Cyclase-associated protein is essential for the functioning of the endo-lysosomal system and provides a link to the actin cytoskeleton

Data from mutant analysis in yeast and Dictyostelium indicate a role for the cyclase-associated protein (CAP) in endocytosis and vesicle transport. We have used genetic and biochemical approaches to identify novel interacting partners of Dictyostelium CAP to help explain its molecular interactions in these processes. Cyclase-associated protein associates and interacts with subunits of the highly conserved vacuolar H(+)-ATPase (V-ATPase) and co-localizes to some extent with the V-ATPase. Furthermore, CAP is essential for maintaining the structural organization, integrity and functioning of the endo-lysosomal system, as distribution and morphology of V-ATPase- and Nramp1-decorated membranes were disturbed in a CAP mutant (CAP bsr) accompanied by an increased endosomal pH. Moreover, concanamycin A (CMA), a specific inhibitor of the V-ATPase, had a more severe effect on CAP bsr than on wild-type cells, and the mutant did not show adaptation to the drug. Also, the distribution of green fluorescent protein-CAP was affected upon CMA treatment in the wildtype and recovered after adaptation. Distribution of the V-ATPase in CAP bsr was drastically altered upon hypo-osmotic shock, and growth was slower and reached lower saturation densities in the mutant under hyper-osmotic conditions. Taken together, our data unravel a link of CAP with the actin cytoskeleton and endocytosis and suggest that CAP is an essential component of the endo-lysosomal system in Dictyostelium.     

Brazilian green propolis on Helicobacter pylori infection. a pilot clinical study

Recent in vitro studies suggest that propolis and some of its phenolic components are able to inhibit Helicobacter pylori growth. To date, there are no clinical studies. AIMS: To evaluate the effect of Brazilian green propolis on H. pylori-infected individuals. PATIENTS AND METHODS: Eighteen (11 females, 7 males, mean age 47 years) participants were included. Before treatment, all participants were submitted to gastroscopy, and H. pylori infection was confirmed by histology, urease test, and (13)C-urea breath test (UBT). Participants with UBT showing a delta over baseline (DOB) value higher than 4 per thousand were considered positive for H. pylori infection. Twenty drops from an alcoholic preparation of Brazilian green propolis were administered three times a day for 7 days. Clinical evaluation and UBT were performed at 1-3 days and at 40 days after the end of therapy to evaluate H. pylori suppression or eradication, respectively. RESULTS: All participants took all medication and completed the study. Eighty-three percent of the subjects did not succeed in suppressing or eradicating H. pylori. Two participants reached partial suppression after treatment, but became positive again at UBT performed 40 days after treatment. Another participant presented negative at UBT 40 days after treatment, not confirmed by a second UBT performed 100 days after treatment. CONCLUSIONS: Brazilian green propolis used in popular dose showed minimal effect on H. pylori infection. Larger studies with longer duration, larger dose, and different frequency of administration of propolis extract should be undertaken to define its role on H. pylori therapy.     

Genetic population structure as indirect measure of dispersal ability in a Lake Tanganyika cichlid

Diversification and speciation processes are influenced by intrinsic (ecological specialization, dispersal) and extrinsic (habitat structure and instability) factors, but the effect of ecological characteristics on dispersal is difficult to assess. This study uses mitochondrial control region sequences to investigate the population structure and demographic history of the endemic Lake Tanganyika cichlid Neolamprologus caudopunctatus with a preference for the rock-sand interface along two stretches of continuous, rocky shoreline, and across a sandy bay representing a potential dispersal barrier. Populations along uninterrupted habitat were not differentiated; whereas, the sandy bay separated two reciprocally monophyletic clades. The split between the two clades between 170,000 and 260,000 years BP coincides with a period of rising water level following a major lowstand, and indicates that clades remained isolated throughout subsequent lake level fluctuations. Low long-term effective population sizes were inferred from modest genetic diversity estimates, and may be due to recent population expansions starting from small population sizes 45,000–60,000 years BP. Comparisons with available data from specialized rock-dwelling species of the␣same area suggest that habitat structure and lake level fluctuations determine phylogeographic patterns on large scales, while fine-scale population structure and demography are modulated by species-specific ecologies.     

Synthesis and X-ray crystal structure of [Ag(phendio)<Subscript>2</Subscript>]ClO<Subscript>4</Subscript> (phendio = 1,10-phenanthroline-5,6-dione) and its effects on fungal and mammalian cells

The Cu(II) and Ag(I) complexes, [Cu(phendio)₃](ClO₄)₂4H₂O and [Ag(phendio)₂]ClO₄ (phendio = 1,10-phenanthroline-5,6-dione), are prepared in good yield by reacting phendio with the appropriate metal perchlorate salt. The X-ray crystal structure of the Ag(I) complex shows it to have a pseudo tetrahedral structure. `Metal-free' phendio and the Cu(II) and Ag(I) phendio complexes strongly inhibit the growth of the fungal pathogen Candida albicans, and are more active than their 1,10-phenanthroline analogues. The simple Ag(I) salts, AgCH<sub>3</sub>CO<sub>2</sub>, AgNO<sub>3</sub> and AgClO<sub>4</sub>.H<sub>2</sub>O display superior anti-fungal properties compared to analogous simple Cu(II) and Mn(II) salts, suggesting that the nature of the metal ion strongly influences activity. Exposing C. albicans to `metal-free' phendio, simple Ag(I) salts and [Ag(phendio)₂]ClO₄ causes extensive, non-specific DNA cleavage. `Metal-free' phendio and [Ag(phendio)₂]ClO₄ induce gross distortions in fungal cell morphology and there is evidence for disruption of cell division. Both drugs also exhibit high anti-cancer activity when tested against cultured mammalian cells.     

Biochemical engineering of the N-acyl side chain of sialic acid leads to increased calcium influx from intracellular compartments and promotes differentiation of HL60 cells

Sialylation of glycoconjugates is essential for mammalian cells. Sialic acid is synthesized in the cytosol from N-acetylmannosamine by several consecutive steps. Using N-propanoylmannosamine, a novel precursor of sialic acid, we are able to incorporate unnatural sialic acids with a prolonged N-acyl side chain (e.g., N-propanoylneuraminic acid) into glycoconjugates taking advance of the cellular sialylation machinery. Here, we report that unnatural sialylation of HL60-cells leads to an increased release of intracellular calcium after application of thapsigargin, an inhibitor of SERCA Ca2+-ATPases. Furthermore, this increased intracellular calcium concentration leads to an increased adhesion to fibronectin. Finally, we observed an increase of the lectin galectin-3, a marker of monocytic differentiation of HL60-cells.     

Is the atherosclerotic phenotype of preeclamptic placentas due to altered lipoprotein concentrations and placental lipoprotein receptors? Role of a small-for-gestational-age phenotype

Atherosis of spiral arteries in uteroplacental beds from preeclamptic women resemble those of atherosclerosis, characterized by increased plasma lipids and lipoproteins. We hypothesized that: 1) lipoprotein receptors/transporters in the placenta would be upregulated in preeclampsia, associated with increased maternal and fetal lipoprotein concentrations; and 2) expression of these would be reduced in preeclamptic placentae from women delivering small-for-gestational-age (SGA) infants. Placental biopsies and maternal and umbilical serum samples were taken from 27 normotensive and 24 preeclamptic women. Maternal/umbilical cord serum LDL, HDL, total cholesterol, and triglycerides were measured. Placental mRNA expression of lipoprotein receptors/transporters were quantified using quantitative RT-PCR. Protein localization/expression of LDL receptor-related protein 1 (LRP-1) in the preeclamptic placentae with/without SGA was measured by immunohistochemistry. Placental mRNA expression of all genes except paraoxonase-1 (PON-1), microsomal triglyceride transfer protein (MTTP), and protein disulfide isomerase family A member 2 (PDIA2) were observed. No differences for any lipoprotein receptors/transporters were found between groups; however, in the preeclamptic group placental LRP-1 expression was lower in SGA delivering mothers (n = 7; P = 0.036). LRP-1 protein was localized around fetal vessels and Hofbauer cells. This is the first detailed study of maternal/fetal lipoprotein concentrations and placental lipoprotein receptor mRNA expression in normotensive and preeclamptic pregnancies. These findings do not support a role of altered lipid metabolism in preeclampsia, but may be involved in fetal growth.     

Extracellular, circulating proteasomes and ubiquitin - incidence and relevance

The ubiquitin-proteasome system is the major pathway for intracellular protein degradation and is also deeply involved in the regulation of most basic cellular processes. Its proteolytic core, the 20S proteasome, has found to be attached also to the cell plasma membrane and certain observations are interpreted as to suggest that they may be released into the extracellular medium, e.g. in the alveolar lining fluid, epididymal fluid and possibly during the acrosome reaction. Proteasomes have also been detected in normal human blood plasma and designated circulating proteasomes; these have a comparatively low specific activity, a distinct pattern of subtypes and their exact origin is still enigmatic. In patients suffering from autoimmune diseases, malignant myeloproliferative syndromes, multiple myeloma, acute and chronic lymphatic leukaemia, solid tumour, sepsis or trauma, respectively, the concentration of circulating proteasomes has been found to be elevated, to correlate with the disease state and has even prognostic significance. Similarly, ubiquitin has been discovered as a normal component of human blood and seminal plasma and in ovarian follicular fluid. Increased concentrations were measured in diverse pathological situations, not only in blood plasma but also in cerebrospinal fluid, where it may have neuroprotective effects. As defective spermatozoa are covered with ubiquitin in the epididymal fluid, extracellular ubiquitination is proposed to be a mechanism for quality control in spermatogenesis. Growing evidence exists also for a participation of extracellular proteasomes and ubiquitin in the fertilization process.