One-dimensional double diffusion was applied to determine critical concentrations at which the precipitation of calcium phosphates occurs in reconstituted connective tissue collagen and agar gels at 37 degrees C and in gelatin gels at 25 degrees C. Experiments were performed in the presence of unbuffered 0.15 mol dm-3 NaCl, or 0.15 mol dm-3 NaCl-veronal adjusted to pH 7.4. It was found that critical concentrations of precipitation of both precipitating components, CaCl2 and phosphate buffer (pH 7.4), were equimolar and independent of the ratios of initial concentrations of the components. Critical concentrations of precipitation were not affected by the concentrations and kinds of gels used. The first-formed precipitates showed amorphous structure by X-ray diffraction analyses. Infrared (IR) spectra of the precipitates indicated CaHPO4 . H2O to be their predominant species. The molar Ca/P ratio obtained by chemical analyses was 1.08. This precipitate transformed in time into octacalcium phosphate. In all experiments, two very thin membranes of precipitate were formed in the gel column at the onset of precipitation simultaneously on both sides of the actual disc of precipitate. IR spectra and chemical analyses showed that both membranes were identical to the actual precipitation discs.

Proteins and peptides in mammals are based exclusively on l-amino acids. Recent investigations show that d-amino acids exhibit physiological effects in vivo, despite of their very small quantities. We have investigated the hepatoprotective effects of the l- and d-enantiomers of α-melanocortin peptide (α-MSH). The results showed that peptide-enantiomerism is related to the protective effects of melanocortin peptides in vivo. l-α-MSH exhibited potent hepatoprotective effect in the experimental model of acetaminophen induced hepatotoxicity in male CBA mice, while its d-mirror image was inefficient. Furthermore, the antibody to the l-peptide did not recognize the d-structure. The results indicate that the opposite peptide configuration may be used to modulate its function and metabolism in vivo and in vitro.

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ACKNOWLEDGMENT evaluated and wi(t) is also given by Equation 68. Thus these successive procedures give the terms wn as wn = X/r \~wn-i + (af ~a02) f e-",2,'-r)ti>"_idr| (77) ,=i ao t J0 >The authors thank M. Hattori of Tokyo Institute of Technology for his helpful discussions.

The insoluble immune complexes (ICs) were prepared under the conditions of double immunodiffusion in gel, using the suspension of the ultrasound treated PK-15 cell-line infected with porcine parvovirus (PPV) containing both viral particles and viral proteins, as well as pig or rabbit anti-PPV polyclonal immune sera. The immunodiffusion performed in an agarose gel allows only viral subunits with a molecular mass equal to or less than 1000 kDa, rather than the viral particles, to diffuse through the gel and reach the point where the immunoprecipitate is to be formed. The immunoprecipitation under the conditions of the diffusion ensures the optimal, i.e. equimolar ratio of both immunoprecipitating components, antibody/antigen in the IC. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the Western blot analyses showed the ICs were composed of two proteins, a protein in which molecular mass corresponded to the VP2 of the PPV and a protein with a molecular mass of the IgG. This suggests that the ICs are mainly composed of the VP2 antigen and IgG class antibodies. The potency of the IC-vaccines prepared in the form of a water-in-oil-in-water emulsion was compared with that of a commercially available, inactivated oil vaccine. The vaccination of gilts, 6 weeks before mating, with the IC containing allogeneic pig antibodies, resulted in the development of high and long-lasting anti-PPV antibody titres, similar to those generated by the licenced vaccine (P > 0.01). The content of the virus material administered by the IC was twice lower than that in the licenced vaccine. Neither systemic nor local reactions were observed in the gilts during the period of the trial with the IC vaccine. The number of viable piglets per litter varied between 9 and 12 and no signs of the PPV infection were detected. Rabbits were used as one of the alternative laboratory animal models accepted for the testing of the vaccine against the PPV. The rabbit humoral immune response generated by the IC containing the allogeneic antibodies were higher than that generated by the ICs containing the xenogeneic pig antibodies. It was similar to that generated by two-times higher content of the virus material administered by a commercially available vaccine. The IC-based vaccines belong to non-replicating, subunit vaccines, which are both ecologically convenient and the safest vaccines of all.