Overexpression of Hevea brasiliensis ethylene response factor HbERF‐IXc5 enhances growth and tolerance to abiotic stress and affects laticifer differentiation

Ethylene response factor 1 (ERF1) is an essential integrator of the jasmonate and ethylene signalling pathways coordinating a large number of genes involved in plant defences. Its orthologue in Hevea brasiliensis, HbERF‐IXc5, has been assumed to play a major role in laticifer metabolism and tolerance to harvesting stress for better latex production. This study sets out to establish and characterize rubber transgenic lines overexpressing HbERF‐IXc5. Overexpression of HbERF‐IXc5 dramatically enhanced plant growth and enabled plants to maintain some ecophysiological parameters in response to abiotic stress such as water deficit, cold and salt treatments. This study revealed that HbERF‐IXc5 has rubber‐specific functions compared to Arabidopsis ERF1 as transgenic plants overexpressing HbERF‐IXc5 accumulated more starch and differentiated more latex cells at the histological level. The role of HbERF‐IXc5 in driving the expression of some target genes involved in laticifer differentiation is discussed.     

Role of Immunohistochemistry in Staging Diffuse Large B-cell Lymphoma (DLBCL)

The use of immunohistochemistry (IHC) in staging bone marrow in non-Hodgkin''s lymphoma (NHL) is largely limited to ambiguous cases, particularly those with lymphoid aggregates. Its role in routine clinical practice remains unestablished. This study aimed to determine whether the routine use of IHC in diffuse large B-cell lymphoma (DLBCL) would improve the detection of lymphomatous involvement in the bone marrow. It also sought to determine the impact of IHC on predicting survival compared with routine histological diagnosis using hematoxylin and eosin (H&E), Giemsa, and reticulin staining. The bone marrow trephines of 156 histologically proven DLBCL cases were assessed on routine histology, and IHC using two T-cell markers (CD45RO and CD3), two B-cell markers (CD20 and CD79a), and κ and λ light chains. IHC detected lymphomatous involvement on an additional 11% cases compared with histology alone. Although both routine histology and IHC were good predictors of survival, IHC was better at predicting survival on stepwise multivariate Cox regression analysis. IHC performed routinely on bone marrow trephines has the ability to improve detection of occult lymphoma in experienced hands. Furthermore, it is a better predictor of survival compared with routine histological examination alone. (J Histochem Cytochem 56:893–900, 2008)     

Regulation of Nedd4-2 self-ubiquitination and stability by a PY motif located within its HECT-domain

Ubiquitin ligases play a pivotal role in substrate recognition and ubiquitin transfer, yet little is known about the regulation of their catalytic activity. Nedd4 (neural-precursor-cell-expressed, developmentally down-regulated 4)-2 is an E3 ubiquitin ligase composed of a C2 domain, four WW domains (protein-protein interaction domains containing two conserved tryptophan residues) that bind PY motifs (L/PPXY) and a ubiquitin ligase HECT (homologous with E6-associated protein C-terminus) domain. In the present paper we show that the WW domains of Nedd4-2 bind (weakly) to a PY motif (LPXY) located within its own HECT domain and inhibit auto-ubiquitination. Pulse-chase experiments demonstrated that mutation of the HECT PY-motif decreases the stability of Nedd4-2, suggesting that it is involved in stabilization of this E3 ligase. Interestingly, the HECT PY-motif mutation does not affect ubiquitination or down-regulation of a known Nedd4-2 substrate, ENaC (epithelial sodium channel). ENaC ubiquitination, in turn, appears to promote Nedd4-2 self-ubiquitination. These results support a model in which the inter- or intra-molecular WW-domain-HECT PY-motif interaction stabilizes Nedd4-2 by preventing self-ubiquitination. Substrate binding disrupts this interaction, allowing self-ubiquitination of Nedd4-2 and subsequent degradation, resulting in down-regulation of Nedd4-2 once it has ubiquitinated its target. These findings also point to a novel mechanism employed by a ubiquitin ligase to regulate itself differentially compared with substrate ubiquitination and stability.     

Composition and Chemical Variability of the Needle Oil from Pinus halepensis growing in Corsica

The composition of oil samples isolated from needles of Pinus halepensis growing in three locations in Corsica (Saleccia, Capo di Feno, and Tre Padule) has been investigated by combination of chromatographic (GC with retention indices) and spectroscopic (MS and ¹³C‐NMR) techniques. In total, 35 compounds that accounted for 77 – 100% of the whole composition have been identified. α‐Pinene, myrcene, and (E)‐β‐caryophyllene were the major component followed by α‐humulene and 2‐phenylethyl isovalerate. Various diterpenes have been identified as minor components. 47 Oil samples isolated from pine needles have been analyzed and were differentiated in two groups. Oil samples of the first group (15 samples) contained myrcene (M = 28.1 g/100 g; SD = 10.6) and (E)‐β‐caryophyllene (M = 19.0 g/100 g; SD = 2.2) as major components and diterpenes were absent. All these oil samples were isolated from pine needles harvested in Saleccia. Oil samples of the second group (32 samples) contained mostly (E)‐β‐caryophyllene (M = 28.7 g/100 g; SD = 7.9), α‐pinene (M = 12.3 g/100 g; SD = 3.6), and myrcene (M = 11.7 g/100 g; SD = 7.3). All these oil samples were isolated from pine needles harvested in Capo di Feno and Tre Padule.     

A role for septin 2 in Drp1‐mediated mitochondrial fission

Mitochondria are essential eukaryotic organelles often forming intricate networks. The overall network morphology is determined by mitochondrial fusion and fission. Among the multiple mechanisms that appear to regulate mitochondrial fission, the ER and actin have recently been shown to play an important role by mediating mitochondrial constriction and promoting the action of a key fission factor, the dynamin‐like protein Drp1. Here, we report that the cytoskeletal component septin 2 is involved in Drp1‐dependent mitochondrial fission in mammalian cells. Septin 2 localizes to a subset of mitochondrial constrictions and directly binds Drp1, as shown by immunoprecipitation of the endogenous proteins and by pulldown assays with recombinant proteins. Depletion of septin 2 reduces Drp1 recruitment to mitochondria and results in hyperfused mitochondria and delayed FCCP‐induced fission. Strikingly, septin depletion also affects mitochondrial morphology in Caenorhabditis elegans, strongly suggesting that the role of septins in mitochondrial dynamics is evolutionarily conserved.     

Epidermal growth factor receptors lose ligand binding ability as WI-38 cells progress from short-term to long-term quiescence

WI-38 cells, density arrested for short periods of time, can be stimulated to re-enter the cell cycle by epidermal growth factor (EGF) alone. However, cells density arrested for longer periods have a prolonged prereplicative phase when serum stimulated and cannot be stimulated by EGF alone. Radio-ligand binding studies performed on WI-38 cells showed that actively growing cells bind [¹²⁵I]EGF at relatively low levels that increase to a maximum as the cells become contact inhibited. As the cells enter a state of deeper quiescence, EGF binding falls to one-third to one-fifth the short-term growth arrested levels, remaining constant thereafter. The EGF-receptor complexes internalize more slowly in long-term growth arrested cells, and the rate of ligand association to the receptor is lower than short-term growth arrested cells. The amount of EGF receptor protein in lysates of equal numbers of both short- and long-term quiescent cells remains the same. These results suggest that the failure of long-term growth arrested cells to respond to EGF is not due to dramatic changes in the amount of receptor protein during prolonged quiescence but more likely to an alteration in the ability of these receptors to bind ligand and/or activate the EGF signal transduction pathway. © 1993 Wiley-Liss, Inc.     

The effect of salinity on the reproduction of coastal submerged macrophytes in experimental communities

The effects of salinity on the reproduction of coastal submerged macrophyte species were studied on samples of communities from six seasonal marshes in two outdoor experiments performed in autumn and in spring. The submerged macrophyte communities were submitted to five different salinity levels (0, 1, 2, 4 and 6 g/1 Cl^?1). In a companion paper (Grillas, van Wijck & Bonis 1993) three groups of species were distinguished on the basis of their biomass production over the salinity range 0 to 6 g/1 Cl^?1: (1) glycophytes (non-salt-tolerant species), (2) salt-tolerant species and (3) halo-phytes. This part of the study describes the impact of salinity on the reproduction of the individual species during the two experiments. The species differ in their capacity to reproduce in the autumn; only Zannichelliapedunculata and Tolypella hispánica were able to produce fruits in that season. For all species reproduction was greater in spring and strongly correlated with biomass, except for Chara canescens. Differences in reproductive effort over the salinity range amplified the halophytic nature of Ruppia marítima and Chara canescens and the intolerance of Callitriche truncata and Chara contraria. For the other species, reproductive effort did not differ significantly over the salinity range. Regarding the effect of salinity on biomass and reproductive effort of individual species, there were large differences in the total weight of propagules produced at the community level and in the relative contribution of individual species. The resulting quantitative changes in the species composition of the seed bank could affect the structure of the communities by their effects on the establishment and survival of species populations.     

Evidence for Nerve Growth Factor-Potentiating Activities of the Nonpeptidic Compound SR 57746A in PC12 Cells

Abstract: SR 57746A {1-[2-(naphth-2-yl)ethyl]-4-(3-trifluoromethylphenyl)-1,2,5,6-tetrahydropyridine hydrochloride} exhibits neurotrophic activities in vivo and in vitro. We used the rat pheochromocytoma PC12 cell line to investigate in vitro cellular changes induced by SR 57746A. A significant increase in the percentage of cells bearing neurite-like processes was obtained in cells treated by SR 57746A and nerve growth factor (NGF) compared with NGF treatment alone. SR 57746A added alone, however, had no effect on morphogenesis or on survival of cells in serum-free medium. In contrast, SR 57746A induced a "priming" effect on PC12 cells for neurite outgrowth within 6 h of addition of the protein tyrosine kinase inhibitor genistein. An increase in α-actinin content resulted from treatment with SR 57746A. Expression of NGF-mediated acetylcholinesterase and choline acetyltransferase was enhanced within 5 days by SR 57746A. The molecule also induced rapid F-actin redistribution. Within 2 min of incubation, outgrowth of F-actin-containing filopodia was clearly visible at the cell periphery, as previously shown with NGF. It is interesting that this effect of SR 57746A could be mimicked by protein tyrosine kinase inhibitors and abolished by preincubation with sodium orthovanadate, a protein tyrosine phosphatase inhibitor.     

A family of modular type mannuronan C-5-epimerase genes controls alginate structure in Azotobacter vinelandii

The ToxR protein is a transmembrane protein that regulates the expression of several virulence factors of Vibrio cholerae. Previous analysis of fusion proteins between ToxR and alkaline phosphatase (ToxR-PhoA) suggested that ToxR was active as a dimer. In order to determine whether dimerization of the ToxR periplasmic domain was essential for activity, this domain was replaced by monomeric and dimeric protein domains. Surprisingly, PhoA (dimeric), β-lactamase (monomeric, ToxR–Bla), or the leucine zipper of GCN4 (dimeric, ToxR-GCN4-M) could substitute functionally for the ToxR periplasmic domain. ToxR-GCN4 fusion proteins, in which the ToxR trans-membrane domain was eliminated (ToxR-GCN4-C), were inactive, but an additional fusion protein that contained a heterologous membrane-spanning domain retained activity. Strains containing each of these ToxR fusion proteins were analysed for in vivo colonization properties and response to in vitro growth conditions that are known to affect expression of the ToxR regulon. Strains containing ToxR-GCN4-M and ToxR-Bla responded like wild-type strains to in vitro growth conditions. In the infant-mouse colonization model, strains containing ToxR fusion proteins were all deficient in colonization relative to strains containing wild-type ToxR, and strains containing monomeric ToxR-Bla were most severely outcompeted. These results suggest that, under in vitro conditions, ToxR does not require a dimerized periplasmic domain, but that, under in vivo conditions, the correct conformation of the ToxR periplasmic domain may be more important for function.     

Protective Effect of <Emphasis Type="Italic">Lactobacillus casei</Emphasis> on DMH-Induced Colon Carcinogenesis in Mice

The administration of probiotics is a promising approach to reduce the prevalence of colon cancer, a multifactorial disease, with hereditary factors, as well as environmental lifestyle-related risk factors. Biogenic polyamines, putrescine, spermidine, and spermine are small cationic molecules with great roles in cell proliferation and differentiation as well as regulation of gene expression. Ornithine decarboxylase is the first rate-limiting enzyme for polyamine synthesis, and upregulation of ornithine decarboxylase activity and polyamine metabolism has been associated with abnormal cell proliferation. This paper is focused on studying the protective role of Lactobacillus casei ATCC 393 in a chemically induced mouse model of colon carcinogenesis, directing our attention on aberrant crypt foci as preneoplastic markers, and on polyamine metabolism as a possible key player in carcinogenesis. BALB/c mice were administered 1,2-dimethylhydrazine dihydrochloride (DMH) to induce colon cancer (20 mg/kg body weight, subcutaneous, twice a week for 24 weeks). L. casei ATCC 393 was given orally (10⁶ CFU, twice a week), 2 weeks before DMH administration. Hematoxylin and eosin staining, high-performance liquid chromatography, and Western blotting were used to evaluate aberrant crypt foci, urinary polyamines, and ornithine decarboxylase expression in the colon. The experimental data showed that the preventive administration of L. casei ATCC 393 may delay the onset of cancer as it significantly reduced the number of DMH-induced aberrant crypt foci, the levels of putrescine, and the expression of ornithine decarboxylase. Hence, this probiotic strain has a prospective role in protection against colon carcinogenesis, and its antimutagenic activity may be associated with the maintenance of polyamine metabolism.