Calmodulin-binding proteins also have a calmodulin-like binding site within their structure. The flip-flop model.

The flip-flop model is a mechanistic model proposed to describe how calmodulin activates enzymes. One prediction based upon this model is that calmodulin-activated enzymes would contain a calmodulin-like binding site which, among other attributes, would bind the peptide melittin. Five purified calmodulin-activated enzymes, namely calcineurin, myosin light chain kinase, phosphorylase b kinase, phosphodiesterase, and NAD kinase, were all found to bind biotinylated melittin and to also bind an antimelittin antibody and biotinylated calmodulins. Using gel blots of crude tissue extracts (rat brain and Arabidopsis), most proteins did not bind any of the probes and thus do not have these characteristics. However, among those which bind any of these probes, a strong correlation was found between those proteins which bind biotinylated calmodulins and those which bind melittin and antimelittin. Gel blots of phosphorylase b kinase demonstrate that the alpha, beta, and gamma subunits all bind calmodulin and melittin. A putative calmodulin-like binding site sequence was identified in eight enzymes or subunits which may play an important role in both melittin binding and calmodulin-dependent regulation of these enzymes.     

The effects of maturation on self-induced dynamic body sway frequencies of girls performing acrobatics or classical dance

We investigated the effects of maturation on the dynamic body sways of healthy girls. Prepubertal and postpubertal girls practising professional physical activities requiring a good ability to maintain equilibrium (acrobats and dancers) were asked to stand on a free seesaw platform and the results compared to those for untrained age-matched girls. This platform (stabilometer) allows self-induced body sways. Stabilograms were obtained by a double integration of the angular acceleration from the recordings of the platform sways made with an accelerometer. Fast Fourier transform processing of stabilograms allowed spectral frequency analysis. The total spectrum energy and the energies of three frequency bands (0–0.5 Hz, 0.5–2 Hz, 2–20 Hz) were determined. ANOVA showed that, for all groups of different equilibrium activity and independent of visual input, prepubertal girls had higher energy values than postpubertal girls in the 0- to 0.5-Hz band whereas the opposite was true for 0.5- to 2-Hz band. Ballet dancers were more dependent than acrobats on visual inputs for the regulation of their postural control but were less dependent than untrained girls at both ages. Maturation seemed to shift body sways towards higher frequencies and the utilization of the cues of postural control was different according to the type of equilibrium activity practised by the subjects. Accepted: 7 February 1997     

Chemo-enzymatic synthesis of the Galili epitope Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc on a homogeneously soluble PEG polymer by a multi-enzyme system.

The alpha-Gal trisaccharide Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc 11 was synthesized on a homogeneously soluble polymeric support (polyethylene glycol, PEG) by use of a multi-enzyme system consisting of beta-1,4-galactosyltransferase (EC 2.4.1.38), alpha-1,3-galactosyltransferase (EC 2.4.1.151), sucrose synthase (EC 2.4.1.13) and UDP-glucose-4-epimerase (EC 5.1.3.2). In addition workup was simplified by use of dia-ultrafiltration. Thus the advantages of classic chemistry/enzymology and solid-phase synthesis could be united in one. Subsequent hydrogenolytic cleavage afforded the free alpha-Gal trisaccharide.     

Conformational analysis of the 3'-5'-cyclic dinucleotide d less than pApA greater than by means of molecular mechanics

The conformational properties of the cyclic dinucleotide d less than pApA greater than were studied by means of molecular mechanics calculations in which a multiconformation analysis was combined with minimum energy calculations. In this approach models of possible conformers are built by varying the torsion angles of the molecule systematically. These models are then subjected to energy minimization; in the present investigation use was made of the AMBER Force field. It followed that the lowest energy conformer has a pseudo-two-fold axis of symmetry. In this conformer the deoxyribose sugars adopt a N-type conformation. The conformation of the sugar-phosphate backbone is determined by the following torsion angles: alpha +, beta t, gamma +, epsilon t and zeta +. The conformation of this ringsystem corresponds to the structure derived earlier by means of NMR spectroscopy and X-ray diffraction. The observation of a preference for N-type sugar conformations in this molecule can be explained by the steric hindrance induced between opposite H3' atoms when one sugar is switched from N- to S-type puckers. The sugars can in principle switch from N- to S-type conformations, but this requires at least the transition of gamma + to gamma -. In this process the molecule obtains an extended shape in which the bases switch from a pseudo-axial to a pseudo-equatorial position. The calculations demonstrate that, apart from the results obtained for the lowest energy conformation, the 180 degrees change in the propagation direction of the phosphate backbone can be achieved by several different combinations of the backbone torsion angles. It appeared that in the low energy conformers five higher order correlations are found. The combination of torsion angles which are involved in changes in the propagation direction of the sugar-phosphate backbone in DNA-hairpin loops and in tRNA, are found in the dataset obtained for cyclic d less than pApA greater than. It turns out, that in the available examples, 180 degrees changes in the backbone direction are localized between two adjacent nucleotides.     

Sampling Atriplex bladders: a comparison of methods

Abstract. A major objection to the convenient sampling of Chenopodiacean bladders by brushing in aqueous solutions is the suspected leakage of ions from the leaf blade. To overcome this problem, a new method of sampling bladder hair is described, which also achieves rapid and quantitative separation and, at the same time, yields bladder samples of undoubtedly high purity. Leakage is stopped by the aid of liquid nitrogen. Comparison of this method to removal of bladders by brushing in aquenous solutions effectively confirm the validity of the customary method beyond dispute.     

Lung microsomal p-nitrophenol hydroxylase -- characterization and reconstitution of its activity

1. Formation of catechols from benzene and nitrobenzene have been implicated in the carcinogenic activity of these chemicals. In liver, p-nitrophenol, an intermediate of p-nitrobenzene is enzymatically converted to 4-nitrocatechol. 2. For the first time in this study, the presence of a highly active enzyme catalyzing the formation of 4-nitrocatechol from p-nitrophenol was detected in lung microsomes. The average specific activity of lung p-nitrophenol hydroxylase was found to be 0.494 nmol 4-nitrocatechol formed mg prot-1 min-1. 3. The optimum conditions for sheep lung microsomal p-nitrophenol hydroxylase were established. The maximal activity was noted at pH 6.8. The rate of p-nitrophenol hydroxylation was linear up to 2 mg prot/ml of incubation mixture. The maximal rate of 4-nitrocatechol formation was observed with 0.25 mM p-nitrophenol. 4. The Lineweaver-Burk and Eadie-Hofstee plots were found to be curve-linear. Two different Km values were calculated as 11.6 and 71.4 microM from the Lineweaver-Burk plot and as 10.7 and 74.5 microM from the Eadie-Hofstee plot. This suggested that there were either two forms of enzyme or two different enzymes participating in ortho hydroxylation of p-nitrophenol in lung microsomes. 5. Lung microsomal p-nitrophenol hydroxylase activity of sheep was reconstituted in the presence of purified lung microsomal cytochrome P-450, NADPH dependent cytochrome P-450 reductase and synthetic lipid, phosphatidylcholine dilauroyl.     

Hypoxanthine phosphoribosyltransferase activity in tissues and hypoxanthine concentrations in plasma and CSF of the horse in comparison with other species

1. Plasma hypoxanthine and xanthine concentrations are very low in the horse and low in rat, mouse and greyhound compared to concentrations in beagles, man, sheep and rabbit. 2. Activities in erythrocytes of the main enzyme metabolizing hypoxanthine, hypoxanthine phosphori-bosyltransferase, show a similar pattern (Tax et al., 1976, Comp. Biochem. Physiol. 54B, 209-212); thus low activities have been found where plasma concentrations were low. 3. Hypoxanthine phosphoribosyltransferase activities in horse tissue other than erythrocytes are similar to those in man and rabbit with high activities in brain; this enzyme may therefore be functionally important in equine brain.     

The Hemagglutinin of Bat-Associated Influenza Viruses Is Activated by TMPRSS2 for pH-Dependent Entry into Bat but Not Human Cells

New World bats have recently been discovered to harbor influenza A virus (FLUAV)-related viruses, termed bat-associated influenza A-like viruses (batFLUAV). The internal proteins of batFLUAV are functional in mammalian cells. In contrast, no biological functionality could be demonstrated for the surface proteins, hemagglutinin (HA)-like (HAL) and neuraminidase (NA)-like (NAL), and these proteins need to be replaced by their human counterparts to allow spread of batFLUAV in human cells. Here, we employed rhabdoviral vectors to study the role of HAL and NAL in viral entry. Vectors pseudotyped with batFLUAV-HAL and -NAL were able to enter bat cells but not cells from other mammalian species. Host cell entry was mediated by HAL and was dependent on prior proteolytic activation of HAL and endosomal low pH. In contrast, sialic acids were dispensable for HAL-driven entry. Finally, the type II transmembrane serine protease TMPRSS2 was able to activate HAL for cell entry indicating that batFLUAV can utilize human proteases for HAL activation. Collectively, these results identify viral and cellular factors governing host cell entry driven by batFLUAV surface proteins. They suggest that the absence of a functional receptor precludes entry of batFLUAV into human cells while other prerequisites for entry, HAL activation and protonation, are met in target cells of human origin.