Relationship between Cumulative Temperature and Light Intensity and G93 Parameters of Isoprene Emission for the Tropical Tree Ficus septica.

The most widely used isoprene emission algorithm, G93 formula, estimates instantaneous leaf-level isoprene emission using the basal emission factor and light and temperature dependency parameters. The G93 parameters have been suggested to show variation depending on past weather conditions, but no study has closely examined the relationship between past meteorological data and the algorithm parameters. Here, to examine the influence of the past weather on these parameters, we monitored weather conditions, G93 parameters, isoprene synthase transcripts and protein levels, and MEP pathway metabolites in the tropical tree Ficus septica for 12 days and analyzed their relationship with cumulative temperature and light intensity. Plants were illuminated with varying (ascending and descending) light regimes, and our previously developed Ping-Pong optimization method was used to parameterize G93. The cumulative temperature of the past 5 and 7 days positively correlated with CT2 and α, respectively, while the cumulative light intensity of the past 10 days showed the highest negative correlation with α. Concentrations of MEP pathway metabolites and IspS gene expression increased with increasing cumulative temperature. At best, the cumulative temperature of the past 2 days positively correlated with the MEP pathway metabolites and IspS gene expression, while these factors showed a biphasic positive and negative correlation with cumulative light intensity. Optimized G93 captured well the temperature and light dependency of isoprene emission at the beginning of the experiment; however, its performance significantly decreased for the latter stages of the experimental duration, especially for the descending phase. This was successfully improved through separate optimization of the ascending and descending phases, emphasizing the importance of the optimization of formula parameters and model improvement. These results have important implications for the improvement of isoprene emission algorithms, particularly under the predicted increase in future global temperatures.

Perilla Pomace, a By-product of Oil Extraction, Is Rich in Nutrients and Can Favorably Modulate Lipid Metabolism in Sprague-Dawley Rats.

Perilla pomace, a by-product of oil extraction, is rich in nutrients, such as proteins, but it has not been used for purposes other than livestock feeding. The aim of this study was to determine how perilla pomace modulates glucose and lipid metabolism in Sprague-Dawley rats. Dried perilla pomace was added to diet at a concentration of 16%. One experimental group was administered perilla oil equivalent to that in the perilla pomace. After four weeks, the animals were euthanized, and biochemical parameters were measured. Two experiments were conducted using a low-fat (7% by weight) and a high-fat (21% by weight) diet. Regardless of the level of fat in the diets, no differences in food intake were found among the groups. In the low-fat diet-fed rats (Experiment 1), epididymal adipose tissue weight was slightly, but not significantly, lower in perilla pomace-fed rats than in those fed the control diet. Hepatic triglyceride and cholesterol levels were significantly reduced by perilla pomace compared to those in the control group. Serum lipid profiles (triglycerides and cholesterol) were similar to those in the liver, without statistically significant differences. Perilla pomace significantly diminished hepatic fatty acid synthase (FAS) activity. In high-fat diet-fed rats (Experiment 2), pomace did not significantly lower epididymal adipose tissue weight. Hepatic cholesterol levels were lower in rats on the perilla oil than in control rats. The activity of hepatic enzymes involved in fat oxidation was significantly higher in rats fed the perilla pomace than in those fed the control diet. Collectively, these results show that perilla pomace favorably modulates fat metabolism, and the specific effects depend on the fat content in the diet.

Isolation and characterization of mimosine degrading enzyme from Arthrobacter sp. Ryudai-S1.

Leucaena leucocephala growing in the tropics and subtropics serves as potential forage for livestock because its foliage is rich in protein, fiber, and minerals. However, its use for livestock feed has been hindered by toxic nonprotein amino acid mimosine. Therefore, it is necessary to develop a method to reduce or eliminate mimosine from foliage. A previous study found that the fermentation of L. leucocephala foliage reduced the mimosine content and prompted the authors to isolate potent mimosine degrading microorganisms and characterize the mimosinase for the complete elimination of mimosine in the L. leucocephala foliage. The soil screening of the L. leucocephala tree surroundings led to the isolation of Arthrobacter sp. Ryudai-S1, which can degrade and assimilate mimosine as a nitrogen and carbon source. Mimosinase in this strain was found to be thermostable and showed strong activity. Docking model's inspection and the interaction energy calculation between mimosine-pyridoxal-5'-phosphate (PLP) complex and the active site of this enzyme identified 11 important amino acid residues that stabilized the binding. Of these amino acid residues, mutation experiment suggested that Tyr-263' and Phe-34 stabilizes the substrate binding and play a critical role in guiding the substrate to proper positions to accomplish high catalytic efficacy and selectivity. These observations suggest that Arthrobacter sp. Ryudai-S1 could be potentially useful for the development of L. leucocephala feed with reduced mimosine content.

Perilla pomace obtained from four different varieties have different levels and types of polyphenols and anti-allergic activity.

Perilla frutescens (L.) Britton var. frutescens (egoma in Japan) is a traditional oilseed that has several varieties with different photoperiod responses. Although egoma pomace, industrial waste produced during oil extraction, is a rich source of macro- and micro-nutrients such as protein, fiber, minerals, and polyphenols, it has not yet been used for purposes other than livestock feeding. To find out a better use of perilla pomace and its function, we selected four varieties of egoma originating from different regions with different photoperiod responses: two varieties were from Japan, which are broadly cultivated for oilseed and are highly sensitive to light and temperature. The other two varieties from Nepal, which are tolerant to low light and low temperature. Rosmarinic acid-3-O-glucoside, rosmarinic acid, and apigenin-7-O-glucoside were detected as the main polyphenolic constituents in every variety, while apigenin and luteolin were present only in perilla pomace from Japan. In IgE-sensitized RBL-2H3 cells, polyphenols derived from two varieties of Japan suppressed degranulation of mast cells, but those derived from the two varieties of Nepal did not, indicating that apigenin and luteolin may be in part responsible for the anti-allergic response. In addition, it was found that proteins involved in the degranulation signaling pathway, such as PLCγ2, Syk, and Akt, were less phosphorylated in cells treated with the egoma pomace extracts of Japanese origin. Taken together, pomace from egoma varieties derived from different regions may differently modulate allergic response in part due to the difference in polyphenol composition and may be applied to develop nutraceuticals and functional foods fortified with anti-allergic properties.

Lotus root extract inhibits skin damage through suppression of collagenase production in vitro.

Lotus root is a traditional food ingredient used primarily in Asia and is rich in polyphenols. To determine its potential use in antiphotoaging, polyphenols were extracted from lotus root with 50% ethanol, and the activity of matrix metalloproteinase (MMP) was measured in dermal cells treated with ultraviolet A (UVA). UVA exposure increased the gene expression of IL-1α, the mRNA levels of MMP-1, and hence, the levels of MMP-1 protein in HaCaT cells, whereas cells treated with lotus polyphenol (LP) normalized these values to the control. In the presence of LP at concentrations of 1 and 10 µg/mL, both the secretion of IL-1α and protein levels of MMP-1 in human keratinocyte cells significantly reduced. Similarly, in the LabCyte EPI-MODEL24, irradiation with UVA caused an increase in mRNA expression of IL-1α and MMP-1, which was prevented by adding LP to the cells. Our results with three different skin cells accordingly showed that LP may help maintain skin health through decreased levels of MMP-1 activity via its anti-inflammatory properties.

Molecular cloning of putative chloroplastic cysteine synthase in Leucaena leucocephala.

Cysteine biosynthesis is directed by the successive commitments of serine acetyltransferase, and O-acetylserine (thiol) lyase (OASTL) compounds, which subsequently frame the decameric cysteine synthase complex. The isoforms of OASTL are found in three compartments of the cell: the cytosol, plastid, and mitochondria. In this investigation, we first isolated putative chloroplastic OASTL (Ch-OASTL) from Leucaena leucocephala, and the Ch-OASTL was then expressed in BL21-competent Escherichia coli. The putative Ch-OASTL cDNA clone had 1,543 base pairs with 391 amino acids in its open reading frame and a molecular weight of 41.54 kDa. The purified protein product exhibited cysteine synthesis ability, but not mimosine synthesis activity. However, they both make the common α-aminoacrylate intermediate in their first half reaction scheme with the conventional substrate O-acetyl serine (OAS). Hence, we considered putative Ch-OASTL a cysteine-specific enzyme. Kinetic studies demonstrated that the optimum pH for cysteine synthesis was 7.0, and the optimum temperature was 40 °C. In the cysteine synthesis assay, the Km and kcat values were 838 ± 26 µM and 72.83 s-1 for OAS, respectively, and 60 ± 2 µM and 2.43 s-1 for Na2S, respectively. We can infer that putative Ch-OASTL regulatory role is considered a sensor for sulfur constraint conditions, and it acts as a forerunner of various metabolic compound molecules.

Molecular characterization of mimosinase and cystathionine ß-lyase in the Mimosoideae subfamily member Mimosa pudica.

Mimosinase degrades the non-protein amino acid mimosine and is thought to have evolved from cystathionine ß-lyase (CBL) via gene duplication. However, no study has, to date, compared the molecular characteristics of mimosinase and CBL. We therefore cloned mimosinase and CBL from the Mimosoideae subfamily member Mimosa pudica (Mp) and explored the molecular relationship between mimosinase and CBL for the first time. The recombinant Mp mimosinase degraded both mimosine and cystathionine with a much higher turnover number (kcat) for mimosine compared with cystathionine, and Mp CBL utilized only cystathionine as a substrate. The critical residues implicated in the substrate binding of Arabidopsis thaliana CBL (Tyr-127, Arg-129, Tyr-181, and Arg-440) were highly conserved in both Mp mimosinase and CBL. However, homology modeling and molecular simulation of these enzymes predicted variations in the residues that interact with substrates. A mutation experiment on Mp mimosinase revealed that the disruption of a disulfide bond in the vicinity of the pyridoxal-5'-phosphate domain increased the enzyme's preference toward cystathionine. Treatment of Mp mimosinase with a disulfide-cleavage agent also decreased mimosinase activity. Furthermore, mutation near the conserved binding residue altered the substrate preference between mimosine and cystathionine. Molecular dynamics simulations of Mp mimosinase suggested a closer coordination of the residues that interact with mimosine at the active site compared with cystathionine, indicating a more compact pocket size for mimosine degradation. This study thus may provide new insights into the molecular diversification of CBL, a C-S lyase, into the C-N lyase mimosinase in the Mimosoideae subfamily.

Cytosolic Cysteine Synthase Switch Cysteine and Mimosine Production in Leucaena leucocephala.

In higher plants, multiple copies of the cysteine synthase gene are present for cysteine biosynthesis. Some of these genes also have the potential to produce various kinds of ß-substitute alanine. In the present study, we cloned a 1275-bp cDNA for cytosolic O-acetylserine(thiol)lyase (cysteine synthase) (Cy-OASTL) from Leucaena leucocephala. The purified protein product showed a dual function of cysteine and mimosine synthesis. Kinetics studies showed pH optima of 7.5 and 8.0, while temperature optima of 40 and 35 °C, respectively, for cysteine and mimosine synthesis. The kinetic parameters such as apparent Km, kcat were determined for both cysteine and mimosine synthesis with substrates O-acetylserine (OAS) and Na2S or 3-hydroxy-4-pyridone (3H4P). From the in vitro results with the common substrate OAS, the apparent kcat for Cys production is over sixfold higher than mimosine synthesis and the apparent Km is 3.7 times lower, suggesting Cys synthesis is the favored pathway.

Molecular characterization of cytosolic cysteine synthase in Mimosa pudica.

In the cysteine and mimosine biosynthesis process, O-acetyl-L-serine (OAS) is the common substrate. In the presence of O-acetylserine (thiol) lyase (OASTL, cysteine synthase) the reaction of OAS with sulfide produces cysteine, while with 3-hydroxy-4-pyridone (3H4P) produces mimosine. The enzyme OASTL can either catalyze Cys synthesis or both Cys and mimosine. A cDNA for cytosolic OASTL was cloned from M. pudica for the first time containing 1,410 bp nucleotides. The purified protein product from overexpressed bacterial cells produced Cys only, but not mimosine, indicating it is Cys specific. Kinetic studies revealed that pH and temperature optima for Cys production were 6.5 and 50 °C, respectively. The measured Km, Kcat, and Kcat Km-1 values were 159 ± 21 µM, 33.56 s-1, and 211.07 mM-1s-1 for OAS and 252 ± 25 µM, 32.99 s-1, and 130.91 mM-1s-1 for Na2S according to the in vitro Cys assay. The Cy-OASTL of Mimosa pudica is specific to Cys production, although it contains sensory roles in sulfur assimilation and the reduction network in the intracellular environment of M. pudica.