In vitro dephosphorylation inhibits the activity of soybean lysine-ketoglutarate reductase in a lysine-regulated manner

In plant seeds, the essential amino acid lysine auto-regulates its own level by modulating the activity of its catabolic enzyme lysine-ketoglutarate reductase via an intracellular signaling cascade, mediated by Ca²⁺ and protein phosphorylation/dephosphorylation. In the present report, it has been further tested whether the activity of soybean lysine-ketoglutarate reductase, as well as that of saccharopine dehydrogenase, the second enzyme in the pathway of lysine catabolism, are modulated by direct phosphorylation of the bifunctional polypeptide containing both of these linked activities. Incubation of purified lysine-ketoglutarate reductase/ saccharopine dehydrogenase with casein kinase II resulted in a significant phosphorylation of the bifunctional enzyme. Moreover, in vitro dephosphorylation of the bifunctional polypeptide with alkaline phosphatase significantly inhibited the activity of lysine-ketoglutarate reductase, but not of its linked enzyme saccharopine dehydrogenase. The inhibitory effect of alkaline phosphatase on lysine-ketoglutarate reductase activity was dramatically stimulated by binding of lysine to the enzyme. Our results suggest that in plant seeds, active lysine-ketoglutarate reductase is a phospho-protein, and that its activity is modulated by opposing actions of protein kinases and phosphatases. Moreover, this modulation is subject to a compound regulation by lysine.     

Aluminium causes nutrient imbalance and structural changes in the needles of Scots pine without inducing clear root injuries

The effects of aluminium chloride (AICI3) treatments (50 and 150 mg/l) on 3-year-old Scots pine (Pinus sylvestris L.) seedlings were studied in a sand culture during 2 growing periods in an open field experiment. Even by the end of the first growing period, a decline was observed in the concentrations of Ca, Mg and P within the needles, and of Ca and Mg in the roots. After the second growing period, increased N and K concentrations were observed in the needles of Al-treated seedlings. Both the needles and roots of Al-treated seedlings showed, after the second growing period, a decline in growth and increased concentrations of AI as the amount of AICI3 in the nutrient solution increased. Al-induced changes in needle structure were found to be symptomatic of a nutrient imbalance, particularly of Mg and P. Al-stress did not result in any observable changes in root anatomy or in the number of mycorrhizas. Scots pine proved to be rather resistant to Al-stress, indicating that direct Al-injuries are not likely in the field, though Al-stress may be a contributing factor in the formation of nutrient imbalances.     

Different states of sarcoplasmic reticulum membrane in the presence of acetyl phosphate and adenosine triphosphate.

In the presence of acetyl phosphate, approximately 0.8 extra sulphydryl groups/10⁵ g protein of sarcoplasmic reticulum membrane vesicles are exposed to reaction with $\text{N}$-ethylmaleimide, whereas in the presence of ATP approximately 0.6 groups/10⁵ g protein are protected. Dithiobis (nitrobenzoic acid) reacts with the membrane sulphydryl groups more slowly in the presence of ATP than in the presence of acetyl phosphate or in the absence of substrate. Sarcoplasmic reticulum membrane is degraded by trypsin at a faster rate than normal when acetyl phosphate is present as seen from changes in electrophoretic patterns, ATPase activity and Ca²⁺ uptake capacity, and at a slower rate when ATP is present as seen from the last two properties. These differences in reactivity are interpreted as being due to differences in membrane conformations induced by the two substrates.     

Competitive ability of maize pollen. Intergametophytic effects

Summary Intergametophytic influences on pollen competitive ability were studied by means of the pollen mixture technique: mixtures of pollen from different genetic sources, marked for the presence of the normal or the mutant allele of the opaque-2 gene, were used to pollinate o2o2 female plants. The variation of relative frequency of one of the two kernel types from apex to base of the resulting ear, made it possible to measure the competitive ability by regression coefficients.The effect of the pollen marker on the character was estimated by analyzing the progeny of heterozygous o2/+ plants self-pollinated through four generations. The o2 allele-bearing pollen revealed a slower germination rate than the dominant counterpart; no differences between the two pollen types were observed with regard to tube growth rate.The occurrence of intergametophytic interactions was confirmed: the competitive ability of a line varied according to the genotype of the competing pollen. Thus, this component has to be taken into account in considering male gametophyte fertilization ability.     

Catalytic activities of human liver cytochrome P-450 IIIA4 expressed in Saccharomyces cerevisiae

A human liver cytochrome P-450 (P-450) IIIA4 cDNA clone was inserted behind an alcohol dehydrogenase promoter in the plasmid vector pAAH5 and expressed in Saccharomyces cerevisiae (D12 and AH22 strains). A cytochrome P-450 with typical spectral properties was expressed at a level of approximately 8 x 10(5) molecules/cell in either strain of yeast. The expressed P-450 IIIA4 had the same apparent monomeric Mr as the corresponding protein in human liver microsomes (P-450NF) and could be isolated from yeast microsomes. Catalytic activity of the yeast microsomes toward putative P-450 IIIA4 substrates was seen in the reactions supported by cumene hydroperoxide but was often lower and variable when supported by the physiological donor NADPH. The catalytic activity of purified P-450 IIIA4 was also poor in some systems reconstituted with rabbit liver NADPH-P-450 reductase and best when both the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and a lipid extract (from liver or yeast microsomes) or L-alpha-1,2-dilauroyl-sn-glycero-3-phosphocholine were present. Under these conditions the expressed P-450 IIIA4 was an efficient catalyst for nifedipine oxidation, 6 beta-hydroxylation of testosterone and cortisol, 2-hydroxylation of 17 beta-estradiol and 17 alpha-ethynylestradiol, N-oxygenation and 3-hydroxylation of quinidine, 16 alpha-hydroxylation of dehydroepiandrosterone 3-sulfate, erythromycin N-demethylation, the 10-hydroxylation of (R)-warfarin, the formation of 9,10-dehydrowarfarin from (S)-warfarin, and the activation of aflatoxins B1 and G1, sterigmatocystin, 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (both + and - diastereomers), 3,4-dihydroxy-3,4-dihydrobenz[a]anthracene, 3,4-dihydroxy-3,4-dihydro-7, 12-dimethylbenz[a]anthracene, 9,10-dihydroxy-9,10-dihydrobenzo[b]fluoranthene, 6-aminochrysene, and tris(2,3-dibromopropyl) phosphate to products genotoxic in a Salmonella typhimurium TA1535/pSK1002 system where a chimeric umuC' 'lacZ plasmid is responsive to DNA alkylation. Reaction rates were stimulated by 7,8-benzoflavone and inhibited by rabbit anti-P-450 IIIA (anti-P-450NF), troleandomycin, gestodene, and cimetidine. Evidence was obtained that rates of reduction of ferric P-450 IIIA4 in yeast microsomes and the reconstituted systems are slow and at least partially responsible for the lower rates of catalysis seen in these systems (relative to liver microsomes). The results of these studies with a defined protein clearly demonstrate the ability of P-450 IIIA4 to catalyze regio- and stereoselective oxidations with a diverse group of substrates, and this enzyme appears to be one of the most versatile catalysts in the P-450 family.     

Laser microirradiation of kinetochores in mitotic PtK2 cells

Irradiation of the kinetochore region of PtK₂ chromosomes by laser light of 532 nm was used to study the function of the kinetochore region in chromosome movement and to create artificial micronuclei in cells. When the sister kinetochores of a chromosome were irradiated at prometaphase, the affected chromosome detached from the spindle and exhibited no further directed movements for the duration of mitosis. The chromatids of the chromosome remained attached to one another until anaphase, at which point they separated. No poleward movement of the chromatids was observed, and at telophase they passively moved to one of the daughter cells and were enclosed in a micronucleus. The daughter cell containing the micronucleus was then isolated by micromanipulation and followed through subsequent mitoses. At the next mitosis, two chromosomes, each with two chromatids, condensed in the micronucleus. These chromosomes did not attach to the spindle and showed chromatid separation, but no poleward movements at anaphase. They were again enclosed in micronuclei at telophase. The third generation mitosis was similar to the second. Occasionally, both the irradiation-produced and naturally occurring micronuclei exhibited no chromosome condensation at mitosis. Feulgenstained monolayers of PtK₂ cells with naturally occurring micronuclei showed that some micronuclei stain positive for DNA and others do not. This finding raises questions about the fate of chromosomes in a micronucleus.     

Inhibition of pancreatic lipase by mixed micelles of diethyl p-nitrophenyl phosphate and bile salts

Solubility and Sephadex filtration assays have shown that dissolved diethyl p-nitrophenyl phosphate can be included into bile salt micelles with a partition coefficient of 32 : 1. This inclusion is probably a prerequisite for the organophosphate to inhibit lipase. The essential role played by colipase confirms that the primary step in the inhibition is an interaction of lipase with bile salt containing micelles. Therefore, it appears that the requirements of lipase towards specific substrates and inhibitors are very similar. The inhibition rate strongly depends on the total bile salt concentration and on the micellar concentration of the organophosphate. This effect may be explained, at least qualitatively, by a competition between simple and mixed micelles for the binding of colipase and lipase.     

Lipid status association with 25-hydroxy vitamin D: Cross sectional study of end stage renal disease patients

BackgroundSome observational studies indicate an association of 25-hydroxy vitamin D (25(OH)D) insufficiency and atherogenic cholesterol concentrations. The aim of this study was to investigate relationship between 25(OH)D concentrations and lipid parameters in end stage renal disease (ESRD) patients, separately for predialysis, hemodialysis and peritoneal dialysis patients.MethodsWe have adjusted 25(OH)D concentrations for seasonal variability with cosinor analysis, and performed all further analysis using these corrected 25(OH)D concentrations. Concentrations of 25(OH)D and the lipid parameters were determined in 214 ESRD patients and 50 control group participants. The analysis included the measurement of 25(OH)D by HPLC, apolipoprotein (Apo) AI, ApoB and Lp(a) by nephelometry, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and triglyceride (TG) by spectrophotometry and manually calculated ApoB/ApoAI and LDL-C/HDL-C ratio.ResultsESRD patients with adjusted 25(OH)D concentrations of 50 nmol/L had significantly higher TC (P = 0.005) and ApoAI (P = 0.049). Significantly higher HDLC (P = 0.011) and ApoAI (P = 0.020) were found in hemodialysis patients with the 25(OH)D concentrations of 50 nmol/L. The other analyzed lipid parameters differed significantly between predialysis, hemodialysis and peritoneal dialysis patients with 25(OH)D concentrations of < 50 nmol/L.ConclusionsOur study indicate the significant relationship between 25(OH)D repletion and optimal concentrations of lipid parameters in ESRD patients. Further research is necessary to explain whether joint evaluation of vitamin D status and lipid abnormalities could improve cardiovascular outcome in ESRD patients.