Structural analysis of heparin by methylation and g.l.c.-m.s.: preliminary results

Heparin is a complex mixture of polysaccharides differing in biological activity and structure, and attempts to relate this activity to structure have suffered, owing to a lack of sufficiently sensitive and specific analytical methods. Application of methylation analysis to determination of the structure of heparin is described. Carboxyl-reduced heparin was converted into its pyridinium salt, this was dissolved in Me2SO, and free OH and NH groups were methylated with dimethylsulfinyl anion. Sulfate groups were removed by solvolysis, and after dialysis, the polymer was acetylated and depolymerized by acetolysis. The resulting monosaccharides were converted into alditol acetates, which were separated by capillary, gas-liquid chromatography, and identified by both electron impact and chemical ionization mass spectrometry. Seventeen different monosaccharides were identified in the hydrolyzate. All of the expected internal hexosaminyl and glycosyluronic residues were identified. Although several sugars were identified as nonreducing termini, only a hexosamine 6-sulfate was identified as a reducing-terminus sugar. The results indicate that methylation analysis of heparins and other complex, sulfated glycosaminoglycans is feasible.     

Ligand displacement reactions of oxyhemocyanin: comparison of reactivities of arthropods and molluscs.

The ligand displacement reactions of oxyhemocyanin have been compared over a series of arthropods and molluscs. The arthropods (with the exception of $\text{Limulus}$) are found to be more reactive than the molluscs, (k_cancer = .04 hr^?1, k_busycon = .002 hr^?1, k_limulus ? 10^?4hr^?1 N₃^? reactions). Correlation of the spectral properties of the oxy sites require these to be extremely similar with small differences being associated with shifts in the dd transition energies. The met produced by ligand displacement contains variable amounts of EPR detectable, group 2 exogenous ligand damaged sites (arthropods 25–35%, molluscs 3–9%, $\text{Limulus}$ <1%). A parallel (arthropod > mollusc ～ $\text{Limulus}$ ～ 0) group 2 ligand damaged site is also reported for the half met derivatives.     

Resonance raman studies of a c type algal cytochrome. Deuterium shifts and a comparison with mammalian cytochrome c.

A c type cytochrome isolated from Synechococcus lividus grown on water and 2H2O media, has been studied by resonance Raman spectroscopy. The spectra were taken on the oxidized and reduced protein with excitation within the Soret band at 441.6 nm to determine whether individual resonance Raman bands of the heme shift upon deuterium substitution and also to provide a comparison with the spectra of horse heart cytochrome c. Some of the shifts observed with the deuterated heme c are larger than the corresponding shifts in meso-deuterated metalloporphyrins suggesting mixing of peripheral substituent vibrations with the skeletal modes of the porphyrin macrocycle. The algal cytochrome exhibits resonance Raman spectra roughly similar to those of horse heart cytochrome c, consistent with its optical absorption spectra which is typical of c type cytochromes, although a detailed comparison reveals note-worthy differences between the spectra of the two proteins; this may be a reflection of the effect of non-methionine ligands and protein environment on the vibrations of the c type heme in the algal cytochrome.     

Lennard-Jones potential calculations of the barrier to rotation around the glycosidic C-N linkage in selected purine nucleosides and nucleotides. A direct comparison of the results of 6-12 potential calculations with results of semiempirical molecular orbital studies

Lennard-Jones (6–12) potential energy calculations were performed on the barrier to rotation around the CN glycosidic linkage in purine nucleosides and nucleotides. While such calculations are strictly valid only for relatively non-polar substances the results of these simple and very inexpensive calculations appear to be in reasonable accord with the results of semi-empirical molecular orbital calculations. The effect of the assumed hydrogen van der Waals radius on rotational barrier was examined and use of the accepted value of 1·2 along with the newer 1·0 simultaneously is shown to be useful in determining the source of such barriers. The detailed curves indicated that those molecules found in the syn conformation in the solid state possessed relatively small barriers to rotation. Adenine nucleotides and nucleosides invariably were indicated to have very large barriers to rotation with notable exception of the 3′-O-acetyladenosine and 2′ and 3′-AMP. A systematic variation of the C(8) substituent from H to F to Cl to Br in guanosine and 8-bromoguanosine indicates that the preference of the syn conformation is primarily due to steric factors. However, a minor barrier which appears to be due to the fluorine electronegativity is also noticed when the C(8)F passes near the ribose O(1′)atom.Inclusion of electrostatic interaction (monopole-monopole) in the potential barrier calculation indicated a minor contribution from this term even in the molecules possessing considerable charge asymmetry.     

Chemical taxonomy of Torrubiella s. lat.: zeorin as a marker of Conoideocrella

The insect pathogens in the genus Torrubiella s. lat. were recently divided into new genera based on molecular phylogenetic characters. Isolates collected at various locations in Thailand, were tested for their productivity of a hopane-type triterpene, zeorin (6α,22-dihydroxyhopane), when cultured in potato dextrose broth under static conditions. Among the 49 strains of Torrubiella s. lat. species, Conoideocrella luteorostrata (ten strains) and C. tenuis (seven strains), all collected on scale insects (Hemiptera), produced zeorin, whereas another six strains of Orbiocrella petchii (which was recently removed from Torrubiella) failed in the detection of this secondary metabolite. All other Torrubiella s. lat. (26 strains), collected on other insect hosts including leafhoppers (eight strains), Lepidoptera (one strain), and spiders (17 strains), produced no detectable zeorin. Paecilomyces cinnamomeus (nine strains), the anamorph of C. luteorostrata, also produced zeorin. These results correspond with the recent taxonomic reclassification based on multigene phylogeny.     

Human visual pigments: microspectrophotometric results from the eyes of seven persons

The material for this work was obtained from seven eyes removed because of malignant growths. Foveal and parafoveal samples of the retinas were taken and transverse measurements were made of the absorbance spectra of the outer segments of the rods and cones, using a Liebman microspectrophotometer. Four kinds of spectra were obtained with absorbance peaks at the following wavelengths: rods, 496.3 +/- 2.3 nm (n = 39); red cones, 558.4 +/- 5.2 nm (n = 58); green cones, 530.8 +/- 3.5 nm (n = 45); blue cones, 419.0 +/- 3.6 nm (n = 5). The distribution of the peaks was unimodal for the rods. For the red and green cones, however, there was evidence for bimodal distributions, with sub-population maxima at 563.2 +/- 3.1 nm (n = 27) and 554.2 +/- 2.3 nm (n = 31) for the reds and at 533.7 +/- 2.1 nm (n = 23) and 527.8 +/- 1.8 nm (n = 22) for the greens. A substantial difference in mean spectral location of the red cones was observed between patient 1 (561 nm) and patient 4 (553 nm). Both patients were classified as normal trichromats by all clinical tests of colour vision but there was a clear difference in their relative sensitivities to long-wave fields. In both direction and magnitude, this difference proved to be that required by the microspectrophotometric results.     

Reactivity of cytochromes c and f with mutant forms of spinach plastocyanin.

The reduction of plastocyanin by cytochromes c and f has been investigated with mutants of spinach plastocyanin in which individual, highly conserved surface residues have been modified. These include Leu-12 and Phe-35 in the 'northern' hydrophobic patch and Tyr-83 and Asp-42 in the 'eastern' acidic patch. The differences observed all involved binding rather than the intrinsic rates of electron transfer. The Glu-12 and Ala-12 mutants showed small but significant decreases in binding constant with cytochrome c, even though the cytochrome is not expected to make contact with the northern face of plastocyanin. These results, and small changes in the EPR parameters, suggested that these mutations cause small conformational changes in surface residues on the eastern face of plastocyanin, transmitted through the copper centre. In the case of cytochrome f, the Glu-12 and Ala-12 mutants also bound less strongly, but Leu12Asn showed a marked increase in binding constant, suggesting that cytochrome f can hydrogen bond directly to Asn-12 in the reaction complex. A surprising result was that the kinetics of reduction of Asp42Asn were not significantly different from wild type, despite the loss of a negative charge.     

Photochemically and chemically activatable antisense oligonucleotides: comparison of their reactivities towards DNA and RNA targets.

Dodecadeoxyribonucleotides derivatized with 1,10-phenanthroline or psoralen were targeted to the point mutation (G<-->U) in codon 12 of the Ha-ras mRNA. DNA and RNA fragments, 27 nucleotides in length, and containing the complementary sequence of the 12mers, were used to compare the reactivity of the activatable dodecamers (cleavage of the target by the phenanthroline-12mer conjugates; photo-induced cross-linking of psoralen-12mer conjugates to the target). The reactivity of the RNA with the dodecamers was weaker than that of the DNA target. With psoralen-substituted oligonucleotides, it was possible to obtain complete discrimination between the mutated target (which contained a psoralen-reactive T(U) in the 12th codon) and the normal target (which contained G at the same position). When longer Ha-ras RNA fragments were used as targets (120 and 820 nucleotides), very little reactivity was observed. Part of the reactivity could be recovered by using 'helper' oligonucleotides that hybridized to adjacent sites on the substrate. A 'helper' chain length greater than 13 was required to improve the reactivity of dodecamers. However, the dodecanucleotides induced RNase H cleavage of the target RNA in the absence of 'helper' oligonucleotide. Therefore, in the absence of the RNase H enzyme, long oligonucleotides are needed to compete with the secondary structures of the mRNA. In contrast, formation of a ternary complex oligonucleotide-mRNA-RNase H led to RNAT cleavage with shorter oligonucleotides.     

Study of the structure of Shigella flexneri o antigen I. Chemical degradation

The lipopolysaccharide antigen of Shigella flexneri F6S serotype 5 appears under the electron microscope as long filaments having a molecular weight of 10·10⁶–45·10⁶. This macromolecular structure is built of repeating units having a sedimentation constant, s_20,w, of 10.2 S and a molecular weight of 250 000 and is linked together by hydrophobic interaction, due to the lipid part of the molecule and by bivalent cations and carboxylic groups. These unit antigen structures are themselves polymerization products built of 1.44-S subunits linked together by weak chemical bonds of the same nature as those binding the units in the antigen molecule. The nature of the degrading agent seems to be important for obtaining the subunit fragments. Some preliminary experiments indicate that the 10.2-S unit antigen structures still retain their biological properties such as phage-receptor function and toxicity.     

Conformation change of cytochrome c. II. Ferricytochrome c refinement at 1.8 A and comparison with the ferrocytochrome structure

The X chromosomal nucleolus organizer of Drosophila hydei contains about 500 ribosomal RNA genes. The 28 S rRNA coding region of about 50% of these genes is interrupted by an intervening sequence of 6.0 × 10³ base-pairs. Restriction enzyme analysis revealed that more than 90% of the rRNA genes with intervening sequences are present as one or a few clusters within the X chromosomal nucleolus organizer. Furthermore, even though X chromosomal rRNA genes show several distinct size classes of non-transcribed spacers, the cluster of repeating units containing an intervening sequence has major spacer lengths of 4.4 × 10³ and 4.6 × 10³ base-pairs; spacers 5.1 × 10³ base-pairs in length are mainly linked with genes lacking the intervening sequence.     

Crystal structure of the catalytic domain of human complement c1s: a serine protease with a handle

C1s is the highly specific modular serine protease that mediates the proteolytic activity of the C1 complex and thereby triggers activation of the complement cascade. The crystal structure of a catalytic fragment from human C1s comprising the second complement control protein (CCP2) module and the chymotrypsin-like serine protease (SP) domain has been determined and refined to 1.7 A resolution. In the areas surrounding the active site, the SP structure reveals a restricted access to subsidiary substrate binding sites that could be responsible for the narrow specificity of C1s. The ellipsoidal CCP2 module is oriented perpendicularly to the surface of the SP domain. This arrangement is maintained through a rigid module-domain interface involving intertwined proline- and tyrosine-rich polypeptide segments. The relative orientation of SP and CCP2 is consistent with the fact that the latter provides additional substrate recognition sites for the C4 substrate. This structure provides a first example of a CCP-SP assembly that is conserved in diverse extracellular proteins. Its implications in the activation mechanism of C1 are discussed.     

The force-length relationship of a muscle-tendon complex: experimental results and model calculations

Models are useful when studying how architectural and physiological properties of muscle-tendon complexes are related to function, because they allow for the simulation of the behaviour of such complexes during natural movements. In the construction of these models, evaluation of their accuracy is an important step. In the present study, a model was constructed to calculate the isometric force-length relationship of the rat extensor digitorum longus muscle-tendon complex. The model is based on the assumption that a muscle-tendon complex is a collection of independent units, each consisting of a muscle fibre in series with a tendon fibre. By intention, values for model parameters were derived indirectly, using only the measured maximal isometric tetanic force, the distance between origin and insertion at which it occurred (optimum lOI) and an estimate of muscle fibre optimal length. The accuracy of the calculated force-length relationship was subsequently evaluated by comparing it to the relationship measured in isometric tetanic contractions of a real complex in the rat. When the length of distal muscle fibres, measured during isometric contraction at optimal lOI of the whole complex, was used as an estimate for muscle fibre optimal length of all muscle fibre-tendon fibre units in the model, the calculated relationship was too narrow. That is, both on the ascending limb and on the descending limb the calculated tetanic force was lower than the measured tetanic force.(ABSTRACT TRUNCATED AT 250 WORDS)     

The extended phenotype(s): a comparison with niche construction theory

While niche construction theory locates animal artefacts in their constructors’ environment, hence treating them as capable of exerting selective pressure on both the constructors and their descendants, the extended phenotype concept assimilates artefacts with their constructors’ genes. Analogous contrasts apply in the case of endoparasite and brood parasite genes influencing host behaviour. The explanatory power of these competing approaches are assessed by re-examining the core chapters of Richard Dawkins’ The Extended Phenotype. Because animal artefacts (chapter 11) have multiple evolutionary consequences for their constructors, the extra-body effects of a gene seemingly include feedback effects on multiple other genes, a result which is more consistent with niche construction theory than with selfish gene theory. In the case of endoparasite genes influencing host behaviour (chapter 12), Dawkins’ argument leaves out what appears to be the key explanatory component, namely the role of the host’s own bodily systems in making it possible for such genes to exist. For action at a distance (chapter 13), it is unclear whether the key genes have extended effects because they sit in the body of the manipulating organism, or alternatively do not have such effects because they sit in the body of its victim. It is argued that niche construction theory offers a superior explanation in all three cases, regardless of whether the extended phenotype concept is interpreted in selfish gene or selfish organism terms.     

Caenorhabditis elegans as a chemical screening tool for the study o f neuromuscular disorders. Manual and semi-automated methods

We previously reported the use of the cheap and fast-growing nematode Caenorhabditis elegans to search for molecules, which reduce muscle degeneration in a model for Duchenne Muscular Dystrophy (DMD). We showed that Prednisone, a steroid that is generally prescribed as a palliative treatment to DMD patients, also reduced muscle degeneration in the C. elegans DMD model. We further showed that this strategy could lead to the discovery of new and unsuspected small molecules, which have been further validated in a mammalian model of DMD, i.e. the mdx mouse model. These proof-of-principles demonstrate that C. elegans can serve as a screening tool to search for drugs against neuromuscular disorders. Here, we report and discuss two methodologies used to screen chemical libraries for drugs against muscle disorders in C. elegans. We first describe a manual method used to find drugs against DMD. We further present a semi-automated method, which is currently in use for the search of drugs against the Schwartz-Jampel Syndrome (SJS). Both assays are simple to implement and can be readily transposed and/or adapted to screens against other muscle/neuromuscular diseases, which can be modeled in the worm. Finally we discuss, with respect to our experience and knowledge, the different parameters that have to be taken into account before choosing one or the other method.