Flow cytometry: an improved method for the selection of highly productive gene-amplified CHO cells using flow cytometry.

In previous work, we clarified the relationship between the productivity and stability of gene-amplified cells and the location of the amplified gene. The location of the amplified gene enabled us to classify resistant cells into two types. One type of resistant cell group, in which the amplified genes were observed near the telomeric region, was named the "telomere type." The other type of cell group, in which the amplified genes were observed in other chromosomal regions, was named the "other type." The phenotypes of these two types of cells are very different. In this experiment, using a fluorescein isothiocyanate-labeled methotrexate (F-MTX) reagent with flow cytometry, we were easily able to distinguish between highly productive cells and the other types of cells. The level of fluorescence differed according to the difference in resistance to MTX. Based on this new finding, highly productive gene-amplified cells could be isolated from heterogeneous gene-amplified cell pools more easily than by the method of limiting-dilution assay. The limiting-dilution method requires several months to obtain highly productive gene-amplified cells, while our flow-cytometry-based method of selection requires only a few weeks.     

Characterization of a phospholipase C beta 2-binding site near the amino-terminal coiled-coil of G protein beta gamma subunits

In previous work (Sankaran, B., Osterhout, J., Wu, D., and Smrcka, A. V. (1998) J. Biol. Chem. 273, 7148-7154), we showed that overlapping peptides, N20K (Asn(564)-Lys(583)) and E20K (Glu(574)-Lys(593)), from the catalytic domain of phospholipase C (PLC) beta2 block Gbetagamma-dependent activation of PLC beta2. The peptides could also be directly cross-linked to betagamma subunits with a heterobifunctional cross-linker succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate. Cross-linking of peptides to Gbeta(1) was inhibited by PLC beta2 but not by alpha(i1)(GDP), indicating that the peptide-binding site on beta(1) represents a binding site for PLC beta2 that does not overlap with the alpha(i1)-binding site. Here we identify the site of peptide cross-linking and thereby define a site for PLC beta2 interaction with beta subunits. Each of the 14 cysteine residues in beta(1) were altered to alanine. The ability of the PLC beta2-derived peptide to cross-link to each betagamma mutant was then analyzed to identify the reactive sulfhydryl moiety on the beta subunit required for the cross-linking reaction. We find that C25A was the only mutation that significantly affected peptide cross-linking. This indicates that the peptide is specifically binding to a region near cysteine 25 of beta(1) which is located in the amino-terminal coiled-coil region of beta(1) and identifies a PLC-binding site distinct from the alpha subunit interaction site.     

Effects of antisense oligodeoxynucleotides against opioid receptors on the receptor mRNA contents

It was demonstrated in the previous study that the microinjection of antisense oligodeoxynucleotide (AS ODN) against mu-opioid receptor (MOR) into periaqueductal gray (PAG) of rat brain selectively decreased the MOR mRNA content in PAG, and the decrease in MOR mRNA content was enhanced by pretreatment of the PAG with MOR AS ODN. In the present investigation, effects of the pretreatment of PAG with AS ODN against kappa- or delta-opioid receptor (KOR or DOR) on the decrease in the MOR mRNA content induced by MOR AS ODN were examined. Both KOR and DOR AS ODNs significantly decreased the target mRNA contents, while they did not significantly change MOR mRNA content. The decrease in MOR mRNA content induced by MOR AS ODN, however, was significantly enhanced by the pretreatment of PAG with either KOR or DOR AS ODNs. Results show that the AS ODN has both the specific target mRNA decreasing action and the nonspecific enhancing action on the AS-induced decrease in the mRNA content.     

Patterning neuronal and glia cells on light-assisted functionalised photoresists

A common photosensitive polymeric material used in semiconductor microlithography (diazo-naphto-quinone/novolak resist) was pattern-exposed with near-UV light to create carboxylic-rich areas on the polymer surface. The patterned surfaces were further functionalised via: (1) the anchorage of peptides for specific cell-attachment or cell-detachment functions; or (2) the diffusion of silicon rich chemical species to achieve the cell detachment. Pairs of antagonistic surface characteristics controlled the cell attachment: (1) amino-rich or carboxylic-rich surfaces; and (2) hydrophilic or hydrophobic surfaces; in which the former promoted the adhesion. It was found that common microlithographic materials and techniques can be upgraded to allow an effective control of the lateral organisation of the artificial arrays of neuronal and glia cells.     

Dynamic analysis of T-lymphocyte function in relation to hepatopathologic changes and effect of interleukin-12 treatment in mice infected with Schistosoma japonicum

We analyzed the dynamics of splenic T-lymphocyte function in relation to hepatopathologic changes in C3H/Hc mice, experimentally infected with Schistosoma japonicum. Vigorous granuloma formation was observed at 7 wk postinfection. At 10 wk postinfection, granuloma formation entered into the down-modulation stage, as represented by the diminished granuloma size. The Th2 response was activated when eggs appeared in the liver, whereas Th1 responses were depressed and the proliferation of T lymphocytes was decreased. The level of IgG antibodies to the worm and egg antigens rose continually after infection. Interleukin-12 treatment of infected mice inhibited Th2 responses and T-cell proliferation, decreased granuloma formation and fibrosis, but had no effect on the fecundity of the worms. These data suggest that egg deposition is the major factor driving Th2 responses, depressing Th1 cytokine expression as well as T-cell proliferation in S. japonicum-infected mice.     

Gradients in cytoplasmic calcium concentration ([Ca2+]i) in migrating human umbilical vein endothelial cells (HUVECs) stimulated by shear-stress

Using a parallel-plate flow-chamber and confocal laser scanning microscopy (CLSM), we studied the distribution and temporal changes in intracellular Ca2+ concentration ([Ca2+]i) in migrating HUVECs stimulated by shear-stress. In the presence or absence of ATP, shear-stress (10 dyne/cm2) caused morphological change and migration of individual HUVECs in the random direction. After 120 minute exposure to shear-stress, 70% of the cells migrated in the direction of flow, whereas, as many as 30% of the cells migrated to the upstream against flow. A nonspecific plasma membrane Ca2+ channel blocker, Ni2+, abolished such responses markedly, suggesting that Ca2+ influx may be essential for shear-stress dependent morphological change and migration of HUVECs. Analysis of [Ca2+]i distribution revealed the appearance of localized [Ca2+]i elevation inside lamellipodium formed in the direction of cell migration. The localized rise in [Ca2+]i might be closely related with morphological change to regulate the direction of cell migration induced by shear-stress.     

High efficient expression of the functional ligand binding site of the inositol 1,4,5-triphosphate receptor in Escherichia coli

Type 1 inositol 1,4,5-trisphosphate receptor (IP3R1), an inositol 1, 4,5-trisphosphate (IP3)-gated Ca2+ release channel, binds IP3 within the N-terminal ligand-binding region. Here we report an improved Escherichia coli expression system in which large amounts of the IP3 binding sites could be efficiently produced as soluble active proteins. We have found that the structures of IP3 binding constructs expressed in E. coli significantly affect their production as soluble protein. Residues 1-604 (T604), which contain the putative protein folding units, yielded about 4.6% of the total soluble fraction. As a result, soluble active T604 would be 19 mg per liter of culture. The affinity for IP3 of T604 (Kd = 45 nM) is comparable to that of the native IP3R1, whereas that of an R441Q mutant is much higher (8.1 nM). This system should provide an invaluable and powerful means to unveil the molecular recognition of IP3R1 for IP3.     

Examination of the reaction of fully reduced cytochrome oxidase with hydrogen peroxide by flow-flash spectroscopy

The reaction of cytochrome c oxidase with hydrogen peroxide has been of great value in generating and characterizing oxygenated species of the enzyme that are identical or similar to those formed during turnover of the enzyme with dioxygen. Most previous studies have utilized relatively low peroxide concentrations (millimolar range). In the current work, these studies have been extended to the examination of the kinetics of the single turnover of the fully reduced enzyme using much higher concentrations of peroxide to avoid limitations by the bimolecular reaction. The flow-flash method is used, in which laser photolysis of the CO adduct of the fully reduced enzyme initiates the reaction following rapid mixing of the enzyme with peroxide, and the reaction is monitored by observing the absorbance changes due to the heme components of the enzyme. The following reaction sequence is deduced from the data. (1) The initial product of the reaction appears to be heme a(3) oxoferryl (Fe(4+)=O(2)(-) + H(2)O). Since the conversion of ferrous to ferryl heme a(3) (Fe(2+) to Fe(4+)) is sufficient for this reaction, presumably Cu(B) remains reduced in the product, along with Cu(A) and heme a. (2) The second phase of the reaction is an internal rearrangement of electrons and protons in which the heme a(3) oxoferryl is reduced to ferric hydroxide (Fe(3+)OH(-)). In about 40% of the population, the electron comes from heme a, and in the remaining 60% of the population, Cu(B) is oxidized. This step has a time constant of about 65 micros. (3) The third apparent phase of the reaction includes two parallel reactions. The population of the enzyme with an electron in the binuclear center reacts with a second molecule of peroxide, forming compound F. The population of the enzyme with the two electrons on heme a and Cu(A) must first transfer an electron to the binuclear center, followed by reaction with a second molecule of peroxide, also yielding compound F. In each of these reaction pathways, the reaction time is 100-200 micros, i.e., much faster than the rate of reaction of peroxide with the fully oxidized enzyme. Thus, hydrogen peroxide is an efficient trap for a single electron in the binuclear center. (4) Compound F is then reduced by the final available electron, again from heme a, at the same rate as observed for the reduction of compound F formed during the reaction of the fully reduced oxidase with dioxygen. The product is the fully oxidized enzyme (heme a(3) Fe(3+)OH(-)), which reacts with a third molecule of hydrogen peroxide, forming compound P. The rate of this final reaction step saturates at high concentrations of peroxide (V(max) = 250 s(-)(1), K(m) = 350 mM). The data indicate a reaction mechanism for the steady-state peroxidase activity of the enzyme which, at pH 7.5, proceeds via the single-electron reduction of the binuclear center followed by reaction with peroxide to form compound F directly, without forming compound P. Peroxide is an efficient trap for the one-electron-reduced state of the binuclear center. The results also suggest that the reaction of hydrogen peroxide to the fully oxidized enzyme may be limited by the presence of hydroxide associated with the heme a(3) ferric species. The reaction of hydrogen peroxide with heme a(3) is very substantially accelerated by the availability of an electron on heme a, which is presumably transferred to the binuclear center concomitant with a proton that can convert the hydroxide to water, which is readily displaced.     

Folding and unfolding of a giant duplex-DNA in a mixed solution with polycations, polyanions and crowding neutral polymers

To understand the conformational behavior of a giant duplex-DNA chain in a mixed solution with various biopolymers with different state of ionization, the higher-order structure of the DNA chain was analyzed with a fluorescence microscope in the presence of polycations (poly-arginine), polyanions (poly-glutamic acid), and neutral polymers (poly-ethylene glycol) as a model for cellular environment. Concentrated medium with neutral polymer induced the discrete folding transition of the DNA. At the threshold condition for the transition, addition of small amounts of either the polycation or the polyanion caused marked structural changes in the folded DNAs. Based on thermodynamic considerations on the experimental results, profile of free energy of a single giant DNA chain was depicted with respect to the size, or the expansion factor alpha, in the three-dimensional structure of the DNA. The effect of the neural crowding polymer on the degree of folding of a single giant DNA chain is discussed in a semi-quantitative manner.