Binding of macrophages and phospholipid flip-flop in supported lipid bilayers

Subclass-specific antibody-dependent interactions (binding and triggering) between macrophages and supported lipid bilayers have been studied. Percentages of mouse macrophage binding (J774 cell line) to the lipid bilayers were dependent on mouse monoclonal IgG subclasses. The efficiencies were as follows: IgG1 = IgG2a greater than IgG2b greater than IgG3. Furthermore, macrophage triggering (spreading) was more efficient on IgG2a- or IgG1-coated lipid bilayers than on IgG2a, IgG3, or non-specific rabbit IgG. The present experiments show also that phospholipid molecules are able to flip-flop from one side of a supported planar bilayer membrane to the other with a half-life of 10 h-1 day at 25 degrees C.     

Purification and characterization of an antibacterial factor from snail mucus

The antibacterial factor from the body surface of the African giant snail, Achatina fulica Férussac, was isolated by DEAE-Toyopearl 650M ion exchange chromatography. The isolated preparation exhibited highly positive antibacterial activity both for the Gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus and for the Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa, but it lost such activity when heated at 75 degrees C for 5 min. The antibacterial factor of the snail mucus was a glycoprotein whose molecular weight (MW) was about 160,000. It was composed of two subunits of MW 70,000-80,000.     

Growth of the malignant and nonmalignant human squamous cells in a protein-free defined medium

Summary A novel protein-free synthetic medium has been developed for the culture of human squamous cell carcinoma cells. This medium, designated PF86-1, supports the serial subcultivation of six out of nine human squamous cell carcinoma cell lines in a protein-free, chemically defined condition without the adapting culture from serum-containing conditions. These cell lines growing in PF86-1 exhibited nearly equal potency to grow in massive culture without noticeable changes in morphology but presented a significantly decreased level of colony forming efficiency when compared with the cells cultured in serum-containing media, suggesting the implication of some autocrine mechanism. Interestingly, this medium supported the growth of normal human squamous cells of oral mucosa and skin for more than 2 mo. in the primary explant culture in spite of high levels of calcium ion concentration, where the overgrowth of fibroblasts as contaminant was not observed. These results suggest that PF86-1 supports the growth of cells derived from epidermal tissues selectively and provides the same defined condition for growth of malignant and nonmalignant human squamous cells. It seems, therefore, that PF86-1 allows investigations on the products of squamous cell carcinoma cells or on the differences of growth mechanisms between normal and neoplastic human squamous cells.     

Efficient production of IGG human monoclonal antibodies by lymphocytes stimulated by lipopolysaccharide,pokeweed mitogen,and interleukin 4

Summary Extensive screening of the mitogens lipopolysaccharide (LPS), pokeweed mitogen (PWM), andStaphylococcus aureus Cowan I (SAC I), alone and in combination and with and without interleukin (IL) was performed forin vitro activation of regional lymph node lymphocytes from lung cancer patients for the production of human IgG, IgM, and IgA. As assessed by electrofusion of the lymphocytes following their exposure to these agents with mouse myeloma cells and incubation of the fused hybridoma, a remarkable stimulatory effect was shown by LPS and particularly by LPS plus IL-4, which was substantially greater than that of either SAC I or PWM with or without various IL. Optimization studies indicated that the addition of PWM to LPS and IL-4 in the culture medium further stimulated the human antibody (Ab) production, and that the optimal formulation for stimulations of human IgG production was a culture medium containing 20 μg/ml of LPS, 1/500 of PWM, and 100 u/ml of IL-4.     

The Expression of Two Splice Variants of Metabotropic Glutamate Receptor Subtype 5 in the Rat Brain and Neuronal Cells During Development

Abstract: We previously reported that a variant with extra amino acid residues exists in the metabotropic glutamate receptor subtype 5 (mGluR5). Either of the two isoforms, named mGluR5b and mGluR5a for the isoforms with and without the inserted sequence, respectively, generated Ca²⁺-activated Cl⁻ current when expressed in Xenopus oocytes. We herein report that these two isoforms are produced by the alternative splicing of the exon skipping type. When examined during the course of postnatal development, the major mGluR5 isotype mRNA was observed to switch from mGluR5a to mGluR5b in the rat hippocampus and the cerebral cortex. We also investigated two cell lines that could be differentiated into neuron-like cells in vitro. Whereas the mGluR5b mRNA was hardly detectable in either undifferentiated or differentiated NG108-15 cells, the relative amounts of the two variant mRNAs changed after the induction of differentiation in the P19 cells. An extracellular application of trans - d,l -1-amino-1,3-cyclopentanedicarboxylate on the neuron-like P19 cells induced intracellular Ca²⁺ mobilization, thus suggesting that the cells could express functional mGluR(s) coupled to phospholipase C and other components that could mediate the signal transduction pathway. This cell line may thus provide a model system for studying both mGluR5 expression and other mGluR-induced phenomena at the molecular level.     

A synaptic modification algorithm in consideration of the generation of rhythmic oscillation in a ring neural network

In consideration of the generation of bursts of nerve impulses (that is, rhythmic oscillation in impulse density) in the ring neural network, a synaptic modification algorithm is newly proposed. Rhythmic oscillation generally occurs in the regular ring network with feedback inhibition and in fact such signals can be observed in the real nervous system. Since, however, various additional connections can cause a disturbance which easily extinguishes the rhythmic oscillation in the network, some function for maintaining the rhythmic oscillation is to be expected to exist in the synapses if such signals play an important part in the nervous system. Our preliminary investigation into the rhythmic oscillation in the regular ring network has led to the selection of the parameters, that is, the average membrane potential (AMP) and the average impulse density (AID) in the synaptic modification algorithm, where the decrease of synaptic strength is supposed to be essential. This synaptic modification algorithm using AMP and AID enables both the rhythmic oscillation and the non-oscillatory state to be dealt with in the algorithm without distinction. Simulation demonstrates cases in which the algorithm catches and holds the rhythmic oscillation in the disturbed ring network where the rhythmic oscillation was previously extinguished.     

Fast atom bombardment tandem mass spectrometric analysis of N-carbamoylamino acids.

Using fast atom bombardment (FAB) and tandem mass spectrometry (MS/MS), we examined 12 synthetic N-carbamoylamino acids (CAA) as tert-butyldimethylsilyl (TBDMS) derivatives. In FAB mass spectrometry and FAB MS/MS, spectra of protonated molecules for CAA provide specific cleavages involving the TBDMS carbamoyl moiety. The daughter scan spectrum of the parent ion indicated that it was useful for structural elucidation and differentiation of structural isomers of CAA. We have also identified each CAA separately in a mixture using a neutral loss scan for characteristic ions. In addition, we demonstrated that CAA in urine samples from patients with ornithine carbamoyl transferase deficiency gave collision-induced dissociation (CID) spectra which correspond well with CID spectra obtained using synthetically prepared CAA.