Biosynthesis of gibberellic acid from milk permeate in repeated batch operation by a mutant Fusarium moniliforme cells immobilized on loofa sponge

Gibberellic acid (GA) production from milk permeate was studied by 28 mutants of Fusarium moniliforme, among which mutant gamma-14 was selected as the best producer. Experiments were carried out in shaker flasks and fermentative process was analyzed with free and immobilized cells. Immobilization of mutant gamma-14 cells onto loofa sponge discs was studied with respect to the optimization of the incubation temperature, initial pH, inoculum size (number of discs) and its reusability for GA production. Best yield of GA (2.40 gl(-1)) was recorded by immobilized cells under optimized cultural conditions (4 immobilized discs, 30 degrees C and pH 5). Data obtained during four reusable cycles showed high stability of GA production and reduction in the initiation time of acid production, resulting in higher levels of GA in shorter time duration. Immobilization of mutant gamma-14 cells onto loofa sponge discs, permitted repeated reuse under the specified fermentation conditions for GA production from milk permeate.     

Apolipoprotein A-I structure and lipid properties in homogeneous, reconstituted spherical and discoidal high density lipoproteins

We prepared a spherical reconstituted high density lipoprotein (rHDL) particle in pure form and compared it with its homogeneous discoidal rHDL precursors, in terms of the structure and stability of the apolipoprotein A-I (apoA-I) component, the dynamics of the surface lipids, and the relative reactivity with lecithin-cholesterol acyltransferase. The apoA-I-structure was examined in the rHDL particles by circular dichroism and fluorescence spectroscopic methods, and the binding of monoclonal antibodies specific for apoA-I epitopes. The stability of apoA-I on the rHDL particles was assessed by the effects of guanidine hydrochloride on the wavelength of maximum intrinsic fluorescence of the apolipoprotein. Lipid dynamics in the acyl chain region and the polarity of the lipid-water interface were investigated by means of fluorescence probes. The conformation of apoA-I in the spherical 93-A rHDL particles was found to be very similar to that in the 96-A rHDL discs but distinct from the apoA-I structure in the 78-A rHDL discs. The stability of apoA-I to denaturation by guanidine hydrochloride was highest in the 93-A rHDL spheres. The experiments on the lipids indicate somewhat more ordered and motionally restricted acyl chains in the spheres, relative to the discs, but a similar surface polarity. These results suggest that the folding and organization of apoA-I on the three particles include protein domains consisting of interacting alpha-helical segments in the carboxyl-terminal region and a globular domain in the amino-terminal region of each apoA-I molecule. The reactivity with lecithin-cholesterol acyltransferase was highest for the 96-A rHDL disc, and 16- and 34-fold lower for the 78-A rHDL disc and the 93-A rHDL sphere, respectively, possibly as a result of differences in apoA-I structure and product inhibition in these particles.     

Action of melittin on the DPPC-cholesterol liquid-ordered phase: a solid state 2H-and 31P-NMR study.

Solid-state deuterium and phosphorus-31 nuclear magnetic resonance studies of deuterium-labeled beta--[2,2',3,4,4',6-2H6]-cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine have been undertaken to monitor the action of melittin on model membranes containing 30 mol% cholesterol, both at the molecular and macroscopic level. Cholesterol totally inhibits the toxin-triggered formation of large unilamellar vesicles and strongly restricts the appearance of small discs. The latter remain stable over a wide temperature range (20-60 degrees C) because of an increase in their cholesterol content as the temperature increases. This process is related to a constant disc hydrophobic thickness of approximately 29 A. The system, when not in the form of discs, appears to be composed of very large vesicles on which melittin promotes magnetically induced ellipsoidal deformation. This deformation is the greatest when the maximum of discs is observed. A model to describe both the disc formation and stability is proposed.     

Genetic stability of cultured hairy roots induced by Agrobacterium rhizogenes on tuber discs of potato cv. Bintje

Agrobacterium rhizogenes induced hairy roots on discs of the tuber tissue of the tetraploid potato cv. Bintje after infection. Early and late generated hairy roots (transformed roots) were excised directly from the tuber discs and analysed for genetic stability by chromosome counts and determination of nuclear DNA content with flow cytometry. Another part was cloned by subculturing on MS-medium without hormones and subsequently analysed. Twenty-one of the 27 primary hairy roots and all 16 subcultured hairy roots were tetraploid thus suggesting genetic stability of transformed roots. Shoot regeneration was observed on hairy roots and therefore, it is suggested that A. rhizogenes transformation can be a suitable system for genetically stable plant regeneration from transformed cells.     

Drosophila damaged DNA binding protein 1 contributes to genome stability in somatic cells

The damaged DNA-binding protein (DDB) complex consists of a heterodimer of p127 (DDB1) and p48 (DDB2) subunits and is believed to have a role in nucleotide excision repair (NER). We used the GAL4-UAS targeted expression system to knock down DDB1 in wing imaginal discs of Drosophila. The knock-down was achieved in transgenic flies using over-expression of inverted repeat RNA of the D-DDB1 gene [UAS-D-DDB1(650)-dsRNA]. As a consequence of RNA interference (RNAi), the fly had a shrunken wing phenotype. The wing spot test showed induced genome instability in transgenic flies with RNAi knock-down of D-DDB1 in wing imaginal discs. When Drosophila larvae with RNAi knock-down of D-DDB1 in wing imaginal discs were treated with the chemical mutagen methyl methanesulfonate (MMS), the frequency of flies with a severely shrunken wing phenotype increased compared to non-treated transgenic flies. These results suggested that DDB1 plays a role in the response to DNA damaged with MMS and in genome stability in Drosophila somatic cells.     

Mitosis in neoplastic and hyperplastic imaginal discs ofDrosophila

Homozygosity for recessive mutations inDrosophila tumour suppressor genes likelethal giant larvae (Igl), lethal giant discs (Igd) orfat (ft) induce uncontrolled cell proliferations in the imaginal discs of the mutant larvae. Imaginal discs of larvae mutant forIgl tumour suppressor gene display neoplastic growths while those mutant forIgd orfat display hyperplastic growths. Results presented in this study reveal that mutant wing imaginal discs with neoplastic or hyperplastic overgrowths display high mitotic activity primarily during the extended period of larval life when their wild-type siblings have already pupariated. Both these categories of overgrowths show overall stability of the karyotypes and only low frequency of aneuploidy. The hyperplastic imaginal discs ofIgd orft mutant larvae displayed normal chromosome condensation. In contrast, the neoplastic imaginal discs ofIgl mutants showed high frequency of mitotic cells with undercondensed chromosomes. In this respect the neoplastic discs resemble malignant neuroblastomas of theIgl larvae which also display undercondensed chromosomes. These results thus suggest an indirect role of the cytoskeletal protein encoded byIgl tumour suppressor gene in aspects of normal chromosome condensation during mitosis.     

SwellGel: a sample preparation affinity chromatography technology for high throughput proteomic applications

Development of high throughput systems for purification and analysis of proteins is essential for the success of today's proteomic research. We have developed an affinity chromatography technology that allows the customization of high capacity/high throughput chromatographic separation of proteins. This technology utilizes selected chromatography media that are dehydrated to form uniform SwellGel discs. Unlike wet resin slurries, these discs are easily adaptable to a variety of custom formats, eliminating problems associated with resin dispensing, equilibration, or leakage. Discs can be made in assorted sizes (resin volume 15 microl-3 ml) dispensed in various formats (384-, 96-, 48-, and 24-well microplates or columns) and different ligands can be attached to the matrix. SwellGel discs rapidly hydrate upon addition of either water or the protein sample, providing dramatically increased capacity compared to coated plates. At the same time, the discs offer greater stability, reproducibility, and ease of handling than standard wet chromatography resins. We previously reported the development of SwellGel for the purification of 6x His- and glutathione-S-transferase (GST)-tagged fusion proteins [Prot. Exp. Purif. 22 (2001) 359-366]. In this paper, we discuss an expanded list of SwellGel stabilized chromatographic methods that have been adapted to high throughput formats for processing protein samples ranging from 10 microl to 10 ml (1 microg to 50 mg protein). Data are presented applying SwellGel discs to high throughput proteomic applications such as affinity tag purification, protein desalting, the removal of abundant proteins from serum including albumin and immunoglobulin, and the isolation of phosphorylated peptides for mass spectrometry.     

The stability of micro-organisms preserved in LENTICULE discs, demonstrated by repeat sample distributions of the EQUAL Scheme for Indicator Organisms (water microbiology)

Aims:  To demonstrate the stability of microorganisms preserved in LENTICULE discs, employed by the EQUAL Scheme for Indicator Organisms.
Methods and Results:  Five sample batches containing several indicator organism parameters were each despatched in two separate distributions at intervals of up to 24 months apart. A comparison of participants' median counts and bar charts of their cumulated results was then made.
Conclusions:  The results demonstrated that LENTICULE discs provided a remarkably stable preparation format for the maintenance of viable organisms in reproducible numbers.
Significance and Impact of the Study:  Complex mixtures of organisms can be maintained in stable numbers for extended periods of time at a freezer temperature of −20°C and satisfactorily transported without the requirement for cold chain conditions.     

Is glycine betaine a non-compatible solute in higher plants that do not accumulate it?

Rape (Brassica napus L. var. oleifera cv. Samourai) leaf discs treated in vitro in the presence of glycine betaine (GB) exhibited very high accumulation of GB, suggesting the operation of a specific uptake system. When further subjected to osmotic upshocks by transfer to PEG 6000 media, the typical osmo-induced proline response of the discs was strongly inhibited. The level of this inhibition was quantitatively related to the amount of GB loaded in the tissues. In contrast, the soluble sugar content increased in GB-treated discs. Surprisingly, viability tests (i.e. membrane stability and 2,3,5-triphenyltetrazolium chloride reduction) indicated a destabilizing effect of GB in these tissues. This is at variance with the relative compatibility of sucrose and proline. In addition, the protein content was lower in GB-treated discs. This could be related to an inhibitory effect on protein synthesis, as demonstrated by radiolabelling of polypeptides with [³⁵S] amino acids. This effect was particularly pronounced on Rubisco large sub-unit synthesis and was still apparent under non-stress conditions. The GB treatment was also followed by the induction or up-regulation of a set of polypeptides, not seen under stress conditions, while the synthesis of osmo-induced polypeptides was not affected by GB. These novel effects of GB lead us to discuss the reasons for its incompatibility in leaf tissues of a non-GB-accumulating species.     

Proline accumulation in canola leaf discs subjected to osmotic stress is related to the loss of chlorophylls and to the decrease of mitochondrial activity

Many stress studies use simplified experimental models like leaf discs, but the validity of such approaches remains a matter of debate, especially when attempts are made to obtain suitable biotests. We report here the analysis of the resistance of canola leaf discs to osmotic stress, as related to the accumulation of proline. For that purpose, the explanted tissues were incubated for 24 h under continuous light and at 28°C on polyethylene glycol solutions of external potentials (Π_ext) ranging from −0.1 to −8.0 MPa. Proline content, water deficit and chlorophyll content were quantified. The cell membrane stability, which is a structural component of viability, was assessed according to electrolyte leakage. Biomembrane oxidative damage was measured through the production of malondialdehyde and the mitochondrial activity was quantified by assessing the ability of the tissues to reduce 2,3,5-triphenyltetrazolium chloride. Although the water deficit of the tissues reached high values (i.e. up to 70%), the cell membrane stability remained high. Furthermore, the oxidative damage to biomembranes was quite low, even in highly dehydrated tissues. In contrast, osmotic stress induced a significant decrease in the chlorophyll content and a strong accumulation of proline. These two processes each reached a maximum at a Π_ext of −3.0 MPa, although the tissues appeared to be viable at even lower Π_ext, suggesting they are not responsible for primary resistance. The mitochondrial activity was strongly decreased in the stressed leaf discs, although this activity was at least partially restored in tissues incubated for 24 h on a non-stressing medium. It appears that the cell components of the osmotic stress resistance assessed observed in this study do not reflect the modes of resistance of an intact plant.     

Biomechanical effect after Coflex and Coflex rivet implantation for segmental instability at surgical and adjacent segments: a finite element analysis

The Coflex device may provide stability to the surgical segment in extension but does not restore stability in other motion. Recently, a modified version called the Coflex rivet has been developed. The effects of Coflex and Coflex rivet implantation on the adjacent segments are still not clear; therefore, the purpose of this study was to investigate the biomechanical differences between Coflex and Coflex rivet implantation by using finite element analyses. The results show that the Coflex implantation can provide stability in extension, lateral bending, and axial rotation at the surgical segment, and it had no influence at adjacent segments except for extension. The Coflex rivet implantation can provide stability in all motions and reduce disc annulus stress at the surgical segment. Therefore, the higher range of motion and stress induced by the Coflex rivet at both adjacent discs may result in adjacent segment degeneration in flexion and extension.     

Biomechanical effect after Coflex and Coflex rivet implantation for segmental instability at surgical and adjacent segments: a finite element analysis

The Coflex device may provide stability to the surgical segment in extension but does not restore stability in other motion. Recently, a modified version called the Coflex rivet has been developed. The effects of Coflex and Coflex rivet implantation on the adjacent segments are still not clear; therefore, the purpose of this study was to investigate the biomechanical differences between Coflex and Coflex rivet implantation by using finite element analyses. The results show that the Coflex implantation can provide stability in extension, lateral bending, and axial rotation at the surgical segment, and it had no influence at adjacent segments except for extension. The Coflex rivet implantation can provide stability in all motions and reduce disc annulus stress at the surgical segment. Therefore, the higher range of motion and stress induced by the Coflex rivet at both adjacent discs may result in adjacent segment degeneration in flexion and extension.     

A practical technique for non-destructive monitoring of soil surface invertebrates for ecological restoration programmes

Summary This study investigates the suitability of using wooden discs as facsimiles for natural fallen logs to non‐destructively monitor invertebrates. Wooden discs cut from tree trunks were placed on the ground at five sites and monitored. A diverse range of species was found under the discs including large numbers of some species. On Quail Island, Pine, Macrocarpa, Oak and Beech discs were compared. Beech discs had significantly fewer invertebrates and recognizable taxonomic units than other disc types. Pine discs had significantly more flatworms than the other three disc types. A comparison of invertebrate numbers when discs were placed directly on soil or grass indicated that larger numbers of individuals and recognizable taxonomic units were found under discs placed on bare soil compared with those on grass. Discs with a soil cavity beneath had significantly fewer individuals and recognizable taxonomic units than discs without cavities. Carabids showed considerable seasonal variation, with highest numbers in spring and lowest in winter, and larger numbers were found under discs at moist sites. Skinks over‐wintered under discs in drier open sites at Lincoln and Quail Island. Disc condition was assessed after 28 months and showed Macrocarpa had deteriorated the most of the four disc types. Wooden discs are a useful tool for the ecological management of terrestrial invertebrate fauna in restoration programmes for monitoring and considerable potential exists for discs to provide habitat for restoration of terrestrial invertebrate taxa.     

Importance of coalescent development of Gracilaria sp. tetraspores

Gracilaria spp. are dominant macroalgae inhabiting the intertidal zone and are exposed to constant wave action and currents. In wild Gracilaria populations, fronds tend to occur in clusters, being grouped close to one another on the shore. It is rare to find thalli growing individually, but the reason for such a clustering development is unknown. In this study we traced the early development of tetraspores of Gracilaria sp. and examined the adhesion strength of discs. We recorded two different tetraspore‐derived disc developmental patterns: in the first pattern, individual tetraspores developed into discs; whereas in the second pattern, multiple tetraspores that had attached in proximity to each other, coalesced to form a single composite disc during early development. We found that more uprights grew from coalesced discs and that the attachment areas of coalesced discs were larger compared with those of individual discs. The adhesion strengths of coalesced discs and individual discs were analyzed. The retention percentage of coalesced discs after treatment with rapid water flow was higher than that of individual discs, suggesting that coalesced discs are better able to withstand wave flow when compared with single‐spore discs. Based on these results, we propose that the clustering of Gracilaria sp. plants at sites within the intertidal zone is a mechanism that enables this macroalgae to survive the wave action and currents that occur in this zone, and that plant recruitment in these areas is enhanced by the coalescence of holdfast discs during early development.     

Mechanical stability of some immobilised preparations for glucose isomerisation in fixed and agitated bed columns

Immobilised preparations mechanical stability was studied by an analysis of particle disintegration in fixed and agitated beds of laboratory reactors. The analysis was carried out by comparing the particle size distributions obtained for three different states within the overall time-course of operation: The initial state, an intermediate state and the final one. It was found that mechanical stability by both fixed bed operation and paddle mixing was high. Agitation by flat discs was shown to cause intensive particle attrition. The relationships observed are discussed with regard to the flow pattern of the various types of reactors.     

Female-specific wing degeneration is triggered by ecdysteroid in cultures of wing discs from the bagworm moth, Eumeta variegata (Insecta: Lepidoptera, Psychidae)

Female adults of the bagworm moth, Eumeta variegata, are completely wingless; by contrast, the male adults have functional wings. Sex-specific differences in the development of wing discs appear to arise during the 8th (penultimate) larval instar. We have previously found that the wing discs of female E. variegata terminate development and disappear during the prepupal period, whereas the wing discs of males continue to develop fully into adult wings. We have investigated the effects of ecdysteroid (20-hydroxyecdysone, 20E) when cultured with larval wing discs, which are normally attached to the larval integument of both male and female larvae. Male wing discs cultured with 20E undergo a remarkable transformation: the discs undergo apolysis and then differentiation. Female wing discs cultured with 20E also undergo apolysis; however, the disc cells enter apoptosis. We have observed condensed chromatin, fragmented nuclei, and secondary lysosomes in the epithelial cells of these female discs. This report establishes that the reduction of female wing discs arises through apoptotic events triggered by ecdysteroid in vitro.     

Cullin-3 regulates pattern formation, external sensory organ development and cell survival during Drosophila development

Ubiquitin-mediated proteolysis regulates the steady-state abundance of proteins and controls cellular homoeostasis by abrupt elimination of key effector proteins. A multienzyme system targets proteins for destruction through the covalent attachment of a multiubiquitin chain. The specificity and timing of protein ubiquitination is controlled by ubiquitin ligases, such as the Skp1-Cullin-F box protein complex. Cullins are major components of SCF complexes, and have been implicated in degradation of key regulatory molecules including Cyclin E, beta-catenin and Cubitus interruptus. Here, we describe the genetic identification and molecular characterisation of the Drosophila Cullin-3 homologue. Perturbation of Cullin-3 function has pleiotropic effects during development, including defects in external sensory organ development, pattern formation and cell growth and survival. Loss or overexpression of Cullin-3 causes an increase or decrease, respectively, in external sensory organ formation, implicating Cullin-3 function in regulating the commitment of cells to the neural fate. We also find that Cullin-3 function modulates Hedgehog signalling by regulating the stability of full-length Cubitus interruptus (Ci155). Loss of Cullin-3 function in eye discs but not other imaginal discs promotes cell-autonomous accumulation of Ci155. Conversely, overexpression of Cullin-3 results in a cell-autonomous stabilisation of Ci155 in wing, haltere and leg, but not eye, imaginal discs suggesting tissue-specific regulation of Cullin-3 function. The diverse nature of Cullin-3 phenotypes highlights the importance of targeted proteolysis during Drosophila development.     

Pupal commitment and its hormonal control in wing imaginal discs

The timing of pupal commitment of the forewing imaginal discs of the silkworm, Bombyx mori, was determined by a transplantation assay using fourth instar larvae. The wing discs were not pupally committed at the time of ecdysis to the fifth instar. Pupal commitment began shortly after the ecdysis and was completed in 14 h. When the discs of newly molted larvae (0-h discs) were cultured in medium containing no hormone, they were pupally committed in 26 h. In vitro exposure of 0-h discs to 20-hydroxyecdysone accelerated the progression of pupal commitment. Methoprene, a juvenile hormone analog (JHA), did not suppress the change in commitment in vitro at physiological concentrations. Thus the wing discs at the time of the molt have lost their sensitivity to JH, and 20E is not a prerequisite for completion of pupal commitment. These results suggest that the change in commitment in the forewing discs may begin before the last larval molt.     

Chitin synthesis in Spodoptera frugiperda wing imaginal discs. III: Role of the peripodial membrane

We determined the contribution of the peripodial membrane to chitin synthesis in cultured wing imaginal discs of Spodoptera frugiperda. This was accomplished by examining chitin synthesis in vitro in intact imaginal discs, in the peripodial membrane, and in imaginal discs in which the peripodial membrane had been injured. Chitin synthesis in peripodial membrane-deprived imaginal discs, peripodial membrane injured imaginal discs, and peripodial membrane fragments was assessed by measuring incorporation of [¹⁴C]GlcNAc after treatment with 20-hydroxyecdysone in tissue culture. Removing or injuring the peripodial membrane resulted in a marked decrease in ecdysteroid-dependent chitin synthesis in these wing discs compared with intact wing discs. In addition, a break in the ecdysteroid treatment of 4 h reduced chitin synthesis in the wing discs substantially. These biochemical experiments were supplemented with ultrastructural and immunocytochemical approaches. A wheat germ agglutinin colloidal gold complex was used to visualize the presence of chitin synthesized by wing discs including the peripodial membrane. These experiments confirmed the importance of an intact peripodial membrane for optimal production of cuticle by the wing pouch. Our results demonstrate that for opti-ma1 production of chitin in tissue culture, wing discs must be treated with 20-hydroxyecdysone for an uninterrupted period of 48 h, and the peripodial membrane of these imaginal discs must be present and uninjured. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.