A New Conformation in Sarcoplasmic Reticulum Calcium Pump and Plasma Membrane Ca2+ Pumps Revealed by a Photoactivatable Phospholipidic Probe

The purpose of this work was to obtain structural information about conformational changes in the membrane region of the sarcoplasmic reticulum (SERCA) and plasma membrane (PMCA) Ca²⁺ pumps. We have assessed changes in the overall exposure of these proteins to surrounding lipids by quantifying the extent of protein labeling by a photoactivatable phosphatidylcholine analog 1-palmitoyl-2-[9-[2′-[¹²⁵I]iodo-4′-(trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]-sn-glycero-3-phosphocholine ([¹²⁵I]TID-PC/16) under different conditions. We determined the following. 1) Incorporation of [¹²⁵I]TID-PC/16 to SERCA decreases 25% when labeling is performed in the presence of Ca²⁺. This decrease in labeling matches qualitatively the decrease in transmembrane surface exposed to the solvent calculated from crystallographic data for SERCA structures. 2) Labeling of PMCA incubated with Ca²⁺ and calmodulin decreases by approximately the same amount. However, incubation with Ca²⁺ alone increases labeling by more than 50%. Addition of C28, a peptide that prevents activation of PMCA by calmodulin, yields similar results. C28 has also been shown to inhibit ATPase SERCA activity. Interestingly, incubation of SERCA with C28 also increases [¹²⁵I]TID-PC/16 incorporation to the protein. These results suggest that in both proteins there are two different E₁ conformations as follows: one that is auto-inhibited and is in contact with a higher amount of lipids (Ca²⁺ + C28 for SERCA and Ca²⁺ alone for PMCA), and one in which the enzyme is fully active (Ca²⁺ for SERCA and Ca²⁺-calmodulin for PMCA) and that exhibits a more compact transmembrane arrangement. These results are the first evidence that there is an autoinhibited conformation in these P-type ATPases, which involves both the cytoplasmic regions and the transmembrane segments.Although membrane proteins constitute more than 20% of the total proteins, the structure of only few of them is known in detail. An important group of integral membrane proteins are ion-motive ATPases. These proteins belong to the family of P-type ATPases, which share in common the formation of an acid-stable phosphorylated intermediate as part of its reaction cycle. Crystallographic information is available for a few members of this family. There are several crystal structures of the Ca²⁺ pump of sarcoplasmic reticulum (SERCA)² revealing different conformations (1–5), and recently, crystal structures of the H⁺-ATPase (6) and of the Na,K-ATPase were reported as well (7).We are interested in obtaining structural information about the plasma membrane calcium pump (PMCA). This pump is an integral part of the Ca²⁺ signaling mechanism (8). It is highly regulated by calmodulin, which activates this protein by binding to an auto-inhibitory region and changing the conformation of the pump from an inhibited state to an activated one (8, 9). Crystallization of PMCA is particularly challenging because there is no natural source from which this protein can be obtained in large quantities. Moreover, the presence of several isoforms in the same tissue further complicates efforts to obtain a homogeneous sample suitable for crystallization.Information about the structure and assembly of the transmembrane domain of an integral membrane protein can also be obtained from the analysis of the lipid-protein interactions. In this work, we have used a hydrophobic photolabeling method to study the noncovalent interactions between PMCA and the surrounding phospholipids under different experimental conditions that lead to known conformations. We employed the photoactivatable phosphatidylcholine analog 1-palmitoyl-2-[9-[2′-[¹²⁵I]iodo-4′-(trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]-sn-glycero-3-phosphocholine ([¹²⁵I]TID-PC/16) that has been previously used to analyze lipid-protein interfaces (10–12). This reagent is located in the phospholipidic milieu, and upon photolysis it reacts indiscriminately with its molecular neighbors. It is thus possible to directly analyze the interaction between a membrane protein and lipids belonging to its immediate environment (13–15). By measuring the amount of labeling of SERCA in conditions that promote conformations for which there are well resolved crystal structures, we were able to validate this photolabeling approach as a convenient tool for analyzing conformational changes within transmembrane regions. Furthermore, using this technique on PMCA and comparing the results obtained for SERCA, we were able to draw structural conclusions about these proteins under activated and inhibited states.     

Cryptic Subtelomeric Rearrangements and X Chromosome Mosaicism: A Study of 565 Apparently Normal Individuals with Fluorescent In Situ Hybridization

Five percent of patients with unexplained mental retardation have been attributed to cryptic unbalanced subtelomeric rearrangements. Half of these affected individuals have inherited the rearrangement from a parent who is a carrier for a balanced translocation. However, the frequency of carriers for cryptic balanced translocations is unknown. To determine this frequency, 565 phenotypically normal unrelated individuals were examined for balanced subtelomeric rearrangements using Fluorescent In Situ hybridization (FISH) probes for all subtelomere regions. While no balanced subtelomeric rearrangements were identified, three females in this study were determined to be mosaic for the X chromosome. Mosaicism for XXX cell lines were observed in the lymphocyte cultures of 3 in 379 women (0.8%), which is a higher frequency than the 1 in 1000 (0.1%) reported for sex chromosome aneuploidies. Our findings suggest that numerical abnormalities of the X chromosome are more common in females than previously reported. Based on a review of the literature, the incidence of cryptic translocation carriers is estimated to be approximately 1/8,000, more than ten-fold higher than the frequency of visible reciprocal translocations.     

Mycobiota and mycotoxins in fermented feed,wheat grains and corn grains in Southeastern Buenos Aires Province,Argentina

Wheat (as bran) and corn (as dry grain or fermented feed) are main ingredients of feedstuffs used in local cattle and pig farms in the South of the Buenos Aires Province (Argentina). Therefore, determining mycobiota and mycotoxins in wheat and corn is of prime importance for developing feed management techniques to optimise animal production and to minimize toxicity. Then, a mycological survey was carried out in the Southeastern part of the Buenos Aires Province, in order to identify the mycobiota and the main mycotoxins present in fermented feed, wheat grain and corn grain samples. Samples were cultured for fungal quantification, isolation and identification, and analysed for deoxynivalenol (DON), zearalenone (ZEA), T-2 toxin and aflatoxins (AFLA). Penicillium (74%), Aspergillus (32%) and Scopulariopsis (21%) were the prevalent genera in fermented feed. Penicillium (70%), Fusarium (47%) and Aspergillus (34%) were the most frequent fungi isolated from corn. Penicillium (42%), Fusarium (27%) and Alternaria (25%) were the most frequently recovered genera from wheat. DON was detected in 59% of the corn samples, in 45% of the wheat samples and in 38% of the silage samples. ZEA was detected in 36% of the corn samples, in 49% of the wheat samples and in 16% of the silage samples. T-2 toxin and aflatoxin B1 were each detected in 4% of the corn samples. Eighteen percent of the fermented feed samples showed T-2 contamination. Fermented feed and wheat samples were negative for AFLA.     

Screening of plant peptidases for the synthesis of arginine-based surfactants

Partially purified preparations with proteolytic activity, obtained from South American native plants, were used as biocatalysts in condensation reactions of N-protected arginine alkyl ester derivatives with decylamine and dodecylamine in low-water content systems. The final products are cationic surfactants with potential application as emulsifiers and preservatives. Most of the proteolytic extracts were obtained from latex of species belonging to the Asclepiadaceae family (araujiain from Araujia hortorum, asclepain c from Asclepias curassavica and funastrain from Funastrum clausum). Hieronymain was obtained from unripe fruits of Bromelia hieronymi (Bromeliaceae). Plant proteases from commercial sources (papain and bromelain) were also tested as catalysts in the same reactions. Araujiain and funastrain furnished good reaction conversions (60–84%, with a ratio synthesis/hydrolysis of 2–5) similar to those obtained with commercial papain. Moreover, araujiain was the biocatalyst which rendered the best conversions (60%) for the synthesis of the two novel Bz-Arg-NH-dodecylamide (Bz-Arg-NHC₁₂) and Bz-Arg-NH-decylamide (Bz-Arg-NHC₁₀) derivatives. Moderate to poor conversions (10–50%, showing a ratio synthesis/hydrolysis of 0.5–1) were achieved with asclepain c, hieronymain and bromelain. The screening presented in this work revealed that, although these are structurally similar, their behavior for the synthesis of this kind of products differ among them.     

Trypanosoma cruzi: Biological characterization of a isolate from an endemic area and its susceptibility to conventional drugs

We describe some biological and molecular characteristics of a Trypanosoma cruzi isolate derived from a Triatomine captured in Nicaragua. PCR based typification showed that this isolate, named Nicaragua, belonged to the lineage Tc I. Nicaragua infected culture cells were treated with allopurinol, showing different behavior according to the cellular compartment, being cardiomyocyte primary cultures more resistant to this drug. The course of the infection in a mice experimental model and its susceptibility to benznidazole and allopurinol was analyzed. In benznidazole treatment, mice reverted the high lethal effect of parasites during the acute infection, however, a few parasites were detected in the heart of 88% of mice 1 year post-infection. Since T. cruzi is a heterogeneous species population it is important to study and characterize different parasites actually circulating in humans in endemic areas. In this work we show that T. cruzi Nicaragua isolate, is sensitive to early benznidazole treatment.     

The Intra-Hippocampal Leucine Administration Impairs Memory Consolidation and LTP Generation in Rats

Leucine accumulates in fluids and tissues of patients affected by maple syrup urine disease, an inherited metabolic disorder, predominantly characterized by neurological dysfunction. Although, a variable degree of cognition/psychomotor delay/mental retardation is found in a considerable number of individuals affected by this deficieny, the mechanisms underlying the neuropathology of these alterations are still not defined. Therefore, the aim of this study was to investigate the effect of acute intra-hippocampal leucine administration in the step-down test in rats. In addition, the leucine effects on the electrophysiological parameter, long-term potentiation generation, and on the activities of the respiratory chain were also investigated. Male Wistar rats were bilaterally administrated with leucine (80 nmol/hippocampus; 160 nmol/rat) or artificial cerebrospinal fluid (controls) into the hippocampus immediately post-training in the behavioral task. Twenty-four hours after training in the step-down test, the latency time was evaluated and afterwards animals were sacrificed for assessing the ex vivo biochemical measurements. Leucine-treated animals showed impairment in memory consolidation and a complete inhibition of long-term potentiation generation at supramaximal stimulation. In addition, a significant increment in complex IV activity was observed in hippocampus from leucine-administered rats. These data strongly indicate that leucine compromise memory consolidation, and that impairment of long-term potentiation generation and unbalance of the respiratory chain may be plausible mechanisms underlying the deleterious leucine effect on cognition.     

Identification of chalcones as in vivo liver monofunctional phase II enzymes inducers

Cancer preventive agents (CPA) are drugs able to suppress the carcinogen metabolic activation or block the formation of ultimate carcinogens. CPA could act through various molecular mechanisms, for example by interfering with the action of procarcinogen. This could be attained by increasing the phase II enzymes levels of quinone reductase (QR) and glutathione S-transferase (GST). New flavonoids, especially chalcones, have been identified as in vivo monofunctional phase II enzymes inducers. Oral administration of chalcone, 4, and both p-methoxy-substituted chalcones, 6 and 14, increased hepatic QR activity with concomitant decrease in CYP1A1 activity, a member of the most important group of phase I enzymes cytochrome P450. Among them, 4 also increased GST activity. While p-bromo-substituted chalcone 8 was the best inducer of QR it decreased hepatic GST expression and cytochrome P450, being the most effective decreasing cytochrome P450-expression. Thienyl-chalcone 20 being the bioisostere of chalcone 4 did not display the same in vivo profile in the phase I level modification. As chalcone 4 its bioisostere, chalcone 20, displayed low DNA strand breakage and absence of mutagenicity. Also, in our preliminary in vivo tumourigenesis/chemopreventive and acute-toxicity studies, chalcones 4, 6 and 8 showed the best behaviours as CPA justifying additional studies that are ongoing.     

Coexpression of virulence and fosfomycin susceptibility in Listeria: molecular basis of an antimicrobial in vitro-in vivo paradox

Discrepancies between resistance in vitro and therapeutic efficacy in vivo are generally attributed to failure of laboratory susceptibility tests to reflect an antibiotic's pharmacokinetic or pharmacodynamic properties. We show here that this phenomenon can result from differential in vitro-in vivo expression of bacterial determinants of antibiotic susceptibility. We found that an in vivo-induced virulence factor, Hpt, also mediates uptake of fosfomycin in Listeria monocytogenes. These bacteria therefore seem resistant to fosfomycin in vitro, although they are in fact susceptible to the antibiotic during infection.     

Cardiovascular risk of young growth-hormone-deficient adolescents. Differences in growth-hormone-treated and untreated patients

OBJECTIVE: To determine whether postprandial lipids, coagulation factors and homocysteine levels are abnormal in young growth hormone (GH)-deficient (GHD) adolescents. METHODS: Fifteen GHD adolescents on GH replacement were studied. Ten untreated GHD adolescents and 15 healthy subjects served as controls. Fasting lipids, lipoprotein(a), fibrinogen, plasminogen activator inhibitor-1, homocysteine, folate and vitamin B12 levels were measured. Cholesterol and triglycerides were measured 4 h after a high fat meal. RESULTS: Fasting and postprandial triglycerides and homocysteine levels of untreated GHD patients were increased compared to those of GH-treated GHD subjects and healthy controls; fibrinogen concentrations were elevated in both treated and untreated adolescents. CONCLUSIONS: GHD adolescents present an abnormal fasting and postprandial lipid profile. In addition, the increased fibrinogen and homocysteine levels are suggestive of the accumulation of cardiovascular risk factors early on in life.