Shedding of glycan‐modifying enzymes by signal peptide peptidase‐like 3 (SPPL3) regulates cellular N‐glycosylation

Protein N‐glycosylation is involved in a variety of physiological and pathophysiological processes such as autoimmunity, tumour progression and metastasis. Signal peptide peptidase‐like 3 (SPPL3) is an intramembrane‐cleaving aspartyl protease of the GxGD type. Its physiological function, however, has remained enigmatic, since presently no physiological substrates have been identified. We demonstrate that SPPL3 alters the pattern of cellular N‐glycosylation by triggering the proteolytic release of active site‐containing ectodomains of glycosidases and glycosyltransferases such as N‐acetylglucosaminyltransferase V, β‐1,3 N‐acetylglucosaminyltransferase 1 and β‐1,4 galactosyltransferase 1. Cleavage of these enzymes leads to a reduction in their cellular activity. In line with that, reduced expression of SPPL3 results in a hyperglycosylation phenotype, whereas elevated SPPL3 expression causes hypoglycosylation. Thus, SPPL3 plays a central role in an evolutionary highly conserved post‐translational process in eukaryotes.     

The Intramembrane Proteases Signal Peptide Peptidase-Like 2a and 2b Have Distinct Functions In Vivo

We reported recently that the presenilin homologue signal peptide peptidase-like 2a (SPPL2a) is essential for B cell development by cleaving the N-terminal fragment (NTF) of the invariant chain (li, CD74). Based on this, we suggested that pharmacological modulation of SPPL2a may represent a novel approach to deplete B cells in autoimmune disorders. With regard to reported overlapping substrate spectra of SPPL2a and its close homologue, SPPL2b, we investigated the role of SPPL2b in CD74 NTF proteolysis and its impact on B and dendritic cell homeostasis. In heterologous expression experiments, SPPL2b was found to cleave CD74 NTF with an efficiency simliar to that of SPPL2a. For in vivo analysis, SPPL2b single-deficient and SPPL2a/SPPL2b double-deficient mice were generated and examined for CD74 NTF turnover/accumulation, B cell maturation and functionality, and dendritic cell homeostasis. We demonstrate that in vivo SPPL2b does not exhibit a physiologically relevant contribution to CD74 proteolysis in B and dendritic cells. Furthermore, we reveal that both proteases exhibit divergent subcellular localizations in B cells and different expression profiles in murine tissues. These findings suggest distinct functions of SPPL2a and SPPL2b and, based on a high abundance of SPPL2b in brain, a physiological role of this protease in the central nervous system.     

Nosocomial Bloodstream Candida Infections in a Tertiary-Care Hospital in South Brazil: A 4-Year Survey

The aims of this study were to evaluate the epidemiology of nosocomial candidemia in a tertiary hospital in South Brazil and the in vitro antifungal susceptibility of isolates. Blood strains from 108 patients were identified by PCR-based method. Some 30.5 % of candidemia were caused by Candida tropicalis, 28.7 % were due to Candida albicans, 24.1 % with Candida parapsilosis sensu stricto, 8.3 % with Candida glabrata sensu lato, 1.8 % involved Candida krusei and 6.6 % with other species. Candidemia was more common in intensive care unit settings (66 %). In vitro susceptibility to antifungal drugs was determined by a microdilution method; and new species-specific clinical breakpoints for fluconazole and voriconazole were applied. Overall susceptibility rates were 100 % for itraconazole, 91 % for fluconazole, 98 % for voriconazole and 99 % for amphotericin B. Fluconazole resistance was mostly among C. parapsilosis sensu stricto isolates (26.9 %). Most of the findings reported here agreed with epidemiological features common to other tertiary hospitals in Brazil; but also revealed some peculiarities, such as a high frequency of C. tropicalis associated with candidemia. Besides, high rate of fluconazole resistance among C. parapsilosis stricto sensu isolates was obtained when applying the new species-specific clinical breakpoints.     

Small molecule inhibitors of protein interaction with glycosaminoglycans (SMIGs), a novel class of bioactive agents with anti-inflammatory properties

Background

Small molecule inhibitors of biologically important protein–glycosaminoglycan (GAG) interactions have yet to be identified.

Methods

Compound libraries were screened in an assay of L-selectin–IgG binding to heparin (a species of heparan sulfate [HS-GAG]). Hits were validated, IC-50s established and direct binding of hits to HS-GAGs was investigated by incubating compounds alone with heparin. Selectivity of inhibitors was assessed in 11 different protein-GAG binding assays. Anti-inflammatory activity of selected compounds was evaluated in animal models.

Results

Screening identified a number of structurally-diverse planar aromatic cationic amines. Scaffolds similar to known GAG binders, chloroquine and tilorone, were also identified. Inhibitors displayed activity also against bovine kidney heparan sulfate. Direct binding of compounds to GAGs was verified by incubating compounds with heparin alone. Selectivity of inhibitors was demonstrated in a panel of 11 heparin binding proteins, including selectins, chemokines (IL-8, IP-10), Beta Amyloid and cytokines (VEGF, IL-6). A number of selected lead compounds showed dose-dependent efficacy in peritonitis, paw edema and delayed type hypersensitivity.

Conclusions

A new class of compounds, SMIGs, inhibits protein–GAG interaction by direct binding to GAGs. Although their IC-50s were in the low micro-molar range, SMIGs binding to HS-GAGs appeared to be stable in physiological conditions, indicating high avidity binding. SMIGs may interfere with major checkpoints for inflammatory and autoimmune events.

General significance

SMIGs are a class of structurally-diverse planar aromatic cationic amines that have an unusual mode of action — inhibiting protein–GAG interactions via direct and stable binding to GAGs. SMIGs may have therapeutic potential in inflammatory and autoimmune disorders.     

Ultrastructural characterization and multilocus sequence analysis (MLSA) of ‘Candidatus Rickettsiella isopodorum’, a new lineage of intracellular bacteria infecting woodlice (Crustacea: Isopoda)

The taxonomic genus Rickettsiella (Gammaproteobacteria; Legionellales) comprises intracellular bacteria associated with a wide range of arthropods including insects, arachnids and crustaceans. The present study provides ultrastructural together with genetic evidence for a Rickettsiella bacterium in the common rough woodlouse, Porcellio scaber (Isopoda, Porcellionidae), occurring in Germany, and shows that this bacterium is very closely related to one of the same genus occurring in California that infects the pill bug, Armadillidium vulgare (Isopoda, Armadillidiidae). Both bacterial isolates displayed the ultrastructural features described previously for crustacean-associated bacteria of the genus Rickettsiella, including the absence of well-defined associated protein crystals; occurrence of the latter is a typical characteristic of infection by this type of bacteria in insects, but has not been reported in crustaceans. A molecular systematic approach combining multilocus sequence analysis (MLSA) with likelihood-based significance testing demonstrated that despite their distant geographic origins, both bacteria form a tight sub-clade within the genus Rickettsiella. In the 16S rRNA gene trees, this sub-clade includes other bacterial sequences from woodlice. Moreover, the bacterial specimens from P. scaber and A. vulgare are found genetically or morphologically different from each of the four currently recognized Rickettsiella species. Therefore, the designation ‘Candidatus Rickettsiella isopodorum’ is introduced for this new lineage of isopod-associated Rickettsiella bacteria.     

Brain size in neonatal and adult Weddell seals: Costs and consequences of having a large brain

Little is known about the ontogeny of brain size in pinnipeds despite potential functional implications of brain substrate (glucose, oxygen) requirements for diving, fasting, growth, and lactation strategies. We measured brain mass (brM) and cranial capacity (CC) in newborn and adult Weddell seals. Neonatal Weddell seals had brM that represented ~70% of adult brM. Weddell seals have the largest neonatal brain, proportional to adult brain, reported for any mammal to date, which is remarkable considering the relatively small size of Weddell seal pups at birth (6%–7% of maternal body mass) compared to neonates of other highly precocial mammals. Provision of sufficient glucose to maintain the large, well‐developed brain of the neonatal Weddell seal has a nontrivial metabolic cost to both pup and mother. We therefore hypothesize that this phenomenon must have functional significance, such as allowing pups to acquire complex under‐ice navigation skills during the period of maternal attendance.     

The effects of genistein and daidzein on cell proliferation kinetics in HT29 colon cancer cells: the expression of CTNNBIP1 (β-catenin), APC (adenomatous polyposis coli) and BIRC5 (survivin)

Soybean isoflavonoids have received significant attention due to their potential anticarcinogenic and antiproliferative effects and possible role in many signal transduction pathways. However, their mechanisms of action and their molecular targets remain to be further elucidated. In this paper, we demonstrated that two soybean isoflavones (genistein and daidzein) reduced the proliferation of the human colon adenocarcinoma grade II cell line (HT-29) at concentrations of 25 and 50–100 μM, respectively. We then investigated the effects of genistein and daidzein by RT-PCR on molecules that involved in tumor development and progression by their regulation of cell proliferation. At a concentration of 50 μM genistein, there was suppressed expression of β-catenin (CTNNBIP1). Neither genistein nor daidzein affected APC (adenomatous polyposis coli) or survivin (BIRC5) expression when cells were treated with concentrations of 10 or 50 μM. These data suggest that the down-regulation of β-catenin by genistein may constitute an important determinant of the suppression of HT-29 cell growth and may be exploited for the prevention and treatment of colon cancer.     

Acute ethanol intake induces mitogen-activated protein kinase activation,platelet-derived growth factor receptor phosphorylation,and oxidative stress in resistance arteries

In the present study, we investigated the role of angiotensin type I (AT₁) receptor in reactive oxygen species (ROS) generation and mitogen-activated protein kinases (MAPK) activation induced by acute ethanol intake in resistance arteries. We also evaluated the effect of ethanol on platelet-derived growth factor receptors (PDGF-R) phosphorylation and the role of this receptor on ROS generation by ethanol. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. Acute ethanol intake did not alter angiotensin I or angiotensin II levels in the rat mesenteric arterial bed (MAB). Ethanol induced vascular oxidative stress, and this response was not prevented by losartan (10 mg/kg; p.o. gavage), a selective AT₁ receptor antagonist. MAB from ethanol-treated rats displayed increased SAPK/JNK and PDGF-R phosphorylation, responses that were not prevented by losartan. The phosphorylation levels of protein kinase B (Akt) and eNOS were not affected by acute ethanol intake. MAB nitrate levels and the reactivity of this tissue to acetylcholine, phenylephrine, and sodium nitroprusside were not affected by ethanol intake. Ethanol did not alter plasma antioxidant capacity, the levels of reduced glutathione, or the activities of superoxide dismutase and catalase in the rat MAB. Short-term effects of ethanol (50 mmol/l) were evaluated in vascular smooth muscle cells (VSMC) isolated from rat MAB. Ethanol increased ROS generation, and this response was not affected by AG1296, a PDGF-R inhibitor, or losartan. Finally, ethanol did not alter MAPK or PDGF-R phosphorylation in cultured VSMC. Our study provides novel evidence that acute ethanol intake induces ROS generation, PDGF-R phosphorylation, and MAPK activation through AT(1)-independent mechanisms in resistance arteries in vivo. MAPK and PDGF-R play a role in vascular signaling and cardiovascular diseases and may contribute to the vascular pathobiology of ethanol.     

Analysis of the abilities of endophytic bacteria associated with banana tree roots to promote plant growth

A total of 40 endophytic bacterial isolates obtained from banana tree roots were characterized for their biotechnological potential for promoting banana tree growth. All isolates had at least one positive feature. Twenty isolates were likely diazotrophs and formed pellicles in nitrogen-free culture medium, and 67% of these isolates belonged to the genus Bacillus sp. The isolates EB-04, EB-169, EB-64, and EB-144 had N fixation abilities as measured by the Kjeldahl method and by an acetylene reduction activity assay. Among the 40 isolates, 37.5% were capable of solubilizing inorganic phosphate and the isolates EB-47 and EB-64 showed the highest solubilization capacity. The isolate EB-53 (Lysinibacillus sp.) had a high solubilization index, whereas 73% of the isolates had low solubilization indices. The synthesis of indole-3-acetic acid (IAA) in the presence of L-tryptophan was detected in 40% of the isolates. The isolate EB-40 (Bacillus sp.) produced the highest amount of IAA (47.88 μg/ml) in medium supplemented with L-tryptophan and was able to synthesize IAA in the absence of L-tryptophan. The isolates EB-126 (Bacillus subtilis) and EB-47 (Bacillus sp.) were able to simultaneously fix nitrogen, solubilize phosphate and produce IAA in vitro. The results of this study demonstrated that the isolates analyzed here had diverse abilities and all have the potential to be used as growth-promoting microbial inoculants for banana trees.     

Phylogenetic structure of a palm community in the central Amazon: changes along a hydro-edaphic gradient

The concepts of phylogenetic community structure (PCS) and phylogenetic niche conservatism (PNC) allow ecologists to address the role of species’ evolutionary history in community assembly. It is important to test the role of historical legacies relative to environmental constraints at local scales, where communities are assembled. We studied phylogenetic structure and niche conservatism for palms (Arecaceae) in the 64-km² Ducke Reserve in the central Amazon, near Manaus. The 72 study plots, each covering 0.1 ha, were distributed regularly in a terra firme forest along a hydro-edaphic gradient. We compared the observed palm PCS with assemblages generated by null models. We also analyzed whether morphological and ecological traits are labile or conserved along the phylogeny and quantified the spatial structure of morphological traits in each plot. We found an overall neutral PCS in combination with low PNC (labile traits), suggesting that evolutionary history poses little constraint on palm community assembly in this Amazonian landscape. Still, there was a tendency towards phylogenetic overdispersion in bottomlands, suggesting competitive exclusion among close relatives or, more likely, environmental filtering acting on convergent traits that affect co-occurrence in flood-prone areas. We conclude that (1) PCS of local communities is random as a whole and morphological traits are overall labile, but that (2) the hydro-edaphic gradient within terra firme forests leads to differences in species co-occurrence so that closely related species occur less often than expected in bottomlands due to diffuse competition among close relatives or environmental filtering on convergent traits.