Effects of physical connection and genetic identity of neighbouring ramets on root-placement patterns in two clonal species

Root-placement patterns were examined in the clonal species Glechoma hederacea and Fragaria vesca when grown with different types of neighbours. Three different patterns were predicted as consequences of different types of interactions between roots: the avoidance pattern if root growth decreases in the presence of neighbouring roots; the intrusive pattern if root growth increases towards neighbouring roots; and the unresponsive pattern if root growth is unaffected by neighbouring roots. Experiments were conducted in which physical connection between ramets, and the genetic identity of neighbouring ramets, were manipulated. The patterns of distribution of entire root systems and elongation rates of individual roots were measured. Root systems and individual roots of G. hederacea avoided contact with roots of neighbouring ramets, irrespective of connection to the neighbour and its genetic or specific identity. In contrast, F. vesca roots grew equally towards and away from intraspecific ramet neighbours and their elongation was stimulated by contact with roots of G. hederacea ramets. These results demonstrate that root-placement patterns of plants grown with different types of neighbours vary between species, and suggest that factors additional to resource depletion could be involved in their development.     

Complement dependent amplification of the innate response to a cognate microbial ligand by the long pentraxin PTX3

The long pentraxin PTX3 is a fluid-phase pattern recognition receptor, which plays a nonredundant role in resistance against selected pathogens. PTX3 has properties similar to Abs; its production is induced by pathogen recognition, it recognizes microbial moieties, activates complement, and facilitates cellular recognition by phagocytes. The mechanisms responsible for the effector function of PTX3 in vivo have not been elucidated. OmpA, a major outer membrane protein of Gram-negative Enterobacteriaceae, is a microbial moiety recognized by PTX3. In the air pouch model, KpOmpA induces an inflammatory response, which is amplified by coadministration of PTX3 in terms of leukocyte recruitment and proinflammatory cytokine production. PTX3 did not affect the inflammatory response to LPS, a microbial moiety not recognized by PTX3. As PTX3 binds to C1q and modulates the activation of the complement cascade, we assessed the involvement of complement in the amplification of the response elicited by KpOmpA and PTX3. Experiments performed using cobra venom factor, C1-esterase inhibitor, and soluble complement receptor 1 indicate that PTX3 amplifies the inflammatory response to KpOmpA through complement activation. The results reported here demonstrate that PTX3 activates a complement-dependent humoral amplification loop of the innate response to a microbial ligand.     

Specific targeting to murine myeloma cells of Cyt1Aa toxin from Bacillus thuringiensis subspecies israelensis

Multiple myeloma is currently an incurable cancer of plasma B cells often characterized by overproduction of abnormally high quantities of a patient-specific, clonotypic immunoglobulin "M-protein." The M-protein is expressed on the cell membrane and secreted into the blood. We previously showed that ligand-toxin conjugates (LTC) incorporating the ribosome-inactivating Ricin-A toxin were very effective in specific cytolysis of the anti-ligand antibody-bearing target cells used as models for multiple myeloma. Here, we report on the incorporation of the membrane-disruptive Cyt1Aa toxin from Bacillus thuringiensis subsp. israelensis into LTCs targeted to murine myeloma cells. Proteolytically activated Cyt1Aa was conjugated chemically or genetically through either its amino or carboxyl termini to the major peptidic epitope VHFFKNIVTPRTP (p87-99) of the myelin basic protein. The recombinant fusion-encoding genes were cloned and expressed in acrystalliferous B. thuringiensis subsp. israelensis through the shuttle vector pHT315. Both chemically conjugated and genetically fused LTCs were toxic to anti-myelin basic protein-expressing murine hybridoma cells, but the recombinant conjugates were more active. LTCs comprising the Cyt1Aa toxin might be useful anticancer agents. As a membrane-acting toxin, Cyt1Aa is not likely to induce development of resistant cell lines.     

Trypanosome MTR4 is involved in rRNA processing

The yeast putative RNA helicase Mtr4p is implicated in exosome-mediated RNA quality control in the nucleus, interacts with the exosome, and is found in the ‘TRAMP’ complex with a yeast nuclear poly(A) polymerase (Trf4p/Pap2p or Trf5p) and a putative RNA-binding protein, Air1p or Air2p. Depletion of the Trypanosoma brucei MTR4-like protein TbMTR4 caused growth arrest and defects in 5.8S rRNA processing similar to those seen after depletion of the exosome. TbNPAPL, a nuclear protein which is a putative homolog of Trf4p/Pap2p, was required for normal cell growth. Depletion of MTR4 resulted in the accumulation of polyadenylated rRNA precursors, while depletion of TbNPAPL had little effect. These results suggest that polyadenylation-dependent nuclear rRNA quality control is conserved in eukaryotic evolution. In contrast, there was no evidence for a trypanosome TRAMP complex since no stable interactions between TbMTR4 and the exosome, TbNPAPL or RNA-binding proteins were detected.     

Comparative analysis of volatiles from Drimys brasiliensis Miers and D. angustifolia Miers (Winteraceae) from Southern Brazil

The essential oils obtained from Southern Brazilian native Drimys brasiliensis Miers and Drimys angustifolia Miers were analyzed by GC and GC/MS. The oils from leaves of both species showed predominance of monoterpenoids, while the oils from stem barks were characterized by sesquiterpenoids. Bicyclogermacrene (20.0% in leaves and 25.4% in stem bark) and drimenol (1.4% in leaves and 26.2% in stem bark) were the most abundant in D. angustifolia, and cyclocolorenone (from 16.0% to 32.3% in fresh and dried leaves and almost 50% in stem bark) in D. brasiliensis. The oil from fruits of D. brasiliensis was also analyzed and presented 31.0% of cyclocolorenone. The predominance of this unusual aromadendrane-type sesquiterpene in D. brasiliensis essential oils could be used as a chemosystematic marker.     

The first agmatine/cadaverine aminopropyl transferase: biochemical and structural characterization of an enzyme involved in polyamine biosynthesis in the hyperthermophilic archaeon Pyrococcus furiosus

We report here the characterization of the first agmatine/cadaverine aminopropyl transferase (ACAPT), the enzyme responsible for polyamine biosynthesis from an archaeon. The gene PF0127 encoding ACAPT in the hyperthermophile Pyrococcus furiosus was cloned and expressed in Escherichia coli, and the recombinant protein was purified to homogeneity. P. furiosus ACAPT is a homodimer of 65 kDa. The broad substrate specificity of the enzyme toward the amine acceptors is unique, as agmatine, 1,3-diaminopropane, putrescine, cadaverine, and sym-nor-spermidine all serve as substrates. While maximal catalytic activity was observed with cadaverine, agmatine was the preferred substrate on the basis of the k(cat)/K(m) value. P. furiosus ACAPT is thermoactive and thermostable with an apparent melting temperature of 108 degrees C that increases to 112 degrees C in the presence of cadaverine. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. The crystal structure of the enzyme determined to 1.8-A resolution confirmed its dimeric nature and provided insight into the proteolytic analyses as well as into mechanisms of thermal stability. Analysis of the polyamine content of P. furiosus showed that spermidine, cadaverine, and sym-nor-spermidine are the major components, with small amounts of sym-nor-spermine and N-(3-aminopropyl)cadaverine (APC). This is the first report in Archaea of an unusual polyamine APC that is proposed to play a role in stress adaptation.     

Tetradecyltrimethylammonium Inhibits <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Hemolytic Phospholipase C Induced by Choline

Pseudomonas aeruginosa expresses hemolytic phospholipase C (PlcH) with choline or under phosphate-limiting conditions. PlcH from these conditions were differently eluted from the Celite-545 column after application of an ammonium sulfate linear reverse gradient. The PlcH from supernatants of bacteria grown in the presence of choline was eluted with 30% ammonium sulfate and was more than 85% inhibited by tetradecyltrimethylammonium. PlcH from supernatants of bacteria grown with succinate and ammonium ions in a low-phosphate medium was eluted as a peak with 10% of salt and was less than 10% inhibited by tetradecyltrimethylammonium. PlcH from low phosphate was purified associated with a protein of 17 kDa. This complex was dissociated and separated on a Sephacryl S-200 column with 1% (w/v) sodium dodecyl sulfate. After this dissociation, the resulting protein of 70 kDa, corresponding to PlcH, was inhibited by tetradecyltrimethylammonium, showing a protection effect of the accompanying protein. RT-PCR analyses showed that in choline media, the plcH gene was expressed independently of plcR. In low-phosphate medium, the plcH gene was expressed as a plcHR operon. Because plcR encodes for chaperone proteins, this result correlates with the observation that PlcH from supernatants of bacteria grown in the presence of choline was purified without an accompanying protein. The consequence of the absence of this chaperone was that tetradecyltrimethylammonium inhibited the PlcH activity.     

Cardiac matrix metalloproteinase-2 expression independently induces marked ventricular remodeling and systolic dysfunction

Although enhanced cardiac matrix metalloproteinase (MMP)-2 synthesis has been associated with ventricular remodeling and failure, whether MMP-2 expression is a direct mediator of this process is unknown. We generated transgenic mice expressing active MMP-2 driven by the alpha-myosin heavy chain promoter. At 4 mo MMP-2 transgenic hearts demonstrated expression of the MMP-2 transgene, myocyte hypertrophy, breakdown of Z-band registration, lysis of myofilaments, disruption of sarcomere and mitochondrial architecture, and cardiac fibroblast proliferation. Hearts from 8-mo-old transgenic mice displayed extensive myocyte disorganization and dropout with replacement fibrosis and perivascular fibrosis. Older transgenic mice also exhibited a massive increase in cardiac MMP-2 expression, representing recruitment of endogenous MMP-2 synthesis, with associated expression of MMP-9 and membrane type 1 MMP. Increases in diastolic [control (C) 33 +/- 3 vs. MMP 51 +/- 12 microl; P = 0.003] and systolic (C 7 +/- 2 vs. MMP 28 +/- 14 microl; P = 0.003) left ventricular (LV) volumes and relatively preserved stroke volume (C 26 +/- 4 vs. MMP 23 +/- 3 microl; P = 0.16) resulted in markedly decreased LV ejection fraction (C 78 +/- 7% vs. MMP 48 +/- 16%; P = 0.0006). Markedly impaired systolic function in the MMP transgenic mice was demonstrated in the reduced preload-adjusted maximal power (C 240 +/- 84 vs. MMP 78 +/- 49 mW/microl(2); P = 0.0003) and decreased end-systolic pressure-volume relation (C 7.5 +/- 1.5 vs. MMP 4.7 +/- 2.0; P = 0.016). Expression of active MMP-2 is sufficient to induce severe ventricular remodeling and systolic dysfunction in the absence of superimposed injury.