Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations. FACS is a technique of choice to purify cell populations of known phenotype. Other bulk methods of purification include panning, complement depletion and magnetic bead separation. However, FACS has several advantages over other available methods. FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density. In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker. FACS allows the purification of individual cells based on size, granularity and fluorescence. In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1). Negative selection of unstained cells is also possible. FACS purification requires a flow cytometer with sorting capacity and the appropriate software. For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser. The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells. The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2). The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb. Sorting parameters can be adjusted depending on the requirement of purity and yield. Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.     

Relative and regional stabilities of the hamster, mouse, rabbit, and bovine prion proteins toward urea unfolding assessed by nuclear magnetic resonance and circular dichroism spectroscopies

The residue-specific urea-induced unfolding patterns of recombinant prion proteins from different species (bovine, rabbit, mouse, and Syrian hamster) were monitored using high-resolution (1)H nuclear magnetic resonance (NMR) spectroscopy. Protein constructs of different lengths, and with and without a His tag attached at the N-terminus, were studied. The various species showed different overall sensitivities toward urea denaturation with stabilities in the following order: hamster ≤ mouse < rabbit < bovine protein. This order is in agreement with recent circular dichroism (CD) spectroscopic measurements for several species [Khan, M. Q. (2010) Proc. Natl. Acad. Sci. U.S.A.107, 19808-19813] and for the bovine protein presented herein. The [urea](1/2) values determined by CD spectroscopy parallel those of the most stable residues observed by NMR spectroscopy. Neither the longer constructs containing an additional hydrophobic region nor the His tag influenced the stability of the structured domain of the constructs studied. The effect of the S174N mutation in rabbit PrP(C) was also investigated. The rank order of the regional stabilities within each protein remained the same for all species. In particular, the residues in the β-sheet region in all four species were more sensitive to urea-induced unfolding than residues in the α2 and α3 helical regions. These observations indicate that the regional specific unfolding pattern is the same for the four mammalian prion proteins studied but militate against the idea that PrP(Sc) formation is linked with the global stability of PrP(C).     

Oligomeric structure of ExbB and ExbB-ExbD isolated from Escherichia coli as revealed by LILBID mass spectrometry

Energy-coupled transporters in the outer membrane of Escherichia coli and other Gram-negative bacteria allow the entry of scarce substrates, toxic proteins, and bacterial viruses (phages) into the cells. The required energy is derived from the proton-motive force of the cytoplasmic membrane, which is coupled to the outer membrane via the ExbB-ExbD-TonB protein complex. Knowledge of the structure of this complex is required to elucidate the mechanisms of energy harvesting in the cytoplasmic membrane and energy transfer to the outer membrane transporters. Here we solubilized an ExbB oligomer and an ExbB-ExbD subcomplex from the cytoplasmic membrane with the detergent undecyl maltoside. Using laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS), we determined at moderate desorption laser energies the oligomeric structure of ExbB to be mainly hexameric (ExbB(6)), with minor amounts of trimeric (ExbB(3)), dimeric (ExbB(2)), and monomeric (ExbB(1)) oligomers. Under the same conditions ExbB-ExbD formed a subcomplex consisting of ExbB(6)ExbD(1), with a minor amount of ExbB(5)ExbD(1). At higher desorption laser intensities, ExbB(1) and ExbD(1) and traces of ExbB(3)ExbD(1), ExbB(2)ExbD(1), ExbB(1)ExbD(1), ExbB(3), and ExbB(2) were observed. Since the ExbB(6) complex and the ExbB(6)ExbD(1) complex remained stable during solubilization and subsequent chromatographic purification on nickel-nitrilotriacetate agarose, Strep-Tactin, and Superdex 200, and during native blue gel electrophoresis, we concluded that ExbB(6) and ExbB(6)ExbD(1) are subcomplexes on which the final complex including TonB is assembled.     

Effects of modified Phycobilin biosynthesis in the Cyanobacterium Synechococcus sp. Strain PCC 7002

The pathway for phycocyanobilin biosynthesis in Synechococcus sp. strain PCC 7002 comprises two enzymes: heme oxygenase and phycocyanobilin synthase (PcyA). The phycobilin content of cells can be modified by overexpressing genes encoding alternative enzymes for biliverdin reduction. Overexpression of the pebAB and HY2 genes, encoding alternative ferredoxin-dependent biliverdin reductases, caused unique effects due to the overproduction of phycoerythrobilin and phytochromobilin, respectively. Colonies overexpressing pebAB became reddish brown and visually resembled strains that naturally produce phycoerythrin. This was almost exclusively due to the replacement of phycocyanobilin by phycoerythrobilin on the phycocyanin α-subunit. This phenotype was unstable, and such strains rapidly reverted to the wild-type appearance, presumably due to strong selective pressure to inactivate pebAB expression. Overproduction of phytochromobilin, synthesized by the Arabidopsis thaliana HY2 product, was tolerated much better. Cells overexpressing HY2 were only slightly less pigmented and blue-green than the wild type. Although the pcyA gene could not be inactivated in the wild type, pcyA was easily inactivated when cells expressed HY2. These results indicate that phytochromobilin can functionally substitute for phycocyanobilin in Synechococcus sp. strain PCC 7002. Although functional phycobilisomes were assembled in this strain, the overall phycobiliprotein content of cells was lower, the efficiency of energy transfer by these phycobilisomes was lower than for wild-type phycobilisomes, and the absorption cross-section of the cells was reduced relative to that of the wild type because of an increased spectral overlap of the modified phycobiliproteins with chlorophyll a. As a result, the strain producing phycobiliproteins carrying phytochromobilin grew much more slowly at low light intensity.     

Gold catalyzed glycosidations for the synthesis of sugar acrylate/acrylamide hybrids and their utility

Propargyl glyco 1,2-orthoesters were exploited for the efficient synthesis of interesting glycomonomers such as glyco-acrylates and acrylamides using gold catalysts. It was observed that propargyl glyco 1,2-orthoesters with hydroxyethyl acrylates gives very good yield of the corresponding glyco-acrylates in a single step in the presence of catalytic amount of gold(III) catalyst; whereas, gold catalyzed glycosidation reaction on hydroxyethyl acrylamides was found to yield the corresponding acrylamidoyl 1,2-orthoester which was then converted to the corresponding glycol-acrylamide in the presence of catalytic amount of TMSOTf. Synthesized glyco-acrylate/acrylamide monomers are shown to undergo thiolate addition as well as free radical polymerization.     

Structural characterization of an immunoenhancing cytotoxic heteroglycan isolated from an edible mushroom Calocybe indica var. APK2

A water-soluble polysaccharide of an edible mushroom Calocybe indica var. APK2 showed immunoenhancing (macrophage, splenocyte, thymocyte, and bone marrow activation) and cytotoxic activity toward HeLa cell lines and found to consist of d-glucose, d-galactose, and l-fucose in a molar ratio of nearly 3:1:1. On the basis of acid hydrolysis, methylation analysis, and NMR studies (¹H, ¹³C, DEPT-135, TOCSY, DQF–COSY, NOESY, ROESY, HMQC, and HMBC), the structure of the repeating unit of the fuco-galacto-glucan was established as:     

Structural characterization and study of immunoenhancing and antioxidant property of a novel polysaccharide isolated from the aqueous extract of a somatic hybrid mushroom of Pleurotus florida and Calocybe indica variety APK2

A water-soluble polysaccharide was isolated from the aqueous extract of the fruit bodies of somatic hybrid PCH9FB, obtained through intergeneric protoplast fusion between the strains Pleurotus florida and Calocybe indica var. On the basis of total acid hydrolysis, the polysaccharide was found to contain galactose, fucose, and glucose in a molar ratio of nearly 2:1:2. Methylation analysis and NMR experiments ((1)H, (13)C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HMQC, and HMBC) showed that the structure of the repeating unit present in the polysaccharide was This molecule showed macrophage, splenocyte, thymocyte activation as well as antioxidant property.     

Development and validation of a robust QSAR model for prediction of carcinogenicity of drugs

Carcinogenicity is one of the toxicological endpoints causing the highest concern. Also, the standard bioassays in rodents used to assess the carcinogenic potential of chemicals and drugs are extremely long, costly and require the sacrifice of large numbers of animals. For these reasons, we have attempted development of a global quantitative structure-activity relationship (QSAR) model using a data set of 1464 compounds (the Galvez data set available from http://www.uv.es/-galvez/tablevi.pdf), including many marketed drugs for their carcinogenesis potential. Though experimental toxicity testing using animal models is unavoidable for new drug candidates at an advanced stage of drug development, yet the developed global QSAR model can in silico predict the carcinogenicity of new drug compounds to provide a tool for initial screening of new drug candidate molecules with reduced number of animal testing, money and time. Considering large number of data points with diverse structural features used for model development (n(training) = 732) and model validation (n(test) = 732), the model developed in this study has an encouraging statistical quality (leave-one-out Q2 = 0.731, R2pred = 0.716). Our developed model suggests that higher lipophilicity values and conjugated ring systems, thioketo and nitro groups contribute positively towards drug carcinogenicity. On the contrary, tertiary and secondary nitrogens, phenolic, enolic and carboxylic OH fragments and presence of three-membered rings reduce the carcinogenicity. Branching, size and shape are found to be crucial factors for drug-induced carcinogenicity. One may consider all these points to reduce carcinogenic potential of the molecules.     

Stability analysis and optimal control of an SIR epidemic model with vaccination

This paper focuses on the study of a nonlinear mathematical SIR epidemic model with a vaccination program. We have discussed the existence and the stability of both the disease free and endemic equilibrium. Vaccine induced reproduction number is determined and the impact of vaccination in reducing the vaccine induced reproduction number is discussed. Then to achieve control of the disease, a control problem is formulated and it is shown that an optimal control exists for our model. The optimality system is derived and solved numerically using the Runge-Kutta fourth order procedure.