Over the past decades, electrospray ionization for mass spectrometry (ESI-MS) has become one of the most commonly employed techniques in analytical chemistry, mainly due to its broad applicability to ...polar and semipolar compounds and the superior selectivity which is achieved in combination with high resolution separation techniques. However, responsiveness of an analytical method also determines its suitability for the quantitation of chemical compounds; and in electrospray ionization for mass spectrometry, it can vary significantly among different analytes with identical solution concentrations. Therefore, we investigated the ESI-response behavior of 56 nitrogen-containing compounds including aromatic amines and pyridines, two compound classes of high importance to both, synthetic organic chemistry as well as to pharmaceutical sciences. These compounds are increasingly analyzed employing ESI mass spectrometry detection due to their polar, basic character. Signal intensities of the peaks from the protonated molecular ion (MH+) were acquired under different conditions and related to compound properties such as basicity, polarity, volatility and molecular size exploring their quantitative impact on ionization efficiency. As a result, we found that though solution basicity of a compound is the main factor initially determining the ESI response of the protonated molecular ion, other factors such as polarity and vaporability become more important under acidic solvent conditions and may nearly outweigh the importance of basicity under these conditions. Moreover, we show that different molecular descriptors may become important when using different types of instruments for such investigations, a fact not detailed so far in the available literature.
Though numerous valuable compounds from red algae already experience high demand in medicine, nutrition, and different branches of industry, these organisms are still recognized as an underexploited ...resource. This study provides a comprehensive characterization of the chemical composition of 15 Arctic red algal species from the perspective of their practical relevance in medicine and the food industry. We show that several virtually unstudied species may be regarded as promising sources of different valuable metabolites and minerals. Thus, several filamentous ceramialean algae (
,
,
) had total protein content of 20-32% of dry weight, which is comparable to or higher than that of already commercially exploited species (
,
sp.). Moreover, ceramialean algae contained high amounts of pigments, macronutrients, and ascorbic acid.
(Gigartinales) accumulated free essential amino acids, taurine, pantothenic acid, and floridoside. Thalli of
and
contained the highest amounts of the nonproteinogenic amino acid β-alanine (9.1 and 3.2 μM g
DW, respectively). Several red algae tend to accumulate heavy metals; although this may limit their application in the food industry, it makes them promising candidates for phytoremediation or the use as bioindicators.
The naturally occurring dipeptide carnosine (β-alanyl-l-histidine) has beneficial effects in different diseases. It is also frequently used as a food supplement to improve exercise performance and ...because of its anti-aging effects. Nevertheless, after oral ingestion, the dipeptide is not detectable in human serum because of rapid degradation by serum carnosinase. At the same time, intact carnosine is excreted in urine up to five hours after intake. Therefore, an unknown compartment protecting the dipeptide from degradation has long been hypothesized. Considering that erythrocytes may constitute this compartment, we investigated the uptake and intracellular amounts of carnosine in human erythrocytes cultivated in the presence of the dipeptide and human serum using liquid chromatography-mass spectrometry. In addition, we studied carnosine's effect on ATP production in red blood cells and on their response to oxidative stress. Our experiments revealed uptake of carnosine into erythrocytes and protection from carnosinase degradation. In addition, no negative effect on ATP production or defense against oxidative stress was observed. In conclusion, our results for the first time demonstrate that erythrocytes can take up carnosine, and, most importantly, thereby prevent its degradation by human serum carnosinase.
Metabolite profiling using gas chromatography coupled to mass spectrometry (GC-MS) is one of the most frequently applied and standardized methods in research projects using metabolomics to analyze ...complex samples. However, more than 20 years after the introduction of non-targeted approaches using GC-MS, there are still unsolved challenges to accurate quantification in such investigations. One particularly difficult aspect in this respect is the occurrence of sample-dependent matrix effects. In this project, we used model compound mixtures of different compositions to simplify the study of the complex interactions between common constituents of biological samples in more detail and subjected those to a frequently applied derivatization protocol for GC-MS analysis, namely trimethylsilylation. We found matrix effects as signal suppression and enhancement of carbohydrates and organic acids not to exceed a factor of ~2, while amino acids can be more affected. Our results suggest that the main reason for our observations may be an incomplete transfer of carbohydrate and organic acid derivatives during the injection process and compound interaction at the start of the separation process. The observed effects were reduced at higher target compound concentrations and by using a more suitable injection-liner geometry.
Leaves from
Fresen (Combretaceae) are commonly used for the treatment of inflammatory conditions, wound healing and bacterial infections in traditional West African medicine. This research focuses on ...the characterization of the phenolic profile and lipophilic compounds of leaves extracts of
. Studies of the in vitro anti-inflammatory activity were performed in TNFα stimulated HaCaT cells and antibacterial activity was evaluated with agar well diffusion and microdilution assays. Antioxidant activity was determined by DPPH and ABTS assays and compared to standards. The phytochemical studies confirmed myricetin-3-
-rhamnoside and myricetin-3-
-glucoside as major components of the leaves extracts, each contributing significantly to the antioxidant activity of the hydrophilic extracts. GC-MS analysis identified 19 substances that were confirmed by comparison with spectral library data and authentic standards.
aqueous leaves extract decreased pro-inflammatory mediators in TNFα stimulated HaCaT cells. Further investigations showed that myricetin-3-
-rhamnoside has an anti-inflammatory effect on IL-8 secretion. In the antimicrobial screening, the largest inhibition zones were found against
,
and
. MIC values resulted in 275.0 µg/mL for
and 385.5 µg/mL for
. The in vitro anti-inflammatory, antibacterial and antioxidant activity supports topical use of
leaves extracts in traditional West African medicine.
The previous studies demonstrated that carnosine (β-alanyl-
l
-histidine) inhibits the growth of tumor cells in vitro and in vivo. Considering carnosine for the treatment of glioblastoma, we ...investigated which proton-coupled oligopeptide transporters (POTs) are present in glioblastoma cells and how they contribute to the uptake of carnosine. Therefore, mRNA expression of the four known POTs (PEPT1, PEPT2, PHT1, and PHT2) was examined in three glioblastoma cell lines, ten primary tumor cell cultures, in freshly isolated tumor tissue and in healthy brain. Using high-performance liquid chromatography coupled to mass spectrometry, the uptake of carnosine was investigated in the presence of competitive inhibitors and after siRNA-mediated knockdown of POTs. Whereas PEPT1 mRNA was not detected in any sample, expression of the three other transporters was significantly increased in tumor tissue compared to healthy brain. In cell culture, PHT1 expression was comparable to expression in tumor tissue, PHT2 exhibited a slightly reduced expression, and PEPT2 expression was reduced to normal brain tissue levels. In the cell line LN405, the competitive inhibitors β-alanyl-
l
-alanine (inhibits all transporters) and
l
-histidine (inhibitor of PHT1/2) both inhibited the uptake of carnosine. SiRNA-mediated knockdown of PHT1 and PHT2 revealed a significantly reduced uptake of carnosine. Interestingly, despite its low expression at the level of mRNA, knockdown of PEPT2 also resulted in decreased uptake. In conclusion, our results demonstrate that the transporters PEPT2, PHT1, and PHT2 are responsible for the uptake of carnosine into glioblastoma cells and full function of all three transporters is required for maximum uptake.
Methane biogenesis in methanogens is mediated by methyl-coenzyme M reductase, an enzyme that is also responsible for the utilization of methane through anaerobic methane oxidation. The enzyme uses an ...ancillary factor called coenzyme Fsub.430, a nickel-containing modified tetrapyrrole that promotes catalysis through a methyl radical/Ni(ii)-thiolate intermediate. However, it is unclear how coenzyme Fsub.430 is synthesized from the common primogenitor uroporphyrinogen iii, incorporating 11 steric centres into the macrocycle, although the pathway must involve chelation, amidation, macrocyclic ring reduction, lactamization and carbocyclic ring formation. Here we identify the proteins that catalyse the biosynthesis of coenzyme Fsub.430 from sirohydrochlorin, termed CfbACfbE, and demonstrate their activity. The research completes our understanding of how the repertoire of tetrapyrrole-based pigments are constructed, permitting the development of recombinant systems to use these metalloprosthetic groups more widely.
Brown algae have an important role in marine environments. With respect to their broad distribution and importance for the environment and human use, brown algae of the order Fucales in particular ...became a model system for physiological and ecological studies. Thus, several fucoids have been extensively studied for their composition on the molecular level. However, research of fucoid physiology and biochemistry so far mostly focused on the adult algae, so a holistic view on the development of these organisms, including the crucial first life stages, is still missing. Therefore, we employed non-targeted metabolite profiling by gas chromatography coupled to mass spectrometry to create a non-biased picture of the early development of the fucoid alga
We found that embryogenic physiology was mainly dominated by a tight regulation of carbon and energy metabolism. The first dramatic changes of zygote metabolism started within 1 h after fertilization, while metabolism of 6-9 days old embryos appeared already close to that of an adult alga, indicated by the intensive production of secondary metabolites and accumulation of mannitol and citric acid. Given the comprehensive description and analysis we obtained in our experiments, our results exhibit an invaluable resource for the design of further experiments related to physiology of early algal development.
We have identified a carboxylesterase produced in liquid cultures of the thermophilic actinomycete Thermobifida fusca KW3 that were supplemented with poly(ethylene terephthalate) fibers. The enzyme ...hydrolyzed highly hydrophobic, synthetic cyclic poly(ethylene terephthalate) trimers with an optimal activity at 60°C and a pH of 6. V max and K m values for the hydrolysis were 9.3 µmol⁻¹ min⁻¹ mg⁻¹ and 0.5 mM, respectively. The esterase showed high specificity towards short and middle chain-length fatty acyl esters of p-nitrophenol. The enzyme retained 37% of its activity after 96 h of incubation at 50°C and a pH of 8. Enzyme inhibition studies and analysis of substitution mutants of the carboxylesterase revealed the typical catalytic mechanism of a serine hydrolase with a catalytic triad composed of serine, glutamic acid, and histidine.
Heme d1 is a modified tetrapyrrole playing an important role in denitrification by acting as the catalytically essential cofactor in the cytochrome cd1 nitrite reductase of many denitrifying ...bacteria. In the course of heme d1 biosynthesis, the two propionate side chains on pyrrole rings A and B of the intermediate 12,18‐didecarboxysiroheme are removed from the tetrapyrrole macrocycle. In the final heme d1 molecule, the propionate groups are replaced by two keto functions. Although it was speculated that the Radical S‐adenosyl‐l‐methionine (SAM) enzyme NirJ might be responsible for the removal of the propionate groups and introduction of the keto functions, this has not been shown experimentally, so far. Here, we demonstrate that NirJ is a Radical SAM enzyme carrying two iron–sulfur clusters. While the N‐terminal 4Fe‐4S cluster is essential for the initial SAM cleavage reaction, it is not required for substrate binding. NirJ tightly binds its substrate 12,18‐didecarboxysiroheme and, thus, can be purified in complex with the substrate. By using the purified NirJ/substrate complex in an in vitro enzyme activity assay, we show that NirJ indeed catalyzes the removal of the two propionate side chains under simultaneous SAM cleavage. However, under the reaction conditions employed, no keto group formation is observed indicating that an additional cofactor or enzyme is needed for this reaction.
The heme d1 biosynthesis enzyme NirJ belongs to the Radical SAM protein family and binds two iron–sulfur clusters. One of the clusters is required for the typical SAM cleavage reaction. Overall NirJ catalyzes the removal of two propionate side chains from the substrate 12,18‐didecarboxysiroheme.
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