Commitment to the First Cortical Reorganization During Conjugation in Stylonychia mytilus: An Argument for Homology with Cortical Development During Binary Fission

The asexual nature of the first cortical reorganization of conjugation in Stylonychia was analyzed by comparing the effect of amputation performed at different stages of early conjugation to that performed on vegetative cells at different stages of the cell cycle. Amputation of vegetative cells delineated a point of commitment to binary fission at 0.51–0.57 of the cell cycle. Cells amputated before this point were induced to undergo the regenerative mode of asexual development, but those amputated after this point continued with binary fission. In parallel, during conjugation a similar commitment was made around the time of formation of tight mating-pairs: early conjugants amputated around this time might undergo regeneration, and those operated on after this stage continued with the first cortical reorganization as in typical conjugants. The two mates of a pair might differ in their response to amputation, suggesting that the timing of commitment to the first cortical reorganization is not related to the events of conjugation, but rather is individually determined in the vegetative cycle of the cells before they pair up in mating. These observations provide support for the notion that the first cortical reorganization of conjugants is homologous to the asexual mode of cortical development in dividers, according to the theory of developmental heterochrony in the sexual reproduction of hypotrichs. The timing of commitment to the first cortical reorganization was found to temporally correlate with the entrance of the micronuclei into meiosis. Since the first cortical reorganization can proceed without the micronucleus, this raises the possibility that initiation of micronuclear meiosis is closely coupled with, and may be determined by, the commitment to the first cortical reorganization.     

Proton transfer dynamics in the nonhomogeneous electric field of a protein.

By adsorption of pyranine (8 hydroxypyrene 1, 3, 6 trisulfonate) to lysozyme we create on the positively charged protein a fluorophoric site with a total charge of -3. Photo dissociation of the dye's hydroxyl proton changes its absorption and fluorescence spectrum, permitting a continuous monitoring of the reprotonation dynamics. Absorbance measurements in the microsecond time scale monitor how the bulk protons penetrate the Coulomb cage of the bound dye. Time-resolved fluorescence monitors how the proton is escaping out of the Coulomb cage of the bound dye. These probe reactions were studied with a series of dye-enzyme complexes where the number of free carboxylate was reduced by amidation, increasing the total charge of the complex from +5 to +12.6. The time-resolved measurements demonstrate the complexity of the electric field in the immediate vicinity of the dye. It is consistent with a negative potential wall (of the anion) surrounded by a positive potential wall of proteinaceous moieties.     

Comparison of Methods for Coccidioidomycosis Complement Fixation

A Laboratory Branch Task Force of the National Communicable Disease Center has proposed a standardized complement fixation procedure (LBCF) and an adaptation of this to microtitration techniques (MT) as uniform methods for performing complement fixation (CF) tests. A common procedure should make CF results from one laboratory more comparable to another. In addition, it would be preferable if the common procedure reproduced the titer levels of a testing procedure which is to be replaced, particularly when valid clinical interpretations have been derived from the latter. Replicated sets of sera were tested by the LBCF, MT, and the standard Smith CF procedure for coccidioidomycosis. Results with all three procedures were highly reproducible within an acceptable one-tube variation of a twofold dilution series, but the frequency of one-tube variations was greater with the MT method than with the other two. There was no statistical difference in the titers obtained with the Smith and LBCF procedures, but there was a significant difference when the MT results were compared to those with the Smith method. The LBCF method should be acceptable as a standardized and uniform CF procedure for coccidioidomycosis, subject to comparative testing between different laboratories.     

Serology of coccidioidomycosis

Summary We believe there is strong evidence to support a continuing search for coccidioidomycosis in new areas, in the Old World as well as in the New World, and in places with a climate and ecology different from the semi-arid conditions of the known endemic areas. Such an investigation would be justified in any population group where there is a high incidence of respiratory disease of unknown etiology. Very satisfactory and practical immunological techniques are available, but the present evidence indicates that the antigens used in these tests should be prepared from strains ofC. immitis recovered from the area to be investigated. Obviously this cannot be done at present, so such a program should be preceeded by an extensive survey of soil samples in the area in order to recover any existing native strains ofC. immitis. Whereas this would be the ideal situation, one could consider initiating the proposed study with coccidioidins prepared from selected strains of this fungus, incorporating as complete a spectrum of known antigens as is possible with our present knowledge, and keeping in mind that even this may not be adequate. We would welcome the opportunity to assist any investigator preparing to undertake a survey for coccidioidomycosis in his country.Paper read at the Eighth International Congresses for Tropical Medicine and Malaria, September 1968, Teheran (Iran).     

Time-resolved study of the inner space of lactose permease

Pyranine (8-hydroxy pyrene-1,3,6-trisulfonate) is a commonly used photoacid that discharges a proton when excited to its first electronic singlet state. Follow-up of its dissociation kinetics reveals the physicochemical properties of its most immediate environment. At vanishing ionic strength the dye adsorbs to the Escherichia coli lactose permease with stoichiometry of 1:1 and an association constant of 2.5 x 10(5) M(-1). The reversal of the binding at high ionic strength and the lower pK value of the bound dye imply that positive charge(s) stabilize the dye in its site. The fluorescence decay curve of the bound dye was measured by time-correlated single photon counting and the measured transient was subjected to kinetic analysis based on the geminate recombination model. The analysis indicated that the binding domain is a cleft (between 9 and 17 A deep) characterized by low activity of water (a((water)) = 0.71), reduced diffusivity of protons, and enhanced electrostatic potential. The binding of pyranine and a substrate are not mutually exclusive; however, when the substrate is added, the dye-binding environment is better solvated. These properties, if attributed to the substrate-conducting pathway, may explain some of the forces operating on the substrate in the cavity. The reduced activities of the water strips the substrate from some of its solvation water molecules and replace them by direct interaction with the protein. In parallel, the lower dielectric constant enhances the binding of the proton to the protein, thus keeping a tight seal that prevents protons from diffusing.