Tumor necrosis factor-alpha in serum of macaques during SIVmac251 acute infection

TNF secretion was explored in sera during acute SIV-infection of cynomolgus macaques. A peak of TNF was detected in sera of animals in concomitance with SIV replication. Likewise, AZT treatment delayed and reduced peaks of viral replication and TNF production. Thus, SIVmac251-infected monkey could be an excellent model to explore the interdigitation existing between HIV and TNF in acute and chronic infection and to develop new therapeutic strategies that target the production of this cytokine or its inductive effects.     

Frequency of the major histocompatibility complex Mamu‐A*01 allele in experimental rhesus macaques in China

Background In Indian rhesus macaques, the major histocompatibility complex Mamu gene, especially the Mamu‐A*01 allele, plays an important role in simian immunodeficiency virus susceptibility and disease progression. The Mamu‐A*01 allele is one of the protective genes mostly being studied in simian acquired immunodeficiency syndrome. Methods PCR was used to amplify the Mamu‐A*01 allele in 130 Chinese‐origin rhesus macaques. Identification of the allele was then confirmed by sequencing and IFN‐γ ELISPOT assay. Results The Mamu‐A*01 allele was detected in 3.85% (5 of 130) of the experimental Chinese‐origin rhesus macaques. The sequence homology reached 99.1% in comparison with Indian rhesus macaques. A significantly large number of spots were observed in Mamu‐A*01‐positive monkeys when analyzed by ELISPOT with Gag181‐189 epitope stimulation. Conclusions Our study suggests that Mamu‐A*01‐positive Chinese‐origin rhesus monkeys are suitable for use in AIDS studies.     

Morphologic and immunophenotypic characteristics of malignant lymphomas in SIV-infected rhesus macaques

Eight cases of extranodal non-Hodgkin's lymphoma in simian immunodeficiency virus (SIV)-infected rhesus macaques, aged 4-9 years, were phenotypically and immunologically characterized, using the updated Kiel classification, in order to determine both the differences and the similarities between these types of lymphoma in immunodeficient rhesus macaques (Macaca mulatta) and man. The high-grade malignant tumors were of B-cell origin, with a predilection for extranodal growth in viscera and periorbital tissues. Immunophenotypical characterization showed that the monkey lymphomas were similar in many aspects to human immunodeficiency virus-associated lymphomas. The number of Ki67 positive cells varied from case to case and ranged from 50 to 90%. A serological screening for the simian equivalent of the Epstein-Barr virus (sEBV) by immunofluorescence assay revealed a prevalence of 92% of the sEBV antibodies in our cohort. The presence of Ebstein-Barr virus nuclear antigen (EBNA-2) could be demonstrated by immunohistochemistry in four out of eight cases. In situ hybridization revealed the presence of small EBV-encoded RNAs (EBER-1, EBER-2) in six of the eight cases. Further studies should define the precise role of herpesvirus infection for lymphomagenesis in SIV-induced immunodeficiency.     

Blood group A glycolipid (Ax) with globo-series structure which is specific for blood group A1 erythrocytes: one of the chemical bases for A1 and A2 distinction

A new blood group A-active glycolipid fraction, termed Ax, showing a chromatographic mobility between Aa and Ab was found in blood group A1 erythrocytes but not in A2 erythrocytes. Ax was identified by its conversion to "globo H" by alpha-N-acetylgalactosaminidase and by 1H-NMR spectroscopy as GalNAc alpha l----3[Fuc alpha l----2]Gal beta l----3GalNAc beta l----3Gal alpha l----4Gal beta l----4Glc beta l----lCer. Globo-H (Fuc alpha l----2Gal beta l----3GalNac beta l----3Gal alpha l----4Gal beta l----4Glc beta l----lCer) was found in blood group A, and O but not in A1 erythrocytes. Thus, one of the A1-specific determinants must be an A determinant carried by globo-series structure.     

Sterols of Saccharomyces cerevisiae erg6 Knockout Mutant Expressing the Pneumocystis carinii S‐Adenosylmethionine:Sterol C‐24 Methyltransferase

The AIDS‐associated lung pathogen Pneumocystis is classified as a fungus although Pneumocystis has several distinct features such as the absence of ergosterol, the major sterol of most fungi. The Pneumocystis carinii S‐adenosylmethionine:sterol C24‐methyltransferase (SAM:SMT) enzyme, coded by the erg6 gene, transfers either one or two methyl groups to the C‐24 position of the sterol side chain producing both C₂₈ and C₂₉ 24‐alkylsterols in approximately the same proportions, whereas most fungal SAM:SMT transfer only one methyl group to the side chain. The sterol compositions of wild‐type Sacchromyces cerevisiae, the erg6 knockout mutant (Δerg6), and Δerg6 expressing the P. carinii or the S. cerevisiae erg6 gene were analyzed by a variety of chromatographic and spectroscopic procedures to examine functional complementation in the yeast expression system. Detailed sterol analyses were obtained using high performance liquid chromatography and proton nuclear magnetic resonance spectroscopy (¹H‐NMR). The P. carinii SAM:SMT in the Δerg6 restored its ability to produce the C₂₈ sterol ergosterol as the major sterol, and also resulted in low levels of C₂₉ sterols. This indicates that while the P. carinii SAM:SMT in the yeast Δerg6 cells was able to transfer a second methyl group to the side chain, the action of Δ²⁴⁽²⁸⁾‐sterol reductase (coded by the erg4 gene) in the yeast cells prevented the formation and accumulation of as many C₂₉ sterols as that found in P. carinii.     

Prediction of retention volumes and UV spectra of peptides in reversed phase HPLC

A method for calculating retention volumes of linear peptides with known primary structures and the values of their UV absorption at chosen wavelengths in reversed phase HPLC are described. These parameters are calculated for every peptide on the basis of the contributions of its amino acid residues determining its degree of retention and its UV spectrum. The contribution values are experimentally found from chromatograms of the free amino acids obtained by multiwavelength photometric detection under the conditions of the peptide chromatography. Thirty peptides have been chromatographed for the evaluation of the proposed method, and the correlation coefficients between the calculated and the experimental retention volumes have been found to be 0.95.     

海藻糖生产菌株筛选过程中产物鉴定的研究

在海藻糖生产菌的筛选过程中,微生物胞内酶转化淀粉生成的产物复杂,将产物逐一纯化是非常烦琐的,但又必须确证产物中是否含有海藻糖。本文将薄层层析、高效液相电喷雾电离质谱联用及核磁共振等分析手段综合应用于海藻糖生产菌株的筛选,在酶反应产物不必被纯化的前提下,准确、快捷地鉴定了酶反应产物中的未知糖组分,最终证明食尼古丁节杆菌（Arthrobacter nicotinovorus）D97利用淀粉或麦芽寡糖的酶反应产物中含有海藻糖。该方法在筛选海藻糖及其它功能性葡二糖生产菌株时较为严密。     

Binding of benzo[a]pyrene at the 1,3,6 positions to nucleic acids in vivo on mouse skin and in vitro with rat liver microsomes and nuclei

Loss of tritium from specific positions in [³H,¹⁴C] aromatic hydrocarbons can elucidate their binding site(s) to DNA and RNA and indicate the mechanism of activation. Studies of tritium loss from [6-³H,¹⁴C]benzo[a]pyrene(B[a]P), [1,3-³H,¹⁴C]B[a]P, [1,3,6-³H,¹⁴C]B[a]P, [6,7-³H,¹⁴C]B[a]P, and [7-³H,¹⁴C]B[a]P were conducted in vitro using liver nuclei and microsomes from 3-methylcholanthrene-induced Sprague-Dawley rats and in vivo on the skin of Charles River CD-1 mice. The relative loss of tritium from $\text{[}{}^3\text{H}\text{,}{}^{14}\text{C}\text{]}\text{B}\text{[a]}\text{P}$ was measured after binding to skin DNA and RNA, to nuclear DNA, and to native and denatured calf thymus and rat liver DNA's and poly(G) by microsomal activation. In skin, nuclei, and microsomes plus native DNA, virtually all B[a]P binding occurred at positions 1,3 and 6; while with microsomes plus denatured DNA or poly(G), B[a]P showed no binding at the 6 position and a small amount at the 1 and 3 positions. In vivo and with nuclei, binding at the 6 position predominated. Little loss of tritium from the 7 position was seen; this was expected because binding at this position is not thought to occur. This confirms the interpretation of loss of tritium as an indication of binding at a given position. These results demonstrate that the use of microsomes to activate B[a]P is not a valid model system for delineating the in vivo mechanism of B[a]P activation, and support previous evidence for one-electron oxidation as the mechanism of activation of hydrocarbons in binding to nucleic acids.     

Resonance Rayleigh scattering technique as a detection method for the RP‐HPLC determination of local anaesthetics in human urine

A highly selective and sensitive method of reversed phase high‐performance liquid chromatography (RP‐HPLC) coupled with resonance Rayleigh scattering (RRS) was developed for the determination of procaine, bupivacaine and tetracaine. Separation of three local anaesthetics was achieved at 35 °C on a C₁₈ column. The mobile phase was 30: 70 (v/v) acetonitrile/triethylamine–phosphoric acid buffer (pH 2.9) at flow rate of 0.3 mL/min. The RRS detection was conducted by taking advantage of the strong RRS enhancement of the local anaesthetics with erythrosine reaction in an acidic medium. Under optimum conditions, the limit of detection (S/N = 3) values were in the range of 2.4–11.2 ng/mL. Recoveries from spiked human urine samples were 95.8%–104.5%. The proposed method applied to the determination of local anaesthetics in human urine achieved satisfactory results. In addition, the mechanism of the reaction is fully discussed. Copyright © 2016 John Wiley & Sons, Ltd.     

雌激素在大鼠杏仁核与纹状体多巴胺代谢中作用的差异

为探讨雌激素对大鼠杏仁核（amygdala,Amy）与纹状体（striatum,Str）多巴胺（dopamine,DA）代谢的作用,本实验采用离体电化学检测技术--高效液相色谱法（high performance liquid chromatography,HPLC）测定正常雌鼠及经雌激素处理的去卵巢（ovariectomy,OVX）雌鼠Amy和Sr的DA及其代谢产物的组织含量。实验结果显示,OVX雌鼠经雌激素处理后,可引起Amy的DA及其代谢产物含量减少,而Str的DA及其代谢产物含量不受其影响。OVX雌鼠Amy的DA更新率低于正常及雌激素处理的OVX鼠,Amy组织的DA含量约是Str组织的1/6,而更新率是Str的2倍左右。以上结果提示,雌性大鼠血清雄激素浓度可影响其Amy组织的DA代谢及组织含量,而Str的DA组织含量不因雌激素浓度的改变而变化。     

Development of an LC‐MS method for simultaneous quantitation of amentoflavone and biapigenin,the minor and major biflavones from Hypericum perforatum L., in human plasma and its application to real blood

Introduction – Biflavones of Hypericum perforatum L. are bioactive compounds used in the treatment of inflammation and depression. Determination of amentoflavone and biapigenin from blood is challenging owing to their similar structures and low concentrations. Objective – To develop a rapid, sensitive and accurate method based on liquid‐phase extraction followed by high‐performance liquid chromatography and electrospray ionisation mass spectrometry (HPLC‐ESI‐MS) for quantification of biflavones in human plasma. Methodology – After extraction from blood, the analytes were subjected to HPLC with an XTerra® MS C₁₈ column and a binary mobile phase consisting of 2% formic acid in water and acetonitrile under isocratic elution conditions, with ESI‐MS detection in the negative ion mode and multiple reaction monitoring (MRM). Results – Both calibration curves showed good linearity within the concentration range 1–500 ng/mL. Limits of detection (S/N = 3) were 0.1 ng for pure substances and the limits of quantitation (S/N = 5) were 1.0 ng/mL from analyte‐spiked serum. The grand mean recovery was 90% from several subsamples of each biflavone. The imprecision (RSD) of peak areas was between 5% (intraday) and 10% (interday) for high concentrations (250 ng/mL) and between 10% (intraday) and 15% (interday) for low concentrations (1 ng/mL). Inaccuracy of the mean was less than 20% at the lower limit of quantitation. Conclusion – The developed and validated method for determination of biflavones from human plasma was effectively applied to pharmacokinetic studies of 13 probands and preliminary results indicate biphasic concentration–time curves. Copyright © 2010 John Wiley & Sons, Ltd.     

不同产地烤烟叶中绿原酸和芸香苷的含量分析

为了探索绿原酸和芸香苷的新型植物资源和生物活性,同时为低等级烤烟叶的综合利用开辟有效途径,本文采用高效液相色谱技术,对中国和湖南省不同生态烟区以及不同部位烤烟叶中绿原酸和芸香苷的含量进行了研究。结果表明,东北烟区低等级烤烟叶中的绿原酸和芸香苷含量很高;湖南省湘南烟区烤烟叶中绿原酸和芸香苷含量显著高于湘北和湘中烟区;不同部位烤烟叶中绿原酸含量差异不显著,芸香苷含量差异极显著,并且上部叶>中部叶>下部叶。     

Subcellular Localization of the Enzymes of the Arginine Dihydrolase Pathway in Trichomonas vaginalis and Tritrichomonas foetus

The enzymes of the arginine dihydrolase pathway were demonstrated in Tritrichomonas foetus and their subcellular localization determined for both T. foetus and Trichomonas vaginalis. Ornithine carbamyltransferase (anabolic and catabolic activities), ornithine decarboxylase and carbamate kinase activity were localized predominately (56–80%) in the non sedimentable fraction of both species. A large proportion (35–40%) of the arginine deiminase was, however, recovered in the large granular fraction, and this distribution was unchanged by increasing the ionic strength of the buffer. Upon density gradient centrifugation the particles containing arginine deiminase activity had an isopycnic density of 1.09 g/ml in percoll, and separated from hydrogenosomes (1.18 g/ml) and lysosomes (1.12 g/ml). Arginine deiminase was also the only enzyme of the dihydrolase pathway which demonstrated latency upon treatment of the 1.09 g/ml fraction with non-ionic detergents. The results demonstrate the presence of the arginine dihydrolase pathway in T. foetus and indicate that at least a portion of the arginine deiminase in trichomonads is membrane associated.     

Crystallographic studies for the chiral recognition of the novel chiral stationary phase derived from (+)‐(R)‐18‐crown‐6 tetracarboxylic acid

Chiral discrimination observed in high‐performance liquid chromatography (HPLC) with the novel chiral stationary phase (CSP‐18C6I) derived from (+)‐(R)‐18‐crown‐6 tetracarboxylic acid [(+)‐18C6H₄] was investigated by X‐ray crystallographic analysis of the complex composed of the R‐enantiomer of 1‐(1‐naphthyl)ethylamine (1‐NEA) and (+)‐18C6H₄. Mixtures of 1‐NEA (the R‐ or S‐enantiomer) and (+)‐18C6H₄ were dissolved in methanol‐water (1:1) solution and allowed to stand for crystallization. The R‐enantiomer crystallized with (+)‐18C6H₄ as a co‐crystal, although the S‐enantiomer did not. This result was in good agreement with the enantiomer elution order of 1‐NEA in CSP‐18C6I. The apparent binding constants (K_a) of the enantiomers to the (+)‐18C6H₄ obtained from ¹H‐NMR experiments also supported the above‐mentioned result. The X‐ray crystal structure of the 1:1 complex of the R‐enantiomer and (+)‐18C6H₄ indicated the four sets of hydrogen bond association between the naphthylethylammonium cation and oxygen of polyether ring or carbonyl group of (+)‐18C6H₄. Chirality 11:173–178, 1999. © 1999 Wiley‐Liss, Inc.     

Measurements of the anaplerotic rate in the human cerebral cortex using 13C magnetic resonance spectroscopy and [1-13C] and [2-13C] glucose

Recent studies in rodent and human cerebral cortex have shown that glutamate-glutamine neurotransmitter cycling is rapid and the major pathway of neuronal glutamate repletion. The rate of the cycle remains controversial in humans, because glutamine may come either from cycling or from anaplerosis via glial pyruvate carboxylase. Most studies have determined cycling from isotopic labeling of glutamine and glutamate using a [1-(13)C]glucose tracer, which provides label through neuronal and glial pyruvate dehydrogenase or via glial pyruvate carboxylase. To measure the anaplerotic contribution, we measured (13)C incorporation into glutamate and glutamine in the occipital-parietal region of awake humans while infusing [2-(13)C]glucose, which labels the C2 and C3 positions of glutamine and glutamate exclusively via pyruvate carboxylase. Relative to [1-(13)C]glucose, [2-(13)C]glucose provided little label to C2 and C3 glutamine and glutamate. Metabolic modeling of the labeling data indicated that pyruvate carboxylase accounts for 6 +/- 4% of the rate of glutamine synthesis, or 0.02 micromol/g/min. Comparison with estimates of human brain glutamine efflux suggests that the majority of the pyruvate carboxylase flux is used for replacing glutamate lost due to glial oxidation and therefore can be considered to support neurotransmitter trafficking. These results are consistent with observations made with arterial-venous differences and radiotracer methods.     

Fingerprinting Analysis of Rhizoma Chuanxiong of Commercial Types using 1H Nuclear Magnetic Resonance Spectroscopy and High Performance Liquid Chromatography Method

The ¹H nuclear magnetic resonance (¹H NMR) fingerprints of fractionated non-polar extracts (control substance for a plant drug (CSPD) A) from Rhizoma chuanxiong, the rhizomes of Ligusticum chuanxiong Hort., of seven specimens from different sources were measured on Fourier Transform (FT)-NMR spectrometer and assigned by comparing them with the ¹H NMR spectra of the isolated pure compounds. The ¹H NMR fingerprints showed exclusively characteristic resonance signals of the major special constituents of the plant. Although the differences in the relative intensity of the ¹H NMR signals due to a discrepancy in the ratio of the major constituents among these samples could be confirmed by high performance liquid chromatography analysis, the general features of the ¹H NMR fingerprint established for an authentic sample of the rhizomes of L. chuanxiong exhibited exclusive data from those special compounds and can be used for authenticating L. Chuanxiong species.