The hyper-fluorescent trichome phenotype of the brt1 mutant of Arabidopsis is the result of a defect in a sinapic acid: UDPG glucosyltransferase

Sinapoylmalate is a major phenylpropanoid that is accumulated in Arabidopsis. Its presence causes the adaxial surface of leaves to fluoresce blue under UV light, and mutations that lead to lower levels of sinapoylmalate decrease UV-induced leaf fluorescence. The Arabidopsis bright trichomes 1 (brt1) mutant was first identified in a screen for mutants that exhibit a reduced epidermal fluorescence phenotype; however, subsequent examination of the mutant revealed that its trichomes are hyper-fluorescent. The results from genetic mapping and complementation analyses showed that BRT1 (At3g21560) encodes UGT84A2, a glucosyltransferase previously shown to be capable of using sinapic acid as a substrate. Residual levels of sinapoylmalate and sinapic acid:UDP-glucose glucosyltransferase activity in brt1 leaves suggest that BRT1 is one member of a family of partially redundant glycosyltransferases that function in Arabidopsis sinapate ester biosynthesis. RT-PCR analysis showed that BRT1 is expressed through all stages of plant life cycle, a result consistent with the impact of the brt1 mutation on both leaf sinapoylmalate levels and seed sinapoylcholine content. Finally, the compound accumulated in brt1 trichomes was identified as a sinapic acid-derived polyketide, indicating that when sinapic acid glycosylation is reduced, a portion of it is instead activated to its CoA thioester, which then serves as a substrate for chalcone synthase.     

High-resolution structure of the pleckstrin homology domain of protein kinase b/akt bound to phosphatidylinositol (3,4,5)-trisphosphate

The products of PI 3-kinase activation, PtdIns(3,4,5)P3 and its immediate breakdown product PtdIns(3,4)P2, trigger physiological processes, by interacting with proteins possessing pleckstrin homology (PH) domains. One of the best characterized PtdIns(3,4,5)P3/PtdIns(3,4)P2 effector proteins is protein kinase B (PKB), also known as Akt. PKB possesses a PH domain located at its N terminus, and this domain binds specifically to PtdIns(3,4,5)P3 and PtdIns(3,4)P2 with similar affinity. Following activation of PI 3-kinase, PKB is recruited to the plasma membrane by virtue of its interaction with PtdIns(3,4,5)P3/PtdIns(3,4)P2. PKB is then activated by the 3-phosphoinositide-dependent pro-tein kinase-1 (PDK1), which like PKB, possesses a PtdIns(3,4,5)P3/PtdIns(3,4)P2 binding PH domain. Here, we describe the high-resolution crystal structure of the isolated PH domain of PKB(alpha) in complex with the head group of PtdIns(3,4,5)P3. The head group has a significantly different orientation and location compared to other Ins(1,3,4,5)P4 binding PH domains. Mutagenesis of the basic residues that form ionic interactions with the D3 and D4 phosphate groups reduces or abolishes the ability of PKB to interact with PtdIns(3,4,5)P3 and PtdIns(3,4)P2. The D5 phosphate faces the solvent and forms no significant interactions with any residue on the PH domain, and this explains why PKB interacts with similar affinity with both PtdIns(3,4,5)P3 and PtdIns(3,4)P2.     

The PIF-binding pocket in PDK1 is essential for activation of S6K and SGK, but not PKB

PKB/Akt, S6K1 and SGK are related protein kinases activated in a PI 3-kinase-dependent manner in response to insulin/growth factors signalling. Activation entails phosphorylation of these kinases at two residues, the T-loop and the hydrophobic motif. PDK1 activates S6K, SGK and PKB isoforms by phosphorylating these kinases at their T-loop. We demonstrate that a pocket in the kinase domain of PDK1, termed the 'PIF-binding pocket', plays a key role in mediating the interaction and phosphorylation of S6K1 and SGK1 at their T-loop motif by PDK1. Our data indicate that prior phosphorylation of S6K1 and SGK1 at their hydrophobic motif promotes their interaction with the PIF-binding pocket of PDK1 and their T-loop phosphorylation. Thus, the hydrophobic motif phosphorylation of S6K and SGK converts them into substrates that can be activated by PDK1. In contrast, the PIF-binding pocket of PDK1 is not required for the phosphorylation of PKBalpha by PDK1. The PIF-binding pocket represents a substrate recognition site on a protein kinase that is only required for the phosphorylation of a subset of its physiological substrates.     

Comparison of conductimetric and traditional plating techniques for detecting yeasts in fruit juices

Frozen fruit juice concentrates containing an average microbial population of log₁₀ 1.54 cfu ml^-1 were examined by traditional plating techniques and direct and indirect conductimetry. The initial populations in diluted (1:4) concentrates increased to an average of log₁₀ 3.82 cfu ml^-1 during incubation at 25°C for 24 h. Incubation before plating and subjecting to conductimetric tests also facilitated the resuscitation of cells that may have been freeze-injured. Yeasts were recovered in equal numbers on acidified (pH 3.5) potato dextrose agar and dichloran rose bengal chloramphenicol agar (pH 5.6). Yeasts and bacteria were recovered on orange serum agar. Detection times determined by indirect conductimetry correlated fairly well ( r = -0.73) with populations (cfu ml^-1) detected on traditional plating media. Populations in diluted concentrates which were not incubated before examination were detected conductimetrically in an average of 48.9 h, whereas detection times for diluted concentrates incubated for 24 h at 25°C before testing were reduced to an average of 14.1 h. Examination by conventional (direct) conductimetry required an additional 10–20 h to reach changes in conductance of 5 μS h^-1.     

The molecular defect in a nonlethal variant of osteogenesis imperfecta. Synthesis of pro-alpha 2(I) chains which are not incorporated into trimers of type I procollagen

Cultured fibroblasts were examined from a patient with a nonlethal form of osteogenesis imperfecta. As reported previously (Nicholls, A. C., Pope, F. M., and Schloon, H. (1979) Lancet 1, 1193), the cells synthesized and secreted a type I procollagen which lacked pro-alpha 2(I) chains and consisted of a trimer of pro-alpha 1(I) chains. No pro-alpha 2(I) chains were recovered from the medium under conditions in which nonhelical pro-alpha 1(I) and pro-alpha 2(I) chains were readily detected in the medium of normal fibroblasts incubated with the hydroxylase inhibitor, alpha, alpha'-dipyridyl. Examination of cellular proteins demonstrated that the fibroblasts synthesized both pro-alpha 1(I) and pro-alpha 2(I) chains. The cellular pro-alpha 2(I) chains did not, however, become disulfide-linked into dimers or trimers of pro-alpha chains. Since the association of pro-alpha chains during the biosynthesis of type I procollagen is directed by the conformation of the COOH-terminal propeptides, the data suggest that the pro-alpha 2(I) chains synthesized by the fibroblasts have a mutated structure in the COOH-terminal propeptides which markedly reduces their affinity for pro-alpha 1(I) chains. A further observation was that the ratio of newly synthesized pro-alpha (I):pro-alpha 2(I) chains in the patient's fibroblasts was 7.18 +/- 0.58 S.E. instead of the value of 2.25 +/- 0.16 S.E. seen in control fibroblasts. One possible explanation for the high ratio is that the proband is homozygous for a mutation altering the structure of the pro-alpha 2(I) chain and that a secondary effect of the structural mutation is a decreased rate of synthesis of pro-alpha 2(I) chains.     

The single copy gene coding for human α1 (IV) procollagen is located at the terminal end of the long arm of chromosome 13

Summary Using dual-laser sorted chromosomes and spot-blot analysis, we have previously assigned genomic DNA sequences coding for human 1(IV) procollagen to chromosome 13 (Pihlajaniemi et al. 1985). By in situ hybridization to normal chromosomes and chromosomes with 13q deletions, we now report the localization of this gene to the terminal end of the long arm of chromosome 13. In addition, Southern and slot blot hybridization analysis clearly show that these genomic sequences are present only once per haploid genome.     

Picosecond phase grating spectroscopy of hemoglobin and myoglobin: energetics and dynamics of global protein motion.

Phase grating spectroscopy has been used to follow the optically triggered tertiary structural changes of carboxymyoglobin (MbCO) and carboxyhemoglobin (HbCO). Probe wavelength and temperature dependencies have shown that the grating signal arises from nonthermal density changes induced by the protein structural changes. The material displaced through the protein structural changes leads to the excitation of coherent acoustic modes of the surrounding water. The coupling of the structural changes to the fluid hydrodynamics demonstrates that a global change in the protein structure is occurring in less than 30 ps. The global relaxation is on the same time scale as the local changes in structure in the vicinity of the heme pocket. The observed dynamics for global relaxation and correspondence between the local and global structural changes provides evidence for the involvement of collective modes in the propagation of the initial tertiary conformational changes. The energetics can also be derived from the acoustic signal. For MbCO, the photodissociation process is endothermic by 21 +/- 2 kcal/mol, which corresponds closely to the expected Fe-CO bond enthalpy. In contrast, HbCO dissipates approximately 10 kcal/mol more energy relative to myoglobin during its initial tertiary structural relaxation. The difference in energetics indicates that significantly more energy is stored in the hemoglobin structure and is believed to be related to the quaternary structure of hemoglobin not present in the monomeric form of myoglobin. These findings provide new insight into the biomechanics of conformational changes in proteins and lend support to theoretical models invoking stored strain energy as the driving force for large amplitude correlated motions.