高效液相色谱法定量分析人胎肝细胞上清液及细胞裂解液中7-酮基胆固醇

用高效液相色谱法定量分析了７－酮基胆固醇在胎肝细胞上清液及细胞裂解液中的含量。色谱柱为μ－ＰｏｒａｓｉｌＳｉＯ２柱,流动相为正己烷∶异丙醇（９１∶９,Ｖ／Ｖ）。方法回收率高、色谱重复性好、分析速度快。     

高效液相色谱法测定人血清中5-氟尿嘧啶浓度

建立了测定人血清中5-氟尿嘧啶的反相高效液相色谱法。采用ZorbaxODS柱4.6mm×250mm,预柱YWG-C184.6mm×50mm;甲醇:水（20:80）为流动相,紫外检测波长为273um,喃氟啶为内标定量。回收率为96.85％,田间和日内变异系数分别为537％和438％,血清中最低检测浓度为10μg／L。在0.04～50mg／L浓度范围内线性良好,相关系数为0.9990。     

Immunocytochemical Localization of XRCC1 and γH2AX Foci Induced by Tightly Focused Femtosecond Laser Radiation in Cultured Human Cells

To assess the prospects for using intense femtosecond laser radiation in biomedicine, it is necessary to understand the mechanisms of its action on biological macromolecules, especially on the informational macromolecule—DNA. The aim of this work was to study the immunocytochemical localization of DNA repair protein foci (XRCC1 and γH2AX) induced by tightly focused femtosecond laser radiation in human cancer A549 cells. The results showed that no XRCC1 or γH2AX foci tracks were observed 30 min after cell irradiation with femtosecond pulses of 10¹¹ W∙cm⁻² peak power density. An increase in the pulse power density to 2 × 10¹¹ W∙cm⁻² led to the formation of linear tracks consisting both of XRCC1 and γH2AX protein foci localized in the places where the laser beam passed through the cell nuclei. A further increase in the pulse power density to 4 × 10¹¹ W∙cm⁻² led to the appearance of nuclei with total immunocytochemical staining for XRCC1 and γH2AX on the path of the laser beam. Thus, femtosecond laser radiation can be considered as a tool for local ionization of biological material, and this ionization will lead to similar effects obtained using ionizing radiation.     

The Existing Methods and Novel Approaches in Mycotoxins’ Detection

Mycotoxins represent a wide range of secondary, naturally occurring and practically unavoidable fungal metabolites. They contaminate various agricultural commodities like cereals, maize, peanuts, fruits, and feed at any stage in pre- or post-harvest conditions. Consumption of mycotoxin-contaminated food and feed can cause acute or chronic toxicity in human and animals. The risk that is posed to public health have prompted the need to develop methods of analysis and detection of mycotoxins in food products. Mycotoxins wide range of structural diversity, high chemical stability, and low concentrations in tested samples require robust, effective, and comprehensible detection methods. This review summarizes current methods, such as chromatographic and immunochemical techniques, as well as novel, alternative approaches like biosensors, electronic noses, or molecularly imprinted polymers that have been successfully applied in detection and identification of various mycotoxins in food commodities. In order to highlight the significance of sampling and sample treatment in the analytical process, these steps have been comprehensively described.     

Tetrandrine Suppresses Human Brain Glioblastoma GBM 8401/luc2 Cell-Xenografted Subcutaneous Tumors in Nude Mice In Vivo

Tetrandrine (TET), a bisbenzylisoquinoline (BBI) alkaloid, is isolated from the plant Stephania tetrandra S. Moore and has a wide range of biological activity, including anticancer properties in vitro and in vivo. At first, we established a luciferase-expressing stable clone that was named GBM 8401/luc2 cells. Herein, the primary results indicated that TET reduced the total cell viability and induced cell apoptosis in GBM 8401/luc2 human glioblastoma cells. However, there is no available information showing that TET suppresses glioblastoma cells in vivo. Thus, we investigated the effects and mechanisms of TET on a GBM 8401/luc2 cell-generated tumor in vivo. After the tumor volume reached 100–120 mm³ in subcutaneously xenografted nude mice, all of the mice were randomly divided into three groups: Group I was treated with phosphate-buffered solution (PBS) containing 0.1% dimethyl sulfoxide, Group II with 25 mg/kg of TET, and Group III with 50 mg/kg of TET. All mice were given the oral treatment of PBS or TET by gavage for 21 days, and the body weight and tumor volumes were recorded every 5 days. After treatment, individual tumors, kidneys, livers, and spleens were isolated from each group. The results showed that TET did not affect the body weights, but it significantly decreased the tumor volumes. The TET treatment at 50 mg/kg had a two-fold decrease in tumor volumes than that at 25 mg/kg when compared to the control. TET decreased the total photon flux, and treatment with TET at 50 mg/kg had a lower total photon flux than that at 25 mg/kg, as measured by a Xenogen IVIS imaging system. Moreover, the higher TET treatment had lower tumor volumes and weights than those of the lower dose. The apoptosis-associated protein expression in the tumor section was examined by immunohistochemical analysis, and the results showed that TET treatment reduced the levels of c-FLIP, MCL-1, and XIAP but increased the signals of cleaved-caspase-3, -8, and -9. Furthermore, the hematoxylin and eosin (H & E) staining of kidney, liver, and spleen tissues showed no significant difference between the TET-treated and control groups. Overall, these observations demonstrated that TET suppressed subcutaneous tumor growth in a nude-mice model via the induction of cell apoptosis.     

Garcinielliptone G from Garcinia subelliptica Induces Apoptosis in Acute Leukemia Cells

Cytotoxicity and apoptosis-inducing properties of compounds isolated from Garcinia subelliptica leaves were investigated. The hexane-soluble portion of MeOH extracts of G. subelliptica leaves that showed cytotoxic activity was separated to yield seven compounds 1–7. Chemical structure analysis using NMR spectroscopy and mass spectrometry confirmed that compound 1 was canophyllol, and compounds 2–7 were garcinielliptones N, O, J, G, F, and garcinielliptin oxide, respectively. Among them, garcinielliptone G (5) showed growth inhibition by causing apoptosis in THP-1 and Jurkat cells derived from human acute monocytic leukemia and T lymphocyte cells, respectively. Apoptosis induced by garcinielliptone G (5) was demonstrated by the detection of early apoptotic cells with fluorescein-labeled Annexin V and increases in cleaved caspase-3 and cleaved PARP protein levels. However, the addition of caspase inhibitor Z-VAD-FMK did not affect growth arrest or apoptosis induction. These results suggest that garcinielliptone G (5) can induce both caspase-3 activation and caspase-independent apoptosis. Therefore, garcinielliptone G (5) may be a potential candidate for acute leukemia treatment.     

Film-Forming Spray of Water-Soluble Chitosan Containing Liposome-Coated Human Epidermal Growth Factor for Wound Healing

Human epidermal growth factor (hEGF) has been known to have excellent wound-healing activity. However, direct application to the wound area can lead to low hEGF bioavailability due to protease enzymes or endocytosis. The use of liposomes as coatings and carriers can protect hEGF from degradation by enzymes, chemical reactions, and immune reactions. Sustained release using a matrix polymer can also keep the levels of hEGF in line with the treatment. Therefore, this study aimed to develop a film-forming spray of water-soluble chitosan (FFSWSC) containing hEGF-liposomes as a potential wound dressing. The hEGF-liposomes were prepared using the hydration film method, and the preparation of the FFSWSC was achieved by the ionic gelation method. The hydration film method produced hEGF-liposomes that were round and spread with a Z-average of 219.3 nm and encapsulation efficiency of 99.87%, whereas the film-forming solution, which provided good sprayability, had a formula containing 2% WSC and 3% propylene glycol with a viscosity, spray angle, droplet size, spray weight, and occlusion factor of 21.94 ± 0.05 mPa.s, 73.03 ± 1.28°, 54.25 ± 13.33 µm, 0.14 ± 0.00 g, and 14.57 ± 3.41%, respectively. The pH, viscosity, and particle size of the FFSWSC containing hEGF-liposomes were stable during storage for a month in a climatic chamber (40 ± 2 °C, RH 75 ± 5%). A wound healing activity test on mice revealed that hEGF-liposomes in FFSWSC accelerated wound closure significantly, with a complete wound closure on day 6. Based on the findings, we concluded that FFSWSC containing hEGF-liposomes has the potential to be used as a wound dressing.     

When UDG and hAPE1 Meet Cyclopurines. How (5′R) and (5′S) 5′,8-Cyclo-2′-deoxyadenosine and 5′,8-Cyclo-2′-deoxyguanosine Affect UDG and hAPE1 Activity?

Ionizing radiation is a factor that seriously damages cellular mechanisms/macromolecules, e.g., by inducing damage in the human genome, such as 5′,8-cyclo-2′-deoxypurines (cdPus). CdPus may become a component of clustered DNA lesions (CDL), which are notably unfavorable for the base excision repair system (BER). In this study, the influence of 5′S and 5′R diastereomers of 5′,8-cyclo-2′-deoxyadenosine (cdA) and 5′,8-cyclo-2′-deoxyguanosine (cdG) on the uracil-DNA glycosylase (UDG) and human AP site endonuclease 1 (hAPE1) activity has been taken under consideration. Synthetic oligonucleotides containing 2′-deoxyuridine (dU) and cdPu were used as a model of single-stranded CDL. The activity of the UDG and hAPE1 enzymes decreased in the presence of RcdG compared to ScdG. Contrary to the above, ScdA reduced enzyme activity more than RcdA. The presented results show the influence of cdPus lesions located within CDL on the activity of the initial stages of BER dependently on their position toward dU. Numerous studies have shown the biological importance of cdPus (e.g., as a risk of carcinogenesis). Due to that, it is important to understand how to recognize and eliminate this type of DNA damage from the genome.     

Tissue-Specific Regulation of HNK-1 Biosynthesis by Bisecting GlcNAc

Human natural killer—1 (HNK-1) is a sulfated glyco-epitope regulating cell adhesion and synaptic functions. HNK-1 and its non-sulfated forms, which are specifically expressed in the brain and the kidney, respectively, are distinctly biosynthesized by two homologous glycosyltransferases: GlcAT-P in the brain and GlcAT-S in the kidney. However, it is largely unclear how the activity of these isozymes is regulated in vivo. We recently found that bisecting GlcNAc, a branching sugar in N-glycan, suppresses both GlcAT-P activity and HNK-1 expression in the brain. Here, we observed that the expression of non-sulfated HNK-1 in the kidney is unexpectedly unaltered in mutant mice lacking bisecting GlcNAc. This suggests that the biosynthesis of HNK-1 in the brain and the kidney are differentially regulated by bisecting GlcNAc. Mechanistically, in vitro activity assays demonstrated that bisecting GlcNAc inhibits the activity of GlcAT-P but not that of GlcAT-S. Furthermore, molecular dynamics simulation showed that GlcAT-P binds poorly to bisected N-glycan substrates, whereas GlcAT-S binds similarly to bisected and non-bisected N-glycans. These findings revealed the difference of the highly homologous isozymes for HNK-1 synthesis, highlighting the novel mechanism of the tissue-specific regulation of HNK-1 synthesis by bisecting GlcNAc.     

Quantitative analysis of memantine in human plasma by gas chromatography/negative ion chemical ionization/mass spectrometry

A sensitive and specific method for the determination of memantine in human plasma is presented. Memantine was extracted from plasma and derivatized to the pentafluorobenzoyl derivative in a one-step procedure avoiding any sample concentration steps. Amantadine was used as an internal standard. The compounds were measured by gas chromatography/negative ion chemical ionization mass spectrometry without any further processing. Using this detection mode, the fragment ions at m/z 353 and 325 were obtained at high relative abundance. Calibration graphs were linear over the range 0.117-30 ng ml(-1). At the limit of quantification (LOQ), the inter-assay precision was 2.00% and the intra-assay variability was 3.22%. The accuracy at the LOQ showed deviations of -1.42% (intra-assay) and -2.47% (inter-assay). The method is rugged, rapid and robust and was applied to the batch determination of memantine during pharmacokinetic profiling of the drug.