New procedure of high-voltage electrophoresis in polyacrylamide gel and its application to the sequencing of nucleic acids.

Addition of primary organic amines, such as n-butylamine, to the mobile phase altered the capacity factors and selectivity of benzo[a]pyrene metabolites obtained with reverse-phase high pressure liquid chromatography on an ODS column. Separation of benzo[a]pyrene phenols in particular was improved with 8 of the 10 available metabolites resolved, including those known to be biologically produced. The method offers sufficiently improved resolution or convenience that it should prove useful in comparative studies of metabolism of benzo[a]-pyrene and other polynuclear aromatic hydrocarbons. Applying the method to analysis of benzo[a]pyrene metabolites produced in vitro by hepatic microsomes from the marine fish Stenotomus versicolor indicated the principal phenolic derivatives produced by this fish were 1-hydroxy-, 3-hydroxy-, 7-hydroxy-, and 9-hydroxybenzo[a]pyrene.     

Zero-cost modification of bright field microscopes for imaging phase gradient on cells: Schlieren optics

A simple, zero-cost, reversible modification of a bright field microscope permits visualization of phase gradients in cells by transmitted illumination, yielding a Nomarski-like effect. This modification, based on schlieren optics, is simultaneously compatible with high-aperture epi-illumination fluorescence excitation. For many objectives that are intended for use in fluorescence work, but are unavailable in phase contrast versions, the modification provides a simple means for locating cells in culture with good image contrast and resolution.     

The effects of ligands on enzymic carboxyl-methylation of neurophysins

The methyl-acceptor activities of bovine neurophysins I and II for the enzyme protein carboxymethylase (EC 2.1.1.24) were found to be similar and as high as for other previously identified, biologically active protein substrates. Effects on the rate of methylation of these neurophysins were investigated with the posterior pituitary hormone ligands, oxytocin and vasopressin, and the hormone-related tripeptide ligand, methionyl-tyrosyl-phenylalaninamide. An increase in the rate of neurophysin II methylation was observed with both oxytocin and tripeptide. This ligand-induced response did not occur with either native neurophysin I or disulfide-scrambled neurophysin II.     

Mobility measurement by analysis of fluorescence photobleaching recovery kinetics.

Fluorescence photobleaching recovery (FPR) denotes a method for measuring two-dimensional lateral mobility of fluorescent particles, for example, the motion of fluorescently labeled molecules in approximately 10 mum2 regions of a single cell surface. A small spot on the fluorescent surface is photobleached by a brief exposure to an intense focused laser beam, and the subsequent recovery of the fluorescence is monitored by the same, but attenuated, laser beam. Recovery occurs by replenishment of intact fluorophore in the bleached spot by lateral transport from the surrounding surface. We present the theoretical basis and some practical guidelines for simple, rigorous analysis of FPR experiments. Information obtainable from FPR experiments includes: (a) identification of transport process type, i.e. the admixture of random diffusion and uniform directed flow; (b) determination of the absolute mobility coefficient, i.e. the diffusion constant and/or flow velocity; and (c) the fraction of total fluorophore which is mobile. To illustrate the experimental method and to verify the theory for diffusion, we describe some model experiments on aqueous solutions of rhodamine 6G.     

Dynamics of fluorescence marker concentration as a probe of mobility.

We have developed an effective experimental system for the characterization of molecular and structural mobility. It incorporates a modified fluorescence microscope geometry and a variety of analytical techniques to measure effective diffusion coefficients ranging over almost six orders of magnitude, from less than 10(-11) cm2/s to greater than 10(-6) cm2/s. Two principal techniques, fluorescence correlation spectroscopy (FCS) and fluorescence photobleaching recovery (FPR), are employed. In the FPR technique, translational transport rates are measured by monitoring the evolution of a spatial inhomogeneity of fluorescence that is produced photochemically in a microscopic volume by a short burst of intense laser radiation. In contrast, FCS uses laser-induced fluorescence to probe the spontaneous concentration fluctuations in microscopic sample volumes. The kinetics are analyzed by computing time-correlation functions of the stochastic fluctuations of the measured fluorescence intensity. The optical system and digital photocount correlator designed around a dedicated minicomputer are described and discussed. The general power of these techniques is demonstrated with examples from studies conducted on bulk solutions, lipid bilayer membranes, and mammalian cell plasma membranes.     

A SENSITIVE ENZYMATIC-ISOTOPIC METHOD FOR THE ANALYSIS OF TYRAMINE IN BRAIN AND OTHER TISSUES

Abstract— An enzymatic-isotopic assay for the measurement of tyramine with a sensitivity of 1.0 ng has been developed. Using this assay, the endogenous content of tyramine in various tissues from adult rats has been determined. The highest tyramine content was found in rat heart atria, followed by salivary gland, kidney, and brain. Within the brain the distribution of tyramine is heterogeneous and the highest tyramine content was localized in the striatum.     

A branching process model of gene amplification following chromosome breakage.

We have devised a mathematical model of gene amplification utilizing recent experimental observations concerning dihydrofolate reductase (DHFR) gene amplification in CHO cells. The mathematical model, based on a biological model which proposes that acentric elements are the initial intermediates in gene amplification, includes the following features: (1) initiation of amplification by chromosomal breakage to produce an acentric structure; (2) replication of acentric DNA, once per cell cycle; (3) dissociation of replicated acentric DNA; (4) unequal segregation of acentric DNA fragments to daughter cells at mitosis; (5) subsequent reintegration of acentric fragments into chromosomes. These processes are assumed to be independent for each element present in a cell at a given time. Thus, processes of unequal segregation and integration may occur in parallel, not necessarily in a unique sequence, and may be reiterated in one or multiple cell cycles. These events are described mathematically as a Galton-Watson branching process with denumerable infinity of object types. This mathematical model qualitatively and quantitatively reproduces the major elements of the dynamical behavior of DHFR genes observed experimentally. The agreement between the mathematical model and the experimental data lends credence to the biological model proposed by Windle et al. (1991), including the importance of chromosome breakage and subsequent gene deletion resulting from resection of the broken chromosome ends as initial events in gene amplification.     

ECOLOGIC DIFFERENCES HAVE SEPARATED PINUS REMORATA AND P. MURICATA SINCE THE EARLY PLEISTOCENE

Pure stands of Pinus remorata regularly occur in more equable sites than those occupied by P. muricata. The fossil record suggests that this relationship has existed since at least the earliest Pleistocene. It apparently was the spread of drier, less equable, postglacial climate that opened new, intermediate coastal sites that favored hybrids of P. muricata × P. remorata, the latter a relict species closely allied to P. radiala var. cedrosensis.     

Isolation and preliminary characterization of temperature-sensitive mutants of poliovirus type 1.

We isolated six temperature-sensitive mutants of poliovirus type 1 (Mahoney) by hydroxylamine mutagenesis and replica plating at 31, 33 (permissive), and 39 degrees C (restrictive). One of these mutants, designated tsB9, was chosen for more detailed examination. tsB9 accumulated 25% of the wild-type amount of virus-specific RNA at the restrictive temperature. We found that tsB9 was not able to synthesize mature, 35S single-stranded RNA at the restrictive temperature. In spite of the absence of significant RNA synthesis, tsB9 retained the ability to inhibit host protein synthesis during infection at 39 degrees C at about the same rate as wild-type virus.     

High-performance liquid chromatographic separation of lipoxygenase isozymes in crude soybean extracts

A mixture of the soybean lipoxygenase isozymes purified by conventional methods was readily resolved by high-performance liquid chromatography using a SynChropak AX-300 anion-exchange column. Analysis of crude soybean extract by this procedure showed the presence of four different lipoxygenase activities. Mutant soybeans lacking in the isozymes lipoxygenase-1 and -3 were used to test the application of this procedure.