Nanosecond time-resolved fluorescence kinetic studies of the 5,5'-dithiobis(2-nitrobenzoic acid) reaction with enzyme I of the phosphoenolpyruvate:glycose phosphotransferase system.

Enzyme I of the bacterial phosphotransferase system is a protein component which undergoes a temperature-dependent monomer/dimer equilibrium. Reaction of sulfhydryl residues with SH-specific reagents inhibits both activity and dimerization. There are four cysteine residues available in each subunit, one of which (Cys 502) is proximate to one of the two tryptophan residues (Trp 498).

Nanosecond time-resolved fluorescence measurements during protein denaturation.

A procedure is described by which the information available from nanosecond time-resolved fluorescence measurements can be used to study rates of reactions taking place on time scales of seconds to hours. A pulse fluorometer was modified so as to obtain a series of short sequential data collections which were rapidly stored on computer disk files. As an application of this new methodology, the unfolding of horse liver alcohol dehydrogenase under acid conditions was monitored by changes in the decay parameters of the intrinsic fluorescence.

Anisotropy decay associated fluorescence spectra and analysis of rotational heterogeneity. 2. 1,6-Diphenyl-1,3,5-hexatriene in lipid bilayers.

The application of a new spectroscopic tool [Knutson, J. R., Davenport, L.

Anisotropy decay associated fluorescence spectra and analysis of rotational heterogeneity. 1. Theory and applications.

Individual fluorescence spectra for species in a heterogeneous system can be determined by using differences between the rotational correlation times of those components. Each spectrum derived is associated with a particular fluorescence anisotropy decay function; hence, they are anisotropy decay associated spectra (ADAS). We have previously shown [Knutson, J.

Excited-state proton transfer of equilenin and dihydroequilenin: interaction with bilayer vesicles.

The two-state excited-state proton-transfer process for d-equilenin [d-3-hydroxyestra-1,3,-5(10),6,8-pentaen-17-one] and dihydroequilenin is found to depend both on pH and on proton acceptor concentration. Both the protonated and deprotonated forms of the excited molecule are fluorescent. As is the case for 2-naphthol, the excited-state pKa (pKa*) is substantially lower than the ground-state pKa.

Studies on the mechanism of action of halogenated aromatic hydrocarbons.

The halogenated aromatic hydrocarbons (dibenzo-p-dioxins, dibenzofurans, azo[xy]benzenes and biphenyls), a group of toxic chemicals in the environment, (a) are approximate isostereomers; (b) produce a similar pattern of biologic responses and (c) appear to act by a common mechanism. These compounds reversibly bind to a soluble receptor protein to initiate a coordinate gene expression, analogous to the action of steroid hormones. This receptor controls two distinct and dissociable pleiotropic responses: (a) the induction of microsomal monooxygenase activity and other drug metabolizing enzymes and (b) morphologic (i.

Histologic changes produced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in the skin of mice carrying mutations that affect the integument.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) produces epidermal hyperplasia and hyperkeratosis, squamous metaplasia of the sebaceous gland, and keratinized cyst formation in 8 strains of mice with the recessive mutation, hairless (hr/hr). The extent of these histologic changes is dependent on the genetic background. No cutaneous lesions are produced in haired (hr/+) mice.

2,3,7,8-Tetrachlorodibenzo-p-dioxin: examination of biochemical effects involved in the proliferation and differentiation of XB cells.

XB, a cell line derived from a mouse teratoma, differentiates into stratified squamous epithelium when incubated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). To examine the possible biochemical mediators of this response, we compared the effects produced by TCDD to those elicited by other compounds which stimulate epidermal proliferation and/or differentiation in mice. XB/3T3 cultures keratinize when incubated with cholera toxin, epidermal growth factor (EGF), or TCDD, but not 12-0-tetradecanoylphorbol-13-acetate (TPA).

Decay-associated fluorescence spectra and the heterogeneous emission of alcohol dehydrogenase.

A procedure is described for using nanosecond time resolved fluorescence decay data to obtain decay-associated fluorescence spectra. It is demonstrated that the individual fluorescence spectra of two or more components in a mixture can be extracted without prior knowledge of their spectral shapes or degree of overlap. The procedure is also of value for eliminating scattered light artifacts in the fluorescence spectra of turbid samples.

Response of murine epidermis to 2,3,7,8-tetrachlorodibenzo-p-dioxin: interaction of the ah and hr loci.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related halogenated aromatic hydrocarbons produce epidermal hyperplasia, hyperkeratosis and sebaceous gland metaplasia in the skin of mice bearing the recessive mutation (hr/hr) hairless. This response is mediated through the cytosol receptor protein: the structure-activity relationship for receptor binding corresponds to that for production of the skin lesion, and these histopathological changes segregate with the genetic polymorphism at the Ah locus, the locus determining the cytosol receptor. In HRS/J mice, an inbred strain segregating for the hr locus, both hairless (hr/hr) and haired (hr/+) mice possess the high-affinity cytosol receptor and respond to TCDD with the induction of epidermal aryl hydrocarbon hydroxylase activity, a receptor-mediated biochemical response; however, only hr/hr mice develop the proliferative/metaplastic skin response.

Escherichia vulneris: a new species of Enterobacteriaceae associated with human wounds.

The name Escherichia vulneris sp. nov. (formerly called Alma group 1 and Enteric group 1 by the Centers for Disease Control and API group 2 by Analytab Products, Inc.

2,3,7,8-tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity.

In this review, we have examined the biochemical and toxic responses produced by halogenated aromatic hydrocarbons and have tried to develop a model for their mechanism of action. These compounds bind to a cellular receptor and evoke a sustained pleiotropic response. In many tissues this response consists of the expression of a battery of enzymes which are, for the most part, involved in drug metabolism, but in other tissues, those which develop toxicity, an additional set of genes is expressed which effects cellular involution, division, and/or differentiation.

Hindered depolarizing rotations of perylene in lipid bilayers. Detection by lifetime-resolved fluorescence anisotropy measurements.

Oxygen quenching of perylene fluorescence was used to vary its fluorescence lifetime. Steady-state fluorescence anisotropy measurements under these quenching conditions were used to investigate the diffusive motions of perylene in the isotropic solvent propylene glycol and in lipid bilayers. These lifetime-resolved anisotropy measurements indicate that the anisotropy of perylene in propylene glycol decays to zero at times long compared to its fluorescence lifetime.

The dynamics of the hostage taker: some major variants.

This paper represents a unique effort to elucidate the dynamics of two major types of politically motivated hostage takers. The data presented are derived from an ongoing research project that is gathering extensive interview and psychological test materials from prisoners convicted of politically motivated crimes, both within the U.S.

Influence of PCPA, shock level, and home-cage conditions on shock-induced aggression.

In a series of experiments, the effect of parachlorophenylalanine (PCPA) on shock-induced fighting was assessed rats raised and maintained under either a 12-hr alternating light-dark cycle (LD) or constant light conditions (LL). PCPA increased shock-induced aggression only in LL groups when testing was accomplished using a 2 mA shock; PCPA resulted in increased aggression in groups from the LD condition only when testing was done at 1 mA. A procedure that used castrated and intact cagemates to manipulate home-cage social experience provided evidence for a role for social experience in determining differences between LL and LD reared rats in shock-induced aggression.

Cellular polyamine depletion reduces DNA synthesis in isolated lymphocyte nuclei.

The accumulation of putrescine, spermidine and spermine in activated bovine lymphocytes was blocked by the combined action of two inhibitors of polyamine biosynthesis, methylglyoxal bis(guanylhydrazone) (MGBG) and alpha-methylornithine. Lymphocytes were cultured under three conditions: (1) alpha-methylornithine alone, (2) MGBG alone, or (3) alpha-methylornithine plus MGBG. DNA synthesis in nuclei isolated from these cells was reduced from control rates by approx.