Exploring abiotic stress on asynchronous protein metabolism in single kernels of wheat studied by NMR spectroscopy and chemometrics

Extreme climate events are being recognized as important factorsin the effects on crop growth and yield. Increased climaticvariability leads to more frequent extreme conditions whichmay result in crops being exposed to more than one extreme eventwithin a growing season. The aim of this study was to examinethe implications of different drought treatments on the proteinfractions in grains of winter wheat using ¹H nuclear magneticresonance spectroscopy followed by chemometric analysis. Triticumaestivum L. cv. Vinjett was studied in a semi-field experimentand subjected to drought episodes either at terminal spikelet,during grain-filling or at both stages. Principal componenttrajectories of the total protein content and the protein fractionsof flour as well as the ¹H NMR spectra of single wheat kernels,wheat flour, and wheat methanol extracts were analysed to elucidatethe metabolic development during grain-filling. The resultsfrom both the ¹H NMR spectra of methanol extracts and the ¹HHR-MAS NMR of single kernels showed that a single drought eventduring the generative stage had as strong an influence on proteinmetabolism as two consecutive events of drought. By contrast,a drought event at the vegetative growth stage had little effecton the parameters investigated. For the first time, ¹H HR-MASNMR spectra of grains taken during grain-filling were analysedby an advanced multiway model. In addition to the results fromthe chemical protein analysis and the ¹H HR-MAS NMR spectraof single kernels indicating that protein metabolism is influencedby multiple drought events, the ¹H NMR spectra of the methanolextracts of flour from mature grains revealed that the amountof fumaric acid is particularly sensitive to water deficits. Key words: Chemometrics, drought, fumaric acid, grain-filling, HR-MAS NMR, PARAFAC, PCA trajectory, single kernel, wheat Received 19 August 2008; Revised 14 October 2008 Accepted 24 October 2008     

Characterization of a cholecystokinin 8-generating endoprotease purified from rat brain synaptosomes.

An endoproteolytic activity that specifically cleaves CCK 33, producing CCK 8, has been purified from a rat brain synaptosome preparation. The purification, which included anion exchange, chromatofocusing, hydroxyapatite, and gel filtration chromatography, resulted in a greater than 3000-fold increase in specific activity. This neutral endoprotease (pH optimum 8) exists as a 90-kDa species, which can be dissociated into active 40-kDa species. The enzyme is a non-trypsin serine protease, which is inhibited by diisopropyl-fluorophosphate and p-aminobenzamidine but not by soybean trypsin inhibitor, phenylmethylsulfonyl fluoride, aprotinin, or a number of thiol or metalloprotease inhibitors. It is highly substrate-specific and cleaves neither trypsin, enteropeptidase, kallikrein substrates, nor analogues of mono- or dibasic cleavage sites of prohormones other than pro-CCK. The endoprotease will not cleave CCK 12 desulfate or CCK (20-29), although these peptides contain common sequences with CCK-33. The protease does cleave [Glu27]CCK (20-29), a peptide in which the glutamate mimics the negative charge normally present on tyrosine sulfate. This suggests that the negative charge at position 27 is important in substrate recognition. The enzyme will also cleave CCK 33 and CCK (1-21) on the carboxyl-terminal side of a single lysine residue in position 11. The subcellular location and specificity of this endoprotease make it a good candidate for a CCK-processing protease.     

Mice Deficient in Phosphofructokinase‐M Have Greatly Decreased Fat Stores

Synthesis of triacylglycerol requires the glucose‐derived glycerol component, and glucose uptake has been viewed as the rate‐limiting step in glucose metabolism in adipocytes. Furthermore, adipose tissue contains all three isoforms of the glycolytic enzyme phosphofructokinase (PFK). We here report that mice deficient in the muscle isoform PFK‐M have greatly reduced fat stores. Mice with disrupted activity of the PFK‐M distal promoter were obtained from Lexicon Pharmaceuticals, developed from OmniBank OST#56064. Intra‐abdominal fat was measured by magnetic resonance imaging of the methylene proton signal. Lipogenesis from labeled glucose was measured in isolated adipocytes. Lipolysis (glycerol and free fatty acid release) was measured in perifused adipocytes. Intra‐abdominal fat in PFK‐M–deficient female mice (5–10 months old) was 17 ± 3% of that of wild‐type littermates (n = 4; P < 0.02). Epididymal fat weight in 15 animals (7–9.5 months) was 34 ± 4% of control littermate (P < 0.002), with 10–30% lower body weight. Basal and insulin‐stimulated lipogenesis in PFK‐M–deficient epididymal adipocytes was 40% of the rates in cells from heterozygous littermates (n = 3; P < 0.05). The rate of isoproterenol‐stimulated lipolysis in wild‐type adipocytes declined ～10% after 1 h and 50% after 2 h; in PFK‐M–deficient cells it declined much more rapidly, 50% in 1 h and 90% in 2 h, and lipolytic oscillations appeared to be damped (n = 4). These results indicate an important role for PFK‐M in adipose metabolism. This may be related to the ability of this isoform to generate glycolytic oscillations, because such oscillations may enhance the production of the triacylglycerol precursor α‐glycerophosphate.     

Characteristics of treeline plant communities in Alaska

The treeline in Alaska is usually mapped as the limit of white spruce Picea glauca (Moench) Voss along the south slope of the Brooks Range and in western Alaska and as the limit of Sitka spruce Picea sitchensis (Bong.) Carr. on Kodiak Island and in the Alaska Peninsula. In some localities black spruce Picea mariana (Mill.) B.S.P. and paper birch Betula papyrifera Marsh. form components of the treeline community with white spruce. Of special interest are the groves of balsam poplar Populus balsamifera L. occurring on the north slope of the Brooks Range and to the west and southwest of the spruce treeline. There is some question as to whether trees survived in unglaciated portions of Alaska during the last glacial period. Spruce trees became prevalent about 10000 yr ago and may still be expanding to their climatic limits. In some areas of Alaska the treeline was more expanded during the Hypsithermal period than it is at present, but in other regions evidence for such an expansion is lacking. Treeline in some areas seems to have been stable for the last several centuries, but is has been expanding rapidly for the last 40 yr in central and western Alaska and for a longer period on Kodiak Island.     

Alteration of the insulin-like growth factor axis during in vitro differentiation of the human osteosarcoma cell line HOS 58

The insulin-like growth factors I and II (IGF-I, IGF-II), their receptors, and high affinity binding proteins (IGFBPs) represent a family of cellular modulators that play essential roles in the development and differentiation of cells and tissues including the skeleton. Recently, the human osteosarcoma cell line HOS 58 cells were used as an in vitro model of osteoblast differentiation characterized by (i) a rapid proliferation rate in low-density cells that decreased continuously with time of culture and (ii) an increasing secretion of matrix proteins during their in vitro differentiation. In the present paper, HOS 58 cells with low cell density at early time points of the in vitro differentiation (i) displayed a low expression of IGF-I and -II; (ii) synthesized low levels of IGFBP-2, -3, -4, and -5, but (iii) showed high expression levels of both the type I and II IGF receptors. During the in vitro differentiation of HOS 58 cells, IGF-I and -II expressions increased continuously in parallel with an upregulation of IGFBP-2, -3, -4, and -5 whereas the IGF-I receptor and IGF-II/M6P receptor mRNA were downregulated. In conclusion, the high proliferative activity in low cell density HOS 58 cells was associated with high mRNA levels of the IGF-IR, but low concentrations of IGFBP-2. The rate of proliferation of HOS 58 cells continuously decreased during cultivation in parallel with a decline in IGF-IR expression, but increase of mitoinhibitory IGFBP-2. These data are indicative for a role of the IGF axis during the in vitro differentiation of HOS 58 cells.     

The Relationship of Ectopic Lipid Accumulation to Cardiac and Vascular Function in Obesity and Metabolic Syndrome

Storage of lipid in ectopic depots outside of abdominal visceral and subcutaneous stores, including within the pericardium and liver, has been associated with obesity, insulin resistance, and cardiovascular risk. We sought to determine whether anatomically distinct ectopic depots were physiologically correlated and site‐specific effects upon cardiovascular function could be identified. Obese subjects (n = 28) with metabolic syndrome but without known atherosclerotic disease and healthy controls (n = 18) underwent magnetic resonance imaging (MRI) and proton MR spectroscopy (MRS) to quantify pericardial and periaortic lipid volumes, cardiac function, aortic compliance, and intrahepatic lipid content. Fasting plasma lipoproteins, glucose, insulin, and free‐fatty acids were measured. Pericardial and intrahepatic (P < 0.01) and periaortic (P < 0.05) lipid volumes were increased in obese subjects vs. controls and were strongly and positively correlated (P ≤ 0.01) but independent of BMI (P = NS) among obese subjects. Intrahepatic lipid was associated with insulin resistance (P < 0.01) and triglycerides (P < 0.05), whereas pericardial and periaortic lipid were not (P = NS). Periaortic and pericardial lipid positively correlated to free‐fatty acids (P ≤ 0.01) and negatively correlated to high‐density lipoprotein (HDL) cholesterol (P < 0.05). Pericardial lipid negatively correlated to cardiac output (P = 0.03) and stroke volume (P = 0.01) but not to left ventricular ejection fraction (P = 0.46). None of the ectopic depots correlated to aortic compliance. In conclusion, ectopic storage of lipid in anatomically distinct depots appeared tightly correlated but independent of body size. Site‐specific functional abnormalities were observed for pericardial but not periaortic lipid. These findings underscore the utility of MRI to assess individual differences in ectopic lipid that are not predictable from BMI.     

Atorvastatin stimulates the production of osteoprotegerin by human osteoblasts

Recently, HMG-CoA reductase inhibitors (statins), potent inhibitors of cholesterol biosynthesis, have been linked to protective effects on bone metabolism. Because of their widespread use, prevention of bone loss and fractures would be a desirable side effect. However, the mechanisms how statins may affect bone metabolism are poorly defined. Here, we evaluated the effect of atorvastatin on osteoblastic production of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG), cytokines that are essential for osteoclast cell biology. While RANKL enhances osteoclast formation and activation, thereby, promoting bone loss, OPG acts as a soluble decoy receptor and antagonizes the effects of RANKL. In primary human osteoblasts (hOB), atorvastatin increased OPG mRNA levels and protein secretion by hOB by up to three fold in a dose-dependent manner with a maximum effect at 10(-6) M (P < 0.001). Time course experiments indicated a time-dependent stimulatory effect of atorvastatin on OPG mRNA levels after 24 h and on OPG protein secretion after 48-72 h (P < 0.001). Treatment of hOB with substrates of cholesterol biosynthesis that are downstream of the HMG-CoA reductase reaction (mevalonate, geranylgeranyl pyrophosphate) reversed atorvastatin-induced enhancement of OPG production. Of note, atorvastatin abrogated the inhibitory effect of glucocorticoids on OPG production. Treatment of hOB with atorvastatin enhanced the expression of osteoblastic differentiation markers, alkaline phosphatase and osteocalcin. In summary, our data suggest that atorvastatin enhances osteoblastic differentiation and production of OPG. This may contribute to the bone-sparing effects of statins.