Kinetics of the diacetyl and 2,3-pentanedione reduction by diacetyl reductase (α-diketone reductase (NAD)) from Staphylococcus aureus

The kinetic mechanism of diacetyl and 2,3-pentanedione reduction by diacetyl reductase from Staphylococcus aureus was investigated. The shape of the primary double reciprocal plots, the product inhibition pattern, and the features of the inhibition by a substrate analogue (acetone) show that diacetyl is reduced via an Ordered Bi-Bi mechanism, and 2,3-pentanedione by an Ordered Bi-Bi or Theorell-Chance mechanism. NADH is the leading substrate in both reactions.Affinity constants for the coenzyme and the substrates and inhibition constants for NAD, acetoin, and acetone were also calculated. This enzyme has a high affinity for NADH; K_m (31–50 μM) and K_s (20–27 μM) for this compound are around one-tenth of the NADH intracellular concentration. Therefore, it must operate in vivo saturated with the coenzyme. This condition is not adequate to play the role, formerly proposed for diacetyl reductases, of regulating the equilibrium between oxidized and reduced forms of pyridine-nucleotides.     

Time dependence of transmembrane potential changes and intracellular calcium flux in stimulated human monocytes

An important characteristic of the functional differentiation of the blood monocyte is the development of its capacity to recognize and respond to stimuli. This ability is mediated to a large extent by specific receptor glycoproteins located on the cell surface. Stimulation of mononuclear phagocytes via these receptors results in a rapid rise in intracellular Ca++ concentration, accompanied or followed by a change in membrane potential, generation of oxidative products, degranulation, and effector functions such as phagocytosis, aggregation, or locomotion. While the development of these characteristics is difficult to characterize in vivo, several investigators have demonstrated in vitro changes in these cells that correlate with the development of effector function. To examine the mechanisms of specific membrane-stimulus interactions of monocytes as they differentiate into macrophage-like cells, we studied the responses of human monocytes and of monocytes incubated in serum-containing medium for up to 96 hr to the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP). Freshly isolated monocytes exhibited little change in transmembrane potential following stimulation with an optimal concentration of peptide and underwent a significant increase only after 48 hr in culture. While constant resting intracellular Ca++ concentrations were maintained during the culture period, intracellular Ca++ levels following fMLP stimulation increased with with incubation in serum, for up to 96 hr. In contrast, fMLP-induced respiratory burst activity increased from 0 to 24 hr in culture; it remained elevated at 48 hr but declined again by 96 hr. Incubation of the cells for 24 hr increased their random (unstimulated) motility in modified Boyden chambers but did not alter the cells' directed (chemotactic) response to fMLP in comparison to the response of freshly isolated monocytes. Peptide binding to the cells did not increase during the incubation period, indicating that an increase in receptor number or in affinity for fMLP was not responsible for the enhanced responsiveness to fMLP as incubation time increased. These studies indicate that incubation of monocytes in serum-containing medium leads to a complex, altered series of responses to fMLP that correlate with the differentiation of the original monocytes in vitro and may relate to the in vivo differentiation of monocytes to macrophages.     

Comparison of the Effects of Forskolin and Dibutyryl Cyclic AMP in Neuroblastoma Cells: Evidence that Some of the Actions of Dibutyryl Cyclic AMP Are Mediated by Butyrate

We have compared the effects of forskolin, N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (dibutyryl cyclic AMP, Bt2-cAMP), and butyrate on several aspects of neuroblastoma cell physiology. The morphology of Neuro 2A cells was similar after incubation with forskolin and Bt2-cAMP, which caused extensive neurite outgrowth, whereas in the presence of butyrate some rudimentary neurites were formed but they were not nearly as extensive. All compounds produced a dose-dependent inhibition of cell proliferation, but the effect of Bt2-cAMP was more marked than that caused by forskolin, thus showing that the effect of Bt2-cAMP is due partially to the butyrate released. Acetylcholinesterase activity was lower in the cells incubated with butyrate or Bt2-cAMP than in untreated cells or in forskolin-treated cells. This suggests that cyclic AMP does not play a role in the regulation of this enzyme. Bt2-cAMP produced histone acetylation, a well-known effect of butyrate in cultured cells, whereas forskolin did not affect this modification. Consequently, the levels of thyroid hormone receptor, a nuclear protein whose concentration is regulated by butyrate through changes in acetylation of chromatin proteins, were decreased in cells incubated with Bt2-cAMP or butyrate, but were unaffected by forskolin. Butyrate elevated the concentration of histone H1(0), a protein that increases in neuroblastoma cells as a result of different treatments that block cell division. The concentration of H1(0) in the cells treated with Bt2-cAMP was at a level intermediate between that found after treatment with butyrate and with forskolin. The present results clearly indicate that some of the effects of Bt2-cAMP on neuroblastoma cells can be attributed to the butyryl moiety of this compound rather than to the cyclic nucleotide itself.     

Estimation of coefficient of coancestry using molecular markers in maize

Summary The coefficient of coancestry (f_AB) between individuals A and B is the classical measure of genetic relationship. f_AB is determined from pedigree records and is the probability that random alleles at the same locus in A and B are copies of the same ancestral allele or identical by descent (ibd). Recently, the proportion of molecular marker variants shared between A and B (S_AB) has been used to measure genetic relationship. But S_AB is an upwardly-biased estimator of f_AB, especially between distantly-related lines. f_AB, S_AB, and adjusted (to remove bias) estimates of molecular marker similarity (f _AB ^M ) were compared. RFLP banding patterns at 46 probe-restriction enzyme combinations were obtained for 23 maize inbred lines derived from the Iowa Stiff Stalk Synthetic (BSSS) maize (Zea mays L.) population, and for 4 non-BSSS lines. f _AB ^M was estimated as , where _A (or _B) was the average proportion of RFLP variants shared between inbred A (or inbred B) and the non-BSSS lines. The average f_AB among 253 pairwise combinations of BSSS lines was 0.212, whereas the average S_AB was 0.397. The average f _AB ^M was 0.162, indicating that the upward bias in S_AB was effectively removed. S_AB and f_AB were significantly different ( = 0.05) in 76.3% of the comparisons, whereas 24.9% of the f _AB ^M values differed significantly from f_AB. The latter result suggests that selection and/or drift were present during inbred line development and that f_AB may not be an accurate measure of the true proportion of ibd alleles between two lines. Cluster analyses based on S _AB ^M and f _AB ^M grouped lines according to pedigree, although several exceptions were noted. The presence of shared molecular marker variants between unrelated lines must be considered when setting S_AB-based minimum distances for varietal protection. Under simplified conditions, more than 250 molecular marker loci are necessary to obtain sufficiently precise estimates of coefficient of coancestry using molecular markers.A contribution from Limagrain Genetics, a Group Limagrain company     

Identification of high affinity membrane-bound fatty acid-binding proteins using a photoreactive fatty acid

A photoaffinity labeling method was developed to identify and characterize high affinity fatty acid-binding proteins in membranes. The specific labeling of these sites requires the use of low concentrations (nanomolar) of the photoreactive fatty acid 11-m-diazirinophenoxy-[11-³H]undecanoate. It was delivered as a bovine serum albumin (BSA) complex which serves as a reservoir for fatty acid and thus allows precise control of unbound fatty acid concentrations. ThefadL protein ofE. coli, which is required for fatty acid permeation of its outer membrane, was labeled by the photoreactive fatty acid neither specifically nor saturably when the probe was added in the absence of BSA; however when a nanomolar concentration of the uncomplexed probe was maintained in the presence of BSA, the labeling of thefadL protein was highly specific and saturable. This photoaffinity labeling method was also used to characterize a 22 kDa, high affinity fatty acid-binding protein which we have recently identified in the plasma membrane of 3T3-L1 adipocytes. This protein bound the probe with a K_d of 216 nM. The approach described is easily capable of identifying membrane-bound fatty acid-binding proteins and can distinguish between those of high and low affinities for fatty acids. It represents a general method for the identification and characterization of fatty acid-binding proteins.Abbreviations BSA Bovine Serum Albumin - DAP m-Diazirinophenoxy - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis     

Seronegative spondyloarthropathies and diffuse idiopathic skeletal hyperostosis in ancient northern Chile

Bioarchaeological research of ancient Amerindians was undertaken to test the hypothesis that seronegative spondyloarthropathies (SNS) and diffuse idiopathic skeletal hyperostosis (DISH) existed in prehistoric South Americans. An osteological-radiographic model was developed from clinical literature and systematically applied to 504 archaeological human remains housed at the Universidad de Tarapacá in Arica, Chile, to search for evidence of these arthritides. The results showed that SNS existed with an average frequency of 7% for the adult sample and DISH averaged 4% in individuals over 40 years old. It was found that the antiquity of SNS date back at least 5,000 years in both New World and Old World populations. In contrast, the antiquity of DISH in the Americas is not clear because no previous studies have dealt with this subject; however, this research finds mild DISH cases dating back 4,000 years in northern Chile. It was also found that SNS and DISH exhibit a trend of increasing incidence with the advent of agro-pastoral activities and village formation. © 1993 Wiley-Liss, Inc.     

Residues 377-389 from the δ subunit ofTorpedo californica acetylcholine receptor are located in the cytoplasmic surface

Torpedo californica acetylcholine receptor (AcChR) enriched, sealed vesicles have been specifically labeled on the cytoplasmic surface with pyridoxal 5′-phosphate (Perez-Ramirez, B., and Martinez-Carrion, M., 1989,Biochemistry 28, 5034–5040). After chromatography of the peptide fragments produced by tryptin digestion of labeled AcChR, several fractions containing the phosphopyridoxyl label were obtained. Edman degradation identified one of the fractions, with sequence SRSELMFEKQSER, as corresponding to residues 377–389 in theδ subunit (primary structure). The latter must be a cytoplasmic region of this transmembranous protein, and residueδK385 must reside in a water-soluble exposed domain of the cytosolic side of the membrane. Introduction of phosphopyridoxyl residues allows for their potential use as probes of conformational changes in the cytosolic surface of the receptor molecule.