Analysis of common antigen of flagella in Bacillus cereus and Bacillus thuringiensis

Abstract Flagellar antigen of Bacillus cereus H.1 was purified and tested for serodiagnostic antigen by ELISA. The antibody against the flagellar antigen of B. cereus H.1 reacted not only with the homologous specific antigen but also reacted with the flagellar antigens of 23 strains of B. cereus . This common flagellar antigen of B. cereus was found to be due to 61-kDa protein by SDS-PAGE and immunoblot assay. Monoclonal antibody H15A5 against common antigenic epitope of B. cereus also reacted with flagellar antigens of 21 strains of Bacillus thuringiensis by ELISA. This monoclonal antibody reacted with the 61-kDa protein of the flagella of B. cereus H.1 and H.2 and B. thuringiensis Kurstaki HD1, Alesti and Aizawai juroi by immunoblot analysis. These results indicated that the common antigenic epitope of the 61-kDa protein existed in the flagella both of B. cereus and B. thuringiensis .     

Electron microscopic studies of the endocytotic process of cationized ferritin in cultured human retinal pigment epithelial cells

Summary The endocytotic process in cultured human RPE cells was observed after 1 min, 20 min, and 2 h incubation with cationized ferritin. Within 1 min the ferritin particles were seen to attach to the cell membrane, especially between microvilli. Uncoated and coated pits could be recognized on the cell membranes, and uncoated and coated endocytotic vesicles were found in the cytoplasm after 20 min of incubation. These vesicles were surrounded by abundant microfilaments and had no visible membranes. Loss of membrane may be an initial step in the process of developing into the irregular clumps of ferritin particles found inside the plasma membrane. With time, more irregular clumps of ferritin, smaller than the particles introduced during incubation, appeared just beneath the cell membrane. Lysosomes were adjacent to the clumps of ferritin particles associated with microtobules and finally degraded these particles. The phagolysosomes containing many particles were surrounded by many microtubules. Small ferritin particles surrounded but had not entered the rough endoplasmic reticulums, and no particles were seen either around or in the Golgi apparatus. Presented at the 7th International Congress of Eye Research, Nagoya, Japan, 27 September 1986.     

Comparative assessment of the resistance of the unstirred water layer to solute transport between two different intestinal perfusion systems

The resistance of the unstirred water layer to solute transport was estimated in two different intestinal single-pass perfusion systems for a comparative study, using D-glucose as a model compound. One is a well established perfusion system in anesthetized rats as a standard (system A). The other is the one in unanesthetized rats for comparison (system B). It was demonstrated that in system B as well as in system A the resistance of the unstirred water layer to D-glucose transport should be taken into account and this resistance, accordingly, the effective thickness of the unstirred water layer (delta) which is assumed to be in proportion to its resistance, could be described as a function of the perfusion rate by using a film model. The delta decreased with increasing perfusion rate and was larger in system A than in system B at each perfusion rate; 785 microns in system A versus 319 microns in system B at the perfusion rate of 0.16 ml/min and 337 microns versus 184 micron at that of 2.95 ml/min. Thus in system B the effective thickness, accordingly, the resistance, of the unstirred water layer was reduced to about 50% of that in system A, but the resistance of the unstirred water layer could still account for 85% of the total resistance at the maximum as far as D-glucose absorption was concerned, while 93% in system A. These results suggest that, compared with perfusion experiments in anesthetized rats (system A), the resistance of the unstirred water layer is reduced but cannot be left out of consideration even if perfusion experiments are performed in unanesthetized rats (system B). And the lower resistance of the unstirred water layer in system B was attributed to a turbulent flow in contrary to a laminar flow in system A.     

Determination of kinetic parameters of a carrier-mediated transport in the perfused intestine by two-dimensional laminar flow model: effects of the unstirred water layer

The kinetic parameters of a carrier-mediated transport for D-glucose and for taurocholate were determined from rat in situ intestinal single perfusion experiments. The true parameters were obtained by the two-dimensional laminar flow model, in which the solute concentration at the aqueous-intestinal membrane interface can be calculated numerically without assuming the aqueous diffusion layer, discriminating the effects of the unstirred water layer. The true Michaelis constant was 4.5 mM for D-glucose and 1.5 mM for taurocholate. The true maximal transport velocity was 3.4 nmol/s per cm2 for D-glucose and 0.29 nmol/s per cm2 for taurocholate. The apparent Michaelis constant was raised by the factor of 6.6 for D-glucose and 3.6 for taurocholate due to the effects of the unstirred water layer. The maximal transport velocity was relatively unaffected by the unstirred water layer in both compounds. The values of the effective (operational) thickness of the unstirred water layer were compatible with those reported previously by employing various experimental methods. The kinetic parameters obtained in vitro everted sacs, for comparison, almost coincided with the true ones in situ. Therefore, the two-dimensional laminar flow model is shown to be valid not only for determining the kinetic parameters of a carrier-mediated transport in situ but also for predicting the absorption rate in situ from the uptake rate in vitro.     

Determination of the membrane permeability coefficient and the reflection coefficient by the two-dimensional laminar flow model for intestinal perfusion experiments

We performed single perfusion experiments in the small intestine of rats in order to prove that the two-dimensional laminar flow model is suitable to determine the membrane permeability coefficient and the reflection coefficient. We used progesterone as an aqueous-diffusion-limited drug, urea as a membrane transport-limited drug and the tritiated water as an intermediate substance. The membrane permeability coefficient for progesterone was calculated to be 3.6 X 10(-4) cm/s. This value did not change when the thickness of the aqueous diffusion layer was altered by increasing the perfusion rate 10-fold. It was directly demonstrated that the two-dimensional laminar flow model was suitable to analyze the data of intestinal perfusion experiments. Membrane permeability coefficients for urea and tritiated water were determined to be 3.4 X 10(-5) cm/s and 8.9 X 10(-5) cm/s, respectively. In the presence of water absorption with the hypotonic perfusion solution, the reflection coefficient for urea was 0.84. This value is thought to be theoretically reasonable, suggesting the usefullness of the two-dimensional laminar flow model to obtain the reflection coefficient in the intestinal membrane.     

Local light stimulation of melanophores of a teleost, Zacco temmincki

Responses of melanophores of the teleost, Zacco temmincki, to local light stimulation were examined in preparations of isolated scales. The melanophores induced the aggregation of melanosomes in darkness and their dispersion in light. Local illumination of a melanophore in the melanosome-dispersed state inhibited centripetal migration of melanosomes only in the stimulated area. Local illumination of a pigment-free branch of a melanophore with aggregated melanosomes generally brought about pigment dispersion into the stimulated area. However, when that area was at a significant distance from the edge of the central melanosome mass, the melanosomes never migrated into the irradiated area. Local illumination of the centrosphere of a cell inhibited the full aggregation of melanosomes in the dispersed and aggregated state. The degree of the inhibition depended on the size of the irradiated area. The results suggest that photoreceptive sites are distributed over the whole of a cell, and that the movements of melanosomes are regulated locally in a very precise manner.     

Pharmacokinetic study of exogenously administered ß-endorphin using a rapid radioreceptor assay in rats

A rapid radioreceptor assay for measuring ß-endorphin (ß-EP) in unextracted serum has been developed. The method is based upon the inhibition by ß-EP of ³H-naloxone binding to the specific receptors on rat brain membranes, prepared in a stable form of pellets. Effect of serum on the assay was minimized by adding pooled serum to the equal dilution of total serum in the assay mixture. Pharmacokinetic analysis of pharmacologically active ß-EP equivalents (ß-EP eq.) in rats was performed using this method. The serum disappearance of ß-EP eq. after iv administration followed a biexponential decline and pharmacokinetic parameters were calculated by a two-compartment open model. The half-lives of α-phase and ß-phase were 2.6 ± 0.5 min and 6.2 ± 1.6 hr (mean ± SE; n=6), respectively. The volume of the central compartment (V₁) and that of steady-state (Vd_ss) were 67 ± 16 and 480 ± 75 ml/kg (mean ± SE; n=6), respectively. The total body serum clearance (CL_tot) was 2.1 ± 0.9 ml/min/kg (mean ± SE; n=6). The serum disappearance curve of ß-EP eq. obtained in the present study was similar to that previously reported by Houghten et al. (Proc. Natl. Acad. Sci. U.S.A. $\text{77}$, 4588–4591 (1980)), in which the disapperance of total radioactivity of tritiated ß-EP in rats was examined.     

A New Enzymatic Cycling Technique for Glutamate Determination in Brain Microdialysates

A quantitative analysis of glutamate in brain dialysate was made by using an enzymatic cycling technique. This method made it possible to measure the concentration of glutamate in dialysate collected at 30-s intervals. Dialysates were collected from Mongolian gerbil hippocampus before, during, and after two 90-s ischemic insults at an interval of 5 min. An extracellular increase in levels of glutamate was already observed in samples collected during a 30-60 s period after the onset of each ischemia, and the levels of glutamate were maximal at the end of each period of ischemia (approximately a fourfold increase). The increased levels of glutamate rapidly returned almost to preischemic levels by 30 s of recirculation. This method will provide more precise information about temporal changes in the extracellular glutamate concentration in the brain during ischemia.