High-lysine corn produced by the combination of enhanced lysine biosynthesis and reduced zein accumulation

Corn is one of the major crops in the world, but its low lysine content is often problematic for animal consumption. While exogenous lysine supplementation is still the most common solution for today's feed corn, high-lysine corn has been developed through genetic research and biotechnology. Reducing the lysine-poor seed storage proteins, zeins, or expressing a deregulated lysine biosynthetic enzyme, CordapA, has shown increased total lysine or free lysine content in the grains of modified corn plants, respectively. Here, by combining these two approaches through genetic crosses, the total lysine content has more than doubled in F1 progeny. We also observe a synergy between the transgenic zein reduction and the enhanced lysine biosynthesis by CordapA expression. The zein reduction plants are found to accumulate higher levels of aspartate, asparagine and glutamate, and therefore, provide excess precursors for the enhanced lysine biosynthesis.     

Activation of pyrophosphate:fructose-6-phosphate 1-phosphotransferase by fructose 2,6-bisphosphate stimulates conversion of hexose phosphates to triose phosphates but does not influence accumulation of carbohydrates in phosphate-deficient tobacco cells

The aim of this work was to investigate the contribution of fructose 2,6-bisphosphate to the regulation of carbohydrate metabolism under phosphate stress. The study exploited heterotrophic tobacco callus lines expressing a modified mammalian 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase that increased the fructose 2,6-bisphosphate content of the tissue. The phosphate status of two transgenic and one untransformed cell line was perturbed by incubation with 2-deoxyglucose, a phosphate-sequestering agent, and by growth of callus on phosphate-depleted media. 31P-NMR spectroscopy confirmed that both treatments decreased cellular levels of inorganic phosphate and phosphorylated metabolites. Despite large decreases in the amounts of phosphate esters, UDPglucose and adenylates in response to phosphate deficiency, the fructose 2,6-bisphosphate content of each line was unaffected by 2-deoxyglucose and increased during growth on phosphate-limited media. Short-term treatment of callus with 2-deoxyglucose had only minor effects on the carbohydrate status of each line, whereas long-term phosphate deficiency caused an increase in starch and a decrease in soluble sugar content in both transgenic and control lines. There were no consistent differences between the three callus lines in metabolism of [U-14C]glucose in response to incubation with 2-deoxyglucose. In contrast, there was a decrease in partitioning of label into glycolytic products (particularly organic acids) in untransformed callus during growth on phosphate-depleted medium. This decrease was greatly attenuated in the transgenic lines with increased fructose 2,6-bisphosphate content. This suggests that the conversion of hexose phosphates to triose phosphates is constrained under phosphate-deficient conditions, and that this restriction can be relieved by activation of pyrophosphate:fructose-6-phosphate 1-phosphotransferase. However, since the transgenic and control lines did not differ in the extent to which the carbohydrate content changed in response to growth on phosphate-depleted media, it is concluded that an increase in flux through pyrophosphate:fructose-6-phosphate 1-phosphotransferase is not a major component of the metabolic response of heterotrophic tobacco cells to phosphate deficiency.     

Monitoring flux through the oxidative pentose phosphate pathway using [1-14C]gluconate

The aim of this work was to examine the metabolism of exogenous gluconate by a 4-day-old cell suspension culture of Arabidopsis thaliana (L.) Heynh. Release of (14)CO(2) from [1-(14)C]gluconate was dependent on the concentration in the medium and could be resolved into a substrate-saturable component (apparent K(m) of approximately 0.4 mM) and an unsaturable component. At an external concentration of 0.3 mM, the rate of decarboxylation of applied gluconate was 0.2% of the rate of oxygen consumption by the cells. There was no effect of 0.3 mM gluconate on the rate of oxygen consumption, or on the rate of (14)CO(2) release from either [1-(14)C]glucose or [6-(14)C]glucose by the culture. The following observations argue that gluconate taken up by the cells is metabolised by direct phosphorylation to 6-phosphogluconate and subsequent decarboxylation through 6-phosphogluconate dehydrogenase. First, more than 95% of the label released from [1-(14)C]gluconate during metabolism by the cell culture was recovered as (14)CO(2). Secondly, inhibition of the oxidative pentose phosphate pathway (OPPP) by treatment with 6-aminonicotinamide preferentially inhibited release of (14)CO(2) from [1-(14)C]gluconate relative to that from [1-(14)C]glucose. Thirdly, perturbation of glucose metabolism by glucosamine did not affect (14)CO(2) from [1-(14)C]gluconate. Fourth, stimulation of the OPPP by phenazine methosulphate stimulated release of (14)CO(2) from [1-(14)C]gluconate to a far greater extent than that from [1-(14)C]glucose. It is proposed that measurement of (14)CO(2) from [1-(14)C]gluconate provides a simple and sensitive technique for monitoring flux through the OPPP pathway in plants.     

Brain repair by endogenous progenitors

Stem cells within the adult brain can be stimulated by injury and growth factor treatment to replace damaged neurons, even neurons that are not normally generated in adults. Coupled with recent insights into the mechanism by which Nogo inhibits axonal regeneration, this discovery may inspire new treatments for central nervous system injuries and neurodegenerative diseases.     

Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: challenging the gene dosage effect hypothesis (Part II)

Summary.  Down syndrome (DS) is the most common genetic cause of mental retardation. To explain the impact of extra chromosome 21 in the pathology of DS, gene dosage effect hypothesis has been proposed, but several investigators including our group have challenged this hypothesis. Although analysis of the sequence of chromosome 21 has been essentially completed, the molecular and biochemical mechanisms underlying the pathology are still unknown. We therefore investigated expression levels of six proteins encoded on chromosome 21 (HACS1, DYRK1A, αA-crystallin, FTCD, GARS-AIRS-GART, and CBS) in fetal cerebral cortex from DS and controls at 18–19 weeks of gestational age using Western blot analysis. Protein expression of HACS1 was significantly and remarkably decreased in DS, and the expression levels of five proteins were comparable between DS and controls suggesting that the gene dosage effect hypothesis is not sufficient to fully explain the DS phenotype. We are continuing to quantify proteins whose genes are encoded on chromosome 21 in order to provide a better understanding of the pathobiochemistry of DS at the protein level. Received July 1, 2002 Accepted July 19, 2002 Published online November 14, 2002 Acknowledgement This work was supported, in part (Dr. D. Patterson), by the National Institute of Child Health and Human Development (NICHD; HD17449). Authors' address: Prof. Dr. Gert Lubec, CChem, FRSC (UK), Department of Pediatrics, University of Vienna, Waehringer Guertel 18, A-1090 Vienna, Austria, Fax: +43-1-40400-3194, E-mail: gert.lubec@akh-wien.ac.at Abbreviations: DS, Down syndrome; HACS1, hematopoietic adapter containing Src homology 3 domain and sterile α motifs; DYRK1A, dual specificity tyrosine phosphorylated and regulated kinase; αA-crystallin, alpha crystallin subunit A; FTCD, formi-minotransferase cyclodeaminase; GARS-AIRS-GART, glycinamide ribonucleotide synthetase-aminoimidazole ribonucleotide synthetase-glycinamide ribonucleotide formyltransferase; CBS, cystathionine β-synthase; NSE, neuron specific enolase; GFAP, glial fibrillary acidic protein     

Diversity of type II restriction endonucleases that require two DNA recognition sites

Orthodox Type IIP restriction endonucleases, which are commonly used in molecular biological work, recognize a single palindromic DNA recognition sequence and cleave within or near this sequence. Several new studies have reported on structural and biochemical peculiarities of restriction endonucleases that differ from the orthodox in that they require two copies of a particular DNA recognition sequence to cleave the DNA. These two sites requiring restriction endonucleases belong to different subtypes of Type II restriction endonucleases, namely Types IIE, IIF and IIS. We compare enzymes of these three types with regard to their DNA recognition and cleavage properties. The simultaneous recognition of two identical DNA sites by these restriction endonucleases ensures that single unmethylated recognition sites do not lead to chromosomal DNA cleavage, and might reflect evolutionary connections to other DNA processing proteins that specifically function with two sites.     

Distinct mechanisms mediate the initial and sustained phases of cell migration in epidermal growth factor receptor-overexpressing cells

Elevated levels of epidermal growth factor receptor (EGFR) are predictive of increased invasion and metastasis in many human cancers. In the present study, we have shown that two distinct pathways regulate cell migration in EGFR-overexpressing invasive cells such as MDA 468 breast cancer cells: mitogen-activated protein kinase (MAPK or ERK 1 and 2) pathways play a major role in early stages to cell migration; and protein kinase C delta isoforms (PKC-delta) play a significant role in later stages of sustained cell migration. Inhibition of MAPK activity with MAP kinase kinase (MEK) inhibitor PD98059 blocks early stages of cell migration (up to 4 h); however, cells revert back to enhanced cell migration after 4 h. While inhibition of PKC-delta activity with rottlerin or dominant-negative PKC-delta expression blocks sustained cell migration after 4 h and up to 12 h, the combination of MAPK and PKC inhibitors completely blocked transforming growth factor alpha (TGF-alpha)-induced cell migration in EGFR-overexpressing breast cancer cells. However, inhibition of MAPK activity completely blocked cell migration in low EGFR-expressing non-invasive breast cancer cells such as MCF-7 cells. Forced overexpression of EGFR in MCF-7 cells (EGFR/MCF-7 cells) resulted in cell migration patterns seen in MDA 468 cells, that is, MAPK pathways play a major role in early stages to cell migration, and PKC-delta plays a major role in later stages of sustained cell migration. The above data demonstrate that EGFR-overexpressing invasive cells have the ability to compensate the loss of MAPK-mediated signaling through activation of PKC-delta signaling for cell migration, which plays a major role in invasion and metastasis. In addition, data suggest that inhibition of MAPK and PKC-delta signaling pathways should abrogate cell migration and invasion in EGFR-overexpressing human breast cancer cells.     

Overexpression of human heme oxygenase-1 attenuates endothelial cell sloughing in experimental diabetes

Heme oxygenase (HO)-1 represents a key defense mechanism against oxidative injury. Hyperglycemia produces oxidative stress and various perturbations of cell physiology. The effect of streptozotocin (STZ)-induced diabetes on aortic HO activity, heme content, the number of circulating endothelial cells, and urinary 8-epi-isoprostane PGF2alpha (8-Epi) levels in control rats and rats overexpressing or underexpressing HO-1 was measured. HO activity was decreased in hyperglycemic rats. Hyperglycemia increased urinary 8-Epi, and this increase was augmented in rats underexpressing HO-1 and diminished in rats overexpressing HO-1. The number of detached endothelial cells and O2- formation increased in diabetic rats and in hyperglycemic animals underexpressing HO-1 and decreased in diabetic animals overexpressing HO-1 compared with controls. These data demonstrate that HO-1 gene transfer in hyperglycemic rats brings about a reduction in O2- production and a decrease in endothelial cell sloughing. Upregulation of HO-1 decreases oxidant production and endothelial cell damage and shedding and may attenuate vascular complications in diabetes.     

Correlation between thyroid hormone status and hepatic hyperplasia and hypertrophy caused by the peroxisome proliferator-activated receptor alpha agonist Wy-14,643

BACKGROUND: The metabolic inhibitor rotenone inhibits hepatocellular proliferation and the incidence of liver cancer resulting from exposure to the PPARalpha agonist Wy-14,643, via unknown mechanisms. Since the absence of thyroid hormones diminishes hepatomegaly, an early biomarker for the hepatocarcinogenicity induced by PPARalpha agonists, this study was undertaken to investigate whether rotenone might interference with the ability of Wy-14,643 to alter the animal thyroid status. METHODS: Male B6C3F1 mice were given Wy-14,643 (100 ppm), rotenone (600 ppm) or a mixture of both, in the feed for 7 days. Bromodeoxyuridine (BrDU), marker of cell replication, was delivered through subcutaneously implanted osmotic mini-pumps. At the end of the experiment, sera were collected and corticosterone and thyroid hormone levels were measured by solid-phase radioimmunoassay kits. In addition, liver tissue samples were stained immunohistochemically for BrDU to determine percentages of labeled cells. Further, cell surface area was determined from images generated by a Zeiss Axioplan microscope equipped with a plan Neofluar x40 0.75 na objective. Tracings of individual hepatocyte perimeters were then analyzed and cell-surface areas were calculated using MicroMeasure FL-4000. RESULTS: Wy-14,643 caused a significant increase in liver weights, hepatocyte BrDU labeling index (LI), and hepatocyte surface area. In animals which received both Wy-14,643 and rotenone simultaneously, all of these effects were significantly less pronounced compared with mice that received Wy-14,643 alone. Rotenone alone decreased liver weights, LI and surface area. The Free Thyroid Index (FTI), which provides an accurate reflection of the animal's thyroid status, was 5.0 +/- 0.3 in control mice. In animals exposed to rotenone, these values decreased to 2.0 +/- 0.9, but in animals which received Wy-14,643, levels increased significantly to 7.7 +/- 0.9. FTI values decreased to 3.4 +/- 0.8 in mice receiving both rotenone and Wy-14,643. CONCLUSION: A strong correlation was observed between the animal thyroid status and both, hepatocyte proliferation (r2 = 0.62), and hepatocyte surface area (r2 = 0.83). These results support the hypothesis that the thyroid status of the animal plays a role in PPARalpha-induced hepatocellular proliferation and liver cell enlargement. Both these events are known to contribute to the expression of liver cancer in response to the activation of PPARalpha.     

Fructose 2,6-bisphosphate activates pyrophosphate: fructose-6-phosphate 1-phosphotransferase and increases triose phosphate to hexose phosphate cycling in heterotrophic cells

The aim of this work was to establish the influence of fructose 2,6-bisphosphate (Fru-2,6-P₂) on non-photosynthetic carbohydrate metabolism in plants. Heterotrophic callus lines exhibiting elevated levels of Fru-2,6-P₂ were generated from transgenic tobacco (Nicotiana tabacum L.) plants expressing a modified rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Lines containing increased amounts of Fru-2,6-P₂ had lower levels of hexose phosphates and higher levels of 3-phosphoglycerate than the untransformed control cultures. There was also a greater redistribution of label into the C6 position of sucrose and fructose, following incubation with [1-¹³C]glucose, in the lines possessing the highest amounts of Fru-2,6-P₂, indicating a greater re-synthesis of hexose phosphates from triose phosphates in these lines. Despite these changes, there were no marked differences between lines in the metabolism of ¹⁴C-substrates, the rate of oxygen uptake, carbohydrate accumulation or nucleotide pool sizes. These data provide direct evidence that physiologically relevant changes in the level of Fru-2,6-P₂ can affect pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) activity in vivo, and are consistent with PFP operating in a net glycolytic direction in the heterotrophic culture. However, the results also show that activating PFP has little direct effect on heterotrophic carbohydrate metabolism beyond increasing the rate of cycling between hexose phosphates and triose phosphates. Received: 29 March 2000 / Accepted: 13 June 2000