Two-photon absorption spectra of carotenoids compounds

Carotenoids are biosynthetic organic pigments that constitute an important class of one-dimensional pi-conjugated organic molecules with enormous potential for application in biophotonic devices. In this context, we studied the degenerate two-photon absorption (2PA) cross-section spectra of two carotenoid compounds (beta-carotene and beta-apo-8'-carotenal) employing the conventional and white-light-continuum Z-scan techniques and quantum chemistry calculations. Because carotenoids coexist at room temperature as a mixture of isomers, the 2PA spectra reported here are due to samples containing a distribution of isomers, presenting distinct conjugation length and conformation. We show that these compounds present a defined structure on the 2PA spectra, that peaks at 650 nm with an absorption cross-section of approximately 5000 GM, for both compounds. In addition, we observed a 2PA band at 990 nm for beta-apo-8'-carotenal, which was attributed to a overlapping of I(I)B(u) +-like and 2(I)Ag(-)-like states, which are strongly one- and two-photon allowed, respectively. Spectroscopic parameters of the electronic transitions to singlet-excited states, which are directly related to photophysical properties of these compounds, were obtained by fitting the 2PA spectra using the sum-over-states approach. The analysis and interpretations of the 2PA spectra of the investigated carotenoids were supported by theoretical predictions of one- and two-photon transitions carried out using the response functions formalism within the density functional theory framework, using the long-range corrected CAM-B3LYP functional. (C) 2011 American Institute of Physics. [doi:10.1063/1.3590157]

Effects of margarines and butter consumption on lipid profiles, inflammation markers and lipid transfer to HDL particles in free-living subjects with the metabolic syndrome

Objective: Our purpose was to examine the effects of daily servings of butter, no-trans-fat margarine and plant sterol margarine, within recommended amounts, on plasma lipids, apolipoproteins (Apos), biomarkers of inflammation and endothelial dysfunction, and on the transfer of lipids to HDL particles in free-living subjects with the metabolic syndrome. Methods: This was a randomized, single-blind study where 53 metabolic syndrome subjects (62% women, mean age 54 years) received isocaloric servings of butter, no-trans-fat margarine or plant sterol margarine in addition to their usual diets for 5 weeks. The main outcome measures were plasma lipids, Apo, inflammatory and endothelial dysfunction markers (CRP, IL-6, CD40L or E-selectin), small dense LDL cholesterol concentrations and in vitro radioactive lipid transfer from cholesterol-rich emulsions to HDL. Difference among groups was evaluated by analysis of variance. Results: There was a significant reduction in Apo-B (-10.4 %, P = 0.043) and in the Apo-B/Apo-A-1 ratio (-11.1%, P = 0.034) with plant sterol margarine. No changes in plasma lipids were noticed with butter and no-trans-fat margarine. Transfer rates of lipids to HDL were reduced in the no-trans-fat margarine group: triglycerides -42.0%, (P<0.001 vs butter and sterol margarine) and free cholesterol -16.2% (P = 0.006 vs sterol margarine). No significant effects were noted on the concentrations of inflammatory and endothelial dysfunction markers among the groups. Conclusions: In free-living subjects with the metabolic syndrome consumption of plant sterol and no-trans-fat margarines within recommended amounts reduced, respectively, Apo-B concentrations and the ability of HDL to accept lipids. European Journal of Clinical Nutrition (2010) 64, 1141-1149; doi:10.1038/ejcn.2010.122; published online 21 July 2010

Atorvastatin effects on SREBF1a and SCAP gene expression in mononuclear cells and its relation with lowering-lipids response

Background: The transcription factors SREBP1 and SCAP are involved in intracellular cholesterol homeostasis. Polymorphisms of these genes have been associated with variations on serum lipid levels and response to statins that are potent cholesterol-lowering drugs. We evaluated the effects of atorvastatin on SREBF1a and SCAP mRNA expression in peripheral blood mononuclear cells (PBMC) and a possible association with gene polymorphisms and lowering-cholesterol response. Methods: Fifty-nine hypercholesterolemic patients were treated with atorvastatin (10 mg/day for 4 weeks). Serum lipid profile and mRNA expression in PBMC were assessed before and after the treatment. Gene expression was quantified by real-time PCR using GAPD as endogenous reference and mRNA expression in HepG2 cells as calibrator. SREBF1 -36delG and SCAP A2386G polymorphisms were detected by PCR-RFLP. Results: Our results showed that transcription of SREBF1a and SCAP was coordinately regulated by atorvastatin (r=0.595, p<0.001), and that reduction in SCAP transcription was associated with the 2386AA genotype (p=0.019). Individuals who responded to atorvastatin with a downregulation of SCAP had also a lower triglyceride compared to those who responded to atorvastatin with an upregulation of SCAP. Conclusion: Atorvastatin has differential effects on SREBF1a and SCAP mRNA expression in PBMC that are associated with baseline transcription levels, triglycerides response to atorvastatin and SCAP A2386G polymorphism. (c) 2008 Elsevier B.V. All rights reserved.

Mitochondrial energy metabolism and redox responses to hypertriglyceridemia

In this work we review recent findings that explain how mitochondrial bioenergetic functions and redox state respond to a hyperlipidemic in vivo environment and may contribute to the maintenance of a normal metabolic phenotype. The experimental model utilized to evidence these adaptive mechanisms is especially useful for these studies since it exhibits genetic hypertriglyceridemia and avoids complications introduced by high fat diets. Liver from hypertrigliceridemic (HTG) mice have a greater content of glycerolipids together with increased mitochondrial free fatty acid oxidation. HTG liver mitochondria have a higher resting respiration rate but normal oxidative phosphorylation efficiency. This is achieved by higher activity of the mitochondrial potassium channel sensitive to ATP (mitoK(ATP)). The mild uncoupling mediated by mitoK(ATP) accelerates respiration rates and reduces reactive oxygen species generation. Although this response is not sufficient to inhibit lipid induced extra-mitochondrial oxidative stress in whole liver cells it avoids amplification of this redox imbalance. Furthermore, higher mitoK(ATP) activity increases liver, brain and whole body metabolic rates. These mitochondrial adaptations may explain why these HTG mice do not develop insulin resistance and obesity even under a severe hyperlipidemic state. On the contrary, when long term high fat diets are employed, insulin resistance, fatty liver and obesity develop and mitochondrial adaptations are inefficient to counteract energy and redox imbalances.

Structural complexes of human adenine phosphoribosyltransferase reveal novel features of the APRT catalytic mechanism

Adenine phosphoribosyltransferase (APRT) is an important enzyme component of the purine recycling pathway. Parasitic protozoa of the order Kinetoplastida are unable to synthesize purines de novo and use the salvage pathway for the synthesis of purine bases rendering this biosynthetic pathway an attractive target for antiparasitic drug design. The recombinant human adenine phosphoribosyltransferase (hAPRT) structure was resolved in the presence of AMP in the active site to 1.76 angstrom resolution and with the substrates PRPP and adenine simultaneously bound to the catalytic site to 1.83 angstrom resolution. An additional structure was solved containing one subunit of the dimer in the apo-form to 2.10 angstrom resolution. Comparisons of these three hAPRT structures with other `type I` PRTases revealed several important features of this class of enzymes. Our data indicate that the flexible loop structure adopts an open conformation before and after binding of both substrates adenine and PRPR Comparative analyses presented here provide structural evidence to propose the role of Glu 104 as the residue that abstracts the proton of adenine N9 atom before its nucleophilic attack on the PRPP anomeric carbon. This work leads to new insights to the understanding of the APRT catalytic mechanism.

Removal of a cysteine ligand from rubredoxin: assembly of Fe2S2 and Fe(S-Cys)(3)(OH) centres

The electron transfer protein rubredoxin from Clostridium pasteurianum contains an Fe(S-Cys)(4) active site. Mutant proteins C9G, C9A, C42G and C42A, in which cysteine ligands are replaced by non-ligating Gly or Ala residues, have been expressed in Escherichia coli. The C42A protein expresses with a (Fe2S2)-S-III cluster in place. In contrast, the other proteins are isolated in colourless forms, although a (Fe2S2)-S-III cluster may be assembled in the C42G protein via incubation with Fe-III and sulfide. The four mutant proteins were isolated as stable mononuclear Hg-II forms which were converted to unstable mononuclear Fe-III preparations that contain both holo and apo protein. The Fe-III systems were characterized by metal analysis and mass spectrometry and by electronic, electron paramagnetic resonance, X-ray absorption and resonance Raman spectroscopies. The dominant Fe-III form in the C9A preparation is a Fe(S-Cys)(3)(OH) centre, similar to that observed previously in the C6S mutant protein. Related centres are present in the proteins NifU and IscU responsible for assembly and repair of iron-sulfur clusters in both prokaryotic and eukaryotic cells. In addition to Fe(S-Cys)(3)(OH) centres, the C9G, C42G and C42A preparations contain a second four-coordinate Fe-III form in which a ligand appears to be supplied by the protein chain. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-0020355-1.

Ancestry informative markers in Amerindians from Brazilian Amazon

Ancestry informative markers (AIMs) are genetic loci with large frequency differences between the major ethnic groups and are very useful in admixture estimation. However, their frequencies are poorly known within South American indigenous populations, making it difficult to use them in admixture studies with Latin American populations, such as the trihybrid Brazilian population. To minimize this problem, the frequencies of the AIMs FY-null RB2300, LPL, AT3-1/1), Sb19.3, APO, and PV92 were determined via PCR and PCR-RFLP in four tribes from Brazilian Amazon (Tikuna, Kashinawa, Baniwa, and Kanamari), to evaluate their potential for discriminating indigenous populations from Europeans and Africans, as well as discriminating each tribe from the others. Although capable of differentiating tribes, as evidenced by the exact test of population differentiation, a neighbor-joining tree suggests that the AIMs are useless in obtaining reliable reconstructions of the biological relationships and evolutionary history that characterize the villages and tribes studied. The mean allele frequencies from these AIMs were very similar to those observed for North American natives. They discriminated Amerindians from Africans, but not from Europeans. On the other hand, the neighbor-joining dendrogram separated Africans and Europeans from Amerindians with a high statistical support (bootstrap = 0.989). The relatively low diversity (GST = 0.042) among North American natives and Amerindians from Brazilian Amazon agrees with the lack of intra-ethnic variation previously reported for these markers. Despite genetic drift effects, the mean allelic frequencies herein presented could be used as Amerindian parental frequencies in admixture estimates in urban Brazilian populations.

Aerobic Exercise Improves Reverse Cholesterol Transport in Cholesteryl Ester Transfer Protein Transgenic Mice

We analyzed the effect of a 6-week aerobic exercise training program on the in vivo macrophage reverse cholesterol transport (RCT) in human cholesteryl ester transfer protein (CETP) transgenic (CETP-tg) mice. Male CETP-tg mice were randomly assigned to a sedentary group or a carefully supervised exercise training group (treadmill 15 m/min, 30 min sessions, five sessions per week). The levels of plasma lipids were determined by enzymatic methods, and the lipoprotein profile was determined by fast protein liquid chromatography (FPLC). CETP activity was determined by measuring the transfer rate of (14)C-cholesterol from HDL to apo-B containing lipoproteins, using plasma from CETP-tg mice as a source of CETP. The reverse cholesterol transport was determined in vivo by measuring the [(3)H]-cholesterol recovery in plasma and feces (24 and 48 h) and in the liver (48 h) following a peritoneal injection of [(3)H]-cholesterol labeled J774-macrophages into both sedentary and exercise trained mice. The protein levels of liver receptors were determined by immunoblot, and the mRNA levels for liver enzymes were measured using RT-PCR. Exercise training did not significantly affect the levels of plasma lipids or CETP activity. The HDL fraction assessed by FPLC was higher in exercise-trained compared to sedentary mice. In comparison to the sedentary group, a greater recovery of [(3)H]-cholesterol from the injected macrophages was found in the plasma, liver and feces of exercise-trained animals. The latter occurred even with a reduction in the liver CYP7A1 mRNA level in exercised trained animals. Exercise training increased the liver LDL receptor and ABCA-1 protein levels, although the SR-BI protein content was unchanged. The RCT benefit in CETP-tg mice elicited by exercise training helps to elucidate the role of exercise in the prevention of atherosclerosis in humans.

Air pollution and antibodies against modified lipoproteins are associated with atherosclerosis and vascular remodeling in hyperlipemic mice

We analyzed the impact of chronic exposure to urban air pollution on the development of atherosclerosis. Hyperlipemic mice (LDLR(-/-)) were submitted to a high fat diet and air pollution for four months. We measured the susceptibility of LDL to oxidative modifications (TBARS), the presence of anti-oxLDL and an apoB-derived peptide (apoB-D) in blood and the degree of atherosclerosis in the aortic arch. Air pollution increased the susceptibility of LDL to oxidation as well as anti-oxLDL and anti-apo-B levels. These levels were even higher than in mice submitted to a high fat diet and non-polluted air. The lipid content of the atherosclerotic plaques in the aorta was increased in groups with a high cholesterol diet independently of the air quality. However, the thickness of the arterial wall was greater in mice fed a high lipid diet with polluted air. Thus, we conclude that urban air pollution exacerbates the susceptibility of LDL to oxidation, atherogenesis and vascular remodeling in hyperlipemic mice and that an immune response accompanies this process. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Internal Pudendal Artery from Type 2 Diabetic Female Rats Demonstrate Elevated Endothelin-1-Mediated Constriction

Introduction. Diabetes is a risk factor for female sexual dysfunction (FSD). FSD has several etiologies, including a vasculogenic component that could be exacerbated in diabetes. The internal pudendal artery supplies blood to the vagina and clitoris and diabetes-associated functional abnormalities in this vascular bed may contribute to FSD. Aim. The Goto-Kakizaki (GK) rat is a non-obese model of type 2 diabetes with elevated endothelin-1 (ET-1) activity. We hypothesize that female GK rats have diminished sexual responses and that the internal pudendal arteries demonstrate increased ET-1 constrictor sensitivity. Methods. Female Wistar and GK rats were used. Apomorphine (APO)-mediated genital vasocongestive arousal (GVA) was measured. Functional contraction (ET-1 and phenylephrine) and relaxation (acetylcholine, ACh) in the presence or absence of the ETA receptor antagonist (ET(A)R; atrasentan) or Rho-kinase inhibitor (Y-27632) were assessed in the internal pudendal and mesenteric arteries. Protein expression of ET-1 and RhoA/Rho-kinase signaling pathway was determined in the internal pudendal and mesenteric arteries. Main Outcome Measure. APO-mediated GVAs; contraction and relaxation of internal pudendal and mesenteric arteries; ET-1/RhoA/Rho-kinase protein expression. Results. GK rats demonstrated no APO-induced GVAs. Internal pudendal arteries, but not mesenteric arteries, from GK rats exhibited greater contractile sensitivity to ET-1 compared with Wistar arteries. ETAR blockade reduced ET-1-mediated constriction in GK internal pudendal and mesenteric arteries. Rho-kinase inhibition reduced ET-1-mediated constriction of GK internal pudendal but not mesenteric arteries; however, it had no effect on arteries from Wistar rats. RhoA protein expression was elevated in GK internal pudendal arteries. At the highest concentrations, ACh-mediated relaxation was greater in the GK internal pudendal artery; however, no difference was observed in the mesenteric artery. Conclusions. Female GK rats demonstrate decreased sexual responses that may be because of increased constrictor sensitivity to the ET-1/RhoA/Rho-kinase signaling in the internal pudendal artery. Allahdadi KJ, Hannan JL, Ergul A, Tostes RC, and Webb RC. Internal pudendal artery from type 2 diabetic female rats demonstrate elevated endothelin-1-mediated constriction. J Sex Med 2011;8:2472-2483.

Stability improvement of the fatty acid binding protein Sm14 from S. mansoni by Cys replacement: Structural and functional characterization of a vaccine candidate

The Schistosoma mansoni fatty acid binding protein (FABP), SmA, is a vaccine candidate against, S. mansoni and F hepatica. Previously, we demonstrated the importance of a correct fold to achieve protection in immunized animals after cercariae challenge [[10]. C.R.R. Ramos, R.C.R. Figueredo, T.A. Pertinhez, M.M. Vilar, A.L.T.O. Nascimento, M. Tendler, I. Raw, A. Spisni, P.L. Ho, Gene structure and M20T polymorphism of the Schistosoma mansoni Sm14 fatty acid-binding protein: structural, functional and immunoprotection analysis. J. Biol. Chem. 278 (2003) 12745-12751]. Here we show that the reduction of vaccine efficacy over time is due to protein dimerization and subsequent aggregation. We produced the mutants Sm14-M20(C62S) and Sm14M20(C62V) that, as expected, did not dimerize in SDS-PAGE. Molecular dynamics calculations and unfolding experiments highlighted a higher structural stability of these mutants with respect to the wild-type. In addition, we found that the mutated proteins, after thermal denaturation, refolded to their active native molecular architecture as proved by the recovery of the fatty acid binding ability. Sm14-M20(C62V) turned out to be the more stable form over time, providing the basis to determine the first 3D solution structure of a Sm14 protein in its apo-form. Overall, Sm14-M20(C62V) possesses an improved structural stability over time, an essential feature to preserve its immunization capability and, in experimentally immunized animals, it exhibits a protection effect against S. mansoni cercariae infections comparable to the one obtained with the wild-type protein. These facts indicate this protein as a good lead molecule for large-scale production and for developing an effective Sm14 based anti-helminthes vaccine. (C) 2008 Elsevier B.V. All rights reserved.

Identification of an apolipoprotein(e) variant associated with type III hyperlipoproteinaemia in an indigenous Australian

As a result of testing for lipid and apolipoprotein(e) (apo E) phenotype status of an indigenous Australian community, an apo E variant associated with type III hyperlipoproteinaemia has been identified. Apo E phenotype was determined by analysis of VLDL by isoelectric focusing, and genotype on DNA amplified by polymerase chain reaction, using two different restriction enzyme isotyping assays. Phenotypes and genotypes were discordant in samples from two subjects and an abnormal-sized restriction fragment was also observed in their genotyping gel patterns. DNA sequencing studies revealed this was due to a single nucleotide deletion. 3817delC, at amino acid 136 on apo E. This resulted in a new reading frame and the premature termination of the apo E protein due to a stop codon (TGA) at nucleotide 4105. The variant apo E null allele showed a recessive mode of inheritance and, in combination with the E2 allele, resulted in the type III hyperlipoproteinaemic phenotype but when inherited with the E4 allele had no marked effect on plasma lipids.

Transactivation-deficient p73 alpha (p73 Delta exon2) inhibits apoptosis and competes with p53

p73 has recently been identified as a structural and functional homolog of the tumor suppressor protein p53. Overexpression of p53 activates transcription of p53 effector genes, causes growth inhibition and induced apoptosis. We describe here the effects of a tumor-derived truncated transcript of p73 alpha (p73 Delta exon2) on p53 function and on cell death. This transcript, which lacks the acidic N-terminus corresponding to the transactivation domain of p53, was initially detected in a neuroblastoma cell line. Overexpression of p73 Delta exon2 partially protects lymphoblastoid cells against apoptosis induced by anti-Fas antibody or cisplatin. By cotransfecting p73 Delta exon2 with wild-type p53 in the p53 null line Saos 2, we found that this truncated transcript reduces the ability of wild-type p53 to promote apoptosis. This anti-apoptotic effect was also observed when p73 Delta exon2 was co-transfected with full-length p73 (p73 alpha). This was further substantiated by suppression of p53 transactivation of the effector gene p21-Waf1 in p73 Delta exon2 transfected cells and by inhibition of expression of a reporter gene under the control of the p53 promoter. Thus, this truncated form of p73 can act as a dominant-negative agent towards transactivation by p53 and p73 alpha, highlighting the potential implications of these findings for p53 signaling pathway. Furthermore, we demonstrate the existence of a p73 Delta exon2 transcript in a very significant proportion (46%) of breast cancer cell lines. However, a large spectrum of normal and malignant tissues need to be surveyed to determine whether this transdominant p73 variant occurs in a tumor-specific manner.

A purple acid phosphatase from sweet potato contains an antiferromagnetically coupled binuclear Fe-Mn center

A purple acid phosphatase from sweet potato is the first reported example of a protein containing an enzymatically active binuclear Fe-Mn center. Multifield saturation magnetization data over a temperature range of 2 to 200 K indicates that this center is strongly antiferromagnetically coupled. Metal ion analysis shows an excess of iron over manganese. Low temperature EPR spectra reveal only resonances characteristic of high spin Fe(III) centers (Fe(III)-apo and Fe(III)-Zn(II)) and adventitious Cu(II) centers. There were no resonances from either Mn(II) or binuclear Fe-Mn centers. Together with a comparison of spectral properties and sequence homologies between known purple acid phosphatases, the enzymatic and spectroscopic data strongly indicate the presence of catalytic Fe(III)-Mn(II) centers in the active site of the sweet potato enzyme. Because of the strong antiferromagnetism it is likely that the metal ions in the sweet potato enzyme are linked via a mu -oxo bridge, in contrast to other known purple acid phosphatases in which a mu -hydroxo bridge is present. Differences in metal ion composition and bridging may affect substrate specificities leading to the biological function of different purple acid phosphatases.