Dynamic light scattering study of suspensions of purple membrane

Purple membrane from Halobacterium halobium in suspensions has been studied by quasielastic light scattering. The intensity correlation functions of polarized scattered light were measured at various K2 values (K being the magnitude of the scattering vector), and the first cumulant Gamma of the field correlation function G1(tau) was obtained by a cumulant expansion method. The apparent diffusion coefficient Gamma /K2 did not increase monotonically with K2 values and showed a distinct anomaly in an intermediate range of K. A theoretical formulation of G1(tau) for a disc and an extremely oblate ellipsoidal shell of revolution (S. Fujime and K. Kubota, Biophys. Chem. 23 (1985) 1) was applied to the analysis of the spectra, and characteristic features of experimental spectra were well reproduced. It was suggested that a strong interference effect between scattered rays on Gamma /K2 should be attributed to a slight noncircular shape of the purple membrane and that a contribution to Gamma /K2 from membrane flexibility should be taken into account. This study will provide experimental evidence of the feasibility of membrane studies by dynamic light scattering.     

Inhibition of macrophage DNA synthesis by immunomodulators. II. Characterization of the suppression by muramyl dipeptide or lipopolysaccharide [3H]thymidine incorporation into macrophages

Guinea pig peritoneal exudate macrophages actively incorporated [3H]thymidine into trichloroacetic acid-insoluble fraction in vitro. The incorporation of [3H]thymidine was almost completely inhibited by aphidicolin, an inhibitor of DNA polymerase alpha and an autoradiograph showed heavy labeling in nuclei of 15% of macrophage populations. These results indicate that the observed thymidine incorporation was due to a nuclear DNA synthesis. The [3H]thymidine incorporation was markedly suppressed when macrophages were activated by immunoadjuvants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS). The suppression of [3H]thymidine incorporation by MDP was neither due to the decrease in thymidine transport through the cell membrane, nor due to dilution by newly synthesized "cold" thymidine. An autoradiograph revealed that MDP markedly decreased the number of macrophages the nuclei of which were labeled by [3H]thymidine. These results suggest that the suppression of [3H]thymidine incorporation by the immunoadjuvants reflects a true inhibition of DNA synthesis. The inhibition of DNA synthesis by MDP was also observed in vivo. Further, it was strongly suggested that the inhibition was not caused by some mediators, such as prostaglandin E2, released from macrophages stimulated by the immunoadjuvants but caused by a direct triggering of the adjuvants at least at the early stage of activation. Cyclic AMP appears to be involved in the inhibitory reaction.     

Torsional motion of eosin-labeled F-actin as detected in the time-resolved anisotropy decay of the probe in the sub-millisecond time range

The internal motion of F-actin in the time range from 10(-6) to 10(-3) second has been explored by measuring the transient absorption anisotropy of eosin-labeled F-actin using laser flash photolysis. The transient absorption anisotropy of eosin-F-actin at 20 degrees C has a component that decays in the submicrosecond time scale to an anisotropy of about 0.3. This anisotropy then decays with a relaxation time of about 450 microseconds to a residual anisotropy of about 0.1 after 2 ms. When the concentration of eosin-F-actin was varied in the range from 7 to 28 microM, the transient absorption anisotropy curves obtained were almost indistinguishable from each other. These results show that the anisotropy decay arises from internal motion of eosin-F-actin. Analysis of the transient absorption anisotropy curves indicates that the internal motion detected by the decay in anisotropy is primarily a twisting of actin protomers in the F-actin helix; bending of the actin filament makes a minor contribution only to the measured decay. The torsional rigidity calculated from the transient absorption anisotropy is 0.2 X 10(-17) dyn cm2 at 20 degrees C, which is about an order of magnitude smaller than the flexural rigidity determined from previous studies. Thus, we conclude that F-actin is more flexible in twisting than in bending. The calculated root-mean-square fluctuation of the torsional angle between adjacent actin protomers in the actin helix is about 4 degrees at 20 degrees C. We also found that the torsional rigidity is approximately constant in the temperature range from 5 to approximately 35 degrees C, and that the binding of phalloidin does not appreciably affect the torsional motion of F-actin.     

Comparative sequence analysis of rotavirus genomic segment 6--the gene specifying viral subgroups 1 and 2

Cloned DNA copies of rotavirus genomic segment 6 from simian 11 (subgroup 1) and human strain Wa (subgroup 2) rotaviruses have been used to determine the nucleotide sequences of the gene that determines viral subgroup specificity. Both genomic segments are 1,356 nucleotides in length and possess 5'- and 3'-terminal untranslated regions of 23 and 142 nucleotides, respectively. The inferred amino acid sequence reveals VP6 to be a polypeptide of 397 amino acids in which more than 90% of the amino acid sequence is conserved between the two viruses. There are 34 amino acid changes between the subgroup 1 and 2 polypeptides, most clustered in three regions of the molecule at residues 39 through 62, 80 through 122, and 281 through 315.     

Establishment of a tumor-specific immunotherapy model utilizing TNP-reactive helper T cell activity and its application to the autochthonous tumor system

Preinduction of potent hapten-reactive helper T cell activity and subsequent immunization with hapten-coupled syngeneic tumor cells result in enhanced induction of tumor-specific immunity through T-T cell collaboration between anti-hapten helper T cells and tumor-specific effector T cells. On the basis of this augmenting mechanism, a tumor-specific immunotherapy protocol was established in which a growing tumor regresses by utilizing a potent trinitrophenyl (TNP)-helper T cell activity. C3H/He mice were allowed to generate the amplified (more potent) TNP-helper T cell activity by skin painting with trinitrochlorobenzene (TNCB) after pretreatment with cyclophosphamide. Five weeks later, the mice were inoculated intradermally with syngeneic transplantable X5563 tumor cells. When TNCB was injected into X5563 tumor mass, an appreciable number of growing tumors, in the only group of C3H/He mice in which the amplified TNP-helper T cell activity had been generated were observed to regress (regressor mice). These regressor mice were shown to have acquired tumor-specific T cell-mediated immunity. Such immunity was more potent than that acquired in mice whose tumor was simply removed by surgical resection. These results indicate that in situ TNP haptenation of the tumor cells in TNP-primed mice can induce the enhanced tumor-specific immunity leading to the regression of a growing tumor. Most importantly, the present study further investigates the applicability of this TNP immunotherapy protocol to an autochthonous tumor system. The results demonstrate that an appreciable percent of growing methylcholanthrene-induced autochthonous tumors regressed by the above TNP immunotherapy protocol. Thus, the present model provides an effective maneuver for tumor-specific immunotherapy in syngeneic transplantable as well as autochthonous tumor systems.     

Effect of dietary fiber, glucomannan, on absorption of sulfonylurea in man

In order to clarify whether a dietary fiber has any effect upon the intestinal absorption of sulfonylurea, changes in plasma concentration of glibenclamide were determined during a six-hour period in nine healthy volunteers who took 2.5 mg of glibenclamide together with a breakfast and 3.9 g of glucomannan in a form of konjac powder and were compared with those of the control experiment in which the same amount of the hypoglycemic agent was given without the dietary fiber. In the control, mean plasma glibenclamide level increased rapidly, reaching a peak at 60 min and decreased gradually thereafter, whereas an increase in plasma glibenclamide level was blunted in the test experiment, thus plasma concentration of glibenclamide being lower at 30, 60, 90 and 150 min compared with the corresponding value of the control (31.7 +/- 24.5 ng/ml vs 76.4 +/- 25.0 ng/ml at 30 min; 51.3 +/- 35.5 ng/ml vs 120.9 +/- 56.0 ng/ml at 60 min; 60.0 +/- 38.8 ng/ml vs 117.4 +/- 53.1 ng/ml at 90 min; 54.0 +/- 31.5 ng/ml vs 100.7 +/- 46.5 ng/ml at 150 min). Mean plasma glucose concentration was significantly lower at 30 min in the test experiment than in the control despite the lower level of plasma glibenclamide in the former. The results suggest that glucomannan may influence the intestinal absorption of glibenclamide. A dietary fiber must be prescribed in due consideration of these facts.     

The significance of reutilization of surfactant phosphatidylcholine

To assess the magnitude of reutilization of surfactant phosphatidylcholine, 68 3-day-old rabbits were injected intratracheally with a trace dose of [3H]choline-labeled surfactant mixed with [14C]palmitate-labeled synthetic dipalmitoylphosphatidylcholine. After timed kills we measured the total phosphatidylcholine associated counts/min in whole lung and alveolar wash and the specific activities of phosphatidylcholine in the alveolar wash, lamellar bodies, and microsomes isolated from the lung of each rabbit. Using a modification of the compartment analysis of Skinner et al. (Skinner, S. M., Clark, R. E., Baker, N., and Shipley, R. A. (1959) Am. J. Physiol. 196, 238-244), we found that surfactant phosphatidylcholine was reutilized with greater than 90% efficiency. The turnover time of the alveolar wash phosphatidylcholine was estimated to be 10.1 h and 9.3 h as measured by the 3H and 14C labels, respectively. From the ratios of alveolar wash-associated natural to synthetic phosphatidylcholine specific activities and from similar ratios obtained in 30 additional rabbits using [14C]choline-labeled natural surfactant and [3H]choline-labeled dipalmitoylphosphatidylcholine, we showed that phosphatidylcholine was reutilized intact rather than as component parts. Within 6 h of injection, the synthetic dipalmitoylphosphatidylcholine functioned metabolically as that administered in the form of natural surfactant.     

Buried seed populations on the volcano Mt. Usu,northern Japan,ten years after the 1977–78 eruptions

In 1987, a study on buried seed populations was conducted in the crater basin of Mt. Usu, a volcano located in northern Japan, where the vegetation had beeen almost completely destroyed by eruptions in 1977 and 1978. The former vegetation had consisted of grassland and broad-leaved forest. In the areas formerly occupied by this grassland and forest, 2128.0 and 1985.3 seeds per square meter, respectively, were extracted from 12 blocks of the former topsoil using a floattion method. This revealed that many seeds were still viable even after ten years of burial under thick volcanic deposits. The seeds were distributed more in sandy soil than in rocky soil of the former topsoil. Twenty-five species, most of which favored grassland, were detected in both the former grassland and forest. From comparison of α-and β-diversities between the grassland and forest, the structure of the buried seed population was considered likely to have been more diverse in the forest than in the grassland. The determinants of composition of the buried seed populations were discussed with reference to the former vegetation and soil characteristics.     

Contribution of buried seeds to revegetation after eruptions of the volcano Usu,Northern Japan

Soon after the 1977–78 eruptions of the volcano Usu, there were created many gullies in which former topsoil, i.e., well-developed soil accumulated before the eruptions, was eroded in the crater basin, whereas the outside of the gully was covered with thick volcanic deposits. The short-dispersal-seed plants were the most abundant in the inside of gully where the former topsoil was exposed, however, they have not immigrated from external environments. The germination tests of the seeds extracted from the former topsoil demonstrated that viable seeds were buried at 1683.3/m² for 9 years after the eruptions. At least, 12 herbaceous species, e.g.,Rumex obtusifolius, Geum macrophyllum var.sachalinense andPoa annua, were derived from the buried seeds in the former topsoil. Their contribution to revegetation was estimated to be 40.0% in the inside of gully where the former topsoil was exposed in 1983, suggesting that buried seeds are one of the most important seed source for revegetation even in the case of a volcano which produced pumice and ash by eruption.