Molecular dissection of interspecific variation between Gossypium hirsutum and Gossypium barbadense (cotton) by a backcross-self approach: I. Fiber elongation

The current study is the first installment of an effort to explore the secondary gene pool for the enhancement of Upland cotton (Gossypium hirsutum L.) germplasm. We developed advanced-generation backcross populations by first crossing G. hirsutum cv. Tamcot 2111 and G. barbadense cv. Pima S6, then independently backcrossing F₁ plants to the G. hirsutum parent for three cycles. Genome-wide mapping revealed introgressed alleles at an average of 7.3% of loci in each BC₃F₁ plant, collectively representing G. barbadense introgression over about 70% of the genome. Twenty-four BC₃F₁ plants were selfed to generate 24 BC₃F₂ families of 22–172 plants per family (totaling 2,976 plants), which were field-tested for fiber elongation and genetically mapped. One-way analysis of variance detected 22 non-overlapping quantitative trail loci (QTLs) distributed over 15 different chromosomes. The percentage of variance explained by individual loci ranged from 8% to 28%. Although the G. barbadense parent has lower fiber elongation than the G. hirsutum parent, the G. barbadense allele contributed to increased fiber elongation at 64% of the QTLs. Two-way analysis of variance detected significant (P<0.001) among-family genotype effects and genotype×family interactions in two and eight regions, respectively, suggesting that the phenotypic effects of some introgressed chromosomal segments are dependent upon the presence/absence of other chromosomal segments.Electronic Supplementary Material Supplementary material is available for this article at     

Development of photocatalytic biosensor for the evaluation of biochemical oxygen demand

The photocatalytic biosensor of flow system using semiconductor TiO2 was developed to evaluate biochemical oxygen demand (BOD) levels in river water. Photocatalysis of sample was carried out in a photoreactor with TiO2 and a 6W black-light blue fluorescent tube as light source. Sample from a photoreactor outlet was measured by an oxygen electrode with a biofilm. The sensor response of photocatalytic biosensor was between 5 and 10 min depending on concentration of biochemical in the samples. At BOD of 1 mgl-1, the sensor response increased 1.33-fold in comparison with that without photocatalysis. The degradation of tannic acid and humic acid with photocatalysis were 51.8 and 38.4%, respectively. Gum arabic and linear alkylbenzene sulfonate (LAS) were degraded a little, but gave the responses of more than double to the sensor. Free radicals yielded by photocatalysis in a photoreactor did not affect the sensor response because their lifetime is extremely short. Fairly good correlation (r=0.983) between the sensor method and the conventional method was obtained for test samples. This biosensor using photocatalytic pretreatment improved the sensitivity.     

The critical role of calpain versus caspase activation in excitotoxic injury induced by nitric oxide

The pathogenesis of various acute and chronic neurodegenerative disorders has been linked to excitotoxic processes and excess generation of nitric oxide. We investigated the deleterious effects of calpain activation in nitric oxide-elicited neuronal apoptosis. In this model, nitric oxide triggers apoptosis of murine cerebellar granule cells by an excitotoxic mechanism requiring glutamate exocytosis and receptor-mediated intracellular calcium overload. Here, we found that calcium-dependent cysteine proteases, calpains, were activated early in apoptosis of cerebellar granule cells exposed to nitric oxide. Release of the proapoptogenic factors cytochrome c and apoptosis-inducing factor from mitochondria preceded neuronal death. However, caspases-3 was not activated. We observed that procaspase-9 was cleaved by calpains to proteolytically inactive fragments. Inhibition of calpains by different synthetic calpain inhibitors or by adenovirally mediated expression of the calpastatin inhibitory domain prevented mitochondrial release of cytochrome c and apoptosis-inducing factor, calpain-specific proteolysis and neuronal apoptosis. We conclude that (i) signal transduction pathways exist that prevent the entry of neurons into a caspase-dependent death after mitochondrial release of cytochrome c and (ii) that calpain activation links nitric oxide-triggered excitotoxic events with the execution of caspase-independent apoptosis in neurons.     

Tyrosine phosphorylation of Sprouty2 enhances its interaction with c-Cbl and is crucial for its function

Mammalian Sprouty (Spry) proteins are now established as receptor tyrosine kinase-induced modulators of the Ras/mitogen-activated protein kinase pathway. Specifically, hSpry2 inhibits the fibroblast growth factor receptor (FGFR)-induced mitogen-activated protein kinase pathway but conversely prolongs activity of the same pathway following epidermal growth factor (EGF) stimulation, where activated EGF receptors are retained on the cell surface. In this study it is demonstrated that hSpry2 is tyrosine-phosphorylated upon stimulation by either FGFR or EGF and subsequently binds endogenous c-Cbl with high affinity. A conserved motif on hSpry2, together with phosphorylation on tyrosine 55, is required for its enhanced interaction with the SH2-like domain of c-Cbl. A hSpry2 mutant (Y55F) that did not exhibit an enhanced binding with c-Cbl failed to retain EGF receptors on the cell surface. Furthermore, individually mutating hSpry2 residues 52-59 to alanine indicated a tight correlation between their affinity for c-Cbl binding and their inhibition of ERK2 activity in the FGFR pathway. We postulate that tyrosine phosphorylation "activates" hSpry2 by enhancing its interaction with c-Cbl and that this interaction is critical for its physiological function in a signal-specific context.     

Titin determines the Frank-Starling relation in early diastole

Titin, a giant protein spanning half the sarcomere, is responsible for passive and restoring forces in cardiac myofilaments during sarcomere elongation and compression, respectively. In addition, titin has been implicated in the length-dependent activation that occurs in the stretched sarcomere, during the transition from diastole to systole. The purpose of this study was to investigate the role of titin in the length-dependent deactivation that occurs during early diastole, when the myocyte is shortened below slack length. We developed a novel in vitro assay to assess myocyte restoring force (RF) by measuring the velocity of recoil in Triton-permeabilized, unloaded rat cardiomyocytes after rigor-induced sarcomere length (SL) contractions. We compared rigor-induced SL shortening to that following calcium-induced (pCa) contractions. The RF-SL relationship was linearly correlated, and the SL-pCa curve displayed a characteristic sigmoidal curve. The role of titin was defined by treating myocytes with a low concentration of trypsin, which we show selectively degrades titin using mass spectroscopic analysis. Trypsin treatment reduced myocyte RF as shown by a decrease in the slope of the RF-SL relationship, and this was accompanied by a downward and leftward shift of the SL-pCa curve, indicative of sensitization of the myofilaments to calcium. In addition, trypsin digestion did not alter the relationship between SL and interfilament spacing (assessed by cell width) after calcium activation. These data suggest that as the sarcomere shortens below slack length, titin-based restoring forces act to desensitize the myofilaments. Furthermore, in contrast to length-dependent activation at long SLs, length-dependent deactivation does not depend on interfilament spacing. This study demonstrates for the first time the importance of titin-based restoring force in length-dependent deactivation during the early phase of diastole.     

The cysteine-rich sprouty translocation domain targets mitogen-activated protein kinase inhibitory proteins to phosphatidylinositol 4,5-bisphosphate in plasma membranes

Sprouty (Spry) proteins have been revealed as inhibitors of the Ras/mitogen-activated protein kinase (MAPK) cascade, a pathway crucial for developmental processes initiated by activation of various receptor tyrosine kinases. In COS-1 and Swiss 3T3 cells, all Spry isoforms translocate to the plasma membrane, notably ruffles, following activation. Here we show that microinjection of active Rac induced the translocation of Spry isoforms, indicating that the target of the Spry translocation domain (SpryTD) is downstream of active Rac. Targeted disruption of actin polymerization revealed that the SpryTD target appeared upstream of cytoskeletal rearrangements. Accumulated evidence indicated that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] is the likely SpryTD target. Human Spry2TD (hSpry2TD) binds to PtdIns(4,5)P(2) in vesicle-binding assays. hSpry2TD colocalizes with the pleckstrin homology domain of phospholipase Cdelta, which binds PtdIns(4,5)P(2). The plasma membrane localization of hSpry2TD was abolished in ionomycin-treated MDCK cells or when PtdIns(4,5)P(2) was specifically dephosphorylated by overexpression of an engineered, green fluorescent protein-tagged inositol 5-phosphatase. Similarly, Spred, a novel Ras/MAPK inhibitor recently found to contain the conserved cysteine-rich SpryTD, also translocated to peripheral membranes and bound to PtdIns(4,5)P(2). Alignment of the Spry and Spred proteins led us to identify a translocation-defective point mutant, hSpry2 D252. Targeting of hSpry2 to PtdIns(4,5)P(2) was shown to be essential for the down-regulation of Ras/MAPK signaling.     

Drosophila crinkled, mutations of which disrupt morphogenesis and cause lethality, encodes fly myosin VIIA

Myosin VIIs provide motor function for a wide range of eukaryotic processes. We demonstrate that mutations in crinkled (ck) disrupt the Drosophila myosin VIIA heavy chain. The ck/myoVIIA protein is present at a low level throughout fly development and at the same level in heads, thoraxes, and abdomens. Severe ck alleles, likely to be molecular nulls, die as embryos or larvae, but all allelic combinations tested thus far yield a small fraction of adult "escapers" that are weak and infertile. Scanning electron microscopy shows that escapers have defects in bristles and hairs, indicating that this motor protein plays a role in the structure of the actin cytoskeleton. We generate a homology model for the structure of the ck/myosin VIIA head that indicates myosin VIIAs, like myosin IIs, have a spectrin-like, SH3 subdomain fronting their N terminus. In addition, we establish that the two myosin VIIA FERM repeats share high sequence similarity with only the first two subdomains of the three-lobed structure that is typical of canonical FERM domains. Nevertheless, the approximately 100 and approximately 75 amino acids that follow the first two lobes of the first and second FERM domains are highly conserved among myosin VIIs, suggesting that they compose a conserved myosin tail homology 7 (MyTH7) domain that may be an integral part of the FERM domain or may function independently of it. Together, our data suggest a key role for ck/myoVIIA in the formation of cellular projections and other actin-based functions required for viability.     

A 3347-locus genetic recombination map of sequence-tagged sites reveals features of genome organization, transmission and evolution of cotton (Gossypium)

We report genetic maps for diploid (D) and tetraploid (AtDt) Gossypium genomes composed of sequence-tagged sites (STS) that foster structural, functional, and evolutionary genomic studies. The maps include, respectively, 2584 loci at 1.72-cM ( approximately 600 kb) intervals based on 2007 probes (AtDt) and 763 loci at 1.96-cM ( approximately 500 kb) intervals detected by 662 probes (D). Both diploid and tetraploid cottons exhibit negative crossover interference; i.e., double recombinants are unexpectedly abundant. We found no major structural changes between Dt and D chromosomes, but confirmed two reciprocal translocations between At chromosomes and several inversions. Concentrations of probes in corresponding regions of the various genomes may represent centromeres, while genome-specific concentrations may represent heterochromatin. Locus duplication patterns reveal all 13 expected homeologous chromosome sets and lend new support to the possibility that a more ancient polyploidization event may have predated the A-D divergence of 6-11 million years ago. Identification of SSRs within 312 RFLP sequences plus direct mapping of 124 SSRs and exploration for CAPS and SNPs illustrate the "portability" of these STS loci across populations and detection systems useful for marker-assisted improvement of the world's leading fiber crop. These data provide new insights into polyploid evolution and represent a foundation for assembly of a finished sequence of the cotton genome.     

Barriers and Bridges to Prevention and Control of Dengue: The Need for a Social–Ecological Approach

This article critically examines how programs for the prevention and control of dengue fever have been conducted in the absence of an integrated approach, and considers the social and ecological factors influencing their effectiveness. Despite recognition of dengue fever as the most important arboviral disease affecting humans, and in spite of a greater emphasis on community-based control approaches, the burden placed on the communities, countries, and regions affected by this disease continues to rise. In considering historical experience in the Americas and the Asia-Pacific region, as well as the global forces that are exerting new pressures, the important elements of successful control programs are identified as community ownership, partnership with government, leadership, scalability, and control of immature mosquitoes. The key barriers to the exchange of knowledge and the transdisciplinary cooperation necessary for sustainable dengue control are rooted in differences in values among policy-makers, citizens, and scientists and are repeatedly expressed in technical, economic, cultural, geographic, and political dimensions. Through consideration of case studies in Cuba, Guatemala, Singapore, Thailand, Indonesia, and Vietnam, the limitations of control approaches that fail to take into account the complexities of ecological and social systems are presented. Bridges to effective control are identified as the basis for adaptability, both of control programs to the mosquito vector’s changing behavior and of education programs to public, regional and local particularities, as well as transdisciplinarity, community empowerment, the ability to scale local experiences up to the macro-level, and the capacity to learn from experience to achieve sustainability.     

Detection of insect Juvenile hormone III and Its precursors from<Emphasis Type="Italic">in Vitro</Emphasis> plantlets of<Emphasis Type="Italic">Cyperus aromaticus</Emphasis>

Juvenile hormone III (JH III) is one of the five closely related sesquiterpenoid compounds important in the regulation of an insect physiological processes in both the developing and the mature insect. JH III and its biosynthetic intermediates, farnesol and methyl farnesoate were detected in thein vitro Cyperus aromaticus plantlets and the mature plants growing in the field. A higher amount of methyl farnesoate (52.7%) and farnesol (31.4%) were found to be present in thein vitro plantlets as compared to the field grown mother plants which contained lower methyl farnesoate (19.5%) and farnesol (14.8%). On the other hand, the mature mother plant (4 months old) contained a higher percentage of JH III than thein vitro plantlets. The estimated content of JHIII analyzed by gas chromatography indicated that the amount of JHIII in the 10 weeks oldin vitro plantlets and the four months old mature plants of C.aromaticus was 0.076 μg/g and 0.085 μg/g respectively. This study indicated thatin vitro culture system could be a potential tool for the production of this natural derived bio-insecticide and could provide a material source for further investigations of the biosynthesis of JH III inC. aromaticus.