In vivo imaging of reactive oxygen and nitrogen species in inflammation using the luminescent probe L-012

Production of reactive oxygen and nitrogen species (ROS/RNS) is an important part of the inflammatory response, but prolonged elevated levels of ROS/RNS as under chronic inflammation can contribute to the development of disease. Monitoring ROS/RNS in living animals is challenging due to the rapid turnover of ROS/RNS and the limited sensitivity and specificity of ROS/RNS probes. We have explored the use of the chemiluminescent probe L-012 for noninvasive imaging of ROS/RNS production during inflammation in living mice. Various inflammatory conditions were induced, and L-012-dependent luminescence was recorded with an ultrasensitive CCD camera. Strong luminescent signals were observed from different regions of the body corresponding to inflammation. The signal was reduced by administration of the SOD mimetic tempol, the NADPH oxidase inhibitor apocynin, and the inhibitor of nitric oxide synthesis L-NAME, signifying the requirement for the presence of ROS/RNS. Additionally, the L-012 signal was abolished in mice with a mutation in the Ncf1 gene, encoding a protein in the NADPH oxidase complex 2, which generates ROS/RNS during inflammation. In conclusion, L-012 is well distributed in the mouse body and mediates a strong ROS/RNS-dependent luminescent signal in vivo and is useful for monitoring the development and regulation of inflammation in living organisms.     

Conservation genetics of Hebe speciosa (Plantaginaceae) an endangered New Zealand shrub

All extant populations of Hebe speciosa (Plantaginaceae), a threatened endemic New Zealand shrub, were investigated using the amplified fragment length polymorphism technique (AFLP). Genetic diversity indices varied significantly among geographical regions and were positively correlated with population size. Among-population genetic differentiation was high (mean pairwise Φ_ST = 0.47), implying complex historical relationships between disjunct populations and negligible contemporary gene flow. Southern populations exhibited extremely low genetic diversity relative to those found in Northland, suggesting that these populations may be more recent in origin. Patterns of genetic relationship among some populations indicate pre-European Māori dispersal and cultivation. The three northernmost populations were found to contain the majority of the species' remaining genetic diversity and thus, should be a focus for future conservation management. Some southern sites may also be culturally significant as evidence of Māori trade and cultivation of Hebe speciosa .  © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society , 2005, 149 , 229–239.     

大肠杆菌全细胞转化联产L-2-氨基丁酸和D-葡萄糖酸

以大肠杆菌BL21(DE3)为表达宿主,构建两株分别表达L-苏氨酸脱氨酶(LTD,基因来源大肠杆菌)和共表达亮氨酸脱氢酶(LDH,来源蜡样芽孢杆菌)/葡萄糖脱氢酶(GDH,来源枯草芽孢杆菌)的重组大肠杆菌,在此基础上,构建了一种以L-苏氨酸和D-葡萄糖为底物联产L-2-氨基丁酸(L-ABA)和D-葡萄糖酸的全细胞转化系统。通过转化条件(温度、p H、细胞通透性和菌体量)优化,并采用分批补料策略,164 g/L L-苏氨酸和248 g/L D-葡萄糖最终转化得到141.6 g/L的L-ABA和269.4 g/L的D-葡萄糖酸,时空得率分别达到7.1 g/(L?h)和13.5 g/(L?h),得率超过99%。本研究使用价格低廉的大宗化学品高效率生产出有较高附加值的产物,全细胞转化系统无需额外添加昂贵的辅酶,更适用于工业化生产。     

Herpes Simplex Virus 1 Infection Alters the mRNA Translation Processing in L-02 Cells

HSV-1 infection-mediated regulation of mRNA translation in host cells is a systematic and complicated process. Investigation of the details of this mechanism will facilitate understanding of biological variations in the viral replication process and host cells. In this study, a comparative proteomics technology platform was applied by two-dimension electrophoresis of HSV-1 infected normal human L-02 cell and control cell lysates. The observed protein spots were analyzed qualitatively and quantitatively by the PDQuest software package. A number of the different observed protein spots closely associated with cellular protein synthesis were identified by matrix-assisted laser-desorption ionization-time of flight-mass spectrometry （MALDI-TOF-MS）. The expression levels of the RPLP1 protein, which is required for mRNA translation, and KHSRP protein, which is involved in rapid decay of mRNA, were up-regulated, whereas the expression level of RNP H2, which is involved in positive regulation on the mRNA splicing process, was down-regulated. All of these results suggest that HSV-1 infection can influence cellular protein synthesis via modulation of cellular regulatory proteins involved in RNA splicing, translation and decay, resulting in optimisation of viral protein synthesis when cellular protein synthesis is shut off Although there is need for further investigations regarding the detailed mechanisms of cellular protein control, our studies provide new insight into the targeting of varied virus signaling pathways involved in host cellular protein synthesis.     

Detection and determination of reticuline and N-methylcoculaurine in the Annonaceae family using liquid chromatography-tandem mass spectrometry

In Guadeloupe, the French West Indies, there is a high incidence of atypical parkinsonism or progressive supranuclear palsy, and all of the investigated patients had taken herbal tea or tropical fruits of the Annonaceae family. Local inhabitants consume the fruits, and also drink tea made from the leaves. In the present study, we used liquid chromatography-tandem mass spectrometry (LC/MS/MS) with multiple reaction monitoring (MRM) to detect low-molecular-weight neurotoxic benzylisoquinoline derivatives in the Annonaceae family. We detected reticuline and N-methylcoculaurine in every Annona muricata sample examined, except for pulp and seed. They were not detected in sweetsop fruits. Norreticuline was not detected in any sample. These three compounds were toxic to SH-SY5Y neuroblastoma cells and inhibited mitochondrial respiratory complex I. It is possible that uptake of the benzylisoquinoline derivatives reticuline and N-methylcoculaurine and their accumulation in the brain may be related to the pathogenesis of the local endemic disease.     

N5-(L-1-carboxyethyl)-L-ornithine synthase: physical and spectral characterization of the enzyme and its unusual low pKa fluorescent tyrosine residues.

N5-(L-1-carboxyethyl)-L-ornithine synthase [E.C. 1.5.1.24] (CEOS) from Lactococcus lactis has been cloned, expressed, and purified from Escherichia coli in quantities sufficient for characterization by biophysical methods. The NADPH-dependent enzyme is a homotetramer (Mr approximately equal to 140,000) and in the native state is stabilized by noncovalent interactions between the monomers. The far-ultraviolet circular dichroism spectrum shows that the folding pattern of the enzyme is typical of the alpha,beta family of proteins. CEOS contains one tryptophan (Trp) and 19 tyrosines (Tyr) per monomer, and the fluorescence spectrum of the protein shows emission from both Trp and Tyr residues. Relative to N-acetyltyrosinamide, the Tyr quantum yield of the native enzyme is about 0.5. All 19 Tyr residues are titratable and, of these, two exhibit the uncommonly low pKa of approximately 8.5, 11 have pKa approximately 10.75, and the remaining six titrate with pKa approximately 11.3. The two residues with pKa approximately 8.5 contribute approximately 40% of the total tyrosine emission, implying a relative quantum yield >1, probably indicating Tyr-Tyr energy transfer. In the presence of NADPH, Tyr fluorescence is reduced by 40%, and Trp fluorescence is quenched completely. The latter result suggests that the single Trp residue is either at the active site, or in proximity to the sequence GSGNVA, that constitutes the beta alphabeta fold of the nucleotide-binding domain. Chymotrypsin specifically cleaves native CEOS after Phe255. Although inactivated by this single-site cleavage of the subunit, the enzyme retains the capacity to bind NADPH and tetramer stability is maintained. Possible roles in catalysis for the chymotrypsin sensitive loop and for the low pKa Tyr residues are discussed.     

Determination of L-756 423, a novel HIV protease inhibitor, in human plasma and urine using high-performance liquid chromatography with fluorescence detection

A method for the determination of L-756 423, a novel HIV protease inhibitor, in human plasma and urine is described. Plasma and urine samples were extracted using 3M Empore extraction disk cartridges in the C₁₈ and MPC (mixed-phase cation-exchange) formats, respectively. The extract was analyzed using HPLC with fluorescence detection (ex 248 nm, em 300 nm), and included a column switching procedure to reduce run-time. The assay was linear in the concentration range 5 to 1000 ng/ml when 1-ml aliquots of plasma and urine were extracted. Recoveries of L-756 423 were greater than 84% over the calibration curve range using the described sample preparation procedures. Intra-day precision and accuracy for this assay was less than 9% RSD and within 7%, respectively. Inter-day variabilities for the plasma (n=17) and urine (n=10) were less than 5% and 3% for low (15 ng/ml) and high (750 ng/ml) quality control samples. Bovine serum albumin (0.5%) was used as an additive to urine to prevent precipitation of L-756 423 during the storage of clinical samples. The assay was used in support of human clinical trials.     

The simple and rapid quantification method for L-3,4-dihydroxyphenylalanine (L-DOPA) from plant sprout using liquid chromatography-mass spectrometry

L-3,4-dihydroxyphenylalanine (L-DOPA) is one of the important secondary metabolites of plants and has been used for various purposes, such as in clinical treatment for Parkinson’s disease and dopamine-responsive dystonia. In plants, L-DOPA is a precursor of many alkaloids, catecholamines, and melanin; the L-DOPA synthesis pathway is similar to that in mammals. L-DOPA acts as an allelochemical, has an important role in several biological processes, such as stress response and metabolism, in plants. L-DOPA is widely used in the clinical treatment as well as a dietary supplement or psychotropic drug, understanding of biosynthesis of L-DOPA in plant could lead to a stable supply of L-DOPA. This paper describes an improved method for simple and rapid quantification of L-DOPA content using liquid chromatography-tandem mass spectrometry. The standard quantitative methods for L-DOPA require multiple purification steps or relatively large amounts of plant material. In our improved method, quantification of L-DOPA was possible with extract of one–two pieces of cotyledon without any partitioning or column for purification. The endogenous L-DOPA (approximately 4,000 µg g⁻¹ FW (fresh weight)) could be detected from the one pieces of cotyledon of the faba bean sprout using this method. This method was also effective for samples with low endogenous amounts of L-DOPA such as broccoli, Japanese white radish, pea, and red cabbage sprouts. Therefore, this improved method will allow to measurement of L-DOPA content easily and accurately from a small amount of plant tissue and contribute to understanding biosynthesis, catabolism, and transport of L-DOPA.     

Esterase and protease activity of purified guinea pig basophil granules.

Highly purified granules of circulating guinea pig basophilic leukocytes were extracted with 0.1% Triton X-100 to yield a mixture of esterases-proteases including caseinolytic activity. By selective inhibition both trypsin- and chymotrypsin-like serine hydrolases have been identified. Sigmoidal pH dependences for hydrolysis of p-tosyl-L-arginine-methyl ester and N-benzoyl-L-tryosine-ethyl ester were observed for both intact granules and Triton granule extracts. Preliminary studies indicate that the enzymes are not solubilized even after Triton X-100 treatment of the granules.