GTP-dependent secretion from neutrophils is regulated by Cdk5

We have previously shown evidence for the existence of a calcium-independent, GTP-regulated mechanism of secretion from neutrophils, but this secretory mechanism remains to be fully elucidated. Cyclin-dependent kinase 5 (Cdk5), the various substrates of which include Munc18 and synapsin 1, has been implicated in neuronal secretion. Although the Cdk5 activator, p35, and Cdk5-p35 activity are primarily associated with neurons, we report here that p35 also exists in neutrophils and that an active Cdk5-p35 complex is present in these cells. Cdk5-p35 activity in human neutrophils is mostly localized in secretory granules, which show an increase in Cdk5-p35 level and activity upon GTP stimulation. The potent Cdk5 inhibitor, roscovitine, completely blocks GTP-stimulated granule Cdk5 activity, which accompanies lactoferrin secretion from neutrophil-specific granules. Roscovitine also inhibits GTP-induced lactoferrin secretion and surface localization of the secretion markers, CD63 and CD66b, to a certain extent. Furthermore, neutrophils from wild-type mice treated with roscovitine and neutrophils from p35(-/-) mice exhibit comparable surface expression levels of both CD63 and CD66b upon GTP stimulation. Although our data suggest that other molecules control GTP-induced secretion from neutrophils, it is clear that Cdk5-p35 is required to elicit the maximum GTP-induced secretory response. Our observation that multiple proteins in neutrophil granules serve as specific substrates of Cdk5 further supports the premise that the kinase is a key component of the GTP-regulated secretory apparatus in neutrophils.     

A multifaceted genomics approach allows the isolation of the rice Pia-blast resistance gene consisting of two adjacent NBS-LRR protein genes

The Oryza sativa (rice) resistance gene Pia confers resistance to the blast fungus Magnaporthe oryzae carrying the AVR-Pia avirulence gene. To clone Pia, we employed a multifaceted genomics approach. First, we selected 12 R-gene analog (RGA) genes encoding nucleotide binding site-leucine rich repeats (NBS-LRRs) proteins from a region on chromosome 11 that shows linkage to Pia. By using seven rice accessions, we examined the association between Pia phenotypes and DNA polymorphisms in the 10 genes, which revealed three genes (Os11gRGA3-Os11gRGA5) exhibiting a perfect association with the Pia phenotypes. We also screened ethyl methane sulfonate (EMS)-treated mutant lines of the rice cultivar 'Sasanishiki' harboring Pia, and isolated two mutants that lost the Pia phenotype. DNA sequencing of Os11gRGA3-Os11gRGA5 from the two mutant lines identified independent mutations of major effects in Os11gRGA4. The wild-type 'Sasanishiki' allele of Os11gRGA4 (SasRGA4) complemented Pia function in both mutants, suggesting that SasRGA4 is necessary for Pia function. However, when the rice cultivar 'Himenomochi' lacking Pia was transfected with SasRGA4, the Pia phenotype was not recovered. An additional complementation study revealed that the two NBS-LRR-type R genes, SasRGA4 and SasRGA5, that are located next to each other and oriented in the opposite direction are necessary for Pia function. A population genetics analysis of SasRGA4 and SasRGA5 suggests that the two genes are under long-term balancing selection.     

Mutation in OsLMS, a gene encoding a protein with two double-stranded RNA binding motifs, causes lesion mimic phenotype and early senescence in rice (Oryza sativa L.)

The rice (Oryza sativa L.) lesion mimic and senescence (lms) EMS-mutant, identified in a japonica cultivar Hitomebore, is characterized by a spontaneous lesion mimic phenotype during its vegetative growth, an accelerated senescence after flowering, and enhanced resistance to rice blast (Magnaporthe oryzae). To isolate the OsLMS gene, we crossed the lms mutant to Kasalath (indica), and used mutant F(2) plants to initially map the candidate region to about 322-kb on the long arm of chromosome 2. Illumina whole-genome re-sequencing of the mutant and aligning the reads to Hitomebore reference sequence within the candidate region delineated by linkage analysis identified a G to A nucleotide substitution. The mutation corresponded to the exon-intron splicing junction of a novel gene that encodes a carboxyl-terminal domain (CTD) phosphatase domain and two double stranded RNA binding motifs (dsRBM) containing protein. By PCR amplification, we confirmed that the mutation causes splicing error that is predicted to introduce a premature stop codon. RNA interference (RNAi) transgenic lines with suppressed expression of LMS gene exhibited the lesion mimic phenotype, confirming that the mutation identified in LMS is responsible for the mutant phenotype. OsLMS shares a moderate amino-acid similarity to the Arabidopsis FIERY2/CPL1 gene, which is known to control many plant processes such as stress response and development. Consistence with this similarity, the lms mutant shows sensitivity to cold stress at the early growth stage, suggesting that LMS is a negative regulator of stress response in rice.     

Adult survival of Delphastus catalinae (Coleoptera: Coccinellidae), a predator of whiteflies (Hemiptera: Aleyrodidae), on diets of whiteflies, honeydew, and honey

Delphastus catalinae (Horn) is a coccinellid predator that is commercially sold for the management of whiteflies. A study was conducted to assay the effect of selected diets on the survival of adult D. catalinae. Treatments of water (as a control), 10% honey, honeydew, and whiteflies [Bemisia tabaci (Gennadius)] were provided to the beetles in laboratory assays. Newly emerged, unfed adult insects were used at the start of a survival experiment with trials lasting 50 d. Another survival experiment used mixed-aged adults from a greenhouse colony, and the trials lasted 21 d. Survival was poor on a diet of solely water; ~1% survived beyond a week at 26°C. Survival using the newly emerged insects was similar between those fed honeydew and honey diets, but those on the whitefly diet had the greatest survival (~60% on day 50). However, in the experiment with mixed-aged beetles, adults on honey, and whitefly diets performed the same over a 21-d experiment. Excluding those on the water diet, survival of beetles on the various diets ranged from ~50-80% after 21 d. In an open choice assay across 7 h, D. catalinae adults were found on the whitefly diet in a much greater incidence than on the other diets, and the number of beetles found on the whitefly diet increased over time. The data supports that when D. catalinae are employed in greenhouses or fields for whitefly management, during low prey populations, honeydew from the whitefly can help sustain the population of this predator. Moreover, a supplemental food such as a honey solution can help sustain the population of D. catalinae when the prey is decreased to low numbers. These results may help in the development of strategies to enhance the utility of predators for the management of whiteflies.     

Estimating attraction of Syrphidae (Diptera) to flowering plants with interception traps

Syrphidae with predaceous larvae are important predators of aphids and other insects and can be attracted and maintained in agricultural environments by the addition of flowering plants. Malaise interception traps baited with moveable flowering plants are a novel means of surveying for attractive species and can have the advantages of: (1) homogenising experimental site and plant quality, (2) portability, (3) continuous sampling, (4) capacity to simultaneously capture a broad range of insects (including pests) and (5) no requirement for additional sensory cues to be effective. Six of the 10 species of plants tested were relatively attractive (number of syrphids captured in flower-traps/numbers captured in no plant controls). While flower-traps captured more syrphid species than their associated controls, there were no differences between flower-traps and controls in the sizes (head height) or proboscis lengths of the flies collected. There were no significant relationships between relative attractiveness and flower width or depth or with plant height and floral area. Similarly, mean proboscis length of flies taken in flower-baited traps was not correlated with flower width or depth. The absence of the latter relationship may be due to the inability of an interception trap to distinguish between attraction and attraction-then-feeding.     

Novel functional MAR elements of double minute chromosomes in human ovarian cells capable of enhancing gene expression

Double minute chromosomes or double minutes (DMs) are cytogenetic hallmarks of extrachromosomal genomic amplification and play a critical role in tumorigenesis. Amplified copies of oncogenes in DMs have been associated with increased growth and survival of cancer cells but DNA sequences in DMs which are mostly non-coding remain to be characterized. Following sequencing and bioinformatics analyses, we have found 5 novel matrix attachment regions (MARs) in a 682 kb DM in the human ovarian cancer cell line, UACC-1598. By electrophoretic mobility shift assay (EMSA), we determined that all 5 MARs interact with the nuclear matrix in vitro. Furthermore, qPCR analysis revealed that these MARs associate with the nuclear matrix in vivo, indicating that they are functional. Transfection of MARs constructs into human embryonic kidney 293T cells showed significant enhancement of gene expression as measured by luciferase assay, suggesting that the identified MARS, particularly MARs 1 to 4, regulate their target genes in vivo and are potentially involved in DM-mediated oncogene activation.     

Life-history studies of Lydella jalisco (Diptera: Tachinidae), a parasitoid of Eoreuma loftini (Lepidoptera: Pyralidae)

Lydella jalisco Woodley (Diptera: Tachinidae) is a solitary endoparasitoid of the Mexican rice borer, Eoreuma loftini (Dyar) (Lepidoptera: Pyralidae), the key pest of sugarcane in Texas. This parasitoid was re-introduced into Texas in 1998 as part of a classical biological control program. Information on the biology of L. jalisco is scarce and indispensable for its propagation in captivity and understanding of L. jalisco-E. loftini interactions. Parasitoid longevity, reproductive biology and immature development were studied under laboratory conditions. At 22 °C, honey-fed mated adults lived about two weeks whereas lifespan of unmated individuals averaged three weeks. Females of L. jalisco emerged with a complement of eggs that continued to develop during the first days following emergence. Mating was necessary for embryonic development. After copulation, eggs were fertilized and gradually transferred to an elongated ovisac where they incubated for about one week before hatching. Hatching occurred in the female's reproductive tract, an attribute of true ovoviviparous reproduction. First instar larvae were available for oviposition during the female's entire lifetime. Egg load increased with adult female size; mean lifetime potential fecundity was 400 ± 140 eggs per female. Parasitoids successfully developed on second to sixth instar host larvae. However, host size at parasitization positively influenced the size of resultant adult parasitoids as well as the duration of the larval growth period which was shorter as host size increased. Parasitoid larvae did not complete development below 15 °C, whereas parasitoid larval and pupal mortality respectively reached 14 and 75 percent when temperature exceeded 30 °C. The importance of biological and reproductive attributes of L. jalisco for biological control, as well as for rearing are discussed.