Possible participation of calmodulin in stimulation of leucine transport by concanavalin A in human lymphocytes

Stimulation of leucine uptake by addition of concanavalin A, mediated by increase of intracellular free Ca2+ concentration [( Ca2+]), in lymphocytes (Mitsumoto, Y., Sato, K. and Mohri, T. (1988) Biochim. Biophys. Acta 968, 353-358) was abolished by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and chlorpromazine, which inhibited membrane hyperpolarization induced by the mitogen. Quinine (0.5-1 mM) completely inhibited the concanavalin A-induced hyperpolarization and extensively inhibited the induced stimulation of leucine uptake. Based on these results, we suggest that the stimulation of leucine uptake by concanavalin A is largely due to activation of the Ca2+-dependent K+ channel which reinforces negative potential of the plasma membrane and is regulated by calmodulin.     

Sensitive sandwich enzyme immunoassay of human growth hormone (hGH) in serum and urine using monoclonal antibody-coated polystyrene balls

A sensitive sandwich enzyme immunoassay for human growth hormone (hGH) using monoclonal antibody is described. A monoclonal anti-hGH IgG-coated polystyrene ball was incubated with hGH and subsequently with affinity-purified rabbit anti-hGH Fab'-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorimetry using 3-(4-hydroxyphenyl) propionic acid as a substrate. The detection limits of hGH in serum and urine were 1.5 ng/l using 20 microliters of serum and 0.2 ng/l using 0.15 ml of urine, respectively. The specificity and assay precision were satisfactory. hGH levels in serum and urine determined by the present sandwich enzyme immunoassay using monoclonal anti-hGH IgG-coated polystyrene balls were well correlated to those determined by the previous sandwich enzyme immunoassay using rabbit anti-hGH IgG-coated polystyrene balls. Levels of hGH in urine collected as first morning voids from healthy subjects aged 19-28 yr were 6.4 +/- 3.2 (SD) ng/g creatinine. However, the present assay gave lower hGH levels than the previous assay. This was at least partly explained by the fact that hGH in urine was less efficiently bound to monoclonal anti-hGH IgG-polystyrene balls than standard hGH, while the binding of hGH in urine and standard hGH to rabbit anti-hGH IgG-coated polystyrene balls was equally efficient. In addition, gel filtration showed that 22K hGH, a major component, in urine was less efficiently bound to monoclonal anti-hGH IgG-coated polystyrene balls than standard 22K hGH. The nature of hGH in serum and urine remains to be investigated.     

Flagellar movement of human spermatozoa

Flagellar movement of human spermatozoa held by their heads with a micropipette was recorded by means of a video-strobe system. Spermatozoa were studied in normal Hanks' solution, Hanks' solution with increased viscosity, cervical mucus, and hyaluronic acid. When flagellar movement in normal Hanks' solution was observed from the direction parallel to the beating plane, segments of the flagellum in focus did not lie on a straight line but on two diverging dashed lines. The distance between the two dashed lines was about 20% of the bend amplitude in the major beating plane. These observations indicate that flagellar beating of human spermatozoa in normal Hanks' solution is not planar. In contrast, segments of the flagellum in focus lay on a straight line when the spermatozoa were observed in Hanks' solution with increased viscosity, cervical mucus, or hyaluronic acid. In normal Hanks' solution, free swimming spermatozoa rotated constantly around their longitudinal axes with a frequency similar to the beat frequency, whereas little or no rotation of spermatozoa occurred in Hanks' solution with increased viscosity, in cervical mucus, or in hyaluronic acid. We conclude that human spermatozoa in normal Hanks' solution beat with a conical helical waveform having an elliptical cross section, the semiaxes of which have a ratio of 0.2. The three-dimensional geometry of the flagellar movement is responsible for the rotation of the sperm around their longitudinal axes.     

Characterization of interaction between Tb3+ and porcine intestinal brush-border membranes

Effects of ionic strength and temperature on the interaction between Tb3+ and porcine intestinal brush-border membrane vesicles were studied. When Tb3+ was added to the vesicle suspension, Tb3+ fluorescence increased with increasing concentration of Tb3+, showing a saturation. The apparent dissociation constant of one of at least two components of this binding reaction was estimated to be about 12.5 microM at 25 degrees C, pH 7.4. But the affinity of Tb3+ for the membrane vesicles was variable with changes of ionic strength and temperature. The affinity was lowered by addition of KCl to medium and by increase of temperature above 30 degrees C. In addition, temperature-induced change in the affinity of Tb3+ for the membranes was reversible over a temperature range from 13 to 46 degrees C. Temperature-dependence profiles of the excimer formation efficiency of pyrene-labeled membranes and of the harmonic mean of the rotational relaxation times of pyrene molecules in the membranes revealed that the phase transition of the membrane lipids occurs at about 30 degrees C. Based on these results, characteristics of Tb3+ binding to the membranes are discussed in relation to the nature of lipid phase and surface charges of the membranes.     

Studies on cation-induced membrane vesicle aggregation of porcine intestinal brush borders

Protein alterations during the development of the mouse brain were studied by two-dimensional gel electrophoresis. A protein spot with a molecular weight (MW) of 68,000 and pI value of 5.6 was found in the brain of the 11th day of gestation. Between the 12th and the 14th day of gestation, spots with the same MW and lower pI values appeared progressively. Neuraminidase digestion converted the pI of these acidic spots to 5.6. Thus, increased sialylation appeared to occur during this period. This class of molecules became hardly detectable on the 15th day, and disappeared completely after the 16th day. Analogous spots were present in the heart, liver, and stomach of the embryos, although the increased sialylation was not observed in the liver. No adult organs so far examined showed these spots. On the other hand, two polypeptides (MW 55,000, pI 4.7, and 53,000, pI 4.6) appeared in the brain on the 13th day of gestation and persisted throughout the fetal period. After birth, they became hardly detectable. Furthermore, a spot (MW 48,000, pI 4.8) became newly detectable in the brain 4-5 weeks after the birth.     

Effect of ionic strength on the membrane fluidity of rabbit intestinal brush-border membranes. A fluorescence probe study

The effect of ionic strength on the fluidity of rabbit intestinal brush-border membranes has been studied using two fluorescence probes, pyrene and 1-anilino-8-naphthalene sulfonate (ANS). The imposition of a potential gradient on the pyrene-probed membrane vesicles (out greater than in) with increasing NaCl concentration in the medium resulted in a marked enhancement of the excimer formation efficiency, accompanied by a decrease in the ratio of fluorescence intensities of the probe at 392 and 375 nm. Fluorescence polarization of the pyrene-membrane complex is independent of temperature in the absence of salts, while it is dependent on temperature from 10 to 47 degrees C in the presence of salts, as shown by the thermal Perrin plots of polarization. It has been demonstrated that there is a linear relationship between the changes in the pyrene excimer formation efficiency in the membranes and of the values of the binding parameters of ANS for the membranes. From these results, it is suggested that the lipid phase of the membranes becomes more fluid by shielding negatively charged groups of the membrane surface and that there is a fairly close correlation between the membrane organization and the membrane surface charge density.     

Ultrastructural localization of carbohydrates in Reichert's membrane of the mouse

In the present investigation, we examined the role of trophoblast and parietal endoderm cells in the synthesis of carbohydrate-containing components of Reichert's membrane. To eliminate the function of Reichert's membrane as a filter between maternal and embryonal tissues we carried out our examination under in vitro conditions. Parietal yolk sac from mouse embryos on day 9 post coitum (p.c.) were cultivated for 0 to 5 days. Because tannic acid enables a complex formation between carbohydrates and osmium we chose the fixation with this acid for the ultrastructural study. Electron microscopy showed that for assembly of Reichert's membrane, trophoblast cells produce and then release components that were detected as tannic acid-positive granules both in the Reichert's membrane and in the vacuoles of the trophoblast cells. To localize specific carbohydrates we used postembedding-gold-lectin histochemistry on LR-Gold^R-embedded tissues. Strong binding sites for the lectins WGA (Triticum vulgare), RCA I (Ricinus communis) and Con A (Canavalia ensiformis) were observed in Reichert's membrane and trophoblast cells but not in the parietal endoderm cells. The LTA (Lotus tetragonolobus)-binding pattern was positive in the membrane and its adjacent cells but that of the LFA (Limax flavus) was negative in the parietal endoderm cells and very weak in Reichert's membrane and trophoblast cells. Our results demonstrate that trophoblast cells are involved in the construction of Reichert's membrane through the production and release of specific glycoconjugates.