Stirred tank culture of Bacillus thuringiensis H-14 for production of the mosquitocidal δ-endotoxin: mathematical modelling and scaling-up studies

The effect of culture conditions on 948.gif" alt="delta" align="BASELINE" BORDER="0">-endotoxin production by strain S128 of Bacillus thuringiensis H-14 (locally isolated in Egypt) was investigated using a 10l working volume fermenter. Fermentation medium formulated from the inexpensive locally available soya (as a nitrogen source) and molasses (as a carbon source) was used. Aeration, agitation, pH and initial concentration of molasses were chosen as experimental factors and their influence on toxin yield was investigated using 2⁴ central composite experimental design. The mathematical model obtained revealed that the optimal batch cultivation conditions with respect to agitation, pH, and initial concentration of molasses were 325 rev min^–1, 7.1 and 2.1% (w/v) respectively. The mathematical model obtained indicated that by increasing the aeration rate over 0.89 v/v per minute the productivity could still be increased. A simulated scaling-up study, in which the Simplex method was applied, is also presented. The results of this investigation could be of great help for large-scale production of a cheap mosquito-larvicide in developing countries where mosquito-borne diseases are still a serious health and economic problem.     

Stable oxygen isotopes in<Emphasis Type="Italic"> Porites</Emphasis> corals monitor weekly temperature variations in the northern Gulf of Aqaba,Red Sea

In order to assess the ability of Porites corals to accurately record environmental variations, high-resolution (weekly/biweekly) coral 948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁸O records were obtained from four coral colonies from the northern Gulf of Aqaba, which grew at depths of 7, 19, 29, and 42 m along one transect. Adjacent to each colony, hourly temperatures, biweekly salinities, and monthly 948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁸O of seawater were continuously recorded over a period of 14 months (April 1999 to June 2000). Contrary to water temperature, which shows a regular and strong seasonal variation and change with depth, seawater 948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁸O exhibits a weak seasonality and little change with depth. Positive correlations between seawater 948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁸O and salinity were observed. The two parameters were related to each other by the equation 948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁸O _Seawater (, VSMOW) = 0.281 × Salinity – 9.14. The high-resolution coral 948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁸O records from this study show a regular pattern of seasonality and are able to capture fine details of the weekly average temperature records. They resolve more than 95% of the weekly average temperature range. On the other hand, attenuation and amplification of coral seasonal amplitudes were recorded in deep, slow-growing corals, which were not related to environmental effects (temperature and/or seawater 948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁸O) or sampling resolution. We propose that these result from a combined effect of subannual variations in extension rate and variable rates of spine thickening of skeletal structures within the tissue layer. However, no smoothing or distortion of the isotopic signals was observed due to calcification within the tissue layer in shallow-water, fast-growing corals. The calculations from coral 948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁸O calibrations against the in situ measurements show that temperature (T) is related to coral 948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁸O (948.gif" alt="delta" align="BASELINE" BORDER="0"> _c ) and seawater 948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁸O (948.gif" alt="delta" align="BASELINE" BORDER="0"> _w ) by the equation T (°C) = –5.38 (948.gif" alt="delta" align="BASELINE" BORDER="0"> _c – 948.gif" alt="delta" align="BASELINE" BORDER="0"> _w ) –1.08. Our results demonstrate that coral 948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁸O from the northern Gulf of Aqaba is a reliable recorder of temperature variations, and that there is a minor contribution of seawater 948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁸O to this proxy, which could be ignored.     

Calcium Channel α2δ Subunits: Differential Expression, Function, and Drug Binding

Voltage-activated calcium channels are transmembrane proteins that act as transducers of electrical signals into numerous intracellular activities. On the basis of their electrophysiological properties they are classified as high- and low-voltage-activated calcium channels. High-voltage-activated calcium channels are heterooligomeric proteins consisting of a pore-forming alpha1 subunit and auxiliary alpha2delta, beta, and--in some tissues--gamma subunits. Auxiliary subunits support the membrane trafficking of the alpha1 subunit and modulate the kinetic properties of the channel. In particular, the alpha2delta subunit has been shown to modify the biophysical and pharmacological properties of the alpha1 subunit. The alpha2delta subunit is posttranslationally cleaved to form disulfide-linked alpha2 and, delta proteins, both of which are heavily glycosylated. Recently it was shown that at least four genes encode for alpha2delta subunits which are expressed in a tissue-specific manner. Their biophysical properties were characterized in coexpression studies with high- and low-voltage-activated calcium channels. Mutations in the gene encoding alpha2delta-2 have been found to underlie the ducky phenotype. This mouse mutant is a model for absence epilepsy and is characterized by spike wave seizures and cerebellar ataxia. Alpha2delta subunits can also support pharmacological interactions with drugs that are used for the treatment of epilepsy and neuropathic pain.     

Chlorophyll reduction in the seed of Brassica napus with a glutamate 1-semialdehyde aminotransferase antisense gene*

Chlorophyll reduction in the seed of Brassica can be achieved by downregulating its synthesis. To reduce chlorophyll synthesis, we have used a cDNA clone of Brassica napus encoding glutamate 1-semialdehyde aminotransferase (GSA-AT) to make an antisense construct for gene manipulation. Antisense glutamate 1-semialdehyde aminotransferase gene (Gsa) expression, directed by a Brassica napin promoter, was targeted specifically to the embryo of the developing seed. Transformants expressing antisense Gsa showed varying degrees of inhibition resulting in a range of chlorophyll reduction in the seeds. Seed growth and development were not affected by reduction of chlorophyll. Seeds from selfed transgenic plants germinated with high efficiency and growth of seedlings was vigorous. Seedlings from T₂ transgenic lines segregated into three distinctive phenotypes: dark green, light green and yellow, indicating the dominant inheritance of Gsa antisense gene. These transgenic lines have provided useful materials for the development of a low chlorophyll seed variety of B. napus.     

Significance of rooting depth in mire plants: Evidence from natural <Superscript>15</Superscript>N abundance

Variation in stable nitrogen isotope ratios (948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁵N) was assessed for plants comprising two wetland communities, a bog-fen system and a flood plain, in central Japan. 948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁵N of 12 species from the bog-fen system and six species from the flood plain were remarkably variable, ranging from –5.9 to +1.1 and from +3.1 to +8.7, respectively. Phragmites australis exhibited the highest 948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁵N value at both sites. Rooting depth also differed greatly with plant species, ranging from 5cm to over 200cm in the bog-fen system. There was a tendency for plants having deeper root systems to exhibit higher 948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁵N values; plant 948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁵N was positively associated with rooting depth. Moreover, an increasing gradient of peat 948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁵N was found along with depth. This evidence, together with the fact that inorganic nitrogen was depleted under a deep-rooted Phragmites australis stand, strongly suggests that deep-rooted plants actually absorb nitrogen from the deep peat layer. Thus, we successfully demonstrated the diverse traits of nitrogen nutrition among mire plants using stable isotope analysis. The ecological significance of deep rooting in mire plants is that it enables those plants to monopolize nutrients in deep substratum layers. This advantage should compensate for any consequential structural and/or physiological costs. Good evidence of the benefits of deep rooting is provided by the fact that Phragmites australis dominates as a tall mire grass.     

Beaver alter stable carbon isotope ratios of benthic particulate organic matter

Beaver are well known to influence the geomorphology and geochemistry of boreal waters, in addition to being prominent vectors in the selective import of deciduous coarse woody debris (CWD). Because the stable carbon isotope ratios of deciduous trees are lower than those of coniferous trees, the possibility exists that the 948.gif" alt="delta" align="BASELINE" BORDER="0">¹³C values of particulate organic matter near beaver lodges may differ from those characteristic for littoral regions without beaver activity. In agreement with this supposition, 948.gif" alt="delta" align="BASELINE" BORDER="0">¹³C was found to significantly increase in progressively smaller size fractions of CWD collected from near lodges, probably due to the more rapid breakdown, decomposition and disappearance of deciduous material.     

Identification and purification of the 69-kDa intracellular protease involved in the proteolytic processing of the crystal delta-endotoxin of Bacillus thuringiensis subsp. tenebrionis

The dynamics of appearance of intracellular proteases in relation to the synthesis of crystal delta-endotoxin was studied to identify the native intracellular protease(s) involved in the proteolytic processing of the 73-kDa protoxin of Bacillus thuringiensis subsp. tenebrionis. In vitro proteolytic activation of the 73-kDa protoxin indicated the possible role of 69-kDa protease in the proteolytic processing of 73-kDa protoxin. The purified 69-kDa protease was able to cause the proteolytic activation of the 73-kDa protoxin to 68-kDa toxin and this conversion was inhibited by ethylenediamine tetraacetic acid and 1,10-phenanthroline.     

Suppression of polydactyly of the Gli3 mutant (extra toes) by deltaEF1 homozygous mutation

Digit patterning is established through multiple genetic interactions. Delta-crystallin enhancer/E2-box factor (deltaEF1) is a zinc finger and homeodomain containing repressor protein, and is expressed in the posterior half of the forelimb bud and in the entire hindlimb bud during the early stage of limb development. The 6EF1-deficient mutant mice display various skeletal abnormalities, among which inferior ossification and abnormal patterning of autopodial bones are similar to those observed in Hox and Gli gene mutants. Gli3 mutant mice, extra toes (Xt), exhibit pre-axial polydactyly losing the identity of digit I. It is demonstrated here that deltaEF1null(lacZ) homozygosity suppressed formation of the extra digit, uniquely of the hindlimb, in both Gli3XtJ heterozygous and homozygous mutants, but with no restoration of digit I identity. In Gli3XtJ mutants, the Hoxd13 expression domain was expanded more dramatically in homozygotes. In Gli3XtJ;deltaEF1null(lacZ) double homozygous mutants, Hoxd13 expression once expanded in Gli3XtJ homozygous mutant was reduced, more conspicuously in the hindlimbs, which may account for hindlimb-restricted suppression of formation of the extra digit. The data suggest the possibility that the extent of Hoxd13 expression along the distal margin of the limb bud is determinative in defining the digit number.     

Variations in the natural abundance of 15N in ryegrass/white clover shoot material as influenced by cattle grazing

Biological N2 fixation in clover is an important source of N in low external-N input farming systems. Using the natural 15N-abundance method, variations in N2 fixation were investigated in grazed and mowed plots of a ryegrass/white clover field. Ryegrass 948.gif" alt="delta" align="BASELINE" BORDER="0">15N varied considerably, from 0.2 to 5.6 under mowed conditions and from –3.3 to 11.6 under grazed conditions. Variations in 948.gif" alt="delta" align="BASELINE" BORDER="0">15N white clover were lower than in ryegrass, especially in the mowed plots (SE = 0.05, n = 20). The variations in the percentage of nitrogen derived from the atmosphere (%Ndfa) in white clover were highest in the grazed plots where it ranged from 12 to 96% (mean = 64%) compared with the mowed plots where it ranged from 64 to 92% (mean = 79%). Thus, the N2 fixation per unit white clover DM in the grazed ley was lower and more variable than under mowing conditions.Urine from dairy cows equivalent to 0, 200, 400 and 800 kg N ha-1 was applied to a ryegrass/white clover plot 6, 4 or 2 weeks before harvest. Without urine application 948.gif" alt="delta" align="BASELINE" BORDER="0">15N of ryegrass was positive. By increasing urine application (948.gif" alt="delta" align="BASELINE" BORDER="0">15N = –1) two weeks before sampling, the 948.gif" alt="delta" align="BASELINE" BORDER="0">15N of ryegrass decreased strongly to about –7 (P < 0.001). However, this effect was only observed when urine was applied two weeks before sampling. When applying 800 kg N four and six weeks before sampling, 948.gif" alt="delta" align="BASELINE" BORDER="0">15N in ryegrass was not significantly different from the treatment without urine application. White clover 948.gif" alt="delta" align="BASELINE" BORDER="0">15N was unaffected by whatever changes occurred in 948.gif" alt="delta" align="BASELINE" BORDER="0">15N of the plant-available soil N pool (reflected in 948.gif" alt="delta" align="BASELINE" BORDER="0">15N of ryegrass). This indicates that within the time span of this experiment, N2 fixation per unit DM was not affected by urine. Therefore, newly deposited urine may not be the main contributing factor to the variation in %Ndfa found in the grazed fields. This experiment suggested that the natural abundance method can be applied for estimating %Ndfa without disturbance in natural animal-grazed systems.     

A fusion gene coding for two different δ-endotoxinsof Bacillus thuringiensis toxic to Plutella xylostella and usefulfor resistance management

Two truncated Bacillus thuringiensis 948.gif" alt="delta" align="BASELINE" BORDER="0">-endotoxin genes, belonging to the classes cry1Ab and cry1B, and both coding for N-terminal toxic fragments of the corresponding crystal proteins, were translationally fused. Expression of the fusion gene driven by the cry1C promoter in Escherichia coli at a very high level resulted in a protein with enhanced toxicity to the diamondback moth (Plutella xylostella).