Tumor promoter 12-O-tetradecanoylphorbol 13-acetate alters state,fluidity and hydration of 1,2-diacyl-sn-glycero-3-phosphocholine bilayers

Previous results (Castagna et al. (1979) FEBS Lett. 100, 62–66; Fisher et al. (1979) Biochem. Biophys. Res. Commun. 86, 1063–1068) indicated us that the active tumor promoter TPA ($\text{12-O-}\text{tetradecanoylphorbol}$ 13-acetate) decreased fluorescence polarisation of diphenylhexatriene in lymphoblastoid and rat embryo cells. In the present study, experiments aimed at examining the molecular interactions of tumor promoters with cell membrane components are performed with fully hydrated multibilayers of 1,2-diacyl-sn-glycero-3-phosphocholine (DPPC) into which increasing amounts of TPA are inserted. The thermotropic behaviour of both the phospholipid bilayers and the interbilayer water was investigated using the differential scanning calorimetry (DSC) and the approach of Ter-Minassian-Saraga et al. ((1982) J. Colloïd Interface Sci. 81, 369–383). The major effects of the tumor promoter are confined to concentrations up to 20% mol fractions of TPA. In this range of concentrations the incorporation of TPA into liposomes decreases the phase-transition temperature but dit not affect $\text{ΔH}{}_{\mathrm{<mtext>DPPC</mtext>}}$. Furthermore TPA increases the hydration of the multibilayers. Above 20% mol fractions of TPA, a different thermal behaviour of the system which might suggest morphological rearrangements was observed. The lipid state in TPA-treated liposomes was monitored by fluorescence polarisation using diphenylhexatriene as a lipophilic fluorescent probe and the phase-transition temperature was calculated. The phase transition temperatures determined by both methods were in good agreement. The lowering of this temperature and the decay of fluorescence anisotropy of diphenylhexatriene were parallel. Those effects are consistent with the ‘fluidising’ effect of TPA on DPPC.     

Influence of hydrogen bonding on the rotamer distribution of the histidine side chain in peptides: 1H NMR and CD studies.

Both 1H NMR and circular dichroism pH titration studies on histidine, His-Gly, Gly-His and Gly-His-Gly indicate that the side-chain spatial orientation depends strongly on the vicinal charges. The arrangement of the imidazole side-chain (rotamer population) is shown by the histidine beta and beta' and the glycine methylene proton chemical shifts as well as the vicinal 1H-1H coupling constants 3JCalpha-H-beta-H, beta'-H. For His-Gly and Gly-His-Gly a good correlation can be found between the ionization of the glycine COOH group and the increase of rotamer III (g-g) which is also visualized by circular dichroism through an enhancement of the ellipticity at 212 nm. In these two peptides a hydrogen bond between the imidazolium and the carboxylate group is supposed to stabilize rotamer III at pH 4-5.     

The role of beta and alpha adrenergic receptors in the lipolytic effect of catecholamines on dog adipocytes (author's transl)]

Pharmacological properties of adrenergic receptors have been investigated in fat cells isolated from omental adipose tissue of the Dog. From the results, the following points can be stated. 1. Lipolysis is markedly enhanced by isoproterenol. This effect is competitively inhibited by propranolol (a beta-adrenoceptor blocking agent). (Fig 1). 2. The beta 2-sympathomimetic salbutamol is found to have only a slight effect on lipolysis rate (Fig. 2). 3. The epinephrine-induced lipolysis is potentiated by phentolamine (an alpha-adrenoceptor blocking agent) only on large sized adipose cells (mean fat cell size 96.7 +/- 5.3 micrometer; Fig. 5). 4. The isoproterenol-induced lipolysis is partially inhibited by phenylephrine (an alpha-adrenomimetic drug) (Table I). These findings show that beta 1 nature of the receptors involved in the adrenergic control of lipolysis in Dog adipose tissue. Moreover an antilipolytic effect of epinephrine, via alpha-adrenergic receptors, is observed, especially in large adipose cells.     

Excited state properties of bilirubin and its photoproducts using picosecond flourescence and ciruclarly polarized luminescence spectroscopy

We have used picosecond time-correlated photon counting to investigate the excited state decay of bilirubin in dioctadecyldimethylammonium (DODAC) vesicles, human and bovine serum albumin, organic solvents and chloroform/water mixtures. In each case a biexponential fluorescence decay τ₁ of 20–90 ps and 99% initial intensity and τ₂ of 1–2 ns, depending on the system, were observed. The fraction of the longer decay increased with observation wavelength. On irradiation of bilirubin in DODAC vesicles or CHCl₃/H₂O solutions the percentage of the longer fluorescence decay in the total fluorescence increased, on prolonged irradiation this percentage decreased as it did when solutions were left in the dark. These observations are interpreted in terms of bilirubin photoisomerisation from the first singlet state, the longer decays being due to the photoproduct isomers, E-Z, Z-E and E-E. Circularly polarised fluorescence spectra are also suggestive of more than one species contributing to the measured fluorescence. One species has the same conformation as bilirubin, the other, which emits at longer wavelengths, has a different excited state conformation.     

Some aspects of DH-524 (2(3,4-dichlorophenoxy)methyl-2-imidazoline) antagonistic-like actions of ethanol intoxication in rats

Previous research indicates ethanol antagonist properties of DH-524 (2(3,4-dichlorophenoxy)methyl-2-imidazoline) in rats. Our research findings suggest that the drug significantly alters plasma alcohol concentration and hematocrit without changing protein concentration. We suggest that the decreased plasma alcohol concentration is in part due to shifts in body water compartments and thus the reduction in ethanol levels.     

Disaccharide conformational flexibility. II. Molecular dynamics simulations of sucrose

Molecular dynamics simulations have been used to study the motions in vacuum of the disaccharide sucrose. Ensembles of trajectories were calculated for each of the five local minimum energy conformations identified in the adiabatic conformational energy mapping of this molecule. The model sucrose molecules were found to exhibit a variety of motions, although the global minimum energy conformation was found to be dynamically stable, and no transitions away from this structure were observed to occur spontaneously. In all but one of these vacuum trajectories, the intramolecular hydrogen bond between residues was maintained, in accord with recent nmr studies of this molecule in aqueous solution. Considerable flexibility of the furanoid ring was found in the trajectories. No "flips" to the opposite puckering for this ring were found in the simulations starting from the global minimum, although such a transition was observed for a trajectory initiated with one of the higher local minimum energy conformations. Overall, the observed structural fluctuations were consistent with the experimental picture of sucrose as a relatively rigid molecule.     

Phylogenetic Articulation of Uric Acid Evolution in Mammals and How It Informs a Therapeutic Uricase

The role of uric acid during primate evolution has remained elusive ever since it was discovered over 100 years ago that humans have unusually high levels of the small molecule in our serum. It has been difficult to generate a neutral or adaptive explanation in part because the uricase enzyme evolved to become a pseudogene in apes thus masking typical signals of sequence evolution. Adding to the difficulty is a lack of clarity on the functional role of uric acid in apes. One popular hypothesis proposes that uric acid is a potent antioxidant that increased in concentration to compensate for the lack of vitamin C synthesis in primate species ∼65 Ma. Here, we have expanded on our previous work with resurrected ancient uricase proteins to better resolve the reshaping of uricase enzymatic activity prior to ape evolution. Our results suggest that the pivotal death-knell to uricase activity occurred between 20 and 30 Ma despite small sequential modifications to its catalytic efficiency for the tens of millions of years since primates lost their ability to synthesize vitamin C, and thus the two appear uncorrelated. We also use this opportunity to demonstrate how molecular evolution can contribute to biomedicine by presenting ancient uricases to human immune cells that assay for innate reactivity against foreign antigens. A highly stable and highly catalytic ancient uricase is shown to elicit a lower immune response in more human haplotypes than other uricases currently in therapeutic development.     

Insight into the molecular basis of substrate recognition by the wall teichoic acid glycosyltransferase TagA

Wall teichoic acid (WTA) polymers are covalently affixed to the Gram-positive bacterial cell wall and have important functions in cell elongation, cell morphology, biofilm formation, and β-lactam antibiotic resistance. The first committed step in WTA biosynthesis is catalyzed by the TagA glycosyltransferase (also called TarA), a peripheral membrane protein that produces the conserved linkage unit, which joins WTA to the cell wall peptidoglycan. TagA contains a conserved GT26 core domain followed by a C-terminal polypeptide tail that is important for catalysis and membrane binding. Here, we report the crystal structure of the Thermoanaerobacter italicus TagA enzyme bound to UDP-N-acetyl-d-mannosamine, revealing the molecular basis of substrate binding. Native MS experiments support the model that only monomeric TagA is enzymatically active and that it is stabilized by membrane binding. Molecular dynamics simulations and enzyme activity measurements indicate that the C-terminal polypeptide tail facilitates catalysis by encapsulating the UDP-N-acetyl-d-mannosamine substrate, presenting three highly conserved arginine residues to the active site that are important for catalysis (R214, R221, and R224). From these data, we present a mechanistic model of catalysis that ascribes functions for these residues. This work could facilitate the development of new antimicrobial compounds that disrupt WTA biosynthesis in pathogenic bacteria.     

Characterization of the Mouse Rh Blood Group Gene

The Rh blood group system is of clinical importance in blood transfusion and as the cause of hemolytic disease of the newborn. Other than their role as carriers of Rh antigens, very little is known about the function of the Rh polypeptides. As a first step to generate an animal model system in which to study the structure and function of Rh, and to extend the phylogenetic analysis of RH genes, the Rh homologue from Mus musculus was characterized. Comparison of RH from humans and mice revealed 71 and 58% sequence identity at the nucleotide and amino acid levels, respectively. Mouse Rh mRNA encodes a protein which is 1 amino acid longer (418 aa) than that of human (417 aa). Rh protein was detected in mouse erythrocyte membranes and was comparable in size to human Rh. Mouse erythrocytes do not show serologic reactivity with human Rh antibodies, probably because the greatest divergence between the mouse and the human genes was seen in the predicted extracellular loops, while the transmembrane regions were more conserved. The mouse RH locus consists of only one gene, which is important for future genetic manipulation and which also indicates that the RH gene duplication seen in humans has occurred since the mammalian radiation.