The Drosophila Mst ortholog,hippo, restricts growth and cell proliferation and promotes apoptosis

Establishing and maintaining homeostasis is critical to the well-being of an organism and is determined by the balance of cell proliferation and death. Two genes that function together to regulate growth, proliferation, and apoptosis in Drosophila are warts (wts), encoding a serine/threonine kinase, and salvador (sav), encoding a WW domain containing Wts-interacting protein. However, the mechanisms by which sav and wts regulate growth and apoptosis are not well understood. Here, we describe mutations in hippo (hpo), which encodes a protein kinase most related to mammalian Mst1 and Mst2. Like wts and sav, hpo mutations result in increased tissue growth and impaired apoptosis characterized by elevated levels of the cell cycle regulator cyclin E and apoptosis inhibitor DIAP1. Hpo, Sav, and Wts interact physically and functionally, and regulate DIAP1 levels, likely by Hpo-mediated phosphorylation and subsequent degradation. Thus, Hpo links Sav and Wts to a key regulator of apoptosis.     

Cross-spectral analysis of physiological tremor and muscle activity

We investigate the relationship between the extensor electromyogram (EMG) and tremor time series in physiological hand tremor by cross-spectral analysis. Special attention is directed to the phase spectrum and the effects of observational noise. We calculate the theoretical phase spectrum for a second-order linear stochastic process and compare the results to measured tremor data recorded from subjects who did not show a synchronized EMG activity in the corresponding extensor muscle. The results show that physiological tremor is well described by the proposed model and that the measured EMG represents a Newtonian force by which the muscle acts on the hand. Received: 7 October 1996 / Accepted in revised form: 29 January 1998     

Influence of cations, sialic acid and pH on the expansion of films of canine lung surfactant.

Sialic acid (14.6 mug/mg protein) was quantitated in the non-cellular material removed from the lung of Beagle dogs by lavage. Sialic acid did not affect the dynamic surface tension properties of either the total alveolar lipid removed by lavage or of its major lipids, dipalmitoyl lecithin (DPL) and dipalmitoyl phosphatidyl glycerol (DPG). The presence of divalent cation (Ca2+, Mg2+, Mn2+ and Zn2+) or a lowered pH in the subphase medium lowered the surface tension during the expansion phase of the total alveolar lipid film when it was compressed and expanded on a Wilhelmy trough. Films of DPL behaved similarly, but no pH effect was observed with DPG monolayers. The cation effect manifested itself in the same direction as the value of the individual stability constants (Zn2+ greater than Mn2+ greater than Mg2+ greater than Ca2+) which suggests ionic binding of the cations to the phosphate group of the phospholipids. A physiological advantage of such an effect may lie in the conservation of the energetically favorable low surface tension state achieved during film compression with a minimum of surfactant lipid.     

Two types of O-methyltransferase are involved in biosynthesis of anticancer methoxylated 4′-deoxyflavones in Scutellaria baicalensis Georgi

The medicinal plant Scutellaria baicalensis Georgi is rich in specialized 4′-deoxyflavones, which are reported to have many health-promoting properties. We assayed Scutellaria flavones with different methoxyl groups on human cancer cell lines and found that polymethoxylated 4′-deoxyflavones, like skullcapflavone I and tenaxin I have stronger ability to induce apoptosis compared to unmethylated baicalein, showing that methoxylation enhances bioactivity as well as the physical properties of specialized flavones, while having no side-effects on healthy cells. We investigated the formation of methoxylated flavones and found that two O-methyltransferase (OMT) families are active in the roots of S. baicalensis. The Type II OMTs, SbPFOMT2 and SbPFOMT5, decorate one of two adjacent hydroxyl groups on flavones and are responsible for methylation on the C6, 8 and 3′-hydroxyl positions, to form oroxylin A, tenaxin II and chrysoeriol respectively. The Type I OMTs, SbFOMT3, SbFOMT5 and SbFOMT6 account mainly for C7-methoxylation of flavones, but SbFOMT5 can also methylate baicalein on its C5 and C6-hydroxyl positions. The dimethoxylated flavone, skullcapflavone I (found naturally in roots of S. baicalensis) can be produced in yeast by co-expressing SbPFOMT5 plus SbFOMT6 when the appropriately hydroxylated 4′-deoxyflavone substrates are supplied in the medium. Co-expression of SbPFOMT5 plus SbFOMT5 in yeast produced tenaxin I, also found in Scutellaria roots. This work showed that both type I and type II OMT enzymes are involved in biosynthesis of methoxylated flavones in S. baicalensis.     

Directed evolution of AraC for improved compatibility of arabinose- and lactose-inducible promoters

Synthetic biological systems often require multiple, independently inducible promoters in order to control the expression levels of several genes; however, cross talk between the promoters limits this ability. Here, we demonstrate the directed evolution of AraC to construct an arabinose-inducible (P(BAD)) system that is more compatible with IPTG (isopropyl-beta-D-1-thiogalactopyranoside) induction of a lactose-inducible (P(lac)) system. The constructed system is 10 times more sensitive to arabinose and tolerates IPTG significantly better than the wild type. Detailed studies indicate that the AraC dimerization domain and C terminus are important for the increased sensitivity of AraC to arabinose.     

Engineering Escherichia coli for production of C12–C14 polyhydroxyalkanoate from glucose

Demand for sustainable materials motivates the development of microorganisms capable of synthesizing products from renewable substrates. A challenge to commercial production of polyhydroxyalkanoates (PHA), microbially derived polyesters, is engineering metabolic pathways to produce a polymer with the desired monomer composition from an unrelated and renewable source. Here, we demonstrate a metabolic pathway for converting glucose into medium-chain-length (mcl)-PHA composed primarily of 3-hydroxydodecanoate monomers. This pathway combines fatty acid biosynthesis, an acyl-ACP thioesterase to generate desired C₁₂ and C₁₄ fatty acids, β-oxidation for conversion of fatty acids to (R)-3-hydroxyacyl-CoAs, and a PHA polymerase. A key finding is that Escherichia coli expresses multiple copies of enzymes involved in β-oxidation under aerobic conditions. To produce polyhydroxydodecanoate, an acyl-ACP thioesterase (BTE), an enoyl-CoA hydratase (phaJ3), and mcl-PHA polymerase (phaC2) were overexpressed in E. coli ΔfadRABIJ. Yields were improved through expression of an acyl-CoA synthetase resulting in production over 15% CDW – the highest reported production of mcl-PHA of a defined composition from an unrelated carbon source.     

Starch branching enzymes belonging to distinct enzyme families are differentially expressed during pea embryo development

cDNA clones for two isoforms of starch branching enzyme (SBEI and SBEII) have been isolated from pea embryos and sequenced. The deduced amino acid sequences of pea SBEI and SBEII are closely related to starch branching enzymes of maize, rice, potato and cassava and a number of glycogen branching enzymes from yeast, mammals and several prokaryotic species. In comparison with SBEI, the deduced amino acid sequence of SBEII lacks a flexible domain at the N-terminus of the mature protein. This domain is also present in maize SBEII and rice SBEIII and resembles one previously reported for pea granule-bound starch synthase II (GBSSII). However, in each case it is missing from the other isoform of SBE from the same species. On the basis of this structural feature (which exists in some isoforms from both monocots and dicots) and other differences in sequence, SBEs from plants may be divided into two distinct enzyme families. There is strong evidence from our own and other work that the amylopectin products of the enzymes from these two families are qualitatively different. Pea SBEI and SBEII are differentially expressed during embryo development. SBEI is relatively highly expressed in young embryos whilst maximum expression of SBEII occurs in older embryos. The differential expression of isoforms which have distinct catalytic properties means that the contribution of each SBE isoform to starch biosynthesis changes during embryo development. Qualitative measurement of amylopectin from developing and maturing embryos confirms that the nature of amylopectin changes during pea embryo development and that this correlates with the differential expression of SBE isoforms.