Metabolic fitness in relation to genetic variation and leukocyte DNA methylation.

Metabolic syndrome (MetS) is a highly prevalent condition causing increased risk of several life-threatening diseases. MetS has a pronounced hereditary basis but is also influenced by environmental factors, partly through epigenetic mechanisms. In this study, the five phenotypes underlying MetS were incorporated into a continuous score for metabolic fitness (MF), and associations with both genotypic variation and leukocyte DNA methylation were investigated. Baseline MF phenotypes (waist circumference, blood pressure, blood glucose, serum triglycerides, and high-density lipoproteins) of 710 healthy Flemish adults were measured. After a 10 yr period, follow-up measures were derived from 618 of these subjects. Genotyping was performed for 65 preselected MF-related genetic variants. Next, full genetic predisposition scores (GPSs) were calculated, combining genotype scores of multiple genetic variants. Additionally, stepwise GPSs were constructed, including only the most predictive genetic variants for the different MF phenotypes. For a subset of 68 middle-aged men, global and gene-specific DNA methylation was investigated, and a biological pathway analysis was performed. The full GPSs were predictive for some baseline MF phenotypes, but not for changes over time. Only a limited number of genetic variants were significantly predictive individually. On the contrary, global and gene-specific DNA methylation was associated with changes in the MF phenotypes rather than with the baseline measures, indicating that effects of DNA methylation on MF are somewhat delayed. Furthermore, several biological pathways were associated with the MF phenotypes through gene promoter methylation. For CETP, G6PC2, MC4R, and TFAP2B both a genetic and epigenetic relationship was found with MF.

Short communication: Growth of dairy isolates of Geobacillus thermoglucosidans in skim milk depends on lactose degradation products supplied by Anoxybacillus flavithermus as secondary species.

Thermophilic bacilli such as Anoxybacillus and Geobacillus are important contaminants in dairy powder products. Remarkably, one of the common contaminants, Geobacillus thermoglucosidans, showed poor growth in skim milk, whereas significant growth of G. thermoglucosidans was observed in the presence of an Anoxybacillus flavithermus dairy isolate. In the present study, we investigated the underlying reason for this growth dependence of G. thermoglucosidans. Whole-genome sequences of 4 A. flavithermus strains and 4 G. thermoglucosidans strains were acquired, with special attention given to carbohydrate utilization clusters and proteolytic enzymes. Focusing on traits relevant for dairy environments, comparative genomic analysis revealed that all G. thermoglucosidans strains lacked the genes necessary for lactose transport and metabolism, showed poor growth in skim milk, and produced white colonies on X-gal plates, indicating the lack of ß-galactosidase activity. The A. flavithermus isolates scored positive in these tests, consistent with the presence of a putative lactose utilization gene cluster. All tested isolates from both species showed proteolytic activity on milk plate count agar plates. Adding glucose or galactose to liquid skim milk supported growth of G. thermoglucosidans isolates, in line with the presence of the respective monosaccharide utilization gene clusters in the genomes. Analysis by HPLC of A. flavithermus TNO-09.006 culture filtrate indicated that the previously described growth dependence of G. thermoglucosidans in skim milk was based on the supply of glucose and galactose by A. flavithermus TNO-09.006.

Volume replacement during trauma resuscitation: a brief synopsis of current guidelines and recommendations.

INTRODUCTION: Intravascular volume and fluid replacement are still cornerstones to correct fluid deficits during early trauma resuscitation, but optimum strategies remain under debate. METHODS: A synopsis of best current knowledge with reference to the following guidelines and recommendations is presented: (1) The European Guideline on Management of Major Bleeding and Coagulopathy following Trauma (fourth edition), (2) S3 Guideline on Treatment of Patients with Severe and Multiple Injuries [English Version of the German Guideline S3 Leitlinie Polytrauma/Schwerverletzten-Behandlung/AWMF Register-Nr. 012/019 sponsored by the German Society for Trauma Surgery/Deutsche Gesellschaft für Unfallchirurgie (DGU)], and (3) S3 Guideline Intravascular Volume Treatment in the Adult [AWMF Register-Nr 001/020 sponsored by the German Society for Anesthesiology and Intensive Medicine/Deutsche Gesellschaft für Anästhesiologie und Intensivmedizin (DGAI)]. RESULTS AND CONCLUSIONS: Volume replacement at a reduced level in severely injured and bleeding trauma patients is advocated (permissive hypotension) until the bleeding is controlled. ATLS principles with Hb, BE, and/or lactate can assess perfusion, estimate/monitor the extent of bleeding/shock, and guide therapy. Isotonic crystalloid solutions are first-line and specific recommendations apply for patients with TBI.

Expression of gamma-glutamyltranspeptidase in a transformed rat cerebral endothelial cell line.

Brain capillary endothelium in vivo contains high levels of gamma-glutamyltranspeptidase activity. In addition, the presence of this enzyme has been used as a marker of neoplastic cells. Normal rat cerebral endothelial cells in culture exhibit a specific activity for gamma-glutamyltranspeptidase of 2 units/10(6) cells. In vitro transformation of these cells is achieved by the use of an avian retrovirus, Schmidt-Ruppin RSV-strain D. The resultant cell line, designated RCE-T1, demonstrates a significant increase in gamma-glutamyltranspeptidase activity up to 20 units/10(6) cells in early passage levels (9-26) after which enzyme activity declines and returns to normal levels by passage 80. This variation in enzyme activity correlates with histochemical staining for this enzyme. Furthermore, the enzyme activity increases linearly over a 1000-fold range of cell concentrations. Various culture modifications do not influence this pattern of enzyme expression. These parameters include trypsin dissociation, cell freezing, degree of confluency and culture maintenance with serum or with conditioned medium obtained from passage levels exhibiting high or low enzyme activity. RCE-T1 cells will provide a unique model system to study the distribution and regulation of this enzyme during differentiation and viral carcinogenesis.

Can reverse shoulder arthroplasty in post-traumatic revision surgery restore the ability to perform activities of daily living?

BACKGROUND: Failed shoulder arthroplasty and failed internal fixation in fractures of the proximal humerus can benefit from implantation of a reverse total shoulder arthroplasty (RSA). While there is some evidence that RSA can improve function regarding range of motion (ROM), pain, satisfaction, and strength, there is sparse data how this translates into activities of daily living (ADLs). A marker-based 3D video motion analysis system has recently been designed that can measure changes of ROM in dynamic movements in every plane. The hypothesis was that a gain of maximum ROM also translates into the ability to perform ADLs and into a significant increase of ROM in ADLs. MATERIALS AND METHODS: Six consecutive patients (5 women, 1 man; 2× failed arthroplasty, 4× failed open reduction and internal fixation) who received RSA were examined the day before and 1 year after shoulder replacement. A 3D motion analysis system using a novel upper extremity model measured active maximum values and ROM in four ADLs. RESULTS: Comparing the pre- to the 1-year postoperative status, RSA resulted in a significant increase in mean maximum values for active flexion (humerus to thorax) of 37° (S.D. ±23°), from 50 to 87° [P=0.005], and for active abduction averaging of 17° (S.D. ±13°), from 52 to 69° [P=0.027]. The extension decreased significantly by about 8° (S.D. ±16°), from a mean of 39 to 31° [P=0.009]. For active adduction and internal and external rotation, there were trends for improvements, but no significant changes. Only three additional tasks of the ADL (out of 13/24 preoperatively) could be performed after revision surgery. Comparing the preoperative to the postoperative ROM in the ADLs in flexion/extension, ROM improved significantly in one ("tying an apron") of four ADLs. There were no significant changes in the abduction/adduction and internal/external rotation in any ADLs. CONCLUSION: RSA in revision cases significantly improved maximum active flexion and abduction, but decreased extension in this series. However, the patients were only able to use this greater ROM to their benefit in one of four ADLs.

Regulation of amino acid uptake into cerebral microvessels.

The carrier-mediated transport systems, found in cerebral endothelium, are responsible for maintaining the nutrient milieu of the CNS. The objective of this study was to identify what factors might regulate the sodium-dependent A-system of neutral amino acid transport in isolated cerebral microvessels. The uptake of amino acids into isolated microvessels was measured using methylaminoisobutyric acid (MeAIB) a nonhydrolyzable A-analog of the A-system carrier. The results indicated that stimulation of adrenergic (both alpha and beta) receptors significantly (P less than 0.05-0.02) increased the uptake of MeAIB. While the muscarinic agonist carbachol alone did not alter the uptake of MeAIB, it blocked the stimulation evoked by adrenergic agonists. Furthermore, addition of the metal ion, aluminum also significantly (P less than 0.05) increased specific uptake of MeAIB by 95%, when compared to untreated controls. These results indicate that the uptake of neutral amino acids, through the A-system, into cerebral microvessels, can be modulated by adrenergic and cholinergic receptors, as well as the metal ion aluminum. Since the A-system of amino acid transport may control appropriate levels of amino acids in brain, modulation of the uptake of amino acids by adrenergic and cholinergic receptors and by aluminum, may be associated with an imbalance of amino acids and possible neurotransmitter defects in the CNS.

Inhibition by lead of phenylethanolamine-N-methyltransferase.

Phenylethanolamine-N-methyltransferase (PNMT) catalyzes the conversion of norepinephrine to epinephrine. The enzyme, obtained from bovine adrenal gland, was incubated with PbCl2 at 23 degrees for various times prior to assay at 37 degrees. Inhibition developed slowly and reached a maximum after 45 min. In the presence of 4.5 nmoles PbCl2 (15 microM), 5.8 microgram protein was inhibited 50%, and inhibition was complete at 18 nmoles PbCl2 (60 microM). At maximum inhibition the PbCl2: protein ratio was 3.1 nmoles PbCl2/microgram protein. In the presence of PbCl2, the graph of enzyme activity versus protein concentration intercepted the abscissa to the right of the origin, indicating that lead is an irreversible or very slowly reversible inhibitor. The activity of PNMT which had been exposed to PbCl2 (2.6 or 5.2 nmoles PbCl2/microgram protein) was not restored by the addition of EDTA, DL-penicillamine or 1,3-dithiothreitol even when the concentration of the chelator was in 10 to 200-fold mole excess over PbCl2. DL-Penicillamine and 1,3-dithiothreitol were unable to prevent PbCl2 inhibition of the enzyme when combined with PbCl2 1 hr before addition of enzyme. EDTA could prevent 40% of PbCl2 inhibition but a decrease in total enzyme activity was noted in the presence of this chelator. Dialysis of the PbCl2-inhibited enzyme against buffer alone or buffer plus DL-penicillamine did not result in restoration of PNMT activity.

Kinetics of aluminum-induced inhibition of delta-aminolevulinic acid dehydratase in vitro.

Anemia, one consequence of aluminum toxicity, may be due to inhibition of enzymes in the heme biosynthetic pathway. In this study, the in vitro effect of aluminum on rat liver and erythrocyte delta-aminolevulinic acid dehydratase (delta-ALA dehydratase), an enzyme that is sensitive to a number of metal ions, was investigated. The presence of 1-10 microM AlCl3 caused a concentration-dependent inhibition of liver delta-ALA dehydratase activity. The Ki for AlCl3-induced inhibition of delta-ALA dehydratase was 4.1 microM, and 10 microM AlCl3 virtually abolished delta-ALA dehydratase activity (99% inhibition). Erythrocyte delta-ALA dehydratase was also inhibited by similar concentrations of AlCl3 and displayed a Ki of 1.1 microM. AlCl3 (5 microM) decreased the Vmax by 50% but did not change the Km, suggestive of reversible, noncompetitive inhibition. Sodium citrate (50 microM) when added with AlCl3 completely restored delta-ALA dehydratase activity to basal levels. Thus, disruption of delta-ALA dehydratase occurred at low micromolar levels of AlCl3 in vitro, which may help to explain abnormalities in the heme pathway in cases of aluminum poisoning.

Control of [3H]ouabain binding to cerebromicrovascular (Na+ + K+)-ATPase by metal ions and proteins.

The (Na+ + K+)-ATPase is localized to the cerebral endothelium, i.e. the blood-brain barrier, and is important for the maintenance of the brain electrolyte environment. Data from the present study indicate that Pb2+ inhibits the binding of [3H]ouabain to the cerebral microvascular (Na+ + K+)-ATPase in a time- and dose-dependent manner. Pb2(+)-induced inhibition developed slowly with a maximum obtained after 40 min. Inhibition of [3H]ouabain binding to the enzyme was 48% at 10 microM Pb2+ and appeared maximal (89%) at 100 microM Pb2+ when compared to [3H]ouabain binding in untreated microvessels at 40 min. In contrast, 100 microM Al3+ caused a 55% increase in [3H]ouabain binding to the (Na+ + K+)-ATPase, relative to untreated microvessels at 40 min. Insulin or bovine serum albumin stimulated [3H]ouabain binding to the enzyme when added at similar concentrations. However, the addition of both insulin and bovine serum albumin did not result in an additive effect. These results show that insulin exerts a nonspecific effect on [3H]ouabain binding to the (Na+ + K+)-ATPase similar to that evoked by bovine serum albumin. However, the metal ions Pb2+ and Al3+ provoke selective alterations in the cerebromicrovascular (Na+ + K+)-ATPase with Pb2+ inhibiting and Al3+ stimulating [3H]ouabain binding.

Autoradiographic visualization and characterization of [3H]ouabain binding to the Na+,K+-ATPase of rat brain and pineal.

Ouabain binds to the catalytic subunit of Na+,K+-ATPase and specific [3H]ouabain binding can be used as a measure of the number of active enzyme molecules present in a given tissue. Specific [3H]ouabain binding can be demonstrated in frozen, cryostat sections from rat brain and pineal and these sites have the characteristics of Na+,K+-ATPase. Incubations carried out in the absence of ATP or the presence of excess unlabeled ouabain reduces specific binding by greater than or equal to 98%. The addition of K+ or omission of Mg2+ also result in a decrease in specific binding. Strophanthidin, digoxin and digoxigenin displace [3H]ouabain binding with IC50 values of 0.73, 0.48 and 1.4 microM, respectively. Scatchard analyses of specific [3H]ouabain binding in brain sections shows a single class of non-interacting binding sites with an apparent affinity (Kd) of 339 nM and a maximal binding capacity (Bmax) of 34.9 pmol/mg protein. [3H]Ouabain binding is unevenly distributed throughout the brain with the olfactory nuclei, superior colliculus, dentate gyrus, pontine nuclei and pineal gland having a relatively high density of binding sites. The outer layers (1-3) of the cerebral cortex show more labeling than the inner layers (4-6) and most other brain areas have intermediate levels of [3H]ouabain binding sites, whereas white matter has virtually no specific binding. Computer-assisted densitometry was used to measure changes in specific [3H]ouabain binding after kainic acid injection into the caudate nucleus. An initial increase in [3H]ouabain binding was observed at 1 and 24 h after lesioning and a decrease in [3H]ouabain binding was evident by 9 days after lesioning.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations of cerebromicrovascular Na+,K(+)-ATPase activity due to fatty acids and acute hypertension.

Acute hypertension, induced in rats by intravenous injection of angiotensin II, previously has been shown to increase cerebrovascular permeability to macromolecules. The purpose of this study was to examine the effect of acute hypertension on Na+,K(+)-ATPase, the enzyme responsible for controlling ionic permeability of the cerebromicrovascular endothelium. The K(+)-dependent p-nitrophenylphosphatase activity of the cerebromicrovascular Na+,K(+)-ATPase was determined using microvessels prepared from hypertensive and normotensive rats. When compared to controls, a 70% decrease (P < 0.02) in the maximum rate (Vmax) of the Na+,K(+)-ATPase from hypertensive rats was evident with no change in the Michaelis constant (KM). In contrast, gamma-glutamyltranspeptidase, a marker enzyme for cerebral endothelial cells, was not significantly affected. Sodium arachidonate (1-100 microM) inhibited the phosphatase activity of the Na+,K(+)-ATPase in microvessels isolated from both normotensive and hypertensive rats in a dose-dependent manner. Furthermore, poly-unsaturated fatty acids (sodium linoleate and arachidonate) evoked the greatest inhibition of the enzyme, while sodium oleate and sodium palmitate inhibited the Na+,K(+)-ATPase to lesser extents. This regulation of enzyme activity by fatty acids was comparable in control and hypertensive groups. In summary, the data indicate that the cerebromicrovascular Na+,K(+)-ATPase was altered as a consequence of acute hypertension and that poly-unsaturated fatty acids can modulate this enzyme in microvessels derived from hypertensive or control rats.

The effect of aluminum on muscarinic receptors in isolated cerebral microvessels.

Aluminum has been shown to alter transport and enzymatic functions of the blood-brain barrier. In this study, quinuclidinyl benzilate (QNB), a muscarinic antagonist, was used to quantitate possible changes in muscarinic receptor number and/or affinity in isolated cerebral microvessels treated with aluminum. The saturation binding isotherm showed an increase in QNB binding at each ligand concentration (0.031-0.25 nM) in the presence of 10 microM AlCl3. Scatchard analysis of this binding data indicated that microvessels treated with aluminum showed a significant increase (P less than 0.05) in maximum binding capacity (284 +/- 21.5 fmol/mg protein) relative to untreated controls (207 +/- 31 fmol/mg protein), but no change in receptor affinity. Incubation of 0.25 nM QNB with 0.1 microM-100 microM AlCl3 resulted in a dose-dependent increase in specific QNB binding that was statistically significant (P less than 0.05-0.01) at or above 1 microM AlCl3. These data demonstrate that aluminum can affect muscarinic cholinergic receptors present on cerebral endothelial cells and suggest that this metal ion may compromise the integrity of the biochemical blood-brain barrier.