Free Amino Acid Metabolism Research Articles

Effect of excessive nitrogen on levels of amino acids and sugars, and differential response to post-harvest cold storage in potato (Solanum tuberosum L.) tubers

Nitrogen (N) is an important nutrient for increased potato tuber yield. However, excessive N can decrease tuber quality. Furthermore, the impact of optimal and higher N levels of potato tuber metabolic profile at harvest and cold storage remains unclear. This study aimed to investigate the metabolic profiling of free amino acids and sugars in potato tubers affected by different nitrogen levels (optimal, ON; and excessive, EN) at harvest (AH) and cold storage (CS) (~4 °C, 4 weeks) through untargeted GC–TOF–MS, and targeted UHPLC–QqQ–MS. Carbohydrate content and vacuolar invertase activity (IV) were determined. Principal component analysis of metabolite data indicated a distinct separation between ON and EN treatments at harvest and cold storage. Multivariate data analysis revealed that sucrose, reducing sugars, and free asparagine were the most altered metabolites (VIP > 1 and P < 0.05), which were involved in starch and sucrose metabolism, and alanine, aspartate and glutamate metabolism. At harvest, the absolute contents of various free amino acids including asparagine were higher (by 1.3–1.5 fold) in the EN treatment than ON treatment, and this difference was maintained at 4-week cold storage. Under the EN treatment, tuber maturity was reduced, and sucrose accumulation was increased at harvest, while IV was increased after cold storage, reducing sugar also accumulated. These results highlighted the negative effects of EN on free amino acid and sugars metabolism in the post–harvest tubers and provided useful information for understanding the underpinning physiological mechanisms.

Phytohormones enabled endophytic Penicillium funiculosum LHL06 protects Glycine max L. from synergistic toxicity of heavy metals by hormonal and stress-responsive proteins modulation.

This study investigates the stress-mitigating effects of endophytic Penicillium funiculosum LHL06 on soybean roots via modulation of physio-biochemical, molecular, and proteomic responses to combined heavy metal (Ni, Cu, Pb, Cr, and Al) toxicity. Preliminary screening revealed that LHL06 can tolerate and remediate combined heavy metal contamination in its media and upregulate gibberellins (GA1, GA3, GA4, GA7 and GA9) and indole-3-acetic acid (IAA) production. Inoculation of LHL06 resulted in marked reduction of metals uptake in roots and shoots by downregulating heavy metal ATPase genes (GmHMA13, GmHMA14, GmHMA19) and GmMATE1 compared to non-inoculated plants; in turn, this decreased abscisic acid and jasmonic acid levels. Moreover, triggering of free amino acid metabolism in LHL06-inoculated roots significantly upregulated expression of stress-related proteins (glutathione S-transferase L3, isoflavone reductase-like, chalcone isomerase A, NAD(P)H dehydrogenase (quinone), FQR1-like 1 isoform X2, and Peroxidase 3) to combat metals toxicity. Compared to non-inoculated-plants, LHL06-inoculated-plants exhibited higher antioxidant activity and transcript accumulation of glutathione S-transferase (GmGST8 and GmGST3), G6PDH, and GmSOD1[Cu-Zn], which decreased metal-induced reactive oxygen species. Therefore, LHL06-inoculation remediate combined metal contamination in soil, activate signaling network of stress-responsive hormones and antioxidant systems for promoting growth and tolerance, and reduce metal-accumulation, thereby making plants safer for consumption.

Fecal Microbiota and Its Correlation With Fatty Acids and Free Amino Acids Metabolism in Piglets After a Lactobacillus Strain Oral Administration.

Lactobacillus has a positive effect on the host intestinal microbiota. In piglets, dietary supplementation with Lactobacillus affects general health and plays an important role in nutrient digestion and fermentation. However, this association requires further investigation. Here, we studied newborn piglets from 12 litters. The nursed piglets were given a creep feed beginning on day 10 post-partum and weaned at day 30. Piglets were fed either a control basic diet or a diet including supplementation with Lactobacillus reuteri ZLR003 at 6.0 × 106 CFU/g feed. At day 30 and 60, feces samples were taken and used for sequencing of the V3-V4 hypervariable region of the 16S rRNA gene. At day 60, feces samples and serum samples were also taken and used to measure the short chain fatty acids (SCFAs) and to detect long chain fatty acids (LCFAs) and free amino acids (FAAs), respectively. The results revealed that L. reuteri ZLR003 could improve piglet fecal microbiota composition, especially at the end of weaned period. The concentrations of lactic acid and butyric acid in feces were higher, and acetic acid concentration was lower in the L. reuteri ZLR003 group compared with the control group (P < 0.05). The serum polyunsaturated fatty acids C18:2n6c, C18:3n3, C20:4n6, and C22:6n3 were significantly higher (P < 0.05), as were the serum FAAs Gly, Ala, Val, Iso, Asn, Asp, Glu, Met, Phe, and Leu (P < 0.05), in the L. reuteri group compared with the control group. A correlation analysis revealed that the genera Ruminococcaceae_UCG-010 and Ruminococcaceae_UCG-014 had a negative correlation with the SCFAs content in feces, the genus Prevotella_9 had a higher positive correlation with C18:2n6c, and the genera Megasphaera and Mitsuokella had a more positive significant effect on the serum FAAs content in weaned piglets in the L. reuteri ZLR003 group compared with the control group. In conclusion, L. reuteri ZLR003 influenced the fecal microbiota composition of piglets, and its effects were related to the metabolism of SCFAs, LCFAs, and FAAs. Our findings will help facilitate the application of Lactobacillus strains in pig production.

Supplementation of Nitrogen and its Influence on Free Sugars, Amino Acid and Protein Metabolism in Roots and Internodes of Wheat

Effect of different doses of nitrogen (N) (90, 120, 150 and 180 kg Nha–1) on the activities of aminotransferases and alkaline inorganic pyrophosphatase (AIP) in relation to the accumulation of proteins, amino acids and sugars in roots and internodes at 15 and 40 days post anthesis (DPA) stages was studied in six wheat genotypes namely HD 2967, GLU 1101, PBW 343, BW 9022, PH-132–4840 and PBW 550. Supra-optimal N doses (150 kg Nha–1 and 180 kg Nha–1) accentuated glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and alkaline inorganic pyrophosphatase activities in correspondence with an increase in amino acid, protein and sugar content in both roots and internodes in all the six genotypes. Activities of analyzed enzymes were significantly high at 15 days post anthesis (DPA) stage and thereafter declined at maturity (40 DPA) in parallel with decrease in amino acid contents. Maximum activity of GOT, GPT and AIP was observed in HD 2967 and GLU 1101 genotypes along with higher build up of proteins and amino acids which resulted in higher grain yield. Activity of GPT was comparatively high over GOT, indicating its major role towards protein synthesis. Grain filling processes in terms of proteins and amino acids were positively correlated with GOT and GPT activities while sugars were correlated to AIP. Thus, nitrogen acquisition and assimilation resulted in favoured utilization of N in form of amino acid and proteins accumulation while sugar content was also stimulated. Due to immense activities of aminotransferases and higher contents of amino acids and proteins in GLU 1101 and HD 2967 genotypes at optimal dose and higher dose of N, these genotypes hold future potential for developing new cultivars with better grain quality characteristics.

Gut microbiota development in mice is affected by hydrogen peroxide produced from amino acid metabolism during lactation

The development of gut microbiota during infancy is an important event that affects the health status of the host; however, the mechanism governing it is not fully understood. l-Amino acid oxidase 1 (LAO1) is a flavoprotein that catalyzes the oxidative deamination of particular l-amino acids and converts them into keto acids, ammonia, and H2O2. Our previous study showed that LAO1 is present in mouse milk and exerts protection against bacteria by its production of H2O2. The data led us to consider whether LAO1, H2O2, or both could impact infant gut microbiota development via mother's milk consumption in mice. Different gut microbiota profiles were observed in the wild-type (WT) and LAO1-knockout mouse pups. The WT pups' microbiota was relatively simple and composed of only a few dominant bacteria, such as Lactobacillus, whereas the lactating knockout pups had high microbiota diversity. Cross-fostering experiments indicated that WT milk (containing LAO1) has the ability to suppress the diversity of microbiota in pups. We observed that the stomach content of pups fed WT milk had LAO1 proteins and the ability to produce H2O2. Moreover, culture experiments showed that Lactobacillus was abundant in the feces of pups fed WT milk and that Lactobacillus was more resistant to H2O2 than Bifidobacterium and Escherichia. Human breast milk produces very little H2O2, which could be the reason for Lactobacillus not being dominant in the feces of breast-fed human infants. In mouse mother's milk, H2O2 is generated from the process of free amino acid metabolism, and H2O2 may be a key player in regulating the initial acquisition and development of gut microbiota, especially growth of Lactobacillus, during infancy.-Shigeno, Y., Zhang, H., Banno, T., Usuda, K., Nochi, T., Inoue, R., Watanabe, G., Jin, W., Benno, Y., Nagaoka, K. Gut microbiota development in mice is affected by hydrogen peroxide produced from amino acid metabolism during lactation.

Borage oil restores acidic skin pH by up-regulating the activity or expression of filaggrin and enzymes involved in epidermal lactate, free fatty acid, and acidic free amino acid metabolism in essential fatty acid-deficient Guinea pigs

Borage oil (BO) reverses a disrupted epidermal lipid barrier and hyperproliferation in essential fatty acid deficiency (EFAD). However, little is known about its effect on skin pH, which is maintained by epidermal lactate, free fatty acids (FFAs), and free amino acids (FAAs) which is generated by lactate dehydrogenase (LDH), secreted phospholipase A2 (sPLA2), or filaggrin degradation with peptidylarginine deiminase-3 (PADI3). We hypothesized that BO restores skin pH by regulating epidermal lactate, FFA metabolism, or FAA metabolism in EFAD. To test this hypothesis, EFAD was induced in guinea pigs by a hydrogenated coconut oil (HCO) diet for 8 weeks, followed by 2 weeks of a BO diet (group HCO + BO). As controls, groups HCO and BO were fed HCO or BO diets for 10 weeks. In group HCO + BO, skin pH, which was less acidic in group HCO, was restored; and epidermal lactate and total FFAs, including palmitate, stearate, linoleate, arachidate, behenate, and lignocerate, were higher than in group HCO. LDH and sPLA2 (mainly the PLA2G2F isoform) activities and protein expressions were similar between groups HCO + BO and BO. Epidermal acidic FAAs, as well as filaggrin and PADI3 protein and mRNA expressions were higher in group HCO + BO than in group HCO. Oleate, total FAAs including other FAAs, and LDH and sPLA2 mRNA expressions were not altered between groups HCO and HCO + BO. Basic FAAs were not altered among groups. Dietary BO restored acidic skin pH and increased epidermal levels of lactate, most FFAs, and acidic FAAs by up-regulating LDH, sPLA2, filaggrin, and PADI3 activities as well as protein or mRNA expressions in EFAD.

Population resequencing reveals candidate genes associated with salinity adaptation of the Pacific oyster Crassostrea gigas

The Pacific oyster Crassostrea gigas is an important cultivated shellfish. As a euryhaline species, it has evolved adaptive mechanisms responding to the complex and changeable intertidal environment that it inhabits. To investigate the genetic basis of this salinity adaptation mechanism, we conducted a genome-wide association study using phenotypically differentiated populations (hyposalinity and hypersalinity adaptation populations, and control population), and confirmed our results using an independent population, high-resolution melting, and mRNA expression analysis. For the hyposalinity adaptation, we determined 24 genes, including Cg_CLCN7 (chloride channel protein 7) and Cg_AP1 (apoptosis 1 inhibitor), involved in the ion/water channel and transporter mechanisms, free amino acid and reactive oxygen species metabolism, immune responses, and chemical defence. Three SNPs located on these two genes were significantly differentiated between groups, as was Cg_CLCN7. For the hypersalinity adaptation, the biological process for positive regulating the developmental process was enriched. Enriched gene functions were focused on transcriptional regulation, signal transduction, and cell growth and differentiation, including calmodulin (Cg_CaM) and ficolin-2 (Cg_FCN2). These genes and polymorphisms possibly play an important role in oyster hyposalinity and hypersalinity adaptation. They not only further our understanding of salinity adaptation mechanisms but also provide markers for highly adaptable oyster strains suitable for breeding.

The concentration of free amino acids in blood serum of dairy cows with primary ketosis.

Ketosis is a common condition found in the initial stages of lactation in high-yielding dairy cows. The major cause of ketosis is a negative energy balance. During the energy deficiency, proteolysis processes develop parallel to lipolysis. During proteolysis, muscle tissue can be used as a source of amino acid. To date, the participation of amino acids in gluconeogenesis (glucogenic amino acids) and ketogenesis (ketogenic amino acids) has not been determined in detail. This paper presents the study on determination of the parameters of protein and free amino acid metabolism in blood serum of dairy cows with primary ketosis compared to healthy cows. This study contributes to better understanding of the role of amino acids in pathogenesis of ketosis. A total of 30 cows, divided into two groups: experimental (15 cows with ketosis) and control (15 healthy cows), were included in the study. The concentrations of glucose, β-hydroxybutyrate, total protein, albumin, urea, and free amino acids were determined in peripheral blood. Statistically significantly higher concentrations of glutamine, glutamic acid, isoleucine (p≤0.001), and tyrosine (p≤0.05) were found in cows with primary ketosis compared to healthy cows. Significant decrease in the concentrations of asparagine, histidine, methionine, and serine (p≤0.001), alanine, leucine, lysine and proline (p≤0.05) was observed. Significant increase of total ketogenic and glucogenic amino acids (p≤0.05), and an increased ratio of total ketogenic and glucogenic amino acids to total amino acids (p≤0.001) were noted in cows with ketosis. In our study, the changes, in particular observed in amino acid concentration in cows with primary ketosis, indicate its intensive use in both ketogenesis and gluconeogenesis processes. Therefore, a detailed understanding of the role that amino acids play in gluconeogenesis and ketogenesis will improve ketosis diagnostics and monitoring the course of a ketosis episode. Perhaps, the prevention of this disease is possible by balancing the appropriate feed ration in terms of amino acid content.

The dynamics of free amino acids in the grains Tripogon chinensis (Franch.) Hack. when germinating in the conditions of the osmotic stress

The content of free amino acids in seeds and sprouts of T. chinensis in the control and at an osmotic pressure of 5 atm. was determined. The group of acidic and basic amino acids predominates in seeds. An adaptive reaction to a physiological drought is the transformation of the free amino acids metabolism. This is expressed in an increase in the total amount of free amino acids at the osmotic pressure of 5 atm., providing the osmotic component of adaptation. Under the conditions of the osmotic stress, the concentration of acidic and basic amino acids increases up to 48 hours, this may be due to the continued hydrolysis of reserve proteins. Under the influence of the osmotic stress there are changes in the group of amino acids, the metabolic precursor of which is glutamic acid. The amino acids, a part of this group, exhibit protective, signaling properties. Thus, the increase in the arginine concentration and ornithine is noted; this indicates the activation of the ornithine cycle and on the increase of amino acids catabolism. The revealed features can ensure the successful germination of T. chinensis grains under the conditions of the physiological drought.

High throughput sequencing of RNA transcriptomes in Ruditapes philippinarum identifies genes involved in osmotic stress response

Ruditapes philippinarum, is an economically important marine bivalve species. The ability to cope with low salinity stress is quite important for the survival of aquatic species under natural conditions. In this study, the transcriptional response of the Manila clam to low salinity stress was characterized using RNA sequencing. The transcriptomes of a low salinity-treatment group (FRp1, FRp2), which survived under low salinity stress, and control group (SRp1, SRp2), which was not subjected to low salinity stress, were sequenced with the Illumina HiSeq platform. A total of 196,578 unigenes were generated. GO and KEGG analyses revealed that signal transduction, immune response, cellular component organization or biogenesis, and energy production processes were the most highly enriched pathways among the genes that were differentially expressed under low salinity stress. All these pathways could be assigned to the following biological functions in the low salinity tolerant Manila clam: signal response to low salinity stress, antioxidant response, intracellular free amino acid transport and metabolism, energy production and conversion, cell signaling pathways, and regulation of ionic content and cell volume. In summary, this is the first study using high-throughput sequencing to identify gene targets that could explain osmotic regulation mechanisms under salinity stress in R. philippinarum.

Cadmium toxicity induced contrasting patterns of concentrations of free sarcosine, specific amino acids and selected microelements in two Noccaea species.

Cadmium (Cd) toxicity affects numerous metabolic processes in plants. In the presence of Cd, plants accumulate specific amino acids which may be beneficial to developing Cd tolerance. Our study aimed to characterize the changes in the metabolism of selected free amino acids that are associated with Cd tolerance, and investigate the levels of selected microelements in order to relate these changes to the adaptation strategies of two metallophytes—Noccaea caerulescens (Redlschlag, Austria) and Noccaea praecox (Mežica, Slovenia). The plants were exposed to Cd contamination (90 mg Cd/kg soil) for 120 days in a pot experiment. Our results showed higher Cd accumulation in N. praecox compared to N. caerulescens. Cadmium contamination reduced the zinc and nickel levels in both species and a mixed effect was determined for copper and manganese content. Differences in free amino acid metabolism were observed between the two metallophytes growing under Cd-free and Cd-loaded conditions. Under Cd-free conditions, aromatic amino acids (phenylalanine, tryptophan and tyrosine) and branched-chain amino acids (leucine, isoleucine and valine) were accumulated more in the leaves of N. praecox than in N. caerulescens. Cd stress increased the content of these amino acids in both species but this increase was significant only in N. caerulescens leaves. Marked differences in the responses of the two species to Cd stress were shown for alanine, phenylalanine, threonine and sarcosine. Cadmium contamination also induced an increase of threonine as alanine and sarcosine decrease, which was larger in N. caerulescens than in N. praecox. All these factors contribute to the higher adaptation of N. praecox to Cd stress.

Molecular characterization, computational analysis and transcript profiling of glutamate dehydrogenase (gdh) gene of Macrobrachium rosenbergii exposed to saline water

Glutamate dehydrogenase (GDH) plays a key role in the metabolism of free amino acids (FAA) in crustaceans and other metazoans. GDH has been widely implicated a central role in response to hyperosmotic condition. However, the response of GDH gene expression to salinity alterations has not yet been determined in Macrobrachium rosenbergii, a euryhaline prawn. To investigate the possible participatory role of GDH gene in altered salinity conditions, we have cloned and characterized GDH gene from M. rosenbergii (MrGDH) and examined the mRNA expression under salinity exposure. The full-length cDNA of MrGDH contained an open reading frame of 1659 nucleotides. The ELFV and NADP binding sites of GDH protein remained highly conserved with 99% identity within crustacean species. The sequence information of cDNA and gDNA together revealed that the MrGDH gene is encoded by an uninterrupted exon. We have generated the 3D structure of MrGDH protein. The significant expression level of GDH in various tissues of shrimp suggests a functional role of this gene during salinity exposure and adaptation. The results generated in this study validated the MrGDH as a potential biomarker for salinity adaptation and could be the first step towards elucidating mechanistic pathways resides during the salinity exposure and osmoregulation in M. rosenbergii.

Integrated Transcriptome and Metabolic Analyses Reveals Novel Insights into Free Amino Acid Metabolism in Huangjinya Tea Cultivar.

The chlorotic tea variety Huangjinya, a natural mutant, contains enhanced levels of free amino acids in its leaves, which improves the drinking quality of its brewed tea. Consequently, this chlorotic mutant has a higher economic value than the non-chlorotic varieties. However, the molecular mechanisms behind the increased levels of free amino acids in this mutant are mostly unknown, as are the possible effects of this mutation on the overall metabolome and biosynthetic pathways in tea leaves. To gain further insight into the effects of chlorosis on the global metabolome and biosynthetic pathways in this mutant, Huangjinya plants were grown under normal and reduced sunlight, resulting in chlorotic and non-chlorotic leaves, respectively; their leaves were analyzed using transcriptomics as well as targeted and untargeted metabolomics. Approximately 5,000 genes (8.5% of the total analyzed) and ca. 300 metabolites (14.5% of the total detected) were significantly differentially regulated, thus indicating the occurrence of marked effects of light on the biosynthetic pathways in this mutant plant. Considering primary metabolism, including that of sugars, amino acids, and organic acids, significant changes were observed in the expression of genes involved in both nitrogen (N) and carbon metabolism. The suite of changes not only generated an increase in amino acids, including glutamic acid, glutamine, and theanine, but it also elevated the levels of free ammonium, citrate, and α-ketoglutarate, and lowered the levels of mono- and di-saccharides and of caffeine as compared with the non-chlorotic leaves. Taken together, our results suggest that the increased levels of amino acids in the chlorotic vs. non-chlorotic leaves are likely due to increased protein catabolism and/or decreased glycolysis and diminished biosynthesis of nitrogen-containing compounds other than amino acids, including chlorophyll, purines, nucleotides, and alkaloids.

Gene Co-Expression Network Analysis Reveals the Correlation Patterns Among Genes in Euryhaline Adaptation of Crassostrea gigas.

The Pacific oyster Crassostrea gigas is a dominant aquaculture species in many intertidal zones throughout the Pacific and Atlantic Oceans and can tolerate a wide range of salinity. Studying the gene expression profiles of oyster gills had found differentially expressed genes (DEGs) involved in salinity tolerance. A systematic study of cellular response to salinity stress may provide insights into the mechanism of acquired salinity tolerance. Here, weighted gene co-expression network analysis (WGCNA) was carried out using RNA-seq data from gill transcriptome in response to different salinity. A total of 25,463 genes were parsed into 22 gene modules, of which 5 gene modules were identified as salinity-related modules. Brown module was the only one significantly correlated with salinity and free amino acids (FAAs) contents, which was associated with cellular metabolism, biosynthesis of amino acids, oxidation reduction, electron transport, nitrogen compound metabolism, and others. The enriched pathways in brown module were mainly about FAAs metabolism. The other four modules were significantly correlated with certain FAAs, and were over-represented in certain salinity. These results indicated that C. gigas triggered different FAAs in different salinity stress. This study represents the first RNA-seq gene network analysis in oysters responding to different salinity stresses. These results provide a systems-level framework to help understand the complexity of cellular process in response to osmotic stress and show the function and regulated genes of different FAAs at the molecular level.

Effects of algal food quality on free amino acid metabolism of Daphnia

Zooplankton frequently face dietary limitation caused by imbalances between their nutrient requirements and the nutrient content of their phytoplankton food. While insufficient nutrition is acknowledged to affect growth and reproduction of zooplankton, the effect of dietary limitation on other aspects of their metabolism remains poorly studied. One understudied biochemical response associated with nutrient stress is changes to the free amino acid (FAA) composition of body tissues resulting from changes in dietary supply. We examined changes in FAA composition in two cladoceran species (Daphnia magna and Daphnia pulex) in response to stress created by ecologically relevant food conditions: low food quantities, high algal carbon: nitrogen (C:N) ratios, and high algal C: phosphorus (P) ratios. We measured 14 FAA after extracting them from lyophilized whole Daphnia grown under these contrasting algal diets. Generally, low food quantity and low algal P increased body FAA content in both daphnid species while many essential FAA decreased due to N-limitation. While significant responses to diet were seen for each daphnid species, the nature of these responses depended on the type of nutritional stress, the FAA examined, and on the species. Non-parametric discriminant analysis exhibited a high predictive ability to classify unknown zooplankton into the correct diet category. The observed changes in body FAA profiles of Daphnia illustrate strong effects that algal food quality can have on the biochemistry of planktonic consumers. Furthermore these results demonstrate body FAA composition of zooplankton may provide an important source of information about zooplankton nutrient limitation especially combined with other nutrient-dependent molecular responses.

The predominance, biodiversity and biotechnological properties of Kluyveromyces marxianus in the production of Pecorino di Farindola cheese

Pecorino di Farindola is a handicraft cheese made by farmers on small scale using raw ewes' milk and pig rennet. In this study, yeast consortia were evaluated during Pecorino di Farindola making and ripening. Molecular identification of 156 isolates was achieved by a combination of PCR-RFLP of the 5.8S ITS rRNA region and sequencing of the D1/D2 domain of the 26S rRNA gene. Kluyveromyces marxianus was the predominant species, while other species (Pichia kudriavzevii, Candida parapsilosis, Candida glaebosa and Candida zeylanoides) were present only during the early weeks of ripening. Moreover, the isolates were differentiated both by RAPD-PCR and a sequence alignment of D1/D2 26S rRNA gene, revealing different K. marxianus profiles and variants, and suggesting the role of local selective pressure as the origin of distinctive K. marxianus populations. The strains were characterized also on the basis of different dairy properties such as growth temperature, lactose, galactose, lactate and citrate assimilation at different NaCl concentrations, as well as lipolytic and caseinolytic activities. Moreover, 39 selected K. marxianus strains were inoculated in pasteurized whey to evaluate their growth kinetics, besides lactose, lactate and free amino acids metabolism. The growth kinetics distinguished different biotypes and different metabolic behavior were determined. The general picture of K. marxianus population from Pecorino di Farindola shows a high biodiversity at genetic and phenotypic levels that potentially offers many opportunities for new and advanced knowledge at species level, providing in the meantime a good basis to study the relationship between genetic variability and functional diversities.

Dose dependent interactions between estradiol‐17β and triiodothyronine with reference to different physiological and biochemical parameters on liver tissue of common Indian Toad (Bufo melanostictus)

To study the dose dependent interaction between two chemically differentand functionally distinct hormones i.e. estradiol‐17β and tri‐iodothyronine(T3), six different animal groups of common Indian toad (Bufo melanostictus)were injected intramuscularly each with single dose of individual treatment[Estradiol‐17β (E‐40 and E‐80); T3 (T‐20 and T‐40), and combination dose (E‐40+T‐20 and E‐80+T‐40)]. The data clearly demonstrated the interaction between these two hormones with respect to different physiochemical parameters in the liver tissue, such as alkaline phosphates activity, oxygen consumption rate, lipid, cholesterol, RNA, DNA, protein, free amino acid, totalsugar and ascorbic acid content with respect to different days after treatment(0, 1, 5 and 10) as well as different doses of hormonal treatments, individuallyand in combination. The comparative study indicated the dose dependenthormone‐hormone interaction between estradiol‐17β and T3 hormone with respect to synergistic effect on oxygen consumption rate, fluctuation in lipid and cholesterol content, antagonistic effect of T3 on activation of the transcriptional apparatus to synthesize RNA, free amino acid metabolism, antagonistic effect on protein metabolism suggesting the protein catabolic or anabolic nature of T3, the permissive effect of estradiol‐17β upon T3 on sugar content in liver tissue of toad.

Determination of aspartase activity in dairy Propionibacterium strains

Propionic acid bacteria (PAB) are important as starter cultures for the dairy industry in the manufacture of Swiss-type cheeses, in which they are involved in the formation of eyes and are responsible for the typical flavour and aroma. These characteristics are mainly due to the classical propionic acid fermentation, but also the conversion of aspartate to fumarate and ammonia by the enzyme aspartase and the subsequent reduction of fumarate to succinate, which occur in dairy Propionibacterium freudenreichii ssp. shermanii and ssp. freudenreichii starter strains. Additionally, the metabolism of free amino acids may be partly responsible for secondary fermentation and the subsequent split defects in cheese matrix. Here a method for aspartase activity was established and a number of dairy propionibacteria belonging to P. freudenreichii ssp. shermanii and freudenreichii were screened for this enzyme activity. A wide range of aspartase activity could be found in PAB isolates originating from cheese. The majority, i.e. 70% of the 100 isolates tested, showed very low levels of aspartate activity.

Development of quantitative metabolomics for Pichia pastoris

Accurate, reliable and reproducible measurement of intracellular metabolite levels has become important for metabolic studies of microbial cell factories. A first critical step for metabolomic studies is the establishment of an adequate quenching and washing protocol, which ensures effective arrest of all metabolic activity and removal of extracellular metabolites, without causing leakage of metabolites from the cells. Five different procedures based on cold methanol quenching and cell separation by filtration were tested for metabolomics of Pichia pastoris regarding methanol content and temperature of the quenching solution as key parameters. Quantitative evaluation of these protocols was carried out through mass balance analysis, based on metabolite measurements in all sample fractions, those are whole broth, quenched and washed cells, culture filtrate and quenching and washing solution. Finally, the optimal method was used to study the time profiles of free amino acid and central carbon metabolism intermediates in glucose-limited chemostat cultures. Acceptable recoveries (>90%) were obtained for all quenching procedures tested. However, quenching at −27°C in 60% v/v methanol performed slightly better in terms of leakage minimization. We could demonstrate that five residence times under glucose limitation are enough to reach stable intracellular metabolite pools. Moreover, when comparing P. pastoris and S. cerevisiae metabolomes, under the same cultivation conditions, similar metabolite fingerprints were found in both yeasts, except for the lower glycolysis, where the levels of these metabolites in P. pastoris suggested an enzymatic capacity limitation in that part of the metabolism.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-011-0308-1) contains supplementary material, which is available to authorized users.

Indispensable and replaceable amino acids in the blood serum and their relationship with macro- and microelements in the newborns of Fe-deficient anemic mothers (in conditions of an industrial city)

Anemia of the newborn is undoubtedly an actual problem in pediatrics. The imbalance between amino acids and biologically active macro- and microelements participating in hematopoiesis and Fe metabolism play an important role in its pathogenesis. Therefore knowledge of amino acid metabolism in the newborn helps to gain insight into pathogenetic mechanisms and to choose the right therapy. A complex inspection of 169 newborns of 18–34-year-old women was done. The control group consisted of 42 newborns of women with normal pregnancy and parturition. The case group consisted of 127 newborns of women with Fe-deficit anemia. Unquestionably one of the factors of Fe-deficit anemia of women aggravates the gestation period, delivery and after-birth period, and has an injurious effect on the newborn. Changes in the trace element and free amino acid metabolism were interconnected in the “mother–newborn” system as evidenced from correlations between the levels of some amino acids, trace element metabolism and erythropoiesis. The pathogenetic importance of the disturbance of the trace element and amino acid metabolism dictates the choice of the correct therapy of Fe-deficit anemia of pregnant women. The experience and results of our investigations support the necessity of preventive estimation of the content of macro- and microelements and amino acids in the blood serum of newborn to correct metabolic disturbances.