In vitro digestion of purified ß-casein variants A(1), A(2), B, and I: effects on antioxidant and angiotensin-converting enzyme inhibitory capacity.

Genetic polymorphisms of bovine milk proteins affect the protein profile of the milk and, hence, certain technological properties, such as casein (CN) number and cheese yield. However, reports show that such polymorphisms may also affect the health-related properties of milk. Therefore, to gain insight into their digestion pattern and bioactive potential, ß-CN was purified from bovine milk originating from cows homozygous for the variants A(1), A(2), B, and I by a combination of cold storage, ultracentrifugation, and acid precipitation. The purity of the isolated ß-CN was determined by HPLC, variants were verified by mass spectrometry, and molar extinction coefficients at λ=280nm were determined. ß-Casein from each of the variants was subjected to in vitro digestion using pepsin and pancreatic enzymes. Antioxidant and angiotensin-converting enzyme (ACE) inhibitory capacities of the hydrolysates were assessed at 3 stages of digestion and related to that of the undigested samples. Neither molar extinction coefficients nor overall digestibility varied significantly between these 4 variants; however, clear differences in digestion pattern were indicated by gel electrophoresis. In particular, after 60min of pepsin followed by 5min of pancreatic enzyme digestion, one ≈4kDa peptide with the N-terminal sequence (106)H-K-E-M-P-F-P-K- was absent from ß-CN variant B. This is likely a result of the (122)Ser to (122)Arg substitution in variant B introducing a novel trypsin cleavage site, leading to the changed digestion pattern. All investigated ß-CN variants exhibited a significant increase in antioxidant capacity upon digestion, as measured by the Trolox-equivalent antioxidant capacity assay. After 60min of pepsin + 120min of pancreatic enzyme digestion, the accumulated increase in antioxidant capacity was ≈1.7-fold for the 4 ß-CN variants. The ACE inhibitory capacity was also significantly increased by digestion, with the B variant reaching the highest inhibitory capacity at the end of digestion (60min of pepsin + 120min of pancreatic enzymes), possibly because of the observed alternative digestion pattern. These results demonstrate that genetic polymorphisms affect the digestion pattern and bioactivity of milk proteins. Moreover, their capacity for radical scavenging and ACE inhibition is affected by digestion.

Prediagnostic transcriptomic markers of Chronic lymphocytic leukemia reveal perturbations 10 years before diagnosis.

BACKGROUND: B-cell lymphomas are a diverse group of hematological neoplasms with differential etiology and clinical trajectories. Increased insights in the etiology and the discovery of prediagnostic markers have the potential to improve the clinical course of these neoplasms. METHODS: We investigated in a prospective study global gene expression in peripheral blood mononuclear cells of 263 incident B-cell lymphoma cases, diagnosed between 1 and 17 years after blood sample collection, and 439 controls, nested within two European cohorts. RESULTS: Our analyses identified only transcriptomic markers for specific lymphoma subtypes; few markers of multiple myeloma (N = 3), and 745 differentially expressed genes in relation to future risk of chronic lymphocytic leukemia (CLL). The strongest of these associations were consistently found in both cohorts and were related to (B-) cell signaling networks and immune system regulation pathways. CLL markers exhibited very high predictive abilities of disease onset even in cases diagnosed more than 10 years after blood collection. CONCLUSIONS: This is the first investigation on blood cell global gene expression and future risk of B-cell lymphomas. We mainly identified genes in relation to future risk of CLL that are involved in biological pathways, which appear to be mechanistically involved in CLL pathogenesis. Many but not all of the top hits we identified have been reported previously in studies based on tumor tissues, therefore suggesting that a mixture of preclinical and early disease markers can be detected several years before CLL clinical diagnosis.

Association of PHB 1630 C>T and MTHFR 677 C>T polymorphisms with breast and ovarian cancer risk in BRCA1/2 mutation carriers: results from a multicenter study.

BACKGROUND: The variable penetrance of breast cancer in BRCA1/2 mutation carriers suggests that other genetic or environmental factors modify breast cancer risk. Two genes of special interest are prohibitin (PHB) and methylene-tetrahydrofolate reductase (MTHFR), both of which are important either directly or indirectly in maintaining genomic integrity. METHODS: To evaluate the potential role of genetic variants within PHB and MTHFR in breast and ovarian cancer risk, 4102 BRCA1 and 2093 BRCA2 mutation carriers, and 6211 BRCA1 and 2902 BRCA2 carriers from the Consortium of Investigators of Modifiers of BRCA1 and BRCA2 (CIMBA) were genotyped for the PHB 1630 C>T (rs6917) polymorphism and the MTHFR 677 C>T (rs1801133) polymorphism, respectively. RESULTS: There was no evidence of association between the PHB 1630 C>T and MTHFR 677 C>T polymorphisms with either disease for BRCA1 or BRCA2 mutation carriers when breast and ovarian cancer associations were evaluated separately. Analysis that evaluated associations for breast and ovarian cancer simultaneously showed some evidence that BRCA1 mutation carriers who had the rare homozygote genotype (TT) of the PHB 1630 C>T polymorphism were at increased risk of both breast and ovarian cancer (HR 1.50, 95%CI 1.10-2.04 and HR 2.16, 95%CI 1.24-3.76, respectively). However, there was no evidence of association under a multiplicative model for the effect of each minor allele. CONCLUSION: The PHB 1630TT genotype may modify breast and ovarian cancer risks in BRCA1 mutation carriers. This association need to be evaluated in larger series of BRCA1 mutation carriers.

Primary brain tumours and specific serum immunoglobulin E: a case-control study nested in the European Prospective Investigation into Cancer and Nutrition cohort.

BACKGROUND: Case-control studies suggest that patients with allergic diseases have a lower risk of developing glioma but not meningioma or schwannoma. However, those data can be differentially biased. Prospective studies with objective measurements of immunologic biomarkers, like immunoglobulin E (IgE), in blood obtained before cancer diagnosis could help to clarify whether an aetiological association exists. METHODS: The present case-control study nested within the European Prospective Investigation into Cancer and Nutrition (EPIC) measured specific serum IgE as a biomarker for the most common inhalant allergens in 275 glioma, 175 meningioma and 49 schwannoma cases and 963 matched controls using the ImmunoCAP specific IgE test. Subjects with an IgE level ≥0.35 kUA/l (kilo antibody units per litre) were classified as sensitized by allergens. Odds ratios (OR) and 95% confidence intervals (CI) were estimated by adjusted conditional logistic regression models for each tumour subtype. The effect of dose-response relationship was assessed in five increasing IgE level categories to estimate P-values for trend. RESULTS: The risk of glioma was inversely related to allergic sensitization (OR = 0.73; 95% CI 0.51-1.06), especially pronounced in women (OR = 0.53; 95% CI 0.30-0.95). In dose-response analyses, for high-grade glioma, the lowest OR was observed in sera with the highest IgE levels (P for trend = 0.04). No association was seen for meningioma and schwannoma. CONCLUSION: The results, based on serum samples prospectively collected in a cohort study, provide some support for the hypothesis that individuals with allergic sensitization are at reduced risk of glioma and confirm results from previous case-control studies.

Genetic variations in VEGF and VEGFR2 and glioblastoma outcome.

Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) are central components in the development and progression of glioblastoma. To investigate if genetic variation in VEGF and VEGFR2 is associated with glioblastoma prognosis, we examined blood samples from 154 glioblastoma cases collected in Sweden and Denmark between 2000 and 2004. Seventeen tagging single nucleotide polymorphisms (SNPs) in VEGF and 27 in VEGFR2 were genotyped and analysed, covering 90% of the genetic variability within the genes. In VEGF, we found no SNPs associated with survival. In VEGFR2, we found two SNPs significantly associated to survival, namely rs2071559 and rs12502008. However, these results are likely to be false positives due to multiple testing and could not be confirmed in a separate dataset. Overall, this study provides little evidence that VEGF and VEGFR2 polymorphisms are important for glioblastoma survival.

Guidelines on management of low-grade gliomas: report of an EFNS-EANO Task Force.

BACKGROUND: Diffuse infiltrative low-grade gliomas of the cerebral hemispheres in the adult are a group of tumors with distinct clinical, histological and molecular characteristics, and there are still controversies in management. METHODS: The scientific evidence of papers collected from the literature was evaluated and graded according to EFNS guidelines, and recommendations were given accordingly. RESULTS AND CONCLUSIONS: WHO classification recognizes grade II astrocytomas, oligodendrogliomas and oligoastrocytomas. Conventional MRI is used for differential diagnosis, guiding surgery, planning radiotherapy and monitoring treatment response. Advanced imaging techniques can increase the diagnostic accuracy. Younger age, normal neurological examination, oligodendroglial histology and 1p loss are favorable prognostic factors. Prophylactic antiepileptic drugs are not useful, whilst there is no evidence that one drug is better than the others. Total/near total resection can improve seizure control, progression-free and overall survival, whilst reducing the risk of malignant transformation. Early post-operative radiotherapy improves progression-free but not overall survival. Low doses of radiation are as effective as high doses and better tolerated. Modern radiotherapy techniques reduce the risk of late cognitive deficits. Chemotherapy can be useful both at recurrence after radiotherapy and as initial treatment after surgery to delay the risk of late neurotoxicity from large-field radiotherapy. Neurocognitive deficits are frequent and can be caused by the tumor itself, tumor-related epilepsy, treatments and psychological distress.

Personal sampling in parallel with open-face filter cassettes and IOM samplers for inhalable dust--implications for occupational exposure limits.

Parallel personal sampling was carried out with the open-face filter cassette and the IOM sampler for inhalable dust for nine types of organic dust. Parallel samples numbering 749 were obtained from 152 plants. Extremely large values and outliers were disregarded, and the remaining data for each type of dust were divided into subsets according to type of product or work task, and analyzed with the aid of linear regression. The coefficient of regression for each subset ranged between 0.2 and 0.7. Hypothetical occupational exposure limits (OELs) for inhalable dust were calculated based on the linear relation obtained between the dust concentrations measured with the open-face filter cassette and the IOM sampler. The fraction of person days with time-weighted average (TWA) concentrations exceeding the calculated hypothetical OELs for inhalable dust was obtained from the distribution of measured TWA inhalable dust concentrations. Based on the results of this study and the difference in sampling efficiency for large particles between the two samplers, it was concluded that the numerical value of the OEL for inhalable dust may be set at approximately twice the numerical value of the corresponding limit value for "total dust." Additional consideration of recently discovered health effects, and technical and economical factors may result in other numerical values of future OELs for inhalable dust.

Physiological deactivation after two contrasting tasks at a video display terminal: learning vs repetitive data entry .

Two contrasting 90 min VDT work situations were simulated in the laboratory: (1) a machine-paced, repetitive data entry task; and (2) a stimulating, self-paced learning task with successive feedback. Thirty non-smoking male students (20-34 years), without previous experience of VDT work, participated individually in each condition on two consecutive days (balanced order) and in a task-free baseline condition. Self-reports and successive measurements (ambulatory recordings) of systolic and diastolic blood pressure and heart rate were obtained during work and during a subsequent 60 min period of deactivation. Urine samples were obtained after each period for the determination of catecholamines and cortisol. In the baseline condition, measurements were obtained at corresponding times of the day. As expected, the data entry task was associated with self-reports of boredom, irritation, and unpleasantness; the learning task wtih alertness, interest, and ability to concentrate. Similar elevations of physiological measurements occurred in both work situations. However, differences between conditions were found after work. Following data entry, deactivation was slower in five of the six variables (significant for epinephrine).

Exertional heatstroke and muscle metabolism: an in vivo 31P-MRS study.

An impairment of muscle energy metabolism has been suggested as a predisposing factor for, as well as a consequence of exertional heatstroke (EHS). Thirteen young men were investigated 6 months after a well-documented EHS using 31Phosphorus Magnetic Resonance Spectroscopy (31P-MRS). The relative concentrations of ATP, phosphocreatine (PCr), inorganic phosphate (Pi), phosphomonoesters (PME), and the intracellular pH (pHi) were determined at rest, during a graded standardized exercise protocol (360 active plantar flexions) and during recovery. Also the leg tissue blood flow was determined by venous occlusion plethysmography during the MRS procedure. Sixteen age-matched healthy male volunteers served as control group. In resting muscle, there were no significant differences between the groups as regards pHi, Pi/PCr, and ATP/PCr+Pi+PME ratios. During steady state exercise conditions, effective power outputs were similar for both groups at each level of exercise: 20, 35, and 50% of maximal voluntary contraction (MVC) of the calf muscle. No significant differences were shown between the two groups in Pi/PCr, pHi, or changes of leg blood flow at each level of exercise. At 50% MVC, Pi/PCr was 0.48 +/- 0.08 vs 0.47 +/- 0.05 (P = 0.96), pHi was 6.94 +/- 0.03 vs 6.99 +/- 0.02, respectively (P = 0.13). Finally, the rate of PCr resynthesis during recovery was not significantly different between the two groups: t1/2 PCr = 0.58 +/- 0.07 vs 0.50 +/- 0.05 min, respectively (P = 0.35). Therefore, no evidence of an impairment of muscle energy metabolism was shown in the EHS group during a standardized submaximal exercise using 31P-MRS performed 6 months after an EHS.

Insulin receptor tyrosine residues 1162 and 1163 control insulin stimulation of myristoyl-diacylglycerol generation and subsequent activation of glucose transport.

Chinese hamster ovary (CHO) transfectants expressing human insulin receptors that were mutated at tyrosines 1162 and 1163 (CHO-Y2 cells) exhibit decreased insulin stimulation of both receptor tyrosine kinase and 2-deoxyglucose uptake compared with transfectants expressing wild-type human insulin receptors (CHO-R cells). We now provide evidence that insulin stimulation of myristoyl-diacylglycerol (DAG) production is also markedly impaired in CHO-Y2 cells; this is manifested as a decreased responsiveness and sensitivity to insulin as compared with CHO-R and parental CHO cells. Further, we report that (i) the concentration-response curves of insulin-stimulated myristoyl-DAG production and 2-deoxyglucose uptake were superimposable within each of the three cell lines. (ii) The insulin-induced increase in myristoyl-DAG production preceded that in 2-deoxyglucose uptake, and the time course was altered for both responses in CHO-Y2 cells. (iii) Insulin also increased the phosphorylation of a 40-kDa protein known to be a substrate for protein kinase C, but to a much lesser extent in CHO-Y2 cells than in CHO-R cells. (iv) Exogenously added 1,2-dimyristoyl-glycerol and 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) again stimulated both the phosphorylation of the 40-kDa protein and 2-deoxyglucose uptake, but in contrast to insulin, they elicited the same level of response in both CHO-R and CHO-Y2 cells. (v) Finally, in protein kinase C-depleted CHO-R cells, insulin and PMA stimulation of 40-kDa protein phosphorylation as well as PMA stimulation of 2-deoxyglucose uptake were completely abolished whereas insulin-stimulated 2-deoxyglucose uptake was only partially decreased. Taken together, these results suggest that insulin stimulation of 2-deoxyglucose uptake involves myristoyl-DAG production and, at least in part, protein kinase C activation, all three of these processes being controlled by receptor tyrosines 1162 and 1163.

Evidence for the involvement of vicinal sulfhydryl groups in the insulin stimulation of intracellular glucose metabolism in Zajdela hepatoma cells.

Phenylarsine oxide (PhAsO), a dithiol reagent that blocks insulin stimulation of glucose transport in 3T3 L1 cells, also altered insulin stimulation of intracellular glucose metabolism in Zajdela Hepatoma cultured cells. PhAsO (2 microM) similarly inhibited the insulin-induced glycogen and lipid syntheses without modifying the basal level of these processes, cell viability or the ATP content. Prior incubation of the cells with PhAsO did not prevent insulin binding to the cells, or activation of the receptor tyrosine kinase, while it minimally (16%) altered receptor internalization. These results indicate that cellular dithiols located at a post-receptor step are involved in the transduction of the insulin signal to intracellular glucose metabolism.

Insulin action is mimicked by polyclonal antireceptor antibodies that activate the insulin receptor tyrosine kinase.

The insulin-like properties of anti-insulin receptor antibodies (P95 Ab) that have been characterized as being directed against the receptor beta-subunit, were studied as probes to assess the interrelationship between insulin action and receptor phosphorylation. When tested on intact cells, P95 Ab mimicked insulin effects. On isolated fat cells, they stimulated 2-deoxyglucose (2-DG) transport and lipogenesis and the P95 antibody maximal effects (173 and 232% of the control values, respectively) represented about 50% of the maximal effects elicited by insulin (317 and 475% of the control values). On cultured Zajdela hepatoma cells (ZHC cells), P95 Ab also mimicked insulin action on the incorporation of [U-14C]glucose into glycogen (158 and 207% of the control value for antibody- and insulin-treated cells, respectively). In all cases the antibody effects were dose-dependent, specific and, when maximal, were not additive with those elicited by insulin. When tested in a cell-free system, P95 Ab faithfully reproduced insulin action on the phosphorylation of the receptor beta-subunit. The maximal antibody and insulin effects (317 and 328% of the control value, respectively) were not additive. P95 Ab were also equally potent as insulin to stimulate the receptor-mediated phosphorylation of an exogenous substrate (365 and 379% of the control value in P95 antibody- and insulin-treated receptors, respectively). As well, P95 Ab proved as able as insulin in stimulating the tyrosine kinase activity of the receptor (89% of the hormone effect) when the activation was carried out in vivo. Taken together, these results are consistent with a role for the kinase activity of the insulin receptor in mediating the action of insulin.

Detection and characterization of anionic polypeptide fraction binding sites in rat liver plasma membranes and cultured hepatocytes.

The binding of human 125I-labeled 'anionic polypeptidic fraction' (APF) to purified rat liver plasma membranes was studied. The dissociation constant for this binding was 3.0 micrograms protein/mg membrane protein. Binding was competitively inhibited by unlabeled human APF, but not by human LDL (low density lipoproteins). When unlabeled HDL3 was added, binding of labeled APF was competitively reduced to a level between that of unlabeled APF and unlabeled LDL. Experiments with cultured rat hepatocytes confirmed those obtained with liver membranes and suggested the presence in rat liver of saturable APF-binding sites which seem to be specific for APF. The physiologic significance of these APF binding sites is discussed in relation to the fate of cholesterol in the liver.