A recessive mutation, immune deficiency (imd), defines two distinct control pathways in the Drosophila host defense.

In this paper we report a recessive mutation, immune deficiency (imd), that impairs the inducibility of all genes encoding antibacterial peptides during the immune response of Drosophila. When challenged with bacteria, flies carrying this mutation show a lower survival rate than wild-type flies. We also report that, in contrast to the antibacterial peptides, the antifungal peptide drosomycin remains inducible in a homozygous imd mutant background. These results point to the existence of two different pathways leading to the expression of two types of target genes, encoding either the antibacterial peptides or the antifungal peptide drosomycin.

Studies of the lamin proteinase reveal multiple parallel biochemical pathways during apoptotic execution.

Although specific proteinases play a critical role in the active phase of apoptosis, their substrates are largely unknown. We previously identified poly(ADP-ribose) polymerase (PARP) as an apoptosis-associated substrate for proteinase(s) related to interleukin 1 beta-converting enzyme (ICE). Now we have used a cell-free system to characterize proteinase(s) that cleave the nuclear lamins during apoptosis. Lamin cleavage during apoptosis requires the action of a second ICE-like enyzme, which exhibits kinetics of cleavage and a profile of sensitivity to specific inhibitors that is distinct from the PARP proteinase. Thus, multiple ICE-like enzymes are required for apoptotic events in these cell-free extracts. Inhibition of the lamin proteinase with tosyllysine "chloromethyl ketone" blocks nuclear apoptosis prior to the packaging of condensed chromatin into apoptotic bodies. Under these conditions, the nuclear DNA is fully cleaved to a nucleosomal ladder. Our studies reveal that the lamin proteinase and the fragmentation nuclease function in independent parallel pathways during the final stages of apoptotic execution. Neither pathway alone is sufficient for completion of nuclear apoptosis. Instead, the various activities cooperate to drive the disassembly of the nucleus.

Interleukin 4 signals through two related pathways.

The interleukin 4 (IL-4) signaling pathway involves activation, by tyrosine phosphorylation, of two distinct substrates, a signal-transducing factor (STF-IL4) and the IL-4-induced phosphotyrosine substrate (4PS). It is not known whether the IL-4-mediated activation of these substrates occurs via related or distinct signaling pathways. We report that 32D cells, an IL-3-dependent myeloid progenitor cell line in which no phosphorylated 4PS is found, activate high levels of STF-IL4 in response to IL-4. Consistent with the known requirement for 4PS or insulin receptor substrate 1 (IRS-1) in IL-4-mediated mitogenesis, activation of STF-IL4 in 32D cells is not sufficient for IL-4-inducible c-myc expression. In addition, we have examined the ability of 32D cells transfected with different truncation mutants of the human IL-4 receptor to activate Jak-3 kinase and STF-IL4 in response to human IL-4. As in the case of 4PS/IRS-1, we have found that activation of both Jak-3 and STF-IL4 requires the presence of the IL-4 receptor region comprising aa 437-557. The finding that the same region of the IL-4 receptor is required for the induction of both 4PS/IRS-1 and STF-IL4 suggests that the IL-4-stimulated activation of these two substrates might involve common factors.

Retinoid-dependent pathways suppress myocardial cell hypertrophy.

Utilizing an in vitro model system of cardiac muscle cell hypertrophy, we have identified a retinoic acid (RA)-mediated pathway that suppresses the acquisition of specific features of the hypertrophic phenotype after exposure to the alpha-adrenergic receptor agonist phenylephrine. RA at physiological concentrations suppresses the increase in cell size and induction of a genetic marker for hypertrophy, the atrial natriuretic factor (ANF) gene. RA also suppresses endothelin 1 pathways for cardiac muscle cell hypertrophy, but it does not affect the increase in cell size and ANF expression induced by serum stimulation. A trans-activation analysis using a transient transfection assay reveals that neonatal rat ventricular myocardial cells express functional RA receptors of both the retinoic acid receptor and retinoid X receptor (RAR and RXR) subtypes. Using synthetic agonists of RA, which selectively bind to RXR or RAR, our data indicate that RAR/RXR heterodimers mediate suppression of alpha-adrenergic receptor-dependent hypertrophy. These results suggest the possibility that a pathway for suppression of hypertrophy may exist in vivo, which may have potential therapeutic value.

Multimeric complexes of the PML-retinoic acid receptor alpha fusion protein in acute promyelocytic leukemia cells and interference with retinoid and peroxisome-proliferator signaling pathways.

The t(15;17) chromosomal translocation, specific for acute promyelocytic leukemia (APL), fuses the PML gene to the retinoic acid receptor alpha (RAR alpha) gene, resulting in expression of a PML-RAR alpha hybrid protein. In this report, we analyzed the nature of PML-RAR alpha-containing complexes in nuclear protein extracts of t(15;17)-positive cells. We show that endogenous PML-RAR alpha can bind to DNA as a homodimer, in contrast to RAR alpha that requires the retinoid X receptor (RXR) dimerization partner. In addition, these cells contain oligomeric complexes of PML-RAR alpha and endogenous RXR. Treatment with retinoic acid results in a decrease of PML-RAR alpha protein levels and, as a consequence, of DNA binding by the different complexes. Using responsive elements from various hormone signaling pathways, we show that PML-RAR alpha homodimers have altered DNA-binding characteristics when compared to RAR alpha-RXR alpha heterodimers. In transfected Drosophila SL-3 cells that are devoid of endogenous retinoid receptors PML-RAR alpha inhibits transactivation by RAR alpha-RXR alpha heterodimers in a dominant fashion. In addition, we show that both normal retinoid receptors and the PML-RAR alpha hybrid bind and activate the peroxisome proliferator-activated receptor responsive element from the Acyl-CoA oxidase gene, indicating that retinoids and peroxisome proliferator receptors may share common target genes. These properties of PML-RAR alpha may contribute to the transformed phenotype of APL cells.

Disruption of cellular signaling pathways by daunomycin through destabilization of nonlamellar membrane structures.

Albeit anthracyclines are widely used in the treatment of solid tumors and leukemias, their mechanism of action has not been elucidated. The present study gives relevant information about the role of nonlamellar membrane structures in signaling pathways, which could explain how anthracyclines can exert their cytocidal action without entering the cell [Tritton, T. R. & Yee, G. (1982) Science 217, 248-250]. The anthracycline daunomycin reduced the formation of the nonlamellar hexagonal (HII) phase (i.e., the hexagonal phase propensity), stabilizing the bilayer structure of the plasma membrane by a direct interaction with membrane phospholipids. As a consequence, various cellular events involved in signal transduction, such as membrane fusion and membrane association of peripheral proteins [e.g., guanine nucleotide-binding regulatory proteins (G proteins and protein kinase C-alpha beta)], where nonlamellar structures (negative intrinsic monolayer curvature strain) are required, were altered by the presence of daunomycin. Functionally, daunomycin also impaired the expression of the high-affinity state of a G protein-coupled receptor (ternary complex for the alpha 2-adrenergic receptor) due to G-protein dissociation from the plasma membrane. In vivo, daunomycin also decreased the levels of membrane-associated G proteins and protein kinase C-alpha beta in the heart. The occurrence of such nonlamellar structures favors the association of these peripheral proteins with the plasma membrane and prevents daunomycin-induced dissociation. These results reveal an important role of the lipid component of the cell membrane in signal transduction and its alteration by anthracyclines.

Interleukin 4 suppresses c-kit ligand-induced expression of cytosolic phospholipase A2 and prostaglandin endoperoxide synthase 2 and their roles in separate pathways of eicosanoid synthesis in mouse bone marrow-derived mast cells.

Mouse bone marrow-derived mast cells (BMMCs) developed with interleukin 3 (IL-3) can be stimulated by c-kit ligand (KL) and accessory cytokines over a period of hours for direct delayed prostaglandin (PG) generation or over a period of days to prime for augmented IgE-dependent PG and leukotriene (LT) production, as previously reported. We now report that IL-4 is counterregulatory for each of these distinct KL-dependent responses. BMMCs cultured for 4 days with KL + IL-3 or with KL + IL-10 produced 5- to 7-fold more PGD2 and approximately 2-fold more LTC4 in response to IgE-dependent activation than BMMCs maintained in IL-3 alone. IL-4 inhibited the priming for increased IgE-dependent PGD2 and LTC4 production to the level obtained by activation of BMMCs maintained in IL-3 alone with an IC50 of approximately 0.2 ng/ml. IL-4 inhibited the KL-induced increase in expression of cytosolic phospholipase A2 (cPLA2) but had no effect on the incremental expression of PG endoperoxide synthase 1 (PGHS-1) and hematopoietic PGD2 synthase or on the continued baseline expression of 5-lipoxygenase, 5-lipoxygenase activating protein, and LTC4 synthase. BMMCs stimulated by KL + IL-10 for 10 h exhibited a delayed phase of PGD2 generation, which was dependent on de novo induction of PGHS-2. IL-4 inhibited the induction of PGHS-2 expression and the accompanying cytokine-initiated delayed PGD2 generation with an IC50 of approximately 6 ng/ml. IL-4 had no effect on the expression of PGHS-2 and the production of PGD2 elicited by addition of IL-1 beta to the combination of KL + IL-10. IL-4 had no effect on the immediate phase of eicosanoid synthesis elicited by KL alone or by IgE and antigen in BMMCs maintained in IL-3. Thus, the counterregulatory action of IL-4 on eicosanoid generation is highly selective for the induced incremental expression of cPLA2 and the de novo expression of PGHS-2, thereby attenuating time-dependent cytokine-regulated responses to stimulation via Fc epsilon receptor I and stimulation via c-kit, respectively.

Biological response to phorbol ester determined by alternative G1 pathways.

A plethora of extracellular signals is known to induce a common set of immediate early genes. The immediate early response, therefore, must not be sufficient to determine the biological outcome. An example of this is found with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). A potent activator of protein kinase C, TPA can either stimulate or inhibit cell proliferation, depending on the cell type. This cell context-dependent response to TPA is observed with two subclones of NIH 3T3 cells, the P- and the N-3T3 clones. TPA is a mitogen for the P-3T3 but an antimitogen for the N-3T3 cells. The immediate early pathway is activated by TPA in both cell types, indicating that this pathway alone does not activate DNA synthesis. The delayed induction of cyclin D1 expression by TPA is observed only in the P-3T3 cells, correlating with mitogenesis. N-Acetylcysteine does not affect the immediate early pathway but can inhibit the TPA-mediated induction of cyclin D1 and DNA synthesis. In the N-3T3 cells, TPA causes an inhibition of the cyclin E-associated kinase at the G1/S transition, correlating with growth inhibition. The growth-inhibitory activity of TPA is not affected by N-acetylcysteine. Thus, the two TPA-regulated G1 pathways can be distinguished by their sensitivity to N-acetylcysteine. These results demonstrate that TPA can activate alternative G1 pathways. Moreover, the selection of the alternative G1 pathways is determined by the cell context, which, in turn, dictates the biological response to TPA.

Structural conservation of ion conduction pathways in K channels and glutamate receptors.

Single channel recordings demonstrate that ion channels switch stochastically between an open and a closed pore conformation. In search of a structural explanation for this universal open/close behavior, we have uncovered a striking degree of amino acid homology across the pore-forming regions of voltage-gated K channels and glutamate receptors. This suggested that the pores of these otherwise unrelated classes of channels could be structurally conserved. Strong experimental evidence supports a hairpin structure for the pore-forming region of K channels. Consequently, we hypothesized the existence of a similar structure for the pore of glutamate receptors. In ligand-gated channels, the pore is formed by M2, the second of four putative transmembrane segments. A hairpin structure for M2 would affect the subsequent membrane topology, inverting the proposed orientation of the next segments, M3. We have tested this idea for the NR1 subunit of the N-methyl-D-aspartate receptor. Mutations that affected the glycosylation pattern of the NR1 subunit localize both extremes of the M3-M4 linker to the extracellular space. Whole cell currents and apparent agonist affinities were not affected by these mutations. Therefore it can be assumed that they represent the native transmembrane topology. The extracellular assignment of the M3-M4 linker challenged the current topology model by inverting M3. Taken together, the amino acid homology and the new topology suggest that the pore-forming M2 segment of glutamate receptors does not transverse the membrane but, rather, forms a hairpin structure, similar to that found in K channels.

The octadecanoic pathway: signal molecules for the regulation of secondary pathways.

Plant defense against microbial pathogens and herbivores relies heavily on the induction of defense proteins and low molecular weight antibiotics. The signals between perception of the aggression, gene activation, and the subsequent biosynthesis of secondary compounds are assumed to be pentacylic oxylipin derivatives. The rapid, but transient, synthesis of cis-jasmonic acid was demonstrated after insect attack on a food plant and by microbial elicitor addition to plant suspension cultures. This effect is highly specific and not caused by a number of environmental stresses such as light, heavy metals, or cold or heat shock. Elicitation of Eschscholtzia cell cultures also led to a rapid alkalinization of the growth medium prior to jasmonate formation. Inhibition of this alkalinization process by the protein kinase inhibitor staurosporine also inhibited jasmonate formation. The induction of specific enzymes in the benzo[c]phenanthridine alkaloid pathway leading to the antimicrobial sanguinarine was induced to a qualitatively and quantitatively similar extent by fungal elicitor, methyl jasmonate, and its linolenic acid-derived precursor 12-oxophytodienoic acid. It is herein proposed that a second oxylipid cascade may exist in plants starting from linoleic acid via 15,16-dihydro-12-oxophytodienoic acid to 9,10-dihydrojasmonate. Experiments with synthetic trihomojasmonate demonstrated that beta-oxidation is not a prerequisite for biological activity and that 12-oxophytodienoic acid and derivatives are most likely fully active as signal transducers. Octadecanoic acid-derived compounds are essential elements in modulating the synthesis of antibiotic compounds and are thus integral to plant defense.

Involvement of small GTP-binding proteins in defense signal-transduction pathways of higher plants.

Small GTP-binding proteins play a critical role in the regulation of a range of cellular processes--including growth, differentiation, and intracellular transportation. Previously, we isolated a gene, rgp1, encoding a small GTP-binding protein, by differential screening of a rice cDNA library with probe DNAs from rice tissues treated with or without 5-azacytidine, a powerful inhibitor of DNA methylation. To determine the physiological role of rgp1, the coding region was introduced into tobacco plants. Transformants, with rgp1 in either sense or antisense orientations, showed distinct phenotypic changes with reduced apical dominance, dwarfism, and abnormal flower development. These abnormal phenotypes appeared to be associated with the higher levels of endogenous cytokinins that were 6-fold those of wild-type plants. In addition, the transgenic plants produced salicylic acid and salicylic acid-beta-glucoside in an unusual response to wounding, thus conferring increased resistance to tobacco mosaic virus infection. In normal plants, the wound- and pathogen-induced signal-transduction pathways are considered to function independently. However, the wound induction of salicylic acid in the transgenic plants suggests that expression of rgp1 somehow interfered with the normal signaling pathways and resulted in cross-signaling between these distinct transduction systems. The results imply that the defense signal-transduction system consists of a complicated and finely tuned network of several regulatory factors, including cytokinins, salicylic acid, and small GTP-binding proteins.

B-cell activation by crosslinking of surface IgM or ligation of CD40 involves alternative signal pathways and results in different B-cell phenotypes.

Treatment of small resting B cells with soluble F(ab')2 fragments of anti-IgM, an analogue of T-independent type 2 antigens, induced activation characterized by proliferation and the expression of surface CD5. In contrast, B cells induced to proliferate in response to thymus-dependent inductive signals provided by either fixed activated T-helper 2 cells or soluble CD40 ligand-CD8 (CD40L) recombinant protein displayed elevated levels of CD23 (Fc epsilon II receptor) and no surface CD5. Treatment with anti-IgM and CD40L induced higher levels of proliferation and generated a single population of B cells coexpressing minimal amounts of CD5 and only a slight elevation of CD23. Anti-IgM- but not CD40L-mediated activation was highly sensitive to inhibition by cyclosporin A and FK520. Sp-cAMPS, an analogue of cAMP, augmented CD40L and suppressed surface IgM-mediated activation. Taken together these results are interpreted to mean that there is a single population of small resting B cells that can respond to either T-independent type 2 (surface IgM)- or T-dependent (CD40)-mediated activation. In response to different intracellular signals these cells are induced to enter alternative differentiation pathways.

Activation of YRP kinase by v-Src and protein kinase C-mediated signal transduction pathways.

We have previously reported that a serine(threonine) protein kinase that phosphorylates histone H1 in vitro is activated by tyrosine phosphorylation in v-Src-transformed rat 3Y1 fibroblasts. We now refer to this kinase as YRP kinase, for tyrosine-regulated protein kinase. Since YRP kinase may play a role in mediating the growth-stimulatory and morphology-altering effects of v-Src, we have further examined the signal transduction involved in the activation of YRP kinase. Although YRP kinase is constitutively activated in fibroblasts transformed by v-Src, activation of protein kinase C was also found to lead to activation of YRP kinase. Activation of YRP kinase by protein kinase C was found to be potentiated by vanadate treatment or overexpression of c-Src. The activation of YRP kinase by v-Src, however, does not appear to be mediated by protein kinase C, suggesting that YRP kinase can be activated by two separate signal transduction pathways. Transformation of fibroblasts by v-Ras or v-Mil did not result in activation of YRP kinase, indicating that the MAP kinase pathway does not mediate the activation of YRP kinase by v-Src or protein kinase C.

Pathways to quiescence: SHARDS view on the star formation histories of massive quiescent galaxies at 1.0 < z < 1.5

We present star formation histories (SFHs) for a sample of 104 massive (stellar mass M > 10^10 M_⊙) quiescent galaxies (MQGs) at z = 1.0–1.5 from the analysis of spectrophotometric data from the Survey for High-z Absorption Red and Dead Sources (SHARDS) and HST/WFC3 G102 and G141 surveys of the GOODS-North field, jointly with broad-band observations from ultraviolet (UV) to far-infrared (far-IR). The sample is constructed on the basis of rest-frame UVJ colours and specific star formation rates (sSFRs = SFR/Mass). The spectral energy distributions (SEDs) of each galaxy are compared to models assuming a delayed exponentially declining SFH. A Monte Carlo algorithm characterizes the degeneracies, which we are able to break taking advantage of the SHARDS data resolution, by measuring indices such as MgUV and D4000. The population of MQGs shows a duality in their properties. The sample is dominated (85 per cent) by galaxies with young mass-weighted ages, t_M t_M < 2 Gyr, short star formation time-scales, 〈τ〉 ∼ 60–200 Myr, and masses log(M/M_⊙) ∼ 10.5. There is an older population (15 per cent) with t_M t_M = 2–4 Gyr, longer star formation time-scales, 〈τ〉∼ 400 Myr, and larger masses, log(M/M_⊙) ∼ 10.7. The SFHs of our MQGs are consistent with the slope and the location of the main sequence of star-forming galaxies at z > 1.0, when our galaxies were 0.5–1.0 Gyr old. According to these SFHs, all the MQGs experienced a luminous infrared galaxy phase that lasts for ∼500 Myr, and half of them an ultraluminous infrared galaxy phase for ∼100 Myr. We find that the MQG population is almost assembled at z ∼ 1, and continues evolving passively with few additions to the population.

Continuous flow nanocatalysis: reaction pathways in the conversion of levulinic acid to valuable chemicals

The selective production of 2-methyltetrahydrofuran from levulinic acid has been effectively conducted using designed Cu based catalysts and compared with a commercial Pd/C system under microwave irradiation. Optimised conditions for the most active catalysts Cu-MINT (>90% conversion, 75% selectivity to MTHF) and Pd/C (78% conversion, 92% selectivity to MTHF) were further translated into a continuous flow process using the proposed catalysts to find out the deactivation of Cu-MINT under flow conditions (79 vs. 13% conversion with a switch in selectivity to products after 30 min in flow), the high stability of Pd/C (73 vs. 70% conversion at stable selectivity under analogous conditions to those of Cu-MINT) but, most importantly, different relevant pathways to valuable products from levulinic acid depending on the type of catalyst employed.