A carbon-13 nuclear magnetic resonance investigation of the metabolic fluxes associated withy glucose metabolism in human erythrocytes

We have used [2-¹³C]d-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy to investigate metabolic fluxes through the major pathways of glucose metabolism in intact human erythrocytes and to determine the interactions among these pathways under conditions that perturb metabolism. Using the method described, we have been able to measure fluxes through the pentose phosphate pathway, phosphofructokinase, the 2,3-diphosphoglycerate bypass, and phosphoglycerate kinase, as well as glucose uptake, concurrently and in a single experiment. We have measured these fluxes in normal human erythrocytes under the following conditions: (1) fully oxygenated; (2) treated with methylene blue; and (3) deoxygenated. This method makes it possible to monitor various metabolic effects of stresses in normal and pathological states. Not only has ¹³C-NMR spectroscopy proved to be a useful method for measuring in vivo flux through the pentose phosphate pathway, but it has also provided additional information about the cycling of metabolites through the non-oxidative portion of the pentose phosphate pathway. Our evidence from experiments with [1-¹³C]-, [2-¹³C]-, and [3-¹³C]d-glucoses indicates that there is an observable reverse flux of fructose 6-phosphate through the reactions catalyzed by transketolase and transaldolase, even in the presence of a net flux through the pentose phosphate pathway.     

Characterization of phosphoinositide kinases in normal and sickle anaemia red cells

PtdIns and PtdInsP kinases from normal erythrocyte (AA) membranes and sickle cell anaemia erythrocyte (SS) membranes have been characterized. PtdIns kinase was studied in native membranes under conditions in which PtdInsP kinase and PtdInsP phosphatase do not express any activity. Kinetic analysis of the AA and SS PtdIns kinases indicate similar K_m values for PtdIns and ATP but higher V_max values for SS PtdIns kinase. PtdInsP kinase was partially purified from erythrocyte ghosts by NaCl extraction. The kinetic parameters of PtdInsP kinase determined under these conditions were similar in AA and SS NaCl extracts. These data suggest the presence of some effector of PtdIns kinase in SS cell membranes, resulting in a greater activity of the enzyme. This leads consequently, to increase the PtdInsP pool and to activate PtdInsP kinase, in agreement with our previous observations of a greater [³²P]Pi incorporation in both polyphosphoinositides in SS cells relatively to AA cells.     

Molecular and chromosomal evolution in anoas (Bovidae: Bubalus spec.)

As a contribution to their taxonomy, population genetic data on zoo-living anoas are reported, and a review of the history of the captive stock is provided. Four different chromosome numbers of 44, 45, 47 and 48 chromosomes have been found, respectively, when karyotyping captive anoas descending from three breeding lines. The number of chromosome arms is 60 throughout, indicating that Robertsonian rearrangements are responsible for this cytogenetic variation. An electrophoretic comparison of isozymes and blood proteins representing 21 genetic loci revealed polymorphism in seven loci: haemoglobin, glyoxalase, superoxide dismutase, phosphoglucomutase, carbonic anhydrase, glucose phosphate isomerase, and an unidentified acid serum protein. Considering the small number of founder specimens and subsequent inbreeding, allozyme variability appears fairly high in anoas. Genetic distances between zoo populations amount to 0.0505 or less. Southern blot hybridizations of restricted DNA from anoas and African buffaloes with a probe from the DRB-like region of the chimpanzee's MHC class II genes also indicate a low degree of genetic differentiation between mountain and lowland anoas. The relevance of these genetic data for the taxonomic classification of mountain and lowland anoas, and for the conservation of anoas by captive breeding is discussed.     

Non-specific phospholipase C4 hydrolyzes phosphosphingolipids and phosphoglycerolipids and promotes rapeseed growth and yield

Phosphorus is a major nutrient vital for plant growth and development, with a substantial amount of cellular phosphorus being used for the biosynthesis of membrane phospholipids. Here, we report that NON-SPECIFIC PHOSPHOLIPASE C4 (NPC4) in rapeseed (Brassica napus) releases phosphate from phospholipids to promote growth and seed yield, as plants with altered NPC4 levels showed significant changes in seed production under different phosphate conditions. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated knockout of BnaNPC4 led to elevated accumulation of phospholipids and decreased growth, whereas overexpression (OE) of BnaNPC4 resulted in lower phospholipid contents and increased plant growth and seed production. We demonstrate that BnaNPC4 hydrolyzes phosphosphingolipids and phosphoglycerolipids in vitro, and plants with altered BnaNPC4 function displayed changes in their sphingolipid and glycerolipid contents in roots, with a greater change in glycerolipids than sphingolipids in leaves, particularly under phosphate deficiency conditions. In addition, BnaNPC4-OE plants led to the upregulation of genes involved in lipid metabolism, phosphate release, and phosphate transport and an increase in free inorganic phosphate in leaves. These results indicate that BnaNPC4 hydrolyzes phosphosphingolipids and phosphoglycerolipids in rapeseed to enhance phosphate release from membrane phospholipids and promote growth and seed production.     

EXTRACTION,ASSAY AND SOME PROPERTIES OF FLORIDOSIDE PHOSPHATE SYNTHASE FROM PORPHYRA PERFORATA (RHODOPHYTA)1

A suitable method for extraction of floridoside phosphate synthase (FPS, UDP-galactose: sn-3-glycerol phosphate: 1→2′α-D-galactosyl transferase)from Porphyra perforata J. Ag. was developed. Two assay methods for enzyme activity were utilized, one measuring the amount of floridoside formed by using gas-liquid chromatography, the other measuring the sn-3-glycerol phosphate-dependent formation of UDP; both assays gave similar results. FPS is a soluble protein, and FPS activity in the extract as determined by the amount of product formed in vitro compared well with the in vivo rate of floridoside synthesis (4–7 μMmol product formed·h^?1·g^?1 fresh wt). The rate of product formation in vitro was linear up to 45 min and proportional to protein concentration in the assay mixture. The temperature optimum was 30–35° C. FPS was active over a range of pH values from 7.0–8.5. It was stable in concentrated solutions in the presence of 0.3 M ammonium sulfate, but activity was lost in diluted solution (protein concentration below 0.2 mg·mL^?1) or below 0.2 M ion strength. The data suggest that FPS may be an oligomeric protein which occurs free in the cytoplasm or loosely bound to a membrane. It may also be a regulatory protein controlling the overall rate of synthesis of floridoside in vivo.     

Effects of phosphate availability on the root system architecture: large-scale analysis of the natural variation between Arabidopsis accessions

Developmental plasticity is one main adaptative response of plants to the availability of nutrients. In the present study, the naturally occurring variation existing in Arabidopsis for the growth responses to phosphate availability was investigated. Initially details of the effects of phosphate starvation for the four currently used accessions Cvi, Col, Ler and Ws were compared. A set of 10 growth parameters, concerning the aerial part and the root system, was measured in both single‐point and time‐course experiments. The length of the primary root and the number of laterals were found to be consistently reduced by phosphate starvation in all four accessions. These two robust parameters were selected to further screen a set of 73 accessions originating from a wide range of habitats. One‐half of the accessions showed also a reduced primary root and less lateral roots when phosphate‐starved, and 25% were not responsive to phosphate availability. For the last quarter of accessions, phosphate starvation was found to affect only one of the two growth parameters, indicating the occurrence of different adaptative strategies. These accessions appear to offer new tools to investigate the molecular basis of the corresponding growth responses to phosphate availability.     

Restoration of eutrophied shallow softwater lakes based upon carbon and phosphorus limitation

Plant communities from oligotrophic, poorly buffered waters are seriously threatened by both, acidification and eutrophication/alkalinization. Acidification is mainly caused by atmospheric deposition of acidifying substances while eutrophication is often the result of inlet of nutrient enriched, calcareous brook- or groundwater. The plant production in very soft waters is often limited by low levels of inorganic carbon, nitrogen and/or phosphorus. This paper deals with the possibilities for restoration of formerly oligotrophic but now eutrophied and alkalinized softwater systems. Restoration based upon nitrogen limitation is not likely to be successful as the atmospheric deposition of nitrogen in The Netherlands is very high. Phosphorus limitation can also be a problem. One can stop the input of phosphorus and remove the mud layer, but the problem remains that also the deeper mineral sandy sediments are saturated with phosphate. A possible remedy, however, is a combination of carbon- and phosphorus limitation. Many plants from eutrophic environments never occur in very soft waters, probably as a result of carbon limitation. In addition, mobilisation of phosphate is much lower in waters with very low bicarbonate levels. Restoration of a former oligotrophic softwater lake by reducing the inlet of calcareous surface water, in combination with removal of the organic sediment layer, appeared to be very successful. Many endangered plant species such asIsoetes echinospora, Luronium natans, Deschampsia setacea andEchinodorus repens developed spontaneously from the still viable seedbank.     

Biomass relationship to growth and phosphate uptake ofPseudomonas fluorescens,Escherichia coli andAcinetobacter radioresistens in mixed liquor medium

The ability ofPseudomonas fluorescens, Escherichia coli andAcinetobacter radioresistenns to remove phosphate during growth was related to the initial biomass as well as to growth stages and bacterial species. Phosphate was removed by these bacteria under favourable conditions as well as under unfavourable conditions of growth. Experiments showed a relationship between a high initial cell density and phosphate uptake. More phosphate was released than removed when low initial cell densities (10²–10⁵ cells ml^–1) were used. At a high initial biomass concentration (10⁸ cells ml^–1), phosphate was removed during the lag phase and during logarthmic growth byP. fluorescens. Escherichia coli. at high initial biomass concentrations (10⁷ cells ml^–1), accumulated most of the phosphate during the first hour of the lag phase and/or during logarithmic growth and in some cases removed a small quantily of phosphate during the stationary growth phase.Acinetobacter radioresistens, at high initial cell densities (10⁶, 10⁷ cells ml^–1) removed most of phosphate during the first hour of the lag phase and some phosphate during the stationary growth phase.Pseudomonas fluorescens removed phosphate more thanA. radioresistens andE. coli with specific average ranges from 3.00–28.50 mg L^–1 compared to average ranges of 4.92–17.14 mg L^–1 forA. radioresistens and to average ranges of 0.50–8.50 mg L^–1 forE. coli.     

Glutamine repeats and inherited neurodegenerative diseases: molecular aspects

Several dominantly inherited, late onset, neurodegenerative diseases are due to expansion of CAG repeats, leading to expansion of glutamine repeats in the affected proteins. These proteins are of very different sizes and, with one exception, show no sequence homology to known proteins or to each other; their functions are unknown. In some, the glutamine repeat starts near the N-terminus, in another near the middle and in another near the C-terminus, but regardless of these differences, no disease has been observed in individuals with fewer than 37 repeats, and absence of disease has never been found in those with more than 41 repeats. Protein constructs with more than 41 repeats are toxic to E. coli and to CHO cells in culture, and they elicit ataxia in transgenic mice. These observations argue in favour of a distinct change of structure associated with elongation beyond 37–41 glutamine repeats. The review describes experiments designed to find out what these structures might be and how they could influence the properties of the proteins of which they form part. Poly- -glutamines form pleated sheets of β-strands held together by hydrogen bonds between their amides. Incorporation of glutamine repeats into a small protein of known structure made it associate irreversibly into oligomers. That association took place during the folding of the protein molecules and led to their becoming firmly interlocked by either strand- or domain-swapping. Thermodynamic considerations suggest that elongation of glutamine repeats beyond a certain length may lead to a phase change from random coils to hydrogen-bonded hairpins. Possible mechanisms of expansion of CAG repeats are discussed in the light of looped DNA model structures.     

用快速琼脂糖层析纯化磷酸甘油酸激酶及磷酸甘油醛脱氢酶

研究了用快速琼脂糖离子交换层析(DEAE—Fast Flow sepharose)结台PEG 4000／Reppal PES双水相体系从黄豆中分离纯化磷酸甘油酸激酶(PGK)及磷酸甘油醛脱氢酶(GAPDH)。控制床层高度(10～20cm),径向放大具有压降低的优点．设计多点进料取代传统的中心管进料,解决了径向流场不均匀的问题。GAPr)H的总收率及纯化倍数分别为58％和144,PGK的总收率及纯化倍数分别为41％和44。工艺成本为2．92美元／ku GPADH,具有一定的实用价值。