Protein oxidative changes in whole and skim milk after ultraviolet or fluorescent light exposure.

We investigated how protein changes occur, at the primary or higher structural levels, when proteins are exposed to UV or fluorescent (FL) light while in the complex matrix, milk. Whole milk (WM) or skim milk (SM) samples were exposed to FL or UV light from 0 to 24h at 4°C. Protein oxidation was evaluated by the formation of protein carbonyls (PC), dityrosine bond (DiTyr), and changes in molecular weight (protein fragmentation and polymerization). Oxidative changes in AA residues were measured by PC. Dityrosine and N'-formylkynurenine (NFK), a carbonylation derivative of Trp, were measured by fluorometry. Protein carbonyls increased as a function of irradiation time for both WM and SM. The initial rate for PC formation by exposure to FL light (0.25 or 0.27 nmol/h for WM and SM, respectively) was slower than that following exposure to UV light (1.95 or 1.20 nmol/h, respectively). The time course of NFK formation resembled that of PC. After 24h of UV exposure, SM had significantly higher levels of NFK than did WM. In contrast, WM samples irradiated with UV had higher levels of DiTyr than did SM samples, indicating different molecular pathways. The formation of intra- or intermolecular DiTyr bonds could be indicative of changes in the tertiary structure or oligomerization of proteins. The existence of NFK suggests the occurrence of protein fragmentation. Thus, proteolysis and oligomerization were analyzed by sodium dodecyl sulfate-PAGE. After 24h of exposing WM to UV or FL light, all the proteins were affected by both types of light, as evidenced by loss of material in most of the bands. Aggregates were produced only by UV irradiation. Hydrolysis by pepsin and enzyme-induced coagulation by rennet were performed to evaluate altered biological properties of the oxidized proteins. No effect on pepsin digestion or rennet coagulation was found in irradiated SM or WM. The oxidative status of proteins in milk and dairy products is of interest to the dairy industry and consumers. These findings provide knowledge that could be useful in determining the optimal lighting conditions in the dairy industry in general and in cheese making in particular.

Milk fat content measurement by a simple UV spectrophotometric method: an alternative screening method.

We developed a simple microtechnique to measure lipids in milk by UV spectrophotometry. This technique is based upon the property of fatty acids to absorb UV light proportional to their concentration. Samples of powdered or fluid milk (30 or 60 muL) were added to 3 mL of analytic grade ethanol and stored at -20 degrees C for at least 1 h. This procedure precipitates proteins and hydrophobic peptides that interfere with UV measurement. Sample absorbances are then measured at 208 nm in an UV-Vis spectrophotometer. This technique correlated very well against Milko-Scan, a device that measures milk fat by IR spectroscopy, with an r(2) >0.982. Accuracy and precision, evaluated by recovery and replicate assays, are also very acceptable. This method is suitable as a fast, cost-effective alternative screening method to estimate milk fat content in small samples without prior lipid extraction.

VLDL modulates the cytokine secretion profile to a proinflammatory pattern.

Human very-low-density lipoprotein (VLDL) inhibits DNA synthesis in lymphocytes activated by the nonspecific mitogen concanavalin A (Con A). We studied the effects of VLDL on lymphocyte activation (IL-2 receptor expression), cell cycle progression, and production of IL-2 and of IL-4 (a proinflammatory and an anti-inflammatory interleukin, respectively) to understand why an atherogenic lipoprotein inhibits cell proliferation. After 48 h of stimulation with the mitogen, VLDL decreased the population of cells bearing IL-2 receptor and the population of T-cells that progress through the cell cycle, increasing the population of T-cells in G(0)/G(1). Cells cultured in the presence of Con A and VLDL produced higher levels of IL-2 and lower levels of IL-4 than cells cultured without VLDL. These results suggest that VLDL inhibits lymphocyte proliferation by reducing IL-2 receptor and enhancing the levels of IL-2. Probably, one atherogenic effect of VLDL is to modulate the cytokine secretion profile of lymphocytes to a predominantly proinflammatory response.

Lipase production by Penicillium restrictum using solid waste of industrial babassu oil production as substrate.

Lipase, protease, and amylase production by Penicillium restrictum in solid-state fermentation was investigated. The basal medium was an industrial waste of babassu oil (Orbignya oleifera) production. It was enriched with peptone, olive oil, and Tween-80. The supplementation positively influenced both enzyme production and fungal growth. Media enriched with Tween-80 provided the highest protease activity (8.6 U/g), whereas those enriched with peptone and olive oil led to the highest lipase (27.8 U/g) and amylase (31.8 U/g) activities, respectively.

High-density lipoprotein from hypercholesterolemic animals has peroxidized lipids and oligomeric apolipoprotein A-I: its putative role in atherogenesis.

Oxidized lipoproteins have been involved in the pathogenesis of atherosclerosis and atherosclerotic lesions contain oxidized low density lipoprotein. Conversely, the presence of oxidized high density lipoprotein (HDL) in vivo has not been clearly established. Oxidation of HDL in vitro models produces an increase in peroxidized lipids and the appearance of apolipoprotein A-I (apo A-I) oligomers. We investigated the oxidative status of HDL in an in vivo model: the hypercholesterolemic chicken. The HDLs from control and hyperlipemic animals were analyzed for the content of lipid peroxides employing spectroscopic and fluorescence techniques, for the level of apo A-I oligomerization, and for susceptibility to in vitro oxidation. HDL from hypercholesterolemic chickens was more peroxidized (as detected by fluorescence), had higher amount of oligomeric apo A-I, and was oxidized to a greater extent by uv irradiation than that of control animals. We speculate that apo A-I oligomerization could be a key step in the atheroma formation.

High-density lipoprotein aggregated by oxidation induces degeneration of neuronal cells.

We have previously reported that high-density lipoprotein (HDL) exhibits antineuritogenic effects on chicken cerebral cells in culture. In the present study, we show the effects of HDLs, oxidized by UV irradiation or heating, on chicken cerebral neurons in culture. Both treatments produced several physical and chemical changes in the HDLs, i.e., formation of lipid peroxides, enlargement of HDL diameters, an increased exposure of the tryptophan groups of the apolipoprotein A-I to a more hydrophilic environment, formation of bityrosines, and cross-linking of apolipoprotein A-I. When these treatments were performed in the absence of EDTA, most of the modifications described above were more intense and HDLs formed a macroaggregate that displays a rosette-like structure. The aggregated HDLs produced neurodegeneration and death when added to both undifferentiated and differentiated cerebral neurons in culture. This process was accompanied by the disorganization of the cellular microtubular cytoskeleton and hyperphosphorylation of the microtubule-associated protein tau. Native HDL or HDLs treated in the presence of EDTA inhibited the neuritogenesis of undifferentiated neurons but did not show any significant effect on the differentiated neurons in culture. The effects on the cellular cytoskeleton and morphology of aggregated HDLs recall those of the fibrillar beta-amyloid peptide. The present results suggest that aggregated HDLs could participate in neurodegeneration associated with oxidative stress in the CNS.

Two distinguishable fluorescent modes of 1-anilino-8-naphthalenesulfonate bound to human albumin.

We study the interaction of 1-anilino-8-naphthalenesulfonate (ANS) with human (HSA) and bovine serum albumin (BSA) by phase and modulation fluorescence spectroscopy. We determined that both HSA and BSA show one or two distinguishable fluorescent sites, depending of the ANS/serum albumin ratio. At above a 1∶1 ANS/HSA molar ratio, the steady-state emission spectra for ANS can be resolved in two components: component 1, emitting with a lifetime (τ1) of 16 ns and a λ1max of 478 nm, with a quantum yield (фf1) of 0.67, and component 2, with a lifetime (τ2) of 2-4 ns and a λ2max of 483 nm, with an average quantum yield (фf2) of about 0.11. Considering these findings, the binding analysis is fitted with a model of two independent sites. Site 1 has an association constantK as1=0.87×10(6)M(-1) and a capacity of 1.04 mol of ANS/mol of HSA, and site 2 aK as2=0.079×10(6)M(-1) and a capacity of 2.34 mol of ANS/mol of HSA. Analysis of fluorescence lifetime distributions shows that the rigidity of the fluorophore environment at site 1 changes when site 2 is occupied. These findings suggest an interconnection between the two sites and that ligands can stabilize the protein's globular structure. To assess the identity of the ANS binding sites we used diazepam as a marker of the site located at the IIIA HSA subdomain and aspirin as a marker of sites located at the IIIA and IIA HSA subdomains. Both ligands displace ANS only from site 1, suggesting that it corresponds to the binding site located at the IIIA sub-domain of the protein. We determined that theK as values for diazepam and aspirin are 0.113× 10(6) and 0.021×10(6) M (-1) respectively.

High-density lipoprotein inhibits UDP-N-acetylgalactosamine:GM3, N-acetylgalactosaminyltransferase and differentiation of cultured cerebral cells: comparison with a formerly described inhibitor of this enzyme.

We have previously described a thermostable inhibitor of the UDP-N-acetylgalactosamine:GM3,N-acetylgalactosaminyltransferase (GM2 synthase) purified from chicken blood serum. Some properties of the GM2 synthase inhibitory preparation (IP) resemble those of high-density lipoprotein (HDL), i.e., both have a MW of 200,000 in native conditions and are resistant to denaturation by heat. These and other facts prompted us to test the possibility that lipoproteins regulate ganglioside biosynthesis in the CNS. For this purpose, serum lipoprotein fractions were isolated from chicken serum by flotation and were assayed as inhibitors of GM2 synthase activity and of neuron differentiation in culture. HDL (in contrast to fractions containing very low-density or low-density lipoprotein) inhibited GM2 synthase with the same specific activity as IP and inhibited neuron cell differentiation in culture in a similar way. Furthermore, these two preparations also share several other characteristics; i.e., both have the same cholesterol content, the same floating behavior on KBr gradients, and the same polypeptide pattern as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and staining with Coomassie Blue, or after western blot and revealing with an antibody prepared against IP, which is able to diminish the inhibitory effect of this preparation. The results described indicate identity between HDL and IP and suggest that HDL (particularly apolipoprotein A) could play an important role on ganglioside biosynthesis modulation during CNS development. The antineuritogenic effect of HDL described in this study could be of physiological relevance during CNS development and response to injury.

Gangliosides released by the perfused rat liver are associated to high density lipoprotein.

We examine here the delivery of gangliosides from the perfused rat liver into the perfusate. One hour after the administration of [3H]GM1 to recirculating perfused livers, almost 80% of the perfusate radioactive gangliosides were recovered associated to the HDL fraction. This fraction was relatively enriched in radioactive GD1a. The pattern of endogenous gangliosides from perfused livers, rat serum and perfusates were very different: GM3 was the main liver ganglioside, GM1 and GD1a were the most abundant in perfusates being GM3 almost absent; GM3, GM1 and GD1a were present in rat serum in similar proportions. Using a non-recirculating perfusion protocol, radioactive gangliosides were found in the HDL fraction since 15 minutes after the administration of [3H]GM1. These results suggest that rat liver supplies the perfusates with some gangliosides and that they are associated to HDL. These facts arise the possibility that the liver is one of the source of serum gangliosides.

Ganglioside incorporation and release by the isolated perfused rat liver.

The fate of exogenous ganglioside GM1 labelled in the sphingosine moiety, [Sph-3H]GM1, administered as a pulse, in the isolated perfused rat liver was investigated. When a non-recirculating protocol was employed, the amount of radioactivity in the liver and perfusates was found to be dependent on the presence of BSA in the perfusion liquid and on the time elapsed after the administration of the ganglioside. When BSA was added to the perfusion liquid, less radioactivity was found in the liver and more in the perfusate at each time tested, for up to 1 h. The recovery of radioactivity in the perfusates followed a complex course which can be described by three pseudo-first-order kinetic constants. The constants, in order of decreasing velocity, are interpreted as: (a) the dilution of the labelled GM1 by the constant influx of perfusion liquid; (b) the washing off of GM1 loosely bound to the surface of liver cells; (c) the release of gangliosides from the liver. Process (b) was found to be faster in the presence of BSA, probably owing to the ability of BSA to bind gangliosides. The [Sph-3H]GM1 in the liver underwent metabolism, leading to the appearance of products of anabolic (GD1a, GD1b) and catabolic (GM2, GM3) origin; GD1a appeared before GM2 and GM3 but, at times longer than 10 min, GM2 and GM3 showed more radioactivity than GD1a. At a given time the distribution of the radioactivity in the perfusates was quite different from that of the liver. In fact, after 60 min GD1a was the only metabolite present in any amount, the other being GM3, the quantity of which was small. This indicates that the liver is able to release newly synthesized gangliosides quite specifically. When a recirculating protocol was used, there were more catabolites and less GD1a than with the non-recirculating protocol. A possible regulatory role of ganglioside re-internalization on their own metabolism in the liver is postulated.

Synthesis and translocation of gangliosides and glycoproteins during urethane anesthesia.

In this work, we have studied (a) the contents of gangliosides, glycoproteins, and phospholipids of the vesicle and plasma membrane fractions from brains of anesthetized and control rats and chickens and (b) the labeling of gangliosides and glycoproteins in the retina ganglion cell layer and optic tectum of urethane-anesthetized and control chickens after intraocular injection of a labeled N-acetylneuraminic acid precursor and the distribution of the label after subcellular fractionation. We found an increase in the content of gangliosides relative to protein in the vesicle fraction of both anesthetized rats and chickens relative to their controls. Other values were not affected by anesthesia. These results do not reflect a faster synthesis of gangliosides stimulated by urethane, because their rate of labeling was diminished in anesthetized animals. During the 4-h period after the animals were injected intraocularly with the radioactive precursor, the highest values of ganglioside-specific radioactivity were found in the vesicle fraction of control and anesthetized animals; at longer intervals, the specific radioactivity of the vesicle and plasma membrane fractions became rather similar. These data are in accordance with previous studies from this laboratory suggesting that the synthesis of the carbohydrate chain of gangliosides is regulated by the physiological demands made by the neurotransmitting system.