Serum Fractions Research Articles

Improvement of the Fresh Taste Intensity of Processed Clementine Juice by Separate Pasteurization of its Serum and Pulp

This work describes a process to separate the Clementine juice by centrifugation into two fractions, the larger one (`serum fraction') almost lacking of pulp, and the smaller one (`pulp fraction') enriched in pulp, which are independently pasteurized in conventional plate heat exchangers under different conditions. Both fractions were aseptically blended giving a final product, whose fresh taste did not differ from that of the initial nonheated juice. The larger serum fraction was almost free of pectin methylesterase activity and could be pasteurized under minimal heat conditions, just enough to destroy microorganisms, less heat-resistant than enzymes. Since only a minor part of the juice (the pulp fraction) received the usual heat treatment, fresh taste was preserved. Destruction of Lactobacillus plantarum inoculated to the serum fraction was fitted accurately by the Weibull model, with a reduction higher than 5 log cycle at 57.5 °C for 20 s and no counts were found at 60 °C for 10 s. According to this, thermal treatment of the serum fraction (78% of the initial juice) was fixed at 60 °C for 15 s. Sensory evaluation of fresh taste intensity showed no significant differences between serum treated at 60°C for 15 s and fresh serum. The pulp fraction (22%) was pasteurized at 85 °C for 15 s and at 90 °C for 30 s to assure enough enzyme inactivation. No differences were found between the nonpasteurized juice and the blend of pulp and serum pasteurized as explained above, whereas both samples presented fresher taste than the whole juice treated at 90 °C for 30 s.

Composition and physicochemical properties of two protein fractions of bovine blood serum

O sangue animal de abatedouros representa uma fonte importante de ingrediente alimenticio subutilizado. A producao de sangue bovino no Brasil foi aproximadamente 3,6 x 106 tons em 2005. Alem do desperdicio de uma proteina de boa qualidade, a subutilizacao deste subproduto da industria animal e uma fonte importante de poluicao dos recursos hidricos e do solo. A concentracao de proteina albumina (BSA) e globulina (BSG) bovinas secas em spray ou liofilizadas foi, aproximadamente, 85,0%. O score de aminoacidos (EAAS) foi 72,7%, para BSA liofilizada, e 89,3%, para BSG. As propriedades emulsificantes e espumantes de ambas as fracoes proteicas foram altas, principalmente a pH 5,5. A capacidade emulsificante e espumante foi maior para BSA, no entanto, tanto a estabilidade da emulsao quanto a da espuma formada foram melhores para a BSG. A adicao de NaCl apresentou uma tendencia de reduzir as duas propriedades surfactantes, independentemente do pH. Considerando a solubilidade, a estabilidade ao calor, propriedades emulsificante e espumante, tanto a BSA quanto a BSG deveriam ser consideradas bons ingredientes funcionais para a manufatura de produtos alimenticios. O estudo nao apenas confirmou dados ja reportados na literatura, como tambem explorou novas propriedades, o que amplifica o potencial de aplicacoes do soro de sangue bovino.

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Searching biomarker candidates in serum using multidimensional native chromatography. II Method evaluation with Alport syndrome and severe inflammation

Biomarker search using multidimensional native liquid fractionation of serum in microplates was evaluated. From different donors, homologous sample fractions with UV absorbance depending on state of illness were selected, and their constituents were identified and quantitated by MS. Analysis of sera of patients with Alport syndrome and severe inflammation proved the reliability of the method by confirming characteristic alterations. Moreover, 23 new marker candidates were detected for Alport syndrome, some of them being involved in matrix degradation and repair, and 33 new candidates for severe inflammation, among them α1B-glycoprotein cysteine-rich secretory protein and an apparently low molecular-weight albumin variant.

A GC-SIM-MS Method for the Determination of Butylidenephthalide in Rat Plasma and Tissue: Application to the Pharmacokinetic and Tissue Distribution Study

Butylidenephthalide is one of the major active components isolated from Rhizoma Chuanxiong. This paper describes a simple, rapid, specific and sensitive method for the quantification of butylidenephthalide in rat plasma and tissue distribution using a liquid-liquid extraction procedure followed by capillary gas chromatography-selected ion monitoring mode-mass spectrometry (GC-SIM-MS) analysis. The calibration curves were linear over the concentration ranging from 0.02–10.0 µg/mL (r > 0.99) for plasma samples and 0.18–7.25 µg/g (r > 0.99) for the tissue samples. The limit of quantification (LOQ) was 1.0 ng/mL or 1.0 ng/g (ten times signal/noise ratio). Within- and between-day precisions expressed as the relative standard deviation (RSD) for the method were 2.39–2.98% and 2.97–4.26%, respectively. The methods of recovery for all samples were greater than 80% at the low, medium, and high concentrations. The method has been successfully applied to a pharmacokinetics study in rats after an oral administration of Butylidenephthalide with a dose of 20.0 mg/kg. The main pharmacokinetic parameters obtained were T max = (0.22 ± 0.06) h, C max = (3 ± 1) µg/mL, AUC = (32 ± 6) h•µg/mL, and K a = (8.5 ± 0.8)/h. The results showed that the butylidenephthalide was easily absorbed. The concentrations of butylidenephthalide in rat kidney, lung, heart, and cerebellum were higher than those in other organs. To determine free fraction in serum, samples were filtered using ultrafiltration membranes with a molecular weight cut-off of 10,000 Da and extracted using liquid-liquid extraction. The extracts were evaporated and analyzed by GC-MS. The protein binding in rat plasma, human plasma, and human serum albumin were 83 ± 4%, 94 ± 3%, and 89 ± 3%, respectively.

Tumor Length in Prostate Cancer

To evaluate the impact of tumor length and fraction of positive biopsy cores on overall survival, I used the data for 526 patients with prostate cancer. Median follow-up in patients not observed until death was more than 6 years. In a Cox model analysis that included age, serum prostate-specific antigen (PSA) level, grade, and fraction of positive cores, tumor length was the most closely associated with overall survival time (P=6 x 10(-5)); however, the impact of tumor length was mostly for a subset of men with tumors measuring more than 20 mm. Patient age, serum PSA level, Gleason score, fraction of cores with tumor, and tumor length were all significantly codependent variables. For routine cases of prostate cancer, measuring tumor length in the needle cores may be unnecessary. Tumor length may assist studies of long-term outcomes or treatment trials in prostate cancer by reducing baseline variance better than other prognostic variables. For the few patients with unusually large amounts of tumor in biopsy specimens, tumor length may provide a concise indicator for the likelihood of an adverse outcome, especially when the values of other prognostic variables appear by themselves to be less ominous.

Bacterial Peptidoglycan Triggers Candida albicans Hyphal Growth by Directly Activating the Adenylyl Cyclase Cyr1p

Human serum potently induces hyphal development of the polymorphic fungal pathogen Candida albicans, a phenotype that contributes critically to infections. The fungal adenylyl cyclase Cyr1p is a key component of the cAMP/PKA-signaling pathway that controls diverse infection-related traits, including hyphal morphogenesis. However, identity of the serum hyphal inducer(s) and its fungal sensor remain unknown. Our initial analyses of active serum fractions revealed signs of bacterial peptidoglycan (PGN)-like molecules. Here, we show that several purified and synthetic muramyl dipeptides (MDPs), subunits of PGN, can strongly promote C. albicans hyphal growth. Analogous to PGN recognition by the mammalian sensors Nod1 and Nod2 through their leucine-rich-repeat (LRR) domain, we show that MDPs activate Cyr1p by directly binding to its LRR domain. Given the abundance of PGN in the intestine, a natural habitat and invasion site for C. albicans, our findings have important implications for the mechanisms of infection by this pathogen.

Serum immunomodulatory factors in gastrointestinal diseases. A 30-50-kD serum fraction in Crohn's disease capable of modulating lymphocyte activation

We tested the hypothesis that serum factors present in Crohn's disease interfere with the process of lymphocyte activation. The mitogen-induced proliferation and the expression of early activation antigens by normal lymphocytes cultured in the presence of either Crohn's disease sera or sera from different controls were evaluated. The mitogen-induced proliferation was significantly impaired in the presence of Crohn's disease sera. These sera markedly inhibited the mitogen-induced interleukin-2 receptor (IL-2R) expression (48% inhibition), while the effect of sera on the expression of the transferrin receptor and the 4F2 antigen was much less pronounced. Diafiltration experiments showed that the inhibitory effect was confined to a 30-50-kD serum fraction. Such a serum property was not related to the patients' disease activity and disappeared after surgical removal of the affected bowel. The capability of inhibiting the mitogen-induced IL-2R expression was not restricted to Crohn's disease and was observed with sera from other inflammatory and neoplastic gastrointestinal disorders. This study indicates that a marked inhibition of the IL-2R is a mechanism underlying the immunosuppressive property of the serum in Crohn's disease and in other gastrointestinal conditions.

Inhibition of enzyme function by human autoantibodies to an autoantigen pyruvate dehydrogenase E2; different epitope for spontaneous human and induced rabbit autoantibodies

Antibodies to the mitochondrial autoantigen M2, characteristic of the autoimmune liver disease primary biliary cirrhosis (PBC), react with the E2 subunit of the pyruvate dehydrogenase enzyme (PDH-E2). We examined the effect of disease sera on the enzyme activation catalysed by the PDH complex. Inhibition of enzyme activity was observed in 19 of 24 sera of patients with PBC with a level of greater than 90% inhibition in 14 at a serum dilution of 1/50. The onset of inhibition by serum was rapid, within the time of mixing, and the inhibitory activity was shown to reside in the immunoglobulin fraction of the serum. The immunoglobulin fraction of control sera from patients with other liver diseases (n = 26) and healthy persons (n = 8) failed to produce inhibitory activity. In addition sera from four rabbits, intensively immunized with a recombinant human M2 autoantigen, gave anti-M2 reactions by fluorescence, ELISA and immunoblotting, but did not inhibit the activity of PDH. The failure of experimentally induced M2 antibodies in rabbits to inhibit is interesting in view of the reactivity of the natural M2 autoantibodies of PBC with the highly conserved site on the enzyme which carries the essential lipoic acid cofactor.

Detection of glomerular-binding immune elements in murine lupus using a tissue-based ELISA.

The glomerulonephritis in systemic lupus erythematosus (SLE) is presumably triggered by the binding of circulating immune elements (autoantibodies and immune complexes) to the glomerulus; however, the nature of these elements is unclear. In order to detect and characterize such elements, we developed an ELISA using whole intact glomeruli as the substrate. With this assay, glomerular binding activity (GBA) was detected in the serum of MRL lpr, NZB x W, and B x SB mice, but not in non-autoimmune BALB/c mice. Less activity was present in the serum of C3H lpr, C57Bl/6J lpr and AKR lpr animals which develop signs of autoimmunity but only modest renal disease. The GBA in MRL lpr mice contained IgG (subclasses 1, 2a and 2b), but not IgG3, IgM, IgA, or C3. GBA was not significantly decreased by preadsorption of MRL lpr serum by DNA-agarose (although anti-DNA antibodies were). Binding activity in serum, however, was diminished by DNAase treatment. Fractionation of MRL lpr serum over a molecular sizing column showed that GBA eluted in a broad peak. GBA bound to the glomerulus ex vivo in a tissue-specific fashion and was enriched in renal eluates relative to serum in vivo. In sum, the binding activity detected by this assay appeared to be a heterogeneous entity (possibly in part immune complexes containing DNA) which bound specifically to the glomerulus and which appeared to parallel the presence of renal disease. This novel assay system may help elucidate the pathogenesis of SLE nephritis and have utility as a disease marker.

Antibodies to small ribonucleoprotein and to 73-kD heat shock protein: two distinct markers of mixed connective tissue disease.

We set out to discover whether antibodies to small ribonucleoprotein antigens (RNP) and to 73-kD heat shock protein (hsp 73) which have been proposed as markers of mixed connective tissue disease (MCTD) recognize different epitopes. MCTD serum was immunoadsorbed on hsp 73-coupled Sepharose and the affinity retained, and non-retained fractions were checked by immunoblotting for recognition of either purified bovine hsp 73 or calf thymus extract RNPs. The hsp 73 affinity-bound serum fraction recognized hsp 73 but not RNP antigens, the reverse being true for the non-retained fraction. We conclude that anti-hsp 73 and anti-RNPs are distinct markers of MCTD.

Isolation and characterization of a high molecular weight lymphocyte Feγ receptor-blocking factor associated with renal allograft survival

We have confirmed our previous observation that improved human renal allograft survival is associated with the presence in pre-transplant serum of a high molecular weight lymphocyte Fc gamma receptor-blocking factor. Serum fractionation studies suggest that this factor is a complex protein consisting of IgG together with an IgG-binding protein which has an apparent molecular weight of approximately 60 kD.

Gravinol Ameliorates High-Fructose-Induced Metabolic Syndrome through Regulation of Lipid Metabolism and Proinflammatory State in Rats

Using a rat model with fructose-induced metabolic syndrome, the effect of gravinol was investigated. Male Wistar rats were fed a 65% fructose diet and administered 10 or 20 mg of gravinol/kg of body weight/day for 2 weeks. High-level fructose feeding led to hyperglycemia, hyperlipidemia, hypertri-glyceridemia, and hypertension. On the other hand, the administration of gravinol significantly lowered serum glucose and total cholesterol levels. The tail arterial blood pressure was significantly elevated with the high-fructose diet. However, rats given gravinol showed a lower blood pressure as compared with fructose-fed control rats. In addition, the triglyceride (TG) levels in serum and lipoprotein fraction were dose-dependently reduced in rats fed gravinol. The decreases of hepatic TG and total cholesterol by gravinol were responsible for the down-regulation of hepatic sterol regulatory element binding protein (SREBP)-1. However, gravinol did not affect the protein levels of hepatic peroxisome proliferator-activated receptor-alpha and SREBP-2. Moreover, gravinol administration in the fructose-fed rats markedly reduced the glycosylated protein and thiobarbituric acid-reactive substance levels in the serum and hepatic mitochondria, and it inhibited the increase of the cyclooxygenase-2 protein level as a result of the down-regulation of nuclear factor kappa B (NF-kappaB). Furthermore, the decrease of anti-apoptotic bcl-2 protein levels and the increase of pro-apoptotic bax protein levels by the high-fructose diet were reversed by gravinol. These findings suggest that fructose-induced metabolic syndrome is attenuated by gravinol administration, which is associated with the reduction of serum lipids and protection against the proinflammatory state induced by oxidative stress.

Identification of a Domain That Mediates Association of Platelet-activating Factor Acetylhydrolase with High Density Lipoprotein

The plasma form of platelet-activating factor (PAF) acetylhydrolase (PAF-AH), also known as lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) inactivates potent lipid messengers such as PAF and modified phospholipids generated in settings of oxidant stress. In humans, PAF-AH circulates in blood in fully active form and associates with high and low density lipoproteins (HDL and LDL). Several studies suggest that the location of PAF-AH affects both the catalytic efficiency and the function of the enzyme in vivo. The distribution of PAF-AH among lipoproteins varies widely among mammals. Here, we report that mouse and human PAF-AHs associate with human HDL particles of different density. We made use of this observation in the development of a binding assay to identify domains required for association of human PAF-AH with human HDL. Sequence comparisons among species combined with domain-swapping and site-directed mutagenesis studies led us to the identification of C-terminal residues necessary for the association of human PAF-AH with human HDL. Interestingly, the region identified is not conserved among PAF-AHs, suggesting that PAF-AH interacts with HDL particles in a manner that is unique to each species. These findings contribute to our understanding of the mechanisms responsible for association of human PAF-AH with HDL and may facilitate future studies aimed at precisely determining the function of PAF-AH in each lipoprotein particle.

Synaptophysin is an autoantigen in paraneoplastic neuropathy

Paraneoplastic neurological syndromes (PNS) are often associated with antineuronal autoantibodies and many of them could be identified in the recent years. However, there are still new antineuronal binding patterns with yet unidentified autoantigens. We here describe a new autoantibody associated with paraneoplastic sensorimotor and autonomic neuropathy in a patient with small cell lung cancer. In indirect immunofluorescence test, the patient's serum colocalised with the synaptic protein synaptophysin in the cerebellum and myenteric plexus of the gut. Immunoblotting showed a 38 kDa reactivity, which is also the molecular weight of synaptophysin. Therefore a Western Blot with recombinant synaptophysin has been used and revealed reactivity of the serum against synaptophysin. In patients with non-paraneoplastic neuropathies or healthy controls, anti-synaptophysin autoantibodies were not detectable. In 20 SCLC patients without neurological syndromes, two patients had low-titer anti-synaptophysin autoantibodies. The patient's serum and IgG fraction showed cytotoxicity to primary cultured myenteric plexus neurons. We conclude that synaptophysin is an autoantigen in paraneoplastic neurological syndromes.

GLYCATED LDL IN THE DEVELOPMENT OF ATHEROSCLEROTIC LESIONS – THE ROLE OF SCAVENGER RECEPTORS

The role of CETP in the development of atherosclerosis is debatable, and few data exist regarding the total impact of CETP inhibition on cholesterol efflux.Acceptor capacities of whole serum and HDL subfractions separated by HPLC were compared using 2 different cell systems. Subjects with CETP deficiency (2 homozygous, 1 compound heterozygous, and 5 heterozygous) were analyzed along with 10 normolipidemic controls. The fractional efflux from cholesterol-labeled Fu5AH hepatoma cells was determined to be SR-BI mediated. The efflux difference between control and liver X receptor (LXR) agonist-induced ABCA1-upregulated J774 macrophages was considered as a measure of ABCA1-mediated efflux.For the Fu5AH cell system, the total acceptor capacities of whole serum and HPLC-separated HDL fraction 2 obtained from the homozygous subjects were 38% and 116% higher than the corresponding values for the controls, respectively (p < 0.05). For the J774 cell system, the total acceptor capacities of whole serum and HPLC-separated HDL fractions were similar among the CETP-deficient subjects and controls.Serum from homozygous subjects with CETP-null defects exhibited enhanced acceptor capacity via an SR-BI dependent pathway, which is regulated by the middle HPLC-separated HDL fraction. Further, the cholesterol acceptor capacity of serum obtained from patients having complete and partial CETP deficiency was preserved via an ABCA1-dependent pathway.

Stress-induced cardiomyopathy due to chronic psycho-emotional overstrain: A separate nosologic entity or a debut of the ischemic heart disease?

Resume Stress-induced cardiomyopathy due to chronic phsycho-emotional overstrain we call non-ischemic disturbances of repolarisation on ECG or/and cardiac arrhythmias in patients with chronic phsyco-emotional overstrain. The results of the investigation of the main indicators of cholesterol metabolism (such as: total cholesterol, LDL/HDL ratio, LDL, triglycerides) and several genetic markers associated with the early development of atherosclerosis (MTHFR gene, ApoE gene) in engine-drivers with stress-induced cardiomyopathy are presented. Patients with stress-induced cardiomyopathy show more frequent availability of T-allele of MTHFR gene compared with healthy male population of St-Petersburg. Increase of the total cholesterol level and LDL/HDL ratio in patients with the signs of stress-induced cardiomyopathy was found. Our data demonstrate the association between ApoE*4 allele with increased levels of total cholesterol and LDL fraction of blood serum, and the association between ApoE*2 allele with increased level of triglycerides as well. Results: therefore, lipid metabolism disturbances and C677T polymorphism MTHFR gene might take part in developing stress-induced cardiomyopathy. So E*2/E*3/E*4 polymorphism ApoE gene might take part in atherosclerotic leasuned in persons subjected to chronic stress.

Studies on 5α-androst-16-en-3-one binding to porcine serum, plasma and testicular cytosolic fraction and to human serum

The present study evaluated whether a specific androstenone-binding protein is present in porcine and human serum, and in the cytosolic fraction of porcine testis. The binding of [ 3H]-androstenone to serum and testicular cytosol was measured in the absence (total binding) and presence (non-specific binding) of unlabelled androstenone. The optimization of the assay is described. As a part of the assay validation, the binding of [ 3H]-dihydrotestosterone ([ 3H]-DHT) to porcine and human serum was also examined. As expected, specific binding of [ 3H]-DHT was detected in human serum, but not in porcine serum. No specific androstenone-binding protein was detected, either in porcine or human serum, or in the cytosolic fraction of porcine testis. The amount of non-specific binding of [ 3H]-androstenone was slightly lower in porcine serum compared to human serum. Between-animal variations in [ 3H]-androstenone binding were studied in plasma samples from 15 animals with androstenone concentrations ranging from 1.1 to 23.1 ng/mL. Mean values ± standard deviations of binding in these samples were 15.2 ± 0.9% for total binding and 15.9 ± 0.8% for non-specific bindings. Low between-animal variations indicate that androstenone binding does not affect androstenone accumulation in fat.

Isoform Analysis of LC−MS/MS Data from Multidimensional Fractionation of the Serum Proteome

We developed a visualization approach for the identification of protein isoforms, precursor/mature protein combinations, and fragments from LC-MS/MS analysis of multidimensional fractionation of serum and plasma proteins. We also describe a pattern recognition algorithm to automatically detect and flag potentially heterogeneous species of proteins in proteomic experiments that involve extensive fractionation and result in a large number of identified serum or plasma proteins in an experiment. Examples are given of proteins with known isoforms that validate our approach and present a subset of precursor/mature protein pairs that were detected with this approach. Potential applications include identification of differentially expressed isoforms in disease states.

Elevated serum β-glucuronidase reflects hepatic lysosomal fragility following toxic liver injury in rats

The level of serum beta-glucuronidase increases in various pathological conditions, including liver disorders. The aim of this investigation was to study the changes in liver lysosomal membrane stability during experimentally induced hepatic fibrosis that may result in the elevation of serum beta-glucuronidase. Liver injury was induced by intraperitoneal injections of N-nitrosodimethylamine (NDMA) in adult male albino rats over 3 weeks. The progression of fibrosis was evaluated histopathologically as well as by monitoring liver collagen content. Lipid peroxides and beta-glucuronidase levels were measured in the liver homogenate and subcellular fractions on days 0, 7, 14, and 21 after the start of NDMA administration. Serum beta-glucuronidase levels were also determined. A significant increase was observed in beta-glucuronidase levels in the serum, liver homogenate, and subcellular fractions, but not in the nuclear fraction on days 7, 14, and 21 after the start of NDMA administration. Lipid peroxides also increased in the liver homogenate and the lysosomal fraction. The measurement of lysosomal membrane stability revealed a maximum lysosomal fragility on day 21 during NDMA-induced fibrosis. In vitro studies showed that NDMA has no significant effect on liver lysosomal membrane permeability. The results of this investigation demonstrated that lysosomal fragility increases during NDMA-induced hepatic fibrosis, which could be attributed to increased lipid peroxidation of lysosomal membrane. In this study, we also elucidated the mechanism of increased beta-glucuronidase and other lysosomal glycohydrolases in the serum during hepatic fibrosis.