Chimeric molecules with multiple neurotrophic activities reveal structural elements determining the specificities of NGF and BDNF.

Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are two members of a family of neurotrophic factors which show both overlapping and distinct neurotrophic activities. Using site-directed mutagenesis, chimeric molecules were constructed where different combinations of sequences from BDNF replaced the corresponding sequences in NGF. The resulting molecules were transiently expressed in COS cells and conditioned media containing the chimeric proteins were assayed for biological activity in explanted chick sympathetic, spinal and nodose ganglia. Our results show that the biological specificities of the two proteins are obtained by specific combinations of a set of sequences that differ between the two molecules. Some of these combinations allowed us to engineer molecules which display multiple neurotrophic activities recruited from both the NGF and BDNF proteins.     

Effects of gravel size and peat material concentrations on embryo survival and alevin emergence of brown trout,Salmo trutta L.

Embryo survival and alevin emergence pattern of brown trout were studied in simulated redds with different homogeneous gravel sizes and different concentrations of peat material. Optimal survival (95%) occurred in 18 mm gravel and survival decreased with decreasing gravel size. High concentrations (40%) of peat material resulted in low survival (65%). The proportion of premature emerging alevins increased in finer gravels and at high peat concentrations. Premature alevins had a large yolk sac and are probably very vulnerable to predators.     

Comparison of peptidase activities in some fungi pathogenic to arthropods

Peptidases, highly specific toward several synthetic chromogenic peptides, were found in the mycelia of four arthropod pathogenic fungi: Aphanomyces astaci, Beauveria bassiana, Metarrhizium anisopliae, and Paecilomyces farinosus. A. astaci peptidases had high hydrolyzing activities toward most of the peptides, especially those with arginine in the P1 position, while those of B. bassiana and P. farinosus readily hydrolyzed peptides with valine and arginine, as well as proline and tyrosine in the P2 and P1 positions, respectively. The hydrolyzing capacities of M. anisopliae peptidases were similar to A. astaci, but showed lower specific activities. Casein or azocoll was only hydrolyzed by A. astaci peptidases. B. bassiana and M. anisopliae had a very low hydrolyzing capacity toward casein and could not degrade azocoll. P. farinosus had no hydrolyzing activity toward casein or azocoll. Only peptidases from the crayfish pathogen A. astaci could degrade the crayfish cuticle. The peptidase preparations of A. astaci and B. bassiana hydrolyzing MeO-Suc-Arg-Pro-Tyr-pNA or Bz-Phe-Val-Arg-pNA were of the serine type. The possible importance of peptidase activity of arthropod pathogenic fungi in the infection process is discussed.     

Control of Adenovirus Gene Expression: Cellular Gene Products Restrict Expression of Adenovirus Host Range Mutants in Nonpermissive Cells

Adenovirus type 5 (Ad5) host range mutants dl312 and hr-1, with lesions in region E1A (0 to 4.5 map units) of the viral genome, fail to accumulate virus-specific early RNA during infection in HeLa cells. In a recent report, we showed that the addition of anisomycin, a stringent inhibitor of protein synthesis, at 1 h after infection of HeLa cells with hr-1 virus resulted in the accumulation of properly spliced and translatable mRNA from all early regions (M. G. Katze, H. Persson, and L. Philipson, Mol. Cell. Biol. 1:807-813, 1981). Based on these results we proposed a model in which expression of early mutant RNA was achieved through inactivation of a cellular protein normally causing a reduction in the amount of viral RNA. These studies have been extended in the present report, which shows that early viral proteins can be detected in Ad5 dl312- and Ad5 hr-1-infected HeLa cells which have been treated for several hours with anisomycin either shortly after infection or before infection. A pulse of drug treatment also resulted in expression of substantial amounts of adenovirus structural proteins after infection with both Ad5 hr-1 and Ad5 dl312, whereas in drug-free controls no late proteins were detected. The Ad5 hr-1 virus previously reported to be DNA replication negative in nonpermissive HeLa cells was found to replicate its DNA, albeit at low levels, when anisomycin was present either from 1 to 5 h postinfection or for 5 h before infection. When infectious virus production was examined in mutant-infected cells the titer of Ad5 dl312 virus was found to increase at least 500-fold in anisomycin-treated HeLa cells. Taken together, these and our previous results suggest that the block in gene expression characteristic for complementation group I Ad5 host range mutants in HeLa cells can be overcome by inactivating cellular gene products serving as negative regulators of viral gene expression.     

Three-dimensional structure of the apo form of the N-terminal EGF-like module of blood coagulation factor X as determined by NMR spectroscopy and simulated folding.

The three-dimensional structure of a 42-residue fragment containing the N-terminal EGF-like module of blood coagulation factor X was determined by means of 2D NMR spectroscopy and computer simulation. The spectroscopic data consisted of 370 NOE distances and 27 dihedral angle constraints. These were used to generate peptide conformations by molecular dynamics simulation. The simulations used a novel functional form for the constraint potentials and were performed with two time steps to ensure rapid execution. Apart from preliminary runs to aid assignment of NOEs, 60 runs resulted in 13 accepted structures, which have two antiparallel beta sheets, no alpha helices, and five tight turns. There is no hydrophobic cluster. The root mean square deviation for the backbone of the 13 conformations is 0.65 +/- 0.11 A against their mean conformation. About half of the side chains have well-defined structure. The overall conformation is similar to that of murine EGF.     

Sea snake (Microcephalophis gracilis) hemoglobin: Primary structure and relationships to other forms

The hemoglobin of the sea snakeMicrocephalophis gracilis was purified and the primary structure of the α and β chains determined. This is the first sea snake hemoglobin structure characterized, and apparently also the first complete structure of any snake hemoglobin (an α chain of a viper was known), allowing judgments of reptilian variants. Variations between the sea snake form and other reptilian forms are large (52–65 differences for the α chains), of similar order as those between the sea snake and avian (56–65 differences) or human (58 differences) forms. Functionally, 19 residues at α/β contact areas and 7 at heme contacts are exchanged in relation to the human α and β chains. Four positions of the sea snake hemoglobin contain residues thus far unique to this form. However, all replacements appear compatible with conserved overall functional properties.     

The lcrB (yscN/U) gene cluster of Yersinia pseudotuberculosis is involved in Yop secretion and shows high homology to the spa gene clusters of Shigella flexneri and Salmonella typhimurium.

Virulent bacteria of the genus Yersinia secrete a number of virulence determinants called Yops. These proteins lack typical signal sequences and are not posttranslationally processed. Two gene loci have been identified as being involved in the specific Yop secretion system (G. Cornelis, p. 231-265, In C. E. Hormache, C. W. Penn, and C. J. Smythe, ed., Molecular Biology of Bacterial Infection, 1992; S. C. Straley, G. V. Plano, E. Skrzypek, P. L. Haddix, and K. A. Fields, Mol. Microbiol. 8:1005-1010, 1993). Here, we have shown that the lcrB/virB locus (yscN to yscU) encodes gene products essential for Yop secretion. As in previously described secretion apparatus mutants, expression of the Yop proteins was decreased in the yscN/U mutants. An lcrH yscR double mutant expressed the Yops at an increased level but did not secrete Yops into the culture supernatant. The block in Yop expression of the ysc mutants was also circumvented by overexpression of the activator LcrF in trans. Although the Yops were expressed in elevated amounts, the Yops were still not exported. This analysis showed that the ysc mutants were unable to secrete Yops and that they were also affected in the negative Ca(2+)-regulated loop. The yscN/U genes showed remarkably high homology to the spa genes of Shigella flexneri and Salmonella typhimurium with respect to both individual genes and gene organization. These findings indicate that the genes originated from a common ancestor.