Development of the prepupal Verson's gland of the tobacco hornworm, Manduca sexta, and its hormonal control

The segmentally arranged Verson's glands are epidermal derivatives comprised of three cells: the duct, saccule, and secretory cells. The development of these glands was followed through the 5th instar and larval-pupal transition of Manduca sexta. The glands are relatively small during the feeding stage, begin to grow at wandering, and undergo about a 50-fold increase in size during the prepupal period. The increase in size is due mainly to the hypertrophy of the secretory cell which synthesizes a heterogeneous set of proteinaceous secretory products. Three prominent 11 to 12 kiloDalton (kD) polypeptides are made by the pharate fifth larval gland, whereas the pupal gland produces polypeptides ranging from 14 to 75 kD with a major complex at 30 to 34 kD. The secretory product is poured out onto the surface of the new cuticle at the time of ecdysis and contains all of the major proteins detected in extracts of the whole gland. The accumulation of secretory products by the gland occurs during the prepupal peak of ecdysteroid and is blocked if this rise is prevented by abdominal isolation. Infusion of 30 micrograms 20-hydroxyecdysone (20-HE) into such isolated abdomens caused synthesis of the pupal products. Treatment with the juvenile hormone mimic, methoprene, during the fifth instar showed that the commitment of the glands to produce the pupal proteins is independent of and occurs before the overlying epidermis becomes committed to make pupal cuticle.     

Pseudomonas syringae pv. tomato hijacks the Arabidopsis abscisic acid signalling pathway to cause disease

We have found that a major target for effectors secreted by Pseudomonas syringae is the abscisic acid (ABA) signalling pathway. Microarray data identified a prominent group of effector-induced genes that were associated with ABA biosynthesis and also responses to this plant hormone. Genes upregulated by effector delivery share a 42% overlap with ABA-responsive genes and are also components of networks induced by osmotic stress and drought. Strongly induced were NCED3, encoding a key enzyme of ABA biosynthesis, and the abscisic acid insensitive 1 (ABI1) clade of genes encoding protein phosphatases type 2C (PP2Cs) involved in the regulation of ABA signalling. Modification of PP2C expression resulting in ABA insensitivity or hypersensitivity led to restriction or enhanced multiplication of bacteria, respectively. Levels of ABA increased rapidly during bacterial colonisation. Exogenous ABA application enhanced susceptibility, whereas colonisation was reduced in an ABA biosynthetic mutant. Expression of the bacterial effector AvrPtoB in planta modified host ABA signalling. Our data suggest that a major virulence strategy is effector-mediated manipulation of plant hormone homeostasis, which leads to the suppression of defence responses.     

Enumeration of Mycobacterium leprae using real-time PCR

Mycobacterium leprae is not cultivable in axenic media, and direct microscopic enumeration of the bacilli is complex, labor intensive, and suffers from limited sensitivity and specificity. We have developed a real-time PCR assay for quantifying M. leprae DNA in biological samples. Primers were identified to amplify a shared region of the multicopy repeat sequence (RLEP) specific to M. leprae and tested for sensitivity and specificity in the TaqMan format. The assay was specific for M. leprae and able to detect 10 fg of purified M. leprae DNA, or approximately 300 bacteria in infected tissues. We used the RLEP TaqMan PCR to assess the short and long-term growth results of M. leprae in foot pad tissues obtained from conventional mice, a gene knock-out mouse strain, athymic nude mice, as well as from reticuloendothelial tissues of M. leprae-infected nine-banded armadillos. We found excellent correlative results between estimates from RLEP TaqMan PCR and direct microscopic counting (combined r = 0.98). The RLEP TaqMan PCR permitted rapid analysis of batch samples with high reproducibility and is especially valuable for detection of low numbers of bacilli. Molecular enumeration is a rapid, objective and highly reproducible means to estimate the numbers of M. leprae in tissues, and application of the technique can facilitate work with this agent in many laboratories.     

Threonine 22 phosphorylation attenuates Hsp90 interaction with cochaperones and affects its chaperone activity

Heat shock protein 90 (Hsp90) is an essential molecular chaperone whose activity is regulated not only by cochaperones but also by distinct posttranslational modifications. We report here that casein kinase 2 phosphorylates a conserved threonine residue (T22) in α helix-1 of the yeast Hsp90 N-domain both in?vitro and in?vivo. This α helix participates in?a hydrophobic interaction with the catalytic loop in Hsp90's middle domain, helping to stabilize the chaperone's ATPase-competent state. Phosphomimetic mutation of this residue alters Hsp90 ATPase activity and chaperone function and impacts interaction with the cochaperones Aha1 and Cdc37. Overexpression of Aha1 stimulates the ATPase activity, restores cochaperone interactions, and compensates for the functional defects of these Hsp90 mutants.     

Herbivory and drought interact to enhance spatial patterning and diversity in a savanna understory

The combination of abiotic stress and consumer stress can have complex impacts on plant community structure. Effective conservation and management of semi-arid ecosystems requires an understanding of how different stresses interact to structure plant communities. We explored the separate and combined impacts of episodic drought, livestock grazing, and wild ungulate herbivory on species co-occurrence and diversity patterns in a relatively productive, semi-arid Acacia savanna. Specifically, we analyzed 9 years of biannual plant community data from the Kenya long-term exclosure experiment, a broad-scale manipulative experiment that has excluded different combinations of large mammalian herbivores from 18 4-ha plots since 1995. During droughts, we observed low species diversity and random species co-occurrence patterns. However, when rain followed a major drought, areas exposed to moderate cattle grazing displayed high species diversity and evidence of significant species aggregation. These patterns were not apparent in the absence of cattle, even if other large herbivores were present. To explore possible mechanisms, we examined patterns separately for common and rare species. We found that aggregation patterns were likely driven by rare species responding similarly to the availability of open micro-sites. Our results indicate that in a productive, fire-suppressed savanna, the combination of periodic drought and moderate cattle grazing can enhance plant biodiversity and fine-scale spatial heterogeneity by opening up space for species that are otherwise rare or cryptic. Our findings also emphasize that domestic herbivores can have significantly stronger impacts on plant community dynamics than wild herbivores, even in an ecosystem with a long history of grazing.     

Fine structure of degenerating abdominal motor neurons after eclosion in the sphingid moth,Manduca sexta

Summary Ultrastructural aspects of the natural degeneration of a group of six motor neurons in the fourth abdominal ganglion of Manduca sexta are described. These motor neurons innervate intersegmental muscles that degenerate and disappear immediately after adult eclosion. The first detectable changes in the cell bodies appear 12 h after eclosion and include disruption of the endoplasmic reticulum and an increase in the size and number of lamellar bodies. At 32 h the nuclear membranes rupture, and the membranous and granular cytoorganelles segregate in different parts of the cell. At that stage the surrounding glial cells participate in the digestion of material from the degenerating neurons. From 72 h onward the remaining neuronal structures become disrupted, and are finally transformed into a single, large lamellar body (residual body) within the glial profile. The degeneration pattern differs significantly from that of embryonic vertebrate neurons.     

Biological staining: mechanisms and theory

New staining techniques continue to be introduced, and older ones continue to be used and improved. Several factors control specificity, selectivity and visibility of the end product in any procedure using dyes, fluorochromes, inorganic reagents or histochemical reactions applied to sections or similar preparations. Local concentration of the tissue target often determines the intensity of the observed color, as does the fine structure within the object being stained, which may facilitate or impede diffusion of dyes and other reagents. Several contributions to affinity control the specificity of staining. These include electrical forces, which result in accumulation of dye ions in regions of oppositely charged tissue polyions. Weaker short-range attractions (hydrogen bonding, van der Waals forces or hydrophobic bonding, depending on the solvent) hold dyes ions and histochemical end products in contact with their macromolecular substrates. Nonionic forces can also increase visibility of stained sites by causing aggregation of dye molecules. Covalent bonds between dye and tissue result in the strongest binding, such as in methods using Schiff's reagent and possibly also some mordant dyes. The rate at which a reagent gains access to or is removed from targets in a section or other specimen affect what is stained, especially when more then one dye is used, together or sequentially. Rate-controlled staining is greatly influenced by the presence and type of embedding medium, such as a resin, that infiltrates the tissue. The rates of chemical reactions are major determinants of outcome in many histochemical techniques. Selective staining of different organelles within living cells is accomplished mainly with fluorochromes and is controlled by mechanisms different from those that apply to fixed tissues. Quantitative structure-activity relations (QSAR) of such reagents can be derived from such molecular properties as hydrophilic-hydrophobic balance, extent of conjugated bond systems, acid-base properties and ionic charge. The QSAR correlates with staining of endoplasmic reticulum, lysosomes, mitochondria, DNA, or the plasma membranes of living cells.