Discovery and structural characterization of an allosteric inhibitor of bacterial cis‐prenyltransferase

Undecaprenyl pyrophosphate synthase (UPPs) is an essential enzyme in a key bacterial cell wall synthesis pathway. It catalyzes the consecutive condensations of isopentenyl pyrophosphate (IPP) groups on to a trans-farnesyl pyrophosphate (FPP) to produce a C55 isoprenoid, undecaprenyl pyrophosphate (UPP). Here we report the discovery and co-crystal structures of a drug-like UPPs inhibitor in complex with Streptococcus pneumoniae UPPs, with and without substrate FPP, at resolutions of 2.2 and 2.1 Å, respectively. The UPPs inhibitor has a low molecular weight (355 Da), but displays potent inhibition of UPP synthesis in vitro (IC₅₀ 50 nM) that translates into excellent whole cell antimicrobial activity against pathogenic strains of Streptococcal species (MIC₉₀ 0.4 µg mL⁻¹). Interestingly, the inhibitor does not compete with the substrates but rather binds at a site adjacent to the FPP binding site and interacts with the tail of the substrate. Based on the structures, an allosteric inhibition mechanism of UPPs is proposed for this inhibitor. This inhibition mechanism is supported by biochemical and biophysical experiments, and provides a basis for the development of novel antibiotics targeting Streptococcus pneumoniae.     

Effect of light exposure on in vitro germination and germ tube growth of eight species of rust fungi

The effects of light on urediniospore germination and germ tube elongation was studied with eight species of rust fungi that infect ornamental plants or row crops. Exposure of six species of fungi to cool white fluorescent light at 400 or 600 micromol s(-1) m(-2) for 24 h significantly reduced germination with largest decreases typically observed at 600 micromol s(-1) m(-2). Germination and germ tube elongation did not recover during 24 h dark incubation after 18 h exposure to fluorescent light at 600 micromol s(-1) m(-2), indicating the effects were not reversible. Germ tube elongation of all fungi was negatively affected by increased length of exposure to fluorescent light. Increased exposure to fluorescent light differentially affected germination of the fungi with Puccinia hemerocallidis, Phakopsora pachyrhizi, Pucciniastrum vaccinii and Puccinia menthae negatively affected and Puccinia sorghi, Puccinia triticina, Puccinia pelargonii-zonalis and Puccinia iridis relatively unaffected in 10 h incubation. Exposure of Ph. pachyrhizi and P. triticina urediniospores to sunlight rapidly reduced germination and germ tube elongation with no germination observed for Ph. pachyrhizi after 2.5 h. Germ tube elongation but not germination of hydrated urediniospores of Ph. pachyrhizi and P. triticina was significantly reduced compared to dry urediniospores exposed to 10 h fluorescent light followed by 24 h dark incubation. Exposure to fluorescent light (all fungi) or sunlight (two fungi) negatively affected urediniospore germ tube elongation. Differences observed in urediniospore germination between fungi suggest some species have co-evolved with their host for differing light conditions. Our data suggests exposure of urediniospores to strong light could inactivate rust fungi on plant surfaces or in the atmosphere.     

Differential Impact of Lipoxygenase 2 and Jasmonates on Natural and Stress-Induced Senescence in Arabidopsis

Jasmonic acid and related oxylipins are controversially discussed to be involved in regulating the initiation and progression of leaf senescence. To this end, we analyzed profiles of free and esterified oxylipins during natural senescence and upon induction of senescence-like phenotypes by dark treatment and flotation on sorbitol in Arabidopsis (Arabidopsis thaliana). Jasmonic acid and free 12-oxo-phytodienoic acid increased during all three processes, with the strongest increase of jasmonic acid after dark treatment. Arabidopside content only increased considerably in response to sorbitol treatment. Monogalactosyldiacylglycerols and digalactosyldiacylglycerols decreased during these treatments and aging. Lipoxygenase 2-RNA interference (RNAi) plants were generated, which constitutively produce jasmonic acid and 12-oxo-phytodienoic acid but do not exhibit accumulation during natural senescence or upon stress treatment. Chlorophyll loss during aging and upon dark incubation was not altered, suggesting that these oxylipins are not involved in these processes. In contrast, lipoxygenase 2-RNAi lines and the allene oxid synthase-deficient mutant dde2 were less sensitive to sorbitol than the wild type, indicating that oxylipins contribute to the response to sorbitol stress.Senescence is an important, highly regulated process at the end of development. Senescence is characterized by breakdown of organelles and molecules, export and transport of these nutrients to other organs/parts of the organism, and finally programmed cell death of the senescing organ.The process of senescence has been intensively studied in leaves, and morphological as well as molecular changes in senescing leaves have been described. Yellowing as a consequence of chlorophyll and chloroplast degradation is the most obvious process during natural leaf senescence. In addition, gene expression changes dramatically during senescence. Some senescence-associated genes (SAG, SEN) have been reported that are induced during this process, and several of the encoded proteins function in macromolecule degradation, detoxification and defense metabolism, or signal transduction (Gepstein et al., 2003). Based on the degradation of chloroplasts and macromolecules, leaf metabolism changes from carbon assimilation to catabolism (Lim et al., 2007).The initiation and progression of senescence is regulated by endogenous as well as exogenous factors. Among the endogenous factors, the developmental status of the organ and of the whole plant (e.g. age and progress in flowering and seed production) has a great impact on the process of senescence. Different stress factors such as pathogen attack, drought, osmotic stress, heat, cold, ozone, UV light, and shading can induce or accelerate senescence (Quirino et al., 2000). Phytohormones are very important regulators that integrate information about the developmental status and the environmental factors. Cytokinins are antagonistic signals and delay senescence. Endogenous levels of cytokinins decrease during senescence, and exogenous application and transgenic approaches, enhancing endogenous levels of these compounds, lead to delayed senescence (Gan and Amasino, 1995). In contrast, the gaseous phytohormone ethylene is known to induce and accelerate senescence (John et al., 1995). There are also several indications that abscisic acid modulates senescence (van der Graaff et al., 2006). The roles of other phytohormones/signaling compounds such as auxin, salicylic acid, and jasmonates are less clear (Lim et al., 2007).Jasmonates are oxylipin signaling molecules derived from linolenic acid. The term jasmonates comprises 12-oxo-phytodienoic acid (OPDA), jasmonic acid (JA), and derivatives such as the methyl ester and amino acid conjugates of JA. One of the first biological activities described for these compounds was the promotion of senescence in oat (Avena sativa) leaves by methyl jasmonate (MeJa) isolated from Artemisia absinthium (Ueda and Kato, 1980). Later on, the induction of senescence-like phenotypes by exogenous application of MeJa was also found in other plant species (Ueda and Kato, 1980; Weidhase et al., 1987a; He et al., 2002). On the molecular level, this senescence-promoting effect of MeJa is accompanied by chlorophyll loss and decreases in Rubisco and photosynthesis (Weidhase et al., 1987a, 1987b). In addition, expression of some senescence-up-regulated genes is also responsive to JA; examples are SEN1, SEN4, SEN5, SAG12, SAG14, and SAG15 (Park et al., 1998; Schenk et al., 2000; He et al., 2002). Due to the results described above, jasmonates have been described for decades as compounds with senescence-promoting activities, while the function of these compounds in natural senescence in planta was critically discussed (Parthier, 1990; Sembdner and Parthier, 1993; Creelman and Mullet, 1997; Wasternack, 2007; Balbi and Devoto, 2008; Reinbothe et al., 2009). Additional indications for a role of jasmonates in regulating senescence are the transient up-regulation of expression of some enzymes involved in JA biosynthesis, such as allene oxide synthase (AOS) and OPDA reductase 3 (OPR3), and the increase in JA levels during natural senescence (He et al., 2002; van der Graaff et al., 2006). Furthermore, alterations in natural and induced senescence have been reported for some mutants with defects in the JA pathway. The mutant coi1, which is impaired in JA signaling, exhibited delayed chlorophyll loss upon dark incubation of detached leaves (Castillo and Leon, 2008). Plants with reduced expression of the 3-ketoacyl-CoA-thiolase KAT2, which is involved in β-oxidation and JA production, showed delayed yellowing during natural senescence and upon dark incubation of detached leaves (Castillo and Leon, 2008).However, there are also several reports that cast doubt on an important function of JA in senescence. For most mutants in JA biosynthesis or signaling, no differences in natural senescence are apparent (He et al., 2002; Schommer et al., 2008). In addition, mutants defective in the expression of AOS or OPR3 do not show altered senescence-like phenotypes upon dark treatment (Schommer et al., 2008; Kunz et al., 2009). It has to be taken into consideration that the knockout in these mutants has pleiotrophic effects during whole plant development. For example, the leaves of plants with reduced expression of the lipase DGL or of OPR3 are larger (Hyun et al., 2008). In addition, several knockout mutants defective in JA biosynthesis or signaling do not produce fertile flowers (Feys et al., 1994; McConn and Browse, 1996; Sanders et al., 2000; Stintzi and Browse, 2000; Ishiguro et al., 2001; von Malek et al., 2002). These changes in development might affect other developmental processes such as senescence.To investigate the function of jasmonates in senescence in more detail, we compared the oxylipin profile of wild-type leaves during natural senescence and upon stress induction of senescence-like phenotypes. The analysis of lipoxygenase 2 (LOX2)-RNA interference (RNAi) plants, which produce low basal levels of oxylipins but are impaired in the accumulation of OPDA and JA during senescence or in response to stress, indicates that 13-LOX products are not necessary for natural senescence or dark-induced chlorophyll loss but are involved in the response to sorbitol.     

Stem cells: cross-talk and developmental programs

The thesis advanced in this essay is that stem cells-particularly those in the nervous system-are components in a series of inborn 'programs' that not only ensure normal development, but persist throughout life so as to maintain homeostasis in the face of perturbations-both small and great. These programs encode what has come to be called 'plasticity'. The stem cell is one of the repositories of this plasticity. This review examines the evidence that interaction between the neural stem cell (as a prototypical somatic stem cell) and the developing or injured brain is a dynamic, complex, ongoing reciprocal set of interactions where both entities are constantly in flux. We suggest that this interaction can be viewed almost from a 'systems biology' vantage point. We further advance the notion that clones of exogenous stem cells in transplantation paradigms may not only be viewed for their therapeutic potential, but also as biological tools for 'interrogating' the normal or abnormal central nervous system environment, indicating what salient cues (among the many present) are actually guiding the expression of these 'programs'; in other words, using the stem cell as a 'reporter cell'. Based on this type of analysis, we suggest some of the relevant molecular pathways responsible for this 'cross-talk' which, in turn, lead to proliferation, migration, cell genesis, trophic support, protection, guidance, detoxification, rescue, etc. This type of developmental insight, we propose, is required for the development of therapeutic strategies for neurodegenerative disease and other nervous system afflictions in humans. Understanding the relevant molecular pathways of stem cell repair phenotype should be a priority, in our view, for the entire stem cell field.     

Possible involvement of neuronal nitric oxide synthase enzyme in early-phase isoflurane-induced hypotension in rats

This study was conducted to demonstrate the involvement of nitric oxide synthase (NOS) in the early-phase isoflurane-induced hypotension and to ascertain whether this NOS is neuronal NOS (nNOS) or endothelial NOS (eNOS). Mean arterial pressures (MAPs) were directly measured from the femoral arteries of urethane-anesthetized rats. Isoflurane-induced changes in MAP were monitored in rats following pretreatment with vehicle or one of the following NOS inhibitors: L-N^G-monomethyl-L-arginine (L-NMMA), which is non-selective; L-N^G-nitro arginine (L-NOARG), which is more selective for nNOS and eNOS; and 7-nitroindazole (7-NI), which is selective for nNOS. Exposure to 2% isoflurane in oxygen produced a triphasic reduction in MAP, including an early phase in which mean arterial pressure (MAP) fell by 25-30% during the initial 2½ min. This early hypotensive response, but not subsequent phases, was abolished by i.v. pretreatment with either L-NMMA or L-NOARG. The early-phase hypotension was also significantly attenuated by i.p. pretreatment with 7-NI; however, the blockade was not as complete as with L-NMMA or L-NOARG. Cerebella and aorta were removed from vehicle- and 7-NI pretreated rats and assayed for NOS activity by determining the conversion of [¹⁴C]L-arginine to [¹⁴C]L-citrulline. The 7-NI pretreatment significantly reduced NOS activity in the cerebellum but not the aorta. These findings indicate that the early-phase isoflurane-induced hypotension may involve nNOS as well as eNOS. The nNOS may participate in regulation of isoflurane-induced neuronal release of endogenous opioid peptide, which produces a vasodilation that is dependent on NO derived from an action of eNOS.     

Involvement of the metabolic hormones leptin, ghrelin, obestatin, IGF-I and of MAP kinase in control of porcine oocyte maturation

The general aim of our in vitro experiments was to study the role of the metabolic hormones leptin, ghrelin, obestatin and IGF-I and mitogen-activated protein kinase (MAPK)-dependent intracellular mechanisms in the control of nuclear maturation of porcine oocytes. For this purpose, porcine oocytes were isolated from the ovary and cultured in the presence of leptin, ghrelin, obestatin, IGF-I, MAPK blocker PD98059 and the combinations of hormones with PD98059. Proportions of matured oocytes (at metaphase II of meiosis, determined by DAPI staining) and of oocytes containing MAPK/ERK1-2 (determined by immunocytochemistry) were measured before and after culture. It was observed that the majority of oocytes isolated from the ovary before culture were immature and did not contain visible MAPK, but some oocytes were mature, and the majority of these oocytes contained MAPK. Incubation of oocytes resulted in a significant increase in the proportion of matured oocytes and in the percentage of oocytes containing MAPK in both the matured and not matured groups. Addition of IGF-I to the culture medium increased the proportion of matured oocytes, addition of leptin decreased it, and ghrelin and obestatin did not oocyte maturation. Addition of hormones did not affect the expression of MAPK in either immature or mature oocytes. PD98059, when given alone, suppressed the maturation and accumulation of MAPK in both mature and immature oocytes. When given together with hormones, PD98059 was able to reduce the stimulatory effect of IGF-I, to invert the inhibitory action of leptin to stimulatory and to induce the stimulatory action of ghrelin and obestatin on meiosis. IGF-I, ghrelin and obestatin, but not leptin, when given together with PD98059, increased the accumulation of MAPK in both immature and mature oocytes. Association of nuclear maturation and expression of MAPK in oocytes before, but not after culture, as well as the prevention of oocyte maturation by MAPK blocker suggests the involvement of MAPK-dependent intracellular mechanisms in the promotion of reinitiation, but not completion of meiosis. The effect of hormonal additions on meiosis of oocytes suggests that IGF-I is a stimulator, leptin can be an inhibitor, while ghrelin and obestatin probably do not control oocyte maturation. The ability of PD98059 to modify the effect of hormones on oocyte maturation and on MAPK expression suggests possible interference of hormones and MAPK-dependent intracellular mechanisms in oocytes. However, no influence of hormones on MAPK and lack of association between action of hormones and PD98059 on MAPK and meiosis suggest that MAPK is probably not a mediator of effect of IGF-I, leptin, ghrelin and obestatin on porcine oocyte nuclear maturation.     

Changes in function but not oligomeric size are associated with αB-crystallin lysine substitution

αB-Crystallin, ubiquitously expressed in many tissues including the ocular lens, is a small heat shock protein that can prevent protein aggregation. A number of post-translation modifications are reported to modify αB-crystallin function. Recent studies have identified αB-crystallin lysine residues are modified by acetylation and ubiquitination. Therefore, we sought to determine the effects of lysine to alanine substitution on αB-crystallin functions including chaperone activity and modulation of actin polymerization. Analysis of the ten substitution mutants as recombinant proteins indicated all the proteins were soluble and formed oligomeric complexes similar to wildtype protein. Lysozyme aggregation induced by chemical treatment indicated that K82, K90, K121, K166 and K174/K175 were required for efficient chaperone activity. Thermal induction of γ-crystallin aggregation could be prevented by all αB-crystallin substitution mutants. These αB-crystallin mutants also were able to mediate wildtype levels of actin polymerization. Further analysis of two clones with either enhanced or reduced chaperone activity on individual client substrates or actin polymerization indicated both retained broad chaperone activity and anti-apoptotic activity. Collectively, these studies show the requirements for lysine residues in αB-crystallin function.     

HIV-specific CD8+ T cells exhibit markedly reduced levels of Bcl-2 and Bcl-xL

Human immunodeficiency virus-specific CD8(+) T cells are highly sensitive to spontaneous and CD95/Fas-induced apoptosis, and this sensitivity may impair their ability to control HIV infection. To elucidate the mechanism behind this sensitivity, in this study we examined the levels of antiapoptotic molecules Bcl-2 and Bcl-x(L) in HIV-specific CD8(+) T cells from HIV-infected individuals. Bcl-2 expression was markedly decreased in HIV-specific CD8(+) T cells compared with CMV-specific and total CD8(+) T cells from HIV-infected individuals as well as total CD8(+) T cells from healthy donors. CD8(+) T cell Bcl-2 levels inversely correlated with spontaneous and CD95/Fas-induced apoptosis of CD8(+) T cells from HIV-infected individuals. HIV-specific CD8(+) T cells also had significantly lower levels of Bcl-x(L) compared with CMV-specific CD8(+) T cells. Finally, IL-15 induces both Bcl-2 and Bcl-x(L) expression in HIV-specific and total CD8(+) T cells, and this correlated with apoptosis inhibition and increased survival in both short- and long-term cultures. Our data indicate that reduced Bcl-2 and Bcl-x(L) may play an important role in the increased sensitivity to apoptosis of HIV-specific CD8(+) T cells and suggest a possible mechanism by which IL-15 increases their survival.     

Human and chimpanzee gene expression differences replicated in mice fed different diets

Although the human diet is markedly different from the diets of closely related primate species, the influence of diet on phenotypic and genetic differences between humans and other primates is unknown. In this study, we analyzed gene expression in laboratory mice fed diets typical of humans and of chimpanzees. The effects of human diets were found to be significantly different from that of a chimpanzee diet in the mouse liver, but not in the brain. Importantly, 10% of the genes that differ in their expression between humans and chimpanzee livers differed also between the livers of mice fed the human and chimpanzee diets. Furthermore, both the promoter sequences and the amino acid sequences of these diet-related genes carry more differences between humans and chimpanzees than random genes. Our results suggest that the mouse can be used to study at least some aspects of human-specific traits.     

Global Analysis of Apicomplexan Protein S‐Acyl Transferases Reveals an Enzyme Essential for Invasion

The advent of techniques to study palmitoylation on a whole proteome scale has revealed that it is an important reversible modification that plays a role in regulating multiple biological processes. Palmitoylation can control the affinity of a protein for lipid membranes, which allows it to impact protein trafficking, stability, folding, signalling and interactions. The publication of the palmitome of the schizont stage of Plasmodium falciparum implicated a role for palmitoylation in host cell invasion, protein export and organelle biogenesis. However, nothing is known so far about the repertoire of protein S‐acyl transferases (PATs) that catalyse this modification in Apicomplexa. We undertook a comprehensive analysis of the repertoire of Asp‐His‐His‐Cys cysteine‐rich domain (DHHC‐CRD) PAT family in Toxoplasma gondii and Plasmodium berghei by assessing their localization and essentiality. Unlike functional redundancies reported in other eukaryotes, some apicomplexan‐specific DHHCs are essential for parasite growth, and several are targeted to organelles unique to this phylum. Of particular interest is DHHC7, which localizes to rhoptry organelles in all parasites tested, including the major human pathogen P. falciparum. TgDHHC7 interferes with the localization of the rhoptry palmitoylated protein TgARO and affects the apical positioning of the rhoptry organelles. This PAT has a major impact on T. gondii host cell invasion, but not on the parasite's ability to egress.