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PubMed2006

AOX--a functional marker for efficient cell reprogramming under stress?

Functional markers for stress tolerance can be used in plant breeding to identify genotypes with high yield stabilities under various conditions. Thus, a good marker should show a strong correlation with favourable adaptive plant behaviour. The efficient reprogramming of target cells for yield determination is currently considered to be the most important step towards defining abiotic stress tolerance. In this Opinion article, we propose a role for the alternative oxidase (AOX) gene as a marker for genetic variation in cell reprogramming and yield stability. Evidence to support this idea comes from the metabolic role of alternative respiration under stress, the link between AOX activity and differential growth, and the single nucleotide polymorphism recently observed in AOX genes. We propose an innovative, interdisciplinary and global research strategy for future experimentation on AOX genes that could have an application in plant breeding.

Arabidopsis Proteins↗
PubMed2003

Directed evolution of a single-chain class II MHC product by yeast display.

Many autoimmune diseases have been linked to the class II region of the major histocompatibility complex (MHC). The linkage is thought to be a result of autoreactive T cells that recognize self-peptides bound to a product of this locus. For example, T cells from non-obese diabetic mice recognize specific 'diabetogenic' peptides bound to a class II MHC allele called I-A(g7). The I-A(g7) molecule is noted for being unstable and difficult to work with, especially in soluble form. In this work, yeast surface display combined with fluorescence-activated cell sorting was used as a means of directed evolution to engineer stabilized variants of a single-chain form of I-A(g7). A library containing mutations at two residues (positions 56 and 57 of the I-A(g7) beta-chain) that are important in the class II disease associations yielded stabilized mutants with preferences for a glutamic acid at residue 56 and a leucine at residue 57. Random mutation of I-A(g7) followed by selection with an anti-I-A(g7) antibody also yielded stabilized variants with mutations in other residues. The methods described here allow the discovery of novel MHC complexes that could facilitate structural studies and provide new opportunities in the development of diagnostics or antagonists of class II MHC-associated diseases.

Amino Acid Sequence↗
PubMed1985

Technetium-99m galactosyl-neoglycoalbumin: preparation and preclinical studies.

Technetium-99m galactosyl-neoglycoalbumin ([Tc] NGA), a labeled analog ligand to the hepatocyte-specific receptor, hepatic binding protein (HBP), was prepared and tested for labeling yield, stability, biodistribution, toxicity, and dosimetry. The ligand was synthesized by the covalent coupling of a carbohydrate bifunctional reagent, 2-imino-2-ethyloxymethyl-1-thiogalactose, to human serum albumin. Testing in mice and rabbits revealed the product to be nontoxic and apyrogenic. Technetium labeling yields in excess of 95%, by the electrolytic method, did not alter the molecular weight profile of the neoglycoalbumin. The NGA-bound activity remained stable for at least 4 hr. Biodistribution studies in rabbits demonstrated the liver as the single focus of tracer uptake. Dosimetry was based on kinetic studies in three baboons. Absorbed doses to liver, small intestine, urinary bladder wall, and uterus were 0.089, 0.28, 0.56, and 0.88 rad/mCi, respectively. Total body, lens of the eye, red marrow, ovaries, and testes were less than 0.06 rad/mCi. High liver specificity imparted by receptor binding combined with high labeling yield, stability, acceptable dosimetry, and safety provide [Tc]NGA with the attributes required for routine clinical assessment of hepatocyte function.

Albumins↗
PubMed2005

Improved stability and yield of Fv targeted superantigen by introducing both linker and disulfide bond into the targeting moiety.

Bacterial superantigens (SAg) are the most potent activators of human T lymphocytes and recombinant immunotoxin using bacterial SAg shows promising clinical values. To engineer superantigen for immunotherapy of hepatocellular carcinoma, we genetically fused the superantigen staphylococcus enterotoxin A (SEA(D(227)A)) to the single-chain disulfide-stabilized Fv (scdsFv) of anti-hepatoma monoclonal antibody HAb25 through a short peptide GGGSGGS. We expressed this recombinant protein in Escherichia coli and extract it from inclusion bodies. We found purified scdsFv-targeted SAg contains equivalent binding affinity with disulfide-stabilized Fv (dsFv) targeted SAg and single-chain Fvs (scFv) targeted SAg, but more stable and more suitable for large scale production. The MTS(3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliu m, inner salt) assay shows that the scdsFv-targeted SAg also shares the ability to activate a large number of T lymphocytes and has cytotoxic activity on human hepatoma cell line SMMC-7721. Therefore, this novel generation of recombinant immunotoxins using scdsFv has a high potential in hepato cancer treatment and the same strategy may also be applied to other cancer treatments.

Antibodies, Monoclonal↗
PubMed2006

Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output.

Luciferases, which have seen expansive employment as reporter genes in biological research, could also be used in applications where the protein itself is conjugated to ligands to create probes that are appropriate for use in small animal imaging. As the bioluminescence activity of commonly used luciferases is too labile in serum to permit this application, specific mutations of Renilla luciferase, selected using a consensus sequence driven strategy, were screened for their ability to confer stability of activity in serum as well as their light output. Using this information, a total of eight favorable mutations were combined to generate a mutant Renilla luciferase (RLuc8) that, compared with the parental enzyme, is 200-fold more resistant to inactivation in murine serum and exhibits a 4-fold improvement in light output. Results of the mutational analysis were also used to generate a double mutant optimized for use as a reporter gene. The double mutant had half the resistance to inactivation in serum of the native enzyme while yielding a 5-fold improvement in light output. These variants of Renilla luciferase, which exhibit significantly improved properties compared with the native enzyme, will allow enhanced sensitivity in existing luciferase-based assays as well as enable the development of novel probes labeled with the luciferase protein.

Amino Acid Sequence↗
PubMed1999

Green revolution: preparing for the 21st century.

In the 1960s there were large-scale concerns about the world's ability to feed itself. However, widespread adoption of "green revolution" technology led to major increases in food-grain production. Between 1966 and 1990, the population of the densely populated low-income countries grew by 80%, but food production more than doubled. The technological advance that led to the dramatic achievements in world food production over the last 30 years was the development of high-yielding varieties of wheat and rice. These varieties are responsive to fertilizer inputs, are lodging resistant, and their yield potential is 2-3 times that of varieties available prior to the green revolution. In addition, these varieties have multiple resistance to diseases and insects and thus have yield stability. The development of irrigation facilities, the availability of inorganic fertilizers, and benign government policies have all facilitated the adoption of green-revolution technology. In the 1990s, the rate of growth in food-grain production has been lower than the rate of growth in population. If this trend is not reversed, serious food shortages will occur in the next century. To meet the challenge of feeding 8 billion people by 2020, we have to prepare now and develop the technology for raising farm productivity. We have to develop cereal cultivars with higher yield potential and greater yield stability. We must also develop strategies for integrated nutrient management, integrated pest management, and efficient utilization of water and soil resources.

Biotechnology↗
PubMed2000

Molecular genetic manipulation of truncated Cry1C protein synthesis in Bacillus thuringiensis to improve stability and yield.

Cry1 protoxins of Bacillus thuringiensis are insecticidal 135-kDa proteins synthesized and assembled into parasporal crystals during sporulation. After ingestion, these crystals dissolve in the midgut and active toxins with molecular masses of about 65-kDa are released from the N-terminal half of the molecule by midgut proteases. Direct synthesis of the toxin-containing N-terminal half of Cry1 molecules using recombinant DNA techniques results in a low level of unstable truncated proteins that do not crystallize. In the present study, inclusions of truncated Cry1C (Cry1C-t) were obtained by combining genetic elements from other endotoxin genes and operons that enhance Cry protein synthesis and crystallization. Increased levels of Cry1C-t synthesis were achieved by using cyt1A promoters to drive expression of the 5' half of cry1C that included in the construct the 5' cry3A STAB-SD mRNA stabilizing sequence and the 3' stem-loop transcription terminator. RNA dot blot analysis showed that the STAB-SD and 3' transcriptional termination sequences were important for stabilization of truncated cry1C (cry1C-t) mRNA. A low level of cry1C-t mRNA was present when only the cyt1A promoters were used to express cry1C-t, but no accumulation of Cry1C-t was detected in Western blots. The orientation of the transcription terminator was important to enhancing Cry1C-t synthesis. Inclusion of the 20- and 29-kDa helper protein genes in cry1C-t constructs further enhanced synthesis. The Cry1C-t protein was toxic to Spodoptera exigua larvae, though the toxicity (50% lethal concentration [LC(50)] = 13.2 microg/ml) was lower than that of full-length Cry1C (LC(50) = 1.8 microg/ml). However, transformation of the HD1 isolate of B. thuringiensis subsp. kurstaki with the cry1C-t construct enhanced its toxicity to S. exigua as much as fourfold.

Animals↗
PubMed2026

Seed shattering habit in millets and the secrets of the abscission layer - a comprehensive review.

Though seed shattering continues to be a significant barrier affecting yield stability and harvesting efficiency in millets and other grasses, millets are increasingly acknowledged as climate-resilient, nutrient-rich 2007cereal crops with the potential to strengthen global nutritional and food security under the combined pressures of climate change, population growth, and limited natural resources. Since strong artificial selection favoured non-shattering phenotypes during domestication, seed shattering, an adaptive trait in wild species that promotes seed dispersal through the formation and activation of specialised abscission layers, became a distinguishing feature of cultivated cereals. With a focus on the morphological, physiological, hormonal, and genetic modulation of the abscission zone, this article summarizes the state of the art regarding seed shattering in millets. Abscission layer morphology, location, and lignification vary greatly among grasses, from well-defined lignified zones in rice and sorghum to non-lignified and anatomically subtle zones in Setaria and Panicum species. Cell wall-modifying enzymes like polygalacturonases, cellulases, expansins, and pectin methylesterases that mediate middle lamella degradation are modulated by coordinated hormonal signalling involving auxin, ethylene, and abscisic acid, which controls the timing and progression of cell separation at the physiological level. Domestication-related genes, including SH1, qSH1, SH4, and LES1, demonstrate convergent evolutionary mechanisms controlling abscission layer development in a variety of grass lineages at the molecular level. Understanding these regulatory networks has been greatly enhanced by recent developments in transcriptomics, functional genomics, and genome sequencing in both model species and underused millets. The role of millets as climate-smart cereals for sustainable future agriculture is reinforced by the integration of anatomical, physiological, and genetic insights, which offer a solid basis for targeted breeding and genome-editing strategies intended to improve seed retention, enhance yield stability, and increase harvest efficiency.

Abscission Layer↗
PubMed1994

Disruption of Maize Kernel Growth and Development by Heat Stress (Role of Cytokinin/Abscisic Acid Balance).

Temperature stress during kernel development affects maize (Zea mays L.) grain growth and yield stability. Maize kernels (hybrid A619 x W64A) were cultured in vitro at 3 d after pollination and either maintained at 25[deg]C or transferred to 35[deg]C for 4 or 8 d, then returned to 25[deg]C until physiological maturity. Kernel fresh and dry matter accumulation was severely disrupted by the long-term heat stress (8 d at 35[deg]C) and did not recover when transferred back to 25[deg]C, resulting in abortion of 97% of the kernels. Kernels exposed to 35[deg]C for 4 d (short-term heat stress) exhibited a recovery in kernel growth and water content at about 18 d after pollination and kernel abortion was reduced to about 23%. During the cell division phase, abscisic acid (ABA) levels showed a steady decline in the control but maintained a moderate level in the heat-stressed kernels. However, later in development heat-stressed kernels had significantly higher levels of ABA than the control. Cytokinin analysis confirmed a peak in zeatin riboside and zeatin levels in control kernels at 10 to 12d after pollination. In contrast, kernels subjected to 4 d of heat stress had no detectable levels of zeatin and the zeatin riboside peak was reduced by 70% and delayed until 18 d after pollination. The long-term heat-stressed kernels showed low to nondetectable levels of either zeatin riboside or zeatin. Regression analysis of ABA level against cytokinin level during the endosperm cell division phase revealed a highly significant negative correlation in nonstressed kernels but no correlation in kernels exposed to short-term or long-term heat stress. Application of benzyladenine to heat-stressed, growth-chamber-grown plants increased thermotolerance in part by reducing kernel abortion at the tip and middle positions on the ear. These results confirm that shift in hormone balance of kernels is one mechanism by which heat stress disrupts maize kernel development. The maintenance of high levels of cytokinins in the kernels during heat stress appears to be important in increasing thermotolerance and providing yield stability of maize.

Journal Article↗
PubMed1998

Stabilization of charge separation and photochemical misses in photosystem II.

Illumination of photosystem II by a saturating short flash results in a stabilized charge separation in only about 90% of the reaction centers. During a series of flashes, the 10% fraction of "photochemical misses" is randomly redistributed among the centers. This phenomenon is investigated in DCMU-inhibited material, eliminating the contribution to misses due to electron transfer equilibrium on the quinone acceptors. Under such conditions, the miss coefficient is about 5% and is enhanced to about 40% in the presence of hydroxylamine at low pH. When a second flash is fired, its efficiency increases as a function of the time delay after the first flash (turnover experiments). This process involves three distinct time domains: <10 micros, 100 micros, and 10 ms. From a study of the 515-nm field-indicating change, it appears that the increased inefficiency caused by hydroxylamine is not due to a lesser amount of initial charge separation but to a recombination process concomitant with the 100 micros phase of the turnover. The slow turnover phase (10 ms) is not associated with a recombination or any other electron transfer event but reflects a modification of open centers during which their probability to achieve charge stabilization rather than recombination is progressively increased. These results are interpreted in terms of an equilibrium between two conformational states of the centers endowed with different stabilization yield ("good" and "bad " stabilizers). The 100 micros turnover phase is due to the reopening of the bad stabilizers by recombination, and the 10 ms phase accompanies the redistribution of these centers among the two conformational states.

Chlorella↗
PubMed2006

Novel replication-incompetent adenoviral B-group vectors: high vector stability and yield in PER.C6 cells.

Adenoviral vectors based on adenovirus type 35 (rAd35) have the advantage of low natural vector immunity and induce strong, insert-specific T- and B-cell responses, making them prime-candidate vaccine carriers. However, severe vector-genome instability of E1-deleted rAd35 vectors was observed, hampering universal use. The instability of E1-deleted rAd35 vector proved to be caused by low pIX expression induced by removal of the pIX promoter, which was located in the E1B region of B-group viruses. Reinsertion of a minimal pIX promoter resulted in stable vectors able to harbour large DNA inserts (> 5 kb). In addition, it is shown that replacement of the E4-Orf6 region of Ad35 by the E4-Orf6 region of Ad5 resulted in successful propagation of an E1-deleted rAd35 vector on existing E1-complementing cell lines, such as PER.C6 cells. The ability to produce these carriers on PER.C6 contributes significantly to the scale of manufacturing of rAd35-based vaccines. Next, a stable rAd35 vaccine was generated carrying Mycobacterium tuberculosis antigens Ag85A, Ag85B and TB10.4. The antigens were fused directly, resulting in expression of a single polyprotein. This vaccine induced dose-dependent CD4+ and CD8+ T-cell responses against multiple antigens in mice. It is concluded that the described improvements to the rAd35 vector contribute significantly to the further development of rAd35 carriers for mass-vaccination programmes for diseases such as tuberculosis, AIDS and malaria.

Adenoviridae↗
PubMed2006

Topological rearrangement yields structural stabilization and interhelical distance constraints in the Kin.46 self-phosphorylating ribozyme.

The Kin.46 ribozyme catalyzes transfer of the gamma (thio)phosphoryl group of ATP (or ATPgammaS) to the ribozyme's 5' hydroxyl. Single-turnover catalytic activities of topologically rearranged versions of Kin.46 were studied to gain insight into its overall tertiary architecture. The distal ends of stems P3 and P4 were tethered through a single-stranded connection domain that altered the interhelical connectivity. The shortest linkers interfered with catalysis, while seven or more nucleotides (nt) in the linker allowed near-normal catalytic rates, suggesting that a distance of roughly 25-35 A optimally separates the termini of these helices. Activity was maximal when the tether contained 15 nt, at which point the k(cat) (0.016 min(-1)) and Km (1.2 mM) values were identical to those of a nontethered control. The presence of the tether alters Mg(2+) dependence, in that Mg2+ binding appears to be more cooperative in the tethered ribozyme (Hill coefficient 1.4-1.8 versus 0.8 for the nontethered ribozyme). Binding affinity for the ATPgammaS substrate increases at elevated concentrations of Mg2+, particularly for the tethered ribozyme. The tethered ribozyme displays significantly enhanced thermal stability, with a maximum initial velocity (0.126 min(-1)) at 60 degrees C, whereas the nontethered ribozyme has a lower maximum initial velocity (0.051 min(-1)) at 50 degrees C. The tether also significantly reduces the apparent entropy of activation. Both of these effects can be understood in terms of stabilization of the ribozyme in a conformation that is on-path with respect to catalysis, and in terms of facilitating formation of the allosteric activation helix P4.

Adenosine Triphosphate↗
PubMed2003

Acute and maintenance treatment with mood stabilizers.

Treatment of bipolar disorder is complicated by the multiple phases of the illness, dimensional symptomatology that varies considerably across individuals, and a limited spectrum of activity for all mood stabilizers. Randomized, blinded, placebo-controlled studies provide clear guidelines to the overall efficacy of treatments for mania. However, for secondary questions, such as the treatment to employ when lithium or valproate is inadequate as monotherapy, evidence is incomplete, and usually derived from both smaller and less well-designed studies. For mania, the spectrum of efficacy of valproate is broader than for other mood stabilizers. However, many patients obtain inadequate benefit from monotherapy regimens of all mood stabilizers. Recent studies indicate that for patients who develop mania while taking a mood stabilizer, combinations of an antipsychotic and a mood stabilizer yield greater improvement than does continuation of the mood stabilizer alone. Maintenance-treatment studies support the efficacy of lithium, valproate and lamotrigine, although with a different spectrum of benefits and limitations for each. Valproate and lithium provide greater benefits for prevention of manic relapses and control of manic symptomatology than for depression. Several studies indicate actual worsening in depressive aspects of bipolar disorder with lithium treatment. Lamotrigine appears effective in delaying relapse into a new episode, with most benefits limited to delaying time to depression. Lamotrigine has not shown anti-manic activity in placebo-controlled studies. In contrast to traditional antidepressant medications, lamotrigine has not been associated with induction of mania, or of rapid-cycling illness symptomatology. Recent studies reported that carbamazepine was inferior to valproate in acute mania, and inferior to lithium in maintenance treatment. Other putative mood stabilizers have to date yielded negative or inconclusive results in studies in mania. Systematic studies are needed to clarify treatment guidelines for youth with bipolar disorder, and for other special populations, e.g. pregnant women and the elderly.

Antimanic Agents↗
PubMed2005

What it will take to feed 5.0 billion rice consumers in 2030.

Major advances have occurred in rice production due to adoption of green revolution technology. Between 1966 and 2000, the population of densely populated low income countries grew by 90% but rice production increased by 130% from 257 million tons in 1966 to 600 million tons in 2000. However, the population of rice consuming countries continues to grow and it is estimated that we will have to produce 40 more rice in 2030. This increased demand will have to be met from less land, with less water, less labor and fewer chemicals. To meet the challenge of producing more rice from suitable lands we need rice varieties with higher yield potential and greater yield stability. Various strategies for increasing the rice yield potential being employed include: (1) conventional hybridization and selection procedures, (2) ideotype breeding, (3) hybrid breeding, (4) wide hybridization and (5) genetic engineering. Various conventional and biotechnology approach are being employed to develop durable resistance to diseases and insect and for tolerance to abiotic stresses. The availability of the rice genome sequence will now permit identification of the function of each of 60,000 rice genes through functional genomics. Once the function of a gene is identified, it will be possible to develop new rice varieties by introduction of the gene through traditional breeding in combination with marker aided selection or direct engineering of genes into rice varieties.

Agriculture↗
PubMed1980

Phospholipid-dependent assembly of mitochondrial ATPase complex.

1. Phosphatidylcholines of different acyl-chain composition and a preparation of ATPase complex depleted of phospholipids have been employed in order to evaluate the contribution of lipid bilayer to the assembly of this multi-subunit component of mitochondrial membrane. 2. At the minimal requirement for bilayer assembly (dinonanoylphosphatidylcholine, mixtures of lysophosphatidylcholine and phosphatidylcholine), fragments with oligomycin-insensitive ATPase activity are reconstituted. Conformational changes with dislocation of ATPase complex subunits may explain these results. 3. At increased strength of acyl-chain interaction (dilauroylphosphatidylcholine and higher homologues), the damage to the ATPase complex is prevented but this is not sufficient to achieve functional restoration. Bilayers with a tendency to coalesce and fuse aggregate in large amounts with the complex and yield low ATPase reactivation. Bilayers of high stability yield complexes with physiological content of phospholipids and efficient ATPase activity. Transition between these two possibilities is found at sixteen carbon acyl-chains. Only at this chain length does the cholate dialysis procedure of reconstitution become feasible. 4. It is concluded that a minimum of 16 carbon atoms in each chain are required to organize a bilayer structurable to maintain the ATPase complex conformation and to sustain the transmembrane position of the whole assembly.

Adenosine Triphosphatases↗