Showing papers on "Low protein published in 2008"

Apple Pomace: A Versatile Substrate for Biotechnological Applications

Abstract: Apple pomace is the processing waste generated after apple juice manufacturing and represents up to 30% of the original fruit. This solid residue consists of a complex mixture of peel, core, seed, calyx, stem, and soft tissue. This residual material is a poor animal feed supplement because of its extremely low protein content and high amount of sugar. The application of agroindustrial by-products in bioprocesses offers a wide range of alternative substrates, thus helping solve pollution problems related to their disposal. Attempts have been made to use apple pomace to generate several value-added products, such as enzymes, single cell protein, aroma compounds, ethanol, organic acids, polysaccharides, and mushrooms. This article reviews recent developments regarding processes and products that employed apple pomace as a substrate for biotechnological applications.

Akt3 and Mutant V600EB-Raf Cooperate to Promote Early Melanoma Development

Abstract: B-Raf is the most mutated gene in melanoma; however, the mechanism through which it promotes early melanomas remains uncertain. Most nevi contain activated (V600E)B-Raf but few develop into melanoma, and expression in melanocytes is inhibitory with low protein levels present in surviving cells, suggesting unknown cooperative oncogenic events are necessary for melanoma development. Because many melanomas have (V600E)B-Raf and active Akt3, it is possible that these proteins cooperatively facilitate melanocyte transformation. In this study, Akt3 is shown to phosphorylate (V600E)B-Raf to lower its activity as well as that of the downstream mitogen-activated protein kinase (MAPK) pathway to levels promoting early melanoma development. Expression of active Akt3 in early melanoma cells containing (V600E)B-Raf reduced MAPK signaling and promoted anchorage-independent growth. Furthermore, expression of both (V600E)B-Raf and active Akt3 in melanocytes promoted a transformed phenotype. Mechanistically, aberrant Akt3 activity in early melanomas serves to phosphorylate Ser(364) and Ser(428) on (V600E)B-Raf to reduce activity of (V600E)B-Raf to levels that promote rather than inhibit proliferation, which aids melanocytic transformation. Inhibition of (V600E)B-Raf or Akt3 in advanced melanoma cells in which both pathways were active reduced anchorage-independent growth and tumor development in a cooperatively acting manner. Inhibition of Akt3 alone in these cells led to increased MAPK signaling. In summary, these results suggest that activating B-Raf mutations initially promote nevi development, but the resulting high, intense activation of the MAPK pathway inhibits further tumor progression requiring Akt3 activation to bypass this barrier and aid melanoma development.

Peptide separation with immobilized pI strips is an attractive alternative to in-gel protein digestion for proteome analysis.

Abstract: Complex protein mixtures have traditionally been separated by 2-DE. Gorg introduced IPGs as the first dimension of protein separation. In recent years, MS-based proteomics has increasingly become the method of choice for identifying and quantifying large number of proteins. In that technology, to decrease analyte complexity, proteins are often separated by 1-D SDS-gel electrophoresis before online MS analysis. Here, we investigate a recently introduced device for peptide separation with IPGs (Agilent OFFGEL). Loading capacity for optimal peptide focusing is below 100 μg and – similar to 2-D gels – IEF is more efficient in the acidic than the basic pH region. The 24-well fractionation format resulted in about 40% additional peptide identifications but less than 20% additional protein identifications than the 12-well format. Compared to in-gel digestion, peptide IEF consistently identified a third more proteins with equal number of fractions. Low protein starting amounts (10 μg) still resulted in deep proteome coverage. Advantages of the in-gel format include better reliability and robustness. Considering its superior performance, diminished sample and work-up requirements, peptide IEF will become a method of choice for sample preparation in proteomics.

Association analysis using SSR markers to find QTL for seed protein content in soybean

Abstract: Association analysis studies can be used to test for associations between molecular markers and quantitative trait loci (QTL). In this study, a genome-wide scan was performed using 150 simple sequence repeat (SSR) markers to identify QTL associated with seed protein content in soybean. The initial mapping population consisted of two subpopulations of 48 germplasm accessions each, with high or low protein levels based on data from the USDA’s Germplasm Resources Information Network website. Intrachromosomal LD extended up to 50 cM with r2 > 0.1 and 10 cM with r2 > 0.2 across the accessions. An association map consisting of 150 markers was constructed on the basis of differences in allele frequency distributions between the two subpopulations. Eleven putative QTL were identified on the basis of highly significant markers. Nine of these are in regions where protein QTL have been mapped, but the genomic regions containing Satt431 on LG J and Satt551 on LG M have not been reported in previous linkage mapping studies. Furthermore, these new putative protein QTL do not map near any QTL known to affect maturity. Since biased population structure was known to exist in the original association analysis population, association analyses were also conducted on two similar but independent confirmation populations. Satt431 and Satt551 were also significant in those analyses. These results suggest that our association analysis approach could be a useful alternative to linkage mapping for the identification of unreported regions of the soybean genome containing putative QTL.

Antibody responses to parenteral and oral vaccines are impaired by conventional and low protein formulas as compared to breast-feeding.

Abstract: The vaccine response to poliovirus, diphtheria and tetanus toxoids in relation to protein intake was studied in infants, either breast-fed or given low (1.1 g/100 ml) or conventional (1.5 g/100 ml) protein formula. Serum, saliva and faeces antibodies were measured by the enzyme-linked immunosorbent assay. Neutralizing poliovirus antibodies were determined. The serum, saliva and faeces antibody responses in the two formula-fed groups of infants did not differ significantly, but for the low protein formula group which had significantly higher serum neutralizing titres to poliovirus after the second vaccine dose than the conventional formula group. However, the breast-fed group had significantly higher antibody levels than the two formula-fed groups together: serum IgG to diphtheria toxoid (p less than 0.01) and serum neutralization of poliovirus (p less than 0.001) at 21-40 months of age, saliva secretory IgA to tetanus (p less than 0.01), diphtheria toxoid (p less than 0.01) and poliovirus (p less than 0.05), as well as faecal IgM to tetanus toxoid (p less than 0.05) and poliovirus (p less than 0.01 and p less than 0.05) at 3 and 4 months of age. Breast-fed infants thus showed better serum and secretory responses to peroral and parenteral vaccines than the formula-fed, whether with a conventional or low protein content.

Protein restriction during gestation and/or lactation causes adverse transgenerational effects on biometry and glucose metabolism in F1 and F2 progenies of rats.

Abstract: Substantial evidence suggests that poor intrauterine milieu elicited by maternal nutritional disturbance may programme susceptibility in the fetus to later development of chronic diseases, such as obesity, hypertension, cardiovascular disease and diabetes. One of the most interesting features of fetal programming is the evidence from several studies that the consequences may not be limited to the first-generation offspring and that it can be passed transgenerationally. In the present study, female rats (F0) were fed either a normal-protein diet [control diet (C); 19 g of protein/100 g of diet] or a low-protein diet [restricted diet (R); 5 g of protein/100 g of diet]. The offspring were termed according to the period and the types of diet the dams were fed, i.e. CC, RC, CR and RR (first letter indicates the diet during gestation and the second the diet during lactation). At 3 months of age, F1 females were bred to proven males, outside the experiment, to produce F2 offspring. At weaning, F2 offspring were divided by gender. RC1 offspring (with the number indicating the filial generation) were born with low birthweight, but afterwards they had catch-up growth, reaching the weight of the CC1 offspring. The increased glycaemia in RC1 offspring was associated with insulin resistance. CR1 and RR1 offspring had impaired growth with no changes in glucose metabolism. RC2 offspring had high BM (body mass) at birth, which was sustained over the whole experiment in male offspring. The F2 generation had more alteration in glucose metabolism than the F1 generation. CR2 and RC2 offspring had hyperglycaemia accompanied by hyperinsulinaemia and insulin resistance in both genders. CR2 offspring had an increase in body adiposity with hyperleptinaemia. In conclusion, low protein during gestation improves BM, fat mass and growth rate in F1 rats, but has adverse effects on glucose and leptin metabolism, resulting in insulin resistance in adult F1 and F2 offspring. Low protein during lactation has adverse effects on glucose, insulin and leptin metabolism, resulting in insulin resistance in adult F2 offspring. These findings suggest that low protein during gestation and/or lactation can be passed transgenerationally to the second generation.

Effects of feeding low protein diets to piglets on plasma urea nitrogen, faecal ammonia nitrogen, the incidence of diarrhoea and performance after weaning.

Abstract: This study evaluated the effects of feeding pigs low protein (LP) diets for different lengths of time after weaning on indices of protein fermentation, the incidence of postweaning diarrhoea (PWD), growth performance, and total-tract apparent digestibility. Sixty weaner pigs weighing 6.1 +/- 0.13 kg (mean +/- SEM) were used in a completely randomised design having five treatments: (i) a high protein diet (HP, 243 g/kg CP) fed for 14 d after weaning (HP14); (ii) a low protein diet (LP, 173 g CP/kg) fed for 5 d after weaning (LP5); (iii) LP diet fed for 7 d after weaning (LP7); (iv) LP diet fed for 10 d after weaning (LP10), and (v) LP diet fed for 14 d after weaning (LP14). All diets were supplemented with lysine, methionine, tryptophan and threonine, with all LP diets additionally fortified with crystalline isoleucine and valine to conform to a proposed ideal amino acid (AA) pattern. A second-stage diet (215 g CP/kg) was fed to pigs at the conclusion of each treatment. None of the diets contained antimicrobial compounds. Feeding a LP diet, regardless of duration of feeding, decreased plasma urea nitrogen (p 0.05) growth performance up to 106 days after weaning compared to pigs fed the HP diet. Total-tract apparent digestibility of dry matter, energy and crude protein were similar (p > 0.05) between treatments. Our data suggest that feeding a LP diet, supplemented with AA to conform to an ideal AA pattern, for 7-10 days after weaning can reduce PWD in pigs fed antibiotic-free diets without compromising production.

Adult male nutrition and reproductive success in Drosophila melanogaster

Abstract: Explanations for the maintenance of variation in reproductive traits influenced by seminal fluid accessory gland proteins (Acps) in male Drosophila melanogaster include nontransitivity in the outcome of sperm competition and/or condition dependence of the traits involved. We investigated the effects of adult male nutrition (five diets) on the expression of Acp- and sperm- mediated traits. We found novel, nonlinear effects, with females showing lower levels of refractoriness to remating after mating with males held on the lowest and highest yeast diets. There were no significant effects of adult male nutrition on male paternity share, but there was a striking, nonlinear effect on second male progeny production, with males kept on intermediate yeast diets fathering the highest number of offspring. Such “bell shaped” responses of life-history traits to nutrition have only previously been reported for longevity. Consistent with previous reports, males maintained on low protein diets had lower premating success and gained fewer rematings with nonvirgins. We show novel and body size independent effects of adult male nutrition on traits influenced by Acps and sperm, which do not fit current condition-dependent handicap models and can affect the strength of sexual selection acting upon such fitness-related traits.

Free Drug Metabolic Clearance in Elderly People

Abstract: The question of whether metabolic drug clearance is decreased in elderly people has been the subject of considerable debate and is very important because clearance is a determinant of dosing. Drug clearance has been shown to be consistently impaired for flow-limited (high-clearance) drugs, but there have been conflicting results for capacity-limited (low-clearance) drugs. A limitation of the studies of capacity-limited drugs is that most have estimated clearance based on total drug concentrations (protein-bound plus free). Total drug clearance reflects both the intrinsic clearance of free drug and the extent of protein binding. Total clearance is a valid measure for capacity-limited drugs with low protein binding and appears to be consistently impaired in elderly subjects. For phenazone [antipyrine] (fraction unbound [fu] >0.9), seven studies have demonstrated statistical reductions in clearance of 20–52%. For theophylline (fu 0.6), five studies have demonstrated reductions in clearance of 22–35%. For paracetamol [acetaminophen] (fu 0.8), the clearance of which has been quoted as unchanged, four studies have demonstrated reductions in clearance of 19–35%. For highly protein-bound drugs, total clearance is not the appropriate parameter. Free drug clearance is more appropriate since it is independent of changes in protein binding. The literature was reviewed to test the hypothesis that in elderly people, capacity-limited drugs with high protein binding will show decreased free clearance even in the absence of a decrease in total clearance. For these drugs, data for free drug clearance based on measurement of actual free drug concentrations are limited, but suggest that the intrinsic metabolic clearance is impaired in elderly subjects. Four studies of naproxen (fu <0.01) have shown reduced free drug clearance of 50% or more. Two studies of valproic acid (fu 0.1–0.2) have shown reduced free clearance of 39% and 65%. Two studies of ibuprofen (fu <0.01) have shown reduced free clearance of S-ibuprofen of 21% and 28%. There is some indirect evidence for reduced clearance of the highly protein-bound drugs oxaprozin, temazepam, lorazepam, diazepam, phenytoin and warfarin, although studies measuring free concentrations are lacking. Together, the above studies support the hypothesis that the intrinsic metabolic drug clearance is impaired in elderly subjects, in the order of 20–60%, and that this effect is masked if highly protein-bound drugs are assessed using total drug clearance. If the findings are confirmed in future well-designed studies of free drug clearance, there are profound and beneficial implications for dosing of drugs in elderly people. Lower doses are likely to achieve appropriate concentrations, allowing full efficacy but decreased dose-related adverse effects.

Lean mass loss is associated with low protein intake during dietary-induced weight loss in postmenopausal women.

Abstract: The health and quality-of-life implications of overweight and obesity span all ages in the United States. We investigated the association between dietary protein intake and loss of lean mass during weight loss in postmenopausal women through a retrospective analysis of a 20-week randomized, controlled diet and exercise intervention in women aged 50 to 70 years. Weight loss was achieved by differing levels of caloric restriction and exercise. The diet-only group reduced caloric intake by 2,800 kcal/week, and the exercise groups reduced caloric intake by 2,400 kcal/week and expended ∼400 kcal/week through aerobic exercise. Total and appendicular lean mass was measured using dual energy x-ray absorptiometry. Linear regression analysis was used to examine the association between changes in lean mass and appendicular lean mass and dietary protein intake. Average weight loss was 10.8±4.0 kg, with an average of 32% of total weight lost as lean mass. Protein intake averaged 0.62 g/kg body weight/day (range=0.47 to 0.8 g/kg body weight/day). Participants who consumed higher amounts of dietary protein lost less lean mass and appendicular lean mass ( r =0.3, P =0.01 and r =0.41, P

Use of a renal-specific oral supplement by haemodialysis patients with low protein intake does not increase the need for phosphate binders and may prevent a decline in nutritional status and quality of life

Abstract: Background. Protein-energy wasting is a frequent and debilitating condition in maintenance dialysis. We randomly tested if an energy-dense, phosphate-restricted, renal-specific oral supplement couldmaintain adequate nutritional intake and prevent malnutrition in maintenance haemodialysis patients with insufficient intake. Methods. Eighty-six patients were assigned to a standard care (CTRL) group or were prescribed two 125-ml packs of Renilon 7.5 R daily for 3 months (SUPP). Dietary intake, serum (S) albumin, prealbumin, protein nitrogen appearance(nPNA), C-reactive protein, subjective global assessment(SGA) and quality of life (QOL) were recorded at baseline and after 3 months. Results. While intention to treat analysis (ITT) did not reveal strong statistically significant changes in dietary intake between groups, per protocol (PP) analysis showed that theSUPP group increased protein (P < 0.01) and energy (P <0.01) intakes. In contrast, protein and energy intakes further deteriorated in the CTRL group (PP). Although there was no difference in serum albumin and prealbumin changes between groups, in the total population serum albumin and prealbumin changes were positively associated with the increment in protein intake (r = 0.29, P = 0.01 and r = 0.27, P = 0.02, respectively). The SUPP group did not increase phosphate intake, phosphataemia remained unaffected, and the use of phosphate binders remained stable or decreased. The SUPP group exhibited improved SGA and QOL (P < 0.05). Conclusion. This study shows that providing maintenance haemodialysis patientswith insufficient intake with a renal- specific oral supplement may prevent deterioration in nutritional indices and QOL without increasing the need for phosphate binders.

Hard Red Spring Wheat Response to Row Spacing, Seeding Rate, and Nitrogen

Abstract: Row spacing, plant density, and N application timing can be manipulated to optimize plant growth and spatial distribution, therefore maximizing sunlight, nutrients, soil water use efficiency and grain yield. A 2-yr field study to evaluate the effects of four seeding rates (108,215, 323, and 430 seeds m -2 ), two row spacings (15 and 30 cm), and three N treatments (FA1, 100% at seeding; FA2,50% at seeding and 50% at tiller formation; and FA3,50% at seeding and 50% at shoot elongation) on grain yield ofMcNeal hard red spring wheat (Triticum aestivum L.) was conducted in central Montana. Spring wheat accumulated greater biomass at a faster rate under the 15-cm row spacing than the 30-cm row spacing. Grain yield was 410 and 412 kg ha -1 greater at 15-cm than at 30-cm row spacings in 2004 and 2005, and the yield increase was primarily attributed to 44 and 40 more spikes m -2 at 15-cm than at 30-cm row spacing in 2004 and 2005, respectively. Grain yield was not significantly affected by the N treatments, thus all N should be applied at seeding. The optimum seeding rate was 215 seeds m -2 . Tillers at higher seeding rates had larger phyllochrons and greater mortalities. Low protein content was found in FA3 and high seeding rate treatments in 2005. Narrow row spacing is recommended for high spring wheat yield in the northern Great Plains. This yield increase cannot be achieved by increasing seeding rate at wide row spacing.

Prosurvival Effect of DHCR24/Seladin-1 in Acute and Chronic Responses to Oxidative Stress

Abstract: The 3β-hydroxysterol-Δ24 reductase (DHCR24) is a broadly expressed oxidoreductase, sharing homologies with a family of flavin adenine dinucleotide-dependent reductases (26). The dhcr24 gene is the human orthologue of the diminuto/dwarf1, initially identified in plants, where it is required for the synthesis of brassinosteroids, a group of plant sterols that are essential for normal growth and development (4, 18, 23). In mammals, DHCR24 plays an indispensable role in cholesterol biosynthesis, catalyzing the conversion of desmosterol to cholesterol (6, 26, 27). However, DHCR24 was also described in a different context: dhcr24 expression was shown to be down-regulated in brain areas affected by Alzheimer's disease (11) and was therefore named seladin-1, the selective Alzheimer's disease indicator 1, suggesting an association of DHCR24/seladin-1 with the selective vulnerability of neurons in the affected brain areas. Conversely, high levels of DHCR24/seladin-1 exert protective functions, conferring resistance against oxidative stress and protecting cells from apoptotic cell death (2, 9, 11, 16). Endogenous DHCR24/seladin-1 levels are highly up-regulated upon acute oxidative stress (3, 28), while expression declines to very low levels upon chronic exposure (3), suggesting that DHCR24/seladin-1 plays a role in integrating cellular responses to oxidative stress. However, the precise molecular mechanism for this protective effect is not known. Intriguingly, recent findings identified an interaction between DHCR24/seladin-1 and the tumor suppressor protein p53. Following oncogenic and oxidative stress, seladin-1 binds to p53 in fibroblasts, thus displacing the E3 ubiquitin ligase Mdm2 from p53, which results in p53 accumulation (28). These data argue in favor of a potential tumor suppressor role of DHCR24/seladin-1, indicating that low protein levels enhance p53 degradation and thus prevent senescence in cellular response to Ras/p53-mediated oncogenic signaling (28). However, it was shown that DHCR24 activity of seladin-1, i.e., the oxidoreductase activity of the protein in cholesterol biosynthesis, is not required for p53 binding and the p53-dependent oxidative stress response (28). Naturally high transcription levels of DHCR24/seladin-1 and gene transfer of DHCR24/seladin-1 cDNA into various cell lines were associated with elevated cholesterol concentrations (3, 9), suggesting a possible role for cholesterol in the protection process. We recently showed that overexpression of DHCR24/seladin-1 in human neuroblastoma SH-SY5Y cells leads to elevated levels of cellular and membrane cholesterol, thereby enhancing the formation of lipid rafts in these cells (6). Oxidative stress leads to the production of reactive oxygen species which attack lipid membrane constituents such as unsaturated phospholipids, glycolipids, and cholesterol, resulting in cellular dysfunction and cell death (10). During apoptosis, sterol regulatory element-binding proteins (SREBPs), which regulate expression of genes involved in lipid and cholesterol homeostasis (30), were shown to be activated by caspase cleavage (21). These observations have led to the hypothesis that cholesterol may be required in the early stages of apoptosis to maintain plasma membrane integrity (25). Moreover, plasma membrane compartments rich in cholesterol may participate in signal transduction pathways activated upon oxidative stress, and thus enhance prosurvival pathways, while cholesterol depletion appears to increase apoptotic events triggered by hydrogen peroxide treatment (29). In addition, methyl-β-cyclodextrin-mediated cholesterol depletion causes apoptosis, which is associated with caspase-3 activation and Akt inactivation, while cholesterol reload replenishes rafts on the cell surface and restores Akt activation and cell viability (13, 29). In the present work, we analyzed the role of DHCR24/seladin-1 in integrating cellular responses to oxidative stress by employing primary neurons and neuroblastoma SH-SY5Y cells. We found that DHCR24/seladin-1 expression is up-regulated in an acute response and down-regulated in a long-term response to oxidative stress. In this context, protective effects of high DHCR24/seladin-1 levels were mediated by increased cellular cholesterol concentrations, which resulted from a generally enhanced cholesterol biosynthesis after exposure to oxidative stress. In contrast, protection against oxidative stress mediated by low levels of DHCR24/seladin-1 was associated with reduced levels of p53 and elevated p53 ubiquitination, while overexpression of DHCR24/seladin-1 stabilized p53, independent of DHCR24 activity and cholesterol concentrations. These findings reveal a dual capacity of DHCR24/seladin-1, which appears to be involved in two mechanistically different prosurvival effects, exerting an acute response as well as a late response to oxidative stress.

Salt-induced protein phase transitions in drying drops.

Abstract: Protein phase transitions in drying sessile drops of protein-salt-water colloidal systems were studied by means of optical and atom-force microscopy. The following sequence of events was observed during drop drying: attachment of a drop to a glass support; redistribution of colloidal phase due to hydrodynamic centrifugal stream; protein ring formation around the edge; formation of protein spatial structures inside a protein ring that pass into gel in the middle of the drop; salt crystallization in the shrinking gel. It was assumed that rapid drying of a protein ring over the circle of high colloidal volume fraction and low strength of interparticle attraction leads to formation of colloidal glass, whereas gel forms only in the middle of the drop at very low protein volume fraction and strong attraction between the particles. Before gelation, colloidal particles form fractal clusters. In dried drops of salt-free protein solutions, no visual protein structures were observed. Structural evolution of protein in sessile drying drops of protein-salt aqueous colloidal solutions is discussed on the basis of experimental data.

Isoleucine and valine supplementation of a low-protein corn-wheat-soybean meal-based diet for piglets: growth performance and nitrogen balance.

Abstract: The effects of Ile and Val supplementa- tion of a low-CP, corn-wheat-soybean meal-based pig- let diet on growth performance, incidence of diarrhea, and N balance were studied using 60 Landrace × Duroc male piglets in a 4-wk experiment. The 60 individually caged piglets were divided into 5 dietary treatments, each consisting of 12 piglets. Diet 1 was a positive con- trol diet (20% CP); diet 2 was a low-CP negative control diet (17% CP); diets 3, 4, and 5 were low-CP diets to which Ile, Val, or the combination of Ile and Val were added, respectively. All diets were supplemented with Lys, Met, Thr, and Trp to provide the required concen- trations of these AA according to the 1998 NRC. Aver- age daily gain and ADFI were similar among pigs fed the positive control, Val-added, and the Val plus Ile- added diets. On wk-2 and wk-4, fecal score was greater (softer feces) in piglets fed the 20% CP level compared with the remaining treatments (P < 0.01). Nitrogen in- take was decreased (P < 0.0001) in pigs fed diets con- taining low levels of CP compared with pigs fed the 20% CP diet. Fecal N excretion (g/d) was decreased (P < 0.05) in piglets fed low-CP diets at wk 1 and wk 4 of feeding, and in urine at wk 4 of feeding. Crude pro- tein levels or AA supplementation had no effect on N retention efficiencies. These results indicate that the supplementation of Val alone, or in combination with Ile, to a low-CP piglet diet with adequate levels of Lys, Met, Thr, and Trp is necessary to achieve maximum performance in pigs consuming corn-wheat-soybean meal-based diets.

Aetiology and management of extrahepatic portal vein obstruction in children: King's College Hospital experience.

Abstract: Objective: To study a single-centre experience of the management of extrahepatic portal vein obstruction (EHPVO) in children during the last 3 decades. Materials and Methods: The medical records of 108 children (67 male, median age 4.75 years, range = 1 day–16.3 years) presenting with EHPVO between 1979 and 2005 were reviewed retrospectively. Results: Extended prothrombotic screening performed in 30 patients revealed low protein C activity (6 patients), low free protein S (2), and a positive lupus anticoagulant (1); factor V Leiden mutations and the JAK2V617F mutation were not identified. Associated congenital anomalies were found in 26 of the 108 children (24%). Clinical presentation included splenomegaly in 98 (91%) and ascites in 3 (3%). Elevation of liver enzymes and prolonged international normalized ratio were seen in 13 (12%) and 14 (13%) children, respectively. Haematological parameters of hypersplenism were present in 13 (12%). Bleeding occurred in 83 (77%) patients with a median age of 4.58 (0.02–16.37) years. On first endoscopy, oesophageal varices were present in 92 patients; of those subjects, 70 (76%) received sclerotherapy, 5 (5%) had band ligation, and 16 (17%) received both. Complications of endoscopy occurred in 34 (37%) patients: oesophageal ulcers in 16, oesophageal stricture in 10, both in 7, and erosive gastritis in 1. Seventeen (16%) children underwent shunt surgery for uncontrolled bleeding at a median age of 9.7 (5.2–23.7) years. Conclusions: The aetiology of EHPVO in the majority of patients remains unknown. Sclerotherapy and banding are effective treatments for bleeding varices with good long-term outcome. Procoagulant state is an infrequent cause of EHPVO in children.

Protein Extraction for Two-Dimensional Gel Electrophoresis of Proteomic Profiling in Turfgrass

Abstract: Protein extraction for two-dimensional gel electrophoresis (2-DE) from plant samples is challenging due to low protein content and high level of contaminants. Proteomic research in turfgrass is limited by the lack of efficient protein extraction methods. To establish an effective protocol of protein extraction suitable for 2-DE analysis in turfgrasses, four protein extraction methods (chloroform/acetone, tris-base/acetone, phenol/ammonium acetate, and trichloroacetic acid [TCA]/acetone methods) were evaluated for creeping bentgrass (Agrostis stolonifera L.). Proteins were extracted from leaves and roots of mature plants using the above four methods and separated by 2-DE. The TCA/acetone extraction had higher protein yield and resolution of protein separation for leaves, compared to the other three methods. For roots, both the TCA and phenol methods had higher protein yields and number of protein spots than the other two methods. The phenol method had more protein spots and higher spot intensities in the low molecular weight (Mr) region or high isoelectric point (pI) region than the TCA extraction, while more protein spots and higher spot intensity in the region of high Mr were detected by the TCA method than by the phenol method. Our results suggested that the TCA method was the most effective among the four methods for leaves, and a combination of the TCA and phenol methods may provide enhanced proteomic information for roots in turfgrass.