It all began with an error: the nomofungin/communesin story

The communesin/nomofungin/perophoramidine story is an impressive example of how biogenetic considerations can lead to the correction of structural misassignments and inspire synthetic chemists with new, fruitful ideas. Intensive studies by a number of research groups culminated in the total synthesis of perophoramidine by the Funk research group in 2004. In 2007, Qin and co-workers completed the first total synthesis of a communesin.     

A synthetic approach to nomofungin/communesin B

[reaction: see text] A highly stereoselective intramolecular cycloaddition of an indole tethered to an aza-ortho-xylylene intermediate effects the rapid construction of a substantial portion of the ring system of the cytotoxic natural product communesin B. An analogous cycloaddition involving an ortho-quinone methide intermediate provides an adduct that clearly revealed that the structural assignment for nomofungin was in error.     

The structural and synthetic implications of the biosynthesis of the calycanthaceous alkaloids, the communesins, and nomofungin

A comparison is made between the calycanthaceous alkaloids, nomofungin, and the communesins using structural and biosynthetic information from studies of the former to shed light on the structural ambiguity of the two latter species. Also, a novel biosynthetic approach for the communesins is presented that involves coupling of tryptamine with the ergot alkaloid aurantioclavine that is suitable for synthetic emulation. Preliminary synthetic studies and intermediates are reported.     

Biomimetic approach to communesin B (a.k.a. nomofungin)

The development of an approach to the alkaloid communesin B (2) is presented. The approach is based on considerations of a possible biosynthetic sequence involving an oxidative coupling of tryptamine with a derivative of the ergot alkaloid aurantioclavine. Structure revision is also suggested for the recently isolated microfilament disrupting alkaloid nomofungin.     

Adhesion of filamentous microbial cells on paper covered with ionic polymers by radiation polymerization

The adhesion of filamentous microbial cells such as Trichoderma reesei was studied by using carriers covered with polymers which were prepared by the radiation polymerization of ionic monomers. The weight of the cells adhering to the carriers increased with increasing cationic monomer content, indicating that the surface of the polymers prepared from cationic and hydrophobic monomers is suitable for the adhesion of the cells. The production of cellulase in the cells adhered to polymers from cationic monomers was higher than that in cells adhered to polymers prepared from anionic monomers. The growth of the cells adhered to the surfaces of the polymers was affected by the hydrophilicity of the polymers.     

Human lymphocyte sorting by gravitational field-flow fractionation

Interest in biological studies on various cell types for many biomedical applications, from research to patient treatments, is constantly increasing. The ability to discriminate (sort) and/or quantify distinct subpopulations of cells has become increasingly important. For instance, not only detection but also the highest depletion of neoplastic cells from normal cells is an important requisite in the autologous transplantation of lymphocytes for blood cancer treatments. In this work, gravitational field-flow fractionation (GrFFF) is shown to be effective for sorting a heterogeneous mixture of human, living lymphocytes constituted of neoplastic B cells from a Burkitt lymphoma cell line and healthy T and B lymphocytes from blood samples. GrFFF does not require the use of fluorescent immunotags for sorting cells, and the sorted cells can be collected for their further characterization. Flow cytometry was used to assess the viability of the cells collected, and to evaluate the cell fractionation achieved. A low amount of neoplastic B lymphocytes (less than 2%) was found in a specific fraction obtained by GrFFF. The high depletion from neoplastic cells (more than 98%) was confirmed by a clonogenicity test.     

Comparative Toxicity of Fly Ash: An In Vitro Study

Fly ash produced during coal combustion is one of the major sources of air and water pollution, but the data on the impact of micrometer-size fly ash particles on human cells is still incomplete. Fly ash samples were collected from several electric power stations in the United States (Rockdale, TX; Dolet Hill, Mansfield, LA; Rockport, IN; Muskogee, OK) and from a metallurgic plant located in the Russian Federation (Chelyabinsk Electro-Metallurgical Works OJSC). The particles were characterized using dynamic light scattering, atomic force, and hyperspectral microscopy. According to chemical composition, the fly ash studied was ferro-alumino-silicate mineral containing substantial quantities of Ca, Mg, and a negligible concentration of K, Na, Mn, and Sr. The toxicity of the fly ash microparticles was assessed in vitro using HeLa cells (human cervical cancer cells) and Jurkat cells (immortalized human T lymphocytes). Incubation of cells with different concentrations of fly ash resulted in a dose-dependent decrease in cell viability for all fly ash variants. The most prominent cytotoxic effect in HeLa cells was produced by the ash particles from Rockdale, while the least was produced by the fly ash from Chelyabinsk. In Jurkat cells, the lowest toxicity was observed for fly ash collected from Rockport, Dolet Hill and Muscogee plants. The fly ash from Rockdale and Chelyabinsk induced DNA damage in HeLa cells, as revealed by the single cell electrophoresis, and disrupted the normal nuclear morphology. The interaction of fly ash microparticles of different origins with cells was visualized using dark-field microscopy and hyperspectral imaging. The size of ash particles appeared to be an important determinant of their toxicity, and the smallest fly ash particles from Chelyabinsk turned out to be the most cytotoxic to Jukart cells and the most genotoxic to HeLa cells.     

Studies on the Antiviral Activities in vitro of Polysaccharide from Eucheuma striatum

To assay the antiviral activities on HSV-1 and CVB3 in vitro of the polysaccharide from Eucheuma striatum, its antiviral mechanism was explored. Vero cells were infected by HSV-1 and CVB3, and they were cultured with serial dilutions of polysaccharide. The cells cytotoxicity of Polysaccharide was evaluated by the MTT method. The inhibitory effects were evaluated by the cytopathic effect (CPE). Its antiviral mechanism was researched by the method of giving samples in different time. The polysaccharide could inhibit the CPE of cells infected by HSV-1 and CVB3. It showed low cytotoxicity on vero cells. Its antiviral activities were better than those of acyclovir and ribavirin which were run in parallel as the positive control samples. The polysaccharide from Eucheuma striatum has potent antiviral activities. Its antiviral mechanism is that it can prevent the virus from absorbing to the cell surface.     

Capillary electrophoretic determination of apoptosis of HeLa cells induced by trichosanthin

Apoptosis is a distinct mode of cell death that is responsible for deletion of cells in normal tissues; it also occurs in specific pathologic contexts. The observation of apoptosis is very important in the research of cancer and cancer therapy. The traditional observation method of apoptosis was agarose gel electrophoresis, which is depending on the determination of ladder-liking DNA fragments extracted from apoptotic cells. It is time-consuming and low-sensitive. Recently, the sieving capillary electrophoresis has been used to detect apoptosis too. However, the problem of DNA fragments contamination is still existing. Here, we have developed a capillary electrophoresis method that could detect apoptosis of whole cell directly and do not need to extract DNA fragments from cells. Apoptosis of adherent cell HeLa cell of carcinoma induced by cyclophosphamide was used as the model to establish the method. The effluence of medicine concentration on apoptosis of cells was studied in detail. It was also found that the method could detect the change of cells in the early period of apoptosis. The induction of apoptosis of HeLa cell by trichosanthin was determined with the method, and the result of flow cytometry was also proved that trichosanthin could result in apoptosis of HeLa cells.     

Photodynamic Efficiency of Protoporphyrin IX: Comparison of Endogenous Protoporphyrin IX Induced by 5-Aminolevulinic Acid and Exogenous Porphyrin IX

Abstract— Photodynamic efficiency of protoporphyrin IX (PP) accumulated in HeLa cells by the incubation of PP with HeLa cells was compared with that of accumulated PP formed from 5-aminolevulinic acid (ALA) as a precursor. The ALA-induced PP was photodynamically more efficient than exogenous PP. The difference is caused by monomelic PP concentration and PP localization site in HeLa cells. Exogenous PP was accumulated mainly in plasma membrane, and the membrane was strongly damaged by irradiation. The ALA-induced PP was selectively accumulated in mitochondria and inactivated the mitochondrial function by irradiation.     

扇贝多肽经由aSMase-JNK通路抑制UVA诱导HaCaT细胞凋亡

建立紫外线A(UVA)辐射损伤HaCaT细胞的病理模型, 从酸性鞘磷脂酶-JNK信号通路的角度研究扇贝多肽(Polypeptide from Chlamys farreri, PCF)抑制UVA诱导HaCaT细胞凋亡的分子机制. 采用Hoechst 33258染色结合琼脂糖凝胶电泳分析细胞凋亡; 用RT-PCR法和细胞免疫荧光染色检测胞内酸性鞘磷脂酶(acid sphingomyelinase, aSMase)的表达; 蛋白印迹法检测细胞内JNK及磷酸化JNK的蛋白水平. 结果表明, PCF可明显地抑制UVA诱导的HaCaT细胞凋亡; aSMase抑制剂Desipramine和JNK抑制剂SP600125均可阻断UVA引起的细胞凋亡; PCF的浓度在1.42~5.68 mmol/L范围内可依赖性地抑制UVA辐射后细胞内aSMase的表达量以及JNK蛋白的磷酸化; 预先加入Desipramine则抑制UVA引起的JNK蛋白的磷酸化. 表明PCF通过阻断aSMase-JNK通路来抑制UVA诱导HaCaT细胞凋亡.     

A first step towards practical single cell proteomics: a microfluidic antibody capture chip with TIRF detection

We have developed a generic platform to undertake the analysis of protein copy number from single cells. The approach described here is 'all-optical' whereby single cells are manipulated into separate analysis chambers using an optical trap; single cells are lysed by a shock wave caused by laser-induced microcavitation, and the protein released from a single cell is measured by total internal reflection microscopy as it is bound to micro-printed antibody spots within the device. The platform was tested using GFP transfected cells and the relative precision of the measurement method was determined to be 88%. Single cell measurements were also made on a breast cancer cell line to measure the relative levels of unlabelled human tumour suppressor protein p53 using a chip incorporating an antibody sandwich assay format. These results suggest that this is a viable method for measuring relative protein levels in single cells.     

Comparison between direct and indirect immunofluorescence method for determination of somatic cell count

A sensitive indirect immunofluorescence (IF) assay for bovine neutrophil and somatic cell counting was developed. The obtained indirect IF was compared with direct IF. The direct IF method uses a single-fluorophore-conjugated antibody directed against the target of interest. The indirect IF method uses two antibodies. The primary antibody is unconjugated. The secondary antibody is directed against the primary and has fluorescent marker. Calibration curves for somatic cells (SC) in buffer model solutions were obtained by both methods, using fluorescence spectrophotometry. The measured linear range of somatic cells by the direct method was from 2?×?10⁴ to 3?×?10⁶ cells and by the indirect method was from 3?×?10⁴ to 3?×?10⁶ cells. The signal obtained by the indirect method was higher than the direct method at low concentrations of SC (from 30,000 to 100 000 cells/mL). That signal amplification probably is due to more than one fluorescent secondary antibody coupling to bound primary antibody. The same effect is observed by fluorescence microscopy. Consequently, the indirect IF method is more sensitive than the direct IF method. Conjugated primary antibodies are more expensive than their unconjugated counterparts, and secondary antibodies are relatively inexpensive. Therefore, using the same conjugated secondary antibody to detect different primary antibodies is cost-effective. Furthermore, a two-color staining microscopic procedure was proposed for simultaneous estimation of total SC count and neutrophil cell count.     

Development of a regenerable cell culture system that senses and releases dead cells

We developed a rapidly regenerable cell culture system in which the cell culture substrate detects cell death and selectively releases the dead cells. This culture material was achieved by combining a detector that responds to the signal from the dead cells and an actuator to release the dead cells. Benzo-18-crown-6-acrylamide (BCAm) with a pendant crown ether receptor was used as the sensor to recognize cellular signals and N-isopropylacrylamide (NIPAM) was used as the actuator. This copolymer of NIPAM and BCAm can respond to potassium ions and change its nature from hydrophobic to hydrophilic at the culture temperature of 37 degrees C. Living cells concentrate potassium ion internally; when cells die, potassium ions are released. The polymer surface recognizes the potassium ions released from the dead cells, the NIPAM hydrates, and the dead cells are selectively detached. This in vitro culture system is a novel one in which artificial culture materials work cooperatively with cellular metabolism by responding to this signal from the cells, thereby realizing in vitro tissue regeneration partly mimicking the mechanisms of in vivo homeostasis.     

关于宫颈癌变细胞红外光谱测定方法的研究

提出了一种测定癌变细胞的新方法，利用高分辨傅里叶变换红外光谱（ＦＴＩＲ），对正常人的细胞和千余名宫颈癌患者的细胞进行了对比研究，初步得出该法能在分子水平上揭示出肿瘤细胞与正常细胞的明显差别，并且通过谱图解析，可直接阐明引起谱图变化的主要原因、细胞癌变的可能机理，从而对病程进行预测，为肿瘤疾病的早期诊断展现了良好的前景。     

Affinophoresis of red blood cells: surface antigen-specific acceleration of electrophoresis

A method employing the technique of affinophoresis to increase the electrophoretic mobility of specific cells according to their surface antigens was developed. Red blood cells were treated consecutively with the maximum subagglutinating dose of an anti-red blood cell serum, a biotinylated second antibody, avidin and finally with a negatively charged biotin-affinophore which was prepared by coupling biotin to polylysine (average degree of polymerization, 270 or 1150), followed by complete succinylation. The electrophoretic mobility of cells was analyzed with an automatic cell electrophoresis analyzer. The use of a homologous anti-serum increased the electrophoretic mobility of rabbit, human and rat blood cells by 2.9, 1.7 and 1.6 times, respectively. A larger affinophore containing fewer biotin moieties was more effective. In the case of a mixture of red blood cells from two species, cells from only one species could be accelerated by using homologous antiserum, e.g., affinophoresis of a mixture of human and rat red blood cells by using either homologous antiserum gave two separate peaks on the histogram, whereas a single peak would be obtained in usual electrophoresis because there is little difference in the original migration velocities of the two cell types.     

用于识别不同细胞蛋白质组的噬菌体抗体芯片

将4个鼠源噬菌体抗体克隆和1个人源噬菌体抗体克隆偶联到羧基终止的硅片表面,制成分析型模型芯片.挑选健康人体淋巴细胞为正常细胞的代表, HeLa细胞为肿瘤细胞的代表,提取细胞的全部蛋白质并用荧光染料Cy3标记,与制成的分析芯片反应,得到了不同的结合图谱.实验结果表明,以噬菌体抗体为分子感受器的分析芯片可用于识别不同细胞的蛋白质组.     

Elastic properties of chemically modified baker's yeast cells studied by AFM

Chemical pretreatment is widely used to facilitate transformation of living cells when foreign components are introduced into a cell through the cell wall. The influence of appropriate chemicals on the wall properties and mechanism of transformation is still a matter of intensive studies. Saccharomyces cerevisiae cells (also known as baker's yeast) were investigated by atomic force microscopy (AFM). The cell walls were modified by lithium acetate and dithiothreitol. The AFM imaging was performed in liquid water‐based environment. The living cells were fixed by trapping into the holes of a polycarbonate membrane. Mechanical and morphological properties of initial intact cells and treated cells were investigated. The increased stiffness of the chemically treated cells was observed. As deduced from the applied theoretical Hertz‐Sneddon model, the treated cells show completely different response mechanism to applied mechanical pressure in comparison with the intact cells. Also, the increased roughness of the cell wall of the treated yeasts was observed. Copyright © 2011 John Wiley & Sons, Ltd.     

Oxygen sensitivity of red cell membrane transporters revisited

In this paper, we provide an update on O2-dependent membrane transport in red cells. O2-sensitive membrane transport was compared in nucleated (chicken) and enucleated (human) red cells, to investigate effects on organic (glucose transporter [GLUT]) and inorganic (K(+)-Cl- cotransporter [KCC]/Na(+)-K(+)-2Cl- cotransporter [NKCC]) transporters, to study the response of so-called "housekeeping" transporters (Na+/K+ pump and anion exchanger [AE]) and, finally, to compare O2 sensitivity in normal human red cells with those from sickle cell patients. The Na+/K+ pump showed no change in activity between oxygenated and deoxygenated cells in any of the samples. KCC in normal human red cells had the greatest O2 sensitivity, being stimulated some 20-fold on oxygenation. It was more modestly stimulated by O2 in chicken red cells and HbS cells. By contrast, NKCC was stimulated by deoxygenation in all cases. GLUT showed little response to O2 tension, other than a small stimulation in deoxygenated chicken red cells. Finally, AE1 was stimulated by oxygenation in HbA cells, but this stimulation by O2 was absent in HbS cells and pink ghosts prepared from HbA cells. The significance of these findings is discussed.