Crystallographic determination of the structures of human alpha-thrombin complexed with BMS-186282 and BMS-189090.

The crystallographic structures of the ternary complexes of human alpha-thrombin with hirugen (a sulfated hirudin fragment) and the small-molecule active site thrombin inhibitors BMS-186282 and BMS-189090 have been determined at 2.6 and 2.8 A. In both cases, the inhibitors, which adopt very similar bound conformations, bind in an antiparallel beta-strand arrangement relative to the thrombin main chain in a manner like that reported for PPACK, D-Phe-Pro-Arg-CH2Cl. They do, however, exhibit differences in the binding of the alkyl guanidine moiety in the specificity pocket. Numerous hydrophilic and hydrophobic interactions serve to stabilize the inhibitors in the binding pocket. Although PPACK forms covalent bonds to both serine and the histidine of the catalytic triad of thrombin, neither BMS-186282 nor BMS-189090 bind covalently and only BMS-186282 forms a hydrogen bond to the serine of the catalytic triad. Both inhibitors bind with high affinity (Ki = 79 nM and 3.6 nM, respectively) and are highly selective for thrombin over trypsin and other serine proteases.     

Vibrio cholerae O139 persists in Dhaka,Bangladesh since 1993

BackgroundAfter a multi-country Asian outbreak of cholera due to Vibrio cholerae serogroup O139 which started in 1992, it is rarely detected from any country in Asia and has not been detected from patients in Africa.Methodology/Principal findingsWe extracted surveillance data from the Dhaka and Matlab Hospitals of International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) to review trends in isolation of Vibrio cholerae O139 in Bangladesh. Data from the Dhaka Hospital is a 2% sample of > 100,000 diarrhoeal patients treated annually. Data from the Matlab Hospital includes all diarrhoeal patients who hail from the villages included in the Matlab Health and Demographic Surveillance System. Vibrio cholerae O139 was first isolated in Dhaka in 1993 and had been isolated every year since then except for a gap between 2005 and 2008. An average of thirteen isolates was detected annually from the Dhaka Hospital during the last ten years, yielding an estimated 650 cases annually at this hospital. During the last ten years, cases due to serogroup O139 represented 0.47% of all cholera cases; the others being due to serogroup O1. No cases with serogroup O139 were identified at Matlab since 2006. Clinical signs and symptoms of cholera due to serogroup O139 were similar to cases due to serogroup O1 though more of the O139 cases were not dehydrated. Most isolates of O139 remained sensitive to tetracycline, ciprofloxacin, and azithromycin, but they became resistant to erythromycin starting in 2009.Conclusions/SignificanceCholera due to Vibrio cholerae serogroup O139 continues to cause typical cholera in Dhaka, Bangladesh.     

Biochemical and functional characterization of anti-HIV antibody-ELP fusion proteins from transgenic plants

The stability and recovery of recombinant proteins expressed in plants are improved by fusion to elastin-like peptides (ELPs). In order to test the suitability of ELP for the production of pharmaceutical proteins, transgenic plants were created that individually expressed the light and heavy chains of the broadly neutralizing anti-human immunodeficiency virus type 1 (anti-HIV-1) monoclonal antibody 2F5, which is being evaluated as a microbicide component. The antibody chains were expressed both with and without a C-terminal ELP fusion. Crossing these plants in all combinations resulted in transgenic lines producing the full antibody in four formats, with ELP on either the light or heavy chains, on both or on neither. Characterization of the affinity-purified antibodies by surface plasmon resonance spectroscopy showed that the kinetic binding parameters were identical to those of a Chinese hamster ovary (CHO) cell counterpart lacking ELP. N -Glycan analysis showed that all four derivatives contained predominantly oligo-mannose-type N -glycans and that the ELP fusions had no significant effect on N -glycan structure. It was concluded that ELP fusion to the light chain, heavy chain or both chains of a plant-derived antibody had no adverse affects on protein quality, but had a positive impact on the yield. ELP fusions do not interfere with folding, assembly, trafficking in the secretory pathway or post-translational modification, but enhance stability whilst at the same time simplifying recovery.     

Irradiance-dependent regulation of gravitropism by red light in protonemata of the moss Ceratodon purpureus

Apical cells of protonemata of the moss Ceratodon purpureus (Hedw.) Brid. are negatively gravitropic in the dark and positively phototropic in red light. Various fluence rates of unilateral red light were tested to determine whether both tropisms operate simultaneously. At irradiances ≥140 nmol m⁻² s⁻¹ no gravitropism could be detected and phototropism predominated, despite the presence of amyloplast sedimentation. Gravitropism occurred at irradiances lower than 140 nmol m⁻² s⁻¹ with most cells oriented above the horizontal but not upright. At these low fluence rates, phototropism was indistinct at 1 g but apparent in microgravity, indicating that gravitropism and phototropism compete at 1 g. The frequency of protonemata that were negatively phototropic varied with the fluence rate and the duration of illumination, as well as with the position of the apical cell before illumination. These data show that the fluence rate of red light regulates whether gravitropism is allowed or completely repressed, and that it influences the polarity of phototropism and the extent to which apical cells are aligned in the light path. Received: 19 January 1999 / Accepted: 19 March 1999     

Flavobacterium johnsoniae as a model organism for characterizing biopolymer utilization in oligotrophic freshwater environments

Biopolymers are important substrates for heterotrophic bacteria in oligotrophic freshwater environments, but information on bacterial growth kinetics with biopolymers is scarce. The objective of this study was to characterize bacterial biopolymer utilization in these environments by assessing the growth kinetics of Flavobacterium johnsoniae strain A3, which is specialized in utilizing biopolymers at μg liter⁻¹ levels. Growth of strain A3 with amylopectin, xyloglucan, gelatin, maltose, or fructose at 0 to 200 μg C liter⁻¹ in tap water followed Monod or Teissier kinetics, whereas growth with laminarin followed Teissier kinetics. Classification of the specific affinity of strain A3 for the tested substrates resulted in the following affinity order: laminarin (7.9 × 10⁻² liter·μg⁻¹ of C·h⁻¹) ≫ maltose > amylopectin ≈ gelatin ≈ xyloglucan > fructose (0.69 × 10⁻² liter·μg⁻¹ of C·h⁻¹). No specific affinity could be determined for proline, but it appeared to be high. Extracellular degradation controlled growth with amylopectin, xyloglucan, or gelatin but not with laminarin, which could explain the higher affinity for laminarin. The main degradation products were oligosaccharides or oligopeptides, because only some individual monosaccharides and amino acids promoted growth. A higher yield and a lower ATP cell⁻¹ level was achieved at ≤10 μg C liter⁻¹ than at >10 μg C liter⁻¹ with every substrate except gelatin. The high specific affinities of strain A3 for different biopolymers confirm that some representatives of the classes Cytophagia-Flavobacteria are highly adapted to growth with these compounds at μg liter⁻¹ levels and support the hypothesis that Cytophagia-Flavobacteria play an important role in biopolymer degradation in (ultra)oligotrophic freshwater environments.     

Focus on Water: Leaf Shrinkage with Dehydration: Coordination with Hydraulic Vulnerability and Drought Tolerance

Leaf shrinkage with dehydration has attracted attention for over 100 years, especially as it becomes visibly extreme during drought. However, little has been known of its correlation with physiology. Computer simulations of the leaf hydraulic system showed that a reduction of hydraulic conductance of the mesophyll pathways outside the xylem would cause a strong decline of leaf hydraulic conductance (K_leaf). For 14 diverse species, we tested the hypothesis that shrinkage during dehydration (i.e. in whole leaf, cell and airspace thickness, and leaf area) is associated with reduction in K_leaf at declining leaf water potential (Ψ_leaf). We tested hypotheses for the linkage of leaf shrinkage with structural and physiological water relations parameters, including modulus of elasticity, osmotic pressure at full turgor, turgor loss point (TLP), and cuticular conductance. Species originating from moist habitats showed substantial shrinkage during dehydration before reaching TLP, in contrast with species originating from dry habitats. Across species, the decline of K_leaf with mild dehydration (i.e. the initial slope of the K_leaf versus Ψ_leaf curve) correlated with the decline of leaf thickness (the slope of the leaf thickness versus Ψ_leaf curve), as expected based on predictions from computer simulations. Leaf thickness shrinkage before TLP correlated across species with lower modulus of elasticity and with less negative osmotic pressure at full turgor, as did leaf area shrinkage between full turgor and oven desiccation. These findings point to a role for leaf shrinkage in hydraulic decline during mild dehydration, with potential impacts on drought adaptation for cells and leaves, influencing plant ecological distributions.As leaves open their stomata to capture CO₂ for photosynthesis, water is lost to transpiration, which needs to be replaced by flow through the hydraulic system. The leaf hydraulic system has two components, which act essentially in series: the pathways for water movement through the xylem from the petiole to leaf minor veins, and those through the living bundle sheath and mesophyll cells to the sites of evaporation (Tyree and Zimmermann, 2002; Sack et al., 2004; Sack and Holbrook, 2006). The decline in leaf hydraulic conductance (K_leaf) with dehydration may thus depend on both components. The importance of the xylem component is well established. Vein xylem embolism and cell collapse have been observed in dehydrating leaves (Salleo et al., 2001; Cochard et al., 2004a; Johnson et al., 2009), and computer modeling and experimental work showed that species with high major vein length per leaf area (VLA; i.e. for the first three vein-branching orders) were more resistant to hydraulic decline, providing more pathways around embolisms (Scoffoni et al., 2011). However, the physical impacts of dehydration on the extraxylem pathways have not been studied, even though in turgid leaves these pathways account for 26% to 88% of leaf hydraulic resistance (i.e. of 1/K_leaf), depending on species (Sack et al., 2003a; Cochard et al., 2004b). The aim of this study was to determine whether leaf shrinkage during dehydration relates to the decline of K_leaf as well as the structural determinants of leaf shrinkage.The shrinkage of leaves with dehydration has drawn attention for over 100 years. Leaves shrink in their area (Bogue, 1892; Gardner and Ehlig, 1965; Jones, 1973; Tang and Boyer, 2007; Blonder et al., 2012) and, considered in relative terms, even more strongly in their thickness (Fig. 1; Meidner, 1952; Gardner and Ehlig, 1965; Downey and Miller, 1971; Syvertsen and Levy, 1982; Saini and Rathore, 1983; Burquez, 1987; McBurney, 1992; Sancho-Knapik et al., 2010, 2011). Leaves fluctuate in thickness daily and seasonally according to transpiration (Kadoya et al., 1975; Tyree and Cameron, 1977; Fensom and Donald, 1982; Rozema et al., 1987; Ogaya and Peñuelas, 2006; Seelig et al., 2012). Indeed, the relation of leaf thickness to water status is so tight that using leaf thickness to guide irrigation has led to water savings of up to 45% (Seelig et al., 2012).Open in a separate windowFigure 1.Sketches of a fully turgid leaf (A) versus a strongly dehydrated leaf (B; drawings based on leaf cross sections of sunflower in Fellows and Boyer, 1978). Note the strong reduction in leaf thickness, cell thickness, and intercellular airspaces in the dehydrated leaf. Epidermal cells are shrunk in the dehydrated leaf, inducing whole-leaf area shrinkage. Note that this sketch represents shrinkage for a typical drought-sensitive species. Many species such as oaks (Quercus spp.) will experience less thickness shrinkage and an increase in intercellular airspace (see “Discussion”). [See online article for color version of this figure.]Previous studies of leaf shrinkage with progressive dehydration have tended to focus on single or few species. These studies showed that thickness declines with water status in two phases. Before the bulk leaf turgor loss point (TLP; leaf water potential [Ψ_leaf] at TLP) is reached, the slope of leaf thickness versus Ψ_leaf or relative water content (RWC) is shallower than past TLP for most species (Meidner, 1955, Kennedy and Booth, 1958, Burquez, 1987, McBurney, 1992, Sancho-Knapik et al., 2010, 2011). This is because before TLP, declining Ψ_leaf is strongly driven by declines in turgor pressure, which have a relatively low impact on cell and airspace volume, whereas past the TLP, declining Ψ_leaf depends only on solute concentration, which increases in inverse proportion as cell water volume declines while airspaces may shrink or expand (Tyree and Hammel, 1972, Sancho-Knapik et al., 2011). However, the steepness of the slope of leaf thickness versus Ψ_leaf before TLP seems to vary strongly across species (Meidner, 1955; Kennedy and Booth, 1958; Fellows and Boyer, 1978; Burquez, 1987; Colpitts and Coleman, 1997; Sancho-Knapik et al., 2010).A high leaf cell volume and turgor is crucial to physiological processes (Boyer, 1968; Lawlor and Cornic, 2002). Shrinkage may affect cell connectivity and water transport (Sancho-Knapik et al., 2011). However, no studies have tested for a possible relationship of leaf shrinkage with the decline of K_leaf during dehydration. Such an association would arise if, across species, shrinkage occurred simultaneously with vein xylem embolism or if tissue shrinkage led to declines in the extraxylem hydraulic conductance.To refine our hypotheses, we modified a computer model of the leaf hydraulic system (Cochard et al., 2004b; McKown et al., 2010; Scoffoni et al., 2011) to predict the impact of losses of xylem and extraxylem conductance on the response of K_leaf to dehydration. We characterized the degree of leaf shrinkage in thickness, in the thickness of cells and airspaces within the leaf, and in leaf area for 14 species diverse in phylogeny, leaf traits, and drought tolerance. We hypothesized that loss of extraxylem hydraulic conductance should have a greater impact on K_leaf at less negative water potentials when xylem tensions are too weak to trigger embolism and induce dramatic declines in K_leaf. We hypothesized that species with greater degrees of shrinkage before TLP would experience greater loss of K_leaf. Furthermore, we hypothesized that species from moist habitats would have greater degrees of shrinkage.For insight into the mechanisms and consequences of leaf shrinkage, we also investigated the relationships of 18 indices of leaf shrinkage with a wide range of aspects of leaf structure and composition, including gross morphology, leaf venation architecture, parameters of pressure-volume curves, and leaf water storage. We hypothesized that, across species, shrinkage in whole leaf, cell, and intercellular airspace thickness would be lower for species with greater allocation to structural rigidity and osmotic concentration, and thus shrinkage would be positively correlated with a lower modulus of elasticity (ε), less negative osmotic pressure at full turgor (π_o), lower leaf mass per area (LMA), and lower leaf density. Additionally, we tested the longstanding hypothesis that species with higher major VLA and/or minor VLA (i.e. the fourth and higher vein-branching orders) would shrink less in area and/or thickness with dehydration (Gardner and Ehlig, 1965). Finally, we tested the ability of dehydrated leaves to recover in size with rehydration. We hypothesized that recovery would be greater for mildly than for strongly dehydrated leaves and that species with greater leaf shrinkage would be better able to recover from shrinkage.     

Amyloplasts as possible statoliths in gravitropic protonemata of the moss Ceratodon purpureus

The kinetics of gravitropism and of amyloplast sedimentation were studied in dark-grown protonemata of the moss Ceratodon purpureus (Hedw.) Brid. The protonemata grew straight up at a rate of 20–25 m·h^– in nutrient-supplemented agar. After they were oriented to the horizontal, upward curvature was first detected after 1–1.5 h and reached 84° by 24 h. The tip cells exhibited an amyloplast zonation, with a tip cluster of nonsedimenting amyloplasts, an amyloplast-free zone, and a zone with pronounced amyloplast sedimentation. This latter zone appears specialized more for lateral than for axial sedimentation since amyloplasts sediment to the lower wall in horizontal protonemata but do not fall to the basal wall in vertical protonemata. Amyloplast sedimentation started within 15 min of gravistimulation; this is within the 12–17-min presentation time. The data support the hypothesis that some amyloplasts function as statoliths in these cells.This work was supported by the National Aeronautics and Space Administration grant NAGW-780. We thank Professor E. Hartmann and J. Schwuchow for providing Ceratodon cultures, Dr. John Z. Kiss and Jeff Young for valuable discussions, and Professor Rainer Hertel (University of Freiburg, FRG) for bringing this material to our attention.     

Climatic sensitivity of species’ vegetative and reproductive phenology in a Hawaiian montane wet forest

Understanding how tropical tree phenology (i.e., the timing and amount of seed and leaf production) responds to climate is vital for predicting how climate change may alter ecological functioning of tropical forests. We examined the effects of temperature, rainfall, and photosynthetically active radiation (PAR) on seed phenology of four dominant species and community-level leaf phenology in a montane wet forest on the island of Hawaiʻi using monthly data collected over ~ 6 years. We expected that species phenologies would be better explained by variation in temperature and PAR than rainfall because rainfall at this site is not limiting. The best-fit model for all four species included temperature, rainfall, and PAR. For three species, including two foundational species of Hawaiian forests (Acacia koa and Metrosideros polymorpha), seed production declined with increasing maximum temperatures and increased with rainfall. Relationships with PAR were the most variable across all four species. Community-level leaf litterfall decreased with minimum temperatures, increased with rainfall, and showed a peak at PAR of ~ 400 μmol/m²s⁻¹. There was considerable variation in monthly seed and leaf production not explained by climatic factors, and there was some evidence for a mediating effect of daylength. Thus, the impact of future climate change on this forest will depend on how climate change interacts with other factors such as daylength, biotic, and/or evolutionary constraints. Our results nonetheless provide insight into how climate change may affect different species in unique ways with potential consequences for shifts in species distributions and community composition.     

Synthesis and biological activity of N-aryl-2-aminothiazoles: potent pan inhibitors of cyclin-dependent kinases

N-Aryl aminothiazoles 6-9 were prepared from 2-bromothiazole 5 and found to be CDK inhibitors. In cells they act as potent cytotoxic agents. Selectivity for CDK1, CDK2, and CDK4 was dependent of the nature of the N-aryl group and distinct from the CDK2 selective N-acyl analogues. The N-2-pyridyl analogues 7 and 19 showed pan CDK inhibitory activity. Elaborated analogues 19 and 23 exhibited anticancer activity in mice against P388 murine leukemia. The solid-state structure of 7 bound to CDK2 shows a similar binding mode to the N-acyl analogues.     

Thyroid receptor ligands. Part 2: Thyromimetics with improved selectivity for the thyroid hormone receptor beta

A set of thyromimetics having improved selectivity for TR-beta1 were prepared by replacing the 3'-isopropyl group of 2 and 3 with substituents having increased steric bulk. From this limited SAR study, the most potent and selective compounds identified were derived from 2 and contained a 3'-phenyl moiety bearing small hydrophobic groups meta to the biphenyl link. X-ray crystal data of 15c complexed with TR-beta1 LBD shows methionine 442 to be displaced by the bulky R3' phenyl ethyl amide side chain. Movement of this amino acid side chain provides an expanded pocket for the bulky side chain while the ligand-receptor complex retains full agonist activity.