Characterisation of the intracellular-glutamate decarboxylase system (GAD): analysis of its function, transcription and role in the acid resistance of various strains of Listeria monocytogenes

The glutamate decarboxylase (GAD) system is important for the acid resistance of Listeria monocytogenes. We previously showed that under acidic conditions, glutamate (Glt)/γ-aminobutyrate (GABA) antiport is impaired in minimal media but not in rich ones, like brain heart infusion. Here we demonstrate that this behavior is more complex and it is subject to strain and medium variation. Despite the impaired Glt/GABA antiport, cells accumulate intracellular GABA (GABA(i)) as a standard response against acid in any medium, and this occurs in all strains tested. Since these systems can occur independently of one another, we refer to them as the extracellular (GAD(e)) and intracellular (GAD(i)) systems. We show here that GAD(i) contributes to acid resistance since in a ΔgadD1D2 mutant, reduced GABA(i) accumulation coincided with a 3.2-log-unit reduction in survival at pH 3.0 compared to that of wild-type strain LO28. Among 20 different strains, the GAD(i) system was found to remove 23.11% ± 18.87% of the protons removed by the overall GAD system. Furthermore, the GAD(i) system is activated at milder pH values (4.5 to 5.0) than the GAD(e) system (pH 4.0 to 4.5), suggesting that GAD(i) is the more responsive of the two and the first line of defense against acid. Through functional genomics, we found a major role for GadD2 in the function of GAD(i), while that of GadD1 was minor. Furthermore, the transcription of the gad genes in three common reference strains (10403S, LO28, and EGD-e) during an acid challenge correlated well with their relative acid sensitivity. No transcriptional upregulation of the gadT2D2 operon, which is the most important component of the GAD system, was observed, while gadD3 transcription was the highest among all gad genes in all strains. In this study, we present a revised model for the function of the GAD system and highlight the important role of GAD(i) in the acid resistance of L. monocytogenes.

Thiamine plays a critical role in the acid tolerance of Listeria monocytogenes

Understanding the molecular basis of acid tolerance in the food-borne pathogen Listeria monocytogenes is important as this property contributes to survival in the food-chain and enhances survival within infected hosts. The aim of this study was to identify genes contributing to acid tolerance in L. monocytogenes using transposon mutagenesis and subsequently to elucidate the physiological role of these genes in acid tolerance. One mutant harboring a Tn917 insertion in the thiT gene (formerly lmo1429), which encodes a thiamine (vitamin B1) uptake system, was found to be highly sensitive to acid. The acid-sensitive phenotype associated with loss of this gene was confirmed with an independently isolated mutant, from which the thiT gene was deleted (ΔthiT). Cells of both wild-type and ΔthiT mutant that were thiamine depleted were found to be significantly more acid sensitive than control cultures. Thiamine-depleted cultures failed to produce significant concentrations of acetoin, consistent with the known thiamine dependence of acetolactate synthase, an enzyme required for acetoin synthesis from pyruvate. As acetoin synthesis is a proton-consuming process, we suggest that the acid sensitivity observed in thiamine-depleted cultures may be owing to an inability to produce acetoin.

Salmon consumption during pregnancy alters fatty acid composition and secretory IgA concentration in human breast milk

Fish oil supplementation during pregnancy alters breast milk composition, but there is little information about the impact of oily fish consumption. We determined whether increased salmon consumption during pregnancy alters breast milk fatty acid composition and immune factors. Women (n = 123) who rarely ate oily fish were randomly assigned to consume their habitual diet or to consume 2 portions of farmed salmon per week from 20 wk of pregnancy until delivery. The salmon provided 3.45 g long-chain (LC) (n-3) PUFA/wk. Breast milk fatty acid composition and immune factors [soluble CD14, transforming growth factor-b (TGFb)1, TGFb2, and secretory IgA] were analyzed at 1, 5, 14, and 28 d postpartum (PP). Breast milk from the salmon group had higher proportions of EPA (80%), docosapentaenoic acid (30%), and DHA (90%) on d 5 PP compared with controls (P < 0.01). The LC (n-6) PUFA:LC (n-3) PUFA ratio was lower for the salmon group on all days of PP sampling (P < 0.004), although individual (n-6) PUFA proportions, including arachidonic acid, did not differ. All breast milk immune factors decreased between d 1 and 28 PP (P < 0.001). Breast milk secretory IgA (sIgA) was lower in the salmon group (d 1–28 PP; P = 0.006). Salmon consumption during pregnancy, at the current recommended intakes, increases the LC (n-3) PUFA concentration of breast milk in early lactation, thus improving the supply of these important fatty acids to the breast-fed neonate. The consequence of the lower breast milk concentration of sIgA in the salmon group is not clear.

Immune factors and fatty acid composition in human milk from river/lake, coastal and inland regions of China

Breast milk fatty acid composition may be affected by maternal diet during gestation and lactation. The influence of dietary and breast milk fatty acids on breast milk immune factors is poorly defined. We determined the fatty acid composition and immune factor concentrations of breast milk from women residing in river & lake, coastal, and inland regions of China, which differ in their consumption of lean fish and oily fish. Breast milk samples were collected on days 3 to 5 (colostrum), 14 and 28 post-partum and analysed for soluble CD14 (sCD14), transforming growth factor (TGF)-β1, TGF-β2, secretory immunoglobulin A (sIgA) and fatty acids. The fatty acid composition of breast milk differed between regions and with time post-partum. The concentrations of all four immune factors in breast milk decreased over time, with sCD14, sIgA and TGF-β1 being highest in colostrum in the river & lake region. Breast milk DHA and arachidonic acid (AA) were positively associated, and γ-linolenic acid and EPA negatively associated, with the concentrations of each of the four immune factors. In conclusion, breast milk fatty acids and immune factors differ between regions in China characterised by different patterns of fish consumption and change during the course of lactation. A higher breast milk DHA and AA concentration is associated with higher concentrations of immune factors in breast milk, suggesting a role for these fatty acids in promoting gastrointestinal and immune maturation of the infant.

The anti-epileptic drug Valproic Acid (VPA) inhibits steroidogenesis in bovine theca and granulosa cells in vitro

Valproic acid (VPA) is used widely to treat epilepsy and bipolar disorder. Women undergoing VPA treatment reportedly have an increased incidence of polycystic ovarian syndrome (PCOS)-like symptoms including hyperandrogenism and oligo- or amenorrhoea. To investigate potential direct effects of VPA on ovarian steroidogenesis we used primary bovine theca (TC) and granulosa (GC) cells maintained under conditions that preserve their 'follicular' phenotype. Effects of VPA (7.8-500 µg/ml) on TC were tested with/without LH. Effects of VPA on GC were tested with/without FSH or IGF analogue. VPA reduced (P<0.0001) both basal (70% suppression; IC(50) 67±10 µg/ml) and LH-induced (93% suppression; IC(50) 58±10 µg/ml) androstenedione secretion by TC. VPA reduced CYP17A1 mRNA abundance (>99% decrease; P<0.0001) with lesser effects on LHR, STAR, CYP11A1 and HSD3B1 mRNA (<90% decrease; P<0.05). VPA only reduced TC progesterone secretion induced by the highest (luteinizing) LH dose tested; TC number was unaffected by VPA. At higher concentrations (125-500 µg/ml) VPA inhibited basal, FSH- and IGF-stimulated estradiol secretion (P<0.0001) by GC without affecting progesterone secretion or cell number. VPA reversed FSH-induced upregulation of CYP19A1 and HSD17B1 mRNA abundance (P<0.001). The potent histone deacetylase (HDAC) inhibitors trichostatin A and scriptaid also suppressed TC androstenedione secretion and granulosal cell oestrogen secretion suggesting that the action of VPA reflects its HDAC inhibitory properties. In conclusion, these findings refute the hypothesis that VPA has a direct stimulatory action on TC androgen output. On the contrary, VPA inhibits both LH-dependent androgen production and FSH/IGF-dependent estradiol production in this in vitro bovine model, likely by inhibition of HDAC.

Epoxide ring opening in a zinc(II) complex in water without any Lewis acid catalyst: formation of only one diastereomer out of 2

The epoxide ring in 5,6-dihydro-5,6-epoxy-1,10-phenanthroline (L) opens up in its reaction with 4-methylaniline and 4-methoxyaniline in water in equimolar proportion at room temperature without any Lewis acid catalyst to give a monohydrate of 6-(4-methyl-phenylamino)-5,6-dihydro-1,10-phenanthrolin-5-ol (L′·H2O) and 6-(4-methoxyphenyl-amino)-5,6-dihydro-1,10-phenanthrolin-5-ol (L″) respectively. Reaction time decreases from 72 to 14 h in boiling water. But the yields become less. Reaction of L with Zn(ClO4)2·6H2O in methanol in 3:1 molar ratio at room temperature affords white [ZnL3](ClO4)2·H2O. The X-ray crystal structure of the acetonitrile solvate [ZnL3](ClO4)2·MeCN has been determined which shows that the metal has a distorted octahedral N6 coordination sphere. [ZnL3](ClO4)2·2H2O reacts with 4-methylaniline and 4-methoxyaniline in boiling water in 1:3 molar proportion in the absence of any Lewis acid catalyst to produce [ZnL′3](ClO4)2·4H2O and [ZnL″3](ClO4)2·H2O, respectively in 1–4 h time in somewhat low yield. In the 1H NMR spectra of [ZnL′3](ClO4)2·4H2O and [ZnL″3](ClO4)2·H2O, only one sharp methyl signal is observed implicating that only one diastereomer out of the 23 possibilities is formed. The same diastereomers are obtained when L′·H2O and L″ are reacted directly with Zn(ClO4)2·6H2O in tetrahydrofuran at room temperature in very good yields. Reactions of L′·H2O and L″ with Ru(phen)2Cl2·2H2O (phen = 1,10-phenanthroline) in equimolar proportion in methanol–water mixture under refluxing condition lead to the isolation of two diastereomers of [Ru(phen)2L′](ClO4)2·2H2O and [Ru(phen)2L″](ClO4)2·2H2O.

Flavour profile of three novel acidic varieties of muskmelon (Cucumis melo L.)

Novel acidic varieties of muskmelon (Cucumis melo L.) are emerging onto the UK market. These melons contain almost twice the amount of citric acid compared to standard melons and are described as ‘zesty and fresh’. This study compared the flavour components of three acidic varieties with a standard Galia-type melon. The volatile and semivolatile compounds were extracted using dynamic headspace extraction (DHE) or solid-phase microextraction (SPME) and solid phase extraction (SPE) respectively, followed by gas chromatography – mass spectrometry (GC-MS) and gas chromatography – olfactometry (GC-O). More than 50 volatile and 50 semivolatile compounds were identified in the headspace and the SPE extracts respectively. GC-O revealed 15 odour-active components in the headspace, with esters being consistently higher in acidic variety. This study showed quantitative and qualitative differences between all four varieties and key differences between acidic varieties and standard melons.

Polysialic acid as a drug carrier: evaluation of a new polysialic acid–epirubicin conjugate and its comparison against established drug carriers

This paper explores the potential of polysialic acid (PSA) as a carrier for low molecular weight anticancer drugs. A PSA–epirubicin (Epi) conjugate was synthesized and compared against Epi conjugates containing established carriers, namely: N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers, poly(ethylene glycol) (PEG) and polyglutamic acid (PGA). Biological assessments in the breast cancer cell line MCF-7 and in the anthracycline resistant MCF-7/DX showed that the PSA–Epi conjugate had the highest activity (40% and 30% cell death in the two cell lines at 1 mM Epi equiv., respectively). FACS studies confirmed internalization of all conjugates by cholesterol-dependent endocytosis. PSA–Epi showed release of Epi (40% at 5 h) when incubated with lysosome extracts. In vivo evaluation showed that all conjugates had a significantly longer half-life compared to free Epi. This study also allowed an investigation on the effect of the polymeric carrier on the biological activity of a conjugate, with the biodegradability of the carrier emerging as an important feature.

Functional γ-aminobutyrate shunt in Listeria monocytogenes: role in acid tolerance and succinate biosynthesis

Listeria monocytogenes, the causative agent of human listeriosis, is known for its ability to withstand severe environmental stresses. The glutamate decarboxylase (GAD) system is one of the principal systems utilized by the bacterium to cope with acid stress, a reaction that produces γ-aminobutyrate (GABA) from glutamate. Recently, we have shown that GABA can accumulate intracellularly under acidic conditions, even under conditions where no extracellular glutamate-GABA exchange is detectable. The GABA shunt, a pathway that metabolizes GABA to succinate, has been described for several other bacterial genera, and the present study sought to determine whether L. monocytogenes has this metabolic capacity, which, if present, could provide a possible route for succinate biosynthesis in L. monocytogenes. Using crude protein extracts from L. monocytogenes EGD-e, we show that this strain exhibits activity for the two main enzyme reactions in the GABA shunt, GABA aminotransferase (GABA-AT) and succinic semialdehyde dehydrogenase (SSDH). Two genes were identified as candidates for encoding these enzyme activities, argD (GABA-AT) and lmo0913 (SSDH). Crude protein extracts prepared from a mutant lacking a functional argD gene significantly reduced GABA-AT activity, while an lmo0913 mutant lost all detectable SSDH activity. The deletion of lmo0913 increased the acid tolerance of EGD-e and showed an increased accumulation of intracellular GABA, suggesting that this pathway plays a significant role in the survival of this pathogen under acidic conditions. This is the first report of such a pathway in the genus Listeria, which highlights an important link between metabolism and acid tolerance and also presents a possible compensatory pathway to partially overcome the incomplete tricarboxylic acid cycle of Listeria.

Lunar and solar FTIR nitric acid measurements at Eureka in winter 2001/2002: comparisons with observations at Thule and Kiruna and with CMAM and SLIMCAT model calculations

For the first time, vertical column measurements of (HNO3) above the Arctic Stratospheric Ozone Observatory (AStrO) at Eureka (80N, 86W), Canada, have been made during polar night using lunar spectra recorded with a Fourier Transform Infrared (FTIR) spectrometer, from October 2001 to March 2002. AStrO is part of the primary Arctic station of the Network for the Detection of Stratospheric Change (NDSC). These measurements were compared with FTIR measurements at two other NDSC Arctic sites: Thule, Greenland (76.5N, 68.8W) and Kiruna, Sweden (67.8N, 20.4E). The measurements were also compared with two atmospheric models: the Canadian Middle Atmosphere Model (CMAM) and SLIMCAT. This is the first time that CMAM HNO3 columns have been compared with observations in the Arctic. Eureka lunar measurements are in good agreement with solar ones made with the same instrument. Eureka and Thule HNO3 columns are consistent within measurement error. Differences among HNO3 columns measured at Kiruna and those measured at Eureka and Thule can be explained on the basis of the available sunlight hours and the polar vortex location. The comparison of CMAM HNO3 columns with Eureka and Kiruna data shows good agreement, considering CMAM small inter-annual variability. The warm 2001/02 winter with almost no Polar Stratospheric Clouds (PSCs) makes the comparison of the warm climate version of CMAM with these observations a good test for CMAM under no PSC conditions. SLIMCAT captures the magnitude of HNO3 columns at Eureka, and the day-to-day variability, but generally reports higher HNO3 columns than the CMAM climatological mean columns.

Effects of acid sulphate on DOC release in mineral soils: the influence of SO42−retention and Al release

Dissolved organic carbon (DOC) in acid-sensitive upland waters is dominated by allochthonous inputs from organic-rich soils, yet inter-site variability in soil DOC release to changes in acidity has received scant attention in spite of the reported differences between locations in surface water DOC trends over the last few decades. In a previous paper, we demonstrated that pH-related retention of DOC in O horizon soils was influenced by acid-base status, particularly the exchangeable Al content. In the present paper, we investigate the effect of sulphate additions (0–437 μeq l−1) on DOC release in the mineral B horizon soils from the same locations. Dissolved organic carbon release decreased with declining pH in all soils, although the shape of the pH-DOC relationships differed between locations, reflecting the multiple factors controlling DOC mobility. The release of DOC decreased by 32–91% in the treatment with the largest acid input (437 μeq l−1), with the greatest decreases occurring in soils with very small % base saturation (BS, <3%) and/or large capacity for sulphate (SO42−) retention (up to 35% of added SO42−). The greatest DOC release occurred in the soil with the largest initial base status (12% BS). These results support our earlier conclusions that differences in acid-base status between soils alter the sensitivity of DOC release to similar sulphur deposition declines. However,superimposed on this is the capacity of mineral soils to sorb DOC and SO42−, and more work is needed to determine the fate of sorbed DOC under conditions of increasing pH and decreasing SO42−.

Proteolysis of milk heated at high temperatures by native enzymes analysed by trinitrobenzene sulphonic acid (TNBS) method

Native enzymes play a significant role in proteolysis of milk during storage. This is significant for heat resistant native enzymes. Plasmin is one of the most heat resistant enzymes found in milk. It has been reported to survive several heat treatments, causing spoilage during storage. The aim of this study was to assess susceptibility of high temperature heated milk to proteolysis by native enzymes. The trinitrobenzene sulphonic acid (TNBS) method was used for this purpose. Raw milk was heated at 110, 120, 130,142°C for 2 s and 85°C for 15 s and milk processed at low temperature (85°C /15s) was selected to mimic pasteurisation. TNBS method confirmed that raw milk and milk processed at 85°C /15s were the most proteolysed, whereas treatment of milk at high temperatures (110, 120, 130 and 142°C for 2 s) inactivated the native enzymes. It may thus be concluded that high temperature processing positively affects proteolysis by lowering its susceptibility to spoilage during storage.

Impact of geographical region on urinary metabolomic and plasma fatty acid profiles in subjects with the metabolic syndrome across Europe: the LIPGENE study

The application of metabolomics in multi-centre studies is increasing. The aim of the present study was to assess the effects of geographical location on the metabolic profiles of individuals with the metabolic syndrome. Blood and urine samples were collected from 219 adults from seven European centres participating in the LIPGENE project (Diet, genomics and the metabolic syndrome: an integrated nutrition, agro-food, social and economic analysis). Nutrient intakes, BMI, waist:hip ratio, blood pressure, and plasma glucose, insulin and blood lipid levels were assessed. Plasma fatty acid levels and urine were assessed using a metabolomic technique. The separation of three European geographical groups (NW, northwest; NE, northeast; SW, southwest) was identified using partial least-squares discriminant analysis models for urine (R 2 X: 0•33, Q 2: 0•39) and plasma fatty acid (R 2 X: 0•32, Q 2: 0•60) data. The NW group was characterised by higher levels of urinary hippurate and N-methylnicotinate. The NE group was characterised by higher levels of urinary creatine and citrate and plasma EPA (20 : 5 n-3). The SW group was characterised by higher levels of urinary trimethylamine oxide and lower levels of plasma EPA. The indicators of metabolic health appeared to be consistent across the groups. The SW group had higher intakes of total fat and MUFA compared with both the NW and NE groups (P≤ 0•001). The NE group had higher intakes of fibre and n-3 and n-6 fatty acids compared with both the NW and SW groups (all P< 0•001). It is likely that differences in dietary intakes contributed to the separation of the three groups. Evaluation of geographical factors including diet should be considered in the interpretation of metabolomic data from multi-centre studies.