Growth inhibition in G(1) and altered expression of cyclin D1 and p27(kip-1 )after forced connexin expression in lung and liver carcinoma cells

Gap junctional intercellular communication (GJIC) and connexin expression are frequently decreased in neoplasia and may contribute to defective growth control and loss of differentiated functions. GJIC, in E9 mouse lung carcinoma cells and WB-aB1 neoplastic rat liver epithelial cells, was elevated by forced expression of the gap junction proteins, connexin43 (Cx43) and connexin32 (Cx32), respectively. Transfection of Cx43 into E9 cells increased fluorescent dye-coupling in the transfected clones, E9-2 and E9-3, to levels comparable to the nontransformed sibling cell line, E10, from which E9 cells originated. Transduction of Cx32 into WB-aB1 cells also increased dye-coupling in the clone, WB-a/32-10, to a level that was comparable to the nontransformed sibling cell line, WB-F344. The cell cycle distribution was also affected as a result of forced connexin expression. The percentage of cells in G(1)-phase increased and the percentage in S-phase decreased in E9-2 and WB-a/32-10 cells as compared to E9 and WB-aB1 cells. Concomitantly, these cells exhibited changes in G(1)-phase cell cycle regulators. E9-2 and WB-a/32-10 cells expressed significantly less cyclin D1 and more p27(kip-1) protein than E9 and WB-aB1 cells. Other growth-related properties (expression of platelet-derived growth factor receptor-beta, epidermal growth factor receptor, protein kinase C-alpha, protein kinase A regulatory subunit-Ialpha, and production of nitric oxide in response to a cocktail of pro-inflammatory cytokines) were minimally altered or unaffected. Thus, enhancement of connexin expression and GJIC in neoplastic mouse lung and rat liver epithelial cells restored G(1) growth control. This was associated with decreased expression of cyclin D1 and increased expression of p27(kip-1), but not with changes in other growth-related functions.     

The function of histone lysine methylation related SET domain group proteins in plants

Histone methylation, which is mediated by the histone lysine (K) methyltransferases (HKMTases), is a mechanism associated with many pathways in eukaryotes. Most HKMTases have a conserved SET (Su(var) 3‐9,E(z),Trithorax) domain, while the HKMTases with SET domains are called the SET domain group (SDG) proteins. In plants, only SDG proteins can work as HKMTases. In this review, we introduced the classification of SDG family proteins in plants and the structural characteristics of each subfamily, surmise the functions of SDG family members in plant growth and development processes, including pollen and female gametophyte development, flowering, plant morphology and the responses to stresses. This review will help researchers better understand the SDG proteins and histone methylation in plants and lay a basic foundation for further studies on SDG proteins.     

The Arabidopsis SUVR4 protein is a nucleolar histone methyltransferase with preference for monomethylated H3K9

Proteins containing the evolutionarily conserved SET domain are involved in regulation of eukaryotic gene expression and chromatin structure through their histone lysine methyltransferase (HMTase) activity. The Drosophila SU(VAR)3-9 protein and related proteins of other organisms have been associated with gene repression and heterochromatinization. In Arabidopsis there are 10 SUVH and 5 SUVR genes encoding proteins similar to SU(VAR)3-9, and 4 SUVH proteins have been shown to control heterochromatic silencing by its HMTase activity and by directing DNA methylation. The SUVR proteins differ from the SUVH proteins in their domain structure, and we show that the closely related SUVR1, SUVR2 and SUVR4 proteins contain a novel domain at their N-terminus, and a SUVR specific region preceding the SET domain. Green fluorescent protein (GFP)-fusions of these SUVR proteins preferably localize to the nucleolus, suggesting involvement in regulation of rRNA expression, in contrast to other SET-domain proteins studied so far. A novel HMTase specificity was demonstrated for SUVR4, in that monomethylated histone H3K9 is its preferred substrate in vitro.     

Arabidopsis membrane-anchored ubiquitin-fold (MUB) proteins localize a specific subset of ubiquitin-conjugating (E2) enzymes to the plasma membrane

The covalent attachment of ubiquitin (Ub) to various intracellular proteins plays important roles in altering the function, localization, processing, and degradation of the modified target. A minimal ubiquitylation pathway uses a three-enzyme cascade (E1, E2, and E3) to activate Ub and select target proteins for modification. Although diverse E3 families provide much of the target specificity, several factors have emerged recently that coordinate the subcellular localization of the ubiquitylation machinery. Here, we show that the family of membrane-anchored ubiquitin-fold (MUB) proteins recruits and docks specific E2s to the plasma membrane. Protein interaction screens with Arabidopsis MUBs revealed that interacting E2s are limited to a well defined subgroup that is phylogenetically related to human UbcH5 and yeast Ubc4/5 families. MUBs appear to interact noncovalently with an E2 surface opposite the active site that forms a covalent linkage with Ub. Bimolecular fluorescence complementation demonstrated that MUBs bind simultaneously to the plasma membrane via a prenyl tail and to the E2 in planta. These findings suggest that MUBs contribute subcellular specificity to ubiquitylation by docking the conjugation machinery to the plasma membrane.     

Des-Tyr1-gamma-endorphin (DT gamma E) and des-enkephalin-gamma-endorphin (DE gamma E): plasma profile and brain uptake after systemic administration in the rat

The plasma disappearance, metabolism and uptake in the brain of [3H-Phe4]-DT gamma E and [3H-Lys9]-DE gamma E were investigated following systemic administration of these neuroleptic-like peptides to rats. 3H-DT gamma E, 3H-DE gamma E and their radioactive metabolites in plasma and brain extracts were determined by reversed-phase HPLC. Plasma disappearance of DT gamma E upon intravenous (IV) dosing followed a biphasic pattern with half-lives of 0.7 min (distribution phase) and 5.5 min (elimination phase). For DE gamma E the plasma disappearance curve was best characterized by a one-compartment model since a second elimination phase was hardly detectable by our methods. The corresponding half-life was 0.6 min, probably representative for the initial distribution phase of DE gamma E. Both neuropeptides distributed rapidly over the larger part of the extracellular fluid. Following the IV route of administration, brain uptake of DT gamma E and DE gamma E appeared to be low. Brain levels of DT gamma E decreased from 0.0075% to 0.0031% of the administered dose/g tissue at 2-15.5 min after injection, whereas those of DE gamma E decreased very rapidly from 0.0174% of the dose/g brain tissue to below the detection limit at 2-4.5 min after injection. As compared to the IV route of administration, subcutaneous (SC) injection of DE gamma E resulted into lower but remarkably longer-lasting peptide concentrations in plasma as well as in brain, possibly because of a sustained release from the SC site of injection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamics of epigenetic control in plants via SET domain containing proteins: Structural and functional insights

Plants control expression of their genes in a way that involves manipulating the chromatin structural dynamics in order to adapt to environmental changes and carry out developmental processes. Histone modifications like histone methylation are significant epigenetic marks which profoundly and globally modify chromatin, potentially affecting the expression of several genes. Methylation of histones is catalyzed by histone lysine methyltransferases (HKMTs), that features an evolutionary conserved domain known as SET [Su(var)3–9, E(Z), Trithorax]. This methylation is directed at particular lysine (K) residues on H3 or H4 histone. Plant SET domain group (SDG) proteins are categorized into different classes that have been conserved through evolution, and each class have specificity that influences how the chromatin structure operates. The domains discovered in plant SET domain proteins have typically been linked to protein-protein interactions, suggesting that majority of the SDGs function in complexes. Additionally, SDG-mediated histone mark deposition also affects alternative splicing events. In present review, we discussed the diversity of SDGs in plants including their structural properties. Additionally, we have provided comprehensive summary of the functions of the SDG-domain containing proteins in plant developmental processes and response to environmental stimuli have also been highlighted.     

Within-field manipulation of potato leafhopper (Homoptera: Cicadellidae) and insect predator populations using an uncut alfalfa strip

The effects of a 3-m wide uncut alfalfa strip on the within field distribution of Empoasca fabae (Harris) (Homoptera: Cicadellidae) and several species of insect predators were examined for 3 yr (1998-2000). The objectives were to determine whether this uncut strip would serve as a trap crop for E. fabae and a refuge for insect predators at first harvest. Empoasca fabae and insect predators in the families Coccinellidae, Nabidae, Anthocoridae, Chrysopidae, and Hemerobiidae were collected weekly using sweep samples and sticky traps from the uncut strips and up to 40 m into the alfalfa regrowth. For 2 yr, both small- (0.34 ha) and large-scale (approximately 11.3 ha) field trials showed higher numbers of E. fabae in 73% of the uncut strips for 2-3 wk after harvest. Similarly, the number of insect predators found within <50% of the uncut strips was also higher during the first or second week after harvest. In 1999, however, we did not observe higher numbers of E. fabae in the uncut strips. Differences may be because of higher E. fabae population numbers in 1999 compared with 1998 and 2000. This research provides alfalfa growers a potential cultural management technique for E. fabae while conserving predatory insects.     

E(var)3-9 of Drosophila melanogaster encodes a zinc finger protein

The importance of a gene's natural chromatin environment for its normal expression is poignantly illustrated when a change in chromosome position results in variable gene repression, such as is observed in position effect variegation (PEV) when the Drosophila melanogaster white (omega) gene is juxtaposed with heterochromatin. The Enhancer of variegation 3-9 [E(var)3-9] gene was one of over a hundred loci identified in screens for mutations that dominantly modify PEV. Haploinsufficiency for E(var)3-9 enhances omegam4 variegation, as would be expected from increased heterochromatin formation. To clarify the role of E(var)3-9 in chromosome structure, the gene has been cloned and its mutant alleles characterized. The involvement of E(var)3-9 in structure determination was supported by its reciprocal effects on euchromatic and heterochromatic PEV; E(var)3-9 mutations increased expression of a variegating heterochromatic gene in two tissue types. E(var)3-9 mutations also had a recessive phenotype, maternal effect lethality, which implicated E(var)3-9 function in an essential process during embryogenesis. Both phenotypes of E(var)3-9 mutations were consistent with its proposed function in promoting normal chromosome structure. The cloning of E(var)3-9 by classical genetic methods revealed that it encodes a protein with multiple zinc fingers, but otherwise novel sequence.     

Comparative analysis of SET domain proteins in maize and Arabidopsis reveals multiple duplications preceding the divergence of monocots and dicots

Histone proteins play a central role in chromatin packaging, and modification of histones is associated with chromatin accessibility. SET domain [Su(var)3-9, Enhancer-of-zeste, Trithorax] proteins are one class of proteins that have been implicated in regulating gene expression through histone methylation. The relationships of 22 SET domain proteins from maize (Zea mays) and 32 SET domain proteins from Arabidopsis were evaluated by phylogenetic analysis and domain organization. Our analysis reveals five classes of SET domain proteins in plants that can be further divided into 19 orthology groups. In some cases, such as the Enhancer of zeste-like and trithorax-like proteins, plants and animals contain homologous proteins with a similar organization of domains outside of the SET domain. However, a majority of plant SET domain proteins do not have an animal homolog with similar domain organization, suggesting that plants have unique mechanisms to establish and maintain chromatin states. Although the domains present in plant and animal SET domain proteins often differ, the domains found in the plant proteins have been generally implicated in protein-protein interactions, indicating that most SET domain proteins operate in complexes. Combined analysis of the maize and Arabidopsis SET domain proteins reveals that duplication of SET domain proteins in plants is extensive and has occurred via multiple mechanisms that preceded the divergence of monocots and dicots.     

Total synthesis of the mollu-series glycosyl ceramides alpha-D-Manp-(1----3)-beta-D-Manp-(1----4)-beta-D-Glcp-(1----1)-Cer and alpha-D-Manp-(1----3)-[beta-D-Xylp-(1----2)]-beta-D-Manp-(1----4)-beta- D-Glcp-(1----1)-Cer

The mollu-series glycosphingolipids, O-alpha-D-mannopyranosyl-(1----3)-O-beta-D-mannopyranosyl-(1----4)-O-bet a-D-glucopyranosyl-(1----1)-2-N-tetracosanoyl-(4E)-sphingeni ne and O-alpha-D-mannopyranosyl-(1----3)-O-[beta-D-xylopyranosyl-(1----2])-O- beta-D-mannopyranosyl-(1----4)-O-beta-D-glucopyranosyl-(1----1)-2-N- tetracosanoyl-(4E)-sphingenine, were synthesized for the first time by using 2,3,4-tri-O-acetyl-D-xylopyranosyl trichloroacetimidate, methyl 2,3,4,6-tetra-O-acetyl-1-thio-alpha-D-mannopyranoside, benzyl O-(4,6-di-O-benzyl-beta-D-mannopyranosyl)-(1----4)-2,3,6-tri-O-benzyl-be ta-D- glucopyranoside 9, and (2S,3R,4E)-2-azido-3-O-(tert-butyldiphenylsilyl)-4-octade cene-1,3-diol 6 as the key intermediates. The hexa-O-benzyl disaccharide 9 was prepared by coupling two monosaccharide synthons, namely, 2,3-di-O-allyl-4,6-di-O-benzyl-alpha-D-mannopyranosyl bromide and benzyl 2,3,6-tri-O-benzyl-beta-D-glucopyranoside. It was demonstrated that azide 6 was highly efficient as a synthon for the ceramide part in the coupling with both glycotriaosyl and glycotetraosyl donors, particularly in the presence of trimethylsilyl triflate.     

Heat-induced preferential synthesis and redistribution of HSP 70 and 28 families in Chinese hamster ovary cells

We observed that members of two HSP families (70 and 28 kDa) preferentially redistributed into the nucleus after heating at 45.5 degrees C for 10 min. The rates of synthesis and redistribution of these proteins were different for each member of HSP families during incubation period at 37 degrees C after heat shock. The maximum rates of synthesis of HSP 70 and HSP 28 families, except HSP 28c, were 6-9 hr after heat shock, whereas the maximum rates of redistribution were 3-6 hr after heat shock. These results suggest that the rates of redistribution of these proteins may be dependent on the amount of intracellular proteins as well as the alteration of binding affinity of nucleoproteins following heat shock.     

Immunodetection and localization of protein(s) related to retinal S-antigen (arrestin) in kidney.

S-antigen (arrestin) is a cytosolic protein which regulates phototransduction in retinal rods. A protein immunologically related to S-antigen was identified in fractions from soluble extract of bovine kidney enriched by gel filtration or by immunoaffinity chromatography using a polyclonal antibody to retinal S-antigen. On immunoblots, this protein was recognized by a panel of monoclonal antibodies (mAbs S2D2, S1A3 and S9E2) directed against different S-antigen epitopes and displayed the same apparent molecular mass (48 kDa) as retinal S-antigen. All three mAbs revealed a specific immunoreactivity by indirect immunocytochemical technique on rat kidney sections. The three mAbs recognized some but not all glomerular cells, identified as epithelial cells by immunoelectron microscopy using the mAb S9E2. Both mAbs S2D2 and S1A3 gave a diffuse cytoplasmic staining in all tubule cells. Proximal tubule cells exhibited a weak immunoreactivity, whereas distal and collecting tubule cells were strongly labeled. In contrast, the mAb S9E2 immunoreaction was restricted to a cell subpopulation from distal and collecting tubules corresponding to intercalated cells identified by immunoelectron microscopy. With the mAb S9E2, the labeling of proximal tubule cells was localized in the apical region of the cytoplasm. These results suggest that two or more 48-kDa proteins immunologically cross-reactive with retinal S-antigen are present in kidney. The observed pattern of distribution is in keeping with the hypothesis that such proteins could play a role in the regulation of G-protein-related receptors present in renal glomerulus and tubule epithelial cells.     

Free radical oxidation of coriolic acid (13-(S)-hydroxy-9Z,11E-octadecadienoic acid)

The reaction of (13S,9Z,11E)-13-hydroxy-9,11-octadecadienoic acid (1a), one of the major peroxidation products of linoleic acid and an important physiological mediator, with the Fenton reagent (Fe(2+)/EDTA/H(2)O(2)) was investigated. In phosphate buffer, pH 7.4, the reaction proceeded with >80% substrate consumption after 4h to give a defined pattern of products, the major of which were isolated as methyl esters and were subjected to complete spectral characterization. The less polar product was identified as (9Z,11E)-13-oxo-9,11-octadecadienoate (2) methyl ester (40% yield). Based on 2D NMR analysis the other two major products were formulated as (11E)-9,10-epoxy-13-hydroxy-11-octadecenoate (3) methyl ester (15% yield) and (10E)-9-hydroxy-13-oxo-10-octadecenoate (4) methyl ester (10% yield). Mechanistic experiments, including deuterium labeling, were consistent with a free radical oxidation pathway involving as the primary event H-atom abstraction at C-13, as inferred from loss of the original S configuration in the reaction products. Overall, these results provide the first insight into the products formed by oxidation of 1a with the Fenton reagent, and hint at novel formation pathways of the hydroxyepoxide 3 and hydroxyketone 4 of potential (patho)physiological relevance in settings of oxidative stress.     

用RAPD法研究火炬松愈伤组织发生的遗传特性(英文)

体外培养对于植物的快速繁殖是非常有效的。和其它一些松果类硬木植物一样,火炬松的体外培养成功率却一直很低。本工作研究了不同的基本培养基和低温条件对于火炬松Ｊ－５６, Ｓ－１００３, ａｎｄ Ｅ－４４０等三个品系的成熟合子胚形成愈伤组织、分化出芽、成苗的影响。在不同的基本培养基条件下芽分化的程度差异很大。合子胚经过９－１２周培养,开始分化,形成具有器官发生的愈伤组织（Ｆｉｇ．２ａ）。分化后３周,开始诱导出芽（Ｆｉｇ．２ｂ）,芽的生长快慢不同（Ｆｉｇ．２ｃ,ｄ）。同一个愈伤组织上会生出几个芽来（Ｆｉｇ．２ｅ）。在添加有ＩＢＡ和ＢＡ的ＴＥ培养基上芽生长最快（Ｆｉｇ．１）。低温条件持续 １５天,能增加芽的数量和分化的程度（Ｔａｂｌｅ１）。上述培养基中增加ＧＡ３时表明,ＧＡ３对于根的诱导有决定性的作用。将９８株再生苗转移到特殊的混合土壤上;成活了７５株苗（Ｆｉｇ．２ｆ）。以这三种火炬松的再生苗尖为材料制备ＤＮＡ。用２０个引物进行ＲＡＰＤ分析,结果表明：这三种火炬松苗的扩增产物是相同的（Ｆｉｇ．２ｇ,ｈ＆ｉ）。这说明：用愈伤组织克隆植株的过程中没有引起植物遗传变异。     

Comparative analysis of anti-restriction activities of ArdA (ColIb-P9) and Ocr (T7) proteins

Anti-restriction proteins ArdA and Ocr are specific inhibitors of type I restriction-modification enzymes. The IncI1 transmissible plasmid ColIb-P9 ardA and bacteriophage T7 0.3(ocr) genes were cloned in pUC18 vector. Both ArdA (ColIb-P9) and Ocr (T7) proteins inhibit both restriction and modification activities of the type I restriction-modification enzyme (EcoKI) in Escherichia coli K12 cells. ColIb-P9 ardA, T7 0.3(ocr), and the Photorhabdus luminescens luxCDABE genes were cloned in pZ-series vectors with the P(ltetO-1) promoter, which is tightly repressible by the TetR repressor. Controlling the expression of the lux-genes encoding bacterial luciferase demonstrates that the P(ltetO-1) promoter can be regulated over an up to 5000-fold range by supplying anhydrotetracycline to the E. coli MG1655Z1 tetR(+) cells. Effectiveness of the anti-restriction activity of the ArdA and Ocr proteins depended on the intracellular concentration. It is shown that the dissociation constants K(d) for ArdA and Ocr proteins with EcoKI enzyme differ 1700-fold: K(d) (Ocr) = 10(-10) M, K(d) (ArdA) = 1.7.10(-7) M.