Repletion of copper-deficient rats with dietary copper restores duodenal hephaestin protein and iron absorption

Copper (Cu) deficiency in rats reduces the relative concentration of duodenal hephaestin (Hp), reduces iron (Fe) absorption, and causes anemia. An experiment was conducted to determine whether these effects could be reversed by dietary Cu repletion. Five groups of eight weanling male rats each were used. Group 1 was fed a Cu-adequate diet (5.0 mg Cu/kg; CuA) and Group 2 was fed a Cu-deficient diet (0.25 mg Cu/kg; CuD) for 28 days. The rats were fed 1.0 g each of their respective diets labeled with 59Fe (37 kBq/g), and the amount of label retained was measured one week later by whole-body-counting (WBC). Group 3 was fed a CuA diet and Groups 4 and 5 were fed a CuD diet for 28 days. Group 5 was then fed the CuA diet for another week while Groups 3 and 4 continued on their previous regimens. Rats in Groups 3, 4, and 5 were fed 1.0 g of diet labeled with 59Fe, and the amount of label retained was measured by WBC one week later. Rats were killed and duodenal enterocytes isolated for Hp protein analysis, whole blood was analyzed for hematological parameters, and various organs for 59Fe content. CuD rats absorbed less (P<0.05) Fe than CuA rats, the relative amount of duodenal Hp was less (P<0.05) in CuD rats, and the CuD rats developed anemia. After the CuD rats had been repleted with Cu for one week, Fe retention rose to values even higher (P<0.05) than those in CuA rats. After two weeks, the relative amount of duodenal Hp was higher (P<0.05) than normal, and most signs of anemia were reversed. Liver 59Fe was elevated in CuD rats, but was restored to normal upon Cu repletion. These findings suggest a strong association between duodenal Hp abundance and Fe absorption in the CuD rat, and that reduced Fe absorption is an important factor in the cause of anemia.     

Molecular determinants of vanilloid sensitivity in TRPV1

Vanilloid receptor 1 (TRPV1), a membrane-associated cation channel, is activated by the pungent vanilloid from chili peppers, capsaicin, and the ultra potent vanilloid from Euphorbia resinifera, resiniferatoxin (RTX), as well as by physical stimuli (heat and protons) and proposed endogenous ligands (anandamide, N-arachidonyldopamine, N-oleoyldopamine, and products of lipoxygenase). Only limited information is available in TRPV1 on the residues that contribute to vanilloid activation. Interestingly, rabbits have been suggested to be insensitive to capsaicin and have been shown to lack detectable [(3)H]RTX binding in membranes prepared from their dorsal root ganglia. We have cloned rabbit TRPV1 (oTRPV1) and report that it exhibits high homology to rat and human TRPV1. Like its mammalian orthologs, oTRPV1 is selectively expressed in sensory neurons and is sensitive to protons and heat activation but is 100-fold less sensitive to vanilloid activation than either rat or human. Here we identify key residues (Met(547) and Thr(550)) in transmembrane regions 3 and 4 (TM3/4) of rat and human TRPV1 that confer vanilloid sensitivity, [(3)H]RTX binding and competitive antagonist binding to rabbit TRPV1. We also show that these residues differentially affect ligand recognition as well as the assays of functional response versus ligand binding. Furthermore, these residues account for the reported pharmacological differences of RTX, PPAHV (phorbol 12-phenyl-acetate 13-acetate 20-homovanillate) and capsazepine between human and rat TRPV1. Based on our data we propose a model of the TM3/4 region of TRPV1 bound to capsaicin or RTX that may aid in the development of potent TRPV1 antagonists with utility in the treatment of sensory disorders.     

Mediation by indole analogues of electron transfer during oxygen activation in variants of Escherichia coli ribonucleotide reductase R2 lacking the electron-shuttling tryptophan 48

Activation of dioxygen by the carboxylate-bridged diiron(II) cluster in the R2 subunit of class I ribonucleotide reductase from Escherichia coli results in the one-electron oxidation of tyrosine 122 (Y122) to a stable radical (Y122*). A key step in this reaction is the rapid transfer of a single electron from a near-surface residue, tryptophan 48 (W48), to an adduct between O(2) and diiron(II) cluster to generate a readily reducible cation radical (W48(+)(*)) and the formally Fe(IV)Fe(III) intermediate known as cluster X. Previous work showed that this electron injection step is blocked in the R2 variant with W48 replaced by phenylalanine [Krebs, C., Chen, S., Baldwin, J., Ley, B. A., Patel, U., Edmondson, D. E., Huynh, B. H., and Bollinger, J. M., Jr. (2000) J. Am. Chem. Soc. 122, 12207-12219]. In this study, we show that substitution of W48 with alanine similarly disables the electron transfer (ET) but also permits its chemical mediation by indole compounds. In the presence of an indole mediator, O(2) activation in the R2-W48A variant produces approximately 1 equiv of stable Y122* and more than 1 equiv of the normal (micro-oxo)diiron(III) product. In the absence of a mediator, the variant protein generates primarily altered Fe(III) products and only one-fourth as much stable Y122* because, as previously reported for R2-W48F, most of the Y122* that is produced decays as a consequence of the inability of the protein to mediate reductive quenching of one of the two oxidizing equivalents of the initial diiron(II)-O(2) complex. Mediation of ET is effective in W48A variants containing additional substitutions that also impact the reaction mechanism or outcome. In the reaction of R2-W48A/F208Y, the presence of mediator suppresses formation of the Y208-derived diiron(III)-catecholate product (which is predominant in R2-F208Y in the absence of reductants) in favor of Y122*. In the reaction of R2-W48A/D84E, the presence of mediator affects the outcome of decay of the peroxodiiron(III) intermediate known to accumulate in D84E variants, increasing the yield of Y122* by as much as 2.2-fold to a final value of 0.75 equiv and suppressing formation of a 490 nm absorbing product that results from decay of the two-electron oxidized intermediate in the absence of a functional ET apparatus.     

Cystitis cystica and recurrent urinary tract infections in children

The pathogenesis of recurrent urinary tract infections (UTIs) in preschool children with anatomically correct urinary tract (UT) is rather obscure. In girls, the bladder wall changes of cystitis cystica (CC) may be per se responsible for UTIs recurrence. During the 20-year period, 127 preschool children (125 girls; median age: 6.1 years) with CC, in whom UT anomalies were excluded, were diagnosed. The mean duration of UTIs symptoms prior to diagnosis was 3.31 +/- 2.51 years. Cystoscopical findings were labelled as mild, moderate and severe in 22.8%, 39.4% and 37.8% of patients, respectively. Following the confirmation of CC, long-term chemoprophylaxis with sulfamethoxazole-trimethoprim/nitrofurantoin was administered. A one year UTI-free period after chemoprophylaxis discontinuation was defined as therapeutic success. With 2.5 years median duration of regular chemoprophylaxis this goal was achieved in 58 children mainly with mild/ moderate CC. Thirty children from "improved/unchanged" group taking regular prophylaxis had significant reduction of UTIs ("improved"). Only 12 children belonging to the same group taking regular prophylaxis and all children with irregular prophylaxis had approximately the same number of UTIs as before treatment ("unchanged"). The "improved/unchanged" outcomes were predominantly found in children with severe form of CC. Although urodynamic disturbances detected in more than 50% of patients in whom urodynamics was performed were not found influential on the disease outcome, they could be responsible for its development. The results of our study suggest that regular and long-lasting chemoprophylaxis remains a basis for successful treatment for majority of patients with CC, even those with severe forms. If not treated properly with chemoprophylactic agents and without fair compliance in taking drugs, the disease is prone to recurrent UTIs.     

Loss of escape-related giant neurons in a spiny lobster, Panulirus argus

When attacked, many decapod crustaceans perform tailflips, which are triggered by a neural circuit that includes lateral giant interneurons, medial giant interneurons, and fast flexor motor giant neurons (MoGs). Slipper lobsters (Scyllaridae) lack these giant neurons, and it has been hypothesized that behavioral (e.g., digging) and morphological (e.g., flattening and armor) specializations in this group caused the loss of escape-related giant neurons. To test this hypothesis, we examined a species of spiny lobster, Panulirus argus. Spiny lobsters belong to the sister taxon of the scyllarids, but they have a more crayfish-like morphology than scyllarids and were predicted to have escape-related giant neurons. Ventral nerve cords of P. argus were examined using paraffin-embedded sections and cobalt backfills. We found no escape-related giant neurons and no large axon profiles in the dorsal region of the nerve cord of P. argus. Cobalt backfills showed one fewer fast flexor motor neuron than in species with MoGs and none of the fast flexor motor neurons show any of the anatomical specializations of MoGs. This suggests that all palinuran species lack this giant escape circuit, and that the loss of rapid escape behavior preceded, and may have driven, alternative predator avoidance and anti-predator strategies in palinurans.     

Anatomical Characteristics of Cherry Rootstocks as Possible Preselecting Tools for Prediction of Tree Vigor

An anatomical study of roots and stems of five self-rooted cherry rootstocks with different growth control potentials was performed to compare their structure and xylem anatomy. The aim was to correlate anatomical parameters with rootstock dwarfing potential and theoretical hydraulic conductance (k _h), and to evaluate the potential application of anatomical characteristics in the preselection process for prediction of ultimate tree vigor. One of the mechanisms of water transport efficiency reduction in dwarfing rootstock stems is from the rootstock xylem anatomy. Anatomical parameters of ??Gisela 5?? and ??Mazzard?? were typical for dwarfing and vigorous rootstocks, respectively, and were thus suggested as reference rootstocks. Significantly greater vessel diameter and frequency were found in invigorating and dwarfing rootstocks, respectively. Higher k _h was obtained in roots, compared to stems, due to significantly larger vascular elements. Dwarfing rootstocks had lower k _h due to small vessel lumens and percentage and, to a lesser extent, because of low wood/cortex ratios or percentage of wood. A higher percentage of wood or xylem in cherry roots and stems was not always positively correlated with their conductivity and vigor. Thus, these parameters cannot be reliably used in prediction of the ultimate vigor, although this method was previously suggested for some other fruit tree species. The most reliable anatomical parameters for that purpose proved to be vessel frequency, vessel lumen area, and percentage of vessels on wood cross section. These characteristics could thus be an effective way to estimate dwarfing capacity and could be applied in rootstock selection and breeding programs.     

Performance of LBSap Vaccine after Intradermal Challenge with L. infantum and Saliva of Lu. longipalpis: Immunogenicity and Parasitological Evaluation

In the last decade, the search for new vaccines against canine visceral leishmaniasis has intensified. However, the pattern related to immune protection during long periods after experimental infection in vaccine trials is still not fully understood. Herein, we investigated the immunogenicity and parasitological levels after intradermal challenge with Leishmania infantum plus salivary gland extract in dogs immunized with a vaccine composed of L. braziliensis antigens plus saponin as an adjuvant (LBSap vaccine). The LBSap vaccine elicited higher levels of total anti-Leishmania IgG as well as both IgG1 and IgG2. Furthermore, dogs vaccinated had increased levels of lymphocytes, particularly circulating B cells (CD21⁺) and both CD4⁺ and CD8⁺ T lymphocytes. LBSap also elicited an intense in vitro cell proliferation associated with higher levels of CD4⁺ T lymphocytes specific for vaccine soluble antigen and soluble lysate of L. infantum antigen even 885 days after experimental challenge. Furthermore, LBSap vaccinated dogs presented high IFN-γ and low IL-10 and TGF-β1 expression in spleen with significant reduction of parasite load in this tissue. Overall, our results validate the potential of LBSap vaccine to protect against L. infantum experimental infection and strongly support further evaluation of efficiency of LBSap against CVL in natural infection conditions.     

Trypanosoma cruzi: Induction of benznidazole resistance in vivo and its modulation by in vitro culturing and mice infection

Through a continuous in vivo drug pressure protocol, using mice as experimental model, we induced benznidazole resistance in Trypanosoma cruzi stocks. Full resistance was obtained for four out of five T. cruzi stocks analyzed. However, the number of benznidazole doses (40–180), as well as the time (4–18 months) necessary to induce resistance varied among the different T. cruzi stocks. The resistance phenotype remained stable after T. cruzi stocks has been maintained by 12 passages in mice (six months) and in acellular culture for the same time. However, the maintenance of resistant parasite for 12 months in acellular culture induces a reduction in its level of benznidazole resistance, while no alteration was detected in parasite maintained for the same time in mice. The data showed the stability of the resistance acquired by drug pressure, but suggest the possibility of reversible changes in the resistance levels after maintenance for long time in acellular culture.     

Cytokine release and expression induced by OmpA proteins from the Gram-negative anaerobes, Porphyromonas asaccharolytica and Bacteroides fragilis

OmpA proteins from Gram-negative anaerobes Porphyromonas asaccharolytica and Bacteroides fragilis induced release and expression of IL-1alpha, tumor necrosis factor (TNF)-alpha, IFN-gamma, IL-6, and IL-10 from murine splenocytes in vitro in a dose-dependent fashion. The release of the cytokines induced by B. fragilis Bf-OmpA was at much lower levels compared with P. asaccharolytica Omp-PA; Bf-OmpA did not induce release of IL-10. Omp-PA and Bf-OmpA were able to upregulate mRNA expression of the tested cytokines. The results obtained with refolded Bf-OmpA were similar to those with native Bf-OmpA. The data presented in this research demonstrate for the first time that Omps from anaerobic bacteria can induce the release of cytokines, suggesting that Omp-PA and Bf-OmpA may play important roles in the pathogenic processes of these bacteria.