Targeted modification of homogalacturonan by transgenic expression of a fungal polygalacturonase alters plant growth

Pectins are a highly complex family of cell wall polysaccharides comprised of homogalacturonan (HGA), rhamnogalacturonan I and rhamnogalacturonan II. We have specifically modified HGA in both tobacco (Nicotiana tabacum) and Arabidopsis by expressing the endopolygalacturonase II of Aspergillus niger (AnPGII). Cell walls of transgenic tobacco plants showed a 25% reduction in GalUA content as compared with the wild type and a reduced content of deesterified HGA as detected by antibody labeling. Neutral sugars remained unchanged apart from a slight increase of Rha, Ara, and Gal. Both transgenic tobacco and Arabidopsis were dwarfed, indicating that unesterified HGA is a critical factor for plant cell growth. The dwarf phenotypes were associated with AnPGII activity as demonstrated by the observation that the mutant phenotype of tobacco was completely reverted by crossing the dwarfed plants with plants expressing PGIP2, a strong inhibitor of AnPGII. The mutant phenotype in Arabidopsis did not appear when transformation was performed with a gene encoding AnPGII inactivated by site directed mutagenesis.     

Abi1 is essential for the formation and activation of a WAVE2 signalling complex

WAVE2 belongs to a family of proteins that mediates actin reorganization by relaying signals from Rac to the Arp2/3 complex, resulting in lamellipodia protrusion. WAVE2 displays Arp2/3-dependent actin nucleation activity in vitro, and does not bind directly to Rac. Instead, it forms macromolecular complexes that have been reported to exert both positive and negative modes of regulation. How these complexes are assembled, localized and activated in vivo remains to be established. Here we use tandem mass spectrometry to identify an Abi1-based complex containing WAVE2, Nap1 (Nck-associated protein) and PIR121. Abi1 interacts directly with the WHD domain of WAVE2, increases WAVE2 actin polymerization activity and mediates the assembly of a WAVE2-Abi1-Nap1-PIR121 complex. The WAVE2-Abi1-Nap1-PIR121 complex is as active as the WAVE2-Abi1 sub-complex in stimulating Arp2/3, and after Rac activation it is re-localized to the leading edge of ruffles in vivo. Consistently, inhibition of Abi1 by RNA interference (RNAi) abrogates Rac-dependent lamellipodia protrusion. Thus, Abi1 orchestrates the proper assembly of the WAVE2 complex and mediates its activation at the leading edge in vivo.     

Drugs against leishmaniasis: a synergy of technology and partnerships

To date, there are no vaccines against any of the major parasitic diseases, and chemotherapy is the main weapon in our arsenal. There is an urgent need for better drugs against Leishmania. With the completion of the human genome sequence and soon that of Leishmania, for the first time we have the opportunity to identify novel chemotherapeutic treatments. This requires the exploitation of a variety of technologies. The major challenge is to take the process from discovery of drug candidates all the way along the arduous path to the marketplace. A crucial component will be the forging of partnerships between the pharmaceutical industry and publicly funded scientists to ensure that the promise of the current revolution in biology lives up to our hopes and expectations.     

Protection by herpes simplex virus glycoprotein D against Fas-mediated apoptosis: role of nuclear factor kappaB

Signals involved in protection against apoptosis by herpes simplex virus 1 (HSV-1) were investigated. Using U937 monocytoid cells as an experimental model, we have demonstrated that HSV-1 rendered these cells resistant to Fas-induced apoptosis promptly after infection. UV-inactivated virus as well as the envelope glycoprotein D (gD) of HSV-1, by itself, exerted a protective effect on Fas-induced apoptosis. NF-kappaB was activated by gD, and protection against Fas-mediated apoptosis by gD was abolished in cells stably transfected with a dominant negative mutant I-kappaBalpha, indicating that NF-kappaB activation plays a role in the antiapoptotic activity of gD in our experimental model. Moreover, NF-kappaB-dependent protection against Fas-mediated apoptosis was associated with decreased levels of caspase-8 activity and with the up-regulation of intracellular antiapoptotic proteins.     

3D structure of Sulfolobus solfataricus carboxypeptidase developed by molecular modeling is confirmed by site-directed mutagenesis and small angle X-ray scattering

Sulfolobus solfataricus carboxypeptidase (CPSso) is a thermostable zinc-metalloenzyme with a M(r) of 43,000. Taking into account the experimentally determined zinc content of one ion per subunit, we developed two alternative 3D models, starting from the available structures of Thermoactinomyces vulgaris carboxypeptidase (Model A) and Pseudomonas carboxypeptidase G2 (Model B). The former enzyme is monomeric and has one metal ion in the active site, while the latter is dimeric and has two bound zinc ions. The two models were computed by exploiting the structural alignment of the one zinc- with the two zinc-containing active sites of the two templates, and with a threading procedure. Both computed structures resembled the respective template, with only one bound zinc with tetrahedric coordination in the active site. With these models, two different quaternary structures can be modeled: one using Model A with a hexameric symmetry, the other from Model B with a tetrameric symmetry. Mutagenesis experiments directed toward the residues putatively involved in metal chelation in either of the models disproved Model A and supported Model B, in which the metal-binding site comprises His(108), Asp(109), and His(168). We also identified Glu(142) as the acidic residue interacting with the water molecule occupying the fourth chelation site. Furthermore, the overall fold and the oligomeric structure of the molecule was validated by small angle x-ray scattering (SAXS). An ab initio original approach was used to reconstruct the shape of the CPSso in solution from the experimental curves. The results clearly support a tetrameric structure. The Monte Carlo method was then used to compare the crystallographic coordinates of the possible quaternary structures for CPSso with the SAXS profiles. The fitting procedure showed that only the model built using the Pseudomonas carboxypeptidase G2 structure as a template fitted the experimental data.     

Imazalil-cyclomaltoheptaose (beta-cyclodextrin) inclusion complex: preparation by supercritical carbon dioxide and 13C CPMAS and 1H NMR characterization

An inclusion complex between imazalil (IMZ), a selected fungicide, and cyclomaltoheptaose (beta-cyclodextrin, betaCD) was obtained using supercritical fluid carbon dioxide. The best preparation conditions were determined, and the inclusion complex was investigated by means of 1H NMR spectroscopy in aqueous solution and 13C CPMAS NMR spectroscopy in the solid state. Information on the geometry of the betaCD/IMZ complex was obtained from ROESY spectroscopy, while the dynamics of the inclusion complex in the kilohertz range was obtained from the proton spin-lattice relaxation times in the rotating frame, T(1rho) (1H).     

Day-night variation in aggressive behavior among psychiatric inpatients

Self-directed aggressive behaviors of human beings show a 24h pattern. The aim of this study was to evaluate if violence of psychiatric inpatients against one another and hospital staff varies over the 24h. The clock time occurrence of 334 episodes of assault behaviors by 119 psychiatric inpatients (78 males and 41 females, mean age 34.8 ± 11.3 years) committed during a 5-year span in the psychiatric unit of the university-based hospital of Ferrara, Italy, was evaluated. The clock time of each event was categorized by hour during the 24h and into one of four 6h intervals for analysis of temporal variation by cosinor and χ² tests, respectively. A significant 24h variation, characterized by an early afternoon peak, was detected irrespective of gender and number (single vs. repeated) of episodes committed. Changes during the 24h in ward activity, patient contact, and endogenous circadian rhythms are likely to contribute to the observed 24h pattern, although further study is needed to confirm our findings and to define causal factors. (Chronobiology International, 18(3), 503-511, 2001)     

Morning preference in onset of symptomatic third-degree atrioventricular heart block

The aim of this study was to examine 24h patterning in the symptoms indicative of third-degree atrio-ventricular (AV) heart block. We found a total of 227 cases (126 men and 101 women) of third-degree AV block that had been diagnosed by the Emergency Medical Department of the St. Anna Hospital in Ferrara, Italy between 1990 and 2001. Determination of the hour of onset of symptomatic third-degree AV block, however, was possible and listed in the records of only 161 or 70.9% of the cases (92 men and 69 women). The onset time of every event was categorized into one of four 6h spans of the 24h: night (00:00-05:59h), morning (06:00-11:59h), afternoon (12:00-17:59h), and evening (18:00-23:59h). The onset of the symptoms of third-degree AV block in the sample of 161 cases was significantly greater in the morning between 06:00 and 11:59h than any other 6h span of the day and night (chi2-test; p < 0.001). The same phenomenon was substantiated in the subgroup of the 92 males (chi2; p < 0.0001), although it could not be detected for the smaller subgroup of 69 women. The 24h pattern, with morning preference, in the onset of symptomatic third-degree AV block is similar to the one in sudden cardiac death and cardiogenic cardiac arrest. The etiology of the 24h pattern in symptomatic AV block is unknown; it may be an expression of intrinsic biological rhythmicity within the heart tissue or its control system, and/or the timing of environmental triggers resulting in coronary ischemia.