[Enantiomeric separation of D, L-isoleucine in citric acid-zinc (â ¡) medium by capillary electrophoresis].

The enantiomeric separation of unmodified D,L-isoleucine was achieved in citric acid-zinc(Ⅱ) medium by capillary electrophoresis (CE) with contactless conductivity detector (C4D). In the conventional chiral separation methods of amino acid, a chiral complex used as the chiral selector was added into the eluent in order to yield a chiral environment. However, in this study a non-chiral solution, i. e. 2.8 mmol/L NaOH+0.8 mmol/L citric acid+2.0 mmol/L zinc acetate was used as the running buffer, and the citric acid-zinc(Ⅱ) acted the role of a chiral selector. Under the optimum experimental conditions:uncoated fused-silica capillary (45 cm×50 µm, Leff=40 cm), separation voltage of +13 kV, electrokinetic injection of 11 kV×8 s, the enantiomers of D,L-isoleucine were baseline separated within 8 min with the resolutions (Rs) of 2.0. The calibration curve of each enantiomer showed good linearity in the range from 1.0 mg/L to 20 mg/L, with the limits of detection of 0.40 mg/L. The intra-and inter-day precisions were examined. The RSDs of peak area and migration time were found to be below 5.0% and 2.5% (n=6), respectively, indicating good repeatability (intra-day) and reproducibility (inter-day) of the method. Interference experiment was also tested. As a result, other common amino acids did not interfere with the detection. The proposed method provided a potential new way to further investigate the enantioseparation of unmodified or native amino acids.

Trigonella foenum-graecum Seed Extract, 4-Hydroxyisoleucine, and Metformin Stimulate Proximal Insulin Signaling and Increase Expression of Glycogenic Enzymes and GLUT2 in HepG2 Cells.

BACKGROUND: Fenugreek (Trigonella foenum-graecum) is globally recognized for its medicinal properties and hypoglycemic effects. The seed extract as well as its active compound, 4-hydroxyisoleucine (4-OH-Ile), have been shown to reduce hyperglycemic insulin resistance. The mechanism by which this occurs has not been investigated in human liver cells (HepG2) in comparison to the antihyperglycemic drug, metformin. METHODS: We investigated the effects of an aqueous fenugreek seed extract (FSE), 4-OH-Ile, and metformin in HepG2 cells relative to insulin as a positive control. Cells were treated with FSE and 4-OH-Ile at 100 ng/mL under normoglycemic (5 mM glucose) and hyperglycemic (30 mM glucose) conditions for 72 hr. Tyrosine phosphorylation of insulin receptor-ß (IR-ß), protein kinase B (Akt), glycogen synthase kinase-3α/ß (GSK-3α/ß), and glucose transporter 2 (GLUT2) was determined by western blotting. Gene expression of sterol regulatory element-binding protein 1c (SREBP1c), GLUT2, glycogen synthase (GS), and glucokinase (GK) was evaluated by quantitative polymerase chain reaction, and supernatant glucose levels were measured using the Piccolo biochemistry analyzer. RESULTS: Under normo- and hyperglycemic conditions, FSE, 4-OH-Ile, insulin (100 ng/mL), and metformin (2 mM) caused a significant increase in phosphorylation of IR-ß, Akt, GSK-3α/ß, and GLUT2. Glucose uptake, however, was most significantly increased in FSE-treated cells during both conditions. FSE induced the most significant changes in downstream insulin signaling, GS, GK, SREBP1c, and GLUT2 expression compared to 4-OH-Ile, metformin, and insulin. In addition, FSE significantly increased glucose uptake. CONCLUSIONS: Collectively, these findings provide a mechanism by which FSE exerts antihyperglycemic effects similar to metformin and insulin that occurs via enhanced insulin signaling, gene expression, and increasing glucose uptake.

4-Hydroxyisoleucine improves insulin resistance in HepG2 cells by decreasing TNF-α and regulating the expression of insulin signal transduction proteins.

Previous studies have indicated that 4­hydroxyisoleucine (4­HIL) improves insulin resistance, however, the underlying mechanisms remain to be elucidated. In the present study, the molecular mechanisms underlying how 4­HIL improves insulin resistance in hepatocytes were examined. HepG2 cells were co­cultured with insulin and a high glucose concentration to obtain insulin­resistant (IR) HepG2 cells. Insulin sensitivity was determined by measuring the glucose uptake rate. The IR HepG2 cells were treated with different concentrations of 4­HIL to determine its effect on IR Hep2 cells. The levels of tumor necrosis factor­α (TNF­α) were measured by an enzyme­linked immunosorbent assay and protein levels of TNF­α converting enzyme (TACE)/tissue inhibitor of metalloproteinase 3 (TIMP3), insulin receptor substrate (IRS)­1, IRS­2, phosphorylated (p)­IRS­1 (Ser307) and glucose transporter type 4 (GLUT4) were measured by western blot analysis. The results of the present study demonstrated that insulin­induced glucose uptake was reduced in IR HepG2 cells; however, this reduction was reversed by 4­HIL in a dose­dependent manner. 4­HIL achieved this effect by downregulating the expression of TNF­α and TACE, and upregulating the expression of TIMP3 in IR HepG2 cells. In addition, 4­HIL increased the expression of the insulin transduction regulators IRS­1 and GLUT4, and decreased the expression of p­IRS­1 (Ser307), without affecting the expression of IRS­2. The present study suggests that 4­HIL improved insulin resistance in HepG2 cells by the following mechanisms: 4­HIL reduced TNF­α levels by affecting the protein expression of the TACE/TIMP3 system and 4­HIL stimulated the expression of IRS­1 and GLUT4, but inhibited the expression of p­IRS­1 (Ser307).

Experimental evaluation of the effect of a modified port-location mode on the performance of a three-zone simulated moving-bed process for the separation of valine and isoleucine.

The removal of isoleucine from valine has been a key issue in the stage of valine crystallization, which is the final step in the valine production process in industry. To address this issue, a three-zone simulated moving-bed (SMB) process for the separation of valine and isoleucine has been developed previously. However, the previous process, which was based on a classical port-location mode, had some limitations in throughput and valine product concentration. In this study, a three-zone SMB process based on a modified port-location mode was applied to the separation of valine and isoleucine for the purpose of making a marked improvement in throughput and valine product concentration. Computer simulations and a lab-scale process experiment showed that the modified three-zone SMB for valine separation led to >65% higher throughput and >160% higher valine concentration compared to the previous three-zone SMB for the same separation.

A steroidal molecule present in the egg wax of the tick Rhipicephalus (Boophilus) microplus inhibits bacterial biofilms.

The cattle tick Rhipicephalus (Boophilus) microplus lays eggs in the soil near the roots of grass, or in similar highly moist environments that are prone to biofilm formation. Tick eggs have a protective wax coating that may be a source of nutrients for microorganisms. However, as the eggs remain viable and show no visible signs of microbial colonization, we hypothesized that the coating might have anti-biofilm properties. We show here that the coating inhibits biofilm formation by both Gram-negative and Gram-positive bacteria, though by different mechanisms. We have identified the anti-biofilm molecule as N-(3-sulfooxy-25-cholest-5-en-26-oyl)-L-isoleucine (boophiline), and we show that it inhibits the expression of fliC (flagellin) and cdrA (biofilm scaffold), whose products are necessary for biofilm formation in Pseudomonas aeruginosa. Boophiline is a novel biofilm inhibitor being also effective against Staphylococcus epidermidis biofilm. In our study we show evidences of the boophiline mode of action in the protection of arthropod eggs against biofilm colonization.

Differences in metabolic profiles of planktonic and biofilm cells in Staphylococcus aureus - (1)H Nuclear Magnetic Resonance search for candidate biomarkers.

Staphylococcus aureus is responsible for many types of infections related to biofilm presence. As the early diagnostics remains the best option for prevention of biofilm infections, the aim of the work presented was to search for differences in metabolite patterns of S. aureus ATCC6538 biofilm vs. free-swimming S. aureus planktonic forms. For this purpose, Nuclear Magnetic Resonance (NMR) spectroscopy was applied. Data obtained were supported by means of Scanning Electron Microscopy, quantitative cultures and X-ray computed microtomography. Metabolic trends accompanying S. aureus biofilm formation were found using Principal Component Analysis (PCA). Levels of isoleucine, alanine and 2,3-butanediol were significantly higher in biofilm than in planktonic forms, whereas level of osmoprotectant glycine-betaine was significantly higher in planktonic forms of S. aureus. Results obtained may find future application in clinical diagnostics of S. aureus biofilm-related infections.

Structures and properties of a diastereoisomeric molecular compound of (2S,3S)- and (2R,3S)-N-acetyl-2-amino-3-methylpentanoic acids.

An X-ray crystal structural analysis revealed that (2S,3S)-N-acetyl-2-amino-3-methylpentanoic acid (N-acetyl-L-isoleucine; Ac-L-Ile) and (2R,3S)-N-acetyl-2-amino-3-methylpentanoic acid (N-acetyl-D-alloisoleucine; Ac-D-aIle) formed a molecular compound containing one Ac-L-Ile molecule and one Ac-D-aIle molecule as an unsymmetrical unit. This molecular compound is packed with strong hydrogen bonds forming homogeneous chains consisting of Ac-L-Ile molecules or Ac-D-aIle molecules and weak hydrogen bonds connecting these homogeneous chains in a fashion similar to that observed for Ac-L-Ile and Ac-D-aIle. Recrystallization of an approximately 1:1 mixture of Ac-L-Ile and Ac-D-aIle from water gave an equimolar molecular compound due to its lower solubility than that of Ac-D-aIle or especially Ac-L-Ile. The results suggest that the equimolar mixture of Ac-L-Ile and Ac-D-aIle could be obtained from an Ac-L-Ile-excess mixture by recystallization from water.

Synthesis and characterization of synthetic analogs of cinnacidin, a novel phytotoxin from Nectria sp.

BACKGROUND: The novel natural product cinnacidin was isolated from a fungal fermentation extract of Nectria sp. DA060097. The compound was found to contain a cyclopentalenone ring system with an isoleucine subunit linked through an amide bond. Initial biological characterization of cinnacidin suggested promising herbicidal activity. RESULTS: Two synthetic analogs, (2S,3S)-2-[(3RS,3aSR,6aRS)-3-methoxy-4-oxo-3,3a,4,5,6,6a-hexahydropentalen-1-ylcarbamoyl]-3-methylvaleric acid and benzyl (2S,3S)-2-[(3RS,3aSR,6aRS)-3-methoxy-4-oxo-3,3a,4, 5,6,6a-hexahydropentalen-1-ylcarbamoyl]-3-methylvalerate, were prepared for further characterization, and their herbicidal activities were compared with that of cinnacidin. CONCLUSIONS: The synthetic compounds were highly phytotoxic on a range of weeds. Based on the symptoms in treated plants, the mode of action of these compounds is suggested to be similar to that of coronatine and jasmonic acid. Coronatine was more active against warm-season grasses, while the cinnacidin benzyl ester analog was more effective on cool-season grasses. In a seedling growth bioassay conducted on bentgrass, the cinnacidin analog was equivalent in activity to coronatine.

4-hydroxyisoleucine an unusual amino acid as antidyslipidemic and antihyperglycemic agent.

Trigonella foenum-graecum, commonly known as fenugreek, is an annual herbaceous plant. From the seeds of T. foenum-graecum an unusual amino acid, 4-hydroxyisoleucine 5, has been isolated, which significantly decreased the plasma triglyceride levels by 33% (P<0.002), total cholesterol (TC) by 22% (P<0.02), and free fatty acids by 14%, accompanied by an increase in HDL-C/TC ratio by 39% in the dyslipidemic hamster model.

Full-capillary sample stacking/sweeping-MEKC for the separation of naphthalene-2,3-dicarboxaldehyde-derivatized tryptophan and isoleucine.

In an attempt to improve the sensitivity of detection in capillary electrophoresis (CE), a novel online sample-concentration method, full-capillary sample stacking (FCSS)/sweeping-micellar electrokinetic chromatography (sweeping-MEKC) mode, is proposed. Naphthalene-2,3-dicarboxaldehyde (NDA)-derivatized tryptophan and isoleucine were selected as model compounds. In the initial step, the weakly acidic compounds, dissolved in a low-conductivity buffer (35.1 microS/cm; apparent ph (pH*) in a mixed solution of acetonitrile/methanol/water, 4.6), fill the entire capillary, two vials of a high-conductivity buffer (2.06 mS/cm; pH* 2.0) are placed on each end, and a negative polarity is then applied. Under these conditions, the direction of the electroosmotic flow (EOF) is toward the inlet. Meanwhile, the anionic analytes move in the reverse direction and are neutralized and stacked at the boundary of a dynamic pH-junction (between the sample matrix and the nonmicellar background solution (BGS)). When the sample concentration is completed, the BGS is quickly changed to solutions containing SDS-BGS for the subsequent separation. Since the mobility of SDS-analytes is then greater than the EOF, the following steps occur by the sweeping (for focusing) and MEKC (for separation) mode. Using these steps, a full-capillary sample injection/separation can be achieved.

[Study of enantiomeric separation of DL-amino acids on L-isoleucine-type chiral-ligand-exchange stationary phase].

Some underivatized DL-amino acid enantiomers were separated on L-isoleucine-type chiral-ligand-exchange stationary phase (EILE). The effects of contents of copper ion and methanol in mobile phase, temperature of column and flow rate of eluent on the efficiency and resolution of DL-amino acids were investigated in detail. The results showed that Cu(II) ion played an important role in the separation. When there was no Cu(II) ion in mobile phase, the DL-amino acids could not be enantio-separated, while the content of Cu(II) increased from 0.3 mmol/L to 1.2 mmol/L, the efficiency of resolution and retention time decreased. The increases of the content of methanol and column temperature are in favor of the resolution for most of DL-amino acids. The flow rate also affected the resolution results. Good results could be obtained in reducing flow rate but it prolongs the retention time. The mechanisms of different chiral separation results on the new stationary phase under different conditions were also investigated.

A more complex isoleucine aptamer with a cognate triplet.

The simplest RNA that can meet a column affinity selection for isoleucine was previously defined using selection amplification with decreasing numbers of randomized nucleotides. This simplest UAUU motif was a small asymmetric internal loop. Conserved positions of the loop include isoleucine codon and anticodon triplets (Lozupone C., Changayil, S., Majerfeld, I., and Yarus, M. (2003) RNA (N. Y.) 9, 1315-1322). Using new primer sequences, we now select a somewhat more complex isoleucine binding RNA, requiring 4.7 more bits of information to describe. The newly selected structure is a terminal or hairpin loop of 20 nucleotides, 15 being invariant. An information profile shows that the new binding site contains five short functional loop regions joined by less significant single nucleotide positions. Among the important nucleotides is a conserved isoleucine anticodon, supporting the escaped triplet theory, which posits a stereochemical genetic code originating in RNA amino acid binding sites.

Lacticin 3147 favours isoleucine transamination by Lactococcus lactis IFPL359 in a cheese-model system.

The bacteriocin, lacticin 3147, increased isoleucine transamination by Lactococcus lactis IFPL359 in a cheese model system. The formation of alpha-keto-beta-methyl-n-valeric acid and 2-hydroxy-3-methyl-valeric acid increased by three times in cheese slurries at 12 degrees C and cheese aroma intensity increased as well, which corresponded with a higher 2-methylbutanal formation.

WF14865A and B, new cathepsins B and L inhibitors produced by Aphanoascus fulvescens. I. Taxonomy, production, purification and biological properties.

WF14865A and B, novel cathepsins B and L inhibitors, were produced and isolated separately from the culture mycelium of a fungal strain Aphanoascus fulvescens No. 14865. Spectroscopic analysis revealed that both WF14865A and B were composed of trans-epoxysuccinyl moieties, 1-H-imidazole-2-ylamine, and isoleucine or leucine. These compounds inhibited human cathepsins B and L with IC50 values in the range of 8.4 approximately 72nM in vitro. Though their in vitro properties were typical as trans-epoxysuccinyl type inhibitors, they exerted strong bone resorption inhibitory effects in low-calcium-diet-fed mouse model at 3.2 approximately 10 mg/kg.

4-Hydroxyisoleucine: a novel amino acid potentiator of insulin secretion.

We report the characterization of a new insulinotropic compound, 4-hydroxyisoleucine. This amino acid has been extracted and purified from fenugreek seeds, which are known in traditional medicine for their antidiabetic properties. 4-Hydroxyisoleucine increases glucose-induced insulin release, in the concentration range of 100 micromol/l to 1 mmol/l, through a direct effect on isolated islets of Langerhans from both rats and humans. The stimulating effect of 4-hydroxyisoleucine was strictly glucose dependent; indeed, ineffective at low (3 mmol/l) or basal (5 mmol/l) glucose concentrations, the amino acid potentiated the insulin secretion induced by supranormal (6.6-16.7 mmol/l) concentrations of glucose. In addition, in the isolated perfused rat pancreas, we could show 1) that the pattern of insulin secretion induced by 4-hydroxyisoleucine was biphasic, 2) that this effect occurred in the absence of any change in pancreatic alpha- and delta-cell activity, and 3) that the more glucose concentration was increased, the more insulin response was amplified. Moreover, 4-hydroxyisoleucine did not interact with other agonists of insulin secretion (leucine, arginine, tolbutamide, glyceraldehyde). Therefore, we conclude that 4-hydroxyisoleucine insulinotropic activity might, at least in part, account for fenugreek seeds' antidiabetic properties. This secretagogue may be considered as a novel drug with potential interest for the treatment of NIDDM.

Production of isoleucine by overexpression of ilvA in a Corynebacterium lactofermentum threonine producer.

Overproduction of isoleucine, an essential amino acid, was achieved by amplification of the gene encoding threonine dehydratase, the first enzyme in the threonine to isoleucine pathway, in a Corynebacterium lactofermentum threonine producer. Threonine overproduction was previously achieved with C. lactofermentum ATCC 21799, a lysine-hyperproducing strain, by introduction of plasmid pGC42 containing the Corynebacterium homdr and thrB genes (encoding homoserine dehydrogenase and homoserine kinase respectively) under separate promoters. The pGC42 derivative, pGC77, also contains ilvA, which encodes threonine dehydratase. In a shake-flask fermentation, strain 21799(pGC77) produced 15 g/l isoleucine, along with small amounts of lysine and glycine. A molar carbon balance indicates that most of the carbon previously converted to threonine, lysine, glycine and isolecine was incorporated into isoleucine by the new strain. Thus, in our system, simple overexpression of wild-type ilvA sufficed to overcome the effects of feedback inhibition of threonine dehydratase by the end-product, isoleucine.

Incorporation of 15N from L-leucine into isoleucine by rat brain cerebral cortex slices.

The fate of leucine nitrogen in the central nervous system was investigated by incubating rat cerebral cortex slices in the presence of 0.5 mM each of L-[15N]-leucine, L-isoleucine, and L-valine. Analysis of the slices and incubation media for free amino acids by gas chromatography-mass spectrometry revealed that the 15N from leucine is incorporated into isoleucine only. No 15N was detected in valine or any other amino acid. These results suggest that leucine, valine, and their corresponding aminotransferases may be compartmentalized in brain cerebral cortex.

Reaction products formed after strong acid treatment of alpha-amino-delta-hydroxyvaleric acid.

High-voltage paper electrophoresis and analytical and preparative amino acid ion-exchange chromatography were used to investigate the products resulting from strong acid treatment and strong acid treatment and subsequent alkalinization of alpha-amino-delta-hydroxyvaleric acid. After strong acid treatment, alpha-amino-delta-hydroxyvaleric acid and proline, as well as several unidentified components, presumably lactones, were recovered. No definitive identification of the putative unstable lactones was accomplished either by physical-chemical means or by ion-exchange chromatography or high-voltage paper electrophoresis. Nevertheless, after acid treatment and alkalinization, only alpha-amino-delta-hydroxyvaleric acid and proline were recovered, in approximately equal amounts. The formation of such large amounts of proline from the acid treatment and alkalinization of alpha-amino-delta-hydroxyvaleric acid is therefore an important consideration in quantitating the number of gamma-glutamyl phosphate residues present in various proteins.

[Separation of a mixture of L-isoleucine and L-leucine]. / Razdelenie smesi L-izoleitsina i L-leitsina.

The conditions of L-leucine and L-isoleucine esterification were investigated. It was found that chromatographically homogeneous L-isoleucine and L-leucine could be isolated from their mixture during its incomplete esterification. As a result, L-leucine transformed into L-leucine and ester chlorohydrate L-isoleucine into L-isoleucine chlorohydrate. By means of further neutralization of the reaction mixture L-isoleucine with a 80-85% yield was obtained, and with the aid of L-leucine ester hydrolysis L-leucine with a 80-85% yield was produced. The content of the major amino acid following its crystallization from the water-ethanol mixture was 99%.

Alloisoleucine formation in maple syrup urine disease: isotopic evidence for the mechanism.

Of the four possible stereoisomers of isoleucine, only L-alloisoleucine and L-isoleucine were found by capillary gas chromatography in the plasma of two maple syrup urine disease (MSUD) patients, one with classical and one with variant MSUD. The relative plasma concentration ratios of L-alloisoleucine/L-isoleucine were 0.795 +/- 0.025 (+/- 95% confidence limits) and 0.637 +/- 0.016 in the classical- and variant-MSUD patients, respectively. The patients were also studied in the postabsorptive state with a 6-hr continuous infusion of L-[15N]leucine. In each patient plasma leucine 15N enrichment approximated plateau after 150 min, and there was rapid appearance of [15N]isoleucine and [15N]alloisoleucine which were identical at plateau, although in variant-MSUD patient [15N]alloisoleucine enrichment did not equal that of [15N]isoleucine until 240 min of infusion. These results offer direct in vivo evidence for the rapid equilibrium of plasma isoleucine and alloisoleucine through keto-enol tautomerization of alpha-keto-beta-methylvalerate.