Podoplanin (Aggrus), which is a type I transmembrane sialomucin-like glycoprotein, is highly expressed in malignant pleural mesothelioma (MPM). We previously reported the generation of a rat anti-human podoplanin Ab, NZ-1, which inhibited podoplanin-induced platelet aggregation and hematogenous metastasis. In this study, we examined the antitumor effector functions of NZ-1 and NZ-8, a novel rat-human chimeric Ab generated from NZ-1 including Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity against MPM in vitro and in vivo. Immunostaining with NZ-1 showed the expression of podoplanin in 73% (11 out of 15) of MPM cell lines and 92% (33 out of 36) of malignant mesothelioma tissues. NZ-1 could induce potent ADCC against podoplanin-positive MPM cells mediated by rat NK (CD161a+) cells, but not murine splenocytes or human mononuclear cells. Treatment with NZ-1 significantly reduced the growth of s.c. established tumors of MPM cells (ACC-MESO-4 or podoplanin-transfected MSTO-211H) in SCID mice, only when NZ-1 was administered with rat NK cells. In in vivo imaging, NZ-1 efficiently accumulated to xenograft of MPM, and its accumulation continued for 3 wk after systemic administration. Furthermore, NZ-8 preferentially recognized podoplanin expressing in MPM, but not in normal tissues. NZ-8 could induce higher ADCC mediated by human NK cells and complement-dependent cytotoxicity as compared with NZ-1. Treatment with NZ-8 and human NK cells significantly inhibited the growth of MPM cells in vivo. These results strongly suggest that targeting therapy to podoplanin with therapeutic Abs (i.e., NZ-8) derived from NZ-1 might be useful as a novel immunotherapy against MPM.

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The gelation mechanism of polyethylene solutions was investigated by using light scattering and X-ray diffraction techniques in terms of the liquid−liquid phase separation. Three kinds of polyethylene, ultrahigh molecular weight polyethylene (UHMWPE), low molecular weight linear polyethylene (L-LMWPE), and low molecular weight branched polyethylene (B-LMWPE), were used as test specimens. When an incident beam of He−Ne gas laser was directed to the UHMWPE and B-LMWPE solutions quenched to a desired temperature, the logarithm of scattered intensity increased linearly in the initial stage and tended to deviate from this linear relationship in the latter stage. If the linear increase in the initial stage can be analyzed within the framework of the linear theory of spinodal decomposition proposed by Cahn, the gelation is obviously attributed to the phase separation leading to the concentration fluctuation of the solution. Furthermore, in the later stage showing the deviation from the linear relationship, light scattering from the UHMWPE solution with 0.5% concentration under Hv polarization condition showed an X-type pattern, indicating the appearance of optically anisotropic rods. On the other hand, the scattering from the B-LMWPE solution with 3% concentration showed a four-leaf clover pattern, indicating the appearance of optically anisotropic spherulites. No crystallite was confirmed by the X-ray diffraction measurements, when the rods and spherulites appeared. With further lapse of time, the Hv patterns became clearer and the corresponding X-ray diffraction intensity curves showed a very small diffraction peak from the (110) plane. In contrast, for the L-LMWPE solutions, the logarithm of scattered intensity against time showed a rapid increase and tended to level off because of rapid change from slightly transparent to whiten gels. In such a process, any quantitative analysis of the scattered light intensity was impossible. The corresponding X-ray diffraction revealed the strong reflections from the (110) and (200) planes, indicating the rapid formation of stable crystallites. Through a series of experiments for the three kinds of polyethylene solution, it turned out that gelation mechanism of polyethylene solutions is strongly affected by molecular weight and the degree of branching.

To analyze polarized light scattering patterns from gels, an approach is proposed to calculate the scattered intensity. In the proposed model system, difference between polar angles of the principal axes of the and elements, which were defined with respect to the axis along the distance between two elements, was given as a correlation of the distance between the two elements. Furthermore, the azimuthal angle, which makes a projection of the principal axis onto a plane perpendicular to the principal axis of the element, was also given as a correlation of the distance between the two elements. The theoretical calculation was carried out for the scattered intensity under Hv and Vv polarization conditions. The general equations proposed for Hv and Vv scattering were based on a statistical approach for polarized light scattering system. The calculated pattern under the Hv polarization condition showed an X-type pattern and was in good agreement with the pattern observed from polymer gels prepared by quenching their solutions to the desired temperatures.

The relationship between the morphology and the mechanical properties of copolymer ethylene-methyl methacrylate (EMMA) films with different MMA contents was investigated as a function of temperature by using positron annihilation, X-ray diffraction, and 13 C solid-state NMR. To make clear the effect of MMA side groups on the morphology and molecular mobility of EMMA as a function of temperature, the characteristics of EMMA were discussed together with those of copolymer (dimethylamino)ethyl methacrylate (EDAM) and branched polyethylene. Three kinds of transitions were confirmed by positron annihilation: the first at temperatures around -130 °C, the second around -40 °C, and the third around 50 °C. The first and the second transitions were due to the commencement of molecular motions, which can remove trapped electrons from their shallow potentials, while the third transition around 50 °C was attributed to the partial melting of unstable small crystallites. These transitions were in good agreement with the results obtained from differential scanning calorimetry (DSC) and X-ray measurements. Namely, the DSC curves of EMMA provided a peak around -25 to -30 °C corresponding to the second transition and a small peak around 43-48 °C corresponding to the third transition. Lifetime (τ 3) showed no obvious change between branched polyethylene and EMMA-I melt films. This result can be explained within the framework of the spur model of Ps formation in terms of the trapping of positrons (and may be electrons) by the polar groups, -CdO, in EMMA.

ABSTRACT-In cultured bovine adrenal chromaffin cells, pituitary adenylate cylase-activating polypeptide (PACAP) stimulated [14C]catecholamine synthesis from [14C]tyrosine (but not from [14C]DOPA) in a con centration-dependent manner, causing maximal stimulation at 10' M. The stimulatory action of PACAP was not affected by staurosporine (an inhibitor of protein kinase C) or in the cells in which protein kinase C was down-regulated by prolonged exposure to TPA (an activator of protein kinase C), whereas it was partial ly attenuated in Ca"-free medium. PACAP (10-'M) increased the formation of [3H]inositol phosphates, [Ca2+]; and 41 Ca' uptake as well as cAMP. The peptide also stimulated the phosphorylation of tyrosine hydroxylase, the enzyme catalyzing the rate-limiting step in catecholamine synthesis. Catecholamine syn thesis and tyrosine hydroxylase phosphorylation stimulated by the maximal effective concentration of dibutyryl cAMP or high K+, which activates Ca 21 uptake, were further enhanced by PACAP, suggesting that both cAMP and Ca2+-dependent protein kinases may be involved in the stimulation of tyrosine hydroxylase phosphorylation and catecholamine synthesis caused by PACAP.

Three kinds of mechanical relaxations, the α, β, and λ transitions, of ultradrawn polyethylene films were obtained by dynamic mechanical measurements, and the results were analyzed by positron annihilation. The gels were prepared by crystallization from dilute solutions and were then dried. The dried gel films were stretched by up to a draw ratio of 300 times (λ = 300). The temperature dependence of the intensity (I 3) of the long-lived component of the orthopositronium (o-Ps) as well as that of the lifetime (λ3) was observed for undrawn and drawn polyethylene films. For the undrawn film, the peak positions of the γ and β transitions corresponded to the first and second transitions of τ3. The β dispersion was not observed for the films with λ = 50 and 300. The second transition of τ3, corresponding to the β dispersion, was observed for drawn films with λ = 50 but was not observed for the film with λ = 300. A histogram of τ3 for three kinds of specimens showed that the size distribution for the undrawn film (λ = 1) became broader, and the peak position shifted to a larger value of the lifetime (τ3) with increasing temperature. This indicates that the free volume holes within the specimen became bigger and had a wider size distribution with increasing temperature and, at the same time, the molecular motion became more active. For the ultradrawn film with λ = 300, the lifetime distribution at 100 °C showed a much wider and asymmetry profile, and the peak position shifted to a shorter value than those for the other two films with drawing ratios of λ = 1 and λ = 50. This indicated poor activity of a large movement (macro-Brownian motion) of amorphous chains. These experimental results justify that the thermal expansion of the film due to an increase in the molecular mobility was almost zero in a given temperature range from 0 to 120 °C and that the storage modulus at 100 °C was higher than 130 GPa.

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