Molecular characterization of Cryptosporidium isolates from humans and animals in Iran

Isolates of Cryptosporidium spp. from human and animal hosts in Iran were characterized on the basis of both the 18S rRNA gene and the Laxer locus. Three Cryptosporidium species, C. hominis, C. parvum, and C. meleagridis, were recognized, and zoonotically transmitted C. parvum was the predominant species found in humans.     

Circulating immune complexes (IC) and IC-induced levels of GM-CSF are increased in sudanese patients with acute visceral Leishmania donovani infection undergoing sodium stibogluconate treatment: implications for disease pathogenesis

Infection with Leishmania donovani is associated with IL-10 as well as with GM-CSF. Immune complexes (IC) exert important functions by stimulation of monocytes/macrophage-mediated production of pro- and anti-inflammatory cytokines in rheumatic diseases. In this investigation, we have explored IC-induced cytokine production during Leishmania infection. Sera from 43 patients with visceral leishmaniasis (VL), 17 patients with post-kala-azar dermal leishmaniasis, and 20 healthy Sudanese controls were precipitated with polyethylene glycol (PEG). The PEG precipitates were added to serum-free PBMC for 20 h,whereupon supernatant levels of IL-1beta, IL-6, IL-10, IL-1 receptor antagonist protein, TNF-alpha, TNF receptor p75, and GM-CSF were investigated using ELISA. Circulating levels of C1q-binding IC were also measured in the serum samples. PEG precipitates from Leishmania-infected patients induced significantly higher levels of GM-CSF (p = 0.0037) and IL-10 (p < 0.0001), as well as of IL-6 (p < 0.0001) and IL-1 receptor antagonist (p = 0.0238) as compared with PEG precipitates from controls. Patients with acute VL as well as VL patients receiving sodium stibogluconate treatment displayed significantly increased levels of PEG precipitate-induced GM-CSF. The induction of GM-CSF by circulating IC was especially prominent in acute VL patients receiving sodium stibogluconate treatment; ANOVA revealed significant interaction between disease activity and treatment for PEG precipitate-induced levels of GM-CSF (disease activity, p = 0.0006; treatment, p = 0.0005; interaction, p = 0.0046). Parallel associations were determined for C1q-binding immune complexes, but not for any cytokine other than GM-CSF. The importance of IC-induced GM-CSF in leishmaniasis warrants further study.     

A model-based parametric study of impact force during running

This paper deals with the impact force during foot-ground impact activities such as the running. A previously developed model is used for this study. The model is a lumped-parameter one consisting of four masses connected to each other via linear springs and viscous dampers. A shoe-specific nonlinear function is used for representation of the ground reaction force. The authors have previously showed that the previous version of the model as well as its simulation is incorrect. This paper slightly modifies the previous model so as it is able to produce results in agreement with the experiments. Then, the modified model is simulated for two typical shoe types. A parametric study is also conducted. The parametric study concerns with the effects of masses, mass ratios, stiffness constants, and damping coefficients on the dynamics of the impact. It is shown that the impact forces increase as the rigid and wobbling masses increase. However, the increase in the impact forces is not the same for all the masses. It is found that the impact force increases as the touchdown velocities increase. Simulations imply that the variations of the damping coefficients result in larger variations of the impact force compared to the stiffness. The effect of the variation of gravity on the simulated impact force is also explored. It is concluded that both the first and the second peaks of the impact force are increased with gravity. An in-depth discussion is included to compare results of the current paper with results of other investigators.     

Biogeochemical processes in intensive zero-effluent marine fish culture with recirculating aerobic and anaerobic biofilters

The biogeochemical processes that drive nutrient transformations and recycling in organic marine sediment-water environments were studied for 17 months in a zero-effluent intensive recirculating culture system. The system consisted of a 10 m³ gilthead seabream (Sparus aurata) tank coupled to aerobic and anaerobic water treatment elements. Nutrients and alkalinity were measured in the system to quantify the main biogeochemical processes. Fractions of the carbon fed in feed were found in fish (18.3%) and in sludge (11%); the missing carbon was respired by fish (45%) and by aerobic (8.4%) and anaerobic (7.7%) microorganisms. Fractions of the nitrogen fed in feed were found in fish (15.4%) and in sludge (14.3%); the missing nitrogen was eliminated by nitrification-denitrification. Most of the phosphorus and ash fed in feed and not found in fish accumulated within the sludge in the system. The rates of nitrification, denitrification and sulphate reduction increased with time, reaching 0.3 g N m^− 2 d^− 1, 53 g N m^− 2 d^− 1 and 145 g S m^− 2 d^− 1, respectively. Nitrification developed more rapidly than denitrification, leading at first to nitrate accumulation (to 20 mmol NO₃ l^− 1 by day 200) and a decrease in alkalinity. Once denitrification surpassed nitrification, nitrate concentrations decreased, eventually being reduced to < 0.3 mmol NO₃ l^− 1 by day 510, and alkalinity stabilized. Toxic hydrogen sulphide, generated within the anaerobic sludge, was oxidized by oxygen and nitrate as it diffused through the anaerobic-aerobic sediment-water interface. When nitrate levels in the water above the sludge dropped below 2 mmol l^− 1, sulphide was also oxidized in the fluidized bed reactor. Denitrification reduced nitrate in the water, respired (jointly with sulphate reduction) carbon in the sludge, oxidized the hydrogen sulphide, and contributed to stabilization of alkalinity and accumulation of polyphosphate in bacteria as a major sink of labile P.     

Redox-active cysteines of a membrane electron transporter DsbD show dual compartment accessibility

The membrane-embedded domain of the unusual electron transporter DsbD (DsbDbeta) uses two redox-active cysteines to catalyze electron transfer between thioredoxin-fold polypeptides on opposite sides of the bacterial cytoplasmic membrane. How the electrons are transferred across the membrane is unknown. Here, we show that DsbDbeta displays an inherent functional and structural symmetry: first, the two cysteines of DsbDbeta can be alkylated from both the cytoplasm and the periplasm. Second, when the two cysteines are disulfide-bonded, cysteine scanning shows that the C-terminal halves of the cysteine-containing transmembrane segments 1 and 4 are exposed to the aqueous environment while the N-terminal halves are not. Third, proline residues located pseudo-symmetrically around the two cysteines are required for redox activity and accessibility of the cysteines. Fourth, mixed disulfide complexes, apparent intermediates in the electron transfer process, are detected between DsbDbeta and thioredoxin molecules on each side of the membrane. We propose a model where the two redox-active cysteines are located at the center of the membrane, accessible on both sides of the membrane to the thioredoxin proteins.     

Differential membrane binding and diacylglycerol recognition by C1 domains of RasGRPs

RasGRPs (guanine-nucleotide-releasing proteins) are exchange factors for membrane-bound GTPases. All RasGRP family members contain C1 domains which, in other proteins, bind DAG (diacylglycerol) and thus mediate the proximal signal-transduction events induced by this lipid second messenger. The presence of C1 domains suggests that all RasGRPs could be regulated by membrane translocation driven by C1-DAG interactions. This has been demonstrated for RasGRP1 and RasGRP3, but has not been tested directly for RasGRP2, RasGRP4alpha and RasGRP4beta. Sequence alignments indicate that all RasGRP C1 domains have the potential to bind DAG. In cells, the isolated C1 domains of RasGRP1, RasGRP3 and RasGRP4alpha co-localize with membranes and relocalize in response to DAG, whereas the C1 domains of RasGRP2 and RasGRP4beta do not. Only the C1 domains of RasGRP1, RasGRP3 and RasGRP4alpha recognize DAG as a ligand within phospholipid vesicles and do so with differential affinities. Other lipid second messengers were screened as ligands for RasGRP C1 domains, but none was found to serve as an alternative to DAG. All of the RasGRP C1 domains bound to vesicles which contained a high concentration of anionic phospholipids, indicating that this could provide a DAG-independent mechanism for membrane binding by C1 domains. This concept was supported by demonstrating that the C1 domain of RasGRP2 could functionally replace the membrane-binding role of the C1 domain within RasGRP1, despite the inability of the RasGRP2 C1 domain to bind DAG. The RasGRP4beta C1 domain was non-functional when inserted into either RasGRP1 or RasGRP4, implying that the alternative splicing which produces this C1 domain eliminates its contribution to membrane binding.     

Association of PI3K-Akt signaling pathway with digitalis-induced hypertrophy of cardiac myocytes

Our previous studies on cardiac myocytes showed that positive inotropic concentrations of the digitalis drug ouabain activated signaling pathways linked to Na(+)-K(+)-ATPase through Src and epidermal growth factor receptor (EGFR) and led to myocyte hypertrophy. In view of the known involvement of phosphatidylinositol 3-kinase (PI3K)-Akt pathways in cardiac hypertrophy, the aim of the present study was to determine whether these pathways are also linked to cardiac Na(+)-K(+)-ATPase and, if so, to assess their role in ouabain-induced myocyte growth. In a dose- and time-dependent manner, ouabain activated Akt and phosphorylation of its substrates mammalian target of rapamycin and glycogen synthase kinase in neonatal rat cardiac myocytes. Akt activation by ouabain was sensitive to PI3K inhibitors and was also noted in adult myocytes and isolated hearts. Ouabain caused a transient increase of phosphatidylinositol 3,4,5-trisphosphate content of neonatal myocytes, activated class IA, but not class IB, PI3K, and increased coimmunoprecipitation of the alpha-subunit of Na(+)-K(+)-ATPase with the p85 subunit of class IA PI3K. Ouabain-induced activation of ERK1/2 was prevented by Src, EGFR, and MEK inhibitors, but not by PI3K inhibitors. Activation of Akt by ouabain, however, was sensitive to inhibitors of PI3K and Src, but not to inhibitors of EGFR and MEK. Similarly, ouabain-induced myocyte hypertrophy was prevented by PI3K and Src inhibitors, but not by an EGFR inhibitor. These findings 1) establish the linkage of the class IA PI3K-Akt pathway to Na(+)-K(+)-ATPase and the essential role of this linkage to ouabain-induced myocyte hypertrophy and 2) suggest cross talk between these PI3K-Akt pathways and the signaling cascades previously identified to be associated with cardiac Na(+)-K(+)-ATPase.     

Bcl-2 proteins link programmed cell death with growth and morphogenetic adaptations in the fungal plant pathogen Colletotrichum gloeosporioides

Proteins belonging to the Bcl-2 family regulate apoptosis in mammals by controlling mitochondria efflux of cytochrome c and other apoptosis-related proteins. Homologues of human Bcl-2 proteins are found in different metazoan organisms where they play a similar role, while they seem to be absent in plants and fungi. Nonetheless, Bcl-2 protein members can induce or prevent yeast cell death, suggesting that enough functional conservation exists between apoptotic machineries of mammals and fungi. Here we show that induction or prevention of apoptosis by Bcl-2 proteins in the fungal plant pathogen Colletotrichum gloeosporioides is tightly linked with growth and morphogenetic adaptation that occur throughout the entire fungal life cycle. Isolates expressing the pro-apoptotic Bax protein underwent cell death with apoptotic characteristics, and showed alterations in conidial germination that are associated with pathogenic and non-pathogenic life styles. Isolates expressing the anti-apoptotic Bcl-2 protein had prolonged longevity, were protected from Bax-induced cell death, and exhibited enhanced stress resistance. These isolates also had enhanced mycelium and conidia production, and were hyper virulent to host plants. Our findings show that apoptotic-associated machinery regulates morphogenetic switches, which are critical for proper responses and adaptation fungi to different environments.     

Tagging and detection strategies for activity-based proteomics

The field of activity-based proteomics is a relatively new discipline that makes use of small molecules, termed activity-based probes (ABPs), to tag and monitor distinct sets of proteins within a complex proteome. These activity-dependant labels facilitate analysis of systems-wide changes at the level of enzyme activity rather than simple protein abundance. While the use of small molecule inhibitors to label enzyme targets is not a new concept, the past ten years have seen a rapid expansion in the diversity of probe families that have been developed. In addition to increasing the number and types of enzymes that can be targeted by this method, there has also been an increase in the number of methods used to visualize probes once they are bound to target enzymes. In particular, the use of small organic fluorophores has created a wealth of applications for ABPs that range from biochemical profiling of diverse proteomes to direct imaging of active enzymes in live cells and even whole animals. In addition, the advent of new bioorthogonal coupling chemistries now enables a diverse array of tags to be added after targets are labeled with an ABP. This strategy has opened the door to new in vivo applications for activity-based proteomic methods.     

Transcytosis of Streptococcus iniae through skin epithelial barriers: an in vitro study

By constructing a biological model based on in vitro culture of polarized rainbow trout primary skin epithelial cell monolayers, the series of early events that precede Streptococcus iniae infection, particularly colonization and translocation through external barriers, were analyzed. Streptococcus iniae promptly invades skin epithelial cells, but the rapid decline of viable intracellular bacteria points out the limited capability of intracellular survival for this bacterium. Translocation assays, supported by electron microscopy microphotographs, demonstrate that following successful in vitro invasion of skin epithelial cell, the bacterium exists free in the cytoplasm after release from the endosome, and translocates through the skin barrier. Bacterial invasion and transcytosis is not accompanied by apparent cell-line damages or disruption of host cells' tight junctions. It is hypothesized that the phenomenon of epithelial invasion coupled to the rapid translocation through the barrier plays a crucial role in Streptococcus iniae infection.