[Quantitative analysis of the binding of peptides to HLA molecules role of polymolphic HLA molecules in the immune response].

We established HLA-peptide binding assay system, using biotinylated peptide and HLA class II transfectants to make clear the interaction of peptides and HLA molecules. By using this system and T cell proliferation assay, we analyzed the effect of polymorphism of DR4 subtypes on responding to M12(4) peptide which derived from M12 protein of Streptococcus. We found amino acid residue which determines T cell recognition is DR beta-74 (Glutamate to Alanine) on this peptide. Using this system, we expect to understand the mechanisms of immune response associated with HLA class II molecules.

Biophysical and mechanical properties of red and white muscle fibres in fish.

1. The electrical properties of red muscle fibres of the silver carp (Carassius auratus (Linné)) were investigated and compared with those of white fibres. The resting membrane potential of red muscle was -73.1 mV, and of white muscle -82.4 mV. The effective resistance of fibre, time constant and space constant were 1.43 MOmega, 26.6 msec and 2.1 mm for red muscle and 1.0 MOmega, 48.4 msec and 1.9 mm for white muscle.2. The membrane capacitance in red muscle was 2.55 muF/cm(2) and in white muscle was 7.23 muF/cm(2), though the sarcoplasmic reticulum and the transverse tubular system were developed equally in both tissues.3. No rectification could be observed in the current-voltage relation in both the muscles. However, in excess potassium solutions, both types of muscle showed anomalous rectification.4. In red muscle it was rare to trigger a spike by nerve stimulation or by field stimulation of the tissue. However, in white muscle, there appeared graded responses and occasionally spikes were generated with overshoot potentials.5. Excess potassium concentration produced contracture in the red muscle, but even forty times of normal potassium never produced contracture in the white muscle.

Membrane properties of the smooth muscle of guinea-pig ureter.

1. The membrane properties of the guinea-pig ureter were studied in physiological Krebs solution by intra- and extracellular stimulating methods.2. The mean membrane potential was 50 mV. Action potentials triggered by external stimulation were composed of repetitive spikes and a plateau phase.3. The effects of intracellular polarization on the membrane activity elicited by extracellular stimulation were observed. Anodal polarization enhanced the amplitude and the maximum rate of rise of the spike while cathodal polarization reduced them. The number of the spikes, the duration and amplitude of the plateau phase were not changed by polarization of any direction.4. The spikes triggered by intracellular stimulation were mostly graded, but repetitive spikes sometimes continued even after cessation of the stimulation. The effective membrane resistance was 15-23 MOmega and the time constant was 2-3 msec.5. Conduction velocity (V), chronaxie, time constant (tau) and space constant (lambda) of the tissue were measured by extracellular stimulation. These values were as follows: V, 3-6 cm/sec; chronaxie, 20-40 msec; tau, 200-300 msec; lambda, 2.5-3 mm. The conduction of excitation might be related to the cable properties of the tissue.6. The relative refractory period measured by extracellular stimulation was as long as 30 sec. During the relative refractory period dissociation of the slow depolarization and the spikes was observed by successive stimuli.7. The plateau phase was prolonged and the frequency of the spontaneous discharges was increased by treatment with Ba(2+). Tetrodotoxin had no effect on spike activity nor on the plateau phase, but Mn(2+) blocked the membrane activity.

Electrophysiological study of the intestinal smooth muscle of the guinea-pig.

The membrane properties of single cells of intestinal smooth muscle of duodenum, jejunum, ileum, caecum and rectum of guinea-pig have been studied.1. The membrane potentials of longitudinal muscles of the duodenum, jejunum, ileum, caecum and rectum varied from 54 to 56 mV and those of circular muscles of jejunum, ileum, caecum and rectum varied from 57 to 60 mV. The ablated longitudinal muscle had a slightly lower value (50 mV) than the intact one.2. The longitudinal muscle generated spontaneous discharges but these were rare in the circular muscles of the intestine except for the caecum. Overshoot potentials could be observed in all regions of the intestine. The maximum rate of rise of the spontaneously discharging longitudinal muscles varied from 11 to 18 V/sec.3. Not all of the slow potential changes (but at least some) were generated by the nervous elements distributed between the muscle layers and in them.4. The conduction velocities measured from the longitudinal muscles of jejunum and rectum in the presence of tetrodotoxin were 2.1 cm/sec and 4.0 cm/sec respectively.5. Chronaxies of the longitudinal muscles of jejunum and rectum were 2-5 msec and 5-18 msec respectively.6. Intracellular stimulation of the single cells of the duodenum and caecum could trigger a spike, similar to that observed in the taenia coli. The spikes were mostly graded ones; a spike of full size was rarely elicited. When the spikes were triggered, the after-hyperpolarization appeared consistently presumably caused by the increased potassium conductance.7. The effective membrane resistance and the time constant were measured for the longitudinal muscles of the jejunum and rectum. When spikes were generated by intracellular stimulation the observed values were 40-50 MOmega and 3-5 msec in both tissues. These values were the same as those observed in the taenia coli.8. When the time constant of the membrane was measured by the extracellular polarizing method, the longitudinal muscles of the jejunum especially and the rectum had smaller time constants than the taenia coli.9. The differences of conduction velocity and chronaxie of the different regions of the intestine are discussed in relation to the cable properties of the tissues which are directly influenced by the morphological arrangements of the tissues.

Nervous factors influencing the membrane activity of intestinal smooth muscle.

The effects of various chemical agents on the spontaneous membrane activities and those electrically elicited in the smooth muscles of small intestine were investigated.1. The effects of various chemicals on the spontaneously active membrane might be summarized as follows. (a) Cholinergic agents; atropine slightly hyperpolarized the membrane and reduced the amplitude of slow potential changes even in aged preparations. Prostigmine depolarized the membrane, and enhanced the amplitude and prolonged the duration of the slow potential changes. Atropine prevented the actions of prostigmine on the membrane. (b) Ba(2+) depolarized the membrane, and enhanced the amplitude and prolonged the duration of the slow potential changes. The spike frequency was initially increased, then reduced. Atropine and tetrodotoxin partially prevented the action of Ba(2+) on the membrane activities.2. Effects of chemical agents on the membrane activity elicited by electrical stimulation might be summarized as follows. (a) Short pulse stimulation (0.5-1 msec) generated the spike as a direct response of the muscle cell membrane, then it was followed by slow depolarization, delayed hyperpolarization, i.e. the ;inhibitory potential', and post-inhibitory rebound successively. (b) The slow depolarization and the post-inhibitory rebound were reduced in amplitude by treatment with atropine, and enhanced by treatments with prostigmine and Ba(2+). Tetrodotoxin blocked all activities except the spike.3. When repetitive stimulation (20 c/s) was applied to the membrane, the membrane hyperpolarized; then, after 3-5 sec, it gradually depolarized even if the stimulation was continued, and triggered spikes. The hyperpolarization always preceded depolarization. The duration and the amplitude of the delayed depolarization was proportionally increased by the increased intensity and duration of stimulation. Atropine and tetrodotoxin blocked the generation of the post-inhibitory rebound.4. Effects of repetitive stimulation on the stored tissues were observed. The responses to repetitive stimulation of the membrane of muscles which had been stored 50 hr at 4 degrees C, were the same as those observed in the fresh tissue. The response of the tissue which had been stored 100 hr was the same as that observed in the fresh tissue treated with tetrodotoxin, i.e. all activities except the spikes were blocked.

Oral immunization with the saliva-binding region of Streptococcus mutans AgI/II genetically coupled to the cholera toxin B subunit elicits T-helper-cell responses in gut-associated lymphoid tissues.

Mice immunized intragastrically (i.g.) with a genetically constructed chimeric protein consisting of the saliva-binding region (SBR) of Streptococcus mutans AgI/II coupled to cholera toxin (CT) A2 and B subunits (CTA2/B) develop serum immunoglobulin G (IgG) and mucosal IgA antibody responses against AgI/II that are enhanced by the coadministration of CT as an adjuvant. To investigate the development of antigen-specific T cells in the gut-associated lymphoid tissues, mice were immunized i.g. with SBR, SBR-CTA2/B, or SBR-CTA2/B plus CT. AgI/II-specific T cells in Peyer's patches (PP), mesenteric lymph nodes (MLN), and spleen were assayed by lymphoproliferation and flow cytometry for the expression of T-cell surface markers, and cytokine mRNA expression was evaluated by reverse transcription-PCR. T-cell responses were consistent with antibody responses but were detectable after the first immunization. Proliferative responses of PP and MLN cells upon stimulation with AgI/II in vitro were low and delayed in mice given SBR alone, and these cells displayed a mixed type 1 and 2 (or Th0) pattern of cytokine expression. Immunization with SBR-CTA2/B resulted in greater AgI/II-specific proliferative responses in PP cells and an increase in the proportion of CD4+ T cells. Coadministration of CT with SBR-CTA2/B led to greater proliferative responses especially in the MLN cells, which then showed an increase in CD4+ cells. Immunization with SBR-CTA2/B (with or without CT) skewed the cytokine expression pattern in PP and MLN cells toward Th2. The results indicate that T helper cells were induced in gut-associated lymphoid tissues by i.g. immunization with SBR-CTA2/B, concomitantly with and prior to the appearance of circulating and mucosal antibodies.

Characterization of T cell epitopes restricted by HLA-DP9 in streptococcal M12 protein.

Interaction of the HLA-DP9 (DPA1*0201/DPB1*0901) molecule and M protein of serotype 12 (SS95/12) streptococci, a main component of the streptococcal cell wall Ag, has been investigated to decipher peptide-binding capacity and T cell activation in the context of the HLA-DP molecule. Seven antigenic peptides (amino acids 19-25) restricted by the HLA-DP9 molecule were identified in M12 protein, using M12 protein- or peptide-specific T cell lines from naturally exposed individuals. The binding affinity of each peptide to the HLA-DP9 molecule was measured by fluorescence intensity of biotinylated peptides bound to L cell transfectants expressing HLA-DP9, followed by treatment with avidin-fluorescence. Binding of biotinylated peptides to the HLA-DP9 molecule was inhibited by an excess amount of corresponding nonbiotinylated peptides and other nonbiotinylated peptides, indicating that the peptides were bound to the HLA-DP9 molecule at a single binding site. Seven synthetic peptides containing the T cell epitopes restricted by the HLA-DP9 molecule had high binding affinity to the HLA-DP9 molecule. Comparison of the amino acid sequences of truncated analogues that could bind to the HLA-DP9 molecule and/or activate T cells suggested an HLA-DP9-specific binding motif, composed of a positively charged residue (R or K) at position 1, a hydrophobic residue (A, G, or L) at position 6, and another hydrophobic residue (L or V) at position 9. Analysis of single amino acid-substituted analogues suggested that the positively charged amino acid in the motif served as a key anchor residue for binding to the HLA-DP9 molecule, which differs from the binding motif to the HLA-DR molecules.