AKT/PKB signaling: navigating downstream

The serine/threonine kinase Akt, also known as protein kinase B (PKB), is a central node in cell signaling downstream of growth factors, cytokines, and other cellular stimuli. Aberrant loss or gain of Akt activation underlies the pathophysiological properties of a variety of complex diseases, including type-2 diabetes and cancer. Here, we review the molecular properties of Akt and the approaches used to characterize its true cellular targets. In addition, we discuss those Akt substrates that are most likely to contribute to the diverse cellular roles of Akt, which include cell survival, growth, proliferation, angiogenesis, metabolism, and migration.     

Phospholipase C-gamma contains introns shared by src homology 2 domains in many unrelated proteins

Many proteins with novel functions were created by exon shuffling around the time of the metazoan radiation. Phospholipase C-gamma (PLC-gamma) is typical of proteins that appeared at this time, containing several different modules that probably originated elsewhere. To gain insight into both PLC-gamma evolution and structure-function relationships within the Drosophila PLC-gamma encoded by small wing (sl), we cloned and sequenced the PLC-gamma homologs from Drosophila pseudoobscura and D. virilis and compared their gene structure and predicted amino acid sequences with PLC-gamma homologs in other animals. PLC-gamma has been well conserved throughout, although structural differences suggest that the role of tyrosine phosphorylation in enzyme activation differs between vertebrates and invertebrates. Comparison of intron positions demonstrates that extensive intron loss has occurred during invertebrate evolution and also reveals the presence of conserved introns in both the N- and C-terminal PLC-gamma SH2 domains that are present in SH2 domains in many other genes. These and other conserved SH2 introns suggest that the SH2 domains in PLC-gamma are derived from an ancestral domain that was shuffled not only into PLC-gamma, but also into many other unrelated genes during animal evolution.     

Analysis of the promoter-distal region of the tra operon of the F sex factor of Escherichia coli K-12 encoded by EcoRI restriction fragments f17, f19, and f2.

The promoter-distal region of the tra operon of the F sex factor Escherichia coli K-12 was analyzed, using the chimeric plasmid pRS31, which contains the F EcoRI restriction fragments f17, f19, and f2 cloned into the EcoRI site of pSC101. A series of deletion plasmids of pRS31, extending increasing distances from a site in f17 through f19 and ending in f2, were isolated. These plasmids were examined by heteroduplex analysis with the parent DNA, and a restriction map of this region of DNA was constructed. A series of Tn5 insertion derivatives of pRS31 were also isolated and mapped, using both heteroduplex analysis and restriction mapping. Both the insertion and deletion mutants were tested in minicells for the synthesis of radioactively labeled proteins. This allowed the identification of the individual gene products and mapping of the genes. The result is a saturated physical map of this region of DNA from fragment f17 through to the IS3 insertion sequence near the promoter-distal end of f2.     

Identification of VCR, a repeated sequence associated with a locus encoding a hemagglutinin in Vibrio cholerae O1.

We have determined the nucleotide sequence of a 6.3-kb BamHI fragment of the chromosome of Vibrio cholerae 569B that includes the sequence of the mannose-fucose-resistant hemagglutinin reported previously (V.L. Franzon, A. Barker, and P. A. Manning, Infect. Immun. 61:3032-3037, 1993). This region contains nine copies of a 124-bp direct repeat, here named VCR, of imperfect dyad symmetry, that are shown by Southern hybridization to occur at least 60 to 100 times in the V. cholerae O1 chromosome. Large-scale chromosomal mapping suggests that the repeats are confined to about 10% of the chromosome. Related sequences are also found in non-O1 V. cholerae but not in other members of the family Vibrionaceae. However, VCR is unrelated to other previously described repetitive sequences.     

NF-kappa B regulation by I kappa B kinase-2 in rheumatoid arthritis synoviocytes

IkappaB kinase-1 and IkappaB kinase-2 (IKK1 and IKK2; also called IKKalpha and IKKbeta, respectively) are part of the signal complex that regulates NF-kappaB activity in many cell types, including fibroblast-like synoviocytes (FLS). We determined which of these two kinases is responsible for cytokine-induced NF-kappaB activation in synoviocytes and assessed the functional consequences of IKK1 or IKK2 overexpression and inhibition. FLS were infected with adenovirus constructs encoding either wild-type (wt) IKK1 or IKK2, the dominant negative (dn) mutant of both kinases, or a control construct encoding green fluorescence protein. Analysis of the NF-kappaB pathway revealed that cytokine-induced IKK activation, IkappaB degradation, and NF-kappaB activation was prevented in cells expressing the IKK2 dn mutant, whereas baseline NF-kappaB activity was increased by IKK2 wt. In addition, synthesis of IL-6 and IL-8, as well as expression of ICAM-1 and collagenase, was only increased by IKK2 wt, and their cytokine-induced production was abrogated by IKK2 dn mutant. However, the IKK1 dn mutant did not inhibit cytokine-mediated activation of NF-kappaB or any of the functional assays. These data indicate that IKK2 is the key convergence pathway for cytokine-induced NF-kappaB activation. Furthermore, IKK2 regulates adhesion molecule, matrix metalloproteinase, and cytokine production in FLS.     

Mapping and validation of chromosome regions conferring boron toxicity tolerance in wheat (Triticum aestivum)

Boron is an essential plant micro-nutrient which can be phytotoxic to plants if present in soils in high concentration. Boron toxicity has been recognised as an important problem limiting production in the low rainfall areas of southern Australia, West Asia and North Africa. Genetic variation for boron toxicity tolerance in wheat has been well-characterised. The efficiency of breeding for boron toxicity tolerance could be greatly enhanced by the development of molecular markers associated with QTLs for tolerance in wheat. A population of 161 doubled haploids from a cross between the tolerant cultivar Halberd and the moderately sensitive cultivar Cranbrook was used to identify chromosomal regions involved in boron tolerance. A combined RFLP and AFLP linkage map of the Cranbrook x Halberd population was used to identify chromosomal regions involved in the boron tolerance traits measured. Regions on chromosome 7B and 7D were associated with leaf symptom expression. The region on chromosome 7B was also associated with the control of boron uptake and with a reduction in the effect of boron toxicity on root-growth suppression. RFLP markers at the chromosome 7B and 7D loci were shown to be effective in selecting for improved boron tolerance in an alternative genetic background. Halberd alleles at the chromosome 7B locus were associated with the concentration of boron in whole shoots and grain. The concentration of boron in whole shoots and in grain were both related to grain yield in a field trial conducted on soil containing toxic levels of boron. Implications relating to marker-assisted selection for boron toxicity tolerance in wheat are discussed. Received: 3 September 1999 / Accepted: 12 February 2000     

Minimum convex hull mass estimations of complete mounted skeletons

Body mass is a critical parameter used to constrain biomechanical and physiological traits of organisms. Volumetric methods are becoming more common as techniques for estimating the body masses of fossil vertebrates. However, they are often accused of excessive subjective input when estimating the thickness of missing soft tissue. Here, we demonstrate an alternative approach where a minimum convex hull is derived mathematically from the point cloud generated by laser-scanning mounted skeletons. This has the advantage of requiring minimal user intervention and is thus more objective and far quicker. We test this method on 14 relatively large-bodied mammalian skeletons and demonstrate that it consistently underestimates body mass by 21 per cent with minimal scatter around the regression line. We therefore suggest that it is a robust method of estimating body mass where a mounted skeletal reconstruction is available and demonstrate its usage to predict the body mass of one of the largest, relatively complete sauropod dinosaurs: Giraffatitan brancai (previously Brachiosaurus) as 23200 kg.     

Genetic analysis and FISH mapping of the Colourless non-ripening locus of tomato

Cnr (Colourless non-ripening) is a dominant pleiotropic ripening mutation of tomato (Lycopersicon esculentum) which has previously been mapped to the proximal region of tomato chromosome 2. We describe the fine mapping of the Cnr locus using both linkage analysis and fluorescence in situ hybridisation (FISH). Restriction fragment length polymorphism (RFLP)-, amplified restriction fragment polymorphism (AFLP)-, and cleaved amplified polymorphic sequence (CAPS)-based markers, linked to the Cnr locus were mapped onto the long arm of chromosome 2. Detailed linkage analysis indicated that the Cnr locus was likely to lie further away from the top of the long arm than previously thought. This was confirmed by FISH, which was applied to tomato pachytene chromosomes in order to gain an insight into the organisation of hetero- and euchromatin and its relationship to the physical and genetic distances in the Cnr region. Three molecular markers linked to Cnr were unambiguously located by FISH to the long arm of chromosome 2 using individual BAC probes containing these single-copy sequences. The physical order of the markers coincided with that established by genetic analysis. The two AFLP markers most-closely linked to the Cnr locus were located in the euchromatic region 2.7-cM apart. The physical distance between these markers was measured on the pachytene spreads and estimated to be approximately 900 kb, suggesting a bp:cM relationship in this region of chromosome 2 of about 330 kb/cM. This is less than half the average value of 750 kb/cM for the tomato genome. The relationship between genetic and physical distances on chromosome 2 is discussed. Received: 11 January 2001 / Accepted: 30 April 2001     

Relationship of aerobic and anaerobic parameters with 400 m front crawl swimming performance

The aims of the present study were to investigate the relationship of aerobic and anaerobic parameters with 400 m performance, and establish which variable better explains long distance performance in swimming. Twenty-two swimmers (19.1±1.5 years, height 173.9±10.0 cm, body mass 71.2±10.2 kg; 76.6±5.3% of 400 m world record) underwent a lactate minimum test to determine lactate minimum speed (LMS) (i.e., aerobic capacity index). Moreover, the swimmers performed a 400 m maximal effort to determine mean speed (S400m), peak oxygen uptake (V.O2PEAK) and total anaerobic contribution (C_ANA). The C_ANA was assumed as the sum of alactic and lactic contributions. Physiological parameters of 400 m were determined using the backward extrapolation technique (V.O2PEAK and alactic contributions of C_ANA) and blood lactate concentration analysis (lactic anaerobic contributions of C_ANA). The Pearson correlation test and backward multiple regression analysis were used to verify the possible correlations between the physiological indices (predictor factors) and S400m (independent variable) (p < 0.05). Values are presented as mean ± standard deviation. Significant correlations were observed between S400m (1.4±0.1 m·s^-1) and LMS (1.3±0.1 m·s^-1; r = 0.80), V.O2PEAK (4.5±3.9 L·min^-1; r = 0.72) and C_ANA (4.7±1.5 L·O₂; r= 0.44). The best model constructed using multiple regression analysis demonstrated that LMS and V.O2PEAK explained 85% of the 400 m performance variance. When backward multiple regression analysis was performed, C_ANA lost significance. Thus, the results demonstrated that both aerobic parameters (capacity and power) can be used to predict 400 m swimming performance.