New method of immobilization of microbial cells by capture on the surface of insoluble pyridinium-type resin

A new method for the immobilization of microbial cells has been developed. Whole cells of Escherichia coli with aspartase activity were immobilized by capture on the surface of cross-linked poly(N-benzyl-4-vinylpyridinium bromide) containing styrene (BVPS resin), an insoluble pyridinium-type resin. When a suspension of the bacterial cells in buffer solution was passed through a glass column containing beads of BVPS resin, the cells were captured on the resin surface and formed an immobilized cell system. A fixed-bed column reactor containing 300 mg of the bacterial cells immobilized by capture on 10 g of BVPS resin beads was used for the preparation of L-aspartic acid from ammonium fumarate. Continuous operation of tne bioreactor produced L-aspartic acid in a quantitative yield when the influent substrate concentration was 0.1M and the flow rate was 0.41-0.83 bed volumes per hour at pH 7.4-7.7 at 30 degrees C.     

A modulating role of mercurials-sensitive sulfhydryl groups in synaptic GABA receptor binding

The specific binding of GABA (γ-aminobutyric acid) agonist ³H-muscimol, to synaptic membranes from the rat brain showed a significant increase, when the membranous preparations were treated with a low concentration (10^?4–10^?5M) of mercurial sulfhydryl reagents such as p-chloromercuribenzoate and mercuric chloride. This activation in GABA receptor binding was bicuculline-sensitive, and was partially restored by subsequent treatments with 10 mM cysteine, penicillamine, or mercaptoethanol. Scatchard analysis of the binding revealed that this activation was due to the increase in the affinity of both high and low affinity bindings sites but not in the B_max values. On the other hand, the treatment of synaptic membranes with hydrophilic sulfhydryl reagents such as N-ethylmaleimide and iodoacetate had no effect on the binding. These hydrophilic sulfhydryl reagents, however, induced an increase of the binding following the pretreatment of synaptic membranes with 0.01% Triton X-100 or 0.5 U/mg prot. of phospholipase A₂ (EC 3.1.1.4.). These results suggest that mercurials-sensitive sulfhydryl groups, which are normally masked by membrane lipids, may play a modulating role in GABA receptor binding at central synapses.     

Membrane-potential- and surface-potential-induced absorbance changes of merocyanine dyes added to chromatophores from Rhodopseudomonas sphaeroides

(1) Three analogs of merocyanine dyes added to suspensions of chromatophore vesicles showed absorbance changes responding to the change in surface potential induced by salt addition and to the change in membrane potential induced by illumination. (2) The extent of the light-induced absorbance changes of the dyes was linearly related, in the presence and absence of uncouplers, to that of carotenoid spectral shift which is an intrinsic probe of the intramembrane electric field. (3) Comparison of the merocyanine absorbance changes induced by salt addition with those induced by illumination indicated that the surface potential change in the outer surface of chromatophore membranes during illumination was very small. (4) Judging from the spectra of these absorbance and from the low permeabilities of the dyes to membrane, the absorbance change are attributed to change in distribution of the dyes between the medium and the outer surface region in chromatophore membranes. The extent of the light-induced absorbance changes of merocyanine dyes depended on the salt concentration of the medium. The types of dependence were different among three merocyanine analogs. This is explained by the mechanism mentioned above assuming appropriate parameters. It is suggested that, under continuous illumination, an equilibrium of the electrochemical potential of H⁺ is reached between the bulk aqueous phase and the outer surface region in the membrane where the merocyanine dyes are distributed.     

Developmental formation of glutamine synthetase in greening pumpkin cotyledons and its subcellular localization

During the germination of pumpkin (Cucurbita sp. Amakuri Nankin) seeds in dark, the activity of glutamine synthetase in cotyledons gradually increased, reaching a maximum at 5 to 6 days. A measurable enhancement (about 4-fold) of the enzyme activity occurred when the seedlings were exposed to continuous illumination from day 4 up to day 8. Glutamine synthetase activity was detectable only in the cytosolic fraction in the etiolated cotyledons, whereas it was found both in the cytosolic and chloroplast fractions in the green cotyledons. The two isoenzymes of glutamine synthetase have been separated by DEAE-cellulose column chromatography of extracts from the green cotyledons. These data indicate that during the greening process the chloroplastic glutamine synthetase is newly synthesized. The roles of cytosolic and chloroplastic glutamine synthetase in germinating pumpkin cotyledons concerning assimilation of NH₃ are discussed.     

Acceleration of the Decay of Membrane Potential after Energization of Chloroplasts in Intact Zea mays Leaves

The relationship between dissipation of the flash-induced membranepotential across the thylakoid membrane and the high energystate was studied in Zea mays leaves. The dark decay of theflash-induced 515-nm absorbance change was accelerated by shortpreillumination of the leaf. No acceleration of the decay bypreillumination was observed when leaves were incubated in argonor CO² gas or treated with DCMU. These effects of preilluminationand incubation were reversible. The delayed fluorescence from chlorophyll a was reversibly decreasedby incubating leaves in argon or CO² gas, though the modes ofdepression were somewhat different from each other. In leavesincubated in argon or CO² gas, the phase of slow decrease ofthe intensity of prompt fluorescence during illumination reversiblydisappeared. The results suggested that the dissipation of membrane potentialgenerated by a flash was accelerated after the energizationof chloroplasts in leaves, probably by increased H^– permeabilityof the thylakoid membrane. O2 was important in maintaining (indarkness) and forming (under illumination) the high energy statein chloroplasts in intact leaves. (Received October 1, 1980; Accepted December 15, 1980)     

Neurochemical Characteristics of Myelin-like Structure in the Chick Retina

Abstract: Certain characteristics of myelin-like structures in the chick retina were examined morphologically and biochemically. Developmental changes of 2', 3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) in the chick retina and optic nerve were examined. The measurable activity in the retina was first detected at 16 days of incubation and thereafter, it increased rapidly until 4 weeks post-hatching. By contrast, CNPase activity in the optic nerve reached the maximum level at 4 days post-hatching and maintained a constant level thereafter. The purifed myelin fraction from the chick retina showed higher activity of CNPase, whereas its activity in the retinal homogenate was very low. Hence, it was considered that the myelin fraction from the chick retina is similar to that of CNS myelin with respect to CNPase. Protein profiles of the purified myelin fractions isolated from the chick optic tectum, optic nerve, retina and sciatic nerve were analysed by SDS-polyacrylamide gel elec-trophoresis. Myelin fractions from the chick optic tectum and optic nerve contained basic protein (BP) and Folch-Lees proteolipid protein (PLP). Myelin fraction from the chick sciatic nerve contained BP, P2 and two glycoproteins (PO and 23K). In contrast, retinal myelin fraction contained only BP. PLP, PO, 23K and P2 proteins were definitely undetectable. Electron micrographs revealed that some axons in the optic nerve fiber layer of the chick retina were wrapped by a spiral-structured myelin-like sheath, which showed some differences from those of CNS and PNS myelin sheaths. It was suggested that the origin of the myelin-like structure in the chick retina is other than from oligodendroglia or Schwann cells.     

Clasping structure in the double cone of the retina of the turtle (Gyoclemys reevesii)

Summary The double cone of the turtle retina was reconstructed three-dimensionally from electron micrographs of 400 serial sections. Cytoplasm of the accessory cone extends bilaterally to surround the principal cone incompletely. The cytoplasmic extensions are 0.1 m in width and 2 m in length at their longest portions. They arise at approximately the median portion of the ellipsoid (consisting of mitochondria) and terminate at the level of the outer limiting membrane. The possible function of these extensions as a clasping structure is discussed.     

The control of phosphoprotein phosphatase by the second-site phosphorylation of a substrate. Studies with H2B histone as model substrate.

The phosphorylation of Ser-32, in addition to Ser-36 of H2B histone, stimulated the rate of Pi release from Ser-36 by the small form (Mr 31 000) of pig heart phosphoprotein phosphatase both in the absence and presence of 50 mM magnesium acetate. By phosphorylation at Ser-32, the Km value for Ser-36 phosphate in H2B histone was increased from 0.38 microM to 1.16 microM in the absence of magnesium acetate, but not significantly changed (from 37.4 microM to 26.2 microM) in the presence of magnesium acetate. With the large form (Mr 224000) of the phosphoprotein phosphatase, however, the phosphorylation at Ser-32 suppressed the rate of Pi release from Ser-36 both in the absence and presence of magnesium acetate. The Km value of the large form for Ser-36 phosphatase in H2B histone was nevertheless increased by phosphorylation at Ser-32, from 1.2 microM to 5.3 microM in the presence of magnesium acetate, but not changed (from 0.26 microM to 0.23 microM) in the absence of magnesium acetate.     

Ascaris suum: suppression of reaginic and hemagglutinating antibody responses in the mouse by crude extract and maintenance fluid.

Intraperitoneal administration in mice of crude extract (CE) or maintenance fluid (MF) of Ascaris suum in Freund's incomplete adjuvant (FICA) in doses of 200 and 2 (CE) and 4 μg (MF) on Days ?4, 0, and +4 relative to the day of the immunization with 10 μg of hen egg white lysozyme (HL) resulted in the suppression of anti-HL reaginic antibody responses at varying degrees depending on the dose and their time of administration. Hemagglutinating antibody responses were also affected but in a different manner. Treatment with CE on Day ?4 resulted in complete suppression of reaginic antibody responses and some degree of suppression of hemagglutinating antibody responses depending on the size of the CE dose. In mice pretreated with MF, transient suppression was found only for reaginic antibody responses. In mice receiving the treatment of CE on Day 0, 200 μg of CE caused complete suppression of reaginic antibody responses, while 2 μg was less effective. Hemagglutinating antibody responses were also suppressed in proportion to the dose. Simultaneous treatment with MF did not cause any suppression of either reaginic or hemagglutinating antibody responses. In mice treated with CE on Day +4, reaginic antibody responses were not markedly suppressed and hemagglutinating antibody responses were also not altered. In contrast, treatment with MF on Day +4 resulted in suppression of reaginic antibody responses during the whole course of the primary response, but had no effect on hemagglutinating antibody responses. When MF was administered 7 days after the priming, no suppressive effect on the antibody responses was demonstrated. On the other hand, if a lower dose (1 μg of HL) was used for the priming, the effect of MF treatment with Day +4 was more pronounced in the primary reaginic antibody response and the secondary response was also affected. A comparable suppression of hemagglutinating antibody responses also was observed.