Baculoviruses as vectors for foreign gene expression in insect cells

Recombinant DNA technology has enabled the expression of a large number of eukaryotic genes in vitro. There has been growing interest in recent years in the development of baculovirus expression vectors as an easily manipulated gene expression system. Both virus and insect cell culture are relatively easy to handle, and biologically active proteins have been produced abundantly from a variety of eukaryotic, prokaryotic and viral genes. This review describes the molecular biology of baculoviruses and some of the current applications of the baculovirus expression system in insect cells. Specific emphasis is placed on those features relevant to the use of this system in pesticide research.     

A patch—clamp study of the action of a nitromethylene heterocycle insecticide on cockroach neurones growing in vitro

The mode of action of a nitromethylene heterocycle (NMH) insecticide was studied by patch–clamp techniques using cockroach embryonic cultures as an experimental model. Under whole-cell recordings, this compound elicited inward currents resembling those induced by O-acetylcholine (ACh). The reversal potentials for both ACh and the NMH were similar, suggesting that the inward currents induced by both were carried by the same species of ion. Pharmacological investigations of NMH-induced responses revealed that the insecticidal action of this compound is exerted through agonistic action at the nicotinic acetylcholine receptor. Single-channel studies were also performed to study the interaction of NMH with the nicotinic-receptor-coupled ion channel.     

Interleukin-4 and interferon-gamma gene expression in summer pasture-associated obstructive pulmonary disease affected horses

We hypothesised that horses affected with summer pasture-associated obstructive pulmonary disease (SPAOPD) react to an allergen or allergens in their summer environment that is either absent or present at lower levels in their winter environment; and that such allergens stimulate SPAOPD-affected horses to produce a different T helper lymphocyte cytokine profile from that of control horses. The primary objective of this study was to determine the cytokine mRNA profile of T helper lymphocytes obtained from summer pasture-associated obstructive pulmonary disease (SPAOPD) affected horses when 1) the horses were showing signs of disease (summer) and 2) they were in clinical remission (winter). A further objective was to determine the differences between cytokine mRNA T helper lymphocyte profiles of control and affected horses in the summer and winter seasons. Interleukin 4 (IL-4) and interferon-gamma (IFN-gamma) mRNA expression levels were increased in bronchoalveolar lavage fluid (BALF) and peripheral blood mononuclear cell (PBMC) samples of affected horses during disease expression. No significant amounts of IL-5 mRNA were detected in any of the samples. These results suggest that there is an allergic component to SPAOPD of horses and that appropriate manipulation of the immune system could offer hope for treatment and prevention of the disease in the future. Further research studies will be needed to determine the most appropriate treatments to use to alter the antigen-stimulated cytokine profile being expressed by SPAOPD-affected horses or to alter the effects that these cytokines produce.     

Modeling the effect of physiological responses to green pruning on net biomass production of Eucalyptus nitens

Green pruning of Eucalyptus nitens (Deane and Maiden) Maiden increases instantaneous rates of light-saturated CO(2) assimilation (A), and changes patterns of total leaf area and foliage distribution. We investigated the importance of such changes on the rate of recovery of growth following pruning. A simple process-based model was developed to estimate daily net biomass production (G(d)) of three-year-old plantation-grown trees over a 20-month period. The trees had been pruned by removal of 0, 50 or 70% of the length of green crown, equivalent to removal of 0, 55 or 88% of leaf area, respectively, when the plantation verged on canopy closure. Total G(d) was reduced by only 20% immediately following the 50%-pruning treatment, as a result of both the high leaf dark respiration and low A in the portion of the crown removed compared to the top of the crown. Pruning at the time of canopy closure preempted a natural and rapid decline in G(d) of the lower crown. Although leaf area index (L) was approximately 6.0 at the time of pruning, high light interception (95%) occurred with an L of 4.0. The 50%-pruning treatment reduced L to 3.5, but the physiological responses to pruning were sufficient to compensate fully for the reduction in intercepted radiation within 110 days of pruning. The 70%-pruning treatment reduced L to 1.9, and reduced G(d) by 77%, reflecting the removal of branches with high A in the mid and upper crown. Physiological responses to the 70%-pruning treatment were insufficient to increase G(d) to the value of unpruned trees during the study. Model sensitivity analysis showed that increases in A following pruning increased G(d) by 20 and 25% in the 50- and 70%-pruned trees, respectively, 20 months after pruning. Changes in leaf area/foliage distribution had a greater effect on G(d) of 50%-pruned trees (47% increase) than did changes in A. However, the reduction in photosynthetic potential associated with the 70%-pruning treatment resulted in only small changes in leaf area/foliage distribution, which consequently had little effect on G(d). The effects of physiological processes occurring within the crown and in response to green pruning on G(d) are discussed with respect to pruning of plantations.     

Variation of sapflow velocity in Eucalyptus globulus with position in sapwood and use of a correction coefficient

Sapflow sensors were used to investigate variation in sapflow velocity at different positions in the sapwood of three-year-old Eucalyptus globulus ssp. globulus Labill. trees. Sapflow velocity was measured at 5-mm intervals across the sapwood by moving two probe sets simultaneously on two opposite radii. Another probe set placed in a fixed position at right angles to the first two sets acted as a control. A sapflow velocity ratio was defined as the velocity given by each moving sensor divided by that given by the static sensor. Correlation between observations of sapflow velocity at different positions exceeded 95%, and the ratio of velocity between any pair of sensors was constant. We observed radial variation in sapflow velocity across the sapwood with the lowest velocities at the center of the tree. Variation due to sensor position was high implying the need for large numbers of sensors for accurate estimates of sap flux. To overcome this need, we used a correction coefficient, namely a simple weighted average of the sapflow ratios with depth in the sapwood, for each fixed sensor. We recommend the use of three probe sets to estimate the correction coefficient. Subsequently, two probe sets can be placed at two fixed positions for routine measurements of sap flux.     

Growth responses, physiology and decay associated with pruning plantation-grown Eucalyptus globulus Labill. and E. nitens (Deane and Maiden) Maiden

Detailed studies have been undertaken to define pruning regimes for Eucalyptus nitens, but little is known of E. globulus responses to pruning although this is a more commonly planted species. This paper describes experiments that aimed to identify (a) levels of pruning that reduce E. globulus growth, (b) physiological processes contributing to those growth responses, (c) the incidence of decay and factors influencing decay incidence following pruning of E. globulus, and (d) comparative responses of E. nitens and E. globulus to live branch pruning. Results of a field experiment indicated that removal of between 30 and 50% of the crown length was appropriate for E. globulus plantations verging on canopy closure. The significant reduction in height growth associated with removal of 50 or 70% of crown length suggested pruning should remain below 50% of crown length if reduced stem growth of pruned trees was to be avoided. Stem volume was only significantly reduced over the period of the experiment by 70% pruning, but it was estimated that standing volume following removal of 50% of crown length would be reduced by 82 m³ ha⁻¹ over a 20-year rotation if there were no other silvicultural interventions. The growth responses observed were probably related to large reductions in leaf area following 50 or 70% removal of crown length. Trees responded to pruning by changing patterns of biomass partitioning to favour stem growth at the expense of branch growth. A glasshouse study determined that light-saturated net CO₂ uptake (A_max) increased following pruning. E. nitens seedlings had both a higher baseline A_max and higher A_max following pruning than did E. globulus, which could partially explain the greater effect of pruning on E. globulus growth than has been observed for E. nitens in other studies. This result, as well as apparently different patterns of foliage distribution through the crowns of E. globulus and E. nitens, suggested that models of pruning responses need to be parameterised for both species. In addition, a more conservative pruning regime may be appropriate for E. globulus than E. nitens. Pruning increased the frequency of branch traces with decay infection, and there was a trend towards increasing decay outbreaks with increasing pruning severity. Decay outbreaks were more likely to occur following pruning of high angle or larger diameter branches.     

Leaf water relations of Eucalyptus globulus ssp. globulus and E. nitens: seasonal, drought and species effects

In August 1990, a 2-ha plantation was established in an area where rainfall (about 515 mm year(-1)) was insufficient to meet evaporative demand. On nine occasions between September 1991 and April 1993, pressure-volume curves were constructed for irrigated and rainfed Eucalyptus globulus ssp. globulus Labill. and E. nitens (Deane and Maiden) Maiden trees. During the experiment, rainfed trees experienced six periods when predawn water potential was significantly lower than that of irrigated trees. In early spring of 1991 and 1992, osmotic potentials at full turgor and turgor loss point in the irrigated E. nitens were significantly lower than at other times of the year, probably because of winter hardening. Water stress reduced osmotic potential and increased bulk elastic modulus in E. nitens, whereas the reverse occurred in E. globulus. However, treatment differences with respect to changes in osmotic and elastic properties were commonly overshadowed by interspecific differences. These were most apparent at the end of the sixth period of water stress when osmotic potentials at full and zero turgor were significantly higher and bulk elastic modulus and relative water content at turgor loss point were significantly lower in E. globulus than in E. nitens. We conclude that the drought-tolerance responses of E. globulus make it a more suitable species than E. nitens for establishment on sites where moderate water stress is experienced.     

Characterization of normal tidal breathing flow-volume loops for Thoroughbred horses

The purpose of this study was to characterize the normal equine tidal breathing flow-volume loop (TBFVL). The study was performed using 18 healthy Thoroughbred horses. TBFVLs constructed from data collected from resting horses had a typical biphasic inspiratory and expiratory phase. The interindividual variability of the indices used to describe TBFVLs was in the range 16–32%, which is comparable to the variability of other measures of equine pulmonary mechanics. The large variability of these data probably limits the value of resting TBFVL indices for detecting subclinical respiratory conditions in individual horses. Factor analysis of these data revealed that in excess of 90% of the variance of the initial response variables could be explained in terms of three common factors. Varimax rotation of these three common factors provided three subsequent factors that were readily identifiable as (1) a factor describing the time-volume relationships of TBFVLs, responsible for 81% of the total variance, (2) a factor explaining the expiratory portion of the TBFVL, explaining 12% of the variance, and (3) a factor describing the inspiratory portion of the loops, responsible for the remaining 7% of the variance. The analysis also provided standardized factor scoring coefficients for use in subsequent studies using similar experimental techniques.Abbreviations EF₂₅ expiratory flow at expiratory volume plus 25%V _E - EF₅₀ midtidal expiratory flow - f respiratory rate - IF₂₅ inspiratory flow at 25% of inspiratory volume - IF₅₀ midtidal inspiratory flow - MSA measure of sampling adequacy - PEF peak expiratory flow - PIF peak inspiratory flow - TBFVL tidal breathing flow-volume loop - T _E expiratory time - T _I inspiratory time - USPTM ultrasonic pneumotachometer - V _E expiratory volume - V _I inspiratory volume