Alternative binding modes of an inhibitor to two different kinases.

Protein kinases are targets for therapeutic agents designed to intervene in signaling processes in the diseased state. Most kinase inhibitors are directed towards the conserved ATP binding site. Because the essential features of this site are conserved in all eukaryotic protein kinases, it is generally assumed that the same compound will bind in a similar manner to different protein kinases. The inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) is a selective inhibitor for the protein kinase CK2 (IC50 1.6 micro m) (Sarno et al. (2001) FEBS Letts.496, 44-48). Three other kinases [cyclin-dependent protein kinase 2 (CDK2), phosphorylase kinase and glycogen synthase kinase 3beta] exhibit approximately 10-fold weaker affinity for TBB than CK2. We report the crystal structure of TBB in complex with phospho-CDK2-cyclin A at 2.2 A resolution and compare the interactions with those observed for TBB bound to CK2. TBB binds at the ATP binding site of both kinases. In CDK2, each of the four bromine atoms makes polar contacts either to main chain oxygens in the hinge region of the kinase or to water molecules, in addition to several van der Waals contacts. The mode of binding of TBB to CDK2 is different from that to CK2. TBB in CDK2 is displaced more towards the hinge region between the N- and C-terminal lobes and rotated relative to TBB in CK2. The ATP binding pocket is wider in CDK2 than in CK2 resulting in fewer van der Waals contacts but TBB in CK2 does not contact the hinge. The structures show that, despite the conservation of the ATP binding pocket, the inhibitor is able to exploit different recognition features so that the same compound can bind in different ways to the two different kinases.     

Nutritional Evaluation of Terrestrial Invertebrates as Traditional Food in Amazonia1

In tropical areas worldwide, more than 1000 terrestrial species of invertebrates are used as food. For populations of Amazonian areas, different species of insects and other invertebrates serve as nourishing food sources. Here, we document the composition and nutritional quality of some invertebrates consumed in Venezuelan Amazonia, including termites (Isoptera) and earthworms (Glossoscolecidae) eaten by the Ye'Kuana (Makiritare) and caterpillars (Lepidoptera) and grubs (Coleoptera: Scarabaeidae) consumed by the Yanomamo. Consumption of 100 g of any of these invertebrates contributed 1.2–9.4 percent of the daily fat requirement and 26–144 percent of the protein daily requirement for an adult male. The sample of caterpillars regularly eaten by these peoples was rich in beta carotene (provitamin A), and a 100 g ingestion guaranteed 323 percent of the recommended daily intake of vitamin A. The greatest amount of alpha tocopherol was provided by the consumption of the smoked earthworms (36.7%). Earthworms and termites contained high levels of sterols, mainly cholesterol. The consumption of these animals by tropical human populations represents not only an important traditional habit but also, considering their nutritional composition, a substantial contribution to the human diet.     

Age determination of common marmosets (Callithrix jacchus) by radiographic examination of skeletal development

Seventy-four animals were examined radiographically to determine the skeletal development of the common marmoset (Callithrix jacchus) from 6 months of age. Twenty-one epiphyses were examined and five stages of ossification were described for each. The animals were divided into nine groups, according to age, and a table of the stage of ossification and age was produced, which may be used for determining the age of animals of unknown history.     

Pectin changes in samples containing poplar cambium and inner bark in relation to the seasonal cycle

Biochemical changes occurring during the transition between meristematic activity and rest were studied in samples containing cambial cells and their phloem derivatives from Populus x euramericana. Uronic acids represented around 9% of the cell-wall dry matter in spring and 7% in summer and winter. In contrast, a higher content of methylated galacturonic acids was observed during the rest period. The degree of esterification increased from 2% in spring to 35% in winter, indicating an important accumulation of acidic pectins during the active season although the cation content was always very low. Nuclear magnetic resonance spectroscopy of neutral polysaccharides solubilized with boiling water showed that in winter arabinans and xylans were the main carbohydrates. By contrast, in spring and in summer the xylans were very scarce, arabinans being the major neutral polysaccharide, indicating that important modifications occur during the autumn. Histochemical observations of material treated with hot water and EDTA confirmed the low relative pectin content during the rest period. Calcium ions, detected as antimonate salt were scarce. In the cambium, they were located mainly in cell junctions whereas in phloem derivatives these cations were distributed throughout the whole cell wall.Abbreviations Araf arabinofuranose - COSY 2 D homonuclear correlation spectroscopy - NMR nuclear magnetic resonance spectroscopy - NOESY 2 D nuclear Overhauser effect spectroscopy - PATAg periodic acid thiosemicarbazide silver proteinate - PME pectin methylesterase - R wall radial wall - T wall tangential wall - Xylp xylopyranose We acknowledge support from the French Ministry of Research and Technology and also from the European Program Eureka 447/ Eurosilva.     

Detection of complete and partial chromosome gains and losses by comparative genomic in situ hybridization

Comparative genomic in situ hybridization (CGH) provides a new possibility for searching genomes for imbalanced genetic material. Labeled genomic test DNA, prepared from clinical or tumor specimens, is mixed with differently labeled control DNA prepared from cells with normal chromosome complements. The mixed probe is used for chromosomal in situ suppression (CISS) hybridization to normal metaphase spreads (CGH-metaphase spreads). Hybridized test and control DNA sequences are detected via different fluorochromes, e.g., fluorescein isothiocyanate (FITC) and tetraethylrhodamine isothiocyanate (TRITC). The ratios of FITC/TRITC fluorescence intensities for each chromosome or chromosome segment should then reflect its relative copy number in the test genome compared with the control genome, e.g., 0.5 for monosomies, 1 for disomies, 1.5 for trisomies, etc. Initially, model experiments were designed to test the accuracy of fluorescence ratio measurements on single chromosomes. DNAs from up to five human chromosome-specific plasmid libraries were labeled with biotin and digoxigenin in different hapten proportions. Probe mixtures were used for CISS hybridization to normal human metaphase spreads and detected with FITC and TRITC. An epifluorescence microscope equipped with a cooled charge coupled device (CCD) camera was used for image acquisition. Procedures for fluorescence ratio measurements were developed on the basis of commercial image analysis software. For hapten ratios 4/1, 1/1 and 1/4, fluorescence ratio values measured for individual chromosomes could be used as a single reliable parameter for chromosome identification. Our findings indicate (1) a tight correlation of fluorescence ratio values with hapten ratios, and (2) the potential of fluorescence ratio measurements for multiple color chromosome painting. Subsequently, genomic test DNAs, prepared from a patient with Down syndrome, from blood of a patient with Tcell prolymphocytic leukemia, and from cultured cells of a renal papillary carcinoma cell line, were applied in CGH experiments. As expected, significant differences in the fluorescence ratios could be measured for chromosome types present in different copy numbers in these test genomes, including a trisomy of chromosome 21, the smallest autosome of the human complement. In addition, chromosome material involved in partial gains and losses of the different tumors could be mapped to their normal chromosome counterparts in CGH-metaphase spreads. An alternative and simpler evaluation procedure based on visual inspection of CCD images of CGH-metaphase spreads also yielded consistent results from several independent observers. Pitfalls, methodological improvements, and potential applications of CGH analyses are discussed.     

Processing of asparagine-linked oligosaccharides in insect cells. N-Acetylglucosaminyltransferase I and II activities in cultured lepidopteran cells

The levels of ß1,2-N-acetylglucosaminyltransferase(GlcNAc-T) I and II activities in cultured cells from Bombyxmori(Bm-N), Mamestra brassicae (IZD-Mb-0503) and Spodoptera frugiperda(Sf-9 and Sf-21) were investigated. Apart from initial experimentswith Man     

Influence of magnesium deficiency on rates of leaf expansion, starch and sucrose accumulation, and net assimilation in Phaseolus vulgaris

Twenty one-day-old Phaseolus vulgaris 'Saxa'plants were cultured in a growth chamber and the plants supplied with either a complete or a Mg-free nutrient solution. From 6 days after transfer to the Mg-free solution, the rate of increase of the area of the second trifoliate leaf was considerably reduced; by day 11 the sucrose concentration in the first trifoliate leaf had increased 6. 2-fold at the end of the dark period and 4. 6-fold after the light period as compared with the control plants. Corresponding starch concentrations increased 6. 6-fold and 2. 9-fold respectively. After days 5 to 6 the assimilation rates declined in the first trifoliate leaf of the plants showing deficiency, in comparison with the plants fully supplied with nutrients; respiration increased during darkness. The reduction in net assimilation rate was to a great extent reversible after resupply of magnesium.
The reduction of magnesium concentration in the deficient plants was much more marked in the expanding leaves than in the mature primary leaves and roots. Sucrose and starch accumulation did not occur when the first trifoliate leaf was partially shaded, although magnesium concentration, as in the unshaded leaves, was reduced to 13% of that of the control plants. The consequences of magnesium deficiency in the expanding first trifoliate leaf are discussed in terms of the possibility of sink limitation.     

The effect of feeding immature Karoo paralysis ticksIxodes rubicundus (Acari: Ixodidae) on the metabolic rate of the rock elephant shrew

The effect of feeding immature Karoo paralysis ticks (Ixodes rubicundus) on the resting metabolic rate (RMR) of their principal natural host, the rock elephant shrew (Elephantulus myurus), was investigated under laboratory conditions. The elephant shrews were artificially infested with numbers of ticks simulating natural burdens. The RMR of the elephant shrews was quantified by measuring the oxygen consumption in an open through-flow system. The RMR of hosts infested only with larvae did not differ from that of the control group. Those infested with nymphs, or nymphs and larvae, had significantly lower RMR's compared to the control animals. There were no signs of paralysis in any of the infested hosts.     

Factors affecting gibberellic acid production by Fusarium moniliforme in solid-state cultivation on starch

The production of gibberellic acid (GA₃) by Fusarium moniliforme M-7121 in solid-state culture was evaluated in flask cultures as well as in 3-I horizontal rotary reactors. The highest production rate of GA₃ was with 80% (w/v) maize flour mixed with wheat bran. The optimum initial moisture content was inversely dependent on the ambient relative humidity. The initial water activity range for optimal growth and GA₃ accumulation was about 0.98 to 0.99, which is unusually high for a filamentous fungus. A low O₂ concentration resulted in a much decreased GA₃ yield and the appearance of a yellow to reddish pigmentation in the mycelium. The lag phase was short and rapid growth continued for up to 2 days in the rotary reactor, with a maximum specific growth rate of 0.12 h^–1. The maximum rate of GA₃ production occurred during the subsequent 3 to 10 days of incubation and the final GA₃ concentration reached was 18.7 mg to 19.3 mg/g dry culture. The point of maximum GA₃ accumulation after 10 to 12 days of incubation was usually marked by a sharp increase in pH.The authors are with the Department of Microbiology and Biochemistry, University of the Orange Free State, P.O. Box 339, 9300 Bioemfontein, Republic of South Africa