Leishmanicidal activity of Yucatecan medicinal plants on Leishmania species responsible for cutaneous leishmaniasis

The leishmanicidal activity of 15 extracts and 4 pure metabolites obtained from Urechites andrieuxii, Colubrina greggii, Dorstenia contrajerva, and Tridax procumbens was evaluated using the newly developed MTS ({3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay, optimized for promastigotes of Leishmania major, Leishmania tropica, and Leishmania aethiopica, as well as for L. aethiopica axenic amastigotes. The assay was then used for calculating the percentage of viable stationary phase parasites after a 24-hr treatment with each plant extract or pure metabolite. The 3 most active samples, 2 from C. greggii (NCG-5C and DCG-3A) and 1 from T. procumbens (TPZ-2A), showed LD50 values of 62.4, 7.2, and 18.5 microg/ml, respectively, on stationary promastigotes, and of 94.2, 27.1, and 95.2 microg/ml, on amastigotes of L. aethiopica. Moreover, TPZ-2A and DCG-3A significantly reduced the percentage of infected monocyte-derived macrophages (THP-I). The percentage of infected cells decreased from 69.9% +/- 2.5% to 20.8% +/- 2% when the cells were treated with the DCG-3A fraction and to 14.9% +/- 0.5% when treated with TPZ-2A, without significantly decreasing the number of human cells. These findings indicate the presence of potentially bioactive metabolites in the roots of C. greggii and in T. procumbens and reflect the importance of pursuing the bioassay-guided purification of these metabolites.     

Ultrastructure and embryonic development of a syconoid calcareous sponge

Abstract. Recent molecular data suggest that the Porifera is paraphyletic (Calcarea+Silicea) and that the Calcarea is more closely related to the Metazoa than to other sponge groups, thereby implying that a sponge‐like animal gave rise to other metazoans. One ramification of these data is that calcareous sponges could provide clues as to what features are shared among this ancestral metazoan and higher animals. Recent studies describing detailed morphology in the Calcarea are lacking. We have used a combination of microscopy techniques to study the fine structure of Syconcoactum Urban 1905, a cosmopolitan calcareous sponge. The sponge has a distinct polarity, consisting of a single tube with an apically opening osculum. Finger‐like chambers, several hundred micrometers in length, form the sides of the tube. The inner and outer layers of the chamber wall are formed by epithelia characterized by apical–basal polarity and occluding junctions between cells. The outer layer—the pinacoderm—and atrial cavity are lined by plate‐like cells (pinacocytes), and the inner choanoderm is lined by a continuous sheet of choanocytes. Incurrent openings of the sponge are formed by porocytes, tubular cells that join the pinacoderm to the choanoderm. Between these two layers lies a collagenous mesohyl that houses sclerocytes, spicules, amoeboid cells, and a progression of embryonic stages. The morphology of choanocytes and porocytes is plastic. Ostia were closed in sponges that were vigorously shaken and in sponges left in still water for over 30 min. Choanocytes, and in particular collar microvilli, varied in size and shape, depending on their location in the choanocyte chamber. Although some of the odd shapes of choanocytes and their collars can be explained by the development of large embryos first beneath and later on top of the choanocytes, the presence of many fused collar microvilli on choanocytes may reflect peculiarities of the hydrodynamics in large syconoid choanocyte chambers. The unusual formation of a hollow blastula larva and its inversion through the choanocyte epithelium are suggestive of epithelial rather than mesenchymal cell movements. These details illustrate that calcareous sponges have characteristics that allow comparison with other metazoans—one of the reasons they have long been the focus of studies of evolution and development.     

Gastrulation in Calcareous Sponges: In Search of Haeckel's Gastraea

Haeckel's studies of development in calcareous sponges (1872)led him to develop the "Gastraea Theory," which proposes thatthe ancestral mode of germ layer formation, or gastrulation,was by invagination to produce a functional gut. His observationsthat gastrulation in the Calcarea occurs by invagination ofa ciliated larva upon settlement and metamorphosis were supportedby remarkable photomicrographs of the stage by Hammer in 1908.Although no later work found the same stage, these conceptsare repeated in texts today. We have re-examined embryogenesisand metamorphosis in Sycon sp. cf. S. raphanus in order to understandwhen gastrulation occurs. Almost all larvae settle on theirciliated anterior pole and metamorphose into a bilayered juvenilewhose interior cells rapidly differentiate into choanocytesand other cells of the young sponge. After a four-year searchwe have found the transitory stage shown by Hammer in whichthe anterior cells invaginate into the posterior half of thelarva. The hole closes and it is not until some days later thatthe sponge forms an osculum at its apical pole. To understandwhether invagination comprises gastrulation and if the holecan be considered to be a blastopore we have carried out a reviewof the literature dealing with this brief moment in calcaroneansponge development. Despite the intrigue of this type of metamorphosis,we conclude that gastrulation occurs earlier, during formationof the two cellular regions of the larva, and that metamorphosisinvolves the reorganization of these already differentiatedregions. Considering the pivotal position occupied by the Calcareaas the possible sister-group to all other Metazoa, these resultscall for a reassessment of germ layer formation and of the relationshipsof the primary germ layers among basal metazoan phyla.     

The ERM proteins interact with the HOPS complex to regulate the maturation of endosomes

In the degradative pathway, the progression of cargos through endosomal compartments involves a series of fusion and maturation events. The HOPS (homotypic fusion and protein sorting) complex is part of the machinery that promotes the progression from early to late endosomes and lysosomes by regulating the exchange of small GTPases. We report that an interaction between subunits of the HOPS complex and the ERM (ezrin, radixin, moesin) proteins is required for the delivery of EGF receptor (EGFR) to lysosomes. Inhibiting either ERM proteins or the HOPS complex leads to the accumulation of the EGFR into early endosomes, delaying its degradation. This impairment in EGFR trafficking observed in cells depleted of ERM proteins is due to a delay in the recruitment of Rab7 on endosomes. As a consequence, the maturation of endosomes is perturbed as reflected by an accumulation of hybrid compartments positive for both early and late endosomal markers. Thus, ERM proteins represent novel regulators of the HOPS complex in the early to late endosomal maturation.     

SIRT3 deacetylase: the Jekyll and Hyde sirtuin

Mitochondrial deacetylase SIRT3 protects against oxidative damage. In an article published online this month in EMBO reports, it is shown to also aggravate paracetamol-induced liver toxicity, calling for caution in trying to pharmacologically enhance SIRT3 activity.EMBO Rep (2011) advance online publication. doi:10.1038/embor.2011.121Post-translational modifications have crucial roles in regulating the functions of many eukaryotic proteins. Among them, lysine acetylation has been traditionally studied in the context of nuclear histone modifications, and was one of the first to be described as part of the ‘histone code'' hypothesis (Kim et al, 2006). More recently, work from several groups has demonstrated that lysine acetylation also modulates the activity of several non-histone proteins. In this context, this modification seems particularly abundant on mitochondrial proteins (Schwer et al, 2009). However, the way in which acetylation influences enzyme function and metabolic reprogramming in pathological states remains unknown. In an article published online this month in EMBO reports, Sack and colleagues shed new light on the role of mitochondrial SIRT3 deacetylase during paracetamol-induced toxicity, describing the mitochondrial protein aldehyde dehydrogenase 2 (ALDH2) as a new target of SIRT3, and a protective role for protein acetylation in this context (Lu et al, 2011).The sirtuin family of NAD⁺-dependent deacetylases comprises seven mammalian homologues (SIRT1–SIRT7) that have diverse functions and cellular localizations (Finkel et al, 2009). Among them, mitochondrial SIRT3 is the main deacetylase involved in the modulation of mitochondrial metabolic and oxidative-stress regulatory pathways (Schwer et al, 2009). SIRT3 seems to mediate protection against oxidative damage under caloric restriction (Someya et al, 2010), as well as promoting enhanced protection against redox and nutrient-excess stress (Zhong & Mostoslavsky, 2011).…these results raise the tantalizing possibility that—at least in the context of [paracetamol] toxicity—the less SIRT3 the betterAcetaminophen (APAP)—commonly known as paracetamol—is a widely used analgesic and anti-pyretic drug that is safe at therapeutic-dose levels. However, APAP overdose has been linked to liver injury in both humans and mice (Jaeschke & Bajt, 2006), with a high mortality rate due to acute liver failure. Remarkably, this hepatotoxic effect seems to be enhanced by fasting (Whitcomb & Block, 1994), a phenomenon that was poorly understood. In the initial phases of cell injury, a product of APAP oxidation—the highly reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI)—binds to protein cysteine and lysine residues (Zhou et al, 1996), eventually depleting hepatic glutathione and leading to the concomitant hepatotoxicity. Although there has been extensive research, the underlying molecular mechanisms of liver injury have not been fully elucidated.In this new study, Lu and colleagues aimed to decipher the way in which fasting or caloric-restriction exacerbate the redox-stress-dependent toxicity of APAP (Lu et al, 2011). Given the known increase in SIRT3 activity on nutrient deprivation, they proposed that, if protein acetylation inhibits NAPQI binding, SIRT3-mediated deacetylation might aggravate acetaminophen-induced liver injury (AILI).First, they tested whether lack of SIRT3 protects against AILI, by analysing susceptibility to liver injury in SIRT3^+/+ and SIRT3^−/− mice treated with a single toxic dose of APAP under fed and fasted conditions. Strikingly, they found that fasted SIRT3^−/− mice showed less hepatotoxicity than the SIRT3-competent mice. By using two-dimensional gel and immunoblot analyses, they then compared hepatic mitochondrial-protein acetylation profiles between fasted SIRT3^−/− and SIRT3^+/+ mice. In these experiments they identified, among several candidates, ALDH2—a known target of NAPQI, binding to which is known to reduce ALDH2 activity (Landin et al, 1996). This dehydrogenase oxidizes and detoxifies aldehydes—including lipid peroxidation products such as trans-4-hydroxy-2-nonenal (4-HNE; Doorn et al, 2006)—and thus buffers these highly reactive metabolites.…SIRT3 might act as a double-edged sword [raising] a word of caution regarding therapeutic strategies aimed at potentiating SIRT3 activityLu and colleagues then focused on ALDH2. In a series of elegant studies, they demonstrated that ALDH2 is a direct target of SIRT3, and deacetylation of ALDH2 modifies NAPQI binding. Liver mitochondria from SIRT3-deficient mice had increased ALDH2 acetylation, indicating a direct interaction between SIRT3 and ALDH2. ALDH2 was then shown to be a direct target of SIRT3 by using in vitro deacetylation assays. Despite these differences, basal ALDH2 activity remained the same in both genotypes; enzymatic activity was therefore evaluated in response to APAP treatment in fasted mice. Remarkably, SIRT3-deficient mitochondria exhibited approximately 40% higher levels of ALDH2 activity after APAP administration and, consequently, significantly lower levels of 4-HNE adducts were detected, in comparison to SIRT3^+/+ mice. SIRT3 is a known protective factor against oxidative stress; however, these results raise the tantalizing possibility that—at least in the context of APAP toxicity—the less SIRT3 the better.Logically, the next step was to show that the protective effect of SIRT3 deficiency is directly dependent on sustained ALDH2 activity. A marked increased in liver injury in the SIRT3-deficient animals was observed after knockdown of ALDH2 by using a lentiviral short-hairpin RNA approach, supporting their argument. To gain further molecular insight, the authors followed previous observations indicating that binding of NAPQI to ALDH2 diminishes ALDH2 activity (Landin et al, 1996). They hypothesized that SIRT3 might deacetylate ALDH2, in turn increasing its binding to NAPQI and leading to the concomitant inactivation of the protein. Indeed, through elegant SIRT3 gain- and loss-of-function experiments, they demonstrated that SIRT3-dependent deacetylation of ALDH2 enhances binding of the enzyme to NAPQI, whereas SIRT3 inactivation decreases NAPQI binding to ALDH2.The Sack group went one step further and used mass spectrometry to identify ALDH2 Lys 377 as the residue deacetylated by SIRT3. They showed that acetylation of Lys 377 is increased in SIRT3-deficient mice, and a mutant ALDH2 with an acetyl-mimicking mutation (K377Q) exhibited significantly less binding to NAPQI, giving a detailed molecular explanation for the protective effect observed in the absence of this sirtuin.These findings demonstrate that SIRT3-mediated deacetylation of mitochondrial proteins modulates susceptibility to AILI. Furthermore, the identification of ALDH2 as the substrate for SIRT3 deacetylation in this process provides a molecular framework in which to understand the apparent paradox of enhanced APAP toxicity under conditions of fasting or caloric restriction. Fasting induces SIRT3-mediated deacetylation of ALDH2, leading to increased NAPQI binding, which in turn reduces ALDH2 activity. This causes an accumulation of highly reactive adducts, probably contributing to the exacerbated hepatotoxicity observed after APAP treatment under nutrient restriction (Fig 1).Open in a separate windowFigure 1SIRT3-mediated exacerbation of acetaminophen-induced liver injury. SIRT3 deacetylates Lys 377 of ALDH2, making it available for NAPQI binding, which de-activates it. The concomitant reduction in the aldehyde-detoxifying activity of ALDH2 aggravates liver injury. AILI, acetaminophen-induced liver injury; ALDH2, aldehyde dehydrogenase 2; NAPQI, N-acetyl-p-benzoquinoneimine.The toxic effects of AILI have been traditionally addressed by using anti-oxidant therapies based on NAPQI binding to cysteine residues. Surprisingly, the functional outcome of NAPQI binding to lysine residues has not been explored so far, although it was described almost 15 years ago (Zhou et al, 1996). The Sack laboratory approached this issue, providing clear, supportive data for an interesting and provocative hypothesis: although it is widely accepted that SIRT3 has protective, anti-oxidant effects, ALDH2 deacetylation by SIRT3 exacerbates APAP-induced hepatotoxicity. This indicates that SIRT3 might act as a double-edged sword, and raises a word of caution regarding therapeutic strategies aimed at potentiating SIRT3 activity. Although this study provides support for this paradoxical effect, some questions remain. First, is ALDH2 the only SIRT3 substrate involved in this phenotype? The authors show that several other proteins were identified in their study, but their roles remain to be explored. Second, what is the physiological role of SIRT3-mediated ALDH2 deacetylation? Does this modification alter ALDH2 activity under conditions of nutrient stress? If so, how? Third, how general is this phenomenon? Does protein deacetylation modulate the binding of other toxic metabolites to proteins in detoxifying organs, such as the liver? Although answers to these questions await future investigation, one thing is certain: we need to exercise caution when evaluating the therapeutic potential of sirtuin modulators.     

Heat shock-induced SRSF10 dephosphorylation displays thermotolerance mediated by Hsp27

Gene regulation in response to environmental stress is critical for the survival of all organisms. From Saccharomyces cerevisiae to humans, it has been observed that splicing of mRNA precursors is repressed upon heat shock. However, a mild heat pretreatment often prevents splicing inhibition in response to a subsequent and more severe heat shock, a phenomenon called splicing thermotolerance. We have shown previously that the splicing regulator SRSF10 (formerly SRp38) is specifically dephosphorylated by the phosphatase PP1 in response to heat shock and that dephosphorylated SRSF10 is responsible for splicing repression caused by heat shock. Here we report that a mild heat shock protects SRSF10 from dephosphorylation during a second and more severe heat shock. Furthermore, this "thermotolerance" of SRSF10 phosphorylation, like that of splicing, requires de novo protein synthesis, specifically the synthesis of heat shock proteins. Indeed, overexpression of one of these proteins, Hsp27, inhibits SRSF10 dephosphorylation in response to heat shock and does so by interaction with SRSF10. Our data thus provide evidence that splicing thermotolerance is acquired through maintenance of SRSF10 phosphorylation and that this is mediated at least in part by Hsp27.     

Detection of etiological agents of malaria in howler monkeys from Atlantic Forests, rescued in regions of São Paulo city, Brazil

Background In some states of the Brazilian extra‐Amazonian region, such as the Atlantic Forest area, autochthonous human cases of malaria were related to simian malarias and vice versa. Methods To verify the presence of Plasmodium, 50 blood samples of howler monkeys (Alouatta guariba clamitans) rescued from the Metropolitan Region of Saõ Paulo city, where the Atlantic Forest is present, were analyzed. The samples were submitted to microscopy (thin and thick blood smears), enzyme‐linked immunosorbent assays (ELISA), indirect immunofluorescent assay (IFA), and polymerase chain reaction (PCR). Results Only one smear showed forms reminiscent of Plasmodium vivax. In ELISA, the frequencies of antibodies against synthetic peptides corresponding to circumsporozoite protein of P. vivax VK210 ‘classic’ (Pvc), P. vivax VK247, human P. vivax‐like (Pvk and Pvl), P. malariae/P. brasilianum (Pm), and P. falciparum (Pf) were 24.0% (12/50) for Pvc, 8.0% (04/50) for Pvk, 6.0% (03/50) for Pvl, 24.0% (12/50) for Pm, and 28.0% (14/50) for Pf, while the frequency of antibodies against PvMSP119 recombinant proteins was 42.0% (21/50). No serum reacted against PfMSP1‐19. In IFA,the seropositivity of antibodies against asexual forms of P. malariae was 31.3% (15/48). We utilized three PCR protocols to develop a molecular consensus (positive results in, at least, two protocols). The frequency of Plasmodium infections detected by PCR was 18.0% (09/50) for P. vivax, 4.0% (02/50) for P. malariae, and 76.0% (38/50) of samples were negative. The molecular consensus was not seen in 4.0% (02/50) of samples. Conclusions These results suggest that a possible interaction between human and simian malaria coming from a zoonotic cycle cannot be discarded because simians that live in the areas of the Atlantic Forest could play a role as a reservoir for Plasmodium.     

Ezrin ubiquitylation by the E3 ubiquitin ligase, WWP1, and consequent regulation of hepatocyte growth factor receptor activity

The membrane cytoskeleton linker ezrin participates in several functions downstream of the receptor Met in response to Hepatocyte Growth Factor (HGF) stimulation. Here we report a novel interaction of ezrin with a HECT E3 ubiquitin ligase, WWP1/Aip5/Tiul1, a potential oncogene that undergoes genomic amplification and overexpression in human breast and prostate cancers. We show that ezrin binds to the WW domains of WWP1 via the consensus motif PPVY(477) present in ezrin's C-terminus. This association results in the ubiquitylation of ezrin, a process that requires an intact PPVY(477) motif. Interestingly ezrin ubiquitylation does not target the protein for degradation by the proteasome. We find that ezrin ubiquitylation by WWP1 in epithelial cells leads to the upregulation of Met level in absence of HGF stimulation and increases the response of Met to HGF stimulation as measured by the ability of the cells to heal a wound. Interestingly this effect requires ubiquitylated ezrin since it can be rescued, after depletion of endogenous ezrin, by wild type ezrin but not by a mutant of ezrin that cannot be ubiquitylated. Taken together our data reveal a new role for ezrin in Met receptor stability and activity through its association with the E3 ubiquitin ligase WWP1. Given the role of Met in cell proliferation and tumorigenesis, our results may provide a mechanistic basis for understanding the role of ezrin in tumor progression.     

N-glycosylation as novel strategy to improve pharmacokinetic properties of bispecific single-chain diabodies

The therapeutic efficacy of recombinant antibodies such as single-chain Fv fragments and small bispecific or bifunctional molecules is often limited by rapid elimination from the circulation because of their small size. Here, we have investigated the effects of N-glycosylation on the activity and pharmacokinetics of a small bispecific single-chain diabody (scDb CEACD3) developed for the retargeting of cytotoxic T cells to CEA-expressing tumor cells. We could show that the introduction of N-glycosylation sequons into the flanking linker and a C-terminal extension results in the production of N-glycosylated molecules after expression in transfected HEK293 cells. N-Glycosylated scDb variants possessing 3, 6, or 9 N-glycosylation sites, respectively, retained antigen binding activity and bispecificity for target and effector cells as shown in a target cell-dependent IL-2 release assay, although activity was reduced approximately 3-5-fold compared with the unmodified scDb. All N-glycosylated scDb variants exhibited a prolonged circulation time compared with scDb, leading to a 2-3-fold increase of the area under curve (AUC). In comparison, conjugation of a branched 40-kDa PEG chain increased AUC by a factor of 10.6, while a chimeric anti-CEA IgG1 molecule had the longest circulation time with a 17-fold increase in AUC. Thus, N-glycosylation complements the repertoire of strategies to modulate pharmacokinetics of small recombinant antibody molecules by an approach that moderately prolongs circulation time.