Two Arabidopsis Mutants That Overproduce Ethylene Are Affected in the Posttranscriptional Regulation of 1-Aminocyclopropane-1-Carboxylic Acid Synthase1

The Arabidopsis mutants eto1 (ethylene overproducer) and eto3 produce elevated levels of ethylene as etiolated seedlings. Ethylene production in these seedlings peaks at 60 to 96 h, and then declines back to almost wild-type levels. Ethylene overproduction in eto1 and eto3 is limited mainly to etiolated seedlings; light-grown seedlings and various adult tissues produce close to wild-type amounts of ethylene. Several compounds that induce ethylene biosynthesis in wild-type, etiolated seedlings through distinct 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) isoforms were found to act synergistically with eto1 and eto3, as did the ethylene-insensitive mutation etr1 (ethylene resistant), which blocks feedback inhibition of biosynthesis. ACS activity, the rate-limiting step of ethylene biosynthesis, was highly elevated in both eto1 and eto3 mutant seedlings, even though RNA gel-blot analysis demonstrated that the steady-state level of ACS mRNA was not increased, including that of a novel Arabidopsis ACS gene that was identified. Measurements of the conversion of ACC to ethylene by intact seedlings indicated that the mutations did not affect conjugation of ACC or the activity of ACC oxidase, the final step of ethylene biosynthesis. Taken together, these data suggest that the eto1 and eto3 mutations elevate ethylene biosynthesis by affecting the posttranscriptional regulation of ACS.

Ubc9 interacts with a nuclear localization signal and mediates nuclear localization of the paired-like homeobox protein Vsx-1 independent of SUMO-1 modification

Vsx-1 is a paired-like:CVC homeobox gene whose expression is linked to bipolar cell differentiation during zebrafish retinogenesis. We used a yeast two-hybrid screen to identify proteins interacting with Vsx-1 and isolated Ubc9, an enzyme that conjugates the small ubiquitin-like modifier SUMO-1. Despite its interaction with Ubc9, we show that Vsx-1 is not a substrate for SUMO-1 in COS-7 cells or in vitro. When a yeast two-hybrid assay is used, deletion analysis of the interacting domain on Vsx-1 shows that Ubc9 binds to a nuclear localization signal (NLS) at the NH2 terminus of the homeodomain. In SW13 cells, Vsx-1 localizes to the nucleus and is excluded from nucleoli. Deletion of the NLS disrupts this nuclear localization, resulting in a diffuse cytoplasmic distribution of Vsx-1. In SW13 AK1 cells that express low levels of endogenous Ubc9, Vsx-1 accumulates in a perinuclear ring and colocalizes with an endoplasmic reticulum marker. However, NLS-tagged STAT1 protein exhibits normal nuclear localization in both SW13 and SW13 AK1 cells, suggesting that nuclear import is not globally disrupted. Cotransfection of Vsx-1 with Ubc9 restores Vsx-1 nuclear localization in SW3 AK1 cells and demonstrates that Ubc9 is required for the nuclear localization of Vsx-1. Ubc9 continues to restore nuclear localization even after a C93S active site mutation has eliminated its SUMO-1-conjugating ability. These results suggest that Ubc9 mediates the nuclear localization of Vsx-1, and possibly other proteins, through a nonenzymatic mechanism that is independent of SUMO-1 conjugation.

Diazepam-induced changes in sleep: Role of the α1 GABAA receptor subtype

Ligands acting at the benzodiazepine (BZ) site of γ-aminobutyric acid type A (GABAA) receptors currently are the most widely used hypnotics. BZs such as diazepam (Dz) potentiate GABAA receptor activation. To determine the GABAA receptor subtypes that mediate the hypnotic action of Dz wild-type mice and mice that harbor Dz-insensitive α1 GABAA receptors [α1 (H101R) mice] were compared. Sleep latency and the amount of sleep after Dz treatment were not affected by the point mutation. An initial reduction of rapid eye movement (REM) sleep also occurred equally in both genotypes. Furthermore, the Dz-induced changes in the sleep and waking electroencephalogram (EEG) spectra, the increase in power density above 21 Hz in non-REM sleep and waking, and the suppression of slow-wave activity (SWA; EEG power in the 0.75- to 4.0-Hz band) in non-REM sleep were present in both genotypes. Surprisingly, these effects were even more pronounced in α1(H101R) mice and sleep continuity was enhanced by Dz only in the mutants. Interestingly, Dz did not affect the initial surge of SWA at the transitions to sleep, indicating that the SWA-generating mechanisms are not impaired by the BZ. We conclude that the REM sleep inhibiting action of Dz and its effect on the EEG spectra in sleep and waking are mediated by GABAA receptors other than α1, i.e., α2, α3, or α5 GABAA receptors. Because α1 GABAA receptors mediate the sedative action of Dz, our results provide evidence that the hypnotic effect of Dz and its EEG “fingerprint” can be dissociated from its sedative action.

Several Thaumatin-Like Proteins Bind to β-1,3-Glucans1

Pathogenesis-related proteins from intercellular fluid washings of stressed barley (Hordeum vulgare L.) leaves were analyzed to determine their binding to various water-insoluble polysaccharides. Three proteins (19, 16, and 15 kD) bound specifically to several water-insoluble β-1,3-glucans. Binding of the barley proteins to pachyman occurred quickly at 22°C at pH 5.0, even in the presence of 0.5 m NaCl, 0.2 m urea, and 1% (v/v) Triton X-100. Bound barley proteins were released by acidic treatments or by boiling in sodium dodecyl sulfate. Acid-released barley proteins could bind again specifically and singly to pachyman. Water-soluble laminarin and carboxymethyl-pachyman competed for the binding of the barley proteins to pachyman. The N-terminal sequence of the 19-kD barley β-1,3-glucan-binding protein showed near identity to the barley seed protein BP-R and high homology to other thaumatin-like (TL) permatins. The 16-kD barley protein was also homologous to TL proteins, whereas the 15-kD barley protein N-terminal sequence was identical to the pathogenesis-related Hv-1 TL protein. Antifungal barley protein BP-R and corn (Zea mays) zeamatin were isolated by binding to pachyman. Two extracellular proteins from stressed pea (Pisum sativum L.) also bound to pachyman and were homologous to TL proteins.

Does a Low Nitrogen Supply Necessarily Lead to Acclimation of Photosynthesis to Elevated CO2?1

Long-term exposure of plants to elevated partial pressures of CO2 (pCO2) often depresses photosynthetic capacity. The mechanistic basis for this photosynthetic acclimation may involve accumulation of carbohydrate and may be promoted by nutrient limitation. However, our current knowledge is inadequate for making reliable predictions concerning the onset and extent of acclimation. Many studies have sought to investigate the effects of N supply but the methodologies used generally do not allow separation of the direct effects of limited N availability from those caused by a N dilution effect due to accelerated growth at elevated pCO2. To dissociate these interactions, wheat (Triticum aestivum L.) was grown hydroponically and N was added in direct proportion to plant growth. Photosynthesis did not acclimate to elevated pCO2 even when growth was restricted by a low-N relative addition rate. Ribulose-1, 5-bisphosphate carboxylase/oxygenase activity and quantity were maintained, there was no evidence for triose phosphate limitation of photosynthesis, and tissue N content remained within the range recorded for healthy wheat plants. In contrast, wheat grown in sand culture with N supplied at a fixed concentration suffered photosynthetic acclimation at elevated pCO2 in a low-N treatment. This was accompanied by a significant reduction in the quantity of active ribulose-1, 5-bisphosphate carboxylase/oxygenase and leaf N content.

Potent Inhibition of Ribulose-Bisphosphate Carboxylase by an Oxidized Impurity in Ribulose-1,5-Bisphosphate1

Oxidation of d-ribulose-1,5-bisphosphate (ribulose-P2) during synthesis and/or storage produces d-glycero-2,3-pentodiulose-1,5-bisphosphate (pentodiulose-P2), a potent slow, tight-binding inhibitor of spinach (Spinacia oleracea L.) ribulose-P2 carboxylase/oxygenase (Rubisco). Differing degrees of contamination with pentodiulose-P2 caused the decline in Rubisco activity seen during Rubisco assay time courses to vary between different preparations of ribulose-P2. With some ribulose-P2 preparations, this compound can be the dominant cause of the decline, far exceeding the significance of the catalytic by-product, d-xylulose-1,5-bisphosphate. Unlike xylulose-1,5-bisphosphate, pentodiulose-P2 did not appear to be a significant by-product of catalysis by wild-type Rubisco at saturating CO2 concentration. It was produced slowly during frozen storage of ribulose-P2, even at low pH, more rapidly in Rubisco assay buffers at room temperature, and particularly rapidly on deliberate oxidation of ribulose-P2 with Cu2+. Its formation was prevented by the exclusion of transition metals and O2. Pentodiulose-P2 was unstable and decayed to a variety of other less-inhibitory compounds, particularly in the presence of some buffers. However, it formed a tight, stable complex with carbamylated spinach Rubisco, which could be isolated by gel filtration, presumably because its structure mimics that of the enediol intermediate of Rubisco catalysis. Rubisco catalyzes the cleavage of pentodiulose-P2 by H2O2, producing P-glycolate.

Effects of Short- and Long-Term Elevated CO2 on the Expression of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Genes and Carbohydrate Accumulation in Leaves of Arabidopsis thaliana (L.) Heynh.1

To investigate the proposed molecular characteristics of sugar-mediated repression of photosynthetic genes during plant acclimation to elevated CO2, we examined the relationship between the accumulation and metabolism of nonstructural carbohydrates and changes in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) gene expression in leaves of Arabidopsis thaliana exposed to elevated CO2. Long-term growth of Arabidopsis at high CO2 (1000 μL L−1) resulted in a 2-fold increase in nonstructural carbohydrates, a large decrease in the expression of Rubisco protein and in the transcript of rbcL, the gene encoding the large subunit of Rubisco (approximately 35–40%), and an even greater decline in mRNA of rbcS, the gene encoding the small subunit (approximately 60%). This differential response of protein and mRNAs suggests that transcriptional/posttranscriptional processes and protein turnover may determine the final amount of leaf Rubisco protein at high CO2. Analysis of mRNA levels of individual rbcS genes indicated that reduction in total rbcS transcripts was caused by decreased expression of all four rbcS genes. Short-term transfer of Arabidopsis plants grown at ambient CO2 to high CO2 resulted in a decrease in total rbcS mRNA by d 6, whereas Rubisco content and rbcL mRNA decreased by d 9. Transfer to high CO2 reduced the maximum expression level of the primary rbcS genes (1A and, particularly, 3B) by limiting their normal pattern of accumulation through the night period. The decreased nighttime levels of rbcS mRNA were associated with a nocturnal increase in leaf hexoses. We suggest that prolonged nighttime hexose metabolism resulting from exposure to elevated CO2 affects rbcS transcript accumulation and, ultimately, the level of Rubisco protein.

TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors

To achieve an efficient intracellular drug and DNA delivery, attempts were made to target microparticulate drug carriers into cytoplasm bypassing the endocytotic pathway. TAT peptides derived from the HIV-1 TAT protein facilitate intracellular delivery of proteins and small colloidal particles. We demonstrated that relatively large drug carriers, such as 200-nm liposomes, can also be delivered into cells by TAT peptide attached to the liposome surface. Liposomes were fluorescently labeled with membranotropic rhodamine-phosphatidylethanolamine or by entrapping FITC-dextran. Incubation of fluorescent TAT liposomes with mouse Lewis lung carcinoma cells, human breast tumor BT20 cells, and rat cardiac myocyte H9C2 results in intracellular localization of certain liposomes. Steric hindrances for TAT peptide⋅cell interaction (attachment of TAT directly to the liposome surface without spacer or the presence of a high MW polyethylene glycol on the liposome surface) abolish liposome internalization, evidencing the importance of direct contact of TAT peptide with the cell surface. Low temperature or metabolic inhibitors, sodium azide or iodoacetamide, have little influence on the translocation of TAT liposomes into cells, confirming the energy-independent character of this process. The approach may have important implications for drug delivery directly into cell cytoplasm.

Cell-type and promoter-context dependent retinoic acid receptor (RAR) redundancies for RAR beta 2 and Hoxa-1 activation in F9 and P19 cells can be artefactually generated by gene knockouts.

By using RAR type (alpha, beta, or gamma)-specific synthetic retinoids and a pan-retinoic X receptor (RXR)-specific ligand, we have investigated the contribution of RARs and RXRs in the activation of RA target genes and the differentiation of embryonal carcinoma cells. We demonstrate cell-type- and promoter context-dependent functional redundancies that differ between the three RAR types for mediating the induction of RARbeta2 and Hoxa-1 in wild-type, RARgamma-/- and RARalpha-/- F9 cells and in P19 cells. The extent of redundancy between RARs is further modulated by the synergistic activation of RXRs with a pan-RXR agonist. We also demonstrate that the expression of RARbeta2 is auto-inducible in RARgamma-/- but not in wild-type F9 cells, indicating that the functional redundancies observed between RARs in gene disruption studies can be artefactually generated. Thus, even though all three RARs can functionally substitute each other for inducing the expression of RA target genes and cell differentiation, one RAR can cell-specifically override the activity of the other RARs. Interestingly, only RARgamma can mediate the retinoic acid-induced differentiation of wild-type F9 cells, whereas the differentiation of P19 cells can be mediated by either RARalpha or RARgamma.

Human immunodeficiency virus type 1 and 2 Tat proteins specifically interact with RNA polymerase II.

The Tat-responsive region (TAR) element is a critical RNA regulatory element in the human immunodeficiency virus (HIV) long terminal repeat, which is required for activation of gene expression by the transactivator protein Tat. Recently, we demonstrated by gel-retardation analysis that RNA polymerase II binds to TAR RNA and that Tat prevents this binding even when Tat does not bind to TAR RNA. These results suggested that direct interactions between Tat and RNA polymerase II may prevent RNA polymerase II pausing and lead to Tat-mediated increases in transcriptional elongation. To test this possibility, we performed protein interaction studies with RNA polymerase II and both the HIV-1 and the closely related HIV-2 Tat protein. These studies indicated that both the HIV-1 and HIV-2 Tat proteins could specifically interact with RNA polymerase II. Mutagenesis of both HIV-1 and HIV-2 Tat demonstrated that the basic domains of both the HIV-1 and HIV-2 Tat proteins were required for this interaction. Furthermore, "far Western" analysis suggested that the largest subunit of RNA polymerase II was the site for interaction with Tat. The interactions between Tat and RNA polymerase II were of similar magnitude to those detected between RNA polymerase II and the cellular transcription factor RAP30, which stably associates with RNA polymerase II during transcriptional elongation. These studies are consistent with the model that RNA polymerase II is a cellular target for Tat resulting in Tat-mediated increases in transcriptional elongation from the HIV long terminal repeat.

Competence for collagenase gene expression by tissue fibroblasts requires activation of an interleukin 1 alpha autocrine loop.

The enzyme collagenase (EC 3.4.24.7), a key mediator in biological remodeling, can be induced in early-passage fibroblasts by a wide variety of agents and conditions. In contrast, at least some primary tissue fibroblasts are incompetent to synthesize collagenase in response to many of these stimulators. In this study, we investigate mechanisms controlling response to two of the conditions in question: (i) trypsin or cytochalasin B, which disrupt actin stress fibers, or (ii) phorbol 12-myristate 13-acetate (PMA), which activates growth factor signaling pathways. We demonstrate that collagenase expression stimulated by trypsin or cytochalasin B is regulated entirely through an autocrine cytokine, interleukin 1 alpha (IL-1 alpha). The IL-1 alpha intermediate also constitutes the major mechanism by which PMA stimulates collagenase expression, although a second signaling pathway(s) contributes to a minor extent. Elevation of the IL-1 alpha level in response to stimulators is found to be sustained by means of an autocrine feedback loop in early-passage fibroblast cultures. In contrast, fibroblasts freshly isolated from the tissue are incompetent to activate and sustain the IL-1 alpha feedback loop, even though they synthesize collagenase in response to exogenous IL-1. We conclude that this is the reason why tissue fibroblasts are limited, in comparison with subcultured fibroblasts, in their capacity to synthesize collagenase. Activation of the IL-1 alpha feedback loop, therefore, seems likely to be an important mechanism by which resident tissue cells adopt the remodeling phenotype.

Thrombospondin 1 expression in transformed endothelial cells restores a normal phenotype and suppresses their tumorigenesis.

Murine endothelial cells are readily transformed in a single step by the polyomavirus oncogene encoding middle-sized tumor antigen. These cells (bEND.3) form tumors (hemangiomas) in mice which are lethal in newborn animals. The bEND.3 cells rapidly proliferate in culture and express little or no thrombospondin 1 (TS1). To determine the role of TS1 in regulation of endothelial cell phenotype, we stably transfected bEND.3 cells with a human TS1 expression vector. The cells expressing human TS1 were readily identified by their altered morphology and exhibited a slower growth rate and lower saturation density than the parental bEND.3 cells. The TS1-expressing cells also formed aligned cords of cells instead of clumps or cysts in Matrigel. Moreover, while the bEND.3 cells formed large tumors in nude mice within 48 hr, the TS1-expressing cells failed to form tumors even after 1 month. The TS1-transfected cells expressed transforming growth factor beta mRNA and bioactivity at levels similar to those of the parental or vector-transfected bEND.3 cells, indicating that the effects of TS1 expression are not due to the activation of transforming growth factor beta by TS1. TS1 expression resulted in a > 100-fold decrease in net fibrinolytic (urokinase-type plasminogen activator, uPA) activity due to more plasminogen-activator inhibitor 1 and less uPA secretion. TS1 thus appears to be an important regulator of endothelial cell phenotype required for maintaining the quiescent, differentiated state.

Preferential expansion and survival of B lymphocytes based on VH framework 1 and framework 3 expression: "positive" selection in appendix of normal and VH-mutant rabbits.

B cells with a rearranged heavy-chain variable region VHa allotype-encoding VH1 gene segment predominate throughout the life of normal rabbits and appear to be the source of the majority of serum immunoglobulins, which thus bear VHa allotypes. The functional role(s) of these VH framework region (FR) allotypic structures has not been defined. We show here that B cells expressing surface immunoglobulin with VHa2 allotypic specificities are preferentially expanded and positively selected in the appendix of young rabbits. By flow cytometry, a higher proportion of a2+ B cells were progressing through the cell cycle (S/G2/M) compared to a2- B cells, most of which were in the G1/G0 phase of the cell cycle. The majority of appendix B cells in dark zones of germinal centers of normal 6-week-old rabbits were proliferating and very little apoptosis were observed. In contrast, in 6-week-old VH-mutant ali/ali rabbits, little cell proliferation and extensive apoptosis were observed. Nonetheless even in the absence of VH1, B cells with a2-like surface immunoglobulin had developed and expanded in the appendix of 11-week-old mutants. The numbers and tissue localization of B cells undergoing apoptosis then appeared similar to those found in 6-week-old normal appendix. Thus, B cells with immunoglobulin receptors lacking the VHa2 allotypic structures were less likely to undergo clonal expansion and maturation. These data suggest that "positive" selection of B lymphocytes through FR1 and FR3 VHa allotypic structures occurs during their development in the appendix.

Suppression of the breakthrough of human immunodeficiency virus type 1 (HIV-1) in cell culture by thiocarboxanilide derivatives when used individually or in combination with other HIV-1-specific inhibitors (i.e., TSAO derivatives).

Five structurally related thiophene and furane analogues of the oxathiin carboxanilide derivative NSC 615985 (UC84) (designated UC10, UC68, UC81, UC42, and UC16) were identified as potent inhibitors of HIV-1 replication in cell culture and HIV-1 reverse transcriptase activity. These compounds were markedly active against a series of mutant HIV-1 strains, containing the Leu-100-->Ile, Val-106-->Ala, Glu-138-->Lys, or Tyr-181-->Cys mutations in their reverse transcriptase. However, the thiocarboxanilide derivatives selected for mutations at amino acid positions 100 (Leu-->Ile), 101 (Lys-->Ile/Glu), 103 (Lys-->Thr/Asp) and 141 (Gly-->Glu) in the HIV-1 reverse transcriptase. The compounds completely suppressed HIV-1 replication and prevented the emergence of resistant virus strains when used at 1.3-6.6 microM--that is, 10- to 25-fold lower than the concentration required for nevirapine and bis(heteroaryl)piperazine (BHAP) U90152 to do so. If UC42 was combined with the [2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5"-(4"-amino-1",2"- oxathiole-2",2"-dioxide)]-beta-D-pentofuranosyl (TSAO) derivative of N3-methylthymine (TSAO-m3T), virus breakthrough could be prevented for a much longer time, and at much lower concentrations, than if the compounds were used individually. Virus breakthrough could be suppressed for even longer, and at lower drug concentrations, if BHAP was added to the combination of UC42 with TSAO-m3T, which points to the feasibility of two- or three-drug combinations in preventing virus breakthrough and resistance development.

Novel microbicidal agents derived from Naja atra cardiotoxin 1 (CTX-1)

During my PhD course, I focused my research on antimicrobial peptides (AMPs), in particular on the aspects of their computational design and development. This work led to the development of a new family of AMPs that I designed, starting from the amino acid sequence of a snake venom toxin, the cardiotoxin 1 (CTX-1) of Naja atra. Naja atra atra cardiotoxin 1, produced by Chinese cobra snakes belonging to Elapidae family, is included in the three-finger toxin family and exerts high cytotoxicity and antimicrobial activity too. This toxin family is characterized by specific folding of three beta-sheet loops (“fingers”) extending from the central core and by four conserved disulfide bridges. Using as template the first loop of this toxin, different sequences of 20 amino acids linear cationic peptides have been designed in order to avoid toxic effects but to maintain and strengthen the antimicrobial activity. As a result, the sequence NCP-0 (Naja Cardiotoxin Peptide-0) was designed as ancestor and subsequently other 4 variant sequences of NCP0 were developed. These variant sequences have shown microbicidal activity towards a panel of reference strains of Gram-positive and Gram-negative bacteria, fungi and an enveloped virus. In particular, the sequence designed as NCP-3 (Naja Cardiotoxin Peptide-3) and its variants NCP-3a and NCP-3b have shown the best antimicrobial activity together with low cytotoxicity against eukaryotic cells and low hemolytic activity. Bactericidal activity has been demonstrated by minimum bactericidal concentration (MBC) assay at values below 10 μg/ml for Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii ( clinical isolates), Moraxella catharralis ATCC 25238, MRSA ATCC 43400, while towards Staphylococcus aureus ATCC 25923, Enterococcus hirae ATCC 10541 and Streptococcus agalactiae ATCC 13813 the bactericidal activity was demonstrated even below 1.6 μg/ml concentration. This potent antimicrobial activity was confirmed even for unicellular fungi Candida albicans, Candida glabrata and Malassezia pachydermatis (MBC 32.26-6.4 μg/ml), and also against the fast-growing mycobacteria Mycobacterium smegmatis DSMZ 43756 and Mycobacterium fortuitum DSMZ 46621 (MBC 100 μg/ml). Moreover, NCP-3 has shown a virucidal activity on the enveloped virus Bovine Herpesvirus 1 (BoHV1) belonging to herpesviridae family. The bactericidal activity is maintained in a high salt concentration (125 and 250 mM NaCl) medium and PB +20% Mueller Hinton Medium for E. coli, MRSA and Pseudomonas aeruginosa reference strains. Considering these in vitro obtained data, we propose NCP-3 and its variants NCP-3a and NCP-3b as promising antimicrobial candidates. For this reason, the whole novel AMPs family has been protected by a national patent (n°102015000015951).