Mode of action of CryIIA: a Bacillus thuringiensis delta-endotoxin

CryIIA is an effective insecticidal delta-endotoxin produced by several strains of Bacillus thuringiensis. Unlike CryI and CryIIIA-toxins that demonstrate some degree of saturable binding on the brush border of susceptible insects, neither saturable binding nor a saturable binding component was found for CryIIA on the midgut brush border of Helicoverpa zea. CryIIA did not dilute and block CryIA(c) binding, however, CryIA(c) effectively diluted CryIIA and stopped the initial binding of CryIIA to the brush border. These observations suggest that CryIIA and CryIA(c) toxins share a common component for binding on the midgut brush border. CryIIA formed voltage-dependent and not highly cation-selective channels in planar lipid bilayers unlike CryIA(c) and CryIIIA. Both CryIA(c) and CryIIA were stable in the digestive fluids of H. zea, but CryIIA was significantly less soluble than CryIA(c). Despite this difference in solubility, CryIIA arrested the feeding of third instar H. zea as rapidly as did CryIA(c), however, the onset of acute morbidity was delayed for CryIIA. Differences in solubility, binding, and ion channels formed by CryIIA toxin, resulted in reduced bioactivity against H. zea when compared with CryIA(c) but represent a unique mode of action among the delta endotoxins.     

The response of amphibian larvae to environmental change is both consistent and variable

Many environments are undergoing rapid environmental change and there is a need to understand the mechanisms by which species can persist in altered environments. Model systems, such as amphibian metamorphosis, which can be generalized across many types of environmental change and across many species, are a powerful tool for understanding mechanisms that facilitate persistence in altered and disturbed environments. Amphibian larvae respond to environmental change by varying age at metamorphosis, or size at metamorphosis. Differential selection pressures on age or size at metamorphosis may result in a differential response among taxa to environmental change. Using a meta‐analysis, we investigated whether age at metamorphosis, size at metamorphosis, and larval growth rate vary within and among taxonomic families of amphibians in experiments that modified the environmental temperature, density of individuals, food, hydroperiod and the presence of predators. For all environmental factors except predators, the direction of the response was consistent across most of the studied taxa. However, there was considerable variation in effect size both within and among families. Results demonstrate that amphibian metamorphosis is a valuable model system for studying the effects of environmental change. Yet, we stress the need for caution in making generalizations about how individuals respond to environmental factors that have an indirect effect on physiology and require the perception of an environmental cue, such as the presence of predators. Synthesis As the current conditions of the environment are rapidly changing there is a need to understand how organisms respond to environmental change, and whether response of one species can be generalized to other species. Using a meta‐analyses, we tested whether the phenotypic response of amphibian larvae to five types of environmental change is consistent among and within taxonomic families. The phenotypic response to changes in environmental factors was consistent when the environmental factor has a direct effect on physiology, but varies among and within family if the environmental factor has an indirect effect on physiology or requires the perception of an environmental cue.     

Effects of oligo sequence and chemistry on the efficiency of oligodeoxyribonucleotide-mediated mRNA cleavage.

Using the endogenous histone H4 mRNA of Xenopus oocytes as a target, we have previously shown that 20mer oligos complementary to different parts of this sequence vary in their effectiveness at causing mRNA cleavage in vivo, and that some of the RNA can never be cleaved. In this paper we show that the resistant RNA is not localised within one part of the oocyte, and that the relative resistance in vivo of endogenous or synthetic H4 mRNA to the different oligos is preserved in an in vitro assay system using deproteinised RNA. If an prior annealing step is included in vitro, all resistance is abolished. Chemical modification of one oligo by end substitution with methylphosphonate or phosphorothioate residues did not improve cleavage efficiency. Oligos with complete phosphorothioate substitution cause slower cleavage in vivo but persist for longer. Consequently phosphorothioate oligos are effective at lower doses than phosphodiester ones, provided that the incubation time is long enough (24 hours). Increasing oligo length from 20nt to 30nt increases phosphorothioate oligo efficiency over long reaction times in vivo, but decreases efficiency during short in vitro assays. Similar increases in length did not affect phosphodiester oligo performance in vivo, but caused a decrease in efficiency in vitro which was overcome by an annealing step.     

Differential scanning calorimetric study of the thermal denaturation of aspartate transcarbamoylase of Escherichia coli

The thermal denaturation of Escherichia coli aspartate transcarbamoylase (c6r6) in the absence and presence of various ligands has been studied by means of high-sensitivity differential scanning calorimetry (DSC). As previously reported [Vickers, K.P., Donovan, J.W., & Schachman, H.K. (1978) J. Biol. Chem. 253, 8493-8498], the denaturational endotherm consists of two peaks, the lower of which is due to denaturation of the three regulatory, r2, subunits while the upper involves the two catalytic, c3, subunits. The temperature of maximal excess apparent specific heat, tm, of the lower peak is raised from the value of 51.4 degrees C for the isolated subunit to 66.8 degrees C as a result of subunit interactions, whereas tm for the c3 peak is essentially the same in the isolated subunit and in the holoenzyme, indicating that the denatured r2 subunits do not interact with the c3 subunits. The total specific denaturational enthalpy for c6r6, 4.83 +/- 0.16 cal g-1, is significantly larger than the weighted mean, 4.08 cal g-1, of the enthalpies for c3 and r2. The fact that no endotherm is observed when previously scanned protein is rescanned indicates that the denaturation is irreversible, as is also the case with the r2 and c3 subunits. Empirical justification for analyzing the data in terms of equilibrium thermodynamics is cited. The observed DSC curves can be expressed within experimental uncertainty as the sum of five sequential two-state steps. The value of t 1/2, the temperature of half-completion, for each step increases with increasing protein concentration, indicating that some dissociation of the protein takes place during denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human complement factor I: analysis of cDNA-derived primary structure and assignment of its gene to chromosome 4

Factor I is a serine proteinase of complement which together with one of several specific cofactors cleaves activation products of the third and fourth components of complement (C3b and C4b) and modulates the activity of C3 convertase. A heterodimer glycoprotein (Mr = 88,000), factor I is synthesized as a single-chain precursor, prepro-I, which undergoes intracellular proteolytic processing. The human hepatoma line HepG2, however, secretes predominantly the single-chain precursor pro-I. In order to determine the molecular basis for this apparent processing defect, factor I cDNA clones were isolated from a HepG2 mRNA-derived library. Sequencing of the largest insert, HI1971, revealed that it contains 14 base pairs of 5' untranslated region, the complete coding sequence for the 583-residue prepro-I (NH2-signal peptide-heavy chain-linking peptide-light chain-COOH), two polyadenylation signals within the 200-base pair 3' untranslated region, and a portion of poly(A) tail. Analysis of the derived protein structure 1) reveals a mosaic multidomain structure of the heavy chain; 2) demonstrates structural similarity between intracellular conversion of pro-I and activation of other serine proteinase zymogens; and 3) indicates that the light chain of factor I resembles most closely the active subunit of tissue plasminogen activator among all serine proteinases and factor D among complement proteinases. Furthermore, this protein sequence was compared to the sequences of factor I cDNA clones isolated from normal human liver libraries and found to be identical. By exclusion, this defines as cellular the basis for the inefficient processing of pro-I by the HepG2 line. Chromosomal localization by the somatic cell hybrid method maps the factor I gene to chromosome 4.     

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant ^1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.     

Mechanistic studies of melanogenesis: the influence of N‐substitution on dopamine quinone cyclization

The influence of side‐chain structure on the mode of reaction of ortho‐quinone amines has been investigated with a view, ultimately, to developing potential methods of therapeutic intervention by manipulating the early stages of melanogenesis. Four N‐substituted dopamine derivatives have been prepared and quinone formation studied using pulse radiolysis and tyrosinase‐oximetry. Ortho‐quinones with an amide or urea side chain were relatively stable, although evidence for slow formation of isomeric para‐quinomethanes was observed. A thiourea derivative cyclized fairly rapidly (k = 1.7/s) to a product containing a seven‐membered ring, whereas a related amidine gave more rapidly (k ～ 2.5 × 10²/s) a stable spirocyclic product. The results suggest that cyclization of amides, ureas and carbamates (NHCO‐X; X = R, NHR or OR) does not occur and is not, therefore, a viable approach to the formation of tyrosinase‐activated antimelanoma prodrugs. It is also concluded that for N‐acetyldopamine spontaneous ortho‐quinone to para‐quinomethane isomerization is slow.     

Differential pollinator effectiveness and importance in a milkweed (Asclepias, Apocynaceae) hybrid zone

? Premise of the study: Exceptions to the ideal of complete reproductive isolation between species are commonly encountered in diverse plant, animal, and fungal groups, but often the causative ecological processes are poorly understood. In flowering plants, the outcome of hybridization depends in part on the effectiveness of pollinators in interspecific pollen transport. In the Asclepias exaltata and A. syriaca (Apocynaceae) hybrid zone in Shenandoah National Park, Virginia, extensive introgression has been documented. The objectives of this study were to (1) determine the extent of pollinator overlap among A. exaltata, A. syriaca, and their hybrids and (2) identify the insect taxa responsible for hybridization and introgression. ? Methods: We observed focal plants of parental species and hybrids to measure visitation rate, visit duration, and per-visit pollinia removal and deposition, and we calculated pollinator effectiveness and importance. ? Key results: Visitation rates varied significantly between the 2 yr of the study. Overall, Apis mellifera, Bombus sp., and Epargyreus clarus were the most important pollinators. However, Bombus sp. was the only visitor that was observed to both remove and insert pollinia for both parent species as well as hybrids. ? Conclusions: We conclude that Bombus may be a key agent of hybridization and introgression in these sympatric milkweed populations, and hybrids are neither preferred nor selected against by pollinators. Thus, we have identified a potential mechanism for how hybrids act as bridges to gene flow between A. exaltata and A. syriaca. These results provide insights into the breakdown of prezygotic isolating mechanisms.