Free sphingosine formation from endogenous substrates by a liver plasma membrane system with a divalent cation dependence and a neutral pH optimum

Long-chain (sphingoid) bases may serve as another category of "lipid second messenger" because they inhibit protein kinase C and affect multiple cellular functions. Free sphingosine has been found in rat liver (Merrill, A. H., Jr., Wang, E., Mullins, R. E., Jamison, W. C. L., Nimkar, S., and Liotta, D. C. (1988) Anal. Biochem. 171, 373-381); hence, this study determined if liver plasma membranes contain free long-chain bases and have the ability to form them from endogenous enzymes and substrates. Isolated plasma membranes contained 0.45 nmol of sphingosine/mg of protein which, based on the recovery of the membranes, was equivalent to 3.5 +/- 1.2 nmol/g of liver and at least half of the total free sphingosine in liver. When the membranes were incubated at 37 degrees C, the amount increased at an initial rate of 5-25 pmol/min/mg, resulting in a 2-3-fold increase over an hour. Sphingosine formation required divalent cations, was optimal at neutral to alkaline pH, and was temperature-dependent. Activities with these characteristics were not identified in microsomes or lysosomes (lysosomal activities with acidic pH optima were detected, however); hence, they appear to reflect a separate plasma membrane system. Sphingosine formation was stimulated by ceramides either added exogenously or formed endogenously by treating the membranes with sphingomyelinase (but not endoglycoceramidase). Sphingomyelin hydrolysis to ceramide was also observed during incubation of the plasma membranes alone. Some of the properties of this system resembled the neutral sphingomyelinase and ceramidase activities of liver. While the physiological significance of this endogenous sphingosine is not known, this system has the appropriate subcellular location to provide sphingosine as a participant in signal transduction.     

Multiple origins for phenylketonuria in Europe

Phenylketonuria (PKU), a disorder of amino acid metabolism prevalent among Caucasians and other ethnic groups, is caused primarily by a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). PKU is a highly heterogeneous disorder, with more than 60 molecular lesions identified in the PAH gene. The haplotype associations, relative frequencies, and distributions of five prevalent PAH mutations (R158Q, R261Q, IVS10nt546, R408W, and IVS12n1) were established in a comprehensive European sample population and subsequently were examined to determine the potential roles of several genetic mechanisms in explaining the present distribution of the major PKU alleles. Each of these five mutations was strongly associated with only one of the more than 70 chromosomal haplotypes defined by eight RFLPs in or near the PAH gene. These findings suggest that each of these mutations arose through a single founding event that occurred within time periods ranging from several hundred to several thousand years ago. From the significant differences observed in the relative frequencies and distributions of these five alleles throughout Europe, four of these putative founding events could be localized to specific ethnic subgroups. Together, these data suggest that there were multiple, geographically and ethnically distinct origins for PKU within the European population.     

Similar-sized daughter-strand gaps are produced in the leading and lagging strands of DNA in UV-irradiated E. coli uvrA cells.

The nascent DNA synthesized after UV irradiation contained discontinuity, i.e., daughter-strand gaps. The sizes of these gaps produced in the leading and lagging strands of UV-irradiated Escherichia coli cells were determined by using strand-specific DNA probes. The DNA isolated from irradiated uvrA delta(lac-pro) cells was hybridized with the 32P-labeled single-stranded DNA probes. After digestion with S1 nuclease, the sizes of the bound radioactive DNA fragments were determined by electrophoresis in an alkaline agarose gel. It was found that the average size of gaps produced in the leading strand was about 0.12 kb, whereas those produced in the lagging strand was slightly smaller than 0.12 kb. No gaps larger than 0.5 kb were detected.     

Analysis of the serotype-specific epitopes of avian infectious bronchitis virus strains Ark99 and Mass41.

The Ark and Mass serotype-specific epitopes of infectious bronchitis virus were studied by immunofluorescence and immunoprecipitation of mutant and recombinant spike glycoproteins (S protein) expressed in mouse L cells. Serotype-specific monoclonal antibodies could bind to the recombinant proteins of Ark99 and Mass41 expressed from the chimeras in which the N-terminal thirds of the S1 sequences were reciprocally exchanged. Therefore, it appears that the respective serotype-specific epitopes of both strains were localized within the C-terminal two-thirds of the S1 proteins. Deletion and insertion of a five-amino-acid fragment on the S1 proteins of Ark99 and Mass41, altered the serotype-specific epitopes. This result implies that the five-amino-acid insertion on the S1 protein of the Ark serotype was involved in determining the conformation of the protein, probably acting as a spacer. In addition, it appears that an interaction between sequences of the N-terminal third and the remaining portion of the S1 protein determines the tertiary structure of the protein as well as the conformation of the serotype-specific epitope.     

Torque generation in the flagellar motor of Escherichia coli: evidence of a direct role for FliG but not for FliM or FliN.

Among the many proteins needed for assembly and function of bacterial flagella, FliG, FliM, and FliN have attracted special attention because mutant phenotypes suggest that they are needed not only for flagellar assembly but also for torque generation and for controlling the direction of motor rotation. A role for these proteins in torque generation is suggested by the existence of mutations in each of them that produce the Mot- (or paralyzed) phenotype, in which flagella are assembled and appear normal but do not rotate. The presumption is that Mot- defects cause paralysis by specifically disrupting functions essential for torque generation, while preserving the features of a protein needed for flagellar assembly. Here, we present evidence that the reported mot mutations in fliM and fliN do not disrupt torque-generating functions specifically but, instead, affect the incorporation of proteins into the flagellum. The fliM and fliN mutants are immotile at normal expression levels but become motile when the mutant proteins and/or other, evidently interacting flagellar proteins are overexpressed. In contrast, many of the reported fliG mot mutations abolish motility at all expression levels, while permitting flagellar assembly, and thus appear to disrupt torque generation specifically. These mutations are clustered in a segment of about 100 residues at the carboxyl terminus of FliG. A slightly larger carboxyl-terminal segment of 126 residues accumulates in the cells when expressed alone and thus probably constitutes a stable, independently folded domain. We suggest that the carboxyl-terminal domain of FliG functions specifically in torque generation, forming the rotor portion of the site of energy transduction in the flagellar motor.     

Effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic acetylcholine receptors on transmembrane potentials and currents in guinea pig ventricular myocytes

The effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic receptor on transmembrane potentials and currents in guinea pig single ventricular cells were analyzed using whole-cell patch clamp technique. These effects were compared with those of the muscarinic receptor agonists carbachol and acetylcholine. The antibodies shortened the action potential duration in a dose-dependent manner. By using a ramp or step rectangular pulse protocol, it was found that the antibodies increased the outward K⁺ current and decreased the inward basal I Ca significantly. The reversal potential of both carbachol-and antibody-induced extra currents were close to –80 mV, being in proximity to the calculated E_k of –90 mV. A -adrenergic receptor agonist, isoprenaline, prolonged the action potential and increased the overshoot which could be inhibited by both antibody and carbachol. Isoprenaline increased inward I_ca and outward I_k simultaneously. Both antibody and carbachol could significantly reduce the isoprenaline-stimulated I_Ca but not the isoprenaline-stimulated I_k. The antibody- or carbachol-induced outward K⁺ current and the depressant effects of antibody and carbachol on isoprenaline-stimulated I_ca were partially antagonized by atropine. These results suggest that the anti-M2 muscarinic receptor antibodies display a stimulatory activity similar to muscarinic receptor agonist on the receptor-mediated electrophysiological events.     

Incorporation of Molecular Oxygen and Water during Enzymatic Oxidation of Cyanide by Pseudomonas fluorescens NCIMB 11764

Cell extracts (high-speed [150,000 x g] supernatants) from Pseudomonas fluorescens NCIMB 11764 catalyzed the oxidation of cyanide to CO(inf2) (and NH(inf3)). Conversion was both oxygen and NADH dependent, with 1 mol of each being consumed per mol of cyanide degraded. Analysis of (sup13)CO(inf2) by mass spectrometry indicated that one atom each of isotopically labelled oxygen 18 from molecular oxygen and water were incorporated during enzymatic conversion. The results confirm earlier reports of oxygenase-mediated cyanide conversion in this organism. A reaction pathway for cyanide oxidation involving initial monooxygenation followed by hydrolysis of a hypothetical oxygenated intermediate to CO(inf2) (and NH(inf3)) is proposed.     

On-line monitoring of intracellular ATP concentration in Escherichia coli fermentations

A method was developed to provide a real-time measurement of intracellular adenosine 5'-triphosophate (ATP) concentrations in growing Escherichia coli. The bacteria to be monitored must first be modified by inserting the cDNA for firefly luciferase expressed from a constitutive promoter. Such a construct leads to constant specific activity of firefly luciferase during both the lag phase and exponential growth. When the luciferase substrate, D-luciferin, is added to the medium, ATP within the cells is utilized in the luciferase-catalyzed reaction that produces light. The light is carried from the bioreactor to a computer-based detector by an optical fiber. The detected per cell light emission varies during exponential growth. Analysis of cytoplasm extracts shows that this variance is related to changes in the ATP concentration, which ranges from 1 to 6 times the literature value for K(M). Experimental analyses demonstrated that inner filter effects are not a significant factor affecting the use of this system. The method was tested in a benchtop fermentor at cell densities above 13 g/L dry cell weight. A correction factor based on the accumulated light data is calculated and used in real time to account for consumption of luciferin from the culture broth by the light producing reaction. Dissolved oxygen concentrations must be kept above 15% of air saturation to ensure constant light output, but no detectable increase in oxygen demand is seen. The method does not significantly affect growth or production rates. (c) 1996 John Wiley & Sons, Inc.