Ca2+ is Required by Clubroot Resistant Turnip Cells for Transient Increases in PAL Activity that Follow Inoculation with Plasmodiophora brassicae

Phenylalanine ammonia‐lyase (PAL, EC 4.3.1.5) activity in clubroot disease‐resistant turnip calli was transiently increased by 20 h after the inoculation with Plasmodiophora brassicae spores. The magnitude of the increase in PAL activity was four to six times higher than constitutive PAL activity. There was no transient increase in PAL activity in susceptible calli. Preincubation of calli in Ca²⁺‐free medium or the removal of Ca²⁺ from cell surfaces by ethylene glycol bis(2‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid‐chelation, completely inhibited induced PAL activity. The influx of exogenous Ca²⁺ into cells appears necessary for this pathogen induced PAL activity. Verapamil and the calmodulin inhibitor W7 almost completely inhibited induced PAL activity at 1 and 0.1 mm , respectively. Neomycin, ruthenium red and (1‐(6‐[(17β‐3‐Methoxyestra‐1,3,5‐(10)‐trien‐17‐yl)amino]hexyl)‐1H‐pyrrole‐2,5‐dione) did not inhibit induced PAL activity. Thus, verapamil and N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulphonamide hydrochloride‐sensitive Ca²⁺‐mediated signalling process appear necessary for P. brassicae induced PAL activity. As the protein synthesis inhibitor cycloheximide (CHX) blocked the induced increasing PAL activity, de novo synthesis of PAL appears to be required for turnip cell defence reactions against P. brassicae.     

The lactose transport protein is a cooperative dimer with two sugar translocation pathways.

The Major Facilitator Superfamily lactose transport protein (LacS) undergoes reversible self-association in the detergent-solubilized state, and is present in the membrane as a dimer. We determined the functional unit for proton motive force (Deltap)-driven lactose uptake and lactose/methyl-beta-D-galactopyranoside equilibrium exchange in a proteoliposomal system in which a single cysteine mutant, LacS-C67, defective in Deltap-driven uptake, was co-reconstituted with fully functional cysteine-less protein, LacS-cl. From the quadratic relationship between the uptake activity and the ratio of LacS-C67/LacS-cl, we conclude that the dimeric state of LacS is required for Deltap-driven uptake. N-ethylmaleimide (NEM) treatment of proteoliposomes abolished the LacS-C67 exchange activity but left the LacS-cl unaffected. After NEM treatment, the exchange activity decreased linearly with increasing ratios of LacS-C67/LacS-cl, suggesting that the monomeric state of LacS is sufficient for this mode of transport. We propose that the two subunits of LacS are functionally coupled in the step associated with conformational reorientation of the empty binding site, a step unique for Deltap-driven uptake.     

Exploitation of Arabidopsis-tomato synteny to construct a high-resolution map of the ovatecontaining region in tomato chromosome 2.

High-resolution genetic and physical maps were constructed for the region of chromosome 2 containing the major fruit-shape locus ovate. A total of 3,000 NIL F2 and F3 NILs derived from Lycopersicon esculentum cv. Yellow Pear (TA503) x L. pennellii (a wild tomato) were used to position ovate adjacent to the marker TG645 and flanked by markers TX700 and BA10R (a 0.03-cM interval). BAC libraries and a BIBAC library were screened with the closest marker, TG645. Genetic mapping with the ends of isolated BAC clones revealed that two BAC clones (100 and 140 kb) both contained the ovate locus. Screening of sequences from these BAC clones revealed synteny between this segment of tomato chromosome 2 and the chromosome-4 region of Arabidopsis containing the BAC clone ATAP22. Microsynteny between the two genomes was exploited to find additional markers near the ovate locus. The placement of ovate on a BAC clone will now allow cloning of this locus and, hence, may open the door to understanding the molecular basis of fruit development and also facilitate the genetic engineering of fruit-shape characteristics. This also represents the first time that microsynteny with Arabidopsis has been exploited for positional cloning purposes in a different plant family.     

An effector of Ypt6p binds the SNARE Tlg1p and mediates selective fusion of vesicles with late Golgi membranes.

Membrane traffic requires vesicles to fuse with a specific target, and SNARE proteins and Rab/Ypt GTPases contribute to this specificity. In the yeast Saccharomyces cerevisae, the Rab/Ypt GTPase Ypt6p is required for fusion of endosome-derived vesicles with the late Golgi. We have shown previously that activation of Ypt6p depends on its exchange factor, Ric1p-Rgp1p, a peripheral membrane protein complex restricted to the Golgi. We show here that a conserved trimeric protein complex, VFT (Vps52/53/54), binds directly to Ypt6p:GTP. Localization of VFT to the Golgi requires Ypt6p, but is unaffected in gos1 and tlg1 mutants, in which late Golgi integral membrane proteins, including SNAREs, are mislocalized. The VFT complex also binds directly to the N-terminal domain of the SNARE Tlg1p, both in vitro and in vivo, in a Ypt6p-independent manner. We suggest that the VFT complex links vesicles containing Tlg1p to their target, which is defined by the local activation of Ypt6p.     

Specificity of GlcNAc-PI de-N-acetylase of GPI biosynthesis and synthesis of parasite-specific suicide substrate inhibitors.

The substrate specificities of Trypanosoma brucei and human (HeLa) GlcNAc-PI de-N-acetylases were determined using 24 substrate analogues. The results show the following. (i) The de-N-acetylases show little specificity for the lipid moiety of GlcNAc-PI. (ii) The 3'-OH group of the GlcNAc residue is essential for substrate recognition whereas the 6'-OH group is dispensable and the 4'-OH, while not required for recognition, cannot be epimerized or substituted. (iii) The parasite enzyme can act on analogues containing betaGlcNAc or aromatic N-acyl groups, whereas the human enzyme cannot. (iv) Three GlcNR-PI analogues are de-N-acetylase inhibitors, one of which is a suicide inhibitor. (v) The suicide inhibitor most likely forms a carbamate or thiocarbamate ester to an active site hydroxy-amino acid or Cys or residue such that inhibition is reversed by certain nucleophiles. These and previous results were used to design two potent (IC50 = 8 nM) parasite-specific suicide substrate inhibitors. These are potential lead compounds for the development of anti-protozoan parasite drugs.     

Crystal structure of a dynamin GTPase domain in both nucleotide-free and GDP-bound forms.

Dynamins form a family of multidomain GTPases involved in endocytosis, vesicle trafficking and maintenance of mitochondrial morphology. In contrast to the classical switch GTPases, a force-generating function has been suggested for dynamins. Here we report the 2.3 A crystal structure of the nucleotide-free and GDP-bound GTPase domain of Dictyostelium discoideum dynamin A. The GTPase domain is the most highly conserved region among dynamins. The globular structure contains the G-protein core fold, which is extended from a six-stranded beta-sheet to an eight-stranded one by a 55 amino acid insertion. This topologically unique insertion distinguishes dynamins from other subfamilies of GTP-binding proteins. An additional N-terminal helix interacts with the C-terminal helix of the GTPase domain, forming a hydrophobic groove, which could be occupied by C-terminal parts of dynamin not present in our construct. The lack of major conformational changes between the nucleotide-free and the GDP-bound state suggests that mechanochemical rearrangements in dynamin occur during GTP binding, GTP hydrolysis or phosphate release and are not linked to loss of GDP.     

Female marmoset monkeys (Callithrix jacchus) can be identified from the chemical composition of their scent marks.

The present study analyzed 42 organic solvent extracts of scent mark pools from five dominant female common marmosets by gas chromatography (GC) and combined GC and mass spectrometry. We determined whether there were qualitative or quantitative differences between the chemical composition of scent marks from individual females. Gas chromatography and mass spectral analysis detected the same 162 chemicals in 86% (36/42) of scent mark pools from five dominant females. This near identical chemical composition of scent marks suggested there were few, if any, qualitative differences between the chemical composition of scent marks from individual females. Instead, quantitative differences in scent may provide the key factor distinguishing individual females. Using the relative concentration of highly volatile chemicals detected by GC in scent marks, linear discriminant analysis classified scent mark pools to their correct donor approximately 91% of the time. Such highly reliable statistical matching of scent to donor suggested that each individual female common marmoset has a unique ratio of highly volatile chemicals in their scent marks which may permit individual identification of females from odors in their scent alone.     

PYCNOGENOL chewing gum minimizes gingival bleeding and plaque formation.

PYCNOGENOL is an antioxidant phytochemical shown to have antiinflammatory activity in both the in vitro and in vivo models. This study compared the effects of chewing gums with and without PYCNOGENOL on gingival bleeding and plaque formation in 40 human subjects. In this double-blind study, subjects were assigned randomly to receive either control gums without PYCNOGENOL or experimental gums containng 5 mg PYCNOGENOL. Subjects used chewing gums for 14 days. Gingival bleeding and plaque scores were taken before and after the experiment. PYCNOGENOL chewing gums significantly reduced gingival bleeding, while no changes were noted in bleeding indexes in control subjects who used regular chewing gums. Subjects using regular control gums had significant increases of dental plaque accumulation during the two-week period. No increases in plaque accumulation were noted in subjects using PYCNOGENOL chewing gums. The data of this study suggest that the use of Pycnogenol chewing gums can minimize gingival bleeding and plaque accumulation.