Production of monoclonal antibodies against calmodulin by in vitro immunization of spleen cells

Monoclonal antibodies against the highly conserved ubiquitous calcium- binding protein, calmodulin (CaM), were produced by immunization of mouse primary spleen cell cultures. Dissociated spleen cells were cultured for 5 d in the presence of mixed thymocyte culture conditioned media (TCM) and purified bovine testes CaM (50 ng-1 mg). Following immunization, cells were fused with mouse myeloma cells (SP2/0, Ag 8.653) and cultured for 2-3 wk before initial screening for antibody. In five independent immunizations there was a range of 25-44% of the initial polyclonal cultures which produced antibodies reacting with purified CaM as determined by immunoassay. 80% of the cloned hybridoma produced IgM immunoglobulins while the remaining clones were IgG producers. This ratio was changed to 50% IgM and 50% IgG by subsequent extension of the in vitro immunization periods and reduced amounts of antigen and extended in vitro culturing. In vitro immunization introduces a new dimension to monoclonal antibody production where limited antigen or poorly antigenic proteins are of interest. The monoclonal antibodies produced in this study have enabled us to to selectively localize CaM in association with distinct subcellular structures, mitochondria, stress fibers, centrioles, and the mitotic spindle.     

Isolation and identification of a fucose-containing ganglioside from bovine thyroid gland

Bovine thyroid glands are known to contain a complex array of gangliosides. One of the predominant gangliosides was isolated and analyzed by gas-liquid chromatography and mass spectrometry. The carbohydrate composition was fucose, N-acetylneuraminic acid, galactose, N-acetylgalactosamine, and glucose in molar ratios of 1:1:2:1:1. The structure of the ganglioside was identified as:

     

An analysis of the x-ray interchain peak profile in dipalmitoylglycerophosphocholine

The primary X-ray peak profile characterizing the interchain structure in the dipalmitoylglycerophosphocholine membrane has been measured as a function of temperature. The scattering between 23 and 34.6° C is characterized by an asymmetric crystalline reflection accounting for 85% of the total intensity, the remaining 15% being liquid-like in character. At a pre-transition temperature of 34.6° C, the reflection profile becomes (nearly) symmetrical, indicating a change in tilt angle of the chains with respect to the membrane surface. This change is accompanied by an increase of 20% in the amount of liquid-like scattering, indicating that the pre-transition mechanism includes a partial melting of the chains. At the melting point, 41.5° C, the crystalline reflection disappears, and the liquid component of the scattering increases to a point where it includes all the scattered intensity. The relative values of the integrated intensities at each temperature are tabulated, and the significance of the peak widths and shapes are discussed.     

GANGLIOSIDE COMPOSITION AND BIOSYNTHESIS IN CULTURED CELLS DERIVED FROM CNS

Abstract— Cultured mouse neuroblastoma cells (clone N18) contained a homologous series of gangliosides, G_M3, G_M2, G_M1 and G_D1a; the total lipid bound sialic acid (LBSA) was 3.3 nmol per mg of protein, of which G_D1a comprised two-thirds. In contrast, neonatal hamster astrocytes (clone NN) and human glioblastoma cells (Cox clone) contained mainly G_M3, which represented 95% of the 2 nmol of LBSA per mg protein in these cells. When the cells were grown in the presence of [¹⁴C]galactose, label was incorporated into all of the gangliosides isolated from the cells. The labeling pattern corresponded to the ganglioside composition of the cell lines; G_D1a was more extensively labeled in N18 cells and G_M3 was the major labeled ganglioside extracted from glial cells. In addition to in rivo biosynthesis, in vitro synthesis of gangliosides was also determined. The activities of five glycosyltransferases of the ganglioside biosynthetic pathway were measured in homogenates of the three cell lines. The neuroblastoma cells contained all five enzyme activities whereas the two glial cell lines were deficient in UDP- N -acetylgalactosamine: G_M3 N -acetylgalactosaminyltransferase activity, which catalyzes the synthesis of G_M2 from G_M3. The results indicated that cells of neuronal origin contain the more complex gangliosides associated with CNS and the requisite biosynthetic enzymes and that cells of glial origin are missing these complex gangliosides and the key glycosyltransferase required for their synthesis.     

Biofeedback heart rate training during exercise

Eighteen healthy human subjects participated in weekly sessions of five 10-minute trials of walking on a treadmill at 2.5 mph and 6% grade. Eight experimental subjects received beat-to-beat heart rate biofeedback during the exercise and were instructed to try to lower their heart rates; ten control subjects did not receive feedback. By the end of 5 weeks (25 trials), the experimental group showed a significantly lower mean heart rate (96.8 vs. 108.6 bpm), systolic blood pressure (114.0 vs. 131.3 mmHg), and rate-pressure product (11.0×10³ vs. 14.3×10³ bpm-mmHg) during exercise than the control group. These differences were maintained after crossover of the feedback provision for five more weeks.     

Ethanol production at 45°C by Kluyveromyces marxianus IMB3 immobilized in magnetically responsive alginate matrices

Summary The thermotolerant yeast, Kluyveromyces marxianus IMB3 produced 11g ethanol/l during growth at 45°C on media containing 4% (w/v) lactose when immobilized in alginate beads whereas the free cells produced 5g ethanol/l. A magnetically responsive biocatalyst, prepared by incorporating Fe₃O₄ into the alginate matrix increased ethanol production to 12g/l in batch-fed reactors. Ethanol concentrations were further increased to a maximum of 18g/l by immobilization of the endogenous K. marxianus -galactosidase to the Fe₃O₄ particles prior to inclusion into the alginate matrix. Maximum ethanol productivity by the system was 87% of the maximum theoretical yield.     

Bioaccumulation of metal cations by Saccharomyces cerevisiae

Yeast cells are capable of accumulation of various heavy metals, preferentially accumulating those of potential toxicity and also those of value. They retain their ability to accumulate heavy metals under a wide range of ambient conditions. In the present study it was shown that yeast cells in suspension accumulate heavy metal cations such as Cu²⁺, Co²⁺. The level of copper accumulation was dependent on the ambient metal concentration and was markedly inhibited by extremes of ambient pH. Temperature (5–40°C) and the presence of the alkali metal sodium had much smaller effects on the level of copper accumulation. This suggests that in waste-waters of pH 5.0–9.0, yeast biomass could provide an effective bioaccumlator for removal and/or recovery of the metal. During bioaccumulation and subsequent processes it is necessary to retain the biomass. It was shown in the present study that this could be achieved by cell immobilization. Immobilization allowed for complete removal of Cu²⁺, Co²⁺, and Cd²⁺ from synthetic metal solutions. The immobilized material could be freed of metals by use of the chelating agent ethylenediamine tetraacetic acid (EDTA) and recycled for further bioaccumulation events with little loss of accumulation capacity.Correspondence to: J. R. Duncan