Investigations into the biochemical processes and regulatory mechanisms of nitrogen (N) utilization can aid in understanding how N is used efficiently in plants. This report describes a deficiency in N utilization in an Arabidopsis (Arabidopsis thaliana) transfer DNA insertion mutant of the mitochondrial folylpolyglutamate synthetase gene DFC, which catalyzes the conjugation of glutamate residues to the tetrahydrofolate during folate synthesis. The mutant seedlings displayed several metabolic changes that are typical of plant responses to low-N stress, including increased levels of starch and anthocyanin synthesis as well as decreased levels of soluble protein and free amino acid, as compared with those in wild-type seedlings when external N was sufficient. More striking changes were observed when dfc seedlings were grown under N-limited conditions, including shorter primary roots, fewer lateral roots, higher levels of glycine and carbon-N ratios, and lower N content than those in wild-type seedlings. Gene expression studies in mutant seedlings revealed altered transcript levels of several genes involved in folate biosynthesis and N metabolism. The biochemical and metabolic changes also suggested that N assimilation is drastically perturbed due to a loss of DFC function. The observation that elevated CO 2 partly rescued the dfc phenotypes suggests that the alterations in N metabolism in dfc may be mainly due to a defect in photorespiration. These results indicate that DFC is required for N utilization in Arabidopsis and provide new insight into a potential interaction between folate and N metabolism. Nitrogen (N) is an essential macronutrient for plants and a major limiting factor for crop growth (Diaz et al., 2006). Investigations into the biochemical processes and regulatory mechanisms of N utilization can aid in understanding how N is used efficiently in plants. Low inorganic N results in numerous perturbations in plant metabolism, such as decreases in nitrate (NO 3 2

Amentoflavone (AMF) is a biflavone found in many herbal dietary supplements. To investigate the pharmacokinetic profile of AMF in rats, a sensitive, simple, and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and used to monitor AMF and its conjugated metabolites in plasma. AMF was administered to rats by oral gavage (po), or by intravenous (iv) or intraperitoneal (ip) injection. Plasma samples (with apiolin as an internal standard) were liquid/liquid extracted after hydrolysis with β-glucuronidase/sulfatase in vitro. Following chromatographic separation on a C18 column with a methanol:water:formic acid (70:30:0.1, v/v/v) mobile phase, AMF and internal standard were determined by electrospray ionization in negative ion mode and their precursor-product ion pairs (m/z 537.1 → 374.9 and m/z 269.2 → 224.9, respectively) were used for measurement. This bioanalytical method was fully validated and showed good linearity (r(2) > 0.99), wide dynamic range (0.93-930 nmol/L), and favorable accuracy and precision. After iv or ip AMF (10 mg/kg) injection, 73.2% ± 6.29% and 70.2% ± 5.18% of the total AMF detected in plasma was present as conjugated metabolites. Furthermore, AMF and AMF conjugates showed similar time courses with no significant differences in the time to reach the maximum plasma concentration (tmax) and terminal half-life (t1/2) (p > 0.05). Following po AMF administration (300 mg/kg), 90.7% ± 8.3% of the total AMF was circulating as conjugated metabolites. When compared with iv administration (with dose correction), the bioavailability of po AMF was very low (0.04% ± 0.01% for free AMF; 0.16% ± 0.04% for conjugated AMF). Collectively, these data provided a preliminary pharmacokinetic profile for AMF that should inform further evaluations of its biological efficacy and preclinical development.

The small molecule fusion inhibitors N-(4-carboxy-3-hydroxyphenyl)-2,5-dimethylpyrrole (NB-2) and N-(3-carboxy-4-hydroxyphenyl)-2,5-dimethylpyrrole (A12) target a hydrophobic pocket of HIV-1 gp41 and have moderate anti-HIV-1 activity. In this paper, we report the design, synthesis, and structure-activity relationship of a group of hybrid molecules in which the pocket-binding domain segment of the C34 peptide was replaced with NB-2 and A12 derivatives. In addition, the synergistic effect between the small molecule and peptide moieties was analyzed, and lead compounds with a novel scaffold were discovered. We found that either the nonpeptide or peptide part alone showed weak activity against HIV-1-mediated cell-cell fusion, but the conjugates properly generated a strong synergistic effect. Among them, conjugates Aoc-βAla-P26 and Noc-βAla-P26 exhibited a low nanomolar IC50 in the cell-cell fusion assay and effectively inhibited T20-sensitive and -resistant HIV-1 strains. Furthermore, the new molecules exhibited better stability against proteinase K digestion than T20 and C34.

The aim of this study was to investigate the metabolic stability and cleavage sites of exendin-4 in rat tissue homogenates, as well as to identify the types of proteases involved in exendin-4 degradation. The stability of exendin-4 in kidney and liver homogenates from rats was evaluated using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) with gradient elution. Furthermore, we used a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and LC-ESI-MS/MS to identify the structures of the major degradation products of exendin-4, and peptidase inhibitors were used to characterize exendin-4 degradation in rat liver and kidney homogenates and to identify the proteases involved in exendin-4 metabolism. Exendin-4 had a half-life of 7.8 and 100.9 min in the kidney and liver homogenate, respectively. The enzymes most likely to be involved in the degradation of exendin-4 were aminopeptidases, serineproteases, and metalloproteases. Exendin-4(15-39) and exendin-4(16-39) were the predominant direct exendin-4 metabolites in the kidney, and the main product of exendin-4 metabolism in the liver was exendin-4(12-39). Our results indicated that the metabolism of exendin-4 involved an initial endoproteolytic cleavage and subsequent exoproteolytic digestion. The degradation of exendin-4 in the kidney and liver homogenates followed distinct patterns, and the primary cleavage sites of exendin-4 degradation in rat kidney homogenates were located after AA-14, and -15, whereas those in rat liver homogenates were located after AA-11.

This study was designed to develop a sensitive, simple and rapid method for the quantitation of morroniside in rat urine using high-performance liquid chromatography-tandem mass spectrometry (LC-MS-MS) and to investigate the excretion of morroniside in rat urine. The mobile phase consisted of water-acetonitrile (gradient elution) at a flow rate of 0.4 mL/min. Detection was performed using positive-ion electrospray ionization in multiple reaction monitoring (MRM) modes. And the detection of morroniside in rat urine by the LC-MS-MS was accurate and precise from 1.0 to 2,500 ng/mL (a correlation coefficient of 0.9953). The recoveries and matrix effects were all in line with the biological sample measurement requirements. The intraday accuracy was 88.68-105.78% with precision of 6.50-11.19% and the interday accuracy was 95.77-102.43% with precision of 7.08-10.40%. Excretion data of morroniside in rat urine indicated that 21.43‰ (i.g.) and 100.35% (i.v.) of the dose administered was excreted as unconverted form, respectively. And the maximal excretion rate was 27.57 and 482.42 μg/h after oral and intravenous administration, respectively. These results indicated that the developed method has satisfactory sensitivity, accuracy and precision for the quantification of morroniside in rat urine.

Morroniside is one of the most important iridoid glycosides in the herbal drug Cornus officinalis Sieb. et Zucc. The current study was designed to investigate the ex vivo and in vivo effects of morroniside on CYP3A activity in rats after treatment with morroniside for 7 days (at 10, 30, 90 mg/kg, i.g.). Morroniside was found to induce CYP3A. According to the ex vivo experiment, the activity of CYP3A was measured by the quantification of 1-hydroxymidazolam, which was the metabolite from CYP3A probe substrate, midazolam. The concentration of 1-hydroxymidazolam was determined by using a validated liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS) method. The levels of messenger RNA (mRNA) and protein of CYP3A were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting analysis, respectively. The pharmacokinetics of midazolam in rats after treatment with morroniside for 7 days (at 10, 30, 90 mg/kg, i.g.) were investigated in vivo. After treatment with morroniside, the activity, mRNA and protein expression of CYP3A were significantly induced and the absorbance and bioavailability of midazolam in rats were reduced. The results indicated that morroniside could induce the activity of CYP3A.

No abstract

Chronic hepatitis B virus (HBV) infection may lead to liver cirrhosis and hepatocellular carcinoma, but few drugs are available for its treatment. Acyclic nucleoside phosphonates (ANPs) have remarkable antivirus activities but are not easily absorbed from the gastrointestinal tract and accumulate in the kidneys, resulting in nephrotoxicity. Therefore, there is a need to find effective liver site-specific prodrugs. The dipivaloyloxymethyl ester of 9-(2-phosphonylmethoxyethyl)adenine (PMEA)-adefovir dipivoxil (ADV)-is a first-line therapy drug for chronic hepatitis B with a low therapeutic index because of renal toxicity and low hepatic uptake. In this study, a series of PMEA derivatives were synthesized to enhance plasma stability and liver release. The metabolic stability of ADV (Chemical I) and its two analogues (Chemicals II and III) was evaluated in rat plasma and liver homogenate in vitro. An ion-pair reverse-phase HPLC-UV method and a hybrid ion trap and high-resolution time-of-flight mass spectrometry (LC-IT-TOF-MS) were used to evaluate the degradation rate of the analogues and to identify their intermediate metabolites, respectively. Chemicals I and II were hydrolyzed by cleavage of the C-O bond to give monoesters. Sufficient enzymatic activation in the liver homogenate through a relatively simple metabolic pathway, in addition to a favorable stability profile in rat plasma, made Chemical II an optimal candidate. Next, six analogues based on the structure of Chemical II were synthesized and evaluated in plasma and liver homogenate. Compared to Chemical II, these compounds generated less active PMEA levels in rat liver homogenate. Therefore, chemical modification of Chemical II may lead to new promising PMEA derivatives with enhanced plasma stability and liver activation.