Differential regulation of cell death programs in males and females by Poly (ADP-Ribose) Polymerase-1 and 17 β estradiol

Cell death can be divided into the anti-inflammatory process of apoptosis and the pro-inflammatory process of necrosis. Necrosis, as apoptosis, is a regulated form of cell death, and Poly-(ADP-Ribose) Polymerase-1 (PARP-1) and Receptor-Interacting Protein (RIP) 1/3 are major mediators. We previously showed that absence or inhibition of PARP-1 protects mice from nephritis, however only the male mice. We therefore hypothesized that there is an inherent difference in the cell death program between the sexes. We show here that in an immune-mediated nephritis model, female mice show increased apoptosis compared to male mice. Treatment of the male mice with estrogens induced apoptosis to levels similar to that in female mice and inhibited necrosis. Although PARP-1 was activated in both male and female mice, PARP-1 inhibition reduced necrosis only in the male mice. We also show that deletion of RIP-3 did not have a sex bias. We demonstrate here that male and female mice are prone to different types of cell death. Our data also suggest that estrogens and PARP-1 are two of the mediators of the sex-bias in cell death. We therefore propose that targeting cell death based on sex will lead to tailored and better treatments for each gender.     

Aromatic Prenylation in Phenazine Biosynthesis: DIHYDROPHENAZINE-1-CARBOXYLATE DIMETHYLALLYLTRANSFERASE FROM STREPTOMYCES ANULATUS*S?

The bacterium Streptomyces anulatus 9663, isolated from the intestine of different arthropods, produces prenylated derivatives of phenazine 1-carboxylic acid. From this organism, we have identified the prenyltransferase gene ppzP. ppzP resides in a gene cluster containing orthologs of all genes known to be involved in phenazine 1-carboxylic acid biosynthesis in Pseudomonas strains as well as genes for the six enzymes required to generate dimethylallyl diphosphate via the mevalonate pathway. This is the first complete gene cluster of a phenazine natural compound from streptomycetes. Heterologous expression of this cluster in Streptomyces coelicolor M512 resulted in the formation of prenylated derivatives of phenazine 1-carboxylic acid. After inactivation of ppzP, only nonprenylated phenazine 1-carboxylic acid was formed. Cloning, overexpression, and purification of PpzP resulted in a 37-kDa soluble protein, which was identified as a 5,10-dihydrophenazine 1-carboxylate dimethylallyltransferase, forming a C–C bond between C-1 of the isoprenoid substrate and C-9 of the aromatic substrate. In contrast to many other prenyltransferases, the reaction of PpzP is independent of the presence of magnesium or other divalent cations. The K_m value for dimethylallyl diphosphate was determined as 116 μm. For dihydro-PCA, half-maximal velocity was observed at 35 μm. K_cat was calculated as 0.435 s^-1. PpzP shows obvious sequence similarity to a recently discovered family of prenyltransferases with aromatic substrates, the ABBA prenyltransferases. The present finding extends the substrate range of this family, previously limited to phenolic compounds, to include also phenazine derivatives.The transfer of isoprenyl moieties to aromatic acceptor molecules gives rise to an astounding diversity of secondary metabolites in bacteria, fungi, and plants, including many compounds that are important in pharmacotherapy. However, surprisingly little biochemical and genetic data are available on the enzymes catalyzing the C-prenylation of aromatic substrates. Recently, a new family of aromatic prenyltransferases was discovered in streptomycetes (1), Gram-positive soil bacteria that are prolific producers of antibiotics and other biologically active compounds (2). The members of this enzyme family show a new type of protein fold with a unique α-β-β-α architecture (3) and were therefore termed ABBA prenyltransferases (1). Only 13 members of this family can be identified by sequence similarity searches in the data base at present, and only four of them have been investigated biochemically (3–6). Up to now, only phenolic compounds have been identified as aromatic substrates of ABBA prenyltransferases. We now report the discovery of a new member of the ABBA prenyltransferase family, catalyzing the transfer of a dimethylallyl moiety to C-9 of 5,10-dihydrophenazine 1-carboxylate (dihydro-PCA).² Streptomyces strains produce many of prenylated phenazines as natural products. For the first time, the present paper reports the identification of a prenyltransferase involved in their biosynthesis.Streptomyces anulatus 9663, isolated from the intestine of different arthropods, produces several prenylated phenazines, among them endophenazine A and B (Fig. 1A) (7). We wanted to investigate which type of prenyltransferase might catalyze the prenylation reaction in endophenazine biosynthesis. In streptomycetes and other microorganisms, genes involved in the biosynthesis of a secondary metabolite are nearly always clustered in a contiguous DNA region. Therefore, the prenyltransferase of endophenazine biosynthesis was expected to be localized in the vicinity of the genes for the biosynthesis of the phenazine core (i.e. of PCA).Open in a separate windowFIGURE 1.A, prenylated phenazines from S. anulatus 9663. B, biosynthetic gene cluster of endophenazine A.In Pseudomonas, an operon of seven genes named phzABCDEFG is responsible for the biosynthesis of PCA (8). The enzyme PhzC catalyzes the condensation of phosphoenolpyruvate and erythrose-4-phosphate (i.e. the first step of the shikimate pathway), and further enzymes of this pathway lead to the intermediate chorismate. PhzD and PhzE catalyze the conversion of chorismate to 2-amino-2-deoxyisochorismate and the subsequent conversion to 2,3-dihydro-3-hydroxyanthranilic acid, respectively. These reactions are well established biochemically. Fewer data are available about the following steps (i.e. dimerization of 2,3-dihydro-3-hydroxyanthranilic acid, several oxidation reactions, and a decarboxylation, ultimately leading to PCA via several instable intermediates). From Pseudomonas, experimental data on the role of PhzF and PhzA/B have been published (8, 9), whereas the role of PhzG is yet unclear. Surprisingly, the only gene cluster for phenazine biosynthesis described so far from streptomycetes (10) was found not to contain a phzF orthologue, raising the question of whether there may be differences in the biosynthesis of phenazines between Pseudomonas and Streptomyces.Screening of a genomic library of the endophenazine producer strain S. anulatus now allowed the identification of the first complete gene cluster of a prenylated phenazine, including the structural gene of dihydro-PCA dimethylallyltransferase.     

Insulin Resistance in Striated Muscle-specific Integrin Receptor ��1-deficient Mice

Integrin receptor plays key roles in mediating both inside-out and outside-in signaling between cells and the extracellular matrix. We have observed that the tissue-specific loss of the integrin β1 subunit in striated muscle results in a near complete loss of integrin β1 subunit protein expression concomitant with a loss of talin and to a lesser extent, a reduction in F-actin content. Muscle-specific integrin β1-deficient mice had no significant difference in food intake, weight gain, fasting glucose, and insulin levels with their littermate controls. However, dynamic analysis of glucose homeostasis using euglycemichyperinsulinemic clamps demonstrated a 44 and 48% reduction of insulin-stimulated glucose infusion rate and glucose clearance, respectively. The whole body insulin resistance resulted from a specific inhibition of skeletal muscle glucose uptake and glycogen synthesis without any significant effect on the insulin suppression of hepatic glucose output or insulin-stimulated glucose uptake in adipose tissue. The reduction in skeletal muscle insulin responsiveness occurred without any change in GLUT4 protein expression levels but was associated with an impairment of the insulin-stimulated protein kinase B/Akt serine 473 phosphorylation but not threonine 308. The inhibition of insulin-stimulated serine 473 phosphorylation occurred concomitantly with a decrease in integrin-linked kinase expression but with no change in the mTOR·Rictor·LST8 complex (mTORC2). These data demonstrate an in vivo crucial role of integrin β1 signaling events in mediating cross-talk to that of insulin action.Integrin receptors are a large family of integral membrane proteins composed of a single α and β subunit assembled into a heterodimeric complex. There are 19 α and 8 β mammalian subunit isoforms that combine to form 25 distinct α,β heterodimeric receptors (1-5). These receptors play multiple critical roles in conveying extracellular signals to intracellular responses (outside-in signaling) as well as altering extracellular matrix interactions based upon intracellular changes (inside-out signaling). Despite the large overall number of integrin receptor complexes, skeletal muscle integrin receptors are limited to seven α subunit subtypes (α1, α3, α4, α5, α6, α7, and αν subunits), all associated with the β1 integrin subunit (6, 7).Several studies have suggested an important cross-talk between extracellular matrix and insulin signaling. For example, engagement of β1 subunit containing integrin receptors was observed to increase insulin-stimulated insulin receptor substrate (IRS)² phosphorylation, IRS-associated phosphatidylinositol 3-kinase, and activation of protein kinase B/Akt (8-11). Integrin receptor regulation of focal adhesion kinase was reported to modulate insulin stimulation of glycogen synthesis, glucose transport, and cytoskeleton organization in cultured hepatocytes and myoblasts (12, 13). Similarly, the integrin-linked kinase (ILK) was suggested to function as one of several potential upstream kinases that phosphorylate and activate Akt (14-18). In this regard small interfering RNA gene silencing of ILK in fibroblasts and conditional ILK gene knockouts in macrophages resulted in a near complete inhibition of insulin-stimulated Akt serine 473 (Ser-473) phosphorylation concomitant with an inhibition of Akt activity and phosphorylation of Akt downstream targets (19). However, a complex composed of mTOR·Rictor·LST8 (termed mTORC2) has been identified in several other studies as the Akt Ser-473 kinase (20, 21). In addition to Ser-473, Akt protein kinase activation also requires phosphorylation on threonine 308 Thr-30 by phosphoinositide-dependent protein kinase, PDK1 (22-24).In vivo, skeletal muscle is the primary tissue responsible for postprandial (insulin-stimulated) glucose disposal that results from the activation of signaling pathways leading to the translocation of the insulin-responsive glucose transporter, GLUT4, from intracellular sites to the cell surface membranes (25, 26). Dysregulation of any step of this process in skeletal muscle results in a state of insulin resistance, thereby predisposing an individual for the development of diabetes (27-33). Although studies described above have utilized a variety of tissue culture cell systems to address the potential involvement of integrin receptor signaling in insulin action, to date there has not been any investigation of integrin function on insulin action or glucose homeostasis in vivo. To address this issue, we have taken advantage of Cre-LoxP technology to inactivate the β1 integrin receptor subunit gene in striated muscle. We have observed that muscle creatine kinase-specific integrin β1 knock-out (MCKItgβ1 KO) mice display a reduction of insulin-stimulated glucose infusion rate and glucose clearance. The impairment of insulin-stimulated skeletal muscle glucose uptake and glycogen synthesis resulted from a decrease in Akt Ser-473 phosphorylation concomitant with a marked reduction in ILK expression. Together, these data demonstrate an important cross-talk between integrin receptor function and insulin action and suggests that ILK may function as an Akt Ser-473 kinase in skeletal muscle.     

Epitope-tagged Receptor Knock-in Mice Reveal That Differential Desensitization of ��2-Adrenergic Responses Is because of Ligand-selective Internalization

Although ligand-selective regulation of G protein-coupled receptor-mediated signaling and trafficking are well documented, little is known about whether ligand-selective effects occur on endogenous receptors or whether such effects modify the signaling response in physiologically relevant cells. Using a gene targeting approach, we generated a knock-in mouse line, in which N-terminal hemagglutinin epitope-tagged α_2A-adrenergic receptor (AR) expression was driven by the endogenous mouse α_2AAR gene locus. Exploiting this mouse line, we evaluated α_2AAR trafficking and α_2AAR-mediated inhibition of Ca²⁺ currents in native sympathetic neurons in response to clonidine and guanfacine, two drugs used for treatment of hypertension, attention deficit and hyperactivity disorder, and enhancement of analgesia through actions on the α_2AAR subtype. We discovered a more rapid desensitization of Ca²⁺ current suppression by clonidine than guanfacine, which paralleled a more marked receptor phosphorylation and endocytosis of α_2AAR evoked by clonidine than by guanfacine. Clonidine-induced α_2AAR desensitization, but not receptor phosphorylation, was attenuated by blockade of endocytosis with concanavalin A, indicating a critical role for internalization of α_2AAR in desensitization to this ligand. Our data on endogenous receptor-mediated signaling and trafficking in native cells reveal not only differential regulation of G protein-coupled receptor endocytosis by different ligands, but also a differential contribution of receptor endocytosis to signaling desensitization. Taken together, our data suggest that these HA-α_2AAR knock-in mice will serve as an important model in developing ligands to favor endocytosis or nonendocytosis of receptors, depending on the target cell and pathophysiology being addressed.G protein-coupled receptors (GPCRs)⁴ represent the largest family of cell surface receptors mediating responses to hormones, cytokines, neurotransmitters, and therapeutic agents (1). In addition to regulating downstream signaling, ligand binding to a receptor can initiate phosphorylation of the active conformation of the receptor by G protein receptor kinases (GRKs) and subsequent binding of arrestins, thus restricting the magnitude and duration of the ligand-evoked signaling responses (2, 3). Binding of arrestins to GPCRs also leads to GPCR internalization (4, 5), a process that has been proposed as a means to desensitize receptor signaling at the cell surface, resensitize receptors, and/or initiate intracellular signaling (6, 7).Different ligands are able to induce distinct signaling and internalization profiles of the same receptor (8-14). However, the lack of available tools to study trafficking of endogenous GPCRs in native target cells has limited our understanding of ligand-selective endocytosis profiles and the relative contribution of receptor endocytosis to desensitization in native biological settings.To specifically test hypotheses regarding ligand-selective effects on GPCR internalization, and functional consequences of this trafficking on signaling, we utilized a homologous recombination gene targeting strategy to introduce a hemagglutinin (HA) epitope-tagged wild type α_2A-adrenergic receptor (AR) into the mouse ADRA2A gene locus (“knock-in”). The α_2AAR is a prototypical GPCR that couples to the G_i/o subfamily of G proteins (15). Studies on genetically engineered mice made null or mutant for the α_2AAR have revealed that this subtype mediates the therapeutic effects of α₂-adrenergic agents on blood pressure, pain perception, volatile anesthetic sparing, analgesia, and working memory enhancement (16-18). Two classic α₂-ligands, clonidine and guanfacine, have been widely used to treat hypertension (19), attention deficit and hyperactivity disorder (20), and to elicit analgesia (19, 21) mediated via the α_2AAR. Clinically guanfacine has a much longer duration of action than clonidine (22-24); this longer duration of action cannot be accounted for by the pharmacokinetic profile of these agents in human beings, as both drugs have a half-life of 12-14 h (25, 26). Because ligand-induced desensitization and trafficking of GPCRs have been implicated as critical mechanisms for modulating response duration in vivo (3), one hypothesis underlying the difference in duration between clonidine and guanfacine is that clonidine provokes accelerated desensitization of the α_2AAR via one or several mechanisms, whereas guanfacine does not. Signaling desensitization in response to these two agonists has not been compared under the same experimental settings. To specifically test this hypothesis, we have exploited our HA-α_2AAR knock-in mice so that we could examine these properties of guanfacine and clonidine in native target cells.We compared internalization of the α_2AAR and inhibition of Ca²⁺ currents induced by clonidine and guanfacine in primary superior cervical ganglia (SCG) neurons, where the α_2AAR is the major adrenergic receptor subtype controlling norepinephrine release and sympathetic tone (17, 27). Our data revealed a differential regulation of α_2AAR trafficking and signaling duration by clonidine versus guanfacine, i.e. clonidine induced a more dramatic desensitization of the α_2AAR than guanfacine, and this desensitization was largely because of α_2AAR internalization. These studies reveal the powerful tool that the HA-α_2AAR knock-in mice provide for identifying endocytosis-dependent and -independent physiological phenomena for this receptor subtype as a first step in defining novel loci for therapeutic intervention in the α_2AAR signaling/trafficking cascade.     

Construction of three new Gateway? expression plasmids for Trypanosoma cruzi

We present here three expression plasmids for Trypanosoma cruzi adapted to the Gateway^® recombination cloning system. Two of these plasmids were designed to express trypanosomal proteins fused to a double tag for tandem affinity purification (TAPtag). The TAPtag and Gateway^® cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid. Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy. The third Gateway adapted vector assayed was the inducible pTcINDEX. When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX).     

Copper��Dopamine Complex Induces Mitochondrial Autophagy Preceding Caspase-independent Apoptotic Cell Death

Parkinsonism is one of the major neurological symptoms in Wilson disease, and young workers who worked in the copper smelting industry also developed Parkinsonism. We have reported the specific neurotoxic action of copper·dopamine complex in neurons with dopamine uptake. Copper·dopamine complex (100 μm) induces cell death in RCSN-3 cells by disrupting the cellular redox state, as demonstrated by a 1.9-fold increase in oxidized glutathione levels and a 56% cell death inhibition in the presence of 500 μm ascorbic acid; disruption of mitochondrial membrane potential with a spherical shape and well preserved morphology determined by transmission electron microscopy; inhibition (72%, p < 0.001) of phosphatidylserine externalization with 5 μm cyclosporine A; lack of caspase-3 activation; formation of autophagic vacuoles containing mitochondria after 2 h; transfection of cells with green fluorescent protein-light chain 3 plasmid showing that 68% of cells presented autophagosome vacuoles; colocalization of positive staining for green fluorescent protein-light chain 3 and Rhod-2AM, a selective indicator of mitochondrial calcium; and DNA laddering after 12-h incubation. These results suggest that the copper·dopamine complex induces mitochondrial autophagy followed by caspase-3-independent apoptotic cell death. However, a different cell death mechanism was observed when 100 μm copper·dopamine complex was incubated in the presence of 100 μm dicoumarol, an inhibitor of NAD(P)H quinone:oxidoreductase (EC 1.6.99.2, also known as DT-diaphorase and NQ01), because a more extensive and rapid cell death was observed. In addition, cyclosporine A had no effect on phosphatidylserine externalization, significant portions of compact chromatin were observed within a vacuolated nuclear membrane, DNA laddering was less pronounced, the mitochondria morphology was more affected, and the number of cells with autophagic vacuoles was a near 4-fold less.A possible role of copper in the neurodegeneration of dopaminergic neurons is supported by the fact that patients with neurological Wilson disease, a copper deposition disorder, display a number of extrapyramidal motor symptoms, including Parkinsonism. The cerebral manifestations in neurological Wilson disease are expressed as bradykinesia, rigidity, tremor, dyskinesia, and dysarthria (1). It has been proposed that neurological Wilson disease can be assigned to the group of secondary Parkinsonian syndromes (2). Interestingly, young workers who worked in the copper smelting industry also developed Parkinsonism (3).Studies performed in rats showed copper (Cu²⁺)-induced degeneration of dopaminergic neurons in the nigrostriatal system. Likewise, it was described that copper neurotoxicity in rat substantia nigra and striatum is dependent on NAD(P)H dehydrogenase inhibition (4, 5). All of these results support a possible role for copper in the neurodegeneration of dopaminergic neurons.The general mechanism of toxicity, induced by the reduced form of copper (Cu⁺) catalyzing the formation of hydroxyl radicals in the presence of hydrogen peroxide through the Fenton reaction, cannot explain the specific degeneration of dopaminergic neurons in Parkinsonism induced in neurological Wilson disease, or in miners working in the copper smelting industry. The selective action of copper can be explained by the ability of copper to form a complex with dopamine, allowing this metal to be transported by cells that have the ability to take up dopamine (6). This specific neurotoxic action of copper in neurons with dopamine uptake is dependent on (i) the ability of copper to form a complex with dopamine (Cu·DA)² (6, 7), (ii) uptake of Cu·DA complex by dopamine transporters, (iii) oxidation of dopamine to aminochrome, and (iv) one-electron reduction of aminochrome by inhibiting NAD(P)H dehydrogenase (6). These findings may explain the selective neurotoxic action of copper in the brain, but they do not explain the cell death mechanism.Currently, cell death is divided into three categories: apoptosis, autophagy, and necrosis. At the current time, only apoptosis and autophagic cell death are generally accepted as being legitimate forms of programmed cell death. Alternative models of cell death have therefore been proposed, including para-apoptosis, mitotic catastrophe, oncosis, and pyroptosis (8–12). Necrosis is characterized mostly by the absence of caspase activation, cytochrome c release, and DNA oligonucleosomal fragmentation. Apoptotic cells are characterized by the formation of blebs, chromatin condensation, DNA oligonucleosomal fragmentation, and exposure of phosphatidylserine on the external membrane. This mode of cell death can be dependent or independent of activation of caspases (13). On the other hand, autophagy can be distinguished from apoptosis by sequestration of bulk cytoplasm and organelles in double or multimembrane autophagic vacuoles that then fuse with the lysosomal system. Some of these described mechanisms are related to neurological diseases such as Parkinson disease (14, 15). Cells can use different methods to activate their own destruction, and more than one death program may be activated at the same time (16, 17).The purpose of this study was to examine the Cu·DA complex-induced cell death mechanism in RCSN-3 cells, a cell line that expresses dopamine, norepinephrine, and serotonin transporters (18, 19).     

Revision of Sternaspis Otto, 1821 (Polychaeta,?Sternaspidae)

To the memory of William Ronald Sendall Sternaspid polychaetes are common and often abundant in soft bottoms in the world oceans. Some authors suggest that only one species should be recognized, whereas others regard a few species as widely distributed in many seas and variable depths from the low intertidal to about 4400 m. There are some problems with species delineation and the distinctive ventro-caudal shield has been disregarded or barely used for identifying species. In order to clarify these issues, the ventral shield is evaluated in specimens from the same locality and its diagnostic potential is confirmed. On this basis, a revision of Sternaspis Otto, 1821 (Polychaeta: Sternaspidae) is presented based upon type materials, or material collected from type localities. The sternaspid body, introvert hooks and shield show three distinct patterns, two genera have seven abdominal segments and tapered introvert hooks, and one genus has eight abdominal segments and spatulate introvert hooks. The ventro-caudal shield has three different patterns: stiff with ribs, and sometimes concentric lines, stiff with feebly-defined ribs but no concentric lines, and soft with firmly adhered sediment particles. Sternaspis is restricted to include species with seven abdominal segments, falcate introvert hooks, and stiff shields, often exhibiting radial ribs, concentric lines or both. Sternaspis includes, besides the type species, Sternaspis thalassemoides Otto, 1821 from the Mediterranean Sea, Sternaspis affinis Stimpson, 1864 from the Northeastern Pacific, Sternaspis africana Augener, 1918, stat. n. from Western Africa, Sternaspis andamanensis sp. n. from the Andaman Sea, Sternaspis costata von Marenzeller, 1879 from Japan, Sternaspis fossor Stimpson, 1853 from the Northwestern Atlantic, Sternaspis islandica Malmgren, 1867 from Iceland, Sternaspis maior Chamberlin, 1919 from the Gulf of California, Sternaspis princeps Selenka, 1885 from New Zealand, Sternaspis rietschi Caullery, 1944 from abyssal depths around Indonesia, Sternaspis scutata (Ranzani, 1817) from the Mediterranean Sea, Sternaspis spinosa Sluiter, 1882 from Indonesia, and Sternaspis thorsoni sp. n. from the Iranian Gulf. Two genera are newly proposed to incorporate the remaining species: Caulleryaspis and Petersenaspis. Caulleryaspis gen. n. is defined by the presence of falcate introvert hooks, seven abdominal segments, and soft shields with sediment particles firmly adhered on them; it includes two species: Caulleryaspis gudmundssoni sp. n. from Iceland and Caulleryaspis laevis (Caullery, 1944) comb. n. from Indonesia. Petersenaspis gen. n. is defined by the presence of spatulate introvert hooks, eight abdominal segments, and stiff shields with poorly defined ribs but no concentric line; it includes Petersenaspis capillata (Nonato, 1966) from Brazil and Petersenaspis palpallatoci sp. n. from the Philippines. Neotypes are proposed for eight species: Sternaspis thalassemoides, Sternaspis affinis, Sternaspis africana, Sternaspis costata, Sternaspis fossor, Sternaspis maior, Sternaspis scutata and Sternaspis spinosa, to stabilize these species-group names, and a lectotype is designated for Sternaspis laevis which is transferred to Caulleryaspis gen. n. The geographic range of most species appears to be much smaller than previously indicated, and for some species additional material in good condition is needed to clarify their distributions. Keys to genera and to all species are also included.     

Effects of Spontaneous Bilayer Curvature on Influenza Virus–mediated Fusion Pores

Cells expressing the hemagglutinin protein of influenza virus were fused to planar bilayer membranes containing the fluorescent lipid probes octadecylrhodamine (R18) or indocarbocyanine (DiI) to investigate whether spontaneous curvature of each monolayer of a target membrane affects the growth of fusion pores. R18 and DiI lowered the transition temperatures for formation of an inverted hexagonal phase, indicating that these probes facilitate the formation of negative curvature structures. The probes are known to translocate from one monolayer of a bilayer membrane to the other in a voltage-dependent manner. The spontaneous curvature of the cis monolayer (facing the cells) or the trans monolayer could therefore be made more negative through control of the polarity of voltage across the planar membrane. Electrical admittance measurements showed that the open times of flickering fusion pores were shorter when probes were in trans monolayers and longer when in cis monolayers compared with times when probe was symmetrically distributed. Open times were the same for probe symmetrically distributed as when probes were not present. Thus, open times were a function of the asymmetry of the spontaneous curvature between the trans and cis monolayers. Enriching the cis monolayer with a negative curvature probe reduced the probability that a small pore would fully enlarge, whereas enriching the trans monolayer promoted enlargement. Lysophosphatidylcholine has positive spontaneous curvature and does not translocate. When lysophosphatidylcholine was placed in trans leaflets of planar membranes, closing of fusion pores was rare. The effects of the negative and positive spontaneous curvature probes do not support the hypothesis that a flickering pore closes from an open state within a hemifusion diaphragm (essentially a “flat” structure). Rather, such effects support the hypothesis that the membrane surrounding the open pore forms a three-dimensional hourglass shape from which the pore flickers shut.     

Metabolism of Uridine 5′-Diphosphate-Glucose in Golgi Vesicles from Pea Stems

Uridine 5′-diphosphate-glucose (UDP-Glc) is transported into the lumen of the Golgi cisternae, where is used for polysaccharide biosynthesis. When Golgi vesicles were incubated with UDP-[³H]Glc, [³H]Glc was rapidly transferred to endogenous acceptors and UDP-Glc was undetectable in Golgi vesicles. This result indicated that a uridine-containing nucleotide was rapidly formed in the Golgi vesicles. Since little is known about the fate of the nucleotide derived from UDP-Glc, we analyzed the metabolism of the nucleotide moiety of UDP-Glc by incubating Golgi vesicles with [α-³²P]UDP-Glc, [β-³²P]UDP-Glc, and [³H]UDP-Glc and identifying the resulting products. After incubation of Golgi vesicles with these radiolabeled substrates we could detect only uridine 5′-monophosphate (UMP) and inorganic phosphate (Pi). UDP could not be detected, suggesting a rapid hydrolysis of UDP by the Golgi UDPase. The by-products of UDP hydrolysis, UMP and Pi, did not accumulate in the lumen, indicating that they were able to exit the Golgi lumen. The exit of UMP was stimulated by UDP-Glc, suggesting the presence of a putative UDP-Glc/UMP antiporter in the Golgi membrane. However, the exit of Pi was not stimulated by UDP-Glc, suggesting that the exit of Pi occurs via an independent membrane transporter.     

Acquired Antibiotic Resistance: Are We Born with It?

The rapid emergence of antibiotic resistance (AR) is a major public health concern. Recent findings on the prevalence of food-borne antibiotic-resistant (ART) commensal bacteria in ready-to-consume food products suggested that daily food consumption likely serves as a major avenue for dissemination of ART bacteria from the food chain to human hosts. To properly assess the impact of various factors, including the food chain, on AR development in hosts, it is important to determine the baseline of ART bacteria in the human gastrointestinal (GI) tract. We thus examined the gut microbiota of 16 infant subjects, from the newborn stage to 1 year of age, who fed on breast milk and/or infant formula during the early stages of development and had no prior exposure to antibiotics. Predominant bacterial populations resistant to several antibiotics and multiple resistance genes were found in the infant GI tracts within the first week of age. Several ART population transitions were also observed in the absence of antibiotic exposure and dietary changes. Representative AR gene pools including tet(M), ermB, sul2, and bla(TEM) were detected in infant subjects. Enterococcus spp., Staphylococcus spp., Klebsiella spp., Streptococcus spp., and Escherichia coli/Shigella spp. were among the identified AR gene carriers. ART bacteria were not detected in the infant formula and infant foods examined, but small numbers of skin-associated ART bacteria were found in certain breast milk samples. The data suggest that the early development of AR in the human gut microbiota is independent of infants' exposure to antibiotics but is likely impacted by exposure to maternal and environmental microbes during and after delivery and that the ART population is significantly amplified within the host even in the absence of antibiotic selective pressure.