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Sugar beet (Beta vulgaris) is an important arable crop, traditionally used for sugar extraction, but more recently, for biofuel production. A wide range of pests, including beet cyst nematode (Heterodera schachtii), root-knot nematodes (Meloidogyne spp.), green peach aphids (Myzus persicae) and beet root maggot (Tetanops myopaeformis), infest the roots or leaves of sugar beet, which leads to yield loss directly or through transmission of beet pathogens such as viruses. Conventional pest control approaches based on chemical application have led to high economic costs. Development of pest-resistant sugar beet varieties could play an important role towards sustainable crop production while minimising environmental impact. Intensive Beta germplasm screening has been fruitful, and genetic lines resistant to nematodes, aphids and root maggot have been identified and integrated into sugar beet breeding programmes. A small number of genes responding to pest attack have been cloned from sugar beet and wild Beta species. This trend will continue towards a detailed understanding of the molecular mechanism of insect-host plant interactions and host resistance. Molecular biotechnological techniques have shown promise in developing transgenic pest resistance varieties at an accelerated speed with high accuracy. The use of transgenic technology is discussed with regard to biodiversity and food safety.

Age and hypertension contribute significantly to cardiac morbidity and mortality, however the importance of each during the progression of hypertrophy is unclear. This investigation examined the effect of age and hypertension on Ca(2+) handling in rat ventricular myocytes by comparing a genetic model of hypertension and cardiac hypertrophy (spontaneously hypertensive rat, SHR) with its normotensive control (Wistar-Kyoto rat, WKY) at 5 and 8 months of age. Experiments were performed on single left ventricular myocytes isolated from SHR or WKY hearts. Intracellular Ca(2+) was measured optically using fura-2 or fluo-3. SHR myocytes had a significantly larger cell width and volume and a significantly decreased cell length/width ratio at 5 and 8 months compared to normotensive controls. Age had no effect on cell length, width, volume or the length/width ratio. Ca(2+) transient amplitude, sarcoplasmic reticulum (SR) Ca(2+) content and contraction amplitude were unaffected by age or hypertrophy. However at 8 months the contribution of the SR to Ca(2+) uptake during relaxation decreased, with a concomitant increase in the contribution of Na(+)/Ca(2+) exchanger (NCX) function to relaxation, in SHR and WKY myocytes. The incidence of non-synchronous SR Ca(2+) release decreased with age but not hypertrophy in SHR and WKY myocytes. These results show that the changes in Ca(2+) handling observed during progression of mild hypertrophy in SHR are the same as those that occur during ageing in normotensive control animals and can, therefore, be ascribed to maturation rather than hypertrophy.

Hypertension-induced cardiac hypertrophy alters the amplitude and time course of the systolic Ca2+transient of subepicardial and subendocardial ventricular myocytes. The present study was designed to elucidate the mechanisms underlying these changes. Myocytes were isolated from the left ventricular subepicardium and subendocardium of 20-wk-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto rats (WKY; control). We monitored intracellular Ca2+using fluo 3 or fura 2; caffeine (20 mmol/l) was used to release Ca2+from the sarcoplasmic reticulum (SR), and Ni2+(10 mM) was used to inhibit Na+/Ca2+exchange (NCX) function. SHR myocytes were significantly larger than those from WKY hearts, consistent with cellular hypertrophy. Subepicardial myocytes from SHR hearts showed larger Ca2+transient amplitude and SR Ca2+content and less Ca2+extrusion via NCX compared with subepicardial WKY myocytes. These parameters did not change in subendocardial myocytes. The time course of decline of the Ca2+transient was the same in all groups of cells, but its time to peak was shorter in subepicardial cells than in subendocardial cells in WKY and SHR and was slightly prolonged in subendocardial SHR cells compared with WKY subendocardial myocytes. It is concluded that the major change in Ca2+cycling during compensated hypertrophy in SHR is a decrease in NCX activity in subepicardial cells; this increases SR Ca2+content and hence Ca2+transient amplitude, thus helping to maintain the strength of contraction in the face of an increased afterload.

A global and transient rise of intracellular Ca2+ (Ca2+i) is central to the operation of pump-leak coupling in the frog early distal tubule (EDT). The endoplasmic reticulum (ER) is the site of this Ca2+ release and reuptake; however, it is likely that other intracellular pools, such as mitochondria, also contribute to cellular Ca2+ homeostasis. The present study was performed to seek evidence of mitochondrial Ca2+ transport in the frog EDT. Experiments were performed on isolated and permeabilized EDT segments from the frog kidney loaded with the low-affinity, Ca2+-sensitive fluorescent indicator, mag-fura-2. Ca2+ uptake in the absence of SarcoEndoplasmic Reticulum Calcium ATPase (SERCA) activity (inhibition by 2,5-di-t-butyl hydroquinone, TBQ) was evident at a bath [Ca2+] of 1 microm, but not at 200 nm, in the presence of ATP. This uptake was sensitive to the protonophore FCCP and the ATP-synthase inhibitor oligomycin. Ca2+ uptake was also stimulated by respiratory substrates; this uptake was enhanced by oligomycin and reversed by the application of FCCP. These findings provide the first evidence of mitochondrial Ca2+ transport in renal tubules, which appears to occur via a low-affinity pathway and which will act as a physiological Ca2+ buffer, protecting the cell from large increases in Ca2+i.

The early distal tubule (EDT) of the frog nephron, similar to the thick ascending limb in mammals, mediates the transepithelial absorption of NaCl. The continued absorption of NaCl in the face of varying Na+load is maintained by coordination of the activity of ion-transporting proteins in the apical and basolateral membranes, so-called pump-leak coupling. Previous studies identified intracellular Ca2+, originating from an intracellular Ca2+store, as playing a key role in pump-leak coupling in the EDT (Cooper GJ, Fowler MR, and Hunter M. Pflügers Arch 442: 243–247, 2001). The purpose of the experiments described in this paper was to identify the intracellular Ca2+storage pools in the renal diluting segment. Store Ca2+movements were monitored by the fluorescence of mag-fura 2 in permeabilized segments of frog EDTs. The presence of both ATP and Ca2+was required to maintain store Ca2+content. Removal of either of these substrates resulted in a passive leak of Ca2+from the stores. The uptake of Ca2+into the store was sensitive to the SERCA inhibitor 2,5-di( tert-butyl) hydroquinone, whereas Ca2+release from the store was stimulated by IP3but not cADPR. Store Ca2+was insensitive to the mitochondrial ATP synthase inhibitor oligomycin, and, under conditions that energized Δψm, the complex 1 inhibitor rotenone and the protonophore FCCP. Ionomycin was able to mobilize store Ca2+following exposure to IP3. These results suggest that the endoplasmic reticulum is a dominant Ca2+store in the frog EDT. A second pool, sensitive to ionomycin but not IP3, may overlap with the IP3-sensitve pool. The data also rule out any contribution by mitochondria to EDT Ca2+cycling.

The activities of transport mechanisms in epithelial cells are generally coordinated in order to minimise disturbances in cellular ion content and volume. Furosemide, a potent inhibitor of transport in the renal diluting segment, up-regulates apical K+ channel activity following the release of calcium from intracellular stores. The signal pathway between furosemide application and this calcium release is not known. Single early distal tubule segments from frog kidney were permeabilised with saponin in order to monitor calcium levels within cytoplasmic stores using the calcium-sensitive dye, mag-fura. The uptake (or release) of calcium to (or from) stores was initiated by adding agents to the bath solution, which is in direct contact with the intracellular organelles. ATP promoted calcium uptake into stores, whereas ATP removal led to a slower, spontaneous calcium release. Following loading, calcium stores could be rapidly depleted by inositol 1,4,5-trisphosphate (IP3), but not ryanodine. Calcium release was evident upon lowering the "intracellular" chloride concentration from 12 to 4 mM, equivalent to the fall in chloride induced by furosemide in intact cells. These results suggest that intracellular chloride may function as a second messenger, mediating cross-talk between the apical membrane and intracellular calcium stores.

The co-transformation of a single plant genome with two independent T-DNA regions provides opportunities for genetic separation in subsequent generations. In an effective strategy, co-delivery events must form a high proportion of the total transformed population. In this study, using the model plant species tobacco (Nicotiana tabacum), it was shown that the frequency of co-transformation within a given To population could be as high as 100% and this was found to be dependent, at least in part, on designing the plasmid vectors so that the kbp size of the first selected T-DNA region was >2-fold that of the designated T-DNA region for co-transfer. Overall, 40-50% of To lines demonstrated the capacity for segregational separation of co-transformed T-DNA regions. Hence, the estimate of the required number of total transformants for such an independent strategy may seem to be as little as 2-fold that for a conventional, single T-DNA strategy, but we strongly temper such estimates with indications that high co-transformation frequencies may be associated with a higher incidence of linkage. In this co-transformation study we used a single (Agrobacterium) strain system in which a single binary plasmid contained either two or three T-DNA regions, each with a selectable marker. This arrangement could reveal that 'read-through' events within the Agrobacterium cells, resulting in the co-transfer of adjacent T-DNA regions as a single linked unit, accounted for up to 20% of co-transformed plant lines. Such read-through co-delivery appeared to be more frequent from the 'supervirulent' EHA101 A. tumefaciens strain, compared to the 'ordinary' LBA4404 strain. By using the binary plasmid with three selectable T-DNA regions, we have been able to consider the frequency of co-integration of a third independent T-DNA within a T0 subpopulation of co-transformants. This was found to be higher than expected. These observations were applied to the co-transfer of (unwanted) plasmid backbone sequences and showed that screening against such sequences may add a significant factor in achieving the desired, final genotype.

Biological scientists are eagerly confronting the challenge of understanding the regulatory mechanisms that control the cell division cycle in eukaryotes. New information will have major implications for the treatment of growth-related diseases and cancer in animals. In plants, cell division has a key role in root and shoot growth as well as in the development of vegetative storage organs and reproductive tissues such as flowers and seeds. Many of the strategies for crop improvement, especially those aimed at increasing yield, involve the manipulation of cell division. This review describes, in some detail, the current status of our understanding of the regulation of cell division in eukaryotes and especially in plants. It also features an outline of some preliminary attempts to exploit transgenesis for manipulation of plant cell division.

Sugar beet cells grown in batch suspension culture have been used to study the regulation of polyamine levels during the transition from a quiescent to a proliferating state. The quiescent state was achieved by maintenance of the phytohormone autonomous cells in the stationary phase of the batch culture cycle. After subculture into fresh medium there was an increase in DNA synthesis which was accompanied by a dramatic increase in cellular polyamine levels. The levels of both free and bound cellular putrescine and spermidine within the cells reached a peak before the onset of the first synchronous division. The levels of putrescine, spermidine and to some extent spermine in the culture medium also increased dramatically shortly after subculture. The increase in polyamines was preceded by a rapid but transient increase in omithine decarboxylase (EC 4.1.1.17) and S-adenosylmethionine decarboxylase (EC 4.1.1.50). Arginine decarboxylase (EC 4.1.1.19) and S-adenosylmethionine synthetase (EC 2.5.1.6) activity did not show the same pattern of cell division-related variation. Inhibition of S-adenosylmethionine biosynthesis with methylglyoxal bis-(guanylhydra-zone) (MGBG) reduced cell division in the suspension culture. Inhibitors of ornithine decarboxylase and arginine decarboxylase individually had little effect on cell division, but in combination led to a reduction in cell division. Addition of polyamines and their precursors to cells in the stationary phase of a batch culture cycle led to the induction of expression of a mitotic cyclin sequence (BvcycII).