well as from six non-serotypable strains. The major outer membrane proteins from each of the serotypes were first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the Laemmli system. Individual proteins within the gel slices were radioiodinated and digested with chymotrypsin, and then their 125I-peptides were separated by electrophoresis and chromatography on cellulose thin-layer plates. The peptide maps obtained by autoradiography were categorized into five different structural classes which correlated with the apparent molecular weights of proteins, i.e., 46 +/- 1K, 41 +/- 1K, 38 +/- 1K, 33 +/- 1K, and 28 +/- 1K. Each of the major outer membrane proteins within a strain had a distinctly different chymotryptic peptide map, indicating significant differences in the primary structure of these proteins. In contrast, outer membrane proteins of the same or very similar molecular weight from different serotype strains had similar, occasionally identical peptide maps, indicating a high degree of structural homology. The unique peptides from proteins of the same structural classes were often hydrophilic, whereas common peptides were often hydrophobic, suggesting that the serotype determinants reside within the variable hydrophilic regions of major outer membrane proteins.     

Effects of Luteolin on Human Breast Cancer Using Gene Expression Array: Inferring Novel Genes

Taraxacum officinale (dandelion) is often used in traditional Chinese medicine for the treatment of cancer; however, the downstream regulatory genes and signaling pathways mediating its effects on breast cancer remain unclear. The present study aimed to explore the effects of luteolin, the main biologically active compound of T. officinale, on gene expression profiles in MDA-MB-231 and MCF-7 breast cancer cells. The results revealed that luteolin effectively inhibited the proliferation and motility of the MDA-MB-231 and MCF-7 cells. The mRNA expression profiles were determined using gene expression array analysis and analyzed using a bioinformatics approach. A total of 41 differentially expressed genes (DEGs) were found in the luteolin-treated MDA-MB-231 and MCF-7 cells. A Gene Ontology analysis revealed that the DEGs, including AP2B1, APP, GPNMB and DLST, mainly functioned as oncogenes. The human protein atlas database also found that AP2B1, APP, GPNMB and DLST were highly expressed in breast cancer and that AP2B1 (cut-off value, 75%) was significantly associated with survival rate (p = 0.044). In addition, a Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the DEGs were involved in T-cell leukemia virus 1 infection and differentiation. On the whole, the findings of the present study provide a scientific basis that may be used to evaluate the potential benefits of luteolin in human breast cancer. Further studies are required, however, to fully elucidate the role of the related molecular pathways.     

Identification of Coffee Genes Expressed During Systemic Acquired Resistance and Incompatible Interaction with Hemileia vastatrix

Coffee genes associated with systemic acquired resistance (SAR) and incompatible reaction against coffee leaf rust inoculation were identified by suppression subtractive hybridization. Analysis of 384 clones of each of the subtracted cDNA libraries identified genes involved in oxidative burst/apoptosis/hypersensitive response, synthesis of antimicrobial proteins, synthesis and transport of antimicrobial metabolites, signal perception and transduction, metabolism of lipids, regulated protein degradation and cell maintenance and development. Induction of distinct sets of genes in the two resistance responses was observed. A wide range of genes involved in defence responses described in other plant species was also found in coffee plants. Semi-quantitative and quantitative RT-PCR analysis of seven selected genes showed differences in their expression profile within 72 h after treatment. Full-length cDNA sequences of two β-1,3-glucanases, one induced during SAR and the other in the incompatible reaction, were obtained by 5' and 3' RACE and the sequence data suggest different properties and cellular localization of the encoded proteins.     

Differential expression during development of ADP-ribosylation factors, 20-kDa guanine nucleotide-binding protein activators of cholera toxin

Cholera toxin exerts its effects on cells in large part through the ADP-ribosylation of guanine nucleotide-binding proteins. Toxin-catalyzed ADP-ribosylation is enhanced by approximately 20-kDa guanine nucleotide-binding proteins termed ADP-ribosylation factors (ARFs), which are allosteric activators of the toxin catalytic unit. Rabbit antiserum against a purified bovine brain ARF (sARF II) reacted on immunoblots with two approximately 20-kDa ARF-like proteins (sARF I and II) in tissue extracts from bovine, rat, frog, and chicken. Levels of ARF were higher in brain than in non-neural tissues. In rat brain, on the second postnatal day, amounts of sARF I and II were similar. By the 10th postnatal day and thereafter, sARF II predominated. Relative levels of ARF determined by immunoreactivity were in agreement with levels assessed in functional assays of cholera toxin-catalyzed ADP-ribosylation. Based on nucleotide and deduced amino acid sequences of human and bovine cDNAs, there appear to be at least six different ARF-like genes. Northern blots of rat brain poly(A)+ RNA were hybridized with cDNA and oligonucleotide probes specific for each of the human and bovine ARF genes. From the second to the 27th postnatal day, ARF 3 mRNA increased, whereas mRNAs for ARFs 2 and 4 decreased; and those for ARFs 1, 5, and 6 were apparently unchanged. Partial amino acid sequence of sARF II is consistent with it being either the ARF 1 or 3 gene product. The developmental changes in rat brain ARF parallel neuronal maturation and synapse formation.     

Production of somatic embryos and shoots from sugarbeet callus: Effects of abscisic acid, other growth regulators, nitrogen source, sucrose concentration and genotype

Summary Two sugarbeet (Beta vulgaris L.) genotypes, REL-1 and REL-2, were used to measure the level of somatic embryo and shoot production from hormone-autonomous callus plated under varied nutrient medium combinations of abscisic acid with the growth regulators 6-benzyladenine, 1-naphthaleneacetic acid, or 2,4-dichlorophenoxyacetic acid, with eight sole nitrogen sources, or with different sucrose concentrations. Clone REL-2 produced embryos up to 35-fold more frequently than clone REL-1. Inclusion of abscisic acid at some concentrations consistently improved embryo production in all experiments and was observed to stimulate shoot production. At some concentrations, 1-naphthaleneacetic acid as well as urea and glutamine stimulated greater embryo production over the control, but only for REL-1, for which there was greater room for improvement. Three and five percent sucrose were superior to 1, 7, and 9%. Higher initial 6-benzyladenine concentration [in the range 0, 0.1−1.0 mg/L (0.44−4.44 μM)] was associated with lower embryo production but greater shoot regeneration for both clones. REL-2 was significantly better than REL-1 in shoot regeneration. The range of embryo production was more than 35-fold between genotypes, whereas the range of physiological effects was no greater than 10-fold. REL-2 has been released to sugarbeet researchers because of its superior embryogenic and shoot regeneration abilities for application in biotechnology.     

Resistin induces monocyte-endothelial cell adhesion by increasing ICAM-1 and VCAM-1 expression in endothelial cells via p38MAPK-dependent pathway

Resistin, firstly reported as an adipocyte-specific hormone, is suggested to be an important link between obesity and diabetes. Recent studies have suggested an association between resistin and atherogenic processes. The adhesion of circulating monocytes to endothelial cells is a critical step in the early stages of atherosclerosis. The purpose of the present study was to investigate the effect of resistin on the adhesion of THP-1 monocytes to human umbilical vein endothelial cells (HUVECs) and the underlying mechanism. Our results showed that resistin caused a significant increase in monocyte adhesion. In exploring the underlying mechanisms of resistin action, we found that resistin-induced monocyte adhesion was blocked by inhibition of p38MAPK activation using SB203580 and SB202190. Furthermore, resistin increased the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) by HUVECs and these effects were also p38MAPK-dependent. Resistin-induced monocyte adhesion was also blocked by monoclonal antibodies against ICAM-1 and VCAM-1. Taken together, these results show that resistin increases both the expression of ICAM-1 and VCAM-1 by endothelial cells and monocyte adhesion to HUVECs via p38MAPK-dependent pathways.     

Evodiamine inhibits in vitro angiogenesis: Implication for antitumorgenicity

Evodiamine, the major bioactive compound isolated from Chinese herbal drug named Wu-Chu-Yu, has been reported to exhibit anti-tumor growth and metastasis. However, the effect of evodiamine on angiogenesis remains to be investigated. We used the fresh medium containing evodiamine or human lung adenocarcinoma cell (CL1 cells) derived conditioned media free of evodiamine to test their capability to induce in vitro angiogenesis, i.e., human umbilical vein endothelial cells (HUVECs) tube formation and invasion. We demonstrated that evodiamine could directly inhibit in vitro HUVECs tube formation and invasion. Locally administered evodiamine also inhibited the in vivo angiogenesis in the chick embryo chorioallantoic membrane (CAM) assay. The gene expression of vascular endothelial growth factor (VEGF) and the p44/p42 mitogen-activated protein kinase (MAPK, ERK) that correlated with endothelial cells angiogenesis were inhibited by evodiamine. We found that the evodiamine-treated CL1 cells derived conditioned medium showed decreased VEGF release and reduced ability of inducing in vitro tube formation. After the collection of conditioned media, the VEGF expression of remaining CL1 cells were determined by Western analyses and revealed that evodiamine decreased VEGF expression. Moreover, administration of recombinant human VEGF(165) (rhVEGF(165)) induced tube formation and ERK phosphorylation by HUVECs, and partially attenuated inhibitory effect of evodiamine. From these results, we suggested that evodiamine is a potent inhibitor of angiogenesis. The mechanism might involve at least the inhibition of VEGF expression, probably through repression of ERK phosphorylation.