Soil morphological control on saline and freshwater lake hydrogeochemistry in the Pantanal of Nhecolandia, Brazil

Joint pedological, geochemical, hydrological and geophysical investigations were performed to study the coexistence or saline and freshwater lakes in close proximity and similar climatic conditions in the Nhecolandia region, Pantanal wetlands in Brazil. The saline lakes are concentrically surrounded by green sandy loam horizons, which cause differential hydrological regimes. Mg-calcite, K-silicates, and amorphous silica precipitate in the soil cover, whereas Mg-silicates and more soluble Na-carbonates are concentrated in the topsoil along the shore of the saline lake. In saline solutions, some minor elements (As, Se) reach values above the water quality recommendations, whereas others are controlled and incorporated in solid phases (Ba, Sr). Locally, the destruction of the sandy loam horizons generates very acidic soil solution (pH similar to 3.5) through a process not yet understood. The soil distributions indicate that some freshwater lakes are former saline lakes. They are invaded by freshwater after destruction of the sandy loam green horizons, then the freshwater becomes enriched in K(+), SO(4)(2-), Fe, Al, and a stream of minor and trace elements. The formation of these green sandy loam horizons in the saline environment and their destruction in the non-saline one emphasizes the dynamic nature of this environment (C) 2008 Elsevier B.V. All rights reserved.

Defective NF-kappaB Activation in Response to LPS in Type 1 Diabetes Dendritic Cells

Dendritic cells (DC) are the potent antigen presenting cells which modulate T cell responses to self or non-self antigens. DC play a significant role in the pathogenesis of autoimmune diseases, inflammation and infection, but also in the maintenance of tolerance. NF-kappaB, particularly RelB is a crucial pathway for myeloid DC differentiation and functional maturation. While the current paradigm is that mature, nuclear RelB+ DC prime T cells for immunity/autoimmunity and immature DC for tolerance, RelB-deficient mice paradoxically develop generalised systemic autoimmune inflammatory disease with myelopoiesis and splenomegaly. Previous studies suggested abnormal DC differentiation in healthy relatives of type 1 diabetes (t1dm) patients. Therefore, we compared NF- kB activation in monocyte-derived DC from t1dm and non-t1dm controls in response to LPS. While resting DC appeared normal, DC from 6 out of 7 t1dm patients but no t2dm or rheumatoid arthritis patients failed to translocate NF- kB subunits to the nucleus in response to LPS, along with a failure to up-regulate expression of cell surface CD40 and MHC class I. NF- kB subunit mRNA increased normally in t1dm DC after LPS. Both the classical or non-canonical NF- kB pathways were affected as both TNF-a and CD40 stimulation led to a similarly abnormal NF- kB response. In contrast, expression of phosphorylated p38 MAPK and pro-inflammatory cytokine production was intact. These abnormalities in NF- kB activation appear to be generally and specifically applicable at a post-translational level in t1dm, and have the capacity to profoundly influence immunoregulation in affected individuals.

Detrusor overactivity is associated with downregulation of large-conductance calcium- and voltage-activated potassium channel protein

Chang S, Gomes CM, Hypolite JA, Marx J, Alanzi J, Zderic SA, Malkowicz B, Wein AJ, Chacko S. Detrusor overactivity is associated with downregulation of large-conductance calcium-and voltage-activated potassium channel protein. Am J Physiol Renal Physiol 298: F1416-F1423, 2010. First published April 14, 2010; doi: 10.1152/ajprenal.00595.2009.-Large-conductance voltage-and calcium-activated potassium (BK) channels have been shown to play a role in detrusor overactivity (DO). The goal of this study was to determine whether bladder outlet obstructioninduced DO is associated with downregulation of BK channels and whether BK channels affect myosin light chain 20 (MLC(20)) phosphorylation in detrusor smooth muscle (DSM). Partial bladder outlet obstruction (PBOO) was surgically induced in male New Zealand White rabbits. The rabbit PBOO model shows decreased voided volumes and increased voiding frequency. DSM from PBOO rabbits also show enhanced spontaneous contractions compared with control. Both BK channel alpha- and beta-subunits were significantly decreased in DSM from PBOO rabbits. Immunostaining shows BK beta mainly expressed in DSM, and its expression is much less in PBOO DSM compared with control DSM. Furthermore, a translational study was performed to see whether the finding discovered in the animal model can be translated to human patients. The urodynamic study demonstrates several overactive DSM contractions during the urine-filling stage in benign prostatic hyperplasia (BPH) patients with DO, while DSM is very quiet in BPH patients without DO. DSM biopsies revealed significantly less BK channel expression at both mRNA and protein levels. The degree of downregulation of the BK beta-subunit was greater than that of the BK alpha-subunit, and the downregulation of BK was only associated with DO, not BPH. Finally, the small interference (si) RNA-mediated downregulation of the BK beta-subunit was employed to study the effect of BK depletion on MLC(20) phosphorylation. siRNA-mediated BK channel reduction was associated with an increased MLC(20) phosphorylation level in cultured DSM cells. In summary, PBOO-induced DO is associated with downregulation of BK channel expression in the rabbit model, and this finding can be translated to human BPH patients with DO. Furthermore, downregulation of the BK channel may contribute to DO by increasing the basal level of MLC(20) phosphorylation.

Fractionation of follicle stimulating hormone charge isoforms in their native form by preparative electrophoresis technology

Complex glycoprotein biopharmaceuticals, such as follicle stimulating hormone (FSH), erythropoietin and tissue plasminogen activator consist of a range of charge isoforms due to the extent of sialic acid capping of the glycoprotein glycans. Sialic acid occupies the terminal position on the oligosaccharide chain, masking the penultimate sugar residue, galactose from recognition and uptake by the hepatocyte asialoglycoprotein receptor. It is therefore well established that the more acidic charge isoforms of glycoprotein biopharmaceuticals have higher in vivo potencies than those of less acidic isoforms due to their longer serum half-life. Current strategies for manipulating glycoprotein charge isoform profile involve cell engineering or altering bioprocesss parameters to optimise expression of more acidic or basic isoforms, rather than downstream separation of isoforms. A method for the purification of a discrete range of bioactive recombinant human FSH (rhFSH) charge isoforms based on Gradiflow(TM) preparative electrophoresis technology is described. Gradiflow(TM) electrophoresis is scaleable, and incorporation into glycoprotein biopharmaceutical production bioprocesses as a potential final step facilitates the production of biopharmaceutical preparations of improved in vivo potency. (C) 2005 Elsevier B.V. All rights reserved.

The systematics and biology of the south African gall-inducing scale insect, Calycicoccus merwei brain (Hemiptera : Coccoidea : Eriococcidae)

The scale insect genus Calycicoccus Brain has a single described species, C. merwei Brain, which is endemic to southeastern South Africa. Females of C. merwei induce small, mostly conical galls on the foliage of their host tree, Apodytes dimidiata E. Meyer ex Arn. (Icacinaceae), which has a wider, mostly coastal distribution, than that currently known for the scale insect. Calycicoccus has been placed in the family Eriococcidae and may be related to the South American genus Aculeococcus Lepage. No other native eriococcid species have been described so far in South Africa, although the family is diverse in other Gondwanan regions. This paper summarizes the biology of C. merwei, redescribes the adult female, describes the adult male, the second-instar female and the first-instar nymphs for the first time, and reconsiders the phylogenetic relationships of the genus. The adult female is shown to have unusual abdominal segmentation, in that segment I is present both dorsally and ventrally, but a segment is absent ventrally on the middle abdomen. First-instar nymphs are sexually dimorphic; males have a larger and relatively narrower body, larger mouthparts, longer antennae and legs, and more thoracic dorsal setae compared with females. Molecular data from nuclear small-subunit ribosomal DNA (18S) and elongation factor 1 alpha (EF-1a) show C. merwei to have no close relatives among the Eriococcidae sampled to date. Instead, the Calycicoccus lineage is part of a polytomy near the base of the Eriococcidae. Molecular dating of the node suggests that the Calycicoccus lineage diverged from other eriococcids more than 100 Mya. These data support the placement of Calycicoccus as the only genus in the subfamily Calycicoccinae Brain.

Leishmania (Viannia) braziliensis identification by PCR in the state of Para, Brazil

The incidence of cutaneous leishmaniasis (CL) is increasing and there is limited surveillance of Leishmania species throughout the world. We identified the species associated with CL in a region of Amazonia, an area recognized for its Leishmania species variability. Clinical findings were analyzed and correlated with the species identified in 93 patients. PCR assays were based on small subunit ribosomal DNA (SSU-rDNA) and G6PD, and were performed in a laboratory located 3,500 km away. Leishmania (V.) braziliensis was identified in 53 patients (57%). The other 40 patients (43%) carried a different species (including six cases of L (L) amazonensis). Molecular methods can be employed, using special media, to allow transport to distant laboratories. L (V.) braziliensis is the most common species in the area of Para. The location of ulcers can suggest CL species (C) 2010 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Giant protein kinases: Domain interactions and structural basis of autoregulation

The myosin-associated giant protein kinases twitchin and titin are composed predominantly of fibronectin- and immunoglobulin-like modules, We report the crystal structures of two autoinhibited twitchin kinase fragments, one from Aplysia and a larger fragment from Caenorhabditis elegans containing an additional C-terminal immunoglobulin-like domain, The structure of the longer fragment shoes that the immunoglobulin domain contacts the protein kinase domain on the opposite side from the catalytic cleft, laterally exposing potential myosin binding residues, Together, the structures reveal the cooperative interactions between the autoregulatory region and the residues from the catalytic domain involved in protein substrate binding, ATP binding, catalysis and the activation loop, and explain the differences between the observed autoinhibitory mechanism and the one found in the structure of calmodulin-dependent kinase I.

Biological activity and metabolic clearance of recombinant human follicle stimulating hormone produced in Sp2/0 myeloma cells

Human follicle stimulating hormone is a pituitary glycoprotein that is essential for the maintenance of ovarian follicle development and testicular spermatogenesis. Like other members of the glycoprotein hormone family, it contains a common a subunit and a hormone specific beta subunit. Each subunit contains two glycosylation sites. The specific structures of the oligosaccharides of human follicle stimulating hormone have been shown to influence both the in vitro and in vivo bioactivity. Since the carbohydrate structure of a protein reflects the glycosylation apparatus of the host cells in which the protein is expressed, we examined the isoform profiles, in vitro bioactivity and metabolic clearance of a preparation of purified recombinant human follicle stimulating hormone derived from a stable, transfected Sp2/0 myeloma cell line, and pituitary human follicle stimulating hormone. Isoelectric focussing and chromatofocussing studies of human follicle stimulating hormone preparations both showed a more basic isoform profile for the recombinant human follicle stimulating hormone compared to that of pituitary human follicle stimulating hormone. The recombinant human follicle stimulating hormone had a significantly higher radioreceptor activity compared to that of pituitary human follicle stimulating hormone, consistent with a greater in vitro potency. Pharmacokinetic studies in rats indicated a similar terminal half life (124 min) to that of the pituitary human follicle stimulating hormone (119 min). Preliminary carbohydrate analysis showed recombinant human follicle stimulating hormone to contain high mannose and/or hybrid type, in addition to complex type carbohydrate chains, terminating with both alpha 2,3 and alpha 2,6 linked sialic acids. These results demonstrate that recombinant human follicle stimulating hormone made in the Sp2/0 myeloma cells is sialylated, has a more basic isoform profile, and has a greater in vitro biological potency compared to those of the pituitary human follicle stimulating hormone.

Antibacterial activity of bovine lactoferrin-derived peptides

Several peptides sharing high sequence homology with lactoferricin B (Lf-cin B) were generated from bovine lactoferrin (Lf) with recombinant chymosin. Two peptides were copurified. one identical to Lf-cin B and another differing from Lf-cin B by the inclusion of a C-terminal alanine (lactoferricin). Two other peptides were copurified from chymosin-hydrolyzed Lf. one differing from Lf-cin B by the inclusion of C-terminal alanyl-leucine and the other being a heterodimer linked by a disulfide bond, These peptides were isolated in a single step from chymosin-hydrolyzed Lf by membrane ton-exchange chromatography and were purified by reverse-phase high-pressure liquid chromatography (HPLC), They were characterized by. N-terminal Edman sequencing, mass spectrometry, and antibacterial activity determination, Pure lactoferricin, prepared from pepsin-hydrolyzed Lf, was purified by standard chromatography techniques, This peptide was analyzed against a number of gram-positive and gram-negative bacteria before and after reduction of its disulfide bond or cleavage after its single methionine residue and was found to inhibit the growth of all the test bacteria at a concentration of 8 mu M or less, Subfragments of lactoferricin were isolated from reduced and cleaved peptide by reverse-phase HPLC, Subfragment 1 (residues I to 10) was active against most of the test microorganisms at concentrations of 10 to 50 mu M. Subfragment 2 (residues 11 to 26) was active against only a few microorganisms at concentrations up to 100 mu M. These antibacterial studies indicate that the activity of lactoferricin Is mainly, but not wholly, due to its N-terminal region.

Identification of intracellular and extracellular domains mediating signal transduction in the inhibitory glycine receptor chloride channel

Fast synaptic neurotransmission is mediated by transmitter-activated conformational changes in ligand-gated ion channel receptors, culminating in opening of the integral ion channel pore. Human hereditary hyperekplexia, or startle disease, is caused by mutations in both the intracellular or extracellular loops flanking the pore-lining M2 domain of the glycine receptor alpha 1 subunit. These flanking domains are designated the M1-M2 loop and the M2-M3 loop respectively. We show that four startle disease mutations and six additional alanine substitution mutations distributed throughout both loops result in uncoupling of the ligand binding sites from the channel activation gate. We therefore conclude that the M1-M2 and M2-M3 loops act in parallel to activate the channel. Their locations strongly suggest that they act as hinges governing allosteric control of the M2 domain. As the members of the ligand-gated ion channel superfamily share a common structure, this signal transduction model may apply to all members of this superfamily.

Identification of the alpha(6) integrin as a candidate receptor for papillomaviruses

Papillomaviruses (PVs) bind in a specific and saturable fashion to a range of epithelial and other cell lines. Treatment of cells with trypsin markedly reduces their ability to bind virus particles, suggesting that binding is mediated via a cell membrane protein. We have investigated the interaction bf human PV type 6b L1 virus-like particles (VLPs) with two epithelial cell lines, CV-1 and HaCaT, which bind VLPs, and a B-cell line (DG75) previously shown not to bind VLPs. Immunoprecipitation of a mixture of PV VLPs with [S-35]methionine-labeled cell extracts and with biotin-labeled cell surface proteins identified four proteins from CV-1 and HaCaT cells of 220, 120, 87, and 35 kDa that reacted with VLPs and were not present in DG75 cells. The alpha(6) beta(4) integrin complex has subunits corresponding to the VLP precipitated proteins, and the tissue distribution of this complex suggested that it was a candidate human PV receptor. Monoclonal antibodies (MAbs) to the alpha(6) or beta(4) integrin subunits precipitated VLPs from a mixture of CV-1 cell proteins and VLPs, whereas MAbs to other integrin subunits did not. An alpha(6) integrin-specific MAb (GoH3) inhibited VLP binding to CV-1 and HaCaT cells, whereas an anti-beta(4) integrin MAb and a range of integrin-specific and other MAbs did not. Furthermore, human laminin, the natural ligand for the alpha(6) beta(4) integrin, was able to block VLP binding. By use of sections of monkey esophagus, the distribution of alpha(6), integrin expression in the basal epithelium was shown to coincide with the distribution of bound VLPs. Taken together, these data suggest that VLPs bind specifically to the alpha(6) integrin subunit and that integrin complexes containing alpha(6) integrin complexed with either beta(1) or beta(4) integrins may act as a receptor for PV binding and entry into epithelial cells.

Protein disulfide isomerase redox-dependent association with p47(phox): evidence for an organizer role in leukocyte NADPH oxidase activation

Mechanisms of leukocyte NADPH oxidase regulation remain actively investigated. We showed previously that vascular and macrophage oxidase complexes are regulated by the associated redox chaperone PDI. Here, we investigated the occurrence and possible underlying mechanisms of PDI-mediated regulation of neutrophil NADPH oxidase. In a semirecombinant cell-free system, PDI inhibitors scrRNase (100 mu g/mL) or bacitracin (1 mM) near totally suppressed superoxide generation. Exogenously incubated, oxidized PDI increased (by similar to 40%), whereas PDIred diminished (by similar to 60%) superoxide generation. No change occurred after incubation with PDI serine-mutated in all four redox cysteines. Moreover, a mimetic CxxC PDI inhibited superoxide production by similar to 70%. Thus, oxidized PDI supports, whereas reduced PDI down-regulates, intrinsic membrane NADPH oxidase complex activity. In whole neutrophils, immunoprecipitation and colocalization experiments demonstrated PDI association with membrane complex subunits and prominent thiol-mediated interaction with p47(phox) in the cytosol fraction. Upon PMA stimulation, PDI was mobilized from azurophilic granules to cytosol but did not further accumulate in membranes, contrarily to p47(phox). PDI-p47(phox) association in cytosol increased concomitantly to opposite redox switches of both proteins; there was marked reductive shift of cytosol PDI and maintainance of predominantly oxidized PDI in the membrane. Pulldown assays further indicated predominant association between PDIred and p47(phox) in cytosol. Incubation of purified PDI (> 80% reduced) and p47(phox) in vitro promoted their arachidonate-dependent association. Such PDI behavior is consistent with a novel cytosolic regulatory loop for oxidase complex (re) cycling. Altogether, PDI seems to exhibit a supportive effect on NADPH oxidase activity by acting as a redox-dependent enzyme complex organizer. J. Leukoc. Biol. 90: 799-810; 2011.