Rapid detection and counting of single bacteria in a wide field using a photon-counting TV camera

Using Escherichia coli as a model bacterium, we tested a photon-counting method for enumeration of bacteria. This method is based on the principle that microscopic sized luminous particles in a wide field can be directly detected and counted using a photon-counting TV camera without the use of a microscope. E. coli cells were labeled with peroxidase and luminescence induced by adding a luminol-based reaction mixture. The number of luminous spots in the TV images was in good agreement with the number of bacterial colonies grown from labeled cells. The results show that our method provides a rapid and easy microbial counting system for such purposes as clinical diagnosis, microbial analysis in food, and environmental assessment.     

Response of Glutamine Synthetase and Glutamate Synthase Isoforms to Nitrogen Sources in Rice Cell Cultures

As a model system with no photorespiration and no long distancetransport, rice cell cultures (Oryza saliva L. cv Sasanishiki)were used to investigate the effect of nitrogen sources on thelevels of isoforms of glutamine synthetase (GS) and glutamatesynthase (GOGAT). Isoforms of GS and GOGAT were analyzed byimmunoblotting methods and their activities in early growthphase of the cells. Cytosolic type GS (41 kDa subunit) and NADH-GOGATwere the major isoforms in the rice cells grown in normal R-2medium. However, contents of plastid type GS (44 kDa subunit)and Fd-GOGAT increased in response to NO_–³ supply. NADH-GOGATactivity also increased following the supply of NO_–³.In vitro translated products from poly(A)⁺RNA prepared fromthe cells showed that the precursor of plastid type GS (49 kDa)was detected at 48 h after the inoculation. Supply of NH₊⁴ resultedin an increase in NADH-GOGAT activity but had no effect on thelevels of Fd-GOGAT, of polypeptides of the plastid type GS orof the corresponding mRNAs. (Received May 30, 1990; Accepted August 23, 1990)     

The role of IL-4 in proliferation and differentiation of human natural killer cells. Study of an IL-4-dependent versus an IL-2-dependent natural killer cell clone

The role of IL-4 in proliferation and differentiation of human NK cells was studied using newly established sublines of an IL-4-dependent NK cell clone (IL4d-NK cells) and an IL-2-dependent NK cell clone (IL2d-NK cells) derived from a parental conditioned medium-dependent NK cell clone (CM-NK cells). IL-4 induced the higher proliferation of CM-NK cells, but abolished their NK activity and decreased CD16 and CD56 Ag expression. In contrast, IL-2 induced the higher NK activity and increased CD16 and CD56 Ag expression. Addition of anti-IL-4 antibody to the culture of CM-NK cells with CM inhibited the proliferation, but slightly increased NK activity, and largely increased CD56 Ag expression. Addition of anti-IL-2 antibody to the culture of CM-NK cells with CM inhibited both proliferation and cytotoxicity. Proliferation of IL4d-NK cells, which is totally dependent on rIL-4, is greater than that of IL2d-NK cells, which was greater than parental CM-NK cells. Morphologically, IL4d-NK cells are small and round, whereas IL2d-NK cells are large and elongated. Anti-IL-4 antibody inhibited proliferation of IL4d-NK but not IL2d-NK cells, whereas anti-IL-2 antibody inhibited that of IL2d-NK but not IL4d-NK cells. IL-2 was not detected in the supernatant from IL4d-NK cells, nor was IL-2-mRNA expressed in IL4d-NK cells. In contrast, IFN-gamma production and protein expression in IL4d- and IL2d-NK cells were detected. NK cell activation markers (CD16 and CD56) were expressed on IL2d-NK cells but not IL4d-NK cells. IL4d-NK cells were not cytotoxic to any tumor cells tested, whereas IL2d-NK cells displayed potent NK activity and lymphokine-activated killer activity. IL4d-NK cells failed to bind K562 tumor cells, whereas one-third of the IL2d-NK cells did. IL4d-NK cells responded to rIL-2, proliferated, and differentiated into cytotoxic NK cells, whereas IL2d-NK cells failed to respond to rIL-4 and died. These results raise a possibility that IL4d-NK cells or IL2d-NK cells primarily represent the immunologic properties of immature or activated types of human NK cells, respectively. Our results provide the first evidence of the capability of IL-4 to support continuous proliferation of a lymphocyte clone with immature NK cell characteristics and to stimulate IFN-gamma production in the clone. IL-4 is suggested as a potential growth factor for certain types of human NK cell progenitors.     

Structure of a growth-blocking peptide present in parasitized insect hemolymph

Last instar larvae of the insect armyworm, Pseudaletia separata, parasitized with the parasitoid wasp, Apanteles kariyai, do not initiate metamorphosis and, ultimately, the wasp larvae emerge from the host larvae about 10 days after parasitization (Tanaka, T., Agui, N., and Hiruma, K. (1987) Gen. Comp. Endocrinol. 67, 364-374). It is necessary for the parasitoid wasp to perturb the armyworm's endocrinological processes that control normal metamorphosis from larvae to pupae. This endocrinological perturbation allows the parasitoid to complete its larval growth before emerging from the host larvae. It is obligatory for the parasitoid larvae to emerge while the host is still in a larval stage because the sclerotized pupal cuticle is impenetrable for the parasitoid larvae. A growth-blocking peptide with repressive activity against juvenile hormone esterase has been proven to exist in the parasitized host larval plasma (Hayakawa, Y. (1990) J. Biol. Chem. 265, 10813-10816). Here, I describe the detailed structure of this peptide and also the corresponding synthetic peptide to confirm this structure.     

Dual-view microscopy with a single camera: real-time imaging of molecular orientations and calcium

A new microscope technique, termed "W" (double view video) microscopy, enables simultaneous observation of two different images of an object through a single video camera or by eye. The image pair may, for example, be transmission and fluorescence, fluorescence at different wavelengths, or mutually perpendicular components of polarized fluorescence. Any video microscope can be converted into a dual imager by simple insertion of a small optical device. The continuous appearance of the dual image assures the best time resolution in existing and future video microscopes. As an application, orientations of actin protomers in individual, moving actin filaments have been imaged at the video rate. Asymmetric calcium influxes into a cell exposed to an intense electric pulse have also been visualized.     

Purification and characterization of recombinant cell surface protein antigen A of Streptococcus sobrinus B13N.

1. It has been reported that immunization of rhesus monkeys with the surface protein antigen I/II from Streptococcus mutans significantly reduced dental caries. 2. The surface protein antigen A (SpaA) from Streptococcus sobrinus is known to correspond antigenically to I/II. MD51 is an Escherichia coli host containing pMD51, a plasmid encoding the SpaA gene from Streptococcus sobrinus B13N. 3. The recombinant SpaA (rSpaA) was purified from cell extracts of Escherichia coli clone MD51. 4. The purified recombinant SpaA was homogeneous with a molecular weight of 210 kDa according to SDS-PAGE and had an isoelectric point of 4.2 based on isoelectric focusing. 5. Amino acid composition of rSpaA showed a relatively high amount of hydrophobic amino acids (39.7%).     

The high affinity receptor for omega-conotoxin represents calcium channels different from those sensitive to dihydropyridines in mammalian brain

A monoclonal antibody recognizing the alpha 2 delta complex of the dihydropyridine (DHP)-sensitive calcium channel of skeletal muscle immunoprecipitated most of the DHP receptor solubilized from bovine and rabbit brains, and bovine cardiac muscle. However, it did not significantly immunoprecipitate the high affinity omega-conotoxin receptor solubilized from these brains. These results indicate that the DHP receptor and the high affinity omega-conotoxin receptor are different molecules in mammalian brain.     

An active site of growth hormone for eliciting the differentiation of preadipose 3T3-F442A cells to adipose cells

In order to elucidate the active sites of growth hormone for eliciting the differentiation of preadipose 3T3-F442A cells to adipocytes, four artificial mutant variants of human growth hormone (hGH) modified in the loop region of amino acid residues 54-74 were prepared in Escherichia coli by site-directed mutagenesis. Although the P59A (replacement of Pro59 with Ala) variant retained almost the same biological- and receptor binding-activity as hGH, the P61A (replacement of Pro61 with Ala) and the P59A-P61A (replacement of both Pro59 and Pro61 with Ala) both exhibited about half the activity, and the delta (62-67) variant (deletion of the residues 62-67) exhibited only about 0.1% the activity of those of intact hGH. The results suggest that Pro61 may be involved in formation of the active conformation of hGH, but Pro59 may not, and that the amino acid residues around 62-67 may be critical for the specific biological features of hGH.     

Antiviral effects of 2',5'oligoadenylates (2-5As), and related compounds

Dephosphorylated "core" of 2',5'-oligoadenylate (2-5A) dimer (A2'p5'A), exogenously added to nonpermeabilized FL cells, inhibited the multiplication of Sindbis virus and vesicular stomatitis virus (VSV). The compound was shown to inhibit viral protein synthesis. The addition of A2'p5'A at the early stage of viral replication was more effective than that at the late stage. In contrast with the core, phosphorylated 2-5A (p5'A2'p5'A and ppp5'A2'p5'A) and 2-5A analogs containing cordycepin (3'-deoxyadenosine) did not show such antiviral effects. The rate of uptake of [3H]ppp5'A 2'p5'A into acid-soluble and acid-insoluble fractions, especially into the acid-insoluble fraction, was faster than that of [3H]A2'p5'A. These results suggest that the difference of antiviral activity between A2'p5'A and ppp5'A2'p5'A does not result from the different rate of uptake by cells, but from the different rate of from acid-soluble to acid-insoluble fractions.     

Maintenance of embedded pig pancreatic pseudo-islets in a collagen gel matrix: Study of the effect of hydrocortisone,a collagenase inhibitor,and nicotnamide on collagenolysis and the morphogenesis of pancreatic islet-cells in collagen gel matrix

Summary We describe a method for maintaining neonatal pig pancreatic isletlike cell clusters (as pseudo-islets) embedded in a collagen gel matrix for long periods. The pseudo-islets were formed from single cells of pig pancreas maintained in a suspension culture and then embedded in pepsin-solubilized type I collagen. When the pseudo-islets were cultured in the collagen matrix, the amount of collagen in the culture decreased gradually during the culture period as soluble hydroxyproline-containing material accumulated in the medium. A low concentration of collagen (0.16%) degraded the collagen gels more rapidly than did high concentrations of collagen (0.64%). The degradation of collagen depended both on the number of pseudo-islets embedded in the gel matrix and on the culture conditions used to maintain them. With added nicotinamide, the accumulation of hydroxyproline decreased in the medium and the structure of the gel matrix was well maintained. Hydrocortisone or a specific inhibitor of collagenase did not decrease the solubilization of embedded pseudo-islet cultures and did not help to maintain their structure. These observation indicate the possible utility of long-term maintenance of pseudo-islets in collagen gel matrix in the presence of nicotinamide. This work was supported by a Grant-in-Aid for Scientific Research in Japan