Detection of Polypeptides Involved in Early Stages of Nodulation in Soybean Roots

Soluble proteins extracted from the roots of nodulating soybean[Glycine max (L.) Merr. cv. T202] and from roots of the non-nodulatingisoline rj₁ of cv. T202 (cv. T201), which had been inoculatedwith Bradyrhizobium japonicum, were analyzed by two-dimensionalpolyacrylamide gel electrophoresis and silver staining, in anattempt to identify polypeptides involved in early stages ofnodulation. Almost identical patterns of polypeptides were generatedby extracts of 3-day-old roots of uninoculated T201 and T202and of inoculated T201 and T202, but a unique spot, correspondingto a polypeptide of 38 kDa was detected in the case of inoculatedroots of T202. Western blotting analysis using "inoculated-T202-rootspecific" antiserum, prepared by titration of antiserum againstproteins from inoculated T202 roots with proteins from inoculatedT201 roots, revealed spots corresponding to polypeptides of26,27, and 33 kDa that were detectable only in the extractsof roots of inoculated T202. However, no unique polypeptidespots were detected in the case of roots of inoculated T201and T202, as compared with those from uninoculated T201 andT202 roots by Western blotting analysis using "inoculated-T201-rootspecific" antiserum prepared by titration of antiserum againstproteins from inoculated T201 roots with proteins from uninoculatedT201 roots. (Received May 27, 1991; Accepted September 30, 1991)     

Action Spectrum for Resetting the Circadian Phototaxis Rhythm in the CW15 Strain of Chlamydomonas: II. Illuminated Cells

The action spectrum for resetting the phase of the circadian clock in Chlamydomonas reinhardtii is different depending upon whether the light stimuli are presented to cells that were in darkness versus dim illumination before stimulation. In this report, we show that phase resetting of illuminated cells appears to be mediated by components of the photosynthetic apparatus. This conclusion is based upon the action spectrum for phase-shifting illuminated cells (which looks like that for photosynthesis) and upon the fact that inhibitors of photosynthetic electron transport also inhibit the light-induced phase shift of illuminated cells. Both of these characteristics differ from that of cells taken from darkness. We, therefore, believe that at least two resetting pathways for this circadian clock exist and that both of these pathways are ecologically significant.     

Manganese, copper, zinc, and iron concentrations and subcellular distribution in two types of skeletal muscle

To clarify trace element distribution in red and white muscle, and to verify two populations of muscle mitochondria, the iron, zinc, copper, and manganese concentrations of whole muscle and their subcellular fractions were determined. The iron, zinc, copper, and manganese concentrations of red muscle were 1.83, 4.31, 2.05, and 1.67 times higher than those of white muscle, respectively. In skeletal muscle subcellular distribution or iron, zinc, and copper were entirely different and that of manganese was relatively similar as compared with those in liver reported previously. The pattern of mineral distribution in all fractions of red muscle was similar to that of white muscle, but their concentrations in some fractions were different between red and white muscle, e.g., iron, zinc, and manganese in supernatant fraction and copper in nuclear and microsomal fractions. The difference between subsarcolemmal and interfibrillar mitochondria were ascertained by the distribution of trace elements.     

Localization of gastrin-releasing peptide (GRP)-like immunoreactivity in the lysosomal compartment of the trigeminal ganglion cells of the rat

Summary Cellular and subcellular localizations of gastrin-releasing peptide-like immunoreactivity (GRP-LI) were examined in the perikarya of trigeminal ganglion cells. By immunolight microscopy using semi-thin sections, GRP-LI was observed in almost all the neuronal somata with various intensity as granular profiles distributing widely in the cytoplasm. By immunoelectron microscopy using ultrathin frozen sections and protein A-gold, GRP-LI was found predominantly in rounded or oval membrane-bound structures which were 300–800 nm in diameter and displayed various electron-density and heterogenous contents. Double-labeling immunoelectron microscopy using antibodies for GRP and cathepsin L, a well-characterized lyosomal proteinase, clearly demonstrated that these GRP-immunoreactive intracytoplasmic structures were lysosomes. In contrast, GRP-LI was detected only occasionally in the large granular vesicles (100–200 nm in diameter). These findings strongly suggest that considerable amount of GRP or GRP-like peptide is subject to intracellular degradation in the lysosome rather than to the regulatory secretion pathway, and this is the reason why no fibers immunoreactive for GRP have been detected in the peripheral sensory field.     

Histochemical study on the maturation of human megakaryocytes using microfluorometry

Summary A microcytofluorometrical DNA measurement was basically studied and was applied to single megakaryocytes previously identified on a Wright-Giemsa stained smear. The smear was first photographed and the location of each megakaryocyte was recorded on a cell map. The smear was then bleached with 50% acid ethanol and absolute methanol, and re-stained with 4,6-diamidino-2-phenylindole (DAPI) reagent (pH 7.4) at 4° C. Nuclear blue fluorescence was observed and the intensity of this fluorescence was proportional to the amount of DNA with the coefficient of variation (CV) of 3.6% when stained for 30 min. After 30 min DAPI staining, the DNA measurement was microcytofluorometrically performed in single megakaryocytes which had been morphologically classified into 4 groups on the basis of cytoplasmic maturation, Bessis' classification, assessed on Wright-Giemsa-stained bone-marrow smears from normal human beings. The histograms of the cells did not show any difference in DNA ploidy distribution among the classes: that is, the DNA histograms disclosed ploidy distribution from 4 N to 64 N with the largest population of 16 N. These findings suggest that nuclear DNA synthesis is completed before platelet production starts. This method is useful for comparing the morphological features and DNA content of single megakaryocytes.     

Alterations in cardiac function and subcellular membrane activities after hypervitaminosis D3

The present study was designed to induce massive accumulation of calcium in the myocardium and to evaluate the effect of calcium overload on myocardial contractile function and biochemical activity of cardiac subcellular membranes. Rats were treated with an oral administration of 500,000 units/kg of vitamin D₃ for 3 consecutive days, and their hearts were sampled on the 5th day for biochemical analysis. On the 4th and 5th days, heart rate, mean aortic pressure, left ventricular systolic pressure and left ventricular dP/dt were significantly lowered in vitamin D₃-treated rats, demonstrating the existence of appreciable myocardial contractile dysfunction. Marked increases in the myocardial calcium (67-fold increase) and mitochondrial calcium contents (24-fold increase) were observed by hypervitaminosis D3. Mitochondrial oxidative phosphorylation and ATPase activity were significantly reduced by this treatment. A decline in sarcolemmal Na⁺, K⁺-ATPase activity was also observed, while relatively minor or insignificant changes in calcium uptake and ATPase activities of sarcoplasmic reticulum were detectable. Electron microscopic examination revealed calcium deposits in the mitochondria after vitamin D₃ treatment. The results suggest that hypervitaminosis D₃ produces massive accumulation of calcium in the myocardium, particularly in the cardiac mitochondrial membrane, which may induce an impairment in the mitochondrial function and eventually may lead to a failure in the cardiac contractile function.     

Further studies on membrane transport with time delay

A theory of cell membrane transport with a time delay which predicts under certain conditions overshoot or oscillatory permeation (Ohshima and Kondo, Biophys. Chem. 33 (1989) 303), is extended with the introduction of a parameter expressing a fraction of solutes inside the cell interior that suffer time delay. It is found that criterion for oscillation depends strongly on this parameter. Results will also be presented for the case of an exponential-type distribution of the delay time.     

Adsorption equilibrium in immuno-affinity chromatography with polyclonal and monoclonal antibodies

The effects of pH, ionic strength, anion species, and antibody concentration on the adsorption equilibrium between immobilized antibodies and antigens were studied by use of anti-BSA, anti-HSA, anti-BlgG, and monoclonal anti-HSA coupled to Sepharose 4B. The polyclonal antibodies possessed average binding affinities of the order of 10(8)M(-1), and the heterogeneity was accounted for by assuming a normal distribution of the free energy of antibody-antigen combination. The monoclonal antibody, on the other hand, showed a homogeneous affinity of the Langmuir type. Bound antigens could be eluted by lowering pH or adding a chaotropic anion, and their purity was very high. The antibody ligand was sufficiently stable for repeated use.     

Thermal Properties of Membrane Lipids from two Cyanobacteria, Anacystis nidulans and Synechococcus sp.

The composition and positional distribution of fatty acids inmonogalactosyldiacylglycerol, digalactosyldiacylglycerol, phosphatidylglyceroland sulphoquinovosyldiacylglycerol from two cyanobacteria, Anacystisnidulans and Synechococcus sp. grown at 25°C have been determinedand compared with measurements of the phase separation temperaturesof the lipids. Only monogalactosyldiacylglycerol in Anacystisand sulphoquinovosyldiacylglycerol in Synechococcus showed phaseseparation temperatures above 0°C. The phase transitiontemperature of a sample of sulphoquinovosyldiacylglycerol containingover 90% of the dihexadecanoyl molecular species has been determinedto be 43°C for the Na⁺ salt and 38°C for the Mg⁺⁺ salt. ^*Deceased. September 14, 1986. (Received June 25, 1986; Accepted August 25, 1986)