Genetic manipulation of 6-phosphofructokinase in potato tubers

The aim of this work was to discover whether genetic manipulation of 6-phosphofructokinase [EC 2.7.1.11; PFK(ATP)] influenced the rate of respiration of tuber tissue of Solanum tuberosum L. Transgenic plants were produced that contained the coding sequence of the Escherichia coli pfkA gene linked to a patatin promoter. Expression of this chimaeric gene in tubers resulted in a 14to 21-fold increase in the maximum catalytic activity of PFK(ATP) without affecting the activities of the other glycolytic enzymes. Tubers, and aged disks of tuber tissue, from transformed plants showed no more than a 30% fall in the content of hexose 6-monophosphates; the other intermediates of glycolysis increased threeto eightfold. Fructose-2,6-bisphosphate was barely detectable in aged disks of transformed tubers. The relative rates of ¹⁴CO₂ production from [1-¹⁴C]-and [6-¹⁴C]-glucose supplied to disks of transformed and control tubers were similar. Oxygen uptake and CO₂ production by aged disks of transformed tubers did not differ significantly from those from control tubers. The same was true of CO₂ production, in air, and in nitrogen, for tuber tissue. It is concluded that PFK(ATP) does not dominate the control of respiration in potato tubers.Abbreviations Fru2,6bisP fructose-2,6-bisphosphate - FW freshweight - GUS -glucuronidase - PFK(ATP) 6-phosphofructokinase - PFK(PPi) pyrophosphate: fructose-6-phosphate 1-phosphotransferase     

The role of the hexose transporter in the chloroplasts of Arabidopsis thaliana L.

The metabolism of wild-type Arabidopsis thaliana L. and its mutant TC265 were compared in order to reveal the role of the chloroplast glucose transporter. Plants were grown in a 12-h photoperiod. From 20 to 40 days after germination, starch per gram fresh weight of shoot in the mutant was four times that in the wild type. The extent of this difference did not alter during this period. Stereological analysis showed that the chloroplasts in the mutant were larger than those in the wild type; the thylakoids appeared to be distorted by the high starch content. [U-¹⁴C]Glucose and [U-¹⁴C]glycerol were supplied, separately, to excised leaves in the dark. [U-¹⁴C]Glucose was a good precursor of sucrose in the wild type and mutant; [U-¹⁴C]glycerol was a poor precursor of sucrose in both. The distribution of ¹⁴C in the wild type was used to calculate that the net flux was from hexose monophosphates to triose phosphates, not vice versa. During the first 4 h of the night the sugar content (75% sucrose, 20% glucose) of the leaves of the mutant dropped sharply, and at all times during the night it was less than that of the wild-type leaves. This drop in sugar coincided with a decrease in the rate of respiration. The growth rate of the mutant was less than that of the wild type. Addition of sucrose restored the rate of respiration at night and increased the rate of growth. It is argued that a major function of the glucose transporter in Arabidopsis chloroplasts is export of the products of starch breakdown that are destined for sucrose synthesis at night.We thank Professor C.R. Somerville for his generous gift of seed of the Arabidopsis mutant TC265. We are also grateful to Mr B. Chapman for assistance with the preparation of the sections for electron microscopy. R.N.T. thanks the Science and Engineering Research Council for a studentship.     

Insect cell density in bioreactor cultures can be estimated from on-line measurements of optical density

Total cell density in growing insect cell suspension cultures was accurately estimated from both off-line and on-line measurements of optical density (OD). The latter measurements were done with an in-situ autoclavable OD sensor. The ability to continuously monitor cell density in insect cell cultures may be useful for the development of a large-scale process for recombinant protein production using baculovirus expression vectors.     

Effect of morphine and abstinence syndrome on [3H]bromoxidine binding to α2-adrenoreceptors in rat brain

At 4 days after the implantation of two subcutaneous 75 mg morphine pellets in the back skin, rats were morphine-dependent. In the three layers studied in the occipital cortex we found that the values of the ₂-adrenergic agonist [³H]bromoxidine binding increased with respect to animals implanted with placebo pellets. Typical behavioral and physiological symptoms of the abstinence syndrome appeared 30 minutes after administration of naloxone, [³H]bromoxidine binding values being similar to those obtained in animals implanted with placebo pellets. The pattern of response of the [³H]bromoxidine binding was similar in the hippocampus and the superficial gray layer of the superior colliculus of the mesencephalon, but the differences were not statistically significant in these areas. This paper concludes that exist brain regional differences in the ₂-adrenoreceptors response under morphine-treatment and possibly under naloxone-induced morphine abstinence syndrome.     

Maximization of recombinant protein yield in the insect cell/baculovirus system by one-time addition of nutrients to high-density batch cultures

Suspension cultures of Sf-9 cells at different stages of growth were infected with a recombinant baculovirus expressing -galactosidase, using a range of multiplicities of infection (MOI) of 0.05 to 50. Following infection, the cells were resuspended either in the medium in which they had been grown or in fresh medium. Specific -galactosidase yields were not markedly affected by either MOI or medium change in cultures infected in early exponential phase (3×10⁶ cells mL^–1). In cultures infected at later growth stages, -galactosidase yields could only be maintained by medium replacement. The possibility that this requirement for medium replacement is due either to the accumulation of an inhibitory byproduct or nutrient limitation was examined. Alanine, a major byproduct of cultured insect cell metabolism, did not significantly reduce recombinant protein yield when added to infected cultures in concentrations of up to 40 mM. Following a factorial design, various nutrient concentrates were added alone or in combination to cultures infected in late exponential phase. Additions that included both yeastolate ultrafiltrate and an amino acid mixture restored specific -galactosidase yields to levels observed at earlier growth stages or in late stages with medium replacement; the addition of these concentrates, by permitting production at higher cell density, led to increases in the volumetric yield of recombinant protein. Together or separately, the concentrates when added to uninfected late exponential phase cultures, lead to a doubling of the maximum total cell protein level normally supported by unamended medium.     

Endophytic fungi alter foraging and dispersal by desert seed-harvesting ants

Endophytic fungi are thought to interact mutualistically with host plants by producing alkaloid metabolites that deter herbivory. Since such fungi are transmitted via seed in some grasses, the presence of endophytes may also protect plants from seed predators. We conducted seed choice experiments for two dominant seed harvesting ants, Pogonomyrmex rugosus in the Sonoran desert and Pogonomyrmex occidentalis at a higher elevation, riparian zone in Arizona, USA. Non-infected fescue (Festuca arundinacea) seeds and seeds infected with the endophytic fungus, Acremonium coenophialum, were presented to ant colonies in three different populations. Infected seeds were harvested less frequently than non-infected seed for the two populations of Pogonomyrmex rugosus but not for the population of Pogonomyrmex occidentalis. We also a conducted seed dispersal experiment for one population of Pogonomyrmex rugosus. Of the seeds that were harvested, most of the colonies discarded more infected seeds into refuse piles than expected by chance. Seeds discarded into refuse piles have greater germination success than surrounding areas. The most important interaction of endophytes and grasses may be deterrence of seed predation and enhancing the probability of germinating in favorable sites, since these processes directly increase plant fitness.     

Plant clonality: Biology and diversity

The current approaches to the study of clonal plants are reviewed. Most studies concentrate at the level of the ramet and clonal fragment exploring the “microscopic” view of clonal plants, dealing with the translocation of resources, clonal integration, plasticity of growth etc. The information gained, by this approach can be used in the understanding of higher levels of organization within the clonal system either with the help of spatially explicit modelling techniques, or by using means and distributions of size within a population instead of studying individual ramets separately. Plant scientists use the term clone with two meanings, viz. (a) a set of physiologically connected, but potentially independent ramets, and (b) a set of genetically identical, but potentially physically separated individuals. The overlap of these terms differs between individual plant species, depending on the extent of physical separation of the ramets and the degree of physiological integration between the ramets; the lower the frequency of ramet separation, the closer are the physiological and genetic concepts of the clone. Three critical areas seem to be neglected in clonal plant research: (a) the interrelationship between hierarchical levels in clonal plants, (b) the particular spatial structure of their environment, and (c) the importance of clonal plants in different ecological communities.     

Fluxes of carbohydrate metabolism in ripening bananas

The major fluxes of carbohydrate metabolism were estimated during starch breakdown by ripening bananas (Musa cavendishii Lamb ex Paxton). Hands of bananas, untreated with ethylene, were allowed to ripen in the dark at 21° C. Production of CO₂ and the contents of starch, sucrose, glucose and fructose of intact fruit were determined for a period of 10 d that included the climacteric. The detailed distribution of label was determined after supplying the following to cores of pulp from climacteric fruit: [U-¹⁴C]-, [1-¹⁴C]-, [3,4-¹⁴C]-and [6-¹⁴C]glucose, [U-¹⁴C]glycerol, ¹⁴CO₂. The data obtained were used to estimate the following fluxes, values given as mol hexose · (g FW)^–1 · h^–1 in parenthesis: starch to hexose monophosphates (5.9) and vice versa (0.4); hexose monophosphates to sucrose (7.7); sucrose to hexose (4.7); hexose to hexose monophosphate (3.8); glycolysis (0.5–1.6); triose phosphate to hexose monophosphates (0.14); oxidative pentose-phosphate pathway (0.48); CO₂ fixation in the dark (0.005). These estimates are related to our understanding of carbohydrate metabolism during ripening.We both thank Mr Richard Trethewey for his constructive criticism: S.A.H. thanks the Managers of the Broodbank Fund for a fellowship.     

Prospects for the genetics of human longevity

Longevity varies between and within species. The existence of species-specific limit to human life-span and its partial heritability indicate the existence of genetic factors that influence the ageing process. Insight into the nature of these genetic factors is provided by evolutionary studies, notably the disposable soma theory, which suggests a central role of energy metabolism in determining life-span. Energy is important in two ways. First, the disposable soma theory indicates that the optimum energy investment in cell maintenance and repair processes will be tuned through natural selection to provide adequate, but not excessive, protection against random molecular damages (e.g. to DNA, proteins). All that is required is that the organism remains in a sound condition through its natural expectation of life in the wild environment, where accidents are the predominant cause of mortality. Secondly, energy is implicated because of the intrinsic vulnerability of mitochondria to damage that may interfere with the normal supply of energy to the cell via the oxidative phosphorylation pathways. Oxidative phosphorylation produces ATP, and as a by-product also produces highly reactive oxygen radicals that can damage many cell structures, including the mitochondria themselves. Several lines of evidence link, on the one hand, oxidative damage to cell ageing, and on the other hand, energy-dependent antioxidant defences to the preservation of cellular homeostasis, and hence, longevity. Models of cellular ageing in vitro allow direct investigation of mechanisms, such as oxidative damage, that contribute to limiting human life-span. The genetic substratum of inter-individual differences in longevity may be unraveled by a two-pronged reverse genetics approach: sibling pair analysis applied to nonagenarian and centenarian siblings, combined with association studies of centenarians, may lead to the identification of genetic influences upon human longevity. These studies have become practicable thanks to recent progress in human genome mapping, especially to the development of microsatellite markers and the integration of genetic and physical maps.