Fluorescence of delta 5,7,9(11),22-ergostatetraen-3 beta-ol in micelles, sterol carrier protein complexes, and plasma membranes

The fluorescent sterol analogue delta 5,7,9(11),22-ergostatetraen-3 beta-ol (dehydroergosterol) was synthesized and purified by reverse-phase high-performance liquid chromatography. Dehydroergosterol in aqueous solution had a critical micelle concentration of 25 nM and a maximum solubility of 1.3 microM as ascertained from fluorescence polarization and light scattering properties, respectively. Several lines of evidence indicated a close molecular interaction of dehydroergosterol with purified rat liver squalene and sterol carrier protein (SCP). SCP increased the maximal solubility of dehydroergosterol in aqueous buffer. The fluorescence emission spectrum of dehydroergosterol was blue shifted upon addition of SCP. The fluorescence lifetime of dehydroergosterol in aqueous buffer was 2.3 ns; addition of SCP resulted in the appearance of a second lifetime component near 12.4 ns. The SCP increased the fluorescence polarization of monomeric dehydroergosterol in aqueous buffer from 0.033 to 0.086. Scatchard analysis of the binding data indicated that dehydroergosterol interacted with purified rat liver SCP with an apparent KD = 0.88 microM and Bmax = 4.8 microM. At maximal binding, 1.0 mol of dehydroergosterol was specifically bound per mole of SCP. The close molecular interaction of dehydroergosterol with SCP was also demonstrated by energy-transfer experiments. The intermolecular distance between SCP and bound dehydroergosterol was evaluated by fluorescence energy transfer from tyrosine residues of SCP to the conjugated triene series of double bonds in dehydroergosterol. The transfer efficiency was 36%, and R, the apparent distance between the tyrosine energy donor and the dehydroergosterol energy acceptor, was 19 A. The significance of these data obtained in vitro for dehydroergosterol interaction with SCP was also tested in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chromosomal localization of the human alpha 1-antitrypsin gene (PI) to 14q31-32.

In situ hybridization of a recombinant cDNA probe containing the human alpha 1-antitrypsin gene to metaphase chromosomes demonstrated significant hybridization to chromosomal segment 14q31-32. A high percentage of cells analyzed (31%) displayed labeling on chromosome 14. Of all labeled sites on chromosome 14, 60% were found on segment 14q31-32. These results refine the previous assignment of the human alpha 1-antitrypsin gene to segment 14q24.1-32.1.     

Stereospecificity of SP1 and SP2 substance P receptors

Previous studies with N-terminal fragments of substance P (SP) have suggested the existence of two separate SP receptor populations. SP1 receptors are found in guinea pig ilea and rat colons. SP2 receptors are found in mouse spinal cords and rat salivary glands. We have now found that substitution of Gly9 in substance P's C-terminal hexapeptide leads to an analog (L-Pro9 SP6-11) which selectively and potently stimulates SP2 receptors. In contrast, substitution of the same residue with D-Proline results in a potent and selective agonist for SP1 receptors. The data dramatically confirm the distinction between SP1 and SP2 receptors and demonstrate that the two receptors have distinct stereochemical architectures.     

Sterol and squalene carrier protein interactions with fluorescent delta 5,7,9(11)-cholestatrien-3 beta-ol

The fluorescent sterol delta 5,7,9(11)-cholestatrien-3 beta-ol (cholestatrienol) was used as an analogue of cholesterol to determine the properties of the sterol in aqueous buffer and the interaction of cholesterol with sterol and squalene carrier protein (SCP). Cholestatrienol was synthesized and purified to a stable product by reverse phase high performance liquid chromatography. The critical micelle concentration of cholestatrienol in aqueous buffer was 1 nM while its maximum solubility was 1.15 microM as ascertained from fluorescence polarization and light scattering properties, respectively. Several lines of evidence indicated a close molecular interaction of cholestatrienol with purified rat liver SCP. The fluorescence emission spectrum of monomeric cholestatrienol in aqueous buffer was blue shifted upon addition of SCP. The fluorescence lifetime of monomeric cholestatrienol in aqueous buffer was increased by SCP from 5 to 12 ns. The SCP increased the fluorescence polarization of monomeric cholestatrienol from 0.002 to 0.38 in aqueous buffer. The close molecular interaction of cholestatrienol with SCP was also demonstrated by energy transfer experiments. Fluorescence energy transfer from tyrosine residues of SCP to the conjugated triene fluorophore in cholestatrienol had a transfer efficiency of 59%. R, the apparent distance between the tyrosine energy donor and the cholestatrienol energy acceptor, was 16.3 A. Binding analysis indicated that cholestatrienol interacted with SCP with an apparent KD = 0.5 microM and a Bmax = 3.54 microM. One mol of cholestatrienol was bound per mol of SCP. These results demonstrate the utility of cholestatrienol not only as a membrane sterol probe molecule but also as a probe for sterol-protein interactions.     

Phosphoenolpyruvate-dependent phosphorylation of a 55-kDa protein of Streptococcus faecalis catalyzed by the phosphotransferase system

Abstract A protein with an M _r of 55000 was isolated from glucose-grown Streptococcus faecalis cells. The protein becomes phosphorylated in a phosphoenolpyruvate-dependent reaction catalyzed by enzyme I and HPr of the bacterial phosphotransferase system. It did not stimulate phosphoenolpyruvate-dependent glucose phosphorylation. Several sugars were tested for their ability to dephosphorylate the phosphorylated protein in the presence of membrane fragments. Even though some of the sugars were able to dephosphorylate phospho-HPr quickly, the factor III-like 55-kDa protein remained phosphorylated. We therefore assumed that this protein is not involved in any sugar uptake reaction but that it exerts a regulatory function in Gram-positive bacteria comparable to the function of factor III specific for glucose in Escherichia coli .     

Banding of Allium Chromosomes Protected Against Dnase Digestion by Dna-Binding Drugs

Metaphase chromosome bands were induced in Allium flavum (Liliaceae) by protecting the chromosomal DN A with DN A-binding compounds of different base specificities against DNase digestion. The bands obtained represented different subsets of C-band heterochromatin.     

The selection mutation equation

Fisher's Fundamental Theorem of Natural Selection is extended to the selection mutation model with mutation rates _ij=_ii.e. depending only on the target gene, by constructing a simple Lyapunov function. For other mutation rates stable limit cycles are possible.     

Transduction analysis of transposon Tn551 insertions in the trp-thy region of the Staphylococcus aureus chromosome.

Previous studies have shown that Tn551, a 5.2-kilobase-pair transposon that determines constitutive resistance to erythromycin, can occupy a variety of chromosomal sites between thy-101 and trp-103 in Staphylococcus aureus 8325. Although many of these insertions were "silent," many others, including lys, thr, met, tyr, and trp, resulted in auxotrophic mutations. The close proximity and erythromycin-resistant phenotypes of the insertions in this region have made their mapping by transformation difficult. Analysis of these sites and similar chemically induced mutations by generalized transduction with phage 80 alpha have defined the order and relationship of these insertion sites and provided a detailed map of this region of the chromosome, including the orientation of the trp operon. The results of this study and a limited phenotypic characterization of the mutants have shown that the divergent pathway from aspartate to lysine, threonine, and methionine, several reactions in tyrosine biosynthesis, and the entire tryptophan operon are determined by this region of the chromosome. The linkage results obtained by transduction have been compared with similar data obtained previously by transformation; this comparison suggests the existence, between thy and lys, of a preferred headful cutting site for transducing phage DNA morphogenesis from the host chromosome.     

Prostaglandin 12 reduces activation of human arterial smooth muscle cells in-vivo

Patients suffering from peripheral vascular disease have been “ultima ratio”-treated with PGI2 at a rate of 5 ng/kg/min for 6 hours a day and 5 consecutive days i.v. 20 of them underwent surgery thereafter as therapy was not sufficient. A histological examination and quantification of vascular tissue revealed that the number of activated smooth muscle cells was significantly lower in treated patients vascular segments than in untreated ones in all the different age groups. A comparable suppression was found in the intima and the media as well. It is thus concluded, that PGI2 inhibits smooth muscle cell proliferation most probably by inhibiting PDGF-release from the platelets and stimulation of smooth muscle cell cAMP. To achieve a more beneficial PGI2-effect at the vascular level, a prolonged PGI2-therapy looks rather promising.