A Bioluminescence Method for the Measurement of l-Glutamate: Applications to the Study of Changes in the Release of l-Glutamate from Lateral Geniculate Nucleus and Superior Colliculus After Visual Cortex Ablation in Rats

We have developed a rapid, simple, specific, and very sensitive bioluminescence method for the measurement of L-glutamate (L-Glu). Oxidation of L-Glu by glutamate dehydrogenase has been coupled with bacterial FMN reductase and luciferase. Light production (i.e., peak height or integral) was linear from less than 0.5 to 500 pmol of L-Glu. Potential interfering substances that may be encountered in brain tissue have been identified. The most potent inhibitors were ascorbate and the biogenic amines. Procedures that conferred long-term stability of the reagent mixture (greater than 8 h) were established. Bioluminescence analysis of L-Glu content in brain tissue extracts, fractions from release experiments, and human CSF corroborated respective results obtained by HPLC analysis. In this study, we have applied the method to monitor changes in the KCl-evoked release of endogenous L-Glu from milligram amounts of brain tissue, i.e., from lateral geniculate nucleus and superior colliculus after visual cortex ablation.     

Haemagglutinating and Hydrophobic Properties of Corynebacterium (Eubacterium) Suis

Corynebacterium (Eubacterium) suis strains from boars and sows haemagglutinated erythrocytes of different animal species (calf, guinea pig, poultry, pig, and human). The haemaigglutination was man nose resistant (MR) and was neither inhibited by L-fucose nor D-galactose. The hydrophobicity measured by salt aggregation test (0.1–0.9 mol/1 (NH4)2SO4) and the hydrophobic interaction chromatography test (90 % retention in octyl sepharose) together with the haemagglutinating activity, indicated the presence of fimbriae on the bacteria. The haemagglutinating and hydrophobic properties were heat-sensitive (60°C for 10 min) suggestive of the presence of a protein structure. Two types of fimbria-tion were demonstrated by electron microscopy. Fetuin and glyco^ protein inhibited the haemagglutination, whereas porcine mucin was without any effect. These results indicate that branched glycoproteins might be important receptors for these fimbriae. The pathogenic aspects of C. suis are discussed, based on recent acquired knowledge of the effect of other pyelonephritogenic bacteria.     

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.     

Predation and potential transfer of pollen in a population of Saxifraga hirculus

Body surface pollen load and gut pollen (pollen diet load) of flower visitors in a Danish population of Saxifraga hirculus were analyzed. The four most frequent visitors, Eurimyia lineata (Syrphidae), Neoascia tenur (Syrphidae), Asindulum nigrum (Mycetophilidae), and Zygaena trifolii (Zygaenidae) had total surface loads (and S. hirculus loads) of 1031 (771), 137 (106), 108 (78), and 318 (27) pollen grains, respectively. Other syrphid visitors had on average a load of 407 (192) pollen grains. Males of E. lineata, N. tenur , and A. nigrum carried more surface pollen than the females; females of E. lineata had a lot of pollen from Ranunculus acris and S. hirculus and males had a lot from S. hirculus . Females of N. tenur carried pollen mostly from S. hirculus, Lotus uliginosus and Galium uliginosum ; males carried pollen mostly from S. hirculus . The surface pollen load of A. nigrum was dominated by S. hirculus pollen, whereas that of Z. trifolii was dominated by Cirsium palustre and Lychnis flos-cuculi pollen. The amount and kind of pollen varied enormously both intraspecifically and interspecifically. Eurimyia lineata, N. tenur and other syrphids had total pollen diet loads (and S. hirculus pollen diet loads) of approximately 14900 (657), 3739 (2327) and 17784 (13040). The average syrphid contained pollen from five plant species, only one to two, however, were quantitatively important. The average diet load in females was nine times that in males in both E. lineata and N. tenur. Eurimyia lineata ate a lot of R. acris; N. tenur ate a lot of S. hirculus and G. uliginosum . Generally, no correlation was observed between size or season and the composition of surface load and diet load. Eurimyia lineata made most of the pollen visits. Thus, the seed set from this Danish population of S. hirculus was dependent on a single syrphid species.     

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.