Phototransformation of the Far-red Light-absorbing Form of Large Pea Phytochrome by Laser Flash Excitation

Phototransformation of the far-red light absorbing form (P_FR)of large pea phytochrome to the red-light absorbing form (P_R)was examined at 2?C after a 715 nm laser flash excitation usinga custom-built multichannel transient spectra analyzer. Themaximum amount of phototransformation intermediates was producedby a pulse of about 50 mJ, which resulted in ca. 65% of P_R obtainedat the photostationary equilibrium. Some flash-induced intermediateswere assumed to return to P_FR in the dark. A difference spectrummeasured at 10 µsec after the flash showed an absorbanceincrease at 651 nm and a decrease at 724 nm. When the samplewas left in darkness after the flash light irradiation, absorbancein the red and far-red region gradually increased, but thatin the green region rapidly decreased. The decay curve of intermediatesmeasured at 554 nm could be resolved into three reaction componentshaving rate constants of 2,500, 590 and 48 sec^–1, respectively.Difference spectra also indicated that a small but significantincrease in absorbance between 370 and 380 nm and a decreasearound 415 nm took place 10–310 µsec after a flash. (Received February 13, 1982; Accepted April 21, 1982)     

Effects of Calcium and Potassium Ions on Phototaxis in Cryptomonas

Effects on positive phototaxis and the cell motility of 7 cationsin 5mM MOPS (morpholinopropane sulfonic acid) buffer (pH 7.0)containing 0.16 mM NaCl, 0.68 mM KCl, 0.5 mM CaCl₂ and 0.16mM MgCl₂ were studied in the unicellular flagellate Cryptomonaswith a photoelectrical measuring apparatus and photomicrography.When calcium ion was removed from the medium by adding 1 mMEGTA (ethylene glycol-bis-(ß-amino-ethylether)-N,N'-tetraaceticacid), the phototactic response was totally inhibited, but theswimming rate was not much affected. The effect of EGTA waspartially reversed by the addition of 1 mM CaCl₂. When 15mMKCl or RbCl was added to the medium, phototaxis was greatlyinhibited, but there was no significant influence on the swimmingrate. Similar but less inhibitory effects were induced in thepresence of NaCl, LiCl and CsCl. KCl-induced inhibition waspartially removed by the addition of 15 mM CaCl₂ or MgCl₂. (Received June 25, 1982; Accepted September 27, 1982)     

Isoforms of C-protein in adult chicken skeletal muscle: detection with monoclonal antibodies

Monoclonal antibodies (McAbs) specific for the C-proteins of chicken pectoralis major and anterior latissimus dorsi (ALD) muscles have been produced and characterized. Antibody specificity was demonstrated by solid phase radioimmunoassay (RIA), immunoblots, and immunofluorescence cytochemistry. Both McAbs MF-1 (or MF-21) and ALD-66 bound to myofibrillar proteins of approximately 150,000 daltons; the former antibody reacted with pectoralis but not ALD myofibrils, whereas the latter recognized ALD but not pectoralis myofibrils. Chromatographic elution of the antigens from DEAE-Sephadex, and their distribution in the A-band, support the conclusion that both of these antibodies recognize variant isoforms of C-protein. Since both McAbs react with a protein of similar molecular weight in the A-band of all myofibrils of the posterior latissimus dorsi (PLD) muscle, we suggest that either another isoform of C-protein exists in the PLD muscle or both pectoralis and ALD-like isoforms coexist in the A-bands of PLD muscle.     

Effects of narrow-beam irradiations with blue and far-red light on the timing of cell division in Adiantum gametophytes

Protonemata of the fern Adiantum capillusveneris L., grown as single-cell filaments under continuous red light, were irradiated with a narrow beam of blue light. Only irradiation of the region containing the nucleus induced cell division. Beams of 30 m in width, which corresponds to the diameter of the nucleus, or wider, were equally effective; beams 10 m wide or less were less effective. The results indicate that the nuclear region is the site of the blue- and near ultraviolet-light-absorbing pigment (P_B-NUV) which mediates the timing effect of cell division. In contrast, the effect of a narrow beam of far-red (FR) light, which delays the onset of the blue-light-induced cell division, was found to be present along the entire length of the protonema cell, including the largely vacuolated basal region of the latter. Polarized FR light having the electrical vector parallel to the protonema axis was less effective than that vibrating in other directions. These observations support the hypothesis that the phytochrome controlling the timing effect is localized in the plasma membrane.     

Random replication and random assortment model for plasmid incompatibility in bacteria

To understand the incompatibility between two related plasmids, both of which replicate in an autonomous state under a common control mechanism, we have developed a model that assumes a random choice mechanism for replication of plasmid copies and their random assortment into daughter cells upon cell division. Segregation kinetics by this model is analyzed mathematically and the number of generations required for segregation is calculated as a function of plasmid copy number per cell. The results obtained offer enough quantitative data to make our model reasonably realistic.     

Construction and characterization of the chimeric enzymes between the Bacillus subtilis cellulase and an alkalophilic Bacillus cellulase

The amino acid sequences of cellulase from Bacillus subtilis (BSC) and that from an alkalophilic Bacillus sp. N-4 (NK1) show significant homology in most parts except for the C-terminal portions. Despite the high homology, the pH activity profiles of the two enzymes are quite different; BSC has its optimum pH at 6-6.5, whereas NK1 is active over a broad pH range from 6 to 10.5. In order to identify the structural features which determine such pH activity profiles, chimeric cellulases between BSC and NK1 were constructed using four restriction sites commonly present within the homologous coding sequences, and were produced in Escherichia coli. The chimeric cellulases showed various chromatographic behaviors, reflecting the origins of their C-terminal regions. The pH activity profiles of the chimeric enzymes in the alkaline range could be classified into either the BSC or NK1 type mainly depending on the origins of the fifth C-terminal regions. In the acidic range, the profile was determined only by the origin of the fourth enzyme region from the N terminus. Comparison of the kinetic parameters between pH 5 and 6 using p-nitrophenyl cellobioside as a substrate indicated that the fourth region is responsible for the pH-dependent change of the kcat value. Only a limited number of amino acids in the fourth region may affect on deprotonation of catalytic residues of the cellulases and modulate the catalytic activity in the acidic pH values.     

Molecular cloning, sequencing, and expression of cDNA for rat liver microsomal aldehyde dehydrogenase.

The cDNA clone for rat liver microsomal aldehyde dehydrogenase (msALDH) was isolated and sequenced. The deduced amino acid sequence consisting of 484 amino acid residues revealed that the carboxyl-terminal region of msALDH has a hydrophobic segment, which is probably important for the insertion of this enzyme into the endoplasmic reticulum membrane. COS-1 cells transfected with the expression vector pcD containing the full-length cDNA showed that the active enzyme was expressed and localized mainly on the cytoplasmic surface of the endoplasmic reticulum membranes. It has been proposed that ALDH isozymes form a superfamily consisting of class 1, 2, and 3 ALDHs (Hempel, J., Harper, K., and Lindahl, R., (1989) Biochemistry 28, 1160-1167). Comparison of the amino acid sequence of rat liver msALDH with those of rat other class ALDHs showed that msALDH was 24.2, 24.0, and 65.5% identical to phenobarbital-inducible ALDH (variant class 1), mitochondrial ALDH (class 2), and tumor-associated ALDH (class 3), respectively. Several amino acid residues common to the other known ALDHs, however, were found to be conserved in msALDH. Based on these results, we proposed to classify msALDH as a new type, class 4 ALDH.     

Effects of TAP-144-SR, a sustained-release formulation of a potent GnRH agonist, on experimental endometriosis in the rat

A new, simple experimental endometriosis model was established by auto-transplanting endometrial tissue fragments beneath kidney capsules in female rats. The transplanted endometrial tissue grew well, forming a fluid-filled cyst, which reached maximal size 2 to 3 weeks after transplantation. The growth and maintenance of the transplants was dependent on the ovary: ovariectomy induced regression of well grown transplants. The therapeutic effects of TAP-144-SR (biodegradable microcapsules of copoly (DL-lactic/glycolic acid) copolymer containing a potent GnRH agonist, TAP-144 (D-Leu6-[des-Gly10-NH2]-GnRH ethylamide, leuprolide acetate) were studied with this rat endometriosis model. A single sc injection of TAP-144-SR (corresponding to 1, 10 or 100 micrograms/kg/day of TAP-144), suppressed the growth of the transplanted endometrial tissues and uterine weight in a dose-dependent manner. At 100 micrograms/kg/day, the suppressive effect was more marked in rats given TAP-144-SR than in those given TAP-144 solution. The extent of suppression was comparable to that caused by ovariectomy. Serum and pituitary concentrations of LH and FSH were also reduced more markedly by the administration of TAP-144-SR than by TAP-144 solution. From these results, the present endometriosis model was found to be useful for the evaluation of compounds with potential therapeutic activity. The sustained-release formulation of TAP-144 seems to be beneficial over its solution in terms of both convenience and efficiency for therapy of patients with endometriosis.     

Factor I-dependent inactivation of human complement C4b of the classical pathway by C3b/C4b receptor (CR1, CD35) and membrane cofactor protein (MCP, CD46).

Proteolytic inactivation of C4b is a crucial step for regulation of the classical complement pathway. A plasma protease factor I and membrane cofactors, C3b/C4b receptor (CR1) and membrane cofactor protein (MCP), participate in the regulation of cell-bound C4b although the physiological potency of these cofactors remains unknown. We have examined the optimal conditions of the factor I-mediated C4b regulatory system using purified cofactors. CR1 being a cofactor at a cofactor/C4b ratio less than 0.1 (w/w), fluid phase C4b, and methylamine-treated C4 (C4ma) were degraded by factor I into C4bi: minimal Cd4 was generated in the fluid phase. Liposome-bound C4b (LAC4b), on the other hand, was degraded into C4c and C4d. CR1 showed two optimal pHs (6.0 and 7.5) for fluid phase C4b, but one (6.0) for LAC4b, and in both cases low conductivity conditions enhanced the C4bi generation. CR1 cofactor activity was barely influenced by the NP-40 concentration. On the other hand, MCP degraded C4b and C4ma, as a factor I-cofactor, more efficiently into C4c and C4d. Though MCP cofactor activity, like that of CR1, was enhanced under low conductivity conditions, it has only one optimal pH, 6.0, in both fluid and solid phases. Furthermore, as in the case of C3b cleavage, a sufficient NP-40 concentration to solubilize membrane was needed for MCP to express full cofactor activity for C4b, in contrast to CR1. MCP was less potent for C4b inactivation than for C3b inactivation, while CR1 acted as a slightly more effective cofactor for C4b cleavage than for C3b cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)