Membrane Lipid Profile Monitored By Mass Spectrometry Detected Differences Between Fresh And Vitrified In Vitro-produced Bovine Embryos.

Summary This study aimed to evaluate the impact of vitrification on membrane lipid profile obtained by mass spectrometry (MS) of in vitro-produced bovine embryos. Matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) has been used to obtain individual embryo membrane lipid profiles. Due to conditions of analysis, mainly membrane lipids, most favorably phosphatidylcholines (PCs) and sphingomyelins (SMs) have been detected. The following ions described by their mass-to-charge ratio (m/z) and respective attribution presented increased relative abundance (1.2-20×) in the vitrified group: 703.5 [SM (16:0) + H]+; 722.5 [PC (40:3) + Na]+; 758.5 [PC (34:2) + H]+; 762.5 [PC (34:0) + H]+; 790.5 [PC (36:0) + H]+ and 810.5 [PC (38:4) + H]+ and/or [PC (36:1) + Na]+. The ion with a m/z 744.5 [PCp (34:1) and/or PCe (34:2)] was 3.4-fold more abundant in the fresh group. Interestingly, ions with m/z 722.5 or 744.5 indicate the presence of lipid species, which are more resistant to enzymatic degradation as they contain fatty acyl residues linked through ether type bonds (alkyl ether or plasmalogens, indicated by the lowercase 'e' and 'p', respectively) to the glycerol structure. The results indicate that cryopreservation impacts the membrane lipid profile, and that these alterations can be properly monitored by MALDI-MS. Membrane lipids can therefore be evaluated by MALDI-MS to monitor the effect of cryopreservation on membrane lipids, and to investigate changes in lipid profile that may reflect the metabolic response to the cryopreservation stress or changes in the environmental conditions.

A Comparison Between Characterization And Biological Properties Of Brazilian Fresh And Aged Propolis.

As propolis is a highly valued bee product, we aimed to verify the quality of aged propolis, investigating their phenolic and flavonoid composition, levels of toxic metals, radical scavenging and antimicrobial activities. Samples of fresh and aged propolis of six different beekeepers, from the same geographical location, were investigated in terms of their phenolic and flavonoid composition and levels of Pb, Cd, and Cr, as well as radical scavenging and antimicrobial activities. The two groups of propolis had similar qualitative composition by HPLC-PDA and ESI(-)-MS. Fresh propolis and aged propolis show no differences when average values of extraction yield, flavonoids, EC50, or MIC were compared and both types of propolis showed good antimicrobial activity at low concentrations. Only levels of phenolic compounds were higher in fresh propolis. The propolis samples considered in this study, aged or fresh, had similar qualitative composition, although they were collected in different periods. Samples only differed in their levels of total phenolic content. Moreover, aged propolis conserves significant radical scavenging and antimicrobial properties. We suggest that aged propolis should not be discarded but explored for alternative applications.

Phenotypic variability in a family with x-linked adrenoleukodystrophy caused by the p.Trp132Ter mutation

X-linked adrenoleukodystrophy (X-ALD) is an inherited disease with clinical heterogeneity varying from presymptomatic individuals to rapidly progressive cerebral ALD forms. This disease is characterized by increased concentration of very long chain fatty acids (VLCFAs) in plasma and in adrenal, testicular and nervous tissues. Affected individuals can be classified in different clinical settings, according to phenotypic expression and age at onset of initial symptoms. Molecular defects in X-ALD individuals usually result from ABCD1 gene mutations. In the present report we describe clinical data and the ABCD1 gene study in two boys affected with the childhood cerebral form that presented with different symptomatic manifestations at diagnosis. In addition, their maternal grandfather had been diagnosed with Addison's disease indicating phenotypic variation for X-ALD within this family. The mutation p.Trp132Ter was identified in both male patients; additionally, three females, out of eleven family members, were found to be heterozygous after screening for this mutation. In the present report, the molecular analysis was especially important since one of the heterozygous females was in first stages of pregnancy. Therefore, depending on the fetus outcome, if male and p.Trp132Ter carrier, storage of the umbilical cord blood should be recommended as hematopoietic stem cell transplantation could be considered as an option for treatment in the future.

A Method for Generation of Bone Marrow-Derived Macrophages from Cryopreserved Mouse Bone Marrow Cells

The broad use of transgenic and gene-targeted mice has established bone marrow-derived macrophages (BMDM) as important mammalian host cells for investigation of the macrophages biology. Over the last decade, extensive research has been done to determine how to freeze and store viable hematopoietic human cells; however, there is no information regarding generation of BMDM from frozen murine bone marrow (BM) cells. Here, we establish a highly efficient protocol to freeze murine BM cells and further generate BMDM. Cryopreserved murine BM cells maintain their potential for BMDM differentiation for more than 6 years. We compared BMDM obtained from fresh and frozen BM cells and found that both are similarly able to trigger the expression of CD80 and CD86 in response to LPS or infection with the intracellular bacteria Legionella pneumophila. Additionally, BMDM obtained from fresh or frozen BM cells equally restrict or support the intracellular multiplication of pathogens such as L. pneumophila and the protozoan parasite Leishmania (L.) amazonensis. Although further investigation are required to support the use of the method for generation of dendritic cells, preliminary experiments indicate that bone marrow-derived dendritic cells can also be generated from cryopreserved BM cells. Overall, the method described and validated herein represents a technical advance as it allows ready and easy generation of BMDM from a stock of frozen BM cells.

Sudan Black B treatment reduces autofluorescence and improves resolution of in situ hybridization specific fluorescent signals of brain sections

Interference by autofluorescence is one of the major concerns of immunofluorescence analysis of in situ hybridization-based diagnostic assays. We present a useful technique that reduces autofluorescent background without affecting the tissue integrity or direct immunofluorescence signals in brain sections. Using six different protocols, such as ammonia/ethanol, Sudan Black B (SBB) in 70% ethanol, photobleaching with UV light and different combinations of them in both formalin-fixed paraffin-embedded and frozen human brain tissue sections, we have found that tissue treatment of SBB in a concentration of 0.1% in 70% ethanol is the best approach to reduce/eliminate tissue autofluorescence and background, while preserving the specific fluorescence hybridization signals. This strategy is a feasible, non-time consuming method that provides a reasonable compromise between total reduction of the tissue autofluorescence and maintenance of specific fluorescent labels.

Influence of magnesium status and magnesium intake on the blood glucose control in patients with type 2 diabetes

Background & aims: This study was undertaken to assess magnesium intake and magnesium status in patients with type 2 diabetes, and to identify the parameters that best predict alterations in fasting glucose and plasma magnesium. Methods: A cross-sectional study was carried out in patients with type 2 diabetes (n = 51; 53.6 +/- 10.5 y) selected within the inclusion factors, at the University Hospital Onofre Lopes. Magnesium intake was assessed by three 24-h recalls. Urine, plasma and erythrocytes magnesium, fasting and 2-h postprandial glucose, HbA1, microalbuminuria, proteinuria, and serum and urine creatinine were measured. Results: Mean magnesium intake (9.37 +/- 1.76 mmol/d), urine magnesium (2.80 +/- 1.51 mmol/d), plasma magnesium (0.71 +/- 0.08 mmol/L) and erythrocyte magnesium (1.92 +/- 0.23 mmol/L) levels were low. Seventy-seven percent of participants presented one or more magnesium status parameters below the cut-off points of 3.00 mmol/L for urine, 0.75 mmol/L for plasma and 1.65 mmol/L for erythrocytes. Subjects presented poor blood glucose control with fasting glucose of 8.1 +/- 3.7 mmol/L, 2-h postprandial glucose of 11.1 +/- 5.1 mmol/L, and HbA1 of 11.4 +/- 3.0%. The parameters that influenced fasting glucose were urine, plasma and dietary magnesium, while plasma magnesium was influenced by creatinine clearance. Conclusions: Magnesium status was influenced by kidney depuration and was altered in patients with type 2 diabetes, and magnesium showed to play an important role in blood glucose control. (C) 2011 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Isolation and Characterization of a Natriuretic Peptide from Crotalus oreganus abyssus (Grand Canyon Rattlesnake) and its Effects on Systemic Blood Pressure and Nitrite Levels

Studies on the therapeutic potential of venom peptides have significantly advanced the development of new peptide drugs. A good example is captopril, a synthetic peptide drug, which acts as an anti-hypertensive and potentiating bradykinin, inhibiting the angiotensin-converting enzyme, whose precursor was isolated from the venom of Bothrops jararacussu. The natriuretic peptide (NPs) family comprises three members, ANP (atrial natriuretic peptide), BNP (B-type natriuretic peptide) and CNP (C-type natriuretic peptide), and has an important role in blood pressure regulation and electrolyte homeostasis. In this study, we describe, for the first time, the isolation and characterization of a novel natriuretic-like peptide (Coa_NP), isolated from Crotalus Oreganus abyssus venom. The peptide has 32 amino acids and its complete sequence is SKRLSNGCFGLKLDRIGAMSGLGCWRLINESK. The Coa_NP has an average molecular mass of 3510.98 Da and its amino acid sequence presents the loop region that is characteristic of natriuretic peptides (17 amino acids, NP domain consensus; CFGXXXDRIXXXSGLGC). Coa_NP is a natriuretic peptide of the ANP/BNP-like family, since the carboxy terminal region of CNP has its own NP domain. The functional experiments showed that Coa_NP produced biological effects similar to those of the other natriuretic peptides: (1) a dose-dependent decrease in mean arterial pressure; (2) significant increases in plasma nitrite levels, and (3) vasorelaxation in thoracic aortic rings that were pre-contracted with phenylephrine. The structural and biological aspects confirm Coa_NP as a natriuretic peptide isolated from snake venom, thus expanding the diversification of venom components.

Metformin pharmacokinetics in nondiabetic pregnant women with polycystic ovary syndrome

The use of metformin throughout gestation by pregnant women with polycystic ovary syndrome (PCOS) significantly reduces the number of first trimester spontaneous abortions and the rate of occurrence of gestational diabetes. The objective of this study was to investigate the pharmacokinetics and the placental transfer of metformin in pregnant women with PCOS. Eight pregnant women with PCOS taking 850 mg metformin every 12 h during the third trimester of pregnancy were evaluated. Maternal blood samples were collected at steady state during the dose interval (0-12 h). Maternal and umbilical cord blood samples were also obtained at delivery. Metformin plasma concentrations were analyzed by high-performance liquid chromatography, and pharmacokinetic parameters were determined using a non-compartmental model. Data are reported as median and minimum and maximum values. Metformin pharmacokinetic parameters were: t(A1/2), 3.8 (2.8-5.4) h; t(max), 2.0 (0.5-3.0) h; C(max), 1.4 (0.5-2.1) mg/L; C(mean), 0.5 (0.2-0.9) mg/L; AUC(0-12), 6.4 (1.1-9.2) mg h/L; Cl/f, 105 (60-274) L/h; Vd/f, 551 (385-1173) L; median fluctuation, 89 (79-95)%. Umbilical/maternal metformin plasma concentration ratios were 0.7 (0.4-1.3). Metformin oral clearance (Cl/f) had increased in our patients relative to nonpregnant healthy volunteers or diabetic patients. Therefore, lower plasma metformin concentrations were observed for nondiabetic pregnant women with PCOS. Future studies should be conducted to demonstrate the therapeutic efficacy of metformin during pregnancy. Caution is warranted as umbilical/maternal metformin plasma concentrations ratios of around 0.7 require metformin dosage adjustment.

Osmoregulation of the Australian freshwater crocodile, Crocodylus johnstoni, in fresh and saline waters

An unusual saltwater population of the "freshwater" crocodilian, Crocodylus johnstoni, was studied in the estuary of the Limmen Bight River in Australia's Northern Territory and compared with populations in permanently freshwater habitats. Crocodiles in the river were found across a large salinity gradient, from fresh water to a salinity of 24 mg.ml-1, more than twice the body fluid concentration. Plasma osmolarity, concentrations of plasma Na+, Cl-, and K+, and exchangeable Na+ pools were all remarkably constant across the salinity spectrum and were not substantially higher or more variable than those in crocodiles from permanently freshwater habitats. Body fluid volumes did not vary; condition factor and hydration status of crocodiles were not correlated with salinity and were not different from those of crocodiles from permanently fresh water. C. johnstoni clearly has considerable powers of osmoregulation in waters of low to medium salinity. Whether this osmoregulatory competence, extends to continuously hyperosmotic environments is not known, but distributional data suggest that C. johnstoni in hyperosmotic conditions may require periodic access to hypoosmotic water. The study demonstrates a physiological capacity for colonisation of at least some estuarine waters by this normally stenohaline freshwater crocodilian.

Homocysteine-lowering treatment with folic acid, cobalamin, and pyridoxine does not reduce blood markers of inflammation, endothelial dysfunction, or hypercoagulability in patients with previous transient ischemic attack or stroke - a randomized substudy

Background and Purpose - Epidemiological and laboratory studies suggest that increasing concentrations of plasma homocysteine ( total homocysteine [tHcy]) accelerate cardiovascular disease by promoting vascular inflammation, endothelial dysfunction, and hypercoagulability. Methods - We conducted a randomized controlled trial in 285 patients with recent transient ischemic attack or stroke to examine the effect of lowering tHcy with folic acid 2 mg, vitamin B-12 0.5 mg, and vitamin B-6 25 mg compared with placebo on laboratory markers of vascular inflammation, endothelial dysfunction, and hypercoagulability. Results - At 6 months after randomization, there was no significant difference in blood concentrations of markers of vascular inflammation (high-sensitivity C-reactive protein [P = 0.32]; soluble CD40L [ P = 0.33]; IL-6 [P = 0.77]), endothelial dysfunction ( vascular cell adhesion molecule-1 [P = 0.27]; intercellular adhesion molecule-1 [P = 0.08]; von Willebrand factor [P = 0.92]), and hypercoagulability (P-selectin [P = 0.33]; prothrombin fragment 1 and 2 [P = 0.81]; D-dimer [P = 0.88]) among patients assigned vitamin therapy compared with placebo despite a 3.7-mumol/L (95% CI, 2.7 to 4.7) reduction in total homocysteine (tHcy). Conclusions - Lowering tHcy by 3.7 mumol/L with folic acid-based multivitamin therapy does not significantly reduce blood concentrations of the biomarkers of inflammation, endothelial dysfunction, or hypercoagulability measured in our study. The possible explanations for our findings are: ( 1) these biomarkers are not sensitive to the effects of lowering tHcy (eg, multiple risk factor interventions may be required); ( 2) elevated tHcy causes cardiovascular disease by mechanisms other than the biomarkers measured; or ( 3) elevated tHcy is a noncausal marker of increased vascular risk.

Interaction of 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate with mimetic membranes and cytotoxic effect on leukemic cells

10-(Octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC) is an alkylphospholipid that can interact with cell membranes because of its amphiphilic character. We describe here the interaction of ODPC with liposomes and its toxicity to leukemic cells with an ED-50 of 5.4, 5.6 and 2.9 pM for 72 h of treatment for inhibition of proliferation of NB4, U937 and K562 cell lines, respectively, and lack of toxicity to normal hematopoietic progenitor cells at concentrations up to 25 pM. The ED-50 for the non-malignant HEK-293 and primary human umbilical vein endothelial cells (HUVEC) was 63.4 and 60.7 mu M, respectively. The critical micellar concentration (CMC) of ODPC was 200 mu M. Dynamic light scattering indicated that dipalmitoylphosphatidylcholine (DPPC) liposome size was affected only above the CMC of ODPC. Differential calorimetric scanning (DCS) of liposomes indicated a critical transition temperature (T(c)) of 41.5 degrees C and an enthalpy (Delta H) variation of 7.3 kcal mol(-1). The presence of 25 mu M ODPC decreased T(c) and Delta H to 393 degrees C and 4.7 kcal mol(-1), respectively. ODPC at 250 mu M destabilized the liposomes (36.3 degrees C. 0.46 kcal mol(-1)). Kinetics of 5(6)-carboxyfluorescein (CF) leakage from different liposome systems indicated that the rate and extent of CF release depended on liposome composition and ODPC concentration and that above the CMC it was instantaneous. Overall, the data indicate that ODPC acts on in vitro membrane systems and leukemia cell lines at concentrations below its CMC, suggesting that it does not act as a detergent and that this effect is dependent on membrane composition. (C) 2010 Elsevier B.V. All rights reserved.

Reductionism and the human duplication thought-experiment

The human duplication thought-experiment is examined, and basic positions concerning the possible outcomes of the experiment are spelled out. A first position sustains supervenience, either from a reductionist or an emergentist perspective, and such views are contrasted. Certain moral aspects of the thought-experiment are then considered, especially in relation to the idea of death. Taking reductionism as a working hypothesis, two possibilities are suggested for investigating the hard problem of qualia: the postulation of some novel sort of physical interaction, and the postulation of a counter-intuitive law of scaling. One possibility for the latter would lead to a violation of supervenience.

CD4+T cells from HIV-1-infected patients recognize wild-type and mutant human immunodeficiency virus-1 protease epitopes

P>Human immunodeficiency virus (HIV)-1 protease is a known target of CD8+ T cell responses, but it is the only HIV-1 protein in which no fully characterized HIV-1 protease CD4 epitopes have been identified to date. We investigated the recognition of HIV-1 protease by CD4+ T cells from 75 HIV-1-infected, protease inhibitor (PI)-treated patients, using the 5,6-carboxyfluorescein diacetate succinimidyl ester-based proliferation assay. In order to identify putative promiscuous CD4+ T cell epitopes, we used the TEPITOPE algorithm to scan the sequence of the HXB2 HIV-1 protease. Protease regions 4-23, 45-64 and 73-95 were identified; 32 sequence variants of the mentioned regions, encoding frequent PI-induced mutations and polymorphisms, were also tested. On average, each peptide bound to five of 15 tested common human leucocyte antigen D-related (HLA-DR) molecules. More than 80% of the patients displayed CD4+ as well as CD8+ T cell recognition of at least one of the protease peptides. All 35 peptides were recognized. The response was not associated with particular HLA-DR or -DQ alleles. Our results thus indicate that protease is a frequent target of CD4+ along with CD8+ proliferative T cell responses by the majority of HIV-1-infected patients under PI therapy. The frequent finding of matching CD4+ and CD8+ T cell responses to the same peptides may indicate that CD4+ T cells provide cognate T cell help for the maintenance of long-living protease-specific functional CD8+ T cells.

PTCH1 gene mutations in exon 17 and loss of heterozygosity on D9S180 microsatellite in sporadic and inherited human basal cell carcinomas

Background Basal cell carcinomas (BCCs) are the most frequent human cancer that results from malignant transformation of basal cells in the epidermis. Gorlin syndrome is a rare inherited autosomal dominant disease that predisposes with multiple BCCs and other birth defects. Both sporadic and inherited BCCs are associated with mutations in the tumor suppressor gene PTCH1, but there is still uncertainty on the role of its homolog PTCH2. Objectives To search for mutations and genomic instability in sporadic and inherited BCCs. Methods DNA obtained from leukocytes and tumor cells was amplified by polymerase chain reaction regarding five exons of PTCH1 and PTCH2 and neighboring microsatellites. Exons were sequenced and compared with the GenBank database. Results Only D9S180, of six microsatellites, showed loss of heterozygosity in three BCCs (two sporadic and one inherited). One sporadic BCC presented the mutation g. 2885G>C in exon 17 of PTCH1, which predicts the substitution p.R962T in an external domain of the protein. In addition, the leukocytes and tumor cells of one patient with Gorlin syndrome showed the mutation g. 2839T>G in the same exon and gene, which predicts a p.E947stop and truncated protein. All control and tumor samples presented IVS9 + 217T in intron 9 of PTCH1. Conclusion Mutations found in the PTCH1 gene and neighboring repetitive sequences may have contributed to the development of the studied BCCs.