Longitudinal Metabolomic Profiling of Amino Acids and Lipids across Healthy Pregnancy

Pregnancy is characterized by a complexity of metabolic processes that may impact fetal development and ultimately, infant health outcomes. However, our understanding of whole body maternal and fetal metabolism during this critical life stage remains incomplete. The objective of this study is to utilize metabolomics to profile longitudinal patterns of fasting maternal metabolites among a cohort of non-diabetic, healthy pregnant women in order to advance our understanding of changes in protein and lipid concentrations across gestation, the biochemical pathways by which they are metabolized and to describe variation in maternal metabolites between ethnic groups. Among 160 pregnant women, amino acids, tricarboxylic acid (TCA) cycle intermediates, keto-bodies and non-esterified fatty acids were detected by liquid chromatography coupled with mass spectrometry, while polar lipids were detected through flow-injected mass spectrometry. The maternal plasma concentration of several essential and non-essential amino acids, long-chain polyunsaturated fatty acids, free carnitine, acetylcarnitine, phosphatidylcholines and sphingomyelins significantly decreased across pregnancy. Concentrations of several TCA intermediates increase as pregnancy progresses, as well as the keto-body β-hydroxybutyrate. Ratios of specific acylcarnitines used as indicators of metabolic pathways suggest a decreased beta-oxidation rate and increased carnitine palmitoyltransferase-1 enzyme activity with advancing gestation. Decreasing amino acid concentrations likely reflects placental uptake and tissue biosynthesis. The absence of any increase in plasma non-esterified fatty acids is unexpected in the catabolic phase of later pregnancy and may reflect enhanced placental fatty acid uptake and utilization for fetal tissue growth. While it appears that energy production through the TCA cycle increases as pregnancy progresses, decreasing patterns of free carnitine and acetylcarnitine as well as increased carnitine palmitoyltransferase-1 rate and β-hydroxybutyrate levels suggest a concomitant upregulation of ketogenesis to ensure sufficient energy supply in the fasting state. Several differences in metabolomic profiles between Hispanic and non-Hispanic women demonstrate phenotypic variations in prenatal metabolism which should be considered in future studies.     

Ribonuclease activity associated with the 60S ribosome-inactivating proteins ricin A, phytolaccin and Shiga toxin

All purified preparations of the ribosome-inactivating proteins ricin A, phytolaccin and Shiga toxin were shown to exhibit ribonuclease activity with 5S or 5.8S rRNA substrates. These toxin species generated reproducible patterns of RNA fragments distinct for each toxin species while multiple preparations of a single toxin species yielded similar RNA fragment patterns. The heat inactivation profile of Shiga toxin was identical for its RNase and protein synthesis inhibitory activities. These data are the first to indicate that the ribosome-inactivating catalytic toxins, in addition to alpha-sarcin, exhibit RNase activity. These results suggest RNase activity may be responsible for ribosome-inactivation catalyzed by ricin, phytolaccin and Shiga toxin proteins.     

Der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast.

The endoplasmic reticulum (ER) of the yeast Saccharomyces cerevisiae contains of proteolytic system able to selectively degrade misfolded lumenal secretory proteins. For examination of the components involved in this degradation process, mutants were isolated. They could be divided into four complementation groups. The mutations led to stabilization of two different substrates for this process. The mutant classes were called ''der'' for ''degradation in the ER''. DER1 was cloned by complementation of the der1-2 mutation. The DER1 gene codes for a novel, hydrophobic protein, that is localized to the ER. Deletion of DER1 abolished degradation of the substrate proteins. The function of the Der1 protein seems to be specifically required for the degradation process associated with the ER. The depletion of Der1 from cells causes neither detectable growth phenotypes nor a general accumulation of unfolded proteins in the ER. In DER1-deleted cells, a substrate protein for ER degradation is retained in the ER by the same mechanism which also retains lumenal ER residents. This suggests that DER1 acts in a process that directly removes protein from the folding environment of the ER.     

Secretory production of an FAD cofactor-containing cytosolic enzyme (sorbitol–xylitol oxidase from Streptomyces coelicolor) using the twin-arginine translocation (Tat) pathway of Corynebacterium glutamicum

Carbohydrate oxidases are biotechnologically interesting enzymes that require a tightly or covalently bound cofactor for activity. Using the industrial workhorse Corynebacterium glutamicum as the expression host, successful secretion of a normally cytosolic FAD cofactor-containing sorbitol–xylitol oxidase from Streptomyces coelicolor was achieved by using the twin-arginine translocation (Tat) protein export machinery for protein translocation across the cytoplasmic membrane. Our results demonstrate for the first time that, also for cofactor-containing proteins, a secretory production strategy is a feasible and promising alternative to conventional intracellular expression strategies.The secretory expression of recombinant proteins can offer significant process advantages over cytosolic production strategies, since secretion into the growth medium greatly facilitates downstream processing and therefore can significantly reduce the costs of producing a desired target protein (Quax, 1997). And, in fact, the enormous secretion capacity of certain Gram-positive bacteria (e.g. various Bacillus species) has been used since many years in industry for the production of mainly host-derived secretory proteins such as proteases and amylases, resulting in amounts of more than 20 g l⁻¹ culture medium (Harwood and Cranenburg, 2008). In contrast, attempts to use Bacillus species for the secretory production of heterologous proteins have often failed or led to disappointing results, a fact that, among other reasons, could in many cases be attributed to the presence of multiple cell wall-associated and secreted proteases that rapidly degraded the heterologous target proteins (Li et al., 2004; Sarvas et al., 2004; Westers et al., 2011). Therefore, an increasing need exists to explore alternative host systems with respect to their ability to express and secrete problematic and/or complex heterologous proteins of biotechnological interest.So far, the Gram-positive bacterium Corynebacterium glutamicum has been used in industry mainly for the production of amino acids and other low-molecular weight compounds (Leuchtenberger et al., 2005; Becker and Wittmann, 2011; Litsanov et al., 2012). However, various recent reports have indicated that C. glutamicum might likewise possess a great potential as an alternative host system for the secretory expression of foreign proteins. Corynebacterium glutamicum belongs to a class of diderm Gram-positive bacteria that, besides the cytoplasmic membrane, possess an additional mycolic acid-containing outer membrane-like structure that acts as an extremely efficient permeability barrier for hydrophilic compounds (Hoffmann et al., 2008; Zuber et al., 2008). Despite this fact, an efficient secretion of various heterologous proteins into the growth medium of this microorganism has been observed (e.g. Billman-Jacobe et al., 1995; Meissner et al., 2007; Kikuchi et al., 2009; Tateno et al., 2009; Tsuchidate et al., 2011).In bacteria, two major export pathways exist for the transport of proteins across the cytoplasmic membrane that fundamentally differ with respect to the folding status of their respective substrate proteins during the actual translocation step. The general secretion (Sec) system transports its substrates in a more or less unfolded state and folding takes places on the trans side of the membrane after the actual transport event (Yuan et al., 2010; du Plessis et al., 2011). In contrast, the alternative twin-arginine translocation (Tat) system translocates its substrates in a fully folded form and therefore provides an attractive alternative for the secretory production of proteins that cannot be produced in a functional form via the Sec route (Brüser, 2007). Carbohydrate oxidases are biotechnologically interesting enzymes (van Hellemond et al., 2006) that are excluded from Sec-dependent secretion since they depend on a tightly or covalently bound cofactor for their activity and, for this reason, require that their folding and cofactor insertion has to take place in the cytosol. Because C. glutamicum has shown to be an excellent host for the Tat-dependent secretion of the cofactor-less model protein GFP (Meissner et al., 2007; Teramoto et al., 2011), we now asked whether it is likewise possible to secrete a cofactor-containing enzyme into the supernatant of C. glutamicum using the same protein export route.As a model protein, we chose the sorbitol–xylitol oxidase (SoXy) from Streptomyces coelicolor, a normally cytosolic enzyme that possesses a covalently bound FAD molecule as cofactor (Heuts et al., 2007; Forneris et al., 2008). FAD is incorporated into the apoprotein in a post-translational and self-catalytic process that only occurs if the polypeptide chain has adopted a correctly folded structure (Heuts et al., 2007; 2009). To direct SoXy into the Tat export pathway of C. glutamicum, we constructed a gene encoding a TorA–SoXy hybrid precursor in which SoXy is fused to the strictly Tat-specific signal peptide of the periplasmic Escherichia coli Tat substrate trimethylamine N-oxide reductase (TorA) (Fig. 1) which, in our previous study, has been proven to be a functional and strictly Tat-specific signal peptide also in C. glutamicum (Meissner et al., 2007). The corresponding torA–soxy gene was cloned into the expression vector pEKEx2 (Eikmanns et al., 1991) under the control of an IPTG-inducible P_tac promotor. After transformation of the resulting plasmid pTorA–SoXy into the C. glutamicum ATCC13032 wild-type strain, two independent colonies of the resulting recombinant C. glutamicum (pTorA–SoXy) strain and, as a control, a colony of a strain that contained the empty expression vector without insert [C. glutamicum (pEKEx2)] were grown in CGXII medium (Keilhauer et al., 1993) at 30°C for 16 h in the presence of 1 mM IPTG. Subsequently, the proteins present in the culture supernatants were analysed by SDS-PAGE followed by staining with Coomassie blue. As shown in Fig. 2, in the supernatants of the pTorA–SoXy-containing cells (lanes 3 and 4), a prominent protein band of approximately 44 kDa can be detected, the size of which is very similar to the calculated molecular mass (44.4 kDa) of SoXy. Since this band is completely lacking in the supernatant of the control strain (lane 2), this strongly suggests that this band corresponds to SoXy that has been secreted into the culture supernatant of C. glutamicum (pTorA–SoXy). And, in fact, this suggestion was subsequently confirmed in a direct way by MALDI-TOF mass spectrometry after extraction of the protein out of the gel followed by tryptic digestion (Schaffer et al., 2001) (data not shown).Open in a separate windowFigure 1The TorA–SoXy hybrid precursor protein. Upper part: Schematic drawing of the relevant part of the pTorA–SoXy expression vector. P_tac, IPTG-inducible tac promotor. RBS, ribosome binding site. To maintain the authentic TorA signal peptidase cleavage site, the first four amino acids of the mature TorA protein (black bar) were retained in the TorA–SoXy fusion protein. White bar: TorA signal peptide (TorA^SP); grey bar: SoXy (amino acids 2–418). Lower part: Amino acid sequence of the signal peptide and early mature region of the TorA–SoXy hybrid precursor. The twin-arginine consensus motif of the TorA signal peptide is underlined. The four amino acids derived from mature TorA are shown in italics. The signal peptidase cleavage site is indicated by an arrowhead.Open in a separate windowFigure 2Secretion of SoXy into the growth medium of C. glutamicum. Cells of C. glutamicum ATCC13032 containing the empty vector pEKEx2 and two independently transformed colonies of C. glutamicum (pTorA–SoXy) were grown overnight in 5 ml of BHI medium (Difco) at 30°C. The cells were washed once with CGXII medium (Keilhauer et al., 1993) and inoculated to an OD₆₀₀ of 0.5 in 5 ml of fresh CGXII medium containing 1 mM IPTG. After 16 h of further growth at 30°C, the supernatant fractions were prepared as described previously (Meissner et al., 2007). Samples corresponding to an equal number of cells were subjected to SDS-PAGE followed by staining with Coomassie blue. Lane 1, molecular mass marker (kDa). Lane 2, C. glutamicum (pEKEx2); lanes 3 and 4, C. glutamicum (pTorA–SoXy). The position of the secreted SoXy protein is indicated by an arrow.Next, the supernatant of C. glutamicum (pTorA–SoXy) was analysed for SoXy enzyme activity by measuring the production of H₂O₂ that is formed during the enzymatic conversion of sorbitol to fructose (Meiattini, 1983). Six hours after induction of gene expression by 1 mM IPTG, an enzymatic activity of 10.3 ± 1.6 nmol min⁻¹ ml⁻¹ could be determined in the supernatant of C. glutamicum (pTorA–SoXy). In contrast, no such activity was found in the supernatant of the control strain C. glutamicum (pEKEx2). From these results we conclude that we have succeeded in the secretion of enzymatically active and therefore FAD cofactor-containing SoXy into the culture supernatant of C. glutamicum.Finally, we examined whether the secretion of SoXy had in fact occurred via the Tat pathway of C. glutamicum. Plasmid pTorA–SoXy was used to transform C. glutamcium ATCC13032 wild type and a C. glutamicum ΔTatAC mutant strain that lacks two essential components of the Tat transport machinery and therefore does not possess a functional Tat translocase (Meissner et al., 2007). The corresponding cells were grown in BHI medium (Difco) at 30°C in the presence of 1 mM IPTG for 6 h. Subsequently, the proteins present in the cellular and the supernatant fractions of the corresponding cells were analysed by SDS-PAGE followed by Western blotting using SoXy-specific antibodies. As shown in Fig. 3, polypeptides corresponding to the unprocessed TorA–SoXy precursor and some minor smaller degradation products of it can be detected in the cellular fractions of both the wild-type and the ΔTatAC deletion strains (lanes 3 and 5). In the supernatant fraction of the Tat⁺ wild-type strain (lane 4), but not that of the ΔTatAC strain (lane 6), a polypeptide corresponding to mature SoXy is present, clearly showing that export of SoXy in the wild-type strain had occurred in a strictly Tat-dependent manner. Another noteworthy finding is the observation that hardly any mature SoXy protein accumulated in the cellular fraction of the Tat⁺ wild-type strain (lane 3), indicating that SoXy is, after its Tat-dependent translocation across the cytoplasmic membrane and processing by signal peptidase, rapidly transported out of the intermembrane space across the mycolic acid-containing outer membrane into the supernatant. However, the mechanism of how proteins cross this additional permeability barrier is completely unknown so far (Bitter et al., 2009).Open in a separate windowFigure 3Transport of TorA–SoXy occurs in a strictly Tat-dependent manner. Plasmid pTorA–SoXy was transformed into C. glutamcium ATCC13032 (Tat⁺) and a C. glutamicum ΔTatAC mutant that lacks a functional Tat translocase (Meissner et al., 2007). As a control, the empty pEKEx2 expression vector was transformed into C. glutamicum ATCC13032 (Tat⁺). The respective strains were grown overnight in 5 ml of BHI medium (Difco) at 30°C. The cells were washed once with BHI and resuspended in 20 ml of fresh BHI medium containing 1 mM IPTG. After 6 h of further growth at 30°C, the cellular (C) and supernatant (S) fractions were prepared as described previously (Meissner et al., 2007). Samples of the C and S fractions were subjected to SDS-PAGE followed by immunoblotting using anti-SoXy antibodies as indicated at the top of the figure. Lanes 1 and 2: C. glutamicum ATCC13032 (pEKEx2); lanes 3 and 4: C. glutamicum ATCC13032 (pTorA–SoXy); lanes 5 and 6: C. glutamicum ΔTatAC (pTorA–SoXy). Asterisk: TorA–SoXy precursor; arrow: secreted SoXy protein. The positions of molecular mass markers (kDa) are indicated at the left margin of the figure.To the best of our knowledge, our results represent the first documented example of the successful secretion of a normally cytosolic, cofactor-containing protein via the Tat pathway in an active form into the culture supernatant of a recombinant expression host. Our results clearly show that, also for this biotechnologically very interesting class of proteins, a secretory production strategy can be a promising alternative to conventional intracellular expression strategies. Besides for SoXy and other FAD-containing carbohydrate oxidases, for which various applications are perceived by industry such as the in situ generation of hydrogen peroxide for bleaching and disinfection performance in technical applications, their use in the food and drink industry, as well as their use in diagnostic applications and carbohydrate biosynthesis processes (Oda and Hiraga, 1998; Murooka and Yamashita, 2001; van Hellemond et al., 2006; Heuts et al., 2007), a secretory production strategy might now be an attractive option also for biotechnologically relevant enzymes that are used as biocatalysts in chemo-enzymatic syntheses and that possess cofactors other than FAD, such as pyridoxal-5′-phosphate (PLP)-dependent ω-transaminases (Mathew and Yun, 2012) or various thiamin diphosphate (TDP)-dependent enzymes (Müller et al., 2009).     

Differences in the Serum Nonesterified Fatty Acid Profile of Young Women Associated with a Recent History of Gestational Diabetes and Overweight/Obesity

BackgroundNonesterified fatty acids (NEFA) play pathophysiological roles in metabolic syndrome and type 2 diabetes (T2D). In this study, we analyzed the fasting NEFA profiles of normoglycemic individuals at risk for T2D (women with a recent history of gestational diabetes (GDM)) in comparison to controls (women after a normoglycemic pregnancy). We also examined the associations of NEFA species with overweight/obesity, body fat distribution and insulin sensitivity.ResultsWomen after GDM had a lower molar percentage of total saturated fatty acids (SFA; 38.55% vs. 40.32%, p = 0.0002) than controls. At an explorative level of significance several NEFA species were associated with post-GDM status (with and without adjustment for body mass index (BMI) and HbA1c): The molar percentages of 14:0, 16:0, 18:0 and 18:4 were reduced, whereas those of 18:1, 18:2, 20:2, 24:4, monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA) and total n-6 NEFA were increased. BMI and the amount of body fat correlated inversely with several SFA and MUFA and positively with various PUFA species over the whole study cohort (abs(ρ)≥0.3 for all). 14:0 was inversely and BMI-independently associated with abdominal visceral adiposity. We saw no correlations of NEFA species with insulin sensitivity and the total NEFA concentration was similar in the post-GDM and the control group.ConclusionIn conclusion, we found alterations in the fasting NEFA profile associated with a recent history of gestational diabetes, a risk marker for T2D. NEFA composition also varied with overweight/obesity and with body fat distribution, but not with insulin sensitivity.     

Circadian communication between unicells?

Populations ofGonyaulax polyedra, in two different phases, about 11 h apart, were mixed, and the intensity of their spontaneous bioluminescence glow recorded for about 2 wk under conditions of constant dim (35±3 μE/m²/s) white light and constant temperature (19.0±0.3°C). The phases and amplitudes of glow signals recorded from mixed cultures were compared with those obtained from the arithmetic sum of the intensity data from two control vials. Peaks in control cultures generally remained separate, but there was a spontaneous increase in the period beginning 6–11 d after the onset of constant conditions. This did not occur in cultures in which the medium was exchanged with fresh medium every 2 d. In the actual mixes of two cultures there was a merging of the two subpeaks in the signal, which did not occur when the medium was exchanged. The results indicate that conditioning of the medium by cells may affect the period of the circadian rhythm and that this might result in a type of communication. Supported by the Deutsche Forschungsgemeinschaft; present address     

Selenoprotein P Status Correlates to Cancer-Specific Mortality in Renal Cancer Patients

Selenium (Se) is an essential trace element for selenoprotein biosynthesis. Selenoproteins have been implicated in cancer risk and tumor development. Selenoprotein P (SePP) serves as the major Se transport protein in blood and as reliable biomarker of Se status in marginally supplied individuals. Among the different malignancies, renal cancer is characterized by a high mortality rate. In this study, we aimed to analyze the Se status in renal cell cancer (RCC) patients and whether it correlates to cancer-specific mortality. To this end, serum samples of RCC patients (n = 41) and controls (n = 21) were retrospectively analyzed. Serum Se and SePP concentrations were measured by X-ray fluorescence and an immunoassay, respectively. Clinical and survival data were compared to serum Se and SePP concentrations as markers of Se status by receiver operating characteristic (ROC) curve and Kaplan-Meier and Cox regression analyses. In our patients, higher tumor grade and tumor stage at diagnosis correlated to lower SePP and Se concentrations. Kaplan-Meier analyses indicated that low Se status at diagnosis (SePP<2.4 mg/l, bottom tertile of patient group) was associated with a poor 5-year survival rate of 20% only. We conclude that SePP and Se concentrations are of prognostic value in RCC and may serve as additional diagnostic biomarkers identifying a Se deficit in kidney cancer patients potentially affecting therapy regimen. As poor Se status was indicative of high mortality odds, we speculate that an adjuvant Se supplementation of Se-deficient RCC patients might be beneficial in order to stabilize their selenoprotein expression hopefully prolonging their survival. However, this assumption needs to be rigorously tested in prospective clinical trials.     

In Vitro Protein Synthesis and Aging in Rhizoctonia solani

A study was made of the ability of cell-free protein synthesis systems from vegetative cells of different age of the fungus Rhizoctonia solani to produce polyphenylalanine. Polyuridylic acid-directed phenylalanine incorporation into peptides decreased linearly with cell age. The 105,000 x g supernatant fluid and ribosomal fractions were equally responsible for the total loss of synthetic activity of the older cells. Initial rates of phenylalanyl-transfer ribonucleic acid (tRNA) synthetase activity decreased with increasing cell age, which accounted for the defect of the supernatant fraction. An accelerated degradation of soluble phenylalanyl-RNA was associated with the ribosomes of the older cells. In vitro systems from cells of different age transferred phenylalanine from phenylalanyl-tRNA to polyphenylalanine at similar rates. Of the 15 specific aminoacyl-tRNA synthetases assayed, 5 increased and 5 decreased in specific activity with increased age; 3 others did not change during aging and 2 were below acceptable detectable levels.