Use Of Whole Cells Research Articles

Delayed rectifier K+ current in embryonic chick heart ventricle.

Single ventricular cells from 7- to 10-day-old embryonic chicks were studied with the use of whole cell, outside-out, and cell-attached patch-clamp techniques. The macroscopic delayed rectifier current, IK, activated at membrane potentials above -25 mV. Peak IK at +40 mV was 103 +/- 20 pA (5.7 +/- 1 microA/cm2). IK was selective for K ions with reversal potentials close to the Nernst equilibrium potentials. The onset of current during a voltage step was sigmoidal and was fit by the function IK = IKoo (1-e-t/tau)2. The peak time constant of activation was greater than 6 s at -25 mV (22 degrees C). Significant inactivation was not observed. IK was blocked by intracellular cesium, extracellular 1 mM 4-aminopyridine, 20 mM tetraethylammonium chloride, and 1 mM barium chloride. Single-channel recordings revealed a K+-selective channel with a slope conductance of 15 pS (extracellular [K+] = 4 mM, intracellular [K+] = 145 mM). An ensemble average of consecutive single-channel traces reproduced the whole cell current. The single-channel density was approximately 0.04/micron 2 based on frequency of patches containing the 15-pS single conductance. Approximately 100 channels/cell would sum to account for the net IK. We have described for the first time a channel underlying the main delayed rectifier current and, as such, a main repolarization current in chick ventricle.

Monoclonal antibodies that bind to the My23 human myeloid cell surface molecule: Epitope analysis and antigen modulation studies

It has previously been shown that the AML-2–23 monoclonal antibody (MoAb) reacts with a glycoprotein on differentiated myeloid cells. The antigen, My23, is released from these cells in culture and is also detectable in normal human plasma. We have now raised a panel of MoAbs against the soluble form of the antigen which reacts with monocytes and calcitriol-treated U937 and HL-60 myeloid cell lines, but not with lymphocytes or undifferentiated U937 and HL-60 cells. As with the AML-2–23 MoAb, the anti-My23 MoAbs immunoprecipitated a 50,000–55,000 protein from calcitriol treated HL-60 cells. Besides binding to cell surface My23, all eight MoAbs as well as the 63D3 MoAb reacted with crude and purified forms of soluble My23. A novel ELISA epitope analysis assay was developed to identify four distinct antigenic determinants on My23. Thus, the soluble and cell surface forms of My23 share several antigenic determinants and are biochemically very similar. Pooled anti-My23 caused selective patching, capping and clearing of My23 from the cell surface. My23 was not associated with cell surface, HLA-DR, HLA-A,B,C, β 2-microglobulin or the Fc receptors for IgG or IgA. These anti-My23 MoAbs should be of importance in antigen functional studies and in clinical treatment protocols for patients with acute myelogenous leukemia. Furthermore, we have shown that a soluble myeloid differentiation antigen can serve as an effective immunogen for the preparation of MoAbs against important cell surface molecules thus obviating many problems inherent in the use of whole cells or detergent lysate-derived immuno-precipitates as immunogens.

Enzyme linked immunosorbent assay for detecting antibody to Trichomonas vaginalis: use of whole cells and aqueous extract as antigen.

An enzyme linked immunosorbent assay (ELISA) for detecting antibody to antigenic Trichomonas vaginalis macromolecules has been identified using whole cells or an aqueous protein extract as antigen. The test was developed under optimum conditions using serum samples from experimental animals. The sensitivity of the ELISA was equal to or greater than that obtained by radioimmunoprecipitation and electrophoresis-fluorography techniques. The ELISA was capable of assessing antibody responses during the development of lesions in animals inoculated subcutaneously and it reproducibly measured the individual classes immunoglobulins directed at T vaginalis. The colorimetric assay was also suitable for showing cross reactivity between trichomonal species as well as between different strains of T vaginalis. Conditions established for monitoring antibody to trichomanads in immunised rabbits or infected mice were equally effective for human materials, such as serum or vaginal washes. Serum from experimental animals or infected people showed high concentrations of IgG, IgA, and IgM antibody to trichomonads. Only antibodies of the IgG and IgA class were detected in vaginal washes from women with acute trichomoniasis. No IgE antibody to trichomonads was found under a variety of conditions in serum samples from patients or experimental animals.

A Modified, Highly Sensitive Enzyme-linked Immunosorbent Assay (ELISA) for Rhizobium meliloti Strain Identification

An evaluation of the ELISA technique was made in order to obtain a very specific and sensitive method for strain identification of Rhizobium meliloti. Antisera against an R. meliloti strain were produced by using as antigen either whole cells or purified lipopolysaccharides from the cell wall. The use of whole cells as antigen gave rise to antibodies which were more sensitive and strain specific than those produced from purified lipopolysaccharides. A further evaluation of the ELISA method was the use of a new type of conjugate consisting of a purified antibody linked to β-galactosidase by using as coupling reagent a hetero-bifunctional reagent, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), which contains two reactive groups directed towards different functional groups. Substrate for this type of conjugate consisted of o-nitrophenyl-β-D-galactopyranoside. This improved technique makes it possible to detect bacteria in samples containing 1 × 103 cells ml−1 whilst retaining strain specificity. Furthermore, preparations of nodules formed by different R. meliloti strains containing 1 × 105 cells ml−1 could be analysed whilst retaining strain specificity. The technique presented thus offers advantages in higher sensitivity and reliability than conventional ELISA techniques.

Adhesion of enterotoxigenic Escherichia coli to immobolized intestinal mucosal preparations: a model for adhesion to mucosal surface components

The ability of enterotoxigenic strains of E. coli to adhere to immobilized mucosal components prepared from the large and small intestines of mice was examined in vitro. Various strains of E. coli were labeled with ( 3H)-acetate and incubated in tissue culture plates containing immobilized mucosal components or bovine serum albumin. E. coli strains which were positive for K88 or K99 antigen, and one E. coli strain isolated from a human urinary tract infection, were shown to adhere readily to large and small intestinal mucosal preparations, but not to bovine serum albumin. E. coli K-12 and a variety of enterotoxigenic strains isolated from humans, including a CFA/1 positive strain, demonstrated little or no ability to adhere to any of the preparations. E. coli adhesion to the mucosal preparations was shown to be mannose-resistant for all E. coli strains tested, but was inhibited by growth of the organisms at 18°C. Adhesion of each of the K88 or K99 positive strains was inhibited by homologous antiserum, but not by heterologous antiserum or normal rabbit serum. The data indicate that the mucosal preparations employed contain receptors for specific bacterial adhesins, and suggest that the use of such preparations may provide an alternative to the use of whole cells both as a source of receptor and as a means of investigating the adhesive properties of E. coli.

Methodologic problems encountered in the assay of proteinases in Lewis lung carcinoma, a mouse metastasizing tumor.

The proteolytic activity in homogenates and extracts of subcellular fractions prepared from subcutaneous Lewis lung carcinoma was determined using proteins and synthetic peptides as substrates. The presence of cathepsin D, plasminogen activator, cathepsin B-, cathepsin G- and elastase-like enzymes was observed. No difference was revealed between the proteolytic activity in homogenates of Lewis lung carcinoma, at the growth stage examined, and in homogenates of normal lung. High specific activities were found in the lysosomal extract, whereas decreasing activities were found in the nuclear extract, the homogenate and the postlysosomal mitochondrial supernatant; no active or trypsin-activatable collagenase activity was detected. The presence in the tumor tissue of these enzymatic activities is in agreement with their proposed role in the process of metastasis. The lack of differences between homogenates of tumor and normal lung tissue suggests that the use of whole cells is required to selectively study tumor proteinases specifically involved in tumor malignancy.

Biosynthetic methods for incorporating positron‐emitting radionuclides into compounds of biomedical interest

AbstractThe progress that has been made in incorporating the short‐lived, positron‐emitting isotopes N‐13 and C‐11, into biologically active compounds by biosynthetic means is reviewed. These methods include the use of whole cells or plant leaves to carry out a sequence of metabolic reactions, such as nitrogen fixation or photosynthesis, and the use of purified enzymes to catalyze specific chemical reactions, to form the labeled compounds.