The Pseudomonas aeruginosa rhlG Gene Encodes an NADPH-Dependent β-Ketoacyl Reductase Which Is Specifically Involved in Rhamnolipid Synthesis

A Pseudomonas aeruginosa gene homologous to the fabG gene, which encodes the NADPH-dependent β-ketoacyl-acyl carrier protein (ACP) reductase required for fatty acid synthesis, was identified. The insertional mutation of this fabG homolog (herein called rhlG) produced no apparent effect on the growth rate and total lipid content of P. aeruginosa cells, but the production of rhamnolipids was completely abrogated. These results suggest that the synthetic pathway for the fatty acid moiety of rhamnolipids is separate from the general fatty acid synthetic pathway, starting with a specific ketoacyl reduction step catalyzed by the RhlG protein. In addition, the synthesis of poly-β-hydroxyalkanoate (PHA) is delayed in this mutant, suggesting that RhlG participates in PHA synthesis, although it is not the only reductase involved in this pathway. Traits regulated by the quorum-sensing response, other than rhamnolipid production, including production of proteases, pyocyanine, and the autoinducer butanoyl-homoserine lactone (PAI-2), were not affected by the rhlG mutation. We conclude that the P. aeruginosa rhlG gene encodes an NADPH-dependent β-ketoacyl reductase absolutely required for the synthesis of the β-hydroxy acid moiety of rhamnolipids and that it has a minor role in PHA production. Expression of rhlG mRNA under different culture conditions is consistent with this conclusion.     

Temporal polyethism in incipient colonies of the primitive termiteZootermopsis angusticollis: A single multiage caste

Division of labor was studied in incipient colonies of the dampwood termite Zootermopsis angusticollisby recording repertoire size, behavior frequencies, and time budgets of larvae. Behavioral repertoire size increased with age: first- and secondinstar larvae were mainly inactive, whereas larvae of the third through seventh instars performed 64–100% of all tasks. The increase in repertoire size from the second to the third instar was abrupt; repertoire size and composition remained more or less constant for older instars. No correlation between age (instars III–VII) and tasks was identified, suggesting that colony labor is performed by a single functional caste that spans the third to the seventh instar without any age-based division of labor. Small colony size, low oviposition rate, simple nest architecture, a lack of spatial association of tasks, and the potential for attaining reproductive status appear to be associated with the lack of age-related behavioral specialization in Z. angusticollis.In effect, the absence of temporal polyethism in this species is likely a consequence of its nesting habits and physiological and developmental constraints.     

Five Entry Points of the Mitochondrially Encoded Subunits in Mammalian Complex I Assembly

Complex I (CI) is the largest enzyme of the mammalian mitochondrial respiratory chain. The biogenesis of the complex is a very complex process due to its large size and number of subunits (45 subunits). The situation is further complicated due to the fact that its subunits have a double genomic origin, as seven of them are encoded by the mitochondrial DNA. Understanding of the assembly process and characterization of the involved factors has advanced very much in the last years. However, until now, a key part of the process, that is, how and at which step the mitochondrially encoded CI subunits (ND subunits) are incorporated in the CI assembly process, was not known. Analyses of several mouse cell lines mutated for three ND subunits allowed us to determine the importance of each one for complex assembly/stability and that there are five different steps within the assembly pathway in which some mitochondrially encoded CI subunit is incorporated.Complex I (CI) (NADH-ubiquinone oxidoreductase; EC 1.6.5.3) is one of the main electron entry points in the mitochondrial respiratory electron transport chain catalyzing the oxidation of NADH to reduce ubiquinone to ubiquinol (31, 39, 40), contributing to the proton motive force to synthesize ATP by the process called oxidative phosphorylation (OXPHOS).CI assembly is a difficult problem to address due to the large size of the complex and its dual genomic nature, as 7 out of its 45 subunits are encoded by the mitochondrial DNA (mtDNA) (10, 11). Until very recently, mammalian CI assembly was explained using two different and apparently contradictory models. One model was proposed by following the time course of formation of CI intermediates in human cells in culture once mitochondrial protein synthesis had recovered after its inhibition by doxycycline (36). Based on these observations, human CI was proposed to be assembled through two different modules corresponding to the membrane and peripheral arms. The other model was proposed after analysis of a cohort of four CI-deficient patients in which seven putative assembly intermediates containing a combination of both peripheral- and membrane arm subunits were identified. Thus, an assembly pathway in which the peripheral- and membrane arm subassemblies came together before the completion of each of the arms was proposed (4). However, the most recent studies have refined the previous models and propose an overlapping view of the process. One study, by green fluorescent protein (GFP) tagging of the NDUFS3 subunit, identified six peripheral-arm intermediates. The second and third smaller NDUFS3-containing subassemblies were accumulated and could not advance into higher-molecular-mass species when mitochondrial protein synthesis was inhibited, thus determining the entry point of the mitochondrially encoded subunits in the CI assembly pathway (37). The most recent study analyzed the incorporation of the mitochondrial subunits in a time course to the fully assembled CI and, on the other hand, the incorporation of the nuclear subunits by importing them into isolated mitochondria (24). Although these two models differ in the order in which some subunits are incorporated, they agree on the general human CI assembly pathway, which takes place via evolutionarily conserved modular subassemblies (14, 25, 28, 37).However, the specific entry points of all the mtDNA-encoded CI subunits (ND subunits) in the CI assembly pathway and their roles in the stability of the complex remained to be clarified. Structural studies related to mutations in the ND subunits in pathological cases have given some hints as to the importance of each of them for CI assembly/stability. In this case, defects in specific ND subunits do not have the same effect: ND1, ND4, and ND6 seem to be fundamental to CI assembly, while ND3 and ND5 are important for its activity but not for assembly. On the other hand, mutations in ND2 alter CI assembly, with abnormal intermediate accumulation (19).In this article, we present new insights into the roles of the ND subunits by using mouse cells deficient for ND4, ND6, and a combination of ND6 and ND5. This study has allowed us to propose the five different entry points by which the mtDNA-encoded subunits are sequentially incorporated into the CI assembly pathway, completing the current view of the process. We conclude that ND4 and ND6 are required for the proper function and assembly of CI, although at different degrees due to their different entry points and roles in the CI assembly pathway.     

Structure and Function Analysis of Therapeutic Monoclonal Antibodies against Dengue Virus Type 2

Dengue virus (DENV) is the most prevalent insect-transmitted viral disease in humans globally, and currently no specific therapy or vaccine is available. Protection against DENV and other related flaviviruses is associated with the development of antibodies against the viral envelope (E) protein. Although prior studies have characterized the neutralizing activity of monoclonal antibodies (MAbs) against DENV type 2 (DENV-2), none have compared simultaneously the inhibitory activity against a genetically diverse range of strains in vitro, the protective capacity in animals, and the localization of epitopes. Here, with the goal of identifying MAbs that can serve as postexposure therapy, we investigated in detail the functional activity of a large panel of new anti-DENV-2 mouse MAbs. Binding sites were mapped by yeast surface display and neutralization escape, cell culture inhibition assays were performed with homologous and heterologous strains, and prophylactic and therapeutic activity was evaluated with two mouse models. Protective MAbs localized to epitopes on the lateral ridge of domain I (DI), the dimer interface, lateral ridge, and fusion loop of DII, and the lateral ridge, C-C′ loop, and A strand of DIII. Several MAbs inefficiently inhibited at least one DENV-2 strain of a distinct genotype, suggesting that recognition of neutralizing epitopes varies with strain diversity. Moreover, antibody potency generally correlated with a narrowed genotype and serotype specificity. Five MAbs functioned efficiently as postexposure therapy when administered as a single dose, even 3 days after intracranial infection of BALB/c mice. Overall, these studies define the structural and functional complexity of antibodies against DENV-2 with protective potential.Dengue virus (DENV), a member of the Flaviviridae family of RNA viruses, is related to several other human pathogens of global concern, including yellow fever and tick-borne, West Nile, and Japanese encephalitis viruses. DENV infection in humans occurs after Aedes aegypti or Aedes albopictus mosquito inoculation and results in clinical disease, ranging from a febrile illness (dengue fever [DF]) to a life-threatening hemorrhagic and capillary leak syndrome (dengue hemorrhagic fever [DHF]/dengue shock syndrome [DSS]). Globally, there is significant diversity among DENV strains, including four distinct serotypes (DENV type 1 [DENV-1], DENV-2, DENV-3, and DENV-4) that differ at the amino acid level by 25 to 40%. Additional complexity occurs within each serotype, as genotypes vary from one another by up to 3% at the amino acid level (21, 49). No approved antiviral treatment is currently available, and several candidate tetravalent vaccines remain in clinical development (reviewed in reference 11). Because of the increased geographic range of its mosquito vectors, urbanization, and international travel, DENV continues to spread worldwide and now causes an estimated 50 to 100 million infections and 250,000 to 500,000 cases of DHF/DSS per year, with 2.5 billion people at risk (68).DENV is an enveloped icosahedral virus with a single-stranded, positive-polarity RNA genome. The 10.7-kb genome is translated as a single polyprotein, which is cleaved into three structural proteins (capsid [C], premembrane/membrane [prM/M], and envelope [E]) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by host and viral proteases. The mature DENV virion is ∼500 Å in diameter, with a highly organized outer protein shell, a 50-Å lipid membrane bilayer, and a nucleocapsid core (26). Mature DENV virions are covered by 90 anti-parallel E protein homodimers, arranged flat along the surface with quasi-icosahedral symmetry. The immature virion, which lacks cleavage of the prM protein, has a rough surface with 60 spikes each composed of three prM-E heterodimers (7, 73). Exposure to mildly acidic conditions in the trans-Golgi network promotes virus maturation through a structural rearrangement of the flavivirus E proteins and cleavage of prM to M by a furin-like protease (29, 66, 69, 70). The ectodomain of DENV E protein is comprised of three discrete domains (34-36, 39). Domain I (DI) is a central, eight-stranded β-barrel, which contains a single N-linked glycan in most DENV strains. DII is a long, finger-like protrusion from DI, with the highly conserved fusion peptide at its distal end and a second N-linked glycan that recognizes DC-SIGN (37, 38, 46, 59). DIII, which adopts an immunoglobulin-like fold, has been suggested to contain cell surface receptor recognition sites (5, 64, 71). Several groups have recently defined contact residues for type-specific, subcomplex-specific, and cross-reactive monoclonal antibodies (MAbs) that recognize DIII of DENV-2 (16, 17, 31, 47, 57, 61). Type-specific MAbs with neutralizing activity against DENV-2 localized to the BC, DE, and FG loops on the lateral ridge of DIII, whereas subcomplex-specific MAbs recognized an adjacent epitope centered on the connecting A strand of DIII at residues K305, K307, and K310.To date, no study has compared the in vitro inhibitory activity of MAbs in cells against a genetically diverse range of DENV-2 strains and their protective capacity in animals. Here, we had the goal of generating strongly neutralizing MAbs that would recognize virtually all DENV-2 strains and function as a possible postexposure therapy. Twenty-four new anti-DENV-2 mouse MAbs were generated with moderate or strong neutralizing activity against the homologous virus in cell culture assays. Binding sites were mapped for the majority of these by yeast surface display, identifying distinct epitopes in regions in DI (lateral ridge), DII (dimer interface, lateral ridge, and fusion loop), and DIII (lateral ridge, C-C′ loop, and A strand). Several MAbs failed to neutralize efficiently at least one DENV-2 strain of a distinct genotype, suggesting that antibody recognition of neutralizing epitopes varies among DENV-2 genotypes.To begin to assess the utility of this new panel of inhibitory MAbs as possible therapeutics against DENV-2, we evaluated their protective capacity in a stringent intracranial challenge model in BALB/c mice. Among the 16 neutralizing MAbs tested in mice, most were protective when given as prophylaxis. Seven of these had postexposure therapeutic activity when administered as a single dose by intraperitoneal route even 3 days after intracranial infection. For the MAbs with the greatest therapeutic potential, protection was confirmed with an antibody-enhanced vascular leakage mouse model (2, 72) of DENV-2 infection.     

Accumulation of Poly (3-Hydroxybutyric Acid) by Some Soil Streptomyces

In a limited-scale survey, 55 soil streptomycetes were screened for the accumulation of poly (3-hydroxybutyrate) [PHB]. Only 18% of the isolates accumulated PHB ranging between 1.9–7.8% of the dry biomass. The promising isolate DBCC-719, identified as Streptomyces griseorubiginosus, accumulated PHB amounting to 9.5% of the mycelial dry mass in the early stationary phase when grown in chemically defined medium with 2% (wt/vol) glucose as the sole source of carbon. Nitrogen-limiting conditions were inhibitory to growth and PHB accumulation. The isolated polymer was highly soluble in chloroform, gave a sharp peak at 235 nm on digestion with concentrated H₂SO₄, and had a characteristic infrared spectrum. Received: 26 March 1999 / Accepted: 3 May 1999     

Amplification mechanisms of inflammation: paracrine stimulation of arachidonic acid mobilization by secreted phospholipase A2 is regulated by cytosolic phospholipase A2-derived hydroperoxyeicosatetraenoic acid

In macrophages and other major immunoinflammatory cells, two phospholipase A(2) (PLA(2)) enzymes act in concert to mobilize arachidonic acid (AA) for immediate PG synthesis, namely group IV cytosolic phospholipase A(2) (cPLA(2)) and a secreted phospholipase A(2) (sPLA(2)). In this study, the molecular mechanism underlying cross-talk between the two PLA(2)s during paracrine signaling has been investigated. U937 macrophage-like cells respond to Con A by releasing AA in a cPLA(2)-dependent manner, and addition of exogenous group V sPLA(2) to the activated cells increases the release. This sPLA(2) effect is abolished if the cells are pretreated with cPLA(2) inhibitors, but is restored by adding exogenous free AA. Inhibitors of cyclooxygenase and 5-lipoxygenase have no effect on the response to sPLA(2). In contrast, ebselen strongly blocks it. Reconstitution experiments conducted in pyrrophenone-treated cells to abolish cPLA(2) activity reveal that 12- and 15-hydroperoxyeicosatetraenoic acid (HPETE) are able to restore the sPLA(2) response to levels found in cells displaying normal cPLA(2) activity. Moreover, 12- and 15-HPETE are able to enhance sPLA(2) activity in vitro, using a natural membrane assay. Neither of these effects is mimicked by 12- or 15-hydroxyeicosatetraenoic acid, indicating that the hydroperoxy group of HPETE is responsible for its biological activity. Collectively, these results establish a role for 12/15-HPETE as an endogenous activator of sPLA(2)-mediated phospholipolysis during paracrine stimulation of macrophages and identify the mechanism that connects sPLA(2) with cPLA(2) for a full AA mobilization response.     

Bromoenol lactone promotes cell death by a mechanism involving phosphatidate phosphohydrolase-1 rather than calcium-independent phospholipase A2

Originally described as a serine protease inhibitor, bromoenol lactone (BEL) has recently been found to potently inhibit Group VI calcium-independent phospholipase A2 (iPLA2). Thus, BEL is widely used to define biological roles of iPLA2 in cells. However, BEL is also known to inhibit another key enzyme of phospholipid metabolism, namely the magnesium-dependent phosphatidate phosphohydrolase-1 (PAP-1). In this work we report that BEL is able to promote apoptosis in a variety of cell lines, including U937, THP-1, and MonoMac (human phagocyte), RAW264.7 (murine macrophage), Jurkat (human T lymphocyte), and GH3 (human pituitary). In these cells, long term treatment with BEL (up to 24 h) results in increased annexin-V binding to the cell surface and nuclear DNA damage, as detected by staining with both DAPI and propidium iodide. At earlier times (2 h), BEL induces the proteolysis of procaspase-9 and procaspase-3 and increases cleavage of poly(ADP-ribose) polymerase. These changes are preceded by variations in the mitochondrial membrane potential. All these effects of BEL are not mimicked by the iPLA2 inhibitor methylarachidonyl fluorophosphonate or by treating the cells with a specific iPLA2 antisense oligonucleotide. However, propranolol, a PAP-1 inhibitor, is able to reproduce these effects, suggesting that it is the inhibition of PAP-1 and not of iPLA2 that is involved in BEL-induced cell death. In support of this view, BEL-induced apoptosis is accompanied by a very strong inhibition of PAP-1-regulated events, such as incorporation of [3H]choline into phospholipids and de novo incorporation of [3H]arachidonic acid into triacylglycerol. Collectively, these results stress the role of PAP-1 as a key enzyme for cell integrity and survival and in turn caution against the use of BEL in studies involving long incubation times, due to the capacity of this drug to induce apoptosis in a variety of cells.     

BFA-sensitive and insensitive exocytic pathways in Entamoeba histolytica trophozoites: their relationship to pathogenesis

Entamoeba histolytica manifests its pathogenicity through several cellular processes triggered by external stimuli that activate signal transduction pathways. The intense secretory activity resulting from stimulation is not correlated with a typical endoplasmic reticulum (ER) or Golgi organization, and little is known in this parasite about endocytic/exocytic pathways. The interactions of trophozoites with fibronectin (FN) and cultured mammalian cells, which elicit secretory activities, were chosen to study mechanisms that regulate cytoplamic traffic. Results showed that Brefeldin A (BFA) induced redistribution of the vesicular network recognized by antibodies against amoebic proteins PDI and ERD2. Furthermore, BFA diminished traffic to the plasma membrane of the beta1 integrin-like FN receptor and the heavy subunit of the Gal/GalNAc lectin, required for adhesion to FN and target cells, respectively. However, BFA did not prevent thiol-proteinase secretion or inhibit the traffic of de novo synthesized proteinases. These data suggest that two distinct transport systems occur in E. histolytica, one similar to classical membrane protein transport and another independent of BFA and inducible by external stimuli. Actin-myosin contractility of the cortical cytoskeleton seems necessary for the final release of exported proteinases and the proper function of the surface proteins involved in adhesion.