A well-characterised peak identification list of MALDI MS profile peaks for human blood serum

MALDI MS profiling, using easily available body fluids such as blood serum, has attracted considerable interest for its potential in clinical applications. Despite the numerous reports on MALDI MS profiling of human serum, there is only scarce information on the identity of the species making up these profiles, particularly in the mass range of larger peptides. Here, we provide a list of more than 90 entries of MALDI MS profile peak identities up to 10 kDa obtained from human blood serum. Various modifications such as phosphorylation were detected among the peptide identifications. The overlap with the few other MALDI MS peak lists published so far was found to be limited and hence our list significantly extends the number of identified peaks commonly found in MALDI MS profiling of human blood serum.

Peptides generated ex vivo from serum proteins by tumor-specific exopeptidases are not useful biomarkers in ovarian cancer

BACKGROUND: The serum peptidome may be a valuable source of diagnostic cancer biomarkers. Previous mass spectrometry (MS) studies have suggested that groups of related peptides discriminatory for different cancer types are generated ex vivo from abundant serum proteins by tumor-specific exopeptidases. We tested 2 complementary serum profiling strategies to see if similar peptides could be found that discriminate ovarian cancer from benign cases and healthy controls. METHODS: We subjected identically collected and processed serum samples from healthy volunteers and patients to automated polypeptide extraction on octadecylsilane-coated magnetic beads and separately on ZipTips before MALDI-TOF MS profiling at 2 centers. The 2 platforms were compared and case control profiling data analyzed to find altered MS peak intensities. We tested models built from training datasets for both methods for their ability to classify a blinded test set. RESULTS: Both profiling platforms had CVs of approximately 15% and could be applied for high-throughput analysis of clinical samples. The 2 methods generated overlapping peptide profiles, with some differences in peak intensity in different mass regions. In cross-validation, models from training data gave diagnostic accuracies up to 87% for discriminating malignant ovarian cancer from healthy controls and up to 81% for discriminating malignant from benign samples. Diagnostic accuracies up to 71% (malignant vs healthy) and up to 65% (malignant vs benign) were obtained when the models were validated on the blinded test set. CONCLUSIONS: For ovarian cancer, altered MALDI-TOF MS peptide profiles alone cannot be used for accurate diagnoses.

A breakthrough in lupin biotechnology: prolific protocolonisation in recalcitrant white lupin (Lupinus albus) triggered by bovine serum albumin

Six nutrient formulations were studied for their efficacy in inducing mitosis in white lupin seedling cotyledon protoplasts of which the formulations of Schafer-Menuhr & Sturmer (AS) and Kao (K8p) were found to be superior over the other four when supplemented with 6-benzylaminopurine and alpha-naphthaleneacetic acid (alpha-NAA). An unltrafiltration treatment of K8p increased mitotic frequency by 130% when compared with the untreated control. Medium enrichment with 0.2% bovine serum albumin (BSA) brought about a dramatic 1341% rise in protoplast division in comparison with BSA-free medium but only when the enrichment was carried out in Kao and Michayluk (KM8p) background containing 2, 4-dichlorophenoxyacetic acid, alpha-NAA and zeatin. A higher number of protocolonies (each proliferating from single protoplast following multiple divisions) were seen in 0.4% BSA. With this breakthrough in white lupin protoplast research, it is now possible to reproducibly obtain protocolonies that was hitherto not possible.

Superovulation and embryo collection in nulliparous Boer goat does immunized against a recombinant ovine alpha-subunit inhibin

With the purpose of eliciting a superovulatory response, 12 adult nulliparous Boer goat does were actively immunized against a recombinant a-subunit of ovine inhibin (roIHN-alpha; two injections of 100 mg 4 weeks apart). Another 12 control Boer goat does were treated with physiological saline and acted as controls. One year later the immunized animals were boostered by the administration of another dose (100 mg) of the immunogen. Following treatment, blood samples were collected twice weekly for the periods of 16 and 12 weeks, respectively, to monitor the inhibin binding ability with the aid of a radio-tracer binding assay. Throughout the experiment, estrus detection was conducted twice daily with the aid of an aproned intact buck. From the first day after treatment to 48 h after standing estrus, ovarian activity was monitored daily by transrectal ultrasonography. On alternate estrous cycles, does were mated and 6 days later flushed transcervically to recover embryos. All goats treated with the roIHN-alpha produced antibodies reactive to the native bovine inhibin tracer-the titre increasing from 2.9 +/- 0.4 to a maximum of 21.9 +/- 2.9% binding after the second injection. The antibody titre gradually subsided over the next 16 weeks. The booster injection restored an elevated antibody titre (11.7 +/- 0.4%), which was maintained until the end of the sampling period 12 weeks later. In the control goats only trace amounts of antibody were recorded throughout the trial. In the roIHN-alpha-immunized goats the number of follicles reaching a diameter of > 4 mm was 14.6 +/- 1.2 per doe. A positive correlation was recorded between the follicle number and antibody titre (r=0.61; P < 0.01). The number of follicles ovulating per doe (6.9 +/- 0.7) followed the same tendency-however, the proportion decreased with increasing follicle numbers. A relatively weak correlation was recorded between the inhibin binding ability and number of ovulations (r=0.27; P < 0.05). In the control goats the majority (92%) of follicles exceeding 4 mm in diameter ovulated (2.5 +/- 0.1 follicles/doe). Embryo collection proved unsatisfactory (42% versus 39% recovery for immunized and control animals, respectively)-presumably because the uterine lumen of the nulliparous does was too narrow to permit effective flushing. In the group of immunized goats the occurrence of short estrous cycles (< 15 days) recorded was 34% versus only 6% in the controls. Overall, immunization of goats against roIHN-alpha led to an almost six-fold increase in number of ovarian follicles, a three-fold increase in ovulations and, despite the low recovery rate, a more than three-fold increase in ova or embryos recovered. It may be concluded that treatment of female goats with roIHN-alpha leads to an inhibin antibody response, accompanied by enhanced ovarian activity. The response was, however, accompanied by a large proportion of retained follicles and a high incidence of short estrous cycles. These problems need to be further investigated before rendering the method fit for application in embryo transfer programs in goats. (C) 2007 Elsevier B.V. All rights reserved.

Numbers of antral follicles during follicular waves in cattle: Evidence for high variation among animals, very high repeatability in individuals, and an inverse association with serum follicle-stimulating hormone concentrations

The extent, causes, and physiological significance of the variation in number of follicles growing during ovarian follicular waves in human beings and cattle are unknown. Therefore, the present study examined the variability and repeatability in numbers of follicles 3 mm or greater in diameter during the follicular waves in bovine estrous cycles, and we determined if the variation in number of follicles during waves was associated with alterations in secretion of FSH, estradiol, inhibin, and insulin-like growth factor I (IGF-I). Dairy cattle were subjected to twice-daily ultrasound analysis to count total number of antral follicles 3 mm or greater in diameter throughout 138 different follicular waves. In another study, blood samples were taken at frequent intervals from cows that consistently had low or very high numbers of follicles during waves and were subjected to immunoassays. Results indicate the following: First, despite an approximately sevenfold variation in number of follicles during waves among animals and marked differences in age, stage of lactation, and season of the year, a very highly repeatable (0.95) number of follicles 3 mm or greater in diameter is maintained during the ovulatory and nonovulatory follicular waves of individuals. Second, variation in number of follicles 3 mm or greater in diameter during waves and the inverse association of number of follicles during waves with FSH are not directly explained by alterations in the patterns of secretion of estradiol, inhibin, or IGF-I. Third, ovarian ultrasound analysis can be used reliably by investigators to identify cattle that consistently have low or high numbers of follicles during waves, thus providing a novel experimental model to determine the causes and physiological significance of the high variation in antral follicle number during follicular waves among single-ovulating species, such as cattle or humans.

Intra-individual variation of serum prostate specific antigen levels in men with benign prostate biopsies

To determine the intra-individual (physiological) variation of prostate-specific antigen (PSA) measurements in men after a benign prostatic biopsy. Sixty-four men were prospectively assessed, all of whom had a benign prostatic biopsy within the preceding 13 months. The degree of intra-individual variability was established by calculating the coefficient of variation on four PSA levels obtained from each patient weekly over a month. Six patients were subsequently diagnosed with prostate cancer and their data are presented separately. In the remaining 58 patients the median (range) individual mean PSA value was 6.3 (0.5-34.1) ng/mL. The median (range) coefficient of variation within the group was 9.5 (2.4-76.1)%. There was a clear linear relationship between mean PSA level and the standard deviation. In 48 of the 63 patients analysed, the coefficient of variation for serum PSA values in the group as a whole was greater than the variation claimed for the assay technique. The significance of the linear relationship between PSA and the standard deviation is discussed, with particular reference to those men who had a benign prostate biopsy.

Cleavage and serum reactivity of the severe acute respiratory syndrome coronavirus spike protein

Severe acute respiratory syndrome (SARS) coronavirus (SCoV) spike (S) protein is the major surface antigen of the virus and is responsible for receptor binding and the generation of neutralizing antibody. To investigate SCoV S protein, full-length and individual domains of S protein were expressed on the surface of insect cells and were characterized for cleavability and reactivity with serum samples obtained from patients during the convalescent phase of SARS. S protein could be cleaved by exogenous trypsin but not by coexpressed furin, suggesting that the protein is not normally processed during infection. Reactivity was evident by both flow cytometry and Western blot assays, but the pattern of reactivity varied according to assay and sequence of the antigen. The antibody response to SCoV S protein involves antibodies to both linear and conformational epitopes, with linear epitopes associated with the carboxyl domain and conformational epitopes associated with the amino terminal domain. Recombinant SCoV S protein appears to be a suitable antigen for the development of an efficient and sensitive diagnostic test for SARS, but our data suggest that assay format and choice of S antigen are important considerations.

Oocyte-mediated suppression of follicle-stimulating hormone- and insulin-like growth factor-induced secretion of steroids and inhibin-related proteins by bovine granulosa cells in vitro: Possible role of transforming growth factor alpha

The objective was to investigate the potential role of the oocyte in modulating proliferation and basal, FSH-induced and insulin-like growth factor (IGF)-induced secretion of inhibin A (inh A), activin A (act A), follistatin (FS), estradiol (E-2), and progesterone (P-4) by mural bovine granulosa cells. Cells from 4- to 6-mm follicles were cultured in serum-free medium containing insulin and androstenedione, and the effects of ovine FSH and IGF analogue (LR3-IGF-1) were tested alone and in the presence of denuded bovine oocytes (2, 8, or 20 per well). Medium was changed every 48 h, cultures were terminated after 144 h, and viable cell number was determined. Results are based on combined data from four independent cultures and are presented for the last time period only when responses were maximal. Both FSH and IGF increased (P < 0.001) secretion of inh A, act A, FS, E-2, and P-4 and raised cell number. In the absence of FSH or IGF, coculture with oocytes had no effect on any of the measured hormones, although cell number was increased up to 1.8-fold (P < 0.0001). Addition of oocytes to FSH-stimulated cells dose-dependently suppressed (P < 0.0001) inh A (6-fold maximum suppression), act A (5.5-fold), FS (3.6-fold), E-2 (4.6-fold), and P-4 (2.4-fold), with suppression increasing with FSH dose. Likewise, oocytes suppressed (P < 0.001) IGF-induced secretion of inh A, act A, FS, and E-2 (P < 0.05) but enhanced IGF-induced P-4 secretion (1.7-fold; P < 0.05). Given the similarity of these oocyte-mediated actions to those we observed previously following epidermal growth factor (EGF) treatment, we used immunocytochemistry to determine whether bovine oocytes express EGF or transforming growth factor (TGF) alpha. Intense staining with TGFalpha antibody (but not with EGF antibody) was detected in oocytes both before and after coculture. Experiments involving addition of TGFalpha to granulosa cells confirmed that the peptide mimicked the effects of oocytes on cell proliferation and on FSH- and IGF-induced hormone secretion. These experiments indicate that bovine oocytes secrete a factor(s) capable of modulating granulosa cell proliferation and responsiveness to FSH and IGF in terms of steroidogenesis and production of inhibin-related peptides, bovine oocytes express TGFalpha but not EGF, and TGFalpha is a prime candidate for mediating the actions of oocytes on bovine granulosa cells.

Prooxidant and antioxidant properties of human serum ultrafiltrates toward LDL: important role of uric acid

Oxidized LDL is present within atherosclerotic lesions, demonstrating a failure of antioxidant protection. A normal human serum ultrafiltrate of M-r below 500 was prepared as a model for the low M-r components of interstitial fluid, and its effects on LDL oxidation were investigated. The ultrafiltrate (0.3%, v/v) was a potent antioxidant for native LDL, but was a strong prooxidant for mildly oxidized LDL when copper, but not a water-soluble azo initiator, was used to oxidize LDL. Adding a lipid hydroperoxide to native LDL induced the antioxidant to prooxidant switch of the ultrafiltrate. Uric acid was identified, using uricase and add-back experiments, as both the major antioxidant and prooxidant within the ultrafiltrate for LDL. The ultrafiltrate or uric acid rapidly reduced Cu2+ to Cu+. The reduction of Cu2+ to Cu+ may help to explain both the antioxidant and prooxidant effects observed. The decreased concentration of Cu2+ would inhibit tocopherol-mediated peroxidation in native LDL, and the generation of Cu+ would promote the rapid breakdown of lipid hydroperoxides in mildly oxidized LDL into lipid radicals. The net effect of the low M-r serum components would therefore depend on the preexisting levels of lipid hydroperoxides in LDL.jlr These findings may help to explain why LDL oxidation occurs in atherosclerotic lesions in the presence of compounds that are usually considered to be antioxidants.

Serum proteomic abnormality predating screen detection of ovarian cancer

Ovarian cancer is characterized by vague, non-specific symptoms, advanced stage at diagnosis and poor overall survival. A nested case control study was undertaken on stored serial serum samples from women who developed ovarian cancer and healthy controls (matched for serum processing and storage conditions as well as attributes such as age) in a pilot randomized controlled trial of ovarian cancer screening. The unique feature of this study is that the women were screened for up to 7 years. The serum samples underwent prefractionation using a reversed-phase batch extraction protocol prior to MALDI-TOF MS data acquisition. Our exploratory analysis shows that combining a single MS peak with CA125 allows statistically significant discrimination at the 5% level between cases and controls up to 12 months in advance of the original diagnosis of ovarian cancer. Such combinations work much better than a single peak or CA125 alone. This paper demonstrates that mass spectra from the low molecular weight serum proteome carry information useful for early detection of ovarian cancer. The next step is to identify the specific biomarkers that make early detection possible.

Interactions of bovine serum albumin with ethylene oxide/butylene oxide copolymers in aqueous solution

The interactions of bovine serum albumin (BSA) with three ethylene oxide/butylene oxide (E/B) copolymers having different block lengths and varying molecular architectures is examined in this study in aqueous solutions. Dynamic light scattering (DLS) indicates the absence of BSA-polymer binding in micellar systems of copolymers with lengthy hydrophilic blocks. On the contrary, stable protein-polyrner aggregates were observed in the case of E18B10 block copolymer. Results from DLS and SAXS suggest the dissociation of E/B copolymer micelles in the presence of protein and the absorption of polymer chains to BSA surface. At high protein loadings, bound BSA adopts a more compact conformation in solution. The secondary structure of the protein remains essentially unaffected even at high polymer concentrations. Raman spectroscopy was used to give insight to the configurations of the bound molecules in concentrated solutions. In the vicinity of the critical gel concentration of E18B10 introduction of BSA can dramatically modify the phase diagram, inducing a gel-sol-gel transition. The overall picture of the interaction diagram of the E18B10-BSA reflects the shrinkage of the suspended particles due to destabilization of micelles induced by BSA and the gelator nature of the globular protein. SAXS and rheology were used to further characterize the structure and flow behavior of the polymer-protein hybrid gels and sols.

Cheesemaking with vegetable coagulants - the use of Cynara L. for the production of ovine milk cheeses

This paper reviews the use of plant extracts as vegetable coagulants for cheesemaking. It covers the plants used as sources of coagulants, with a historical overview and particular emphasis on Cynara species. The genus Cynara L., its composition, milk clotting and proteolytic enzymes (cardosins) and their specificity towards peptide linkages are also described. Cheeses produced in the Iberian Peninsula using Cynara L. as coagulant are documented. Cynara L. is still the most used vegetable coagulant in cheesemaking, and also the most investigated. However, much work remains to be done to understand its action during cheese maturation and further characterization.

Effects of consumption of probiotics and prebiotics on serum lipid levels in humans

The objective of this article is to review existing studies concerning the effects of probiotics and prebiotics on serum cholesterol concentrations, with particular attention on the possible mechanisms of their action. Although not without exception, results from animal and human studies suggest a moderate cholesterol-lowering action of dairy products fermented with appropriate strain(s) of lactic acid bacteria and bifidobacteria. Mechanistically, probiotic bacteria ferment food-derived indigestible carbohydrates to produce short-chain fatty acids in the gut, which can then cause a decrease in the systemic levels of blood lipids by inhibiting hepatic cholesterol synthesis and/or redistributing cholesterol from plasma to the liver. Furthermore, some bacteria may interfere with cholesterol absorption from the gut by deconjugating bile salts and therefore affecting the metabolism of cholesterol, or by directly assimilating cholesterol. For prebiotic substances, the majority of studies have been done with the fructooligosaccharides inulin and oligofructose, and although convincing lipid-lowering effects have been observed in animals, high dose levels had to be used. Reports in humans are few in number. In studies conducted in normal-lipidemic subjects, two reported no effect of inulin or oligofructose on serum lipids, whereas two others reported a significant reduction in serum triglycerides (19 and 27%, respectively) with more modest changes in serum total and LDL cholesterol. At present, data suggest that in hyperlipidemic subjects, any effects that do occur result primarily in reductions in cholesterol, whereas in normal lipidemic subjects, effects on serum triglycerides are the dominant feature.