HCO3- secretion in the esophageal submucosal glands

The mammalian esophagus has the capacity to secrete a HCO(3)(-) and mucin-rich fluid in the esophageal lumen. These secretions originate from the submucosal glands (SMG) and can contribute to esophageal protection against refluxed gastric acid. The cellular mechanisms by which glandular cells achieve these secretions are largely unknown. To study this phenomenon, we used the pH-stat technique to measure luminal alkali secretion in an isolated, perfused pig esophagus preparation. Immunohistochemistry was used to localize receptors and transporters involved in HCO(3)(-) transport. The SMG-bearing esophagus was found to have significant basal alkali secretion, predominantly HCO(3)(-), which averaged 0.21 +/- 0.04 microeq.h(-1).cm(-2). This basal secretion was doubled when stimulated by carbachol but abolished by HCO(3)(-) or Cl(-) removal. Basal- and carbachol-stimulated secretions were also blocked by serosal application of atropine, pirenzipine, DIDS, methazolamide, and ethoxzolamide. The membrane-impermeable carbonic anhydrase inhibitor benzolamide, applied to the serosal bath, partially inhibited basal HCO(3)(-) secretion and blocked the stimulation by carbachol. Immunohistochemistry using antibodies to M(1) cholinergic receptor or carbonic anhydrase-II enzyme showed intense labeling of duct cells and serous demilunes but no labeling of mucous cells. Labeling with an antibody to Na(+)-(HCO(3)(-))(n) (rat kidney NBC) was positive in ducts and serous cells, whereas labeling for Cl(-)/HCO(3)(-) exchanger (AE2) was positive in duct cells but less pronounced in serous cells. These data indicate that duct cells and serous demilunes of SMG play a role in HCO(3)(-) secretion, a process that involves M(1) cholinergic receptor stimulation. HCO(3)(-) transport in these cells is dependent on cytosolic and serosal membrane-bound carbonic anhydrase. HCO(3)(-) secretion is also dependent on serosal Cl(-) and is mediated by DIDS-sensitive transporters, possibly NBC and AE2.     

Use of the paired samples (cerebrospinal fluid and serum) in immunodiagnostic of active and inactive human neurocysticercosis

Paired samples of cerebrospinal fluid (CSF) and serum of 30 patients--10 with active, 10 with inactive neurocysticercosis (NCC), and 10 control subjects--were evaluated by enzyme-linked immunosorbent assay (ELISA) using two Taenia crassiceps metacestode extracts as antigen in order to detect IgG antibodies. In active NCC, high levels of IgG were detected (p < 0.05). The CSF samples showed 80% (CI 72-88) of reactivity in the saline extract (S) and 90% (CI 84-95) in sodium dodecyl sulphate (SDS) and the serum samples were reactive in 90% (CI 84-95) and 100% (CI 98-100) in the S and SDS antigenic extracts, respectively. The use of the paired samples of CSF and serum in active NCC showed equivalent results suggesting that the serum samples could be used as a screening in those patients whose CSF puncture is counter-indicated.     

Purification of Xylitol from Fermented Hemicellulosic Hydrolyzate Using Liquid–Liquid Extraction and Precipitation Techniques

Xylitol was produced by Candida guilliermondii by fermentation of sugarcane bagasse hemicellulosic hydrolysate. Undesirable impurities were extracted from the broth using either ethyl acetate, chloroform or dichloromethane. The best results on clarification of the broth without xylitol loss were obtained with ethyl acetate. When ethanol, acetone or tetrahydrofuran were used for precipitation of impurities, only tetrahydrofuran clarified the fermented broth, but a high xylitol loss (~30%) was observed.     

The ischemic rat heart releases S100B

S100B is an astrocytic protein assessed in cerebrospinal fluid and serum as a biochemical marker of cerebral injuries. However, increasing evidences suggest the influence of extra cerebral sources on its serum levels. Since it was reported that the injured myocardium expresses S100B, we investigated whether the isolated heart releases this protein. The rat hearts were excised and perfused by the Langendorff technique of isolated heart perfusion. After stabilization, 10 hearts (ischemic group) were submitted to 20 minutes of ischemia and 30 minutes of reperfusion, and 5 hearts (control group) were submitted to 50 minutes of perfusion. The perfusion fluid was collected at pre-ischemia, and 0, 5, 10, 15 and 30 min after ischemia (or equivalent in controls) for S100B and cardiac troponin T (a heart injury marker) assays. In the ischemic group, S100B and troponin T levels increased significantly at time 0 min: S100B values [mug/L, median (IQ25/IQ75)] increased from < or = 0.02 (< or = 0.02/0.03) to 0.38 (0.22/0.84), while troponin T values [mug/L, median (IQ25/IQ75)] increased from 0.31 (0.15/0.45) to 2.84 (2.00/3.63). Our results point to the ischemic heart as an extra cerebral source of S100B.     

Synthesis and characterisation of [[μ-(1,7,11,17-tetraaza-2,6,12,16-tetraoxacycloeicosane)][(μ-(aquo)]bis[(acac)(ethanol)nickel(II)]] [BPh4]2

The pale blue title compound, 4, was obtained from the reaction between 1,7,11,17-tetraaza-2,6,12,16-tetraoxacycloeicosane, Ni(acac)₂ and NaBPh₄ in aqueous acetone. X-ray structure determination at 120 K revealed that the dication of the ionic complex, 4, contains two independent octahedral Ni^II centres with trans-Ni₂N₂O₄ chromophores. The macrocyclic ligand and an aqua ligand act as bridges to the two nickel centres: the Ni-O(aquo)-N bond angle is 137.65(17)°. Each Ni centre is bonded to two nitrogens of the macrocycle, to a chelating acac unit, to an ethanol molecule as well as the bridging oxygen of the aqua group. The two nickel atoms sit outside the macrocycle cavity, such that the macrocyclic ligand acts as a canopy for the remainder of the dication. While none of the macrocycle oxygens are involved in the coordination to Ni, they are involved in internal hydrogen bonding with the aqua and ethanol ligands. Magnetic measurements show a paramagnetic behaviour down to 2 K, with an effective moment of 2.8 Bohr magnetons at room temperature.     

Intracellular positioning of isoforms explains an unusually large adenylate kinase gene family in the parasite Trypanosoma brucei

Adenylate kinases occur classically as cytoplasmic and mitochondrial enzymes, but the expression of seven adenylate kinases in the flagellated protozoan parasite Trypanosoma brucei (order, Kinetoplastida; family, Trypanosomatidae) easily exceeds the number of isoforms previously observed within a single cell and raises questions as to their location and function. We show that a requirement to target adenylate kinase into glycosomes, which are unique kinetoplastid-specific microbodies of the peroxisome class in which many reactions of carbohydrate metabolism are compartmentalized, and two different flagellar structures as well as cytoplasm and mitochondrion explains the expansion of this gene family in trypanosomes. The three isoforms that are selectively built into either the flagellar axoneme or the extra-axonemal paraflagellar rod, which is essential for motility, all contain long N-terminal extensions. Biochemical analysis of the only short form trypanosome adenylate kinase revealed that this enzyme catalyzes phosphotransfer of gamma-phosphate from ATP to AMP, CMP, and UMP acceptors; its high activity and specificity toward CMP is likely to reflect an adaptation to very low intracellular cytidine nucleotide pools. Analysis of some of the phosphotransfer network using RNA interference suggests considerable complexity within the homeostasis of cellular energetics. The anchoring of specific adenylate kinases within two distinct flagellar structures provides a paradigm for metabolic organization and efficiency in other flagellates.     

Toxoplasma gondii: isolation of tachyzoites rhoptries and incorporation into Iscom

Rhoptries have been isolated from Toxoplasma gondii tachyzoites by subcellular fractionation in isopynic density sucrose gradient. Five bands were observed, and transmission electron microscopy of these indicated that rhoptries were in band 3. This band had a density of 1.17 g/cm(3). Fraction 1 had membrane structures of the parasite. Fraction 2 contained membranes and mitochondria. Fraction 4 had mostly conoid structure and fraction 5 showed ghosts. The electrophoretic and Western blotting analysis of the fractions indicated the presence of a number of proteins. Iscoms were constructed from band 3, which contained the rhoptry structures. Iscom showed a only protein incorporated of 55 kDa. Isolation of the parasite organelles has got in this work is necessary to identification, characterization, and function elucidation of the organelle proteins.     

The fourth transmembrane segment of the Na,K-ATPase alpha subunit: a systematic mutagenesis study

The Na,K-ATPase is a major ion-motive ATPase of the P-type family responsible for many aspects of cellular homeostasis. To determine the structure of the pathway for cations across the transmembrane portion of the Na,K-ATPase, we mutated 24 residues of the fourth transmembrane segment into cysteine and studied their function and accessibility by exposure to the sulfhydryl reagent 2-aminoethyl-methanethiosulfonate. Accessibility was also examined after treatment with palytoxin, which transforms the Na,K-pump into a cation channel. Of the 24 tested cysteine mutants, seven had no or a much reduced transport function. In particular cysteine mutants of the highly conserved "PEG" motif had a strongly reduced activity. However, most of the non-functional mutants could still be transformed by palytoxin as well as all of the functional mutants. Accessibility, determined as a 2-aminoethyl-methanethiosulfonate-induced reduction of the transport activity or as inhibition of the membrane conductance after palytoxin treatment, was observed for the following positions: Phe(323), Ile(322), Gly(326), Ala(330), Pro(333), Glu(334), and Gly(335). In accordance with a structural model of the Na,K-ATPase obtained by homology modeling with the two published structures of sarcoplasmic and endoplasmic reticulum calcium ATPase (Protein Data Bank codes 1EUL and 1IWO), the results suggest the presence of a cation pathway along the side of the fourth transmembrane segment that faces the space between transmembrane segments 5 and 6. The phenylalanine residue in position 323 has a critical position at the outer mouth of the cation pathway. The residues thought to form the cation binding site II ((333)PEGL) are also part of the accessible wall of the cation pathway opened by palytoxin through the Na,K-pump.     

Tracking Trojan peptides in cells

Trojan peptides or cell-penetrating peptides (CPP) are natural or designed peptides identified as cellular membrane-crossing molecules, in particular through their potency to vehiculate various kinds of compounds to the cytoplasm and nucleus of living cells. The indirect methods used so far to detect these peptides in cells led to controversial hypotheses on the mechanism of their cell entry. Therefore, we have developed a MALDI-TOF mass spectrometry-based quantification method to track these peptides inside cells. This new method is presented in this review.     

The effect of Bulgarian propolis against Trypanosoma cruzi and during its interaction with host cells

Propolis has shown activity against pathogenic microorganisms that cause diseases in humans and animals. The ethanol (Et-Blg) and acetone (Ket-Blg) extracts from a Bulgarian propolis, with known chemical compositions, presented similar activity against tissue culture-derived amastigotes. The treatment of Trypanosoma cruzi-infected skeletal muscle cells with Et-Blg led to a decrease of infection and of the intracellular proliferation of amastigotes, while damage to the host cell was observed only at concentration 12.5 times higher than those affecting the parasite. Ultrastructural analysis of the effect of both extracts in epimastigotes revealed that the main targets were the mitochondrion and reservosomes. Et-Blg also affected the mitochondrion-kinetoplast complex in trypomastigotes, offering a potential target for chemotherapeutic agents.