Expression, Purification, and Initial Characterization of the Recombinant Storage Protein Precursor ofTheobroma cacao

The gene encoding the 67-kDa cocoa storage protein precursor has been cloned fromTheobroma cacaoand expressed inEscherichia coliusing the pET expression system. The recombinant storage protein has been renatured from inclusion bodies at 30°C using 20 m glycine–NaOH buffer, pH 10.0, containing 1 m oxidized glutathione and 0.1% Brij. The renatured protein was purified and demonstrated to adopt a stable native conformation by optical spectroscopy. Secondary structure analysis from circular dichroism indicated the protein to be 23% α-helix and 38% β-sheet, in close agreement with values obtained using a secondary structure prediction program.     

Assessing post-induction cellular response in a recombinant E. coli lactose-inducible system by monitoring β-galactosidase levels

Summary A synthetic lactose-inducible promoter was chosen to study host cell responses to the over-expression of heterologous genes. Fermentations were conducted to compare the effect of induction strategies on the synthesis of -galactosidase versus the production of recombinant protein. The levels of lactose, IPTG and glucose during induction were manipulated to adjust the utilization of lactose as the inducer and/or the carbon source. In addition, the involvement of the gal operon in lactose metabolism was also explored in order to optimize lactose transport and utilization during induction.     

Bulk-phase viscoelastic properties of seawater relationship with plankton components

The viscous and elastic moduli at different shear rates, togetherwith various biological oceanographic properties, were determinedin seawater from different hydrological layers in the southernNorth Sea in June. The biological oceanographic parameters includedPhaeocystis and Noctiluca abundances, chlorophyll a level (Chl),bacteria. HNAN and aggregate volume fraction. The plankton wasjointly dominated by Phaeocyslis sp. and Noctiluca scinullans.Noctiluca abundance showed no correlation with any other biologicalor viscoelastic parameter, but Phaeocystis abundance correlatedstrongly. The other biological parameters correlated with Phaeocystisand with each other positively and mostly significantly. Overall,viscoelasticity correlated more strongly with Chl than withany other biological parameter. For non-microlayer samples,the excess complex (viscoelastic) modulus (µ.Pa) G^*_E =2.0 x Ch1^1–3 (Chl in mg m^–3). Viscous and elasticmoduli also correlated closely with each other. For a givenvalue of Chl. the microlayer samples were 6.5 or 14 times (dependingon the estimation method) more viscoelastic than in bulk-phasesamples. Viscoelasticity in samples of settled benthic ‘fluff’were lower even than bulk-phase samples, but this differencewas not significant. Comparison with Mediterranean data on viscoelasticity(Jenkinson. Oceanol. Acta, 16, 317–334, 1993), using publishedvalues for phytoplankton biomass (Wiadnyana, J. Rech. Océanogr.,17, 1–6, 1992), suggests that the relationship betweenChl (or phytoplankton biomass) and viscoelasticity might begeneral. This apparent biomodification of the viscosity andelasticity of seawater is discussed in relation to its likelyimpact on turbulence and plankton ecology.     

Functional molecular aspects of the NADH dehydrogenases of plant mitochondria

There are multiple routes of NAD(P)H oxidation associated with the inner membrane of plant mitochondria. These are the phosphorylating NADH dehydrogenase, otherwise known as Complex I, and at least four other nonphosphorylating NAD(P)H dehydrogenases. Complex I has been isolated from beetroot, broad bean, and potato mitochondria. It has at least 32 polypeptides associated with it, contains FMN as its prosthetic group, and the purified enzyme is sensitive to inhibition by rotenone. In terms of subunit complexity it appears similar to the mammalian and fungal enzymes. Some polypeptides display antigenic similarity to subunits fromNeurospora crassa but little cross-reactivity to antisera raised against some beef heart complex I subunits. Plant complex I contains eight mitochondrial encoded subunits with the remainder being nuclear-encoded. Two of these mitochondrial-encoded subunits, nad7 and nad9, show homology to corresponding nuclear-encoded subunits inNeurospora crassa (49 and 30 kDa, respectively) and beef heart CI (49 and 31 kDa, respectively), suggesting a marked difference between the assembly of CI from plants and the fungal and mammalian enzymes. As well as complex I, plant mitochondria contain several type-II NAD(P)H dehydrogenases which mediate rotenone-insensitive oxidation of cytosolic and matrix NADH. We have isolated three of these dehydrogenases from beetroot mitochondria which are similar to enzymes isolated from potato mitochondria. Two of these enzymes are single polypeptides (32 and 55 kDa) and appear similar to those found in maize mitochondria, which have been localized to the outside of the inner membrane. The third enzyme appears to be a dimer comprised of two identical 43-kDa subunits. It is this enzyme that we believe contributes to rotenone-insensitive oxidation of matrix NADH. In addition to this type-II dehydrogenases, several observations suggest the presence of a smaller form of CI present in plant mitochondria which is insensitive to rotenone inhibition. We propose that this represents the peripheral arm of CI in plant mitochondria and may participate in nonphosphorylating matrix NADH oxidation.     

Diversity in cyclic sesquiterpene production by Gossypium hirsutum

Major sesquiterpene components of oil of Texas Race Stock 810 of Gossypium hirsutum were - and β-selinene. This is the seventh cyclic terpene type found to date in this genus. Both - and β-selinene, along with aromadendrene, were found but only as minor components of extracts of several domestic cultivars of G. hirsutum.     

Uptake of saline groundwater by plants: An analytical model for semi-arid and arid areas

An analytical model, based on unsaturated zone water and solute balances, was developed to describe the uptake of saline groundwater by plants in dry regions. It was assumed that: i. initially, the profile had low water and salt contents to some depth; ii. both water and solutes move upwards from the water table by piston flow due only to plant water extraction; iii. the uptake of water concentrates solutes in the soil solution until some threshold salinity is reached, above which plants can no longer extract water due to osmotic effects; iv. uptake of the groundwater does not affect the water table level; and v. uptake of groundwater is only limited by transmission of groundwater through the soil. Model predictions were compared with measurements of groundwater uptake made over 15 months at five sites in aEucalyptus forest in a semi-arid area, using independently measured model parameters. Depth and salinity of groundwater, and soil type varied greatly between sites. Predicted groundwater uptake rates were close to measured values, generally being within ∼ 0.1 mm day^-1. Sensitivity analysis showed that groundwater depth and salinity were the main controls on uptake of groundwater, while soil properties appeared to have a lesser effect. The model showed that uptake of groundwater would result in complete salinisation of the soil profile within 4 to 30 yr at the sites studied, unless salts were leached from the soil by rainfall or flood waters. However, a relatively small amount of annual leaching may be sufficient to allow groundwater uptake to continue. Thus groundwaters, even when saline, may be important sources of water to plants in arid and semi-arid areas.     

Swim Stress Increases the Potency of Glycine at the N-Methyl-d-Aspartate Receptor Complex

Abstract: We have previously demonstrated that chronic administration of antidepressants results in a reduction in the potency of glycine to displace 5,7-[³H]dichlorokynurenic acid (5,7-[³H]-DCKA) from the strychnine-insensitive glycine recognition site of the NMDA receptor complex. We now report that exposure of rats to the forced swim test results in a 56% increase in the potency of glycine to displace 5,7-[³H]DCKA from frontal cortical homogenates. These data are consistent with the hypothesis that the forced swim test, a preclinical screen sensitive to acute administration of antidepressant drugs and NMDA receptor antagonists, also results in adaptation of the NMDA receptor complex. Moreover, these data lend further support to the hypothesis that glutamatergic pathways are involved in the neurobiological response to stress and, potentially, in the pathophysiology of depression.     

The isolation of Ant1, a transposable element from Aspergillus niger

A transposable element has been isolated from the industrially important fungus Aspergillus niger (strain N402). The element was identified as an insertion sequence within the coding region of the nitrate reductase gene. It had inserted at a TA site and appeared to have duplicated the target site upon insertion. The isolated element was found to be 4798 by in length and contained 37-bp inverted, imperfect, terminal repeats (ITRs). The sequence of the central region of the element revealed an open reading frame (designated ORF1) which showed similarity, at the amino acid level, to the transposase of the Tc1/mariner class of DNA transposons. Another sequence within the central region of the element showed similarity to the 3 coding and downstream untranslated region of the amyA gene of A. niger. Sequence homology and structural features indicate that this element, which has been named Ant1 (A. niger transposon 1), is related to the Tc1/mariner group of DNA transposons. Ant1 is apparently present as a single copy in strain N402 of A. niger.     

Field-scale biofiltration of gasoline vapors extracted from beneath a leaking underground storage tank

Approximately 15000 L of unleaded gasoline werereleased into the surrounding vadose zone from aleaking underground storage tank. Initialremediation was by soil vapor extraction andcombustion which soon became cost prohibitive, asadded propane was required to reach the combustionlimit of the extracted vapors. As a cost effectivealternative, a field-scale compost based biofilterwas used in conjunction with soil vapor extractionto remediate the vadose zone. The biofilter wasconstructed on site using 4:1 diatomaceousearth:composted horse manure. Results of a fivemonth study showed that the biofilter removedapproximately 90% of total petroleum hydrocarbons(TPH) and >90% of the BTEX compounds (benzene,toluene, ethylbenzene, xylene), achieving thestringent permit requirements set at either 90% TPHreduction or less than 1.36 kg per day of volatileorganic compounds (VOC's) released to theatmosphere. The biofilter showed the capacity toreadily adapt to changing environmental conditionssuch as increased contaminant loading, andvariations in temperature and moisture. Thebacterial population in the biofilter was uniformlydiverse throughout the biofilter, suggesting that aconsortium of bacteria was needed for efficientbiodegradation. The cost of biofilter set up andoperation saved 90% in the first year alone of theoperating expenses incurred by soil vapor extractionand combustion.