Effects of different lipid levels on protozoa population, microbial protein synthesis and rumen degradability in cattle

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Recovery of rat growth and lipid profiles in adult rats subjected to fetal protein malnutrition with a fructose-rich diet

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Growth and metabolism of pacu (Piaractus mesopotamicus Holmberg 1887) juveniles fed diets containing different protein, lipid and carbohydrate levels

To verify the potential of lipids and carbohydrates to spare dietary protein and to understand the intermediary metabolism of interaction of these nutrients in pacu juveniles, an experiment was carried out to evaluate pacu physiological and performance parameters. The experimental design was completely randomized with 12 treatments in a 2 x 2 x 3 factorial arrangement, consisting of diets containing two digestible protein levels (200 and 230 g kg(-1) PD), two lipid levels (40 and 80 g kg(-1)) and three carbohydrate levels (410, 460 and 500 g kg(-1)). Fish-fed 230 g kg(-1) digestable protein (DP) showed increased glycaemia, decreased hepatic glycogen, as well as a smaller intake index and better feed conversion ratio. The higher dietary lipid level (80 g kg(-1)) reduced protein intake and serum protein concentration, increased liver and body fat content, but did not affect growth. At a lipid level of 80 g kg(-1), the increase in dietary carbohydrate levels promoted greater weight gain (WG), crude protein intake (CPI) and better feed conversion ratio (FCR). For fish fed diets containing 40 g kg(-1) lipid, the best energy-productive values (EPV) were obtained at 460 g kg(-1) carbohydrate. Increased levels of the main nutrients in the diets reduced the levels of serum triglycerides, while the increase in energy concentration increased the hepatosomatic (HSI) and glycaemia index values. Pacu used lipids as effectively as carbohydrates in the maximization of protein usage, as long as dietary protein was at a level of 230 g kg(-1) DP. The physiological parameters indicated that the best balance between the DP, dietary lipid and carbohydrate levels within the ranged this trial was obtained at 230, 40 and 460 g kg(-1), respectively, without lower growth.

CHOLESTEROL MODULATION OF LIPID PROTEIN INTERACTIONS IN LIVER MICROSOMAL MEMBRANE - A SPIN LABEL STUDY

ESR spectra of spin probes were used to monitor lipid-protein interactions in native and cholesterol-enriched microsomal membranes. In both systems composite spectra were obtained, one characteristic of bulk bilayer organization and another due to a motionally restricted population, which was ascribed to lipids in a protein microenvironment. Computer spectral subtractions revealed that cholesterol modulates the order/mobility of both populations in opposite ways, i.e., while the lipid bilayer region gives rise to more anisotropic spectra upon cholesterol enrichment, the spectra of the motionally restricted population become indicative of increased mobility and/or decreased order. These events were evidenced by measurement of both effective order parameters and correlation times. The percentages of the motionally restricted component were invariant in native and cholesterol-enriched microsomes. Variable temperature studies also indicated a lack of variation of the percentages of both spectral components, suggesting that the motionally restricted one was not due to protein aggregation. The results correlate well with the effect of cholesterol enrichment on membrane-bound enzyme kinetics and on the behavior of fluorescent probes [Castuma & Brenner (1986) Biochemistry 25, 4733-4738]. Several hypothesis are put forward to explain the molecular mechanism of the cholesterol-induced spectral changes.

Effect of protein, carbohydrate, lipid, and selenium levels on the performance, carcass yield, and blood changes in broilers

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The effect of lipid sources on intake, rumen fermentation parameters and microbial protein synthesis in Nellore steers supplemented with glycerol

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Linker for Activation of T-cell Family Member2 (LAT2) a Lipid Raft Adaptor Protein for AKT Signaling, Is an Early Mediator of Alkylphospholipid Anti-leukemic Activity

Lipid rafts are highly ordered membrane domains rich in cholesterol and sphingolipids that provide a scaffold for signal transduction proteins; altered raft structure has also been implicated in cancer progression. We have shown that 25 mu M 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets high cholesterol domains in model membranes and induces apoptosis in leukemia cells but spares normal hematopoietic and epithelial cells under the same conditions. We performed a quantitative (SILAC) proteomic screening of ODPC targets in a lipid-raft-enriched fraction of leukemic cells to identify early events prior to the initiation of apoptosis. Six proteins, three with demonstrated palmitoylation sites, were reduced in abundance. One, the linker for activation of T-cell family member 2 (LAT2), is an adaptor protein associated with lipid rafts in its palmitoylated form and is specifically expressed in B lymphocytes and myeloid cells. Interestingly, LAT2 is not expressed in K562, a cell line more resistant to ODPC-induced apoptosis. There was an early loss of LAT2 in the lipid-raft-enriched fraction of NB4 cells within 3 h following treatment with 25 mu M ODPC. Subsequent degradation of LAT2 by proteasomes was observed. Twenty-five mu M ODPC inhibited AKT activation via myeloid growth factors, and LAT2 knockdown in NB4 cells by shRNA reproduced this effect. LAT2 knockdown in NB4 cells also decreased cell proliferation and increased cell sensitivity to ODPC (7.5X), perifosine (3X), and arsenic trioxide (8.5X). Taken together, these data indicate that LAT2 is an early mediator of the anti-leukemic activity of alkylphospholipids and arsenic trioxide. Thus, LAT2 may be used as a target for the design of drugs for cancer therapy. Molecular & Cellular Proteomics 11: 10.1074/mcp.M112.019661, 1898-1912, 2012.

The photosynthetic bacterial reaction center in native and artificial envirnoments: effects on light-induced electron transfer

In this thesis we focussed on the characterization of the reaction center (RC) protein purified from the photosynthetic bacterium Rhodobacter sphaeroides. In particular, we discussed the effects of native and artificial environment on the light-induced electron transfer processes. The native environment consist of the inner antenna LH1 complex that copurifies with the RC forming the so called core complex, and the lipid phase tightly associated with it. In parallel, we analyzed the role of saccharidic glassy matrices on the interplay between electron transfer processes and internal protein dynamics. As a different artificial matrix, we incorporated the RC protein in a layer-by-layer structure with a twofold aim: to check the behaviour of the protein in such an unusual environment and to test the response of the system to herbicides. By examining the RC in its native environment, we found that the light-induced charge separated state P+QB - is markedly stabilized (by about 40 meV) in the core complex as compared to the RC-only system over a physiological pH range. We also verified that, as compared to the average composition of the membrane, the core complex copurifies with a tightly bound lipid complement of about 90 phospholipid molecules per RC, which is strongly enriched in cardiolipin. In parallel, a large ubiquinone pool was found in association with the core complex, giving rise to a quinone concentration about ten times larger than the average one in the membrane. Moreover, this quinone pool is fully functional, i.e. it is promptly available at the QB site during multiple turnover excitation of the RC. The latter two observations suggest important heterogeneities and anisotropies in the native membranes which can in principle account for the stabilization of the charge separated state in the core complex. The thermodynamic and kinetic parameters obtained in the RC-LH1 complex are very close to those measured in intact membranes, indicating that the electron transfer properties of the RC in vivo are essentially determined by its local environment. The studies performed by incorporating the RC into saccharidic matrices evidenced the relevance of solvent-protein interactions and dynamical coupling in determining the kinetics of electron transfer processes. The usual approach when studying the interplay between internal motions and protein function consists in freezing the degrees of freedom of the protein at cryogenic temperature. We proved that the “trehalose approach” offers distinct advantages with respect to this traditional methodology. We showed, in fact, that the RC conformational dynamics, coupled to specific electron transfer processes, can be modulated by varying the hydration level of the trehalose matrix at room temperature, thus allowing to disentangle solvent from temperature effects. The comparison between different saccharidic matrices has revealed that the structural and dynamical protein-matrix coupling depends strongly upon the sugar. The analyses performed in RCs embedded in polyelectrolyte multilayers (PEM) structures have shown that the electron transfer from QA - to QB, a conformationally gated process extremely sensitive to the RC environment, can be strongly modulated by the hydration level of the matrix, confirming analogous results obtained for this electron transfer reaction in sugar matrices. We found that PEM-RCs are a very stable system, particularly suitable to study the thermodynamics and kinetics of herbicide binding to the QB site. These features make PEM-RC structures quite promising in the development of herbicide biosensors. The studies discussed in the present thesis have shown that, although the effects on electron transfer induced by the native and artificial environments tested are markedly different, they can be described on the basis of a common kinetic model which takes into account the static conformational heterogeneity of the RC and the interconversion between conformational substates. Interestingly, the same distribution of rate constants (i.e. a Gamma distribution function) can describe charge recombination processes in solutions of purified RC, in RC-LH1 complexes, in wet and dry RC-PEM structures and in glassy saccharidic matrices over a wide range of hydration levels. In conclusion, the results obtained for RCs in different physico-chemical environments emphasize the relevance of the structure/dynamics solvent/protein coupling in determining the energetics and the kinetics of electron transfer processes in a membrane protein complex.

Systematic studies of protein immobilization by surface plasmon field-enhanced fluorescence spectroscopy

The research interest of this study is to investigate surface immobilization strategies for proteins and other biomolecules by the surface plasmon field-enhanced fluorescence spectroscopy (SPFS) technique. The recrystallization features of the S-layer proteins and the possibility of combining the S-layer lattice arrays with other functional molecules make this protein a prime candidate for supramolecular architectures. The recrystallization behavior on gold or on the secondary cell wall polymer (SCWP) was recorded by SPR. The optical thicknesses and surface densities for different protein layers were calculated. In DNA hybridization tests performed in order to discriminate different mismatches, recombinant S-layer-streptavidin fusion protein matrices showed their potential for new microarrays. Moreover, SCWPs coated gold chips, covered with a controlled and oriented assembly of S-layer fusion proteins, represent an even more sensitive fluorescence testing platform. Additionally, S-layer fusion proteins as the matrix for LHCII immobilization strongly demonstrate superiority over routine approaches, proving the possibility of utilizing them as a new strategy for biomolecular coupling. In the study of the SPFS hCG immunoassay, the biophysical and immunological characteristics of this glycoprotein hormone were presented first. After the investigation of the effect of the biotin thiol dilution on the coupling efficiently, the interfacial binding model including the appropriate binary SAM structure and the versatile streptavidin-biotin interaction was chosen as the basic supramolecular architecture for the fabrication of a SPFS-based immunoassay. Next, the affinity characteristics between different antibodies and hCG were measured via an equilibrium binding analysis, which is the first example for the titration of such a high affinity interaction by SPFS. The results agree very well with the constants derived from the literature. Finally, a sandwich assay and a competitive assay were selected as templates for SPFS-based hCG detection, and an excellent LOD of 0.15 mIU/ml was attained via the “one step” sandwich method. Such high sensitivity not only fulfills clinical requirements, but is also better than most other biosensors. Fully understanding how LHCII complexes transfer the sunlight energy directionally and efficiently to the reaction center is potentially useful for constructing biomimetic devices as solar cells. After the introduction of the structural and the spectroscopic features of LHCII, different surface immobilization strategies of LHCII were summarized next. Among them the strategy based on the His-tag and the immobilized metal (ion) affinity chromatography (IMAC) technique were of great interest and resulted in different kinds of home-fabricated His-tag chelating chips. Their substantial protein coupling capacity, maintenance of high biological activity and a remarkably repeatable binding ability on the same chip after regeneration was demonstrated. Moreover, different parameters related to the stability of surface coupled reconstituted complexes, including sucrose, detergent, lipid, oligomerization, temperature and circulation rate, were evaluated in order to standardize the most effective immobilization conditions. In addition, partial lipid bilayers obtained from LHCII contained proteo-liposomes fusion on the surface were observed by the QCM technique. Finally, the inter-complex energy transfer between neighboring LHCIIs on a gold protected silver surface by excitation with a blue laser (λ = 473nm) was recorded for the first time, and the factors influencing the energy transfer efficiency were evaluated.

The coupling between electron transfer and Protein/Solvent Dynamics in Photosynthetic Reaction Centers: Spectroscopic Studies in Amorphous Matrices

We investigated at the molecular level protein/solvent interactions and their relevance in protein function through the use of amorphous matrices at room temperature. As a model protein, we used the bacterial photosynthetic reaction center (RC) of Rhodobacter sphaeroides, a pigment protein complex which catalyzes the light-induced charge separation initiating the conversion of solar into chemical energy. The thermal fluctuations of the RC and its dielectric conformational relaxation following photoexcitation have been probed by analyzing the recombination kinetics of the primary charge-separated (P+QA-) state, using time resolved optical and EPR spectroscopies. We have shown that the RC dynamics coupled to this electron transfer process can be progressively inhibited at room temperature by decreasing the water content of RC films or of RC-trehalose glassy matrices. Extensive dehydration of the amorphous matrices inhibits RC relaxation and interconversion among conformational substates to an extent comparable to that attained at cryogenic temperatures in water-glycerol samples. An isopiestic method has been developed to finely tune the hydration level of the system. We have combined FTIR spectral analysis of the combination and association bands of residual water with differential light-minus-dark FTIR and high-field EPR spectroscopy to gain information on thermodynamics of water sorption, and on structure/dynamics of the residual water molecules, of protein residues and of RC cofactors. The following main conclusions were reached: (i) the RC dynamics is slaved to that of the hydration shell; (ii) in dehydrated trehalose glasses inhibition of protein dynamics is most likely mediated by residual water molecules simultaneously bound to protein residues and sugar molecules at the protein-matrix interface; (iii) the local environment of cofactors is not involved in the conformational dynamics which stabilizes the P+QA-; (iv) this conformational relaxation appears to be rather delocalized over several aminoacidic residues as well as water molecules weakly hydrogen-bonded to the RC.

A molecular approach towards tethered bilayer lipid membranes: synthesis and characterization of novel anchor lipids

Tethered bilayer lipid membranes (tBLMs) are a promising model system for the natural cell membrane. They consist of a lipid bilayer that is covalently coupled to a solid support via a spacer group. In this study, we developed a suitable approach to increase the submembrane space in tBLMs. The challenge is to create a membrane with a lower lipid density in order to increase the membrane fluidity, but to avoid defects that might appear due to an increase in the lateral space within the tethered monolayers. Therefore, various synthetic strategies and different monolayer preparation techniques were examined. Synthetical attempts to achieve a large ion reservoir were made in two directions: increasing the spacer length of the tether lipids and increasing the lateral distribution of the lipids in the monolayer. The first resulted in the synthesis of a small library of tether lipids (DPTT, DPHT and DPOT) characterized by 1H and 13C NMR, FD-MS, ATR, DSC and TGA. The synthetic strategy for their preparation includes synthesis of precursor with a double bond anchor that can be easily modified for different substrates (e.g. metal and metaloxide). Here, the double bond was modified into a thiol group suitable for gold surface. Another approach towards the preparation of homogeneous monolayers with decreased two-dimensional packing density was the synthesis of two novel anchor lipids: DPHDL and DDPTT. DPHDL is “self-diluted” tether lipid containing two lipoic anchor moieties. DDPTT has an extended lipophylic part that should lead to the preparation of diluted, leakage free proximal layers that will facilitate the completion of the bilayer. Our tool-box of tether lipids was completed with two fluorescent labeled lipid precursors with respectively one and two phytanyl chains in the hydrophobic region and a dansyl group as a fluorophore. The use of such fluorescently marked lipids is supposed to give additional information for the lipid distribution on the air-water interface. The Langmuir film balance was used to investigate the monolayer properties of four of the synthesized thiolated anchor lipids. The packing density and mixing behaviour were examined. The results have shown that mixing anchor with free lipids can homogeneously dilute the anchor lipid monolayers. Moreover, an increase in the hydrophylicity (PEG chain length) of the anchor lipids leads to a higher packing density. A decrease in the temperature results in a similar trend. However, increasing the number of phytanyl chains per lipid molecule is shown to decrease the packing density. LB-monolayers based on pure and mixed lipids in different ratio and transfer pressure were tested to form tBLMs with diluted inner layers. A combination of the LB-monolayer transfer with the solvent exchange method accomplished successfully the formation of tBLMs based on pure DPOT. Some preliminary investigations of the electrical sealing properties and protein incorporation of self-assembled DPOT and DDPTT-based tBLMs were conducted. The bilayer formation performed by solvent exchange resulted in membranes with high resistances and low capacitances. The appearance of space beneath the membrane is clearly visible in the impedance spectra expressed by a second RC element. The latter brings the conclusion that the longer spacer in DPOT and the bigger lateral space between the DDPTT molecules in the investigated systems essentially influence the electrical parameters of the membrane. Finally, we could show the functional incorporation of the small ion carrier valinomycin in both types of membranes.