Triphenylmethylphosphonium cation distribution as a measure of hormone-induced alterations in white adipocyte membrane potential

Summary Triphenylmethylphosphonium (TPMP⁺) partitions into the mitochondrial and cytosolic compartments in the rat white adipocyte in a potential-dependent fashion. The relationship between [³H]TPMP⁺ distribution, intracellular cAMP generation and lipolysis in response to hormones and cAMP-mimetic compounds was examined. Half-maximal [³H]TPMP⁺ efflux and glycerol release were produced by 15 and 9nm adrenocorticotropin, 170 and 110nm 1-epinephrine, 70 and 27 m isobutylmethylxanthine and 800 and 750 m dibutyryl cAMP, respectively. Hormone-stimulated cAMP generation was also correlated with [³H]TPMP⁺ efflux and lipolysis in terms of concentration dependency. In kinetic experiments, glycerol release and [³H]TPMP⁺ efflux in response to adrenocorticotropin or cholera toxin proceeded over a similar time course, whereas an earlier rise in cAMP generation was detected.The depolarizing effect of lipolytic compounds was localized to the mitochondrial compartment. When cells were incubated in elevated-[K⁺] _c buffer, the stimulatory effect of dibutyryl cAMP on [³H]TPMP⁺ efflux and lipolysis persisted, suggesting that maintenance of the plasma membrane potential is not critical for demonstration of these responses.When the extracellular concentration of serum albumin, which provides binding sites for free fatty acids, was increased from 1 to 3% an increase in glycerol release and a decrease in [³H]TPMP⁺ efflux was observed. We suggest that intracellular free fatty acid accumulation in response to lipolytic agents causes dissipation of the mitochondrial membrane potential and efflux of [³H]TPMP⁺ from the organelle and cell.     

Gbu Glycine Betaine Porter and Carnitine Uptake in Osmotically Stressed Listeria monocytogenes Cells

The food-borne pathogen Listeria monocytogenes grows actively under high-salt conditions by accumulating compatible solutes such as glycine betaine and carnitine from the medium. We report here that the dominant transport system for glycine betaine uptake, the Gbu porter, may act as a secondary uptake system for carnitine, with a K_m of 4 mM for carnitine uptake and measurable uptake at carnitine concentrations as low as 10 μM. This porter has a K_m for glycine betaine uptake of about 6 μM. The dedicated carnitine porter, OpuC, has a K_m for carnitine uptake of 1 to 3 μM and a V_max of approximately 15 nmol/min/mg of protein. Mutants lacking either opuC or gbu were used to study the effects of four carnitine analogs on growth and uptake of osmolytes. In strain DP-L1044, which had OpuC and the two glycine betaine porters Gbu and BetL, triethylglycine was most effective in inhibiting growth in the presence of glycine betaine, but trigonelline was best at inhibiting growth in the presence of carnitine. Carnitine uptake through OpuC was inhibited by γ-butyrobetaine. Dimethylglycine inhibited both glycine betaine and carnitine uptake through the Gbu porter. Carnitine uptake through the Gbu porter was inhibited by triethylglycine. Glycine betaine uptake through the BetL porter was strongly inhibited by trigonelline and triethylglycine. These results suggest that it is possible to reduce the growth of L. monocytogenes under osmotically stressful conditions by inhibiting glycine betaine and carnitine uptake but that to do so, multiple uptake systems must be affected.     

Molecular and genetic characterization and physical mapping of 11 new markers detecting multiallele restriction fragment length polymorphisms on the short arm of human chromosome 3

Summary Genetic markers with high degrees of polymorphisms are of vital importance in the construction of high resolution (2–4 cM) linkage maps of human chromosomes as specified in the short-term goals of the Human Genome Initiative. In this paper, we report on molecular and genetic characterization and physical localization of 11 new multiallele restriction fragment length polymorphism markers on human chromosome 3p. Ten of these represent three- and four-allele polymorphisms of the base substitution type probably at two adjacent restriction sites. One has been identified as a novel minisatellite sequence comprising a variable copy number tandem repeat array of a G/T-rich 79-bp sequence. This collection of multiallele polymorphic (PIC values: 0.40–0.60) markers should prove valuable and increase the resolution power of the available chromosome 3p genetic markers.     

Proton NMR comparison of noncovalent and covalently cross-linked complexes of cytochrome c peroxidase with horse, tuna, and yeast ferricytochromes c.

Proton NMR spectroscopy at 500 and 361 MHz has been used to characterize the noncovalent or electrostatic complexes of yeast cytochrome c peroxidase (CcP) with horse, tuna, yeast isozyme-1, and yeast isozyme-2 ferricytochromes c and the covalently cross-linked complexes of cytochrome c peroxidase with horse and yeast isozyme-1 ferricytochromes c. Under the conditions employed in this work, the stoichiometry of the predominant complex formed in solution (which totaled greater than 90% of complex formed) was found to be 1:1 in all cases. These studies have elucidated significant differences in the proton NMR absorption spectra and the one-dimensional nuclear Overhauser effect difference spectra of the complexes, depending on the specific species of ferricytochrome c incorporated. In particular, the results indicate that the noncovalent complexes formed between CcP and physiological redox partners (yeast isozyme-1 or yeast isozyme-2 ferricytochromes c) are distinctly different from the noncovalent complexes formed between CcP and ferricytochromes c from horse and tuna. Parallel chemical cross-linking studies carried out using mixtures of cytochrome c peroxidase with horse ferricytochrome c, and cytochrome c peroxidase with yeast isozyme-1 ferricytochrome c further emphasize such cytochrome c-dependent differences, with only the covalently cross-linked complex of physiological redox partners (cytochrome c peroxidase/yeast isozyme-1) displaying NMR spectra characteristic of a heterogeneous mixture of different 1:1 complexes. Finally, one-dimensional nuclear Overhauser effect experiments have proven valuable in selectively and efficiently probing the protein-protein interface in these complexes, including the environment around the cytochrome c heme 3-methyl group and Phe-82.     

A DNA-activated protein kinase from HeLa cell nuclei.

A DNA-activated protein kinase (DNA-PK) was purified from nuclei of HeLa cells. Activity was associated with a single high-molecular-mass (approximately-300,000 Da) polypeptide when analyzed by gel filtration, denaturing polyacrylamide gel electrophoresis, and Western immunoblotting using a monoclonal antibody that also inhibits enzyme activity. Nuclear localization was indicated by subcellular fractionation and confirmed by immunofluorescence on whole cells. Double-stranded DNA stimulated phosphorylation of the 300-kDa polypeptide in purified preparations as well as phosphorylation of the exogenous substrates alpha-casein, simian virus 40 large T antigen, and the human heat shock protein hsp90. Autophosphorylation led to inactivation of the enzyme. The phosphorylation of casein was stimulated over 30-fold by DNA and was specific for serine and threonine residues. Bovine serum albumin and histone H1 were poor substrates for DNA-PK, and no phosphorylation of immunoglobulin G or histones other than H1 was observed. Supercoiled or heat-denatured DNA and synthetic double-stranded RNA or RNA-DNA copolymers did not stimulate casein phosphorylation by DNA-PK. Interaction of the enzyme with DNA in the absence of exogenous substrates was demonstrated by thermal inactivation and gel mobility shifts. These characteristics identify DNA-PK as distinct from other protein kinases described in the literature and suggest that activation by DNA is an important feature of the enzyme's in vivo function.     

Isolation and identification of L-beta-aspartyl-Llysine and L-gamma-glutamyl-L-ornithine from normal human urine.

L-beta-Aspartyl-L-lysine and L-gamma-glutamyl-L-ornithine were isolated from pooled normal human urine and each peptide was shown to be identical with the authentic peptides. The concentrations of these dipeptides in the urine of individual subjects were determined directly by using a new buffer sequence on a standard ion exchange chromatographic amino acid analyzer with a sensitivity of 10(-10) mol. In urine from normal subjects ranging in age from 12 to 64 years, mean values of 1.47 mumol/g of creatinine of L-gamma-glutamyl-L-ornithine and 8,24 mumol/g of creatinine of L-beta-aspartyl-L-lysine were found. The urine of children under 10 years of age contained, relative to creatinine excretion, more L-beta-aspartyl-L-lysine and L-gamma-glutamyl-L-ornithine than that of older children and adults. All urines contained substantially larger concentrations of L-beta-aspartyl-L-lysine than of L-gamma-glutamyl-L-ornithine. Both peptides were found in urine collected after 21 hr of fasting in lower concentrations than found in urine from nonfasting subjects. The urinary concentrations of both peptides did not appear to be influenced by race or sex.     

Experimental study of finite amplitude pulsatile pressure waves in elastic tubes

An experimental study of the propagation of pulsatile pressure waves in an elastic tube was made and results were compared to a theoretical analysis by Lou. The pressure waves were sinusoidally varying acting in a horizontal, longitudinally constrained tube containing water. The independent experimental parameters varied were the pressure wave frequency, pressure wave volume per cycle, static tube pressure and steady flow rate. The wave propagation speeds, measured by non-intrusive techniques, were found to be functions of the wave frequency and the phase angles of the wave elements as theoretically predicted by Lou.     

Phosphorylation of Microtubule-Associated Protein 2 at Distinct Sites by Calmodulin-Dependent and Cyclic-AMP-Dependent Kinases

Microtubule-associated protein 2 (MAP2) is an excellent substrate for both cyclic-AMP (cAMP)-dependent and Ca2+/calmodulin-dependent kinases. A recently purified cytosolic Ca2+/calmodulin-dependent kinase (now designated CaM kinase II) phosphorylates MAP2 as a major substrate. We now report that microtubule-associated cAMP-dependent and calmodulin-dependent protein kinases phosphorylate MAP2 on separate sites. Tryptic phosphopeptide digestion and two-dimensional phosphopeptide mapping revealed 11 major peptides phosphorylated by microtubule-associated cAMP-dependent kinase and five major peptide species phosphorylated by calmodulin-dependent kinase. All 11 of the cAMP-dependently phosphorylated peptides were phosphorylated on serine residues, whereas four of five major peptides phosphorylated by the calmodulin-dependent kinase were phosphorylated on threonine. Only one peptide spot phosphorylated by both kinases was indistinguishable by both migration and phosphoamino acid site. The results indicate that cAMP-dependent and calmodulin-dependent kinases may regulate microtubule and cytoskeletal dynamics by phosphorylation of MAP2 at distinct sites.     

滇产云南松、思茅松松针油的化学成分

云南松(Pinus yunnanensis Fr.)和思茅松〔Pinus kesiya Royle ex Gord.var.langbianensis(A.Chev.)Gaussen〕为云南针叶林主要树种。我们已研究了思茅松松节油的化学成分,为松针油利用提供科学数据,比较松节油与松针油化学成分的同异,并考察α-蒎烯,β-蒎烯的含量与分布的关系,本文用气相色谱/质谱/计算机联用仪,对滇南到滇中水平分布及由低到高垂直分布的八个云南松和九个思茅松松针油油样进行了定性、定量分析。