The Pseudomonas fluorescens lipase has a C-terminal secretion signal and is secreted by a three-component bacterial ABC-exporter system

Both Pseudomonas aeruginosa and Pseudomonas fluorescens secrete a lipase into the extracellular medium. Unlike the lipase of P. aeruginosa, the lipase produced by P. fluorescens does not contain any N-terminal signal sequence. We show that the P. fluorescens lipase is secreted through the signal peptide-independent pathway of the alkaline protease that we previously identified in P. aeruginosa. Secretion of this protease (AprA) is dependent on the presence of three genes located adjacent to the aprA gene, aprD, aprE and aprF. The three secretion functions permit an efficient secretion of P. fluorescens lipase. Inactivation of one of them (AprE) prevented this secretion. In Escherichia coli, the three proteins AprD, AprE, AprF are necessary and sufficient for efficient secretion of lipase to the extracellular medium. The secretion signal is located within the C-terminal part of the lipase sequence and can promote efficient secretion of a passenger protein. Thus the P. fluorescens lipase secretion system belongs to the group of the three-component bacterial ABC-exporter systems.     

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.     

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.     

Suppression of pea nuclear topoisomerase I enzyme activity by pea PCNA

Proliferating cell nuclear antigen (PCNA), a highly conserved DNA polymerase accessory protein of eukary- otic kingdom, has not been studied thoroughly in bio- chemical terms in plants. We describe the isolation of the cDNA encoding PCNA from the pea cDNA library using the PCR approach. The cDNA was used for expression of pea PCNA in bacteria as a fusion protein (GST.PCNA) with the GST tag at the amino terminal end. The GST.PCNA stimulated the partially purified pea DNA polymerases approximately 30-fold. The stimulation was due to the oligomeric form of GST.PCNA. The pea PCNA interacted with the recombinant type I pea topoiso- merase as well as the native pea nuclear topoisomerase I and repressed the DNA relaxation activities. However, the DNA binding activity of Topo I remained undisturbed in the presence of high amounts of PCNA, thereby signify- ing that the catalysis of Topo I was probably affected by PCNA.