Cell attachment to collagen: The ionic requirements

This study is concerned with three aspects of the ionic requirements for cell attachment to collagen; namely (a) the divalent, (b) monovalent cation specificities and (c) pH optima for cell interaction with collagen. The divalent cation requirement for cell attachment to collagen can be sufficed by Ca²⁺, Mg²⁺ and certain transition group metals; whereas Ba²⁺, Sr²⁺, and polyamines are inactive. The pH optimum for cell attachment in this system occurs in the physiological range. The monovalent cation requirement for cell attachment to collagen is satisfied by isotonic NaCl, KCl, LiCl, NH₄Cl, sucrose, and glucose. Pronounced inhibition of cell attachment occurs under both hypertonic and hypotonic conditions.     

A Model of Functional Epistasis and Linkage Disequilibrium in Populations with Overlapping Generations

A model of functional epistasis is proposed in which it is assumed that coupling and repulsion genotypes differ in metabolic efficiency and thus in development time and net fecundity. The implications of this model are investigated for iteroparous populations with fluctuating rates of increase. It is found that the fluctuations in rate of increase can lead to large fluctuations in gamete frequency and D, the coefficient of linkage disequilibrium, but that D will almost always have a value of zero at some point during the populations' demographic cycle. Some of the model populations would be expected to be in a state of linkage disequilibrium only fleetingly: others would exhibit D-cycles interpretable as random fluctuation. Implications of the model for interpretations of existing data on linkage disequilibrium among enzyme loci in Drosophila are discussed.     

IN SITU PRESERVATION OF THE TRANSVERSE FLAGELLUM OF PERIDINIUM CINCTUM (DINOPHYCEAE) FOR SCANNING ELECTRON MICROSCOPY1

Three-dimensional structure of the transverse flagellum was studied by means of scanning electron microscopy (SEM) in Peridinium cinctum (O.F.M.) Ehrenberg. Several fixation and dehydration procedures were compared. Cells fixed, rapidly in 1% osmium tetroxide and critical-point dried showed accurate preservation of structural features seen in living cells; in particular, both transverse and posterior flagella were retained in the furrows on the cell surface. Osmium-fixed and freeze-dried, samples were prone to cellular collapse and loss of flagella. Glutar-aldehyde-fixed and critical-point dried cells showed a layer of material covering the thecal plates, most conspicuously over the girdle region; flagella were absent. Stereo-pair micrographs illustrate the 3-dimensional configuration of the transverse flagellum. Modifications of previous structural models are proposed.     

Intramolecular determination of primary kinetic isotope effects in hydroxylations catalyzed by cytochrome P-450

Isotope effects for hydroxylation reactions catalyzed by cytochrome P-450 have usually been measured by comparing the overall reaction velocities of deuterated and nondeuterated substrates. Since the rate-limiting step is probably not the single reaction involving covalent bond cleavage, such an approach does not yield information about the primary isotope effect. We measured the primary kinetic isotope effect for benzylic hydroxylation by a method utilizing intramolecular competition, using the symmetrical substrate 1,3-diphenylpropane-1,1-d₂. These experiments yield a value of $\text{k}{}_{\mathrm{<mtext>H</mtext>}}\text{k}{}_{\mathrm{D}}\text{= 11}$, a larger effect than has previously been reported for benzylic hydroxylations.     

Mapping of nucleoside phosphorylase (Np-1) and esterase 10 (Es-10) on mouse chromosome 14

A method for detecting two alleles at Np-1 (nucleoside phosphorylase) and three alleles at Es-10 (esterase 10) from mouse blood by cellulose acetate electrophoresis is described. The allelic constitution at these loci for 44 inbred strains and stocks was determined. The location of Np-1 on chromosome 14 was established by backcross experiments in which alleles at Np-1 and Robertsonian translocations were segregating. Es-10 was shown to be linked to Np-1, and the following genetic map of Chr 14 was constructed: centromere-(8.9±4.0 cM)-[Np-1, Wc]-(10.2±1.9 cM)-Es-10-(15.5±3.7 cM)-s. The homologous human loci, NP and ES-D, are not linked.This work was supported by Contract E(11-1)-3267 with the Energy Research and Development Administration, by Contracts NO1-ES4-2156 and NO1-ES4-2159 with the National Institute of Environmental Health Sciences, and by Grants GM 19656 and GM 20919 from the National Institute of General Medical Sciences. D. A. K. was a participant in the 1975 Summer Program for College, Graduate, and Medical Students, which was supported, in part, by the Clark Foundation. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

A simple statistical method for use in kinetic analysis based on Lineweaver-Burk plots

Statistical methods for the analysis of initial-velocity and/or inhibition data are described. They involve application of F tests (i) to determine goodness of fit to the first-order Michaelis-Menten equation, (ii) to predict the reaction mechanism by assessing slope and y-intercept effects in Lineweaver-Burk plots according to the inspection rules of Cleland [Cleland, W. W. (1963) Biochim. Biophys. Acta67, 188–196], (iii) to test the linearity of the replots of slopes or y-intercepts versus the reciprocal of the substrate concentration or the inhibitor concentration, and (iv) to estimate the true K_m or K_i values from these replots. The method serves to fill a gap in the kinetic analysis methodology between the antiquated graphical method and the sophisticated direct computer-fitting of data to a variety of possible rate equations. The entire theoretical and computational format is provided to allow the investigator to apply these statistical tests to his data using only a desk-top calculator.     

Resolution of optical isomers by high-pressure liquid chromatography: The separation of benzo[a]pyrene trans-diol derivatives

The optical isomers of (±)r-7,t-8-dihydroxy-7,8-dihydrobenzo[a]pyrene and its synthetic precursor (±)r-7,t-8-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene were resolved as their di-(−)menthoxyacetates using high-pressure liquid chromatography. Saponification of the resolved diesters yielded the corresponding enantiomers. The specific rotation, CD spectra, and ORD curves are reported. The resolution of these optical isomers permits detailed studies on the enzymatic intermediates and the mechanism of benzo[a]pyrene activation to its carcinogenic form. The method is of general usefulness for the resolution of optical isomers.