The systematic utility of floral and vegetative fragrance in two genera of nyctaginaceae

We examined relationships between fragrance and phylogeny using a number of approaches to coding fragrance data and comparing the hierarchical information in fragrance data with the phylogenetic signal in a DNA sequence data set. We first used distance analyses to determine which coding method(s) best distinguishes species while grouping conspecifics. Results suggest that interspecific differences in fragrance composition were maximized by coding as presence/absence of fragrance compounds and biosynthetic pathways rather than when quantitative information was also included. Useful systematic information came from both compounds and pathways and from fragrance emitted by both floral and vegetative tissues. The coding methods that emerged from the distance analyses as best distinguishing species were then adapted for use in phylogenetic analysis. Although hierarchical signal among fragrance data sets was congruent, this signal was highly incongruent with the phylogenetic signal in the DNA sequence data. Notably, topologies inferred from fragrance data sets were congruent with the DNA topology only in the most distal portions (e.g., sister group pairs or closely related species that had similar fragrance profiles were often recovered by analyses of fragrance). Examination of consistency and retention indices for individual fragrance compounds and pathways as optimized onto one of the most-parsimonious trees inferred from DNA data revealed that although most compounds were homoplastic, some compounds were perfectly congruent with the DNA phylogeny. In particular, compounds and pathways found in a few taxa were less homoplastic than those found in many taxa. Pathways that synthesize few volatiles also seem to have lower homoplasy than those that produce many. Although fragrance data as a whole may not be useful in phylogeny reconstruction, these data can provide additional support for clades reconstructed with other types of characters. Factors other than phylogeny, including pollinator interactions, also likely influence fragrance composition.     

Components of spatial patterning in a serpentine grassland

We analyzed the components of spatial patterning in species abundance in a diverse grassland dominated by early season, annual forbs. Species abundance, soil depth and gopher disturbances were measured by means of a nested spatial design on two 8m×20m plots that differed in the amount of larger scale variability in soil depth. Species distributions at the scale of the 10cm×10cm quadrat were highly clumped, with a decline in clumping with mean abundance. Even with the effect of abundance on clumping removed, species showed differences in the degree of clumping and these differences were consistent between the two plots. The abundance of the species and the frequency of disturbance correlated weakly to moderately with soil depth. Semivariance analyses indicated that the most common species all showed complex spatial patterning at a range of scales. Some of this variation corresponded to patterns of soil depth variation and patterns of gopher disturbance; however, a large amount of the spatial patterning in species abundance remains unexplained.     

Fusicoccin signaling reveals 14-3-3 protein function as a novel step in left-right patterning during amphibian embryogenesis

To gain insight into the molecular mechanisms underlying the control of morphogenetic signals by H+ flux during embryogenesis, we tested Fusicoccin-A (FC), a compound produced by the fungus Fusicoccum amygdali Del. In plant cells, FC complexes with 14-3-3 proteins to activate H+ pumping across the plasma membrane. It has long been thought that FC acts on higher plants only; here, we show that exposing frog embryos to FC during early development specifically results in randomization of the asymmetry of the left-right (LR) axis (heterotaxia). Biochemical and molecular-genetic evidence is presented that 14-3-3-family proteins are an obligate component of Xenopus FC receptors and that perturbation of 14-3-3 protein function results in heterotaxia. The subcellular localization of 14-3-3 mRNAs and proteins reveals novel cytoplasmic destinations, and a left-right asymmetry at the first cell division. Using gain-of-function and loss-of-function experiments, we show that 14-3-3E protein is likely to be an endogenous and extremely early aspect of LR patterning. These data highlight a striking conservation of signaling pathways across kingdoms, suggest common mechanisms of polarity establishment between C. elegans and vertebrate embryos, and uncover a novel entry point into the pathway of left-right asymmetry determination.     

Inhibitors of hepatitis C virus NS3.4A protease 1. Non-charged tetrapeptide variants

Tetrapeptide-based peptidomimetic compounds have been shown to effectively inhibit the hepatitis C virus NS3.4A protease without the need of a charged functionality. An aldehyde is used as a prototype reversible electrophilic warhead. The SAR of the P1 and P2 inhibitor positions is discussed.     

Wsc1 and Mid2 are cell surface sensors for cell wall integrity signaling that act through Rom2, a guanine nucleotide exchange factor for Rho1

Wsc1 and Mid2 are highly O-glycosylated cell surface proteins that reside in the plasma membrane of Saccharomyces cerevisiae. They have been proposed to function as mechanosensors of cell wall stress induced by wall remodeling during vegetative growth and pheromone-induced morphogenesis. These proteins are required for activation of the cell wall integrity signaling pathway that consists of the small G-protein Rho1, protein kinase C (Pkc1), and a mitogen-activated protein kinase cascade. We show here by two-hybrid experiments that the C-terminal cytoplasmic domains of Wsc1 and Mid2 interact with Rom2, a guanine nucleotide exchange factor (GEF) for Rho1. At least with regard to Wsc1, this interaction is mediated by the Rom2 N-terminal domain. This domain is distinct from the Rho1-interacting domain, suggesting that the GEF can interact simultaneously with a sensor and with Rho1. We also demonstrate that extracts from wsc1 and mid2 mutants are deficient in the ability to catalyze GTP loading of Rho1 in vitro, providing evidence that the function of the sensor-Rom2 interaction is to stimulate nucleotide exchange toward this G-protein. In a related line of investigation, we identified the PMT2 gene in a genetic screen for mutations that confer an additive cell lysis defect with a wsc1 null allele. Pmt2 is a member of a six-protein family in yeast that catalyzes the first step in O mannosylation of target proteins. We demonstrate that Mid2 is not mannosylated in a pmt2 mutant and that this modification is important for signaling by Mid2.     

Plasma membrane estrogen receptors signal to antiapoptosis in breast cancer

Chemotherapy or irradiation treatment induces breast cancer cell apoptosis, but this can be limited by estradiol (E2) through unknown mechanisms. To investigate this, we subjected estrogen receptor-expressing human breast cancer cells (MCF-7 and ZR-75-1) to paclitaxel (taxol) or to UV irradiation. Marked increases in cell apoptosis were induced, but these were significantly reversed by incubation with E2. Taxol or UV stimulated c-Jun N-terminal kinase (JNK) activity, which was inhibited by E2. Expression of a dominant-negative Jnk-1 protein strongly prevented taxol- or UV-induced apoptosis, whereas E2 inhibition of apoptosis was reversed by expression of constituitively active Jnk-1. As targets for participation in apoptosis, Bcl-2 and Bcl-xl were phosphorylated in response to JNK activation by taxol or UV; this was prevented by E2. Taxol or UV activated caspase activity in a JNK-dependent fashion and caused the cleavage of procaspase-9 to caspase-9, each inhibited by E2. Independently, the steroid also activated extracellular signal-regulated protein kinase activity, which contributed to the antiapoptotic effects. We report novel and rapid mechanisms by which E2 prevents chemotherapy or radiation-induced apoptosis of breast cancer, probably mediated through the plasma membrane estrogen receptor.     

A new family of adenylyl cyclase genes in the male germline of Drosophila melanogaster

We describe the cloning and characterization of a new gene family of adenylyl cyclase related genes in Drosophila. The five adenylyl cyclase-like genes that define this family are clearly distinct from previously known adenylyl cyclases. One member forms a unique locus on chromosome 3 whereas the other four members form a tightly clustered, tandemly repeated array on chromosome 2. The genes on chromosome 2 are transcribed in the male germline in a doublesex dependent manner and are expressed in postmitotic, meiotic, and early differentiating sperm. These genes therefore provide the first evidence for a role for the cAMP signaling pathway in Drosophila spermatogenesis. Expression from this locus is under the control of the always early, cannonball, meiosis arrest, and spermatocyte arrest genes that are required for the G₂/M transition of meiosis I during spermatogenesis, implying a mechanism for the coordination of differentiation and proliferation. Evidence is also provided for positive selection at the locus on chromosome 2 which suggests this gene family is actively evolving and may play a novel role in spermatogenesis. Received: 26 September 1999 / Accepted: 27 October 1999     

The stability, structural organization, and denaturation of pectate lyase C, a parallel beta-helix protein

Pectate lyase C (pelC) was the first protein in which the parallel beta-helix structure was recognized. The unique features of parallel beta-helix-containing proteins-a relatively simple topology and unusual interactions among side chains-make pelC an interesting protein to study with respect to protein folding. In this paper, we report studies of the unfolding equilibrium of pelC. PelC is unfolded reversibly by gdn-HCl at pH 7 and 5, as monitored by far- and near-UV CD and fluorescence. The coincidence of these spectroscopically detected transitions is consistent with a two-state transition at pH 7, but the three probes are not coincident at pH 5. No evidence was found for a loosely folded intermediate in the transition region at pH 5. At pH 7, the for unfolding is 12.2 kcal/mol, with the midpoint of the transition at 0.99 M gdn-HCl and m = 12.3 kcal/(mol.M). Thus, pelC is unusually stable and has an m value that is much larger than for typical globular proteins. Thermal denaturation of pelC has been studied by differential scanning calorimetry (DSC) and by CD. Although thermal denaturation is not reversible, valid thermodynamic data can be obtained for the unfolding transition. DeltaH(van't Hoff)/DeltaH(cal) is less than 1 for pHs between 5 and 8, with a maximum value of 0.91 at pH 7 decreasing to 0.85 at pH 8 and to 0.68 at pH 5. At all pHs studied, the excess heat capacity can be deconvoluted into two components corresponding to two-state transitions that are nearly coincident at pH 7, but deviate more at higher and lower pH. Thus, pelC appears to consist of two domains that interact strongly and unfold in a cooperative fashion at pH 7, but the cooperativity decreases at higher and lower pH. The crystal structure of pelC shows no obvious domain structure, however.     

Inherited predisposition to mycobacterial infection: historical considerations

Although one third of the world's population is estimated to be infected with Mycobacterium tuberculosis, only one tenth of infected individuals develop clinical disease. There is substantial epidemiological evidence that host genetic factors are important determinants of susceptibility to mycobacterial disease. This paper gives a historical context to the recent exciting advances in the field which have led to the identification of a number of human mycobacterial susceptibility genes.