Red Blood Cell Stiffness and Adhesion Are Species-Specific Properties Strongly Affected by Temperature and Medium Changes in Single Cell Force Spectroscopy

Besides human red blood cells (RBC), a standard model used in AFM-single cell force spectroscopy (SCFS), little is known about apparent Young’s modulus (Ea) or adhesion of animal RBCs displaying distinct cellular features. To close this knowledge gap, we probed chicken, horse, camel, and human fetal RBCs and compared data with human adults serving as a repository for future studies. Additionally, we assessed how measurements are affected under physiological conditions (species-specific temperature in autologous plasma vs. 25 °C in aqueous NaCl solution). In all RBC types, Ea decreased with increasing temperature irrespective of the suspension medium. In mammalian RBCs, adhesion increased with elevated temperatures and scaled with reported membrane sialic acid concentrations. In chicken only adhesion decreased with higher temperature, which we attribute to the lower AE-1 concentration allowing more membrane undulations. Ea decreased further in plasma at every test temperature, and adhesion was completely abolished, pointing to functional cell enlargement by adsorption of plasma components. This halo elevated RBC size by several hundreds of nanometers, blunted the thermal input, and will affect the coupling of RBCs with the flowing plasma. The study evidences the presence of a RBC surface layer and discusses the tremendous effects when RBCs are probed at physiological conditions.     

Protective Effect of 3,4-Dihydroxybenzoic Acid Isolated from Cladophora wrightiana Harvey Against Ultraviolet B Radiation-Induced Cell Damage in Human HaCaT Keratinocytes

The aim of the present study was to elucidate the protective properties of 3,4-dihydroxybenzoic acid (DBA) isolated from Cladophora wrightiana Harvey (a green alga) against ultraviolet B (UVB)-induced damage to human HaCaT keratinocytes. DBA exhibited scavenging actions against the 1,1-diphenyl-2-picrylhydrazyl radical, the superoxide anion, and the hydroxyl radical. Furthermore, DBA decreased the levels of intracellular reactive oxygen species generated by hydrogen peroxide or UVB treatment of the cells. DBA also decreased the UVB-augmented levels of phospho-histone H2A.X and the extent of comet tail formation, which are both indications of DNA damage. In addition, the compound safeguarded keratinocytes from UVB-induced injury by reversing the production of apoptotic bodies, overturning the disruption of mitochondrial membrane potential, increasing the expression of the anti-apoptotic protein, B-cell lymphoma 2, and decreasing the expression of the pro-apoptotic proteins, Bcl-2-associated X and cleaved caspase-3. Taken together, these results demonstrate that DBA isolated from a green alga protects human keratinocytes against UVB-induced oxidative stress and apoptosis.     

Determination of Tiopronin in Human Plasma by SPE then Reversed-Phase HPLC–UV

An accurate, simple and sensitive method based on reversed-phase high-performance liquid chromatography with UV detection has been developed for determination of tiopronin (TP) in human plasma. TP in plasma was reacted with p-bromophenacyl bromide (p-BPB) to give the TP-p-BPB adduct and this derivative was then extracted from the plasma on a silica gel cartridge. Potential interfering compounds were removed by washing with water, and the TP-p-BPB adduct was then eluted with acetonitrile. The organic phase obtained was evaporated to complete dryness under a stream of nitrogen. The residue was dissolved in acetonitrile and this solution was injected on to a reversed-phase ODS HPLC column. The mobile phase was usually the ternary mixture acetonitrile–water–trifluoroacetic acid, 40:59.88:0.12 (v/v). The retention times of TP-p-BPB and the internal standard adduct were 14.4 and 17.9 min, respectively. No interfering peaks were encountered in several blank plasma samples examined. The limit of detection for TP was 12 ng mL^?1. Extraction recovery exceeded 70%. The calibration plot for the TP derivative was linear in the range 40?4000 ng mL^?1, regression coefficient 0.9989, and the coefficient of the variation of the points of the calibration plot was below 10%. The method was validated appropriately and successfully applied to the determination of TP in human plasma.     

毛细管气相色谱法测定健康人外周血单个核细胞磷脂脂肪酸组成

采用毛细管气相色谱法对输注脂肪乳剂的健康志愿者外周血单个核细胞磷脂脂肪酸组成进行了分析测定 ,并将定量结果与输注脂肪乳剂前进行了比较 ,结果表明 :与输注脂肪乳剂前相比 ,连续 7d外周静脉输注 2 0 %脂肪乳剂 (2 5 0mL d) ,外周血单个核细胞数无明显变化 ;外周血单个核细胞磷脂酰乙醇胺中棕榈酸(P <0 .0 5 )和油酸 (P <0 .0 1 )明显增加 ,硬脂酸下降 ;磷脂酰胆碱中棕榈油酸 (P <0 .0 5 )和亚麻酸 (P <0 .0 5 )明显增加 ,而两种磷脂中花生四烯酸及其它多不饱和脂肪酸、饱和脂肪酸与不饱和脂肪酸的比值以及脂肪酸的不饱和指数均未发生明显变化。     

Damage to UV-Sensitive Cells by Short UV in Photographic Flashes

Abstract— Light emitted by electronic photographic flash units is shown to damage bacteria and human skin fibroblasts deficient in repair systems, with survival curves very similar to those produced by 254 nm short UV. The lesions induced by these flashes are as photorepairable by the photolyase enzyme as those induced by 254 nm UV and result in equivalent survival rates. Biological dosimetry performed with microorganisms highly sensitive to UV ( Escherichia coli K12 AB2480, deficient in excision and recombinational-dependent repair systems and Bacillus subtilis UVSSP spores, deficient in excision and in a specific spore repair process) revealed that each 1 ms flash of light from the photographic unit used in this work contained the equivalent of 0.25 J m⁻² of 254 nm UV, when measured at a distance of 7.0 cm. This dose of UV was found to be lethal to both repair-deficient E. coli bacteria and repair-deficient human skin fibroblasts obtained from xeroderma pigmentosum donors, as well as mutagenic in B/r wild-type and HCR-mutant bacteria.     

Sensitive Sandwich Enzyme Immunoassay of Human Interleukin-2 Produced In Vitro by Peripheral Blood Mononuclear Cells

Abstract

A sensitive sandwich enzyme immunoassay for human inter-leukin-2 (hIL-2) using monoclonal antibody IS described. A monoclonai anti-hIL-2 IgG-coated poiystyrene ball was incubated with hIL-2 and subsequently with affinity-purified rabbit anti-hIL-2 Fab'-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorimetry using 3-(4-hydroxyphenyl)propionic acid as a substrate. There was no cross-reaction with interleukins 1α and 1bT, epidermal growth factor, insulin and other protein hormones. The detection limit of hIL-2 was 3 pg/tube or 30 ng/1 using 0.1 ml of cuiture supernatant. When peripheral blood mononuclear cells from healthy subjects aged 22-62 yr were cultured in the absence and presence of phytohemagglutinin P for 48 h, hIL-2 levels in the culture supernatants were <0.03-0.10 μg/1 and 0.18-3.7 μg/1, respectively.     

Effect of Low-Dose Gamma Radiation on HIV Replication in Human Peripheral Blood Mononuclear Cells

Recent studies have demonstrated that UV light and x-irradiation enhance human immunodeficiency virus (HIV) gene expression. There are few published data on related effects of γ-radiation. This may be of clinical relevance, as radiotherapy has been used extensively for the treatment of acquired immunodeficiency syndrome associated conditions. With this in mind, we have studied the effects of γ-radiation on HIV replication in mononuclear cells (MC). These cells were obtained from five seronegative healthy donors, exposed to0–200 cGy γ-radiation, stimulated with phytohemagglutinin-P (PHA-P) for 24 h, infected with a laboratory strain of HIV (HTLV-IIIB, multiplicity of infection = 0.001), then carried in culture for 14 days. Overall, when considering p24 antigen levels on days 7 and 11 in cultures established from cells exposed to 50 cGy, the maximal levels were significantly higher than those measured in the parallel control cultures taken as a whole (P < 0.05), with viral replication enhanced as much as 1000-fold in one case. No significant cytotoxicity was observed following exposure to doses up to 50 cGy. The mechanism of the observed effect remains unknown but may relate to direct gene activation and/or free radical generation, leading to such activation. To date, there is no evidence that viral stimulation occurs following therapeutic radiation in a clinical setting.     

Quantitative Analysis of Genistein in Human Plasma by Online Concentration CE with UV Detection

A rapid capillary electrophoresis method based on online acid barrage stacking has been successfully established for analysis of trace amounts of genistein in human plasma. Genistein was analyzed within 8 min, with 200 mmol L^?1 citric acid (pH 1.7) as acid barrage, and injection times of 180 and 30 s for sample and acid, respectively. Good linearity was obtained in the range 0.05–5 mg L^?1 and the limit of detection was 0.03 mg L^?1. Compared with normal sample injection, this method resulted in more than a fiftyfold improvement in detection sensitivity. The technique has potential for use in studies of genistein metabolism and of exposure levels in humans.     

植物血凝素与金色葡萄球菌肠毒素刺激人外周血单个核细胞的比较研究

比较了植物血凝素(PHA)与金色葡萄球菌肠毒素(SEA)对外周血单个核细胞(PBMCs)的刺激效果。取健康人外周静脉血,分离得到PBMCs,分别加入PHA及SEA进行刺激并孵育12 h,利用原子力显微镜和倒置荧光显微镜观察细胞形态变化,利用量子点联合共聚焦显微镜观察活化后细胞膜表面CD3、CD69的分布情况,利用流式细胞术检测淋巴细胞活化早期细胞分化抗原CD69分子表达的变化情况。结果显示,丝裂原PHA刺激的淋巴细胞大多成群聚集,而超抗原SEA刺激的淋巴细胞大多呈分散状,且二者形态有明显差异;两组活化后淋巴细胞的体积均大于静息组,且活化过程中发生极化作用,迁移淋巴细胞,形成膜突起;丝裂原和超抗原刺激淋巴细胞12 h后,使淋巴细胞表达CD69抗原分子,但在量表达上存在差异性(PHA:39.5%±8.7%;SEA:8.3%±1.8%),抗原分子CD3和CD69在膜表面呈不均匀分布,且SEA活化后的T淋巴细胞表面受体CD3和CD69分子在空间上形成了微结构域;外周单个核细胞中其它非T细胞在丝裂原或者超抗原的刺激下也能表达活化抗原分子CD69。     

Determination of Gemcitabine Triphosphate in Peripheral Blood Mononuclear Cells by Reverse-Phase HPLC

This paper describes a high-performance liquid chromatographic assay coupled with UV detection at 272 nm, to determine the levels of the nucleotide analogue gemcitabine triphosphate, one of the active metabolites of the antitumoral drug gemcitabine, in peripheral blood mononuclear cells. Isocratic ion-pair chromatography on a C₁₈ column was used. Samples were treated with trichloroacetic acid 40%, cleaned and neutralized with freon:trioctylamine (4:1). The method was linear on the concentration range tested, and the evaluated precision was found satisfactory (RSD < 6.8). The detection limit was 31 pmol, and the quantitation limit 102 pmol. The recovery of gemcitabine triphosphate ranged between 95 and 114%. The procedure was used to provide pharmacokinetic data from cancer patients treated with intravenous gemcitabine.     

Sensitive Quantification of Carboxylic Acid Metabolite of Clopidogrel in Human Plasma by LC with UV Detection

A rapid LC method with UV detection was developed for the quantification of carboxylic acid metabolite of clopidogrel in human plasma. Following a simple protein precipitation using a mixture of methanolic solution of ZnSO₄, the analyte and commercially available internal standard were separated using a mobile phase of water–acetonitril (85:15, v/v) adjusted to pH 3.5 on a Chromolith C18 column at a flow rate of 2.5 mL min^?1 with a total retention time of 4 min. Linearity was verified over the range of 20–3,000 ng mL^?1 where the LOQ was 20 ng mL^?1. This method was applied in a pharmacokinetic study.     

DNA Damage Checkpoint Responses in the S Phase of Synchronized Diploid Human Fibroblasts

We investigated the hypothesis that the strength of the activation of the intra‐S DNA damage checkpoint varies within the S phase. Synchronized diploid human fibroblasts were exposed to either 0 or 2.5 J m^?2 UVC in early, mid‐ and late‐S phase. The endpoints measured were the following: (1) radio‐resistant DNA synthesis (RDS), (2) induction of Chk1 phosphorylation, (3) initiation of new replicons and (4) length of replication tracks synthesized after irradiation. RDS analysis showed that global DNA synthesis was inhibited by approximately the same extent (30 ± 12%), regardless of when during S phase the fibroblasts were exposed to UVC. Western blot analysis revealed that the UVC‐induced phosphorylation of checkpoint kinase 1 (Chk1) on serine 345 was high in early and mid S but 10‐fold lower in late S. DNA fiber immunostaining studies indicated that the replication fork displacement rate decreased in irradiated cells at the three time points examined; however, replicon initiation was inhibited strongly in early and mid S, but this response was attenuated in late S. These results suggest that the intra‐S checkpoint activated by UVC‐induced DNA damage is not as robust toward the end of S phase in its inhibition of the latest firing origins in human fibroblasts.     

Role of Calcium in Photodynamically Induced Cell Damage of Human Fibroblasts

Photodynamically induced changes in the cytoplasmic free calcium concentration ([Ca²⁺]_i) and its role in cell damage were investigated in human skin fibroblasts using confocal laser microscopy. Fluorescence and absorbance spectrophotometry measurements indicate that the photosensitizer aluminum phthalocyanine tetrasulfonate (AlPcS₄) binds to the plasma membrane and only after irradiation is able to enter the cells, causing massive morphologic alterations. Upon irradiation of sensitizer-treated cells, the increase in [Ca²⁺]_i is related to the amount of light and extracellular [Ca²⁺]_e. The increase in [Ca²⁺]_i was substantially reduced in the absence of [Ca²⁺]_e. Cell damage or death after photodynamic treatment was prevented and shifted toward higher fluence by increasing [Ca²⁺]_i at high [Ca²⁺]_e and was greater at low [Ca²⁺]_e. Application of Ca²⁺ channel blockers, such as Co²⁺, Cd²⁺ or verapamil, could not prevent the increase of [Ca²⁺]_i. Our results indicate that activation of the photosensitizer, AlPcS₄, causes an influx of Ca²⁺, which protects cells from photodamage. At low [Ca²⁺]_e and high fluence values, release of Ca²⁺ from internal stores probably as a protective measure occurs in order to increase the [Ca²⁺]_i.     

Determination of Cefaclor in Human Plasma by Reversed-Phase High-Performance Liquid Chromatography with UV Detection and Its Application to the Bioequivalence Studies

Abstract

A selective and sensitive reversed-phase high-performance liquid chromatography method was developed and validated for quantitation of cefaclor in human plasma using cefradine as an internal standard. Calibration curve was linear over range of 0.1–20 mg · L^?1. The intra- and inter-run relative standard deviations of the assay were less than 7%. The mean absolute recoveries determined at the concentrations of 0.3, 3.0, 8.0, and 15.0 mg · L^?1 were 69.9%, 69.9%, 77.1% and 72.0%, respectively. The analytical method established was proved to be specific, precise, sensitive, and suitable for applying in the pharmacokinetic and bioequivalence studies of cefaclor in human.     

Spectrally and Time-Resolved Fluorescence Imaging of 22-NBD-Cholesterol in Human Peripheral Blood Mononuclear Cells in Chronic Kidney Disease Patients

The interaction of the fluorescent probe 22-NBD-cholesterol with membranes of human peripheral blood mononuclear cells (PBMC) was tested by time- and spectrally resolved fluorescence imaging to monitor the disturbance of lipid metabolism in chronic kidney disease (CKD) and its treatment with statins. Blood samples from healthy volunteers (HV) and CKD patients, either treated or untreated with statins, were compared. Spectral imaging was done using confocal microscopy at 16 spectral channels in response to 458 nm excitation. Time-resolved imaging was achieved by time-correlated single photon counting (TCSPC) following excitation at 475 nm. The fluorescence of 22-NBD-cholesterol was mostly integrated into plasmatic membrane and/or intracellular membrane but was missing from the nuclear region. The presence of two distinct spectral forms of 22-NBD-cholesterol was uncovered, with significant variations between studied groups. In addition, two fluorescence lifetime components were unmasked, changing in CKD patients treated with statins. The gathered results indicate that 22-NBD-cholesterol may serve as a tool to study changes in the lipid metabolism of patients with CKD to monitor the effect of statin treatment.     

The Induction and Analysis of Micronuclei and Cell Killing by Ultraviolet-B Radiation in Human Peripheral Blood Lymphocytes

The DNA-damaging potential of ultraviolet-B (UVB) radiation was investigated by analyzing the frequency and origin of micronuclei (MN) in cytokinesis-blocked, binucleated (BN) peripheral blood lymphocytes (PBL) and cloning efficiencies (CE) of PBL after exposure to different fluences of UVB. In total, PBL obtained from five normal donors were investigated. The PBL from all donors showed a dose-related, linear-quadratic increase in the frequency of MN per 1000 BN cells and in the frequency of micronucleated BN cells. In two experiments the origin of UVB-induced MN was studied by analyzing MN for the presence or absence of centromeres by applying the MN assay in combination with a centromeric probe and fluorescence in situ hybridization. This revealed, for the first time, that UVB-induced MN were centromere negative, indicating that UVB acted exclusively as a clastogenic agent in the tested dose range. The PBL from all donors showed a clear dose-dependent decrease in CE, after UVB exposure. The UVB-exposed PBL from all donors showed an inverse relationship between the induction of MN and the decrease in CE, but regression analysis revealed no correlation between the induction of MN and the decrease in cell survival. It is concluded that UVB has a clastogenic and cytotoxic effect on PBL.     

Analysis of Pazufloxacin Mesilate in Human Plasma and Urine by LC with Fluorescence and UV Detection,and Its Application to Pharmacokinetic Study

A liquid chromatographic method for analysis of pazufloxacin mesilate in human plasma and urine has been developed and validated for selectivity, sensitivity, accuracy, precision, and stability in pharmacokinetic analysis. The sensitivity of the method was 0.02 μg mL^?1 in plasma and 0.5 μg mL^?1 in urine, with overall intra-day and inter-day precision (RSD < 10%) and accuracy (90–120%) acceptable for clinical pharmacokinetic analysis. Recovery from plasma and urine was 80–110% for both pazufloxacin mesilate and enoxacin, the internal standard. Pazufloxacin was stable in both plasma and urine, with no significant degradation under four different conditions. The method was successfully used in a preliminary study of the bioavailability of pazufloxacin mesilate in healthy human volunteers after intravenous administration of 300 and 500 mg.     

LDPE Transformation by Exposure to Sequential Low-Pressure Plasma and TiO2/UV Photocatalysis

Low-density polyethylene (LDPE) sheets (3.0 ± 0.1 cm) received sequential treatment, first by the action of direct-current low-pressure plasma (DC-LPP) with a 100% oxygen partial pressure, 3.0 × 10⁻² mbar pressure, 600 V DC tension, 5.6 cm distance, 6-min treatment. Then, sheets were submitted to TiO₂ photocatalysis at UV radiation at 254 nm (TiO₂/UV) with a pH value of 4.5 ± 0.2 and a TiO₂ concentration of 1 gL⁻¹. We achieved a complementary effect on the transformation of LDPE films. With the first treatment, ablation was generated, which increased hydrophilicity. With the second treatment, the cavities appeared. The changes in the LDPE sheets’ hydrophobicity were measured using the static contact angle (SCA) technique. The photocatalytic degradation curve at 400 h revealed that the DC-LPP photocatalysis sequential process decreased SCA by 82°. This was achieved by the incorporation of polar groups, which increased hydrophilicity, roughness, and rigidity by 12 and 38%, respectively. These sequential processes could be employed for LDPE and other material biodegradation pretreatment.     

Determination of Pidotimod in Human Plasma by RP-HPLC

Pidotimod, a synthetic dipeptid, has been shown to affect in a positive way some immune functions in humans. It induces an increase in the expression of interleukin-2-T-lymphocyte receptor and can play an important role as an immunostimulating agent, improving immunodefences. The speration and determination of pidotimod in plasma was achieved by using Zorbax Rx-Cl8 colunm. with a guard column.