Chlorophyll reduction in the seed of Brassica napus with a glutamate 1-semialdehyde aminotransferase antisense gene*

Chlorophyll reduction in the seed of Brassica can be achieved by downregulating its synthesis. To reduce chlorophyll synthesis, we have used a cDNA clone of Brassica napus encoding glutamate 1-semialdehyde aminotransferase (GSA-AT) to make an antisense construct for gene manipulation. Antisense glutamate 1-semialdehyde aminotransferase gene (Gsa) expression, directed by a Brassica napin promoter, was targeted specifically to the embryo of the developing seed. Transformants expressing antisense Gsa showed varying degrees of inhibition resulting in a range of chlorophyll reduction in the seeds. Seed growth and development were not affected by reduction of chlorophyll. Seeds from selfed transgenic plants germinated with high efficiency and growth of seedlings was vigorous. Seedlings from T₂ transgenic lines segregated into three distinctive phenotypes: dark green, light green and yellow, indicating the dominant inheritance of Gsa antisense gene. These transgenic lines have provided useful materials for the development of a low chlorophyll seed variety of B. napus.     

Activity profiles of enzymes involved in glutamine and glutamate metabolism in the apple during autumnal senescence

Seasonal changes in glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), and glutamate dehydrogenase (EC 1.4.1.3) were measured in both senescing leaf and bark tissues of ‘Golden Delicious’ apple trees (Malus domestica Borkh.). From the measured enzyme activities we attempted to estimate the in vivo catalytic potentials of the enzymes with special reference to nitrogen mobilization and conservation of senescing apple trees. The cumulative glutamine synthetase activity of leaf tissue was about three times higher than that of bark. The estimated catalytic potential of leaf glutamine synthetase was 800-fold higher than the actual protein nitrogen loss of senescing leaves. The cumulative glutamate synthase activity of bark was about six times higher than that of leaf. The estimated catalytic potential of bark glutamate synthase was 160-times higher than the actual protein nitrogen gain in that tissue. The cumulative glutamate dehydrogenase activities in leaf and bark tissue were approximately the same. However, the catalytic potential of leaf glutamate dehydrogenase was twice that of leaf glutamate synthase. It is thus concluded that the physiological role of glutamine synthetase in senescing leaf tissue is to furnish the amide(s) prior to mobilization of nitrogen to storage tissue. The higher activity of glutamate synthase in bark tissue could provide a mechanism to transform the imported amide nitrogen to amino nitrogen of glutamate for storage protein synthesis. The possible regulatory factors upon the activity of these enzymes in the tissues of senescing apple trees are discussed.     

Effect of fructose-1,6-bisphosphate on glutamate uptake and glutamine synthetase activity in hypotix astrocyte cultures

Astrocytes are important in regulating the microencironment of neurons both by catabolic and synthetic pathways. The glutamine synthetase (GS) activity observed in astrocytes affects neurons by removing toxic substances, NH₃ and glutamate; and by providing an important neuronal substrate, glutamine. This glutamate cycle might play a critical role during periods of hypoxia and ischemia, when an increase in extracellular excitatory amino acids is observed. It was previously shown in our laboratory that fructose-1,6-bisphosphate (FBP) protected cortical astrocyte cultures from hypoxic insult and reduced ATP loss following a prolonged (18–30 hrs) hypoxia. In the present study we established the effects of FBP on the level of glutamate uptake and GS activity under normoxic and hypoxic conditions. Under normoxic conditions, [U-¹⁴C]glutamate uptake and glutamine production were independent of FBP treatment; whereas under hypoxic conditions, the initial increase in glutamate uptake and an overall increase in glutamine production in astrocytes were FBP-dependent. Glutamine synthetase activity was dependent on FBP added during the 22 hours of either normoxic- or hypoxic-treatment, hence significant increases in activity were observed due to FBP regardless of the oxygen/ATP levels in situ. These studies suggest that activation of GS by FBP may provide astrocytic protection against hypoxic injury.     

Mature ferredoxin-NADP reductase with a glutaminyl residue at N-terminus from spinach chloroplasts

When 35%-acetone extract of spinach chloroplasts was separated by SDS-PAGE, ferredoxin-NADP reductase (FNR) appeared as a single band at a molecular mass of 35 kDa. After the polypeptides on the SDS-PAGE plate were electroblotted onto PVDF membrane, the FNR band was cut out and analyzed for N-terminal structure in a gas-phase protein sequencer. Two different FNR peptides were identified: one with glutamine at its N-terminus (Gln-FNR) and the other with -pyroglutamic acid (tFNR) fraction was extracted from chloroplasts with their loosely bound FNR (lFNR) fraction removed in advance. The tFNR fraction contained Gln-FNR only. The Gln-FNR could be highly purified by affinity chromatography using a ferredoxin column. The purified Gln-FNR was digested with arginyl endopeptidase for peptide mapping and partial sequence analysis. Primary structure of Gln-FNR differed from that of lFNR loosely bound FNR - tFNR tightly bound FNR - -pyroglutamic acid at N-terminus     

A second Warburg-like effect in cancer metabolism: The metabolic shift of glutamine-derived nitrogen

Carbon and nitrogen are essential elements for life. Glucose as a carbon source and glutamine as a nitrogen source are important nutrients for cell proliferation. About 100 years ago, it was discovered that cancer cells that have acquired unlimited proliferative capacity and undergone malignant evolution in their host manifest a cancer-specific remodeling of glucose metabolism (the Warburg effect). Only recently, however, was it shown that the metabolism of glutamine-derived nitrogen is substantially shifted from glutaminolysis to nucleotide biosynthesis during malignant progression of cancer—which might be referred to as a “second” Warburg effect. In this review, address the mechanism and relevance of this metabolic shift of glutamine-derived nitrogen in human cancer. We also examine the clinical potential of anticancer therapies that modulate the metabolic pathways of glutamine-derived nitrogen. This shift may be as important as the shift in carbon metabolism, which has long been known as the Warburg effect.     

Identification of the synaptic vesicle glycoprotein 2 receptor binding site in botulinum neurotoxin A

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release by hydrolysing SNARE proteins. The most important serotype BoNT/A employs the synaptic vesicle glycoprotein 2 (SV2) isoforms A-C as neuronal receptors. Here, we identified their binding site by blocking SV2 interaction using monoclonal antibodies with characterised epitopes within the cell binding domain (H_C). The site is located on the backside of the conserved ganglioside binding pocket at the interface of the H_CC and H_CN subdomains. The dimension of the binding pocket was characterised in detail by site directed mutagenesis allowing the development of potent inhibitors as well as modifying receptor binding properties.     

Binding and Functional Folding (BFF): A Physiological Framework for Studying Biomolecular Interactions and Allostery

EF-hand Ca²⁺-binding proteins (CBPs), such as S100 proteins (S100s) and calmodulin (CaM), are signaling proteins that undergo conformational changes upon increasing intracellular Ca²⁺. Upon binding Ca²⁺, S100 proteins and CaM interact with protein targets and induce important biological responses. The Ca²⁺-binding affinity of CaM and most S100s in the absence of target is weak (^CaK_D > 1 μM). However, upon effector protein binding, the Ca²⁺ affinity of these proteins increases via heterotropic allostery (^CaK_D < 1 μM). Because of the high number and micromolar concentrations of EF-hand CBPs in a cell, at any given time, allostery is required physiologically, allowing for (i) proper Ca²⁺ homeostasis and (ii) strict maintenance of Ca²⁺-signaling within a narrow dynamic range of free Ca²⁺ ion concentrations, [Ca²⁺]^free. In this review, mechanisms of allostery are coalesced into an empirical “binding and functional folding (BFF)” physiological framework. At the molecular level, folding (F), binding and folding (BF), and BFF events include all atoms in the biomolecular complex under study. The BFF framework is introduced with two straightforward BFF types for proteins (type 1, concerted; type 2, stepwise) and considers how homologous and nonhomologous amino acid residues of CBPs and their effector protein(s) evolved to provide allosteric tightening of Ca²⁺ and simultaneously determine how specific and relatively promiscuous CBP-target complexes form as both are needed for proper cellular function.     

Critical adjustment of cysteine and glutamine concentrations for improved clonal growth of WI-38 cells

Summary Clonal growth of WI-38 cells with a plating efficiency of 45% has been achieved in a synthetic nutrient mixture (MCDB 102) supplemented with either whole or dialyzed fetal bovine serum. For optimum growth, the concentration of cysteine in the medium must be adjusted precisely. Deviation by a factor of three in either direction from the optimum concentration (9.0×10⁻⁵M) eliminates essentially all clonal growth. A high concentration of glutamine (2.5×10⁻³M) is also needed for, optimum clonal growth. Presented in preliminary form at the 26th Annual Meeting of the Tissue Culture Association, June 4, 1975. This work was supported by Grant No. HD-08181 from the National Institute of Child Health and Human Developement, Grant No. AG-00310 from the National Institute on Aging, and by Contract No. 223-74-1156 from the Bureau of Biologics, Food and Drug Administration.