Quantitative trait loci mapping of polyphenol metabolites from a 'Latham' x 'Glen Moy' red raspberry (Rubus idaeus L) cross.

OBJECTIVE: The objective of this study was to investigate the genetic control of polyphenol accumulation in red raspberry (Rubus idaeus L). METHODS: The levels of total anthocyanins and 37 individual polyphenol metabolites were measured over three years in a raspberry biparental mapping population. Quantitative trait loci (QTLs) for these traits were mapped onto a high-density SNP linkage map. RESULTS: At least one QTL was detected for each trait, with good consistency among the years. On four linkage groups (LG), there were major QTLs affecting several metabolites. On LG1, a QTL had large effects on anthocyanins and flavonols containing a rutinoside or rhamnose group. On LG4, a QTL had large effects on several flavonols and on LG5 and LG6 QTLs had large effects on ellagic acid derivatives. Smaller QTLs were found on LG2 and LG3. CONCLUSION: The identification of robust QTLs for key polyphenols in raspberry provides great potential for marker-assisted breeding for improved levels of potentially health beneficial components.

Defining the osteoarthritis patient: back to the future.

The history of osteoarthritis (OA) is important because it can help broaden our perspective on past and present controversies. The naming of OA, beginning with Heberden's nodes, is itself a fascinating story. According to Albert Hoffa, R. Llewellyn Jones and Archibald Edward Garrod, the name OA was introduced in the mid-nineteenth century by surgeon Richard von Volkmann who distinguished it from rheumatoid arthritis and gout. Others preferred the terms 'chronical rheumatism', 'senile arthritis', 'hypertrophic arthritis' or 'arthritis deformans'. A similar narrative applies to the concept of OA affecting the whole joint vs the 'wear-and-tear' hypothesis, inflammation and the role of the central nervous system (CNS). In the late nineteenth and early twentieth centuries, the Garrods (father and son) and Hermann Senator argued that OA was a whole joint disease, and that inflammation played a major role in its progression. Garrod Jnr and John Spender also linked OA to a neurogenic lesion 'outside the joint'. The remaining twentieth century was no less dynamic, with major advances in basic science, diagnostics, treatments, surgical interventions and technologies. Today, OA is characterized as a multi-disease with inflammation, immune and CNS dysfunction playing central roles in whole joint damage, injury progression, pain and disability. In the current 'omics' era (genomics, proteomics and metabolomics), we owe a great debt to past physicians and surgeons who dared to think 'outside-the-box' to explain and treat OA. Over 130 years later, despite these developments, we still don't fully understand the underlying complexities of OA, and we still don't have a cure.

De novo intracerebral arteriovenous malformations and a review of the theories of their formation.

PURPOSE: Arteriovenous malformations (AVM) are still frequently described as congenital lesions in medical texts despite little evidence existing for their congenital nature. Increasing numbers of case reports of de novo AVMs add weight to the notion that they are dynamic lesions and that they can form postnatally. A thorough review of all reported cases of de novo AVM formation and a review of articles relating to AVM pathogenesis was planned to summarise current research on AVM pathogenesis and provide insight into the future implications for AVM research and treatment. METHODS AND RESULTS: MEDLINE was searched to find 29 cases of de novo AVM formation with prior MRI imaging, nine of which also had prior digital subtraction angiography. A discussion of AVM pathogenesis is undertaken through a review of articles relating to AVM embryology, postnatal angiogenesis, syndromic forms of AVMs and studies of AVM molecular biology and genetics in human and animal models. CONCLUSIONS: There is little evidence for an embryological origin through dysregulated vasculogenesis, whereas there is a raft of evidence to support dysregulated angiogenesis in childhood or even adulthood. Translational implications include risk stratification by biomarkers for predicting haemorrhage and novel therapeutic approaches to suppress AVM proliferation and initiate reversal.

Pulmonary function after hemorrhagic shock and resuscitation in a porcine model.

BACKGROUND: Hemorrhagic shock may trigger an inflammatory response and acute lung injury. The combination adenosine, lidocaine (AL) plus Mg(2+) (ALM) has organ-protective and anti-inflammatory properties with potential benefits in resuscitation.The aims of this study were to investigate: (1) pulmonary function and inflammation after hemorrhagic shock; (2) the effects of ALM/AL on pulmonary function and inflammation. METHODS: Pigs (38 kg) were randomized to: sham + saline (n = 5); sham + ALM/AL (n = 5); hemorrhage control (n = 11); and hemorrhage + ALM/AL (n = 9). Hemorrhage animals bled to a mean arterial pressure (MAP) of 35 mmHg for 90 min, received resuscitation with Ringer's acetate and 20 ml of 7.5% NaCl with ALM to a minimum MAP of 50 mmHg, after 30 min shed blood and 0.9% NaCl with AL were infused. Hemorrhage controls did not receive ALM/AL. Primary endpoints were pulmonary wet/dry ratio, PaO2 /FiO2 ratio (partial pressure of arterial oxygen to the fraction of inspired oxygen), cytokine and protein measurements in bronchoalveolar lavage fluid (BALF) and lung tissue, neutrophil invasion and blood flow in lung tissue. RESULTS: In the hemorrhage groups, wet/dry ratio increased significantly compared with the sham groups. PaO2 /FiO2 ratio decreased during shock but normalized after resuscitation. BALF did not indicate significant pulmonary inflammation, oxidative stress or increased permeability. Intervention with ALM caused a temporary increase in pulmonary vascular resistance and reduced urea diffusion across the alveolar epithelia, but had no effect on wet/dry ratio. CONCLUSION: Hemorrhagic shock and resuscitation did not cause acute lung injury or pulmonary inflammation. The question whether ALM/AL has the potential to attenuate acute lung injury is unanswered.

Persistence of HIV reservoir following successful haematopoietic stem cell transplant for juvenile myelomonocytic leukaemia in a child with perinatally acquired HIV.

This report describes a case of juvenile myelomonocytic leukaemia (JMML) on a background of both perinatally acquired HIV infection and congenital cytomegalovirus, and management of antiretroviral therapy during haematopoietic stem cell transplant. Peripheral blood HIV viral load remained below the lower limit of detection throughout and following transplant and is currently <20 RNA copies/mL. The child is currently in remission from JMML, but HIV DNA remains detectable despite myeloablative conditioning and sustained plasma HIV viral suppression.

Characterisation of phenolic glycolipids from Mycobacterium marinum.

The phenolic glycolipids from two strains of Mycobacterium marinum have been isolated and characterised. The glycolipids from M. marinum MNC 170 were principally glycosides of diacyl C37, C39 and C41 phenolphthiocerols A, but in M. marinum MNC 842, these lipids were accompanied by glycosides of diacyl phenolphthiodiolones A and novel phthiotriols A with the same overall chain-lengths. The main acyl components of the phenolic glycolipids from M. marinum MNC 170 were C26 dimethyl and C27 and C29 trimethyl-branched fatty acids, but in the lipids of M. marinum MNC 842, the C27 trimethyl acid was the only principal component. The sugar composition of all these glycolipids had been previously shown to correspond to 3-O-methylrhamnose.

Metabolites of conjugated isomers of linoleic acid (CLA) in the rat.

Metabolism of conjugated isomers of linoleic acid (CLA) in rats was studied by feeding high quantities of CLA (180 mg/day) for six days to animals that had been reared on a fat-free diet for two weeks. After this period, animals were sacrificed and liver lipids extracted. High-performance liquid chromatography (HPLC) analyses with UV detection revealed the presence of conjugated polyunsaturated fatty acids in the total liver lipid methyl esters. After isolation by HPLC, three fatty acid metabolites were identified by gas liquid chromatography coupled with mass spectrometry as being C20:3 delta 8,12,14, C20:4 delta 5,8,12,14 and C20:4 delta 5,8,11,13. A higher quantity of C20:4 delta 5,8,12,14 compared to C20:4 delta 5,8,11,13 was observed. These must arise from the elongation and desaturation of 18:2 delta 10,12 and 18:2 delta 9,11, respectively.

Daphnia magna can tolerate short-term starvation without major changes in lipid metabolism.

Daphnia magna is a common crustacean that is adapted to brief spells of fasting. Lipids are naturally a major component of their diet and are stored as energy reserves. However, there has been some controversy in the literature on the extent to which dietary lipids are used directly for complex lipid formation in Daphnia. We examined lipid metabolism in D. magna by labeling the animals using [1-14C]acetate and then followed the turnover of radiolabeled lipids during a pulse chase. Daphnia were either fed or maintained without food during the chase period. The decrease in radioactivity during the chase was relatively unaffected by feeding, although there were some differences in the distribution of radioactivity between lipid classes or individual FA. The polar lipids, which were four times better labeled than nonpolar lipids, contained the most radioactivity in the zwitterionic phosphoglycerides, PE and PC. Under the experimental conditions, the turnover of the polar membrane lipids was unaffected by feeding. Within nonpolar lipids, TAG accounted for up to about 80% of the label, followed by DAG. Overall, our data show that D. magna is capable of high rates of lipid radiolabeling de novo and, in addition, is able to use--and indeed may be dependent on--some dietary components such as the PUFA linoleate and alpha-linolenate. The results also clearly show that Daphnia is able to tolerate brief spells of fasting (24 h) with very little change to its lipid metabolism.

Epidural abscess with associated spondylodiscitis following prostatic biopsy.

Spondylodiscitis is often iatrogenic in nature. We report the case of a 69-year-old man presenting with spondylodiscitis and associated epidural abscess following transrectal ultrasonography guided prostate biopsy despite ciprofloxacin cover. To our knowledge, this is the first case of spondylodiscitis secondary to fluoroquinolone resistant Escherichia coli.

Intragastric nitrosation of nicotine is not a significant contributor to nitrosamine exposure.

To determine the potential for intragastric nicotine nitrosation, we carried out a kinetic study of the reaction of nicotine with nitrous acid in aqueous solution. The reaction of nicotine with nitrous acid resulted in the formation of three products, NNA, NNN, and NNK. The three parallel reactions were first order of 10-6 L/mol/s. The optimum pH range for formation of NNA, NNN, and NNK was 2.4 to 3.1. Thiocyanate (100 mM) slightly increased the rate of formation of NNN and NNK but tripled the rate of formation of NNA at pH 3.5 at 37 degrees C. We have also studied the nitrosation of pseudooxynicotine, a bacterial and fungal metabolite of nicotine. This secondary amine nitrosated rapidly to produce NNK. Our proposed mechanism for the conversion of nicotine to NNK includes nine kinetically distinct steps and is in agreement with our experimental results. The rate limiting step involves the formation of nicotine-1',2'-iminium ion. This ion hydrolyzes to form pseudooxynicotine which undergoes rapid, irreversible nitrosation to NNK. Given the very slow rate of nicotine nitrosation, it is unlikely that nicotine itself contributes to exposure to nitroso compounds due to chemically mediated intragastric nitrosation.

Substrate regulation of mitochondrial oxidative phosphorylation in hypercapnic rabbit muscle.

Endurance muscle performance is highly dependent on ATP production from mitochondrial oxidative phosphorylation. To study the role of the mitochondrial oxidative enzymes in muscle fatigue, we analyzed the relationship between the concentrations of substrates associated with ATP synthesis and the muscle performance of electrically stimulated rabbit muscle under CO2-induced acidosis. Two different conditions of pacing-induced muscle performance were produced in the gastrocnemius and soleus muscle groups in anesthetized rabbits by stimulating the sciatic nerve submaximally at two frequencies. Phosphorus nuclear magnetic resonance was used to measure ATP, phosphocreatine, and Pi and to provide data for a calculation of intracellular pH and free ADP. To induce acidosis, the animal was ventilated with 20% CO2. The administration of CO2 effectively reduced the intracellular pH from 6.9 to 6.7 and reduced the isometric tension-time integral (TTI) to below half the value measured in normocapnia at the low pacing frequency. A twofold increase in the pacing frequency resulted in a doubling of the TTI in normocapnia and a tripling of TTI in hypercapnia. The increases in TTI corresponded with increases in free ADP and Pi concentrations. Under the various conditions, all free ADP values were near the in vitro Michaelis-Menten constant (Km) of ADP. The Michaelis-Menten relationship of the oxidative phosphorylative enzymes was applied to the change in substrate concentrations with respect to TTI. From this relationship we observed that the in vivo Km of free ADP was 26 microM, which is close to the in nitro Km, and that Km and maximal reaction velocity did not change under hypercapnia and increased pacing frequency.(ABSTRACT TRUNCATED AT 250 WORDS)

Investigation into factors affecting precision in ion trap mass spectrometry using different scan directions and axial modulation potential amplitudes.

Electrospray ionization mass spectra obtained from different scan directions are observed to be dependent on the axial modulation potential amplitudes used for resonant ejection and on the positive deviation caused by higher even-multipole fields present in most commercial ion traps. The axial modulation voltage influences the dissociation of ions during resonant ejection and the observed mass shifts. The higher even-multipole fields in commercial ion traps are known to influence resonant ejection from the ion trap and can cause a loss in mass resolution for peaks in reverse scan mass spectra compared with that obtained by the forward scan. However, along with the dissociation of ions during resonant ejection causing a loss in resolution, the possibility of resolving an isotopic distribution is also shown to be influenced by the mass shifts caused by the space charge. These mass shifts differ depending on the scan direction employed. A significant loss in resolution can also result from resonant ejection using non-optimal axial modulation voltages. We also present results showing the ejection of ions at betaz = 1/2 using the reverse scan mode without the axial modulation voltage. Ion ejection at betaz = 1/2 is uncommon in commercial (stretched ion traps) with the conventional analytical scan without the use of a frequency of the axial modulation voltage corresponding to this non-linear resonance.

In vivo determination of drug kinetic linearity and individual organ elimination by the accelerated infusion technique.

The objectives of this study were to (1) establish a controlled drug input method, accelerated infusion, for pharmacokinetic analysis in animals and (2) use the accelerated infusion method to determine the biological barriers responsible for the low oral bioavailability of a peptidic compound in rats. The method involves the administration of an infusion at flow rates which increase linearly with time, that is, accelerated infusion (AI). The accuracy of the system in delivering AIs was tested by comparing the observed volumes with the expected volumes. For experiments with the peptidic compound, a solution was administered by AI into the systemic circulation of rats via the carotid artery (IA). The pharmacokinetic linearity of the compound was determined from the linear phase of the concentration-time curve with the systemic clearance (CLs) calculated from the slope of the linear phase. Solutions of the peptidic compound were administered into the portal vein (IPV) or the inferior vena cava (IVC) by AI to assess the hepatic and pulmonary contribution to elimination. AIs into the duodenum (ID) assessed the roles of permeability and metabolism on the absorption of the peptidic compound across the intestinal mucosa. The system was reliable in delivering the desired AI in vitro. The pharmacokinetics of the peptidic compound in rats was linear up to 17 micrograms/ml in blood. Systemic clearance (CLs) of total compound in blood was calculated to be 31 ml/min/kg. Above 17 micrograms/ml, drug concentrations showed an upward deviation from linearity, indicating loss of linearity due to saturation of elimination. Plasma concentration-time profiles for IPV, IVC, and IA infusions were not substantially different. Our conclusions can be summarized as follows: (1) Accelerated infusion is a valuable, reliable approach that can be used to assess pharmacokinetics linearity, first-pass metabolism, and bioavailability. (2) The peptidic compound has a broad, linear kinetic range in rats, and nonlinearity at high concentration is due to saturation of elimination. (3) The compound has low first-pass elimination in the intestinal mucosa, liver, and lung. (4) The factor that determines the oral bioavailability in rats of the peptidic compound used in this study is mucosal permeability.

Analysis of phospholipid molecular species by liquid chromatography--atmospheric pressure chemical ionisation mass spectrometry of diacylglycerol nicotinates.

A method using liquid chromatography - atmospheric pressure chemical ionisation mass spectrometry was evaluated for determining the molecular species composition of phospholipids (phosphatidylcholines from soybean, egg yolk and bovine liver) after conversion to diacylglycerol nicotinate derivatives. The structures could be deduced from pseudo-molecular ions ([MH-123](+)) and three pairs of monoacyl containing fragment ions. All molecular species in mixed peaks were readily identified and many minor components, earlier not encountered in the samples under investigation, were identified. Acyl chain regioisomers were readily distinguished by the ratio of the [MH-RCHCO](+) ions. Molecular species differing only in the position of the double bonds in one polyunsaturated acyl chain were separated on the basis of retention times. A half quantitative estimation of the molecular species composition of complex samples was achieved by a combination of UV detection and, for mixed peaks, the areas of [MH-123](+) ions.

Liquid chromatography with particle-beam electron-impact mass spectrometry of diacylglycerol nicotinates.

Several synthetic diacylglycerols and natural mixtures derived from soybean, egg yolk, and bovine liver phosphatidylcholines were examined in the form of nicotinate derivatives by liquid chromatography with particle-beam electron-impact mass spectrometry. High-performance liquid chromatography was carried out in the reversed-phase mode with a base-deactivated octyl-/octadecylsilyl stationary phase. The nature (size and degree of unsaturation) of the acyl residues was readily determined from the mass spectra. Uniquely, the positions of the double bonds could be deduced, although this became increasingly difficult as the degree of unsaturation of diacylglycerols containing mixed acyl residues increased. Reverse 1,2-diacylglycerol isomers could be distinguished by their mass spectra.

Quantitation of high energy phosphate compounds and metabolic significance in the developing dog brain.

The phosphate metabolites, PCr, ATP, ADP and inorganic phosphate (Pi), were quantitated in the brain of the newborn, neonatal, juvenile and adult dog to investigate the potential control mechanisms responsible for increased ATP demands during development. The concentrations of PCr and Pi were measured in vivo by MRS using the enzymatic-measured ATP as the internal standard. Phosphocreatine values increased during development from 2.08 mmol/kg wet weight in the 0-2 day newborn to 5.11 mmol/kg wet weight in the adult brain and paralleled the increases in the total creatine pool (PCr + Cr) from 4.12 to 10.05 mmol/kg wet weight. Brain ATP concentrations increased approximately 40% during postnatal development; however, when expressed as intracellular concentration, no increase in ATP was apparent due to the age-dependent decrease in extracellular space. The Pi concentration, estimated by MRS, increased significantly during postnatal development with a range of 1.78 to 2.52 mmol/kg wet wt, then decreased to 1.97 mmol/kg wet weight at adulthood. In those developmental stages where total Pi was measured enzymatically on freeze-clamped tissue, the NMR visible Pi comprised about 48 to 93% of the total, with the highest percentage being visible in the newborn brain. The intracellular pH decreased from 7.21 in the newborn to 7.10 in the adult. With development, the free ADP concentration, calculated from the components of the creatine kinase equilibrium, ranged from 27 to 34 microM. These values are close to the apparent in vitro Km of ADP for oxidative phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Age dependence of steady state mitochondrial oxidative metabolism in the in vivo hypoxic dog brain.

Mitochondrial bioenergetics were investigated in newborn, neonatal and adult dog brains during normoxia and hypoxia. The ratio of the rate of ATP synthesis to the maximum synthesis rate (V/Vmax), phosphorylation potential, [ADP] and PCr/Pi, were used to evaluate age related mitochondrial hypoxic tolerance. These indicators were calculated from the phosphorus compounds measured by in vivo 31P MRS quantitatively using ATP as an internal reference. Indicators and substrates of mitochondrial function, V/Vmax, ADP, and Pi reached a peak value during the neonatal (3-21 days) period of development, suggesting that the oxidative metabolism of the neonate is more vulnerable to stress when compared to newborns and adults. Distinction among newborns and neonates became apparent during hypoxia. Newborns (0-2 days old) showed substantial tolerance by maintaining V/Vmax until exposure to severe hypoxia. Older neonates (3-21 days old) showed increases in V/Vmax, [Pi] and [ADP] under less than severe conditions of hypoxia. Adults exhibited low V/Vmax values even during exposure to severe hypoxia, further indicating that mitochondrial oxidative processes are more stable in adults than in newborns and neonates. This study provides evidence that newborns and adults are more capable of maintaining mitochondrial function under conditions of minimal to moderate hypoxia than 3-21 day old neonates.

Genotypic variation in fatty acid content of blackcurrant seeds.

The fatty acid composition and total fatty acid content of seeds from 36 blackcurrant genotypes developed at the Scottish Crop Research Institute were examined. A rapid small-scale procedure, involving homogenization of seeds in toluene followed by sodium methoxide transesterification and gas chromatography, was used. There was considerable variation between genotypes. The gamma-linolenic acid content generally varied from 11 to 19% of the total fatty acids, but three genotypes had higher values of 22-24%, levels previously not reported for blackcurrant seed and similar to those for borage seed. Other nutritionally important fatty acids, stearidonic acid and alpha-linolenic acid, varied from 2 to 4% and 10-19%, respectively. The mean total fatty acid contents ranged from 14 to 23% of the seed, but repeatability was poor. The results are discussed. Blackcurrant seeds are mainly byproducts from juice production, and the study shows the potential for developing blackcurrant genotypes with optimal added value.

Determination of conjugated linoleic acid (CLA) concentrations in milk chocolate.

The fatty acids from a series of milk-chocolate-based confectionery samples were analyzed as methyl esters by GC to determine the presence and amount of conjugated linoleic acid (CLA). A single peak corresponding to the 9-cis,11-trans isomer and ranging from less than 0.1% to nearly 0.2% of the total fatty acids, corresponding to up to 0.3 mg per g of chocolate, was observed. One of the chocolate extracts and a milk extract were subjected to silver ion HPLC and GC-MS in order to confirm the identity of the major isomer and tentatively identity minor isomers.

Novel di-, tri-, and tetraenoic fatty acids with bis-methylene-interrupted double-bond systems from the sponge Haliclona cinerea.

Unusual fatty acids, with up to 34 carbon atoms and containing one or two bis-methylene-interrupted double-bond systems, have been identified in the sponge Haliclona cinerea from the Black Sea. These include the dienes-5,9-16:2, 7,11-18:2, 9,13-20:2, 13,17-24:2, 15,19-26:2, 17,21-28:2, 19,23-30:2 and 21,25-32:2; trienes-5,9,23-30:3, 5,9,24-31:3, 5,9,25-32:3, and 5,9,27-34:3; and the tetraenes-5,9,19,23-30:4, 5,9,21,25-32:4, and 5,9,23,27-34:4. In addition, 5,9,13-eicosatrienoic acid was present. Many of these do not appear to have been described before, and only 5,9-16:2 and 5,9,23-30:3 are found often in sponges. They were identified by using silver-ion high-performance liquid chromatography to simplify the complex mixture of fatty acids for subsequent analysis by gas chromatography-mass spectrometry as picolinyl ester derivatives. Deuteration with Wilkinson's catalyst in homogeneous solution confirmed the structures. We speculate that the di- and tetraenoic fatty acids arise by chain elongation of 5,9-hexadecadienoic acid, also a major component of the lipids, followed by further insertion of double bonds in the 5 and 9 positions. The trienes may be formed from 9-hexadecenoic acid by similar mechanisms.