Dynamics and Distribution of Cyanophages and Their Effect on Marine Synechococcus spp

Cyanophages infecting marine Synechococcus cells were frequently very abundant and were found in every seawater sample along a transect in the western Gulf of Mexico and during a 28-month period in Aransas Pass, Tex. In Aransas Pass their abundance varied seasonally, with the lowest concentrations coincident with cooler water and lower salinity. Along the transect, viruses infecting Synechococcus strains DC2 and SYN48 ranged in concentration from a few hundred per milliliter at 97 m deep and 83 km offshore to ca. 4 x 10 ml near the surface at stations within 18 km of the coast. The highest concentrations occurred at the surface, where salinity decreased from ca. 35.5 to 34 ppt and Synechococcus concentrations were greatest. Viruses infecting strains SNC1, SNC2, and 838BG were distributed in a similar manner but were much less abundant (<10 to >5 x 10 ml). When Synechococcus concentrations exceeded ca. 10 ml, cyanophage concentrations increased markedly (ca. 10 to > 10 ml), suggesting that a minimum host density was required for efficient viral propagation. Data on the decay rate of viral infectivity d (per day), as a function of solar irradiance I (millimoles of quanta per square meter per second), were used to develop a relationship (d = 0.2610I - 0.00718; r = 0.69) for conservatively estimating the destruction of infectious viruses in the mixed layer of two offshore stations. Assuming that virus production balances losses and that the burst size is 250, ca. 5 to 7% of Synechococcus cells would be infected daily by viruses. Calculations based on contact rates between Synechococcus cells and infectious viruses produce similar results (5 to 14%). Moreover, balancing estimates of viral production with contact rates for the farthest offshore station required that most Synechococcus cells be susceptible to infection, that most contacts result in infection, and that the burst size be about 324 viruses per lytic event. In contrast, in nearshore waters, where ca. 80% of Synechococcus cells would be contacted daily by infectious cyanophages, only ca. 1% of the contacts would have to result in infection to balance the estimated virus removal rates. These results indicate that cyanophages are an abundant and dynamic component of marine planktonic communities and are probably responsible for lysing a small but significant portion of the Synechococcus population on a daily basis.     

Detecting Enzymatic Activity in Cells Using Fluorogenic Substrates

Fluorogenic substrates can detect enzymatic activity associated with cells. It is difficult, however, to detect activity within a single cell or in an organelle since hydrolytic substrates yield products that rapidly leak from the cell. Several new solutions are presented including trapping the fluorescent product in membranes, in cell organelles, or as a glutathione conjugate. Novel substrates also are described that directly yield highly fluorescent precipitates at the site of enzymatic activity. These can be used for detecting endogenous activity in cells or for enzyme-amplified histochemical detection. Some of these substrates can be used in live cells.     

Flow cytometric identification of Paclitaxel-accumulating subpopulations

An immunofluorescence procedure was developed for paclitaxel quantification at the single cell level via flow cytometry in Taxus cuspidata suspension cultures. Intracellular staining was validated via fluorescence microscopy. Paclitaxel content of isolated cells and protoplasts was compared to total paclitaxel levels measured via HPLC. Paclitaxel accumulation was significantly increased by elicitation with methyl jasmonate (100 microM) on day 7 post-transfer as compared to unelicited cultures. Maximum accumulation was observed by day 12 post-transfer in both total paclitaxel (approximately 0.25 mg/L) and the percentage of paclitaxel-accumulating cells (approximately 95%). A similar trend was observed with isolated protoplasts, although protoplasts accumulated only ca. 40-75% of the paclitaxel present in single cells. In unelicited cell cultures, a small subpopulation (ca. 3-5%) of single cells was shown to accumulate paclitaxel. Although nearly all cells were observed to accumulate paclitaxel in methyl jasmonate-elicited cell cultures, a high degree of cell-to-cell variation was observed in paclitaxel content. The identified subpopulations represent targets for cell sorting, which may be applied to develop higher-accumulating cell lines. The quantification of single cell paclitaxel content is useful for characterizing production variability in cell cultures and can be utilized to develop rational strategies to increase paclitaxel production.     

Estimating average cellular turnover from 5-bromo-2'-deoxyuridine (BrdU) measurements

Cellular turnover rates in the immune system can be determined by labelling dividing cells with 5-bromo-2'-deoxyuridine (BrdU) or deuterated glucose ((2)H-glucose). To estimate the turnover rate from such measurements one has to fit a particular mathematical model to the data. The biological assumptions underlying various models developed for this purpose are controversial. Here, we fit a series of different models to BrdU data on CD4(+) T cells from SIV(-) and SIV(+) rhesus macaques. We first show that the parameter estimates obtained using these models depend strongly on the details of the model. To resolve this lack of generality we introduce a new parameter for each model, the 'average turnover rate', defined as the cellular death rate averaged over all subpopulations in the model. We show that very different models yield similar estimates of the average turnover rate, i.e. ca. 1% day(-1) in uninfected monkeys and ca. 2% day(-1) in SIV-infected monkeys. Thus, we show that one can use BrdU data from a possibly heterogeneous population of cells to estimate the average turnover rate of that population in a robust manner.     

Efficient transfection of primarily cultured porcine embryonic fibroblasts using the Amaxa Nucleofection system

Porcine embryonic fibroblasts (PEF) are important as donor cells for nuclear transfer for generation of genetically modified pigs. In this study, we determined an optimal protocol for transfection of PEF with the Amaxa Nucleofection system, which directly transfers DNA into the nucleus of cells, and compared its efficiency with conventional lipofection and electroporation. Cell survival and transfection efficiency were assessed using dye-exclusion assay and a green fluorescent protein (GFP) reporter construct, respectively. Our optimized nucleofection parameters yielded survival rates above 60%. Under these conditions, FACS analysis demonstrated that 79% of surviving cells exhibited transgene expression 48 h after nucleofection when program U23 was used. This efficiency was higher than that of transfection of PEFs with electroporation (ca. 3-53%) or lipofection (ca. 3-8%). Transfected cells could be expanded as stably transgene-expressing clones over a month. When porcine nuclear transfer (NT) was performed using stable transformant expressing GFP as a donor cell, 5-6% of reconstituted embryos developed to blastocysts, from which 30-50% of embryos exhibited NT-embryo-derived green fluorescence. Under the conditions evaluated, nucleofection exhibited higher efficiency than conventional electroporation and lipofection, and may be a useful alternative for generation of genetically engineered pigs through nuclear transfer.     

Creatine and creatinine transport in old and young human red blood cells

The time course of creatine influx or efflux as measured in populations of red cells or red cell ghosts with normal age distribution does not follow simple two-compartment kinetics. This suggests that the contributions of individual cells to transport as measured in the populations as a whole are not uniform. In agreement with this inference, fractionation of red cell populations with respect to cell age shows that transport in young cells is considerably faster than in old cells.The dependence of creatine transport on creatine concentration in the medium follows an equation that can be interpreted to represent a super-imposition of a saturable component (apparent K_m = 0.02 mM) and another component that cannot be saturated up to a creatine concentration of 5.0 mM. In contrast to the non-saturable component, the saturable component depends on the energy metabolism of the cell and can be inhibited by β-guanidinopropionic acid and the proteolytic enzyme pronase. This latter finding suggests that the saturable component represents active transport that is mediated by a transport protein. The non-saturable component is little, if at all, dependent on cell age while the saturable component is higher in young cells than in old cells. Phloretin inhibits both components of creatine flux, but the maximal inhibition that can be achieved at high concentration is only 70–80%.Under the experimental conditions used for the study of creatine transport, creatinine equilibration between cells and medium follows the kinetics expected for a steady-state two-compartment system. Creatinine flux is proportional to creatine concentration over the concentration range studied (up to 5 mM). It cannot be inhibited by β-guanidinopropionic acid or pronase.     

Isolement d'une alcool déshydrogénase chez Rhizobium et rôle dans le métabolisme indolique

Rhizabium meliloti contains an alcohol dehydrogenase (E.C.1.1.1.1.) which can be isolated by breaking the cells. This soluble enzyme was purified 16.1-fold by fractional precipitations with ammonium sulfate followed by gel filtration on Sephadex. The activity of the enzyme was tested with various aldehydes as substrates in the presence of NADH. Indole-3-acetaldehyde (IAAld) can be reduced to tryptophol (Tr-ol), and the optimal pH for this reaction is ca. 6.5. The reaction can be reversed, and Tr-ol is oxidised in the presence of NAD, but is was found that the yield was very poor; the optimal pH was ca. 8.6. This alcohol dehydrogenase is responsible for Tr-ol formation in Rhizobium, but under our experimental conditions tryptophol cannot really be considered as a precursor of IAAld and indole-3-acetic acid.     

Coupling of canine serotonin 5-HT(1B) and 5-HT(1D) receptor subtypes to the formation of inositol phosphates by dual interactions with endogenous G(i/o) and recombinant G(alpha15) proteins

Molecular cloning and expression of canine (ca) serotonin 5-HT(1B) and ca 5-HT(1D) receptor subtypes showed that besides the lower binding affinity of ketanserin for the ca 5-HT(1D) receptor, the ligand binding profiles were similar to their human homologues. Site-directed mutagenesis studies suggest that a Gln(189) residue in the second extracellular loop of the ca 5-HT(1D) receptor may partially account for the lower binding affinity of ketanserin. The coupling of ca 5-HT(1B) and ca 5-HT(1D) receptor subtypes to the phospholipase C pathway was analyzed by measuring stimulation of inositol phosphate formation in COS-7 cells. Zolmitriptan potently stimulated (EC(50) = 4.9 nM) the inositol phosphate formation at ca 5-HT(1D) receptors in a fully pertussis toxin (PTX)-dependent manner, whereas only a weak PTX-resistant inositol phosphate response (26-29% at 10 microM zolmitriptan) could be detected for the ca 5-HT(1B) receptor at a similar expression level. In contrast, both ca 5-HT(1B) and ca 5-HT(1D) receptor subtypes yielded a similar maximal magnitude of inositol phosphate formation (300-340% at 10 microM zolmitriptan) upon co-expression with a mouse (m) G(alpha15) protein. PTX treatment and co-expression with a beta-adrenergic receptor kinase C-terminal polypeptide partially (20-46%) abolished the m G(alpha15) protein-dependent ca 5-HT(1B) and ca 5-HT(1D) receptor-mediated stimulation of inositol phosphate formation. This study suggests both 5-HT receptor subtypes can activate betagamma subunits of endogenous G(i/o) proteins besides their coupling to recombinant m G(alpha15) protein.     

Cell-density-dependent lysis and sporulation of Myxococcus xanthus in agarose microbeads.

Vegetative cells of Myxococcus xanthus were immobilized in 25-microns-diameter agarose microbeads and incubated in either growth medium or sporulation buffer. In growth medium, the cells multiplied, glided to the periphery, and then filled the beads. In sporulation buffer, up to 90% of the cells lysed and ca. 50% of the surviving cells formed resistant spores. A strong correlation between sporulation and cell lysis was observed; both phenomena were cell density dependent. Sporulation proficiency was a function of the average number of cells within the bead at the time that sporulation conditions were imposed. A minimum of ca. 4 cells per microbead was necessary for efficient lysis and sporulation to proceed. Increasing this number accelerated the lysis and sporulation process. No lysis occurred when an average of 0.4 cell was entrapped per bead. Entrapping an average of 1.7 cells per bead resulted in 46% lysis and 3% sporulation of survivors, whereas entrapping an average of 4.2 cells per bead yielded 82% lysis and 44% sporulation of the surviving cells. Sporulation and lysis also depended upon the cell density in the culture as a whole. The existence of these two independent cell density parameters (cells per bead and cells per milliliter) suggests that at least two separate cell density signals play a role in controlling sporulation in M. xanthus.     

A rotary molecular motor that can work at near 100% efficiency

A single molecule of F1-ATPase is by itself a rotary motor in which a central gamma-subunit rotates against a surrounding cylinder made of alpha3beta3-subunits. Driven by the three betas that sequentially hydrolyse ATP, the motor rotates in discrete 120 degree steps, as demonstrated in video images of the movement of an actin filament bound, as a marker, to the central gamma-subunit. Over a broad range of load (hydrodynamic friction against the rotating actin filament) and speed, the F1 motor produces a constant torque of ca. 40 pN nm. The work done in a 120 degree step, or the work per ATP molecule, is thus ca. 80 pN nm. In cells, the free energy of ATP hydrolysis is ca. 90 pN nm per ATP molecule, suggesting that the F1 motor can work at near 100% efficiency. We confirmed in vitro that F1 indeed does ca. 80 pN nm of work under the condition where the free energy per ATP is 90 pN nm. The high efficiency may be related to the fully reversible nature of the F1 motor: the ATP synthase, of which F1 is a part, is considered to synthesize ATP from ADP and phosphate by reverse rotation of the F1 motor. Possible mechanisms of F1 rotation are discussed.     

Establishment of a persistent baculovirus infection in a lepidopteran cell line

The paper describes the first overt attempt to establish an insect cell line (Spodoptera frugiperda), persistently infected with its homologous baculovirus. The persistently infected cells were morphologically different and grew to a higher density than the noninfected parent line. The parent line, however, had a shorter doubling time. Persistently infected cells were passaged 40 times over 10 months; they still continued to produce infectious virus and polyhedral inclusion bodies. However, the infectious viral titer was ca. 100 times lower in the persistently infected line than in the parent line; also, the number of inclusion bodies was reduced ca. 98%. Interference with both homologous and heterologous baculoviruses was demonstrated in the persistently infected cell line. Sevently percent of the persistently infected cells contained antigens for S. frugiperda nuclear polyhedrosis virus, ca. 1% of the cells showed infectious viral centers, and ca. 3% of the cells contained inclusion bodies. Although the inclusion bodies from the persistently infected cells were infectious for S. frugiperda larvae, they were about 3 times less infectious than the inclusion bodies produced in the parent line.     

Disulfide-bonded aggregates of heparan sulfate proteoglycans

Heparan sulfate proteoglycans have been isolated from Swiss mouse 3T3 cells by using two nondegradative techniques: extraction with 4 M guanidine or 2.5% 1-butanol. These proteoglycans were separated from copurifying chondroitin sulfate proteoglycans by using ion-exchange chromatography on DEAE-cellulose in the presence of 2 M urea. The purified heparan sulfate proteoglycans are substantially smaller, ca. Mr 20 000, than those isolated from these same cells with trypsin, ca. Mr 720 000 [Johnston, L.S., Keller, K. L., & Keller, J. M. (1979) Biochim. Biophys. Acta 583, 81-94]. However, all of the heparan sulfate proteoglycans extracted by these three methods contain similar glycosaminoglycan chains (Mr 7500) and are derived from the same pool of cell surface associated molecules. The trypsin-released heparan sulfate proteoglycan (ca. Mr 720 000) can be significantly reduced in size (ca. Mr 33 000) under strong denaturing conditions in the presence of the disulfide reducing agent dithiothreitol, which suggests that this form of the molecule is a disulfide-bonded aggregate. The heparan sulfate proteoglycan isolated from the medium also undergoes a significant size reduction in the presence of dithiothreitol, indicating that a similar aggregate is formed as part of the normal release of heparan sulfate proteoglycans into the medium. These results suggest that well-shielded disulfide bonds between individual heparan sulfate proteoglycan monomers may account for the large variation in sizes which has been reported for heparan sulfate proteoglycans isolated from a variety of cells and tissues with a variety of extraction procedures.     

The histologic and functional characterization of enzymatically dispersed intestinal mast cells of nonhuman primates: effects of secretagogues and anti-allergic drugs on histamine secretion

Despite the apparent involvement of gastrointestinal mast cells in hypersensitivity reactions in the mucosa, remarkably little information is available concerning the characteristics of these cells from man and higher animals. To study the characteristics of gastrointestinal mast cells from nonhuman primates, a previously described technique which uses a combination of mechanical and enzymatic methods to obtain mast cells from the tissues of rodents required modification to permit the successful dispersion of normal gastrointestinal tissues of higher animals. This modified procedure, as described in this report, appears to be relatively selective for mast cells located in the mucosal site, and typically yields ca 9 X 10(5) mast cells per gram of tissue. The mucosal mast cells obtained comprised ca 2% of the total nucleated cells, contained approximately 1 pg of histamine per cell, and stained metachromatically with toluidine blue only at low pH. The cells exhibited a dose-dependent release of histamine on challenge with goat anti-human IgE or the ionophores A23187 and Br-x537A but were refractory to the action of compound 48/80. IgE-mediated histamine release from monkey intestinal mast cells differed from that observed from rat intestinal mast cells in that release was inhibited not only by quercetin but also by theophylline. Disodium cromoglycate gave variable results. The data indicate that viable nonhuman primate mucosal mast cells can be obtained for study, and that these cells, although sharing some characteristics of mucosal mast cells from lower species, have distinct and unique properties. The availability of this nonhuman primate model for the study of mast cell function in higher animals should contribute to the understanding of mast cell-mediated diseases in man.     

Collection, storage and transfusion of blood stem cells for the treatment of hemopoietic failure.

Migration of hemopoietic stem cells via the blood to sites of stem cell need is a principle that becomes established during the embryonic development of hemopoiesis and can be observed in the adult whenever bone marrow transplantations are being performed. The regular presence of stem cells in the peripheral blood lends itself to the study of their collection, storage, and use for transfusion purposes in cases of bone marrow failure. Both in dog and in man, granulocyte-macrophage progenitor cells (CFU-C) can be collected by leukapheresis from the blood in large quantities, particularly if the yield is increased by the administration of mobilizing agents such as dextran sulfate, and appear to be an indicator for the presence of stem cells. For collection and storage, a closed plastic bag system has been developed that allows the safe handling of the cells. The loss of CFU-C from freezing and thawing with DMSO as a cryoprotective agent is only 10%-20%. If frozen and thawed mononuclear leukocytes are transfused into 1200 rad whole-body X-irradiated autologous or allogeneic recipient dogs, a hemopoietic take is observed when 0.2 X 10(5) CFU-C are present among the mononuclear leukocytes (MNC). Graft-versus-host disease can be avoided in the allogeneic situation when a purified CFU-C rich cell fraction is being transfused. In man collection and storage of MNC including CFU-C is feasible and may eventually become a therapeutic tool.     

Cytochemical evaluation of gamma-glutamyl-transpeptidase activity in cervical smears

The cervicovaginal smears of 43 patients attending an outpatient service for early cancer detection were cytochemically studied for the presence of gamma-glutamyl-transpeptidase (GGT) in epithelial cells. This was done in order to evaluate such an enzyme phenotype as a marker for cancer development. The results showed that 70% of the 38 patients with a cytologic diagnosis of "inflammatory" or preneoplastic/neoplastic conditions had GGT-positive cells in their smears. None of the five cytologically normal cases showed any epithelial cells with GGT activity. Although most of the GGT-positive cells were metaplastic, some morphologically normal, dysplastic or neoplastic cells also expressed the enzyme. The data suggest that cytochemically detectable transpeptidase activity appears whenever alterations of the normal epithelial microenvironment occurs, but is not necessarily linked to the carcinogenic process. Therefore, cytochemically GGT-positive cells should not be used as an indicator of neoplastic transformation of the cervical epithelium.     

Dynamics and Distribution of Cyanophages and Their Effect on Marine Synechococcus spp.

Cyanophages infecting marine Synechococcus cells were frequently very abundant and were found in every seawater sample along a transect in the western Gulf of Mexico and during a 28-month period in Aransas Pass, Tex. In Aransas Pass their abundance varied seasonally, with the lowest concentrations coincident with cooler water and lower salinity. Along the transect, viruses infecting Synechococcus strains DC2 and SYN48 ranged in concentration from a few hundred per milliliter at 97 m deep and 83 km offshore to ca. 4 × 10⁵ ml^-1 near the surface at stations within 18 km of the coast. The highest concentrations occurred at the surface, where salinity decreased from ca. 35.5 to 34 ppt and Synechococcus concentrations were greatest. Viruses infecting strains SNC1, SNC2, and 838BG were distributed in a similar manner but were much less abundant (<10 to >5 × 10³ ml^-1). When Synechococcus concentrations exceeded ca. 10³ ml^-1, cyanophage concentrations increased markedly (ca. 10² to > 10⁵ ml^-1), suggesting that a minimum host density was required for efficient viral propagation. Data on the decay rate of viral infectivity d (per day), as a function of solar irradiance I (millimoles of quanta per square meter per second), were used to develop a relationship (d = 0.2610I - 0.00718; r² = 0.69) for conservatively estimating the destruction of infectious viruses in the mixed layer of two offshore stations. Assuming that virus production balances losses and that the burst size is 250, ca. 5 to 7% of Synechococcus cells would be infected daily by viruses. Calculations based on contact rates between Synechococcus cells and infectious viruses produce similar results (5 to 14%). Moreover, balancing estimates of viral production with contact rates for the farthest offshore station required that most Synechococcus cells be susceptible to infection, that most contacts result in infection, and that the burst size be about 324 viruses per lytic event. In contrast, in nearshore waters, where ca. 80% of Synechococcus cells would be contacted daily by infectious cyanophages, only ca. 1% of the contacts would have to result in infection to balance the estimated virus removal rates. These results indicate that cyanophages are an abundant and dynamic component of marine planktonic communities and are probably responsible for lysing a small but significant portion of the Synechococcus population on a daily basis.     

Use of aqueous silver to enhance inactivation of coliphage MS-2 by UV disinfection

A synergistic effect between silver and UV radiation has been observed that can appreciably enhance the effectiveness of UV radiation for inactivation of viruses. At a fluence of ca. 40 mJ/cm(2), the synergistic effect between silver and UV was observed at silver concentrations as low as 10 microg/liter (P < 0.0615). At the same fluence, an MS-2 inactivation of ca. 3.5 logs (99.97%) was achieved at a silver concentration of 0.1 mg/liter, a significant improvement (P < 0.0001) over the ca. 1.8-log (98.42%) inactivation of MS-2 at ca. 40 mJ/cm(2) in the absence of silver. Modified Chick-Watson kinetics were used to model the synergistic effect of silver and UV radiation. For an MS-2 inactivation of 4 logs (99.99%), the coefficient of dilution (n) was determined to be 0.31, which suggests that changes in fluence have a greater influence on inactivation than does a proportionate change in silver concentration.     

Naringenin reduces lung metastasis in a breast cancer resection model

Metastasis is the main cause of death in cancer patients. To improve the outcomes of patients undergoing a surgery, new adjuvant therapies that can effectively inhibit metastases have to be developed. Studies have shown that flavonoid naringenin, a natural product that is mainly present in grapes and citrus, may contribute to cancer prevention. It has many advantages compared to traditional chemotherapeutic drugs, such as low toxicity. To determine whether naringenin can also inhibit metastases, a breast cancer resection model that mimics clinical situations was established. We found that orally administered naringenin significantly decreased the number of metastatic tumor cells in the lung and extended the life span of tumor resected mice. Flow cytometry analysis revealed that T cells displayed enhanced antitumor activity in naringenin treated mice, with an increased proportion of IFN-γ and IL-2 expressing T cells. In vitro studies further demonstrated that relief of immunosuppression caused by regulatory T cells might be the fundamental mechanism of metastasis inhibition by naringenin. These results indicate that orally administered naringenin can inhibit the outgrowth of metastases after surgery via regulating host immunity. Thus, naringenin can be an ideal surgical adjuvant therapy for breast cancer patients.     

A transplantable rat Leydig cell tumor--1. LH and prolactin receptors and effects of the endocrine status of the host animal

We have examined the binding capacity and properties (affinity, specificity) of LH and prolactin (Prl) receptors in a transplantable rat Leydig cell tumor (H-540) grown in intact, castrated and hypophysectomized rats. LH receptors in adult rat testis and Prl receptors in the rat ventral prostate were examined simultaneously for comparison. The results can be summarized as follows: The qualitative properties (affinity, specificity) of LH and Prl receptors in tumor Leydig cells appear to be identical to those of corresponding receptors in non-tumor tissues. The levels of LH receptors in tumor Leydig cells are only some 1% of that present in normal Leydig cells from adult rats. Tumor Leydig cells grown in hypophysectomized rats had even lower levels of LH receptors; ca. 1/3 of that found in tumors from intact rats. The levels of Prl receptors in the tumor Leydig cells are almost as high as in normal Leydig cells from adult rats. In tumors grown in hypophysectomized rats, the levels of Prl receptors were much lower (ca. 20%) than in tumors from intact or castrated rats. There were great variations in the number of LH and Prl receptors in individual tumors, and there was a positive correlation (r = 0.88; P less than 0.01) between LH and Prl receptors in individual tumors. No differentiation toward a "LH receptor tumor" or "Prl receptor tumor" was observed. Thus, receptors for LH and Prl in tumor cells are qualitatively normal, but the number is greatly (LH) or moderately (Prl) reduced. These receptors in the tumor Leydig cells are stimulated by pituitary hormones.     

Separation of cell mixtures by immunoaffinity cell partitioning: strategies for low abundance cells

The partitioning of cells in aqueous two-phase systems formed by poly(ethylene glycol) (PEG) and dextran can be changed by incubating the cells with a PEG-modified antibody directed specifically against its surface. We have developed a new approach for immunoaffinity cell partitioning (IACP) in which the antibodies are first reacted with tresylated monomethoxy PEG (TMPEG) in sodium phosphate buffer, pH 7.5, the excess TMPEG is quenched by reaction with bovine serum albumin, and the resulting preparation is used directly for incubation with the cells without any isolation of the monomethoxyPEG (MPEG)-antibody conjugates. We have demonstrated the specificity of this IACP method by showing that MPEG-modified anti-human red blood cell antibody increases the partition of human erythrocytes from the interface to the PEG-rich top phase (up to 100%) but not the partitioning of either neutrophils or HL60 cells. Irrelevant antibodies do not affect the partitioning of red blood cells. The partitioning behaviors of erythrocytes and HL60 cells in mixtures varying from 75 to 10% red blood cells subjected to IACP are similar to those of the pure cell population, i.e., erythrocytes ca. 100% and HL60 cells 3% in top phase. Thus, the population of erythrocytes can be almost completely extracted into the top phase in a single step. The contaminant cells represent only a small percentage (less than 5% in most of the cases) of the cell mixture recovered in top phase. Both cell populations can be completely separated by countercurrent distribution (CCD).(ABSTRACT TRUNCATED AT 250 WORDS)