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Mutant human lysozymes (HLZ) lacking two disulfide bonds were constructed to study the importance of each disulfide bond on oxidative refolding. To avoid destabilization, a calcium-binding site was introduced. Five of the six species of two-disulfide mutants could be obtained with enzymatic activity. Based on the information obtained from refolding and unfolding experiments, the order of importance in oxidative refolding was found to be as follows: SS2(Cys30-Cys116)SS1(Cys6-Cys128) approximately SS3(Cys65-Cys81)SS4(Cys77-Cys95). Without SS2, these mutants refolded with low efficiency or did not refold at all. The bond SS2 is located in the interface of B-and D-helices, and a small hydrophobic cluster is formed near SS2. This cluster may play an important role in the folding process and stabilization, and SS2 may act as a stabilizer through its polypeptide linkage. The bond SS2 is the most important disulfide bond for oxidative folding of lysozymes.

We have investigated the biochemical mechanism of tumor necrosis factor (TNF)-alpha-induced release of plasminogen activator inhibitor-1 (PAI-1) from human umbilical vein endothelial cells (HUVEC). Treatment of HUVEC with TNF-alpha for 3 h resulted in a 2. 8-fold increase in the PAI-1 release compared with control. The increase in PAI-1 release was accompanied by a 133% increase in the intracellular acidic sphingomyelinase (SMase) activity. High-performance liquid chromatographic (HPLC) analysis revealed that the intracellular ceramide levels increased to 126% of the control (P0.05), but the contents of membranous ceramide remained unaltered. We have previously shown that a cell-permeable ceramide analog, N-acetylsphingosine (C2-ceramide) enhances the PAI-1 release from HUVEC. Here, N-acetylsphinganine (C2-dihydroceramide) was found to specifically suppress both C2-ceramide- and TNF-alpha-induced increase in PAI-1 release from HUVEC without affecting the control PAI-1 release. Treatment of HUVEC with staphylococcal SMase that may mimic the activation of the membranous neutral SMase also increased the PAI-1 release. The increase in PAI-1 release by this mechanism was suppressed by a cyclooxygenase inhibitor, aspirin, whereas the inhibitor did not affect TNF-alpha-induced increase in PAI-1 release. Taken together, these findings suggest that TNF-alpha prominently utilizes the lysosomal acidic SMase-ceramide signaling pathway in the induction of PAI-1 release from HUVEC.

We have started to offer drug information to patients with a focus on the initial signs of severe adverse effects. We classified the adverse effects into groups according to the regions of occurrence and edited the initial signs of each group into a compact form to ensure that only the minimum essential information was given out.

Powders and granules consisted of 20% aminophylline were prepared to improve physical properties of aminophylline powder.Two percent of sodium carboxymethyl cellulose (CMCNa), 5% of polyvinylpyrrolidone K-90 (PVP) and 5% of starch paste were used as binders to make granules of 20% aminophylline.Distribution of particle size of all six different aminophylline granules conformed to standards prescribed in Japanese Pharmacopoeia (JP).Values of angle of repose and grouping rate of granules of 20% aminophylline were lower than those of aminophylline powders, indicating that flowabilities of granules were higher than those of powders.The powders and the granules did not change in appearance for 30 days under severe condition (at 30°C, relative humidity of 92%).Content of aminophylline in each preparation was measured by absorption spectrophotmetry at 273nm.Among several preparations we tested, both aminophylline powders and granules with D-mannitol as a diluent were most stable for 30 days under severe condition.Disintegration time of the granules was within 10 minutes in water at 37°C.Dissolution test of the granules by paddle method showed a rapid release of aminophylline into water, indicating that bioavailability of the granules may be similar to that of aminophylline powders.From the results of this study, it is expected that 20% aminophylline granule with D-mannitol as a diluent is useful as a hospital preparation.

In order to obtain information on the compatibility of Berachin Dry Syrup (BE. D. S.) with drugs in common use, two presentation of the mixtures were investigated: powder (27 drugs) and solution (20 drugs) preparation. Forty seven drugs were studied by sensory test, and solution preparation (20 drugs) was investigated for changes in redispersibility, viscosity, pH and buffer capacity curve. BE. D. S. residual amouut (%) was measured by absorptiometry and high-performance liquid chromatography.The changes with time were analyzed by scoring and subject to a time series analysis (by 2 mean vector). A significant change was observed for 6 of the 27 drugs in the wetting under the severe condition (30°C, R. H. 92%), and no change for solution was observed. In solution, a significant change wasn't observed in redispersibility, viscosity, pH, and buffer capacity curve. BE. D. S. was almost stable in 2 weeks under any condition tested.The above results show that BE. D. S. can be kept in the mixtures with various syrups under the best and moderate condition.

For the topical application to the treatment of leukoplakia and lichen planus, four oleaginous oral ointments containing 2% of cepharanthin were prepared with oleaginous bases; white petro1atum with Iiquid paraffin, white petrolatum: plastibase (3:2) with liquid paraffin, white petrolatum: Plastibase (1:1) with liquid paraffin, and plastibase.Consistency of four ointments measured with penetrometer, and spreadability measured with spreadmeter was not changed when these ointments were kept at 20° or 30°C for 21 days.The stabilities of cepharanthin in each ointment measumed with pH meter and spectrophotometer showed that cepharanthin was very stable in each ointment for 21 days at 20°C or 30°C.Adhesion time of each ointment was measured using 1% methylrosaniline chloride as an indicator dye. Among four oleaginous bases, plastibase seemed to be the most useful base for clinical application judging from several sensory tests and clinical tests.With these results it is concluded that cepharanthin oral ointment consisting of plastibase is the most effective to the treatment of leukoPlakia and lichen Plenus among four oleaginous ointments prepared.

Compatibility of sodium valproate (VPA) fine granules with 48 drugs was investigated by sensory test, and VPA residual potency (%) was measured by nonaqueous titration and gas chromatography. The data on change in compatibiiity with time were analyzed by scoring and were subject to a time series analysis. Change in compatibility was observed in 23 of the 48 drugs under the severe conditions (30°, RH92%), and in 12 drugs under the medium conditions (20°, RH75%). In gas chromatography, when combined with ascorbic acid, residual potency (%) of VPA began to decrease sharply on the first day, thus suggesting that such combination be avoided. There is not always correlation between change in appearance and the results of nonaqueous titration and gas chromatography. Since VPA may be administered for as long as 30 days, caution should be exercised in mixing this drug with other agents.