Isolating substrates for an engineered alpha-lytic protease by phage display

Panning of a substrate phage library with an -lytic protease mutant showed that substrate phage display can be used to isolate sequences with improved protease sensitivity even for proteases of relatively broad specificity. Two panning experiments were performed with an engineered -lytic protease mutant known to have a preference for cleavage after His or Met residues. Both experiments led to the isolation of protease-sensitive phage containing linker sequences in which His and Met residues were enriched compared with the initial library. Despite the relatively hydrophobic substrate binding site of the enzyme, the predominant protease-sensitive sequence isolated from the second library panning had the sequence Asp-Ser-Thr-Met. Kinetic studies showed that this sequence was cleaved up to 4.5-fold faster than rationally designed positive controls. Protease-resistant phage particles were also selected and characterized, with the finding that Gly and Pro appeared frequently at the putative P₄ positions, whereas Asp dominated the putative P₁ position.     

NODs: intracellular proteins involved in inflammation and apoptosis

NOD (nucleotide-binding oligomerization domain) proteins are members of a family that includes the apoptosis regulator APAF1 (apoptotic protease activating factor 1), mammalian NOD-LRR (leucine-rich repeat) proteins and plant disease-resistance gene products. Several NOD proteins have been implicated in the induction of nuclear factor-kappaB (NF-kappaB) activity and in the activation of caspases. Two members of the NOD family, NOD1 and NOD2, mediate the recognition of specific bacterial components. Notably, genetic variation in the genes encoding the NOD proteins NOD2, cryopyrin and CIITA (MHC class II transactivator) in humans and Naip5 (neuronal apoptosis inhibitory protein 5) in mice is associated with inflammatory disease or increased susceptibility to bacterial infections. Mammalian NOD proteins seem to function as cytosolic sensors for the induction of apoptosis, as well as for innate recognition of microorganisms and regulation of inflammatory responses.     

Effect of trehalose and sucrose on the hydration and dipole potential of lipid bilayers

The water activity in dimyristoylphosphatidylcholine (DMPC) decreases by 60% when the lipid is dehydrated in the presence of trehalose concentrations higher than 0.02 M. In contrast, sucrose in concentrations 10 times higher produced only a 20% decrease in the water activity in the sample. Titrations of a DMPC solution in chloroform yielded 14 water molecules per lipid when pure water was added and seven water molecules per lipid when the titration was done with 0.025 M trehalose. The same concentrations of sucrose produced a turbid solution, which made it impossible to quantify the number of water molecules per lipid. Lipid monolayers spread on an air/water interface showed a decrease from 480 mV in pure water to 425 mV in 0.1 M trehalose. However, the same concentrations of sucrose produced an increase of less than 100 mV. Results obtained with Fourier transform infrared spectroscopy (FTIR) under the same conditions denoted that trehalose binds to the carbonyl groups, while sucrose showed no specific binding. It is concluded that per lipid molecule, 11 of 14 water molecules can be replaced by three trehalose molecules. About four are displaced by changes in the water activity of the bulk solution, and seven by specific interactions with the phospholipids. In this last case, at least two of them are linked to the carbonyls, and this appears to be the cause of the decrease in the dipole potential of the membrane. In contrast, four sucrose molecules displace only three water molecules per lipid, with no effect on the dipole potential or the carbonyl groups.     

Viscoelastic properties of linseed oil-based medium chain length poly(hydroxyalkanoate) films: effects of epoxidation and curing

Medium-chain-length poly(hydroxyalkanoate) (mcl-PHA) polymers derived from linseed oil (PHA-L) have a relatively small molar mass and contain a high concentration of unsaturated side-chains. As such, these polymers are amorphous and take on the consistency of a viscous liquid at room temperature. In order to increase the application potential of this material, the side-chain olefinic groups of PHA-L were converted to epoxy derivatives (PHA-LE) using m-chloroperoxybenzoic acid (m-CPBA). Epoxidation resulted in a 37% conversion of olefinic to epoxy groups. The epoxy groups enhanced the PHA-LE film susceptibility to crosslinking upon exposure to air. PHA-LE films began to crosslink and stiffen in less than 25 days, whereas PHA-L films began to crosslink between days 50 and 75. The PHA-LE films showed an increase in tensile strength (TS, from 4.8 to 20.7 MPa) and Young's modulus (YM, from 12.9 to 510.6 MPa) between 25 and 100 days. In contrast, PHA-L had a TS of 25.0 MPa and YM of 767.8 MPa after 100 days. Epoxidation helped induce crosslink formation; however, aging for 100 days ultimately resulted in crosslinked films from both PHA-L and PHA-LE with higher strength and durability than the original materials.     

The evolution of nodulation

In this review we will first describe the different steps leading to nodule formation, and these will be compared with processes of non-symbiotic plant development and growth. In general, aspects of both actinorhizal as well as rhizobial symbiosis are described, but in several cases, the emphasis will be on the Rhizobium-legume symbiosis because more knowledge of this system is available. Subsequently, the phylogeny of nodulating plants is described and a comparison is made between several aspects of legume and actinorhizal nodulation. At the end of this paper the relationship between nodule symbiosis and endomycorrhizal symbiosis is described, and it is discussed to what extent the development of root nodules involves unique properties, or whether processes and genes have been recruited from common plant development and the endomycorrhizal symbiosis.     

Abundance and growth of Pinna carnea (Mytiloida: Pinnacea) juveniles in suspended-frame culture

We quantified the abundance of juvenile Pinna carnea at Turpialito, Golfo de Cariaco, Venezuela, using collectors suspended at 8 m depth for 16 months and then evaluated their subsequent growth in suspended enclosures over 14 months in relation to concurrent measures of temperature and planktontonic food availability. All body components showed a rapid increase during the first four-five months. There was a decrease in somatic tissue mass and great variability in growth coincident at first spawning, suggesting a negative effect of gonadal production on somatic growth. A multiple regression analysis indicated a strong relationship between tissue growth and food availability as measured by organic sestonic matter. Although we determinated an adequated spat abundance for culture activities (80 spat per collector-30 x 60 cm), spat collection was limited, mainly from February through April. In addition to high variability in growth and negative influence of environmental factors during unfavorable periods, the muscles production required in 14 months, suggesting that P. carnea is not appropriate for commercial production of muscle. However, the high weight of all edible tissues mass attained (21 g) and the high survival rate in all experimentation (> 96%) suggests that P. carnea is fit for aquaculture.     

Annual distribution and abundance of Ceratium dens (Peridinales: Ceratiaceae) in the Gulf of California, Mexico

Ceratium dens distribution in the Gulf of California was studied based on three oceanographic campaigns, monthly abundance (1995-96) and during three red tide events in Mazatlán Bay. 52 phytoplankton samples with a Bongo net and 64 microns mesh light, during the year 120 water samples with a van Dorn bottle were collected in two sampling stations and from three red tide events. All samples were counted by the inverted-microscope method. The results showed that C. dens was present in the upper Gulf of California and surroundings of Cabo San Lucas. Two peaks were observed during spring and autumn in Mazatlán Bay during 1995-1996. The highest densities (20-360 cells.-l-1) were observed in coastal areas, whereas the lowest densities (1-14 cells.-l-1) were recorded in the open sea. During red tide events in 1985, 1989 and 1997, 144,000-256,000 cells.-l-1, 100,000-400,000 cells.-l-1, and 189,000-592,000 cells.ul-l was observed, respectively. During the red tide events C. dens varied, although sometimes was replaced by Skeletonema costatum or Pseudonitzchia spp and Ceratium furca. C. dens, seems to prefer areas with high productivity.     

Identification of Thai isolates assigned to the genus Gluconobacter based on 16S-23S rDNA ITS restriction analysis

Forty-four Thai isolates phenotypically assigned to the genus Gluconobacter were examined for 16S-23S rDNA ITS restriction analysis by MboII and SduI (=Bsp1286I) digestions. The Thai isolates tested were divided into seven groups: Group I for fourteen isolates, Group IX for one isolate, Group X for two isolates, Group V-2 for four isolates, Group XI for three isolates, Group IV for one isolate, and Group III for nineteen isolates. There were no isolates of either Group II or Group V-1 that were identified as G. cerinus. The isolates of Group III, Group IV, and Group XI were subjected to an additional 16S-23S rDNA ITS restriction analysis by AvaII, TaqI, BsoBI, and BstNI digestions. The isolates of Group III were divided into three groups and two subgroups: Group III-2 for five isolates, Group III-6 for two isolates, and Group III-4, which was divided into two subgroups, Subgroup III-4a for four isolates and Subgroup III-4b for eight isolates. The fourteen isolates of Group I were identified as G. oxydans, and the two isolates of Group X were temporarily identified as G. oxydans. The five isolates of Group III-2 and the one isolate of Group IV were identified as G. frateurii. The remaining twenty-two isolates of Group V-2, Group III-4, Group III-6, Group IX, and Group XI were not identified but are candidates for several new species.     

In vivo antigen stability affects DNA vaccine immunogenicity

The factors that determine the immunogenicity of Ags encoded by viral vaccines or DNA vaccines in vivo are largely unknown. Depending on whether T cell induction occurs via direct presentation of vaccine-encoded epitopes or via one of the different proposed pathways for Ag cross-presentation, the effect of intracellular Ag stability on immunogenicity may possibly vary. However, the influence of Ag stability on CD8(+) T cell induction has not been addressed in clinically relevant vaccine models, nor has the accumulation of vaccine-encoded Ags been monitored in vivo. In this study, we describe the relationship between in vivo Ag stability and immunogenicity of DNA vaccine-encoded Ags. We show that in vivo accumulation of DNA vaccine-encoded Ags is required for the efficient induction of CD8(+) T cell responses. These data suggest that many of the currently used transgene designs in DNA vaccination trials may be suboptimal, and that one should either use pathogen-derived or tumor-associated Ags that are intrinsically stable, or should increase the stability of vaccine-encoded Ags by genetic engineering.