Effects of DNase production, plasmid size, and restriction barriers on transformation of Vibrio cholerae by electroporation and osmotic shock

Attempts to transform wild type strains of V. cholerae with plasmid DNA by traditional osmotic shock methods were not successful. A mutant of V. cholerae that was deficient in extracellular DNase was transformed with plasmid DNA by osmotic shock, demonstrating directly that extracellular DNase is a major barrier to transformation of V. cholerae. Transformation of wild type and DNase-negative strains of V. cholerae was accomplished by electroporation. Efficiency of transformation by electroporation increased with field strength, decreased with plasmid size, and was relatively insensitive to changes in the electrolyte composition of the buffer as long as isotonic sucrose was present. Host-controlled modification/restriction systems also affected transformation efficiency in V. cholerae.     

Discrimination of jittered sonar echoes by the echolocating bat,Eptesicus fuscus: The shape of target images in echolocation

1. Behavioral experiments with jittering echoes examined acoustic images of sonar targets in the echolocating bat, Eptesicus fuscus, along the echo delay or target range axis. Echo phase, amplitude, bandwidth, and signal-to-noise ratio were manipulated to assess the underlying auditory processes for image formation. 2. Fine delay acuity is about 10 ns. Calibration and control procedures indicate that this represents temporal acuity rather than spectral discrimination. Jitter discrimination curves change in phase when the phase of one jittering echo is shifted by 180 degrees relative to the other, showing that echo phase is involved in delay estimation. At an echo detectability index of about 36 dB, fine acuity is 40 ns, which is approximately as predicted for the delay accuracy of an ideal receiver. 3. Compound performance curves for 0 degrees and 180 degrees phase conditions match the crosscorrelation function of the echoes. The locations of both 0 degrees and 180 degrees phase peaks in the performance curves shift along the time axis by an amount that matches neural amplitude-latency trading in Eptesicus, confirming a temporal basis for jitter discrimination.     

Upregulation ofE. coli 38kDa proteins induced by glutaraldehyde and formaldehyde

Exposure of the W3110 strain ofEscherichia coli K12 to low concentrations of glutaraldehyde or formaldehyde results in an unusual pattern of protein expression, as determined by high-resolution, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). A decline in total protein synthesis is accompanied by the upregulation of three proteins of approximate molecular weight 38 kDa. In the presence of 0.1 mM glutaraldehyde this response occurs within the first 5 min of incubation, and with 0.1 mM formaldehyde, within the first 30 min of incubation. The 38 kDa proteins continue to be expressed at high levels until cell death. Comparison of our 2-D PAGE patterns withE. coli gene-protein and plasmid indexes indicates that one of the proteins may be the major gene product of thepyrC locus. This pattern of protein synthesis may indicate a novelE. coli stress response.     

Nasal trigeminal responses to toluene presented by an automated delivery system

An apparatus was developed which permits the automated deliveryof volatile chemical stimuli for use in neurophysiology experiments.A computer-controlled olfactometer, incorporating electronicmass flow controllers (EMFCs) and Teflon-lined solenoid valves,generated and delivered clean or odorized air. Neural and respiratorysignals from the animal were amplified and stored, along withtrial information (e.g. odorant concentration) on a chart recorderand video cassette recorder, both of which were controlled bythe computer. This apparatus was used to measure responses totoluene from the rat ethmoid nerve, a part of the ophthalmicdivision of the trigeminal nerve. Multi-unit responses to thiscompound were first observed at 2000 p.p.m. The magnitude ofthe response increased linearly with logarithmic increases inconcentration up to vapour saturation. Changes in respirationin response to toluene also were observed, although neural responsesoften were seen in the absence of respiratory changes.     

Phytohormone mutants in plant research

The techniques used for the production and identification of plant hormone mutants are described. The properties used to classify these mutants into the broad synthesis and sensitivity categories are discussed, and the genetic considerations needed to allow their effective use in plant hormone research examined. A brief outline of significant recent work on the gibberellin (GA), abscisic acid (ABA), auxin, ethylene, cytokinin and phytochrome mutants is provided. Suggestions for future emphasis are made, particularly relating to an examination of the tissue and ontogenetic specificity of the plant hormone genes.     

Terpenoid precursors of strigol as seed germination stimulants of broomrape (Orobanche ramosa) and witchweed (Striga asiatica)

Strigol and some of its synthetic precursors and analogs are known to be germination stimulants for broomrape (Orobanche ramosa) and witchweed (Striga asiatica). Fifteen synthetic terpenoids, similar in structure to one of the four rings of the strigol molecule, were evaluated in two bioassays as seed germination stimulants with broomrape, and nine were found to be active. Five of the more active compounds contained ester groups. Whereas the study was intended primarily to evaluate forced germination of broomrape by aqueous solutions, the results are almost qualitatively identical for broomrape and witchweed. Monocyclic compounds with chemical structures similar to two of the rings of strigol have now been shown to possess significant bioactivity as germination stimulants.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.     

Neurite outgrowth in response to transfected N-CAM changes during development and is modulated by polysialic acid

We have used monolayers of control 3T3 cells and 3T3 cells transfected with a cDNA encoding human N-CAM as a culture substrate for embryonic chick retinal ganglion cells (RGCs). At embryonic day 6 (E6), but not at E11, RGCs extended longer neurites on monolayers of N-CAM-transfected cells. This loss of RGC responsiveness was not associated with substantial changes in the level of N-CAM expression on RGC growth cones. The neurite outgrowth response from E6 RGCs could be inhibited by removal of N-CAM from the monolayer, by removal of alpha 2-8-linked polysialic acid from neuronal N-CAM, or by antibodies that bind exclusively to chick (neuronal) N-CAM. In contrast, the response was not dependent on neuronal beta 1 integrin function. These data provide substantive evidence for a homophilic binding mechanism directly mediating N-CAM-dependent neurite outgrowth, and suggest that changes in polysialic acid expression on neuronal N-CAM may modulate N-CAM-dependent axonal growth during development.     

Hormonally regulated expression of arginine kinase in Drosophila melanogaster

Summary Arginine kinase (AK) is present throughout the life cycle of Drosophila melanogaster, but there is a sharp, transient peak of AK activity during the prepupal period and a second period of elevated activity at the time of eclosion of the adult. Imaginal discs show the greatest increase in AK activity at the prepupal stage of those tissues assayed. The prepupal peak is not seen when the temperature-sensitive ecdysoneless mutant ecd-1 is shifted to 29° C at mid-third instar larval stage. The peak in activity reappears when ecd-1 is either shifted back to 20° C after 60 h at 29° C or is fed 20-hydroxyecdysone. At the restrictive temperature, imaginal discs from ecd-1 larvae progressively lose AK activity, whereas discs from 20-hydroxyecdysone-fed larvae have a marked increase in AK activity at stage P3 of the prepupal period. These data suggest that the prepupal peak is regulated by the hormone 20-hydroxyecdysone.     

Extraction and quantitation of astaxanthin from Phaffia rhodozyma

Summary The rapid, quantitative release of astaxanthin and other carotenoids from the yeast Phaffia rhodozyma is described. Hashed cells are ruptured with dimethylsulfoxide (DMSO) and carotenoids extracted into an organic solvent. Extraction and spectrophotometric quantitation of total carotenoids is rapid, reproducible and only small volumes (0.1–2 ml) of culture are required. HPLC analysis in normal phase silica gel column indicates that astaxanthin comprises 65–95% of the total pigmented carotenoids of P. rhodozyma.