A path analysis of the causal relationships between red cell glycolytic intermediates, ADP and ATP in sickle cell anemia

The statistical relationships among the glycolytic intermediates (GI)) of the Embden-Meyerhof pathway, adenine nucleotides (ANs) and various hematological measures were estimated for 34 sickle cell anemia patients. Heterogeneity in linear and quadratic regressions of hemoglobin and hematocrit, both singly and jointly, on the GI and AN variables implied 1) that any single formula to standardize optical density measures of the GIs and ANs on a per gram hemoglobin or per liter cell water basis would not uniformly remove hemoglobin and hematocrit effects: 2) that ignoring significant hematological effects could bias the estimates of correlation among GIs and ANs; and 3) that hemoglobin and hematocrit measures do not reflect the same source of variability. The correlations among the GIs and ANs, after adjustment for hematological variability, were analyzed by path analysis to determine which of five proposed path models for cause and effect relationships were compatible with the data. AMP had a greater influence on ADP (coefficient of determination (CD) = 23%) than all the GIs together, while G6P and ADP influenced ATP variability the most (CD = 33% and 12%). The contributions of unknown factors to ADP and ATP variability were large for all models (CD = 56--77%) possibly due to stress of sickle cell disease. The path model with AMP and the four GIs (G6P, F6P, FDP, DHAP) influencing ADP variation, and the same GIs and ADP influencing ATP was the model most compatible with the data.     

Enhancing GDP-fucose production in recombinant Escherichia coli by metabolic pathway engineering

Guanosine 5′-diphosphate (GDP)-fucose is the indispensible donor substrate for fucosyltransferase-catalyzed synthesis of fucose-containing biomolecules, which have been found involving in various biological functions. In this work, the salvage pathway for GDP-fucose biosynthesis from Bacterioides fragilis was introduced into Escherichia coli. Besides, the biosynthesis of guanosine 5′-triphosphate (GTP), an essential substrate for GDP-fucose biosynthesis, was enhanced via overexpression of enzymes involved in the salvage pathway of GTP biosynthesis. The production capacities of metabolically engineered strains bearing different combinations of recombinant enzymes were compared. The shake flask fermentation of the strain expressing Fkp, Gpt, Gmk and Ndk obtained the maximum GDP-fucose content of 4.6 ± 0.22 μmol/g (dry cell mass), which is 4.2 fold that of the strain only expressing Fkp. Through fed-batch fermentation, the GDP-fucose content further rose to 6.6 ± 0.14 μmol/g (dry cell mass). In addition to a better productivity than previous fermentation processes based on the de novo pathway for GDP-fucose biosynthesis, the established schemes in this work also have the advantage to be a potential avenue to GDP-fucose analogs encompassing chemical modification on the fucose residue.     

Guanine nucleotides can activate the insulin-stimulated phosphodiesterase in liver plasma membranes

The insulin-stimulated cyclic AMP phosphodiesterase from liver plasma membranes is shown to be activated upon incubation with guanine nucleotides in the presence of ATP. The non-hydrolysable analogue of ATP, adenylyl imidodiphosphate failed to substitute for ATP in achieving activation. GTP, its non-hydrolysable analogues p[NH]ppG and GTP-gamma-S, as well as GDP, all elicited activation. It is suggested that guanine nucleotides, and probably insulin, exert their effect on this enzyme through a distinct species of guanine nucleotide regulatory protein.     

Behaviour of cyclic nucleotides and Ca2+ levels in liver tissue of rats poisoned by white phosphorus and trichlorobromomethane

The content of hepatic cyclic AMP was increased soon after intoxication by white phosphorus. Its level reached a maximum 4 h after poisoning, but in subsequent phases tended to return to normal. In contrast, the cyclic GMP concentration was altered only 24 and 36 h after treatment with the same hepatotoxin. Similar modifications of cAMP and cGMP content were also detected after poisoning by trichlorobromomethane (CBrCl3). As a consequence, an altered cGMP/cAMP ratio was found in both experimental conditions. Further, the modification of cAMP content after white phosphorus was detected prior to liver damage (steatosis and necrosis), while the highest concentration of the cyclic nucleotide in CBrCl3-poisoned rats was found when fatty liver was already evident. In addition, in phosphorus-poisoned rats, the hepatic content of Ca2+ was found to be unmodified during all phases of the intoxication, while after CBrCl3 a phasic increase of the Ca2+ level was observed at 4, 24 and 36 h.     

Regulation of glutamate dehydrogenase activity by manipulation of nucleotide supply in Daucus carota suspension cultures

The enzyme glutamate dehydrogenase (GDH, EC 1.4.1.2) is ubiquitous in plant species. It is now generally accepted that the primary role of this enzyme is not assimilation of ammonium and it has been suggested that GDH may be important in provision of carbon skeletons under conditions of carbon limitation. In carrot ( Daucus carota L. Chantenay) cell suspension cultures carbon starvation results in de-repression of GDH activity. The regulation of this de-repression has not been investigated. This paper examines the possibility that the availability of adenosine nucleotides is instrumental in the regulation of GDH activity. In repressed cultures the adenosine nucleotides cAMP (0.2 m M ), AMP (0.2 m M ) and ADP (0.4 m M ) caused an increase in GDH activity of 61, 33 and 7%, respectively. ATP (0.2 m M ) had the opposite effect in maintaining repression of GDH. Under de-repressed conditions only cAMP (0.2 m M ) enhanced GDH activity (14%). Inhibition of oxidative phosphorylation using a range of inhibitors resulted in de-repression of GDH and stimulation of respiration. The results from this work indicate that exogenously applied adenosine nucleotides and electron transport inhibitors alter the GDH repression/de-repression status. Addition of these compounds alters or disrupts ATP levels, mimicking carbon depletion. This causes an increase in GDH activity, supporting the idea that GDH may provide carbon skeletons for carbon metabolism and suggesting that ATP status is important in regulation of the enzyme activity.     

Multigenerations Assessment of Dietary Nucleotides Consumption in Weaned Rats

With the multifaceted activities of nucleotides, there is a history of safe consumption of dietary nucleotides (NTs) in the human diet. This study investigated the multigenerations cumulative toxicity on rats’ development after weaning. Weaning rats (F0) were fed with NTs at the dosage of 0.01, 0.04, 0.16, 0.64, and 1.28% (wt/wt) for 90 days and then mated for 1:1 pattern. The offspring was F1. F1 rats were fed with NTs for 90 days after weaning. Afterwards, F1 go on to the second reproductive part. We repeated the above process, until F3 rats were born. We observed the weight, food consumption in the whole experiment, and detected the blood indicators and organ pathology at the terminal. No abnormal reaction, behavior disorder, and organ pathology related to toxic symptom were observed in NTs‐treated groups. Weight gain and diet utilization ratio of males in each NTs group had significant increase after weaning (p < 0.05). With the exception of decrease in uric acid (p < 0.05) of NTs male, there were no differences between the control and NTs groups in blood indicators. NTs could promote early growth and development of rats after weaning, especially in males.     

New insights into the melanophilin (MLPH) gene controlling coat color phenotypes in American mink

The mutation causing the Silverblue color type (pp) is one of the most used recessive mutations within American mink (Neovison vison) fur farming, since it is involved in some of the popular color types such as Violet and Saphire which originate from a combination of recessive mutations. In the present study, the genomic and mRNA sequences of the melanophilin (MLPH) gene were studied in Violet, Silverblue and wild-type (wt) mink animals. Although breeding schemes and previous literature indicates that the Violet (aammpp) phenotype is a triple recessive color type involving the same locus as the Silverblue (pp) color type, our findings indicate different genotypes at the MLPH locus. Upon comparison at genomic level, we identified two deletions of the entire intron 7 and of the 5′ end of intron 8 in the sequence of the Silverblue MLPH gene. When investigating the mRNA, the Silverblue animals completely lack exon 8, which encodes 65 residues, of which 47 define the Myosin Va (MYO5A) binding domain. This may cause the incorrect anchoring of the MLPH protein to MYO5A in Silverblue animals, resulting in an improper pigmentation as seen in diluted phenotypes. Additionally, in the MLPH mRNA of wt, Violet and Silverblue phenotypes, part of intron 8 is retained resulting in a truncated MLPH protein, which is 359 residues long in wt and Violet and 284 residues long in Silverblue. Subsequently, our findings point out that the missing actin-binding domain, in neither of the 3 analyzed phenotypes affects the transport of melanosomes or the consequent final pigmentation. Moreover, the loss of the major part of the MYO5A domain in the Silverblue MLPH protein seems to be the responsible for the dilute phenotype. Based on our genomic DNA data, genetic tests for selecting Silverblue and Violet carrier animals can be performed in American mink.     

Entamoeba histolytica: flavins in axenic organisms.

The individual flavin species of axenic Entamoeba histolytica were assayed: separated riboflavin was assayed by the lumiflavin method; flavin-adenine dinucleotide (FAD), by an enzymatic method; flavin mononucleotide (FMN) was calculated from the difference, total flavin minus FAD and riboflavin. The amount of flavin in micrograms per grams fresh cells follows: total flavin, 7.6 ± 0.9 calculated as riboflavin; riboflavin, 1.6 ± 0.7; FMN, 6.6 ± 0.5; and FAD, 1.2 ± 0.1. Recalculated to nanomoles per milligrams total amebal protein these values were: total flavin, 0.21; riboflavin, 0.04; FMN, 0.15; and FAD, 0.02. The identity of each flavin was confirmed by a paper chromatographic method. Analyses on Panmede, the main source of flavins in the TP-S-1 medium, indicate that it contains all three forms of flavin. Its contribution to growth medium in micrograms per milliliters: riboflavin, 2.1 ± 0.3; FMN, 0.6 ± 0.1; and FAD, 0.4 ± 0.1. The in vivo biosynthesis of FMN and FAD from riboflavin by E. histolytica is demonstrated. A new and convenient method was found to separate riboflavin from flavin nucleotides in tissue extracts.     

Inosine monophosphate dehydrogenase inhibitors. Probes for investigations of the functions of guanine nucleotide binding proteins in intact cells

Taken together, the above reports indicate that the IMP dehydrogenase inhibitors are valuable probes for investigation of the biological functions of guanine nucleotides in intact cells. While these agents have minor effects on levels of other nucleotides and enzymes, non-specific effects can be monitored by addition of guanine or guanosine to provide substrates for the salvage pathway of guanine nucleotide synthesis. The most important question yet to be resolved in employing these agents is why incomplete depletion of intracellular guanine nucleotides results in such dramatic effects on G-protein function. Since the level of GTP in resting cells is approximately 0.5 mM, even a 90% reduction in GTP levels should leave enough nucleotide to adequately activate most known G-proteins, as the latter display high binding affinities for guanine nucleotides in cell free systems. Several explanations have been proposed to account for this disparity. Much of the intracellular guanine nucleotide may be bound or compartmentalized and therefore unable to interact with certain G-proteins. Possibly, G-proteins in the intracellular environment possess a much lower affinity for GTP that they do in cell free system. It may be to the cells' advantage that relatively minor fluctuations in levels of GTP result in pronounced alterations in the biological function of G-proteins as this effect may provide a physiologically important mechanism for the regulation of G-proteins in vivo. Further studies are necessary to clarify the mechanisms involved in the regulation of the biological function of G-proteins and oncogene products by guanine nucleotides in intact cells.