ELECTRON MICROSCOPY OF OSTEOCLASTS IN HEALING FRACTURES OF RAT BONE

Osmium-fixed, undecalcified, callus tissue from healing fractures of rat tibias was sectioned with a diamond knife for study with the electron microscope. Large multinucleated cells were found adjacent to bone. A characteristic labyrinthine infolded border was consistently seen in parts of the cells close to the bone surface. The innermost parts of this "ruffled border" gave rise to vacuoles. The bone surface was always disrupted under the "ruffled border" of the cells. Needle-like crystals were seen at the osseous fringe, within folds in the ruffled border as well as within vacuoles deeper in the cells. Collagen fibers denuded of crystals were never observed. Mitochondria, containing clusters of fine granules, were abundant. The part of the cell away from bone contained rough endoplasmic reticulum and the cell membrane was thrown into irregular microvilli. These observations are discussed in relation to current concepts of osteoclastic resorption of bone.     

The deactivation pathways of the excited-states of the mycosporine-like amino acids shinorine and porphyra-334 in aqueous solution.

In vitro studies on the structurally related mycosporine-like amino acids (MAAs) porphyra-334 and shinorine in aqueous solutions were carried out aiming at their full photochemical and photophysical characterization and expanding the evidence on the assigned UV-photoprotective role of the molecules in vivo. The experiments on shinorine confirmed a high photostability and a poor fluorescence quantum yield, in concordance with previous results on porphyra-334. The estimation of triplet production quantum yields for both MAAs was achieved by laser-flash photolysis measurements. In particular, photosensitization experiments on porphyra-334 support the participation of the triplet state in the photodecomposition mechanism yielding a more precise value of [capital Phi](T). As well, photoacoustic calorimetry experiments allowed the first direct quantification of the nonradiative relaxation pathways of the excited MAAs in solution, corroborating that the vast majority (ca. 97%) of the absorbed energy is promptly delivered to the surroundings as heat, consistently with the low photodecomposition and emission yields observed.     

Ionic iridium-gold and rhodium-gold perfluorobenzenethiolato binuclear complexes: syntheses and solution behavior

By reacting [(C₅Me₅)M(SR_F)₂] (forM = Ir, Rf = C₆F₅ (1a) or C₆F₄H-p (1b); for M = Rh, Rf = C₆F₅ (2a) or C₆F₄H-p (2a)) in toluene with Na[AuCl₄], ionic binuclear compounds with the general formula [(C₅Me₅)M(μ-SR_F)₂AuCl₂]Cl for M = Ir, R = C₆F₅ (3a) or C₆F₄H-p (3a); for M = Rh, R_F = C₆F₅ (4a) or C₆F₄H-p (4b) can be obtained, together with small amounts of [(C₅Me₅)₂Rh₂(μ-SR_F)(μ-Cl)₂]Cl (R_F = C₆F₅ (5a) or C₆F₄H-p (5b)) as by-products when 2a and 2b were used.     

Synthesis and biological evaluation of novel NK-1 tachykinin receptor antagonists: The use of cycloalkyl amino acids as a template

In the course of a program aimed at synthesizing novel, potent NK-1 tachykinin receptor antagonists, we developed upon a bioactive model by comparing the low energy structures of a series of peptide and nonpeptide Substance P antagonists. The comparison was based on the super imposition of the aromatic rings, assuming that the rest of the molecule behaves predominantly as a template to arrange the key aromatic groups in the right spatial position. A series of 2-aminocyclohexane carboxylic acid analogues were then selected as the best templates for reproducing the postulated bioactive structure, leading to several pseudo-peptides with interesting biological activity. According to the molecular modeling, these compounds exhibit a neat parallel facing of the indolyl and naphthyl groups at about 3 Å distance. Ultraviolet absorption and steady state fluorescence measurements support this conclusion, showing a linear correlation between the spectral properties and the binding affinity of these analogues. Stacking of the indole ring with naphthalene gives rise to a complex characterized by a well-defined molar extinction coefficient. Consistently, steady state and lifetime fluorescence measurements suggest that the quenching process is ascribable to ground-state interactions between the chromophores. Implications of the π stacking propensity of aromatic groups in the biological activity of the compounds examined are briefly discussed. © 1995 John Wiley & Sons, Inc.     

Mutations in the X-linked E1 alpha subunit of pyruvate dehydrogenase: exon skipping, insertion of duplicate sequence, and missense mutations leading to the deficiency of the pyruvate dehydrogenase complex.

Human pyruvate dehydrogenase (PDH)-complex deficiency is an inborn error of metabolism that is extremely heterogeneous in its presentation and clinical course. In a study of 14 patients (7 females and 7 males), we have found a mutation in the coding region of the E1 alpha gene in all 14 patients. Two female patients had the same 7-bp deletion at nt 927; another female patient had a 3-bp deletion at nt 931. Another female patient was found to have a deletion of exon 6 in her cDNA. Two other female patients were found to have insertions, one of 13 bp at nt 981 and one of 46 bp at nucleotide 1078. Two male patients were found to have a 4-bp insertion at nucleotide 1163. The remaining six patients all had missense mutations. A male patient and a female patient both had an A1133G mutation. The other missense mutations were C214T, C615A, and C787G (two patients). Five of these mutations are novel mutations, five have been previously reported in other patients, and two were published observations in other patients in an E1 alpha-mutation summary. In the four cases where parent DNA was available, only one mother was found to be a carrier of the same mutation as her child.     

Properties of spinach chloroplast fructose-1,6-diphosphatase

Photosynthetic fructose-1,6-diphosphatase (FDPase) fractions I and II, earlier purified from spinach leaves, show a similar amino acid composition, with the exception of a higher glutamic acid content in the latter. In both fractions glutamic and aspartic acids are the main amino acids. pH activity profiles of fractions I and II are similar, with optima at 8·65–8·70, both showing a high specificity for fructose- 1,6-diphosphate. These two fractions are Mg²⁺-dependent for activity, with an Optimum Mg²⁺ concentration of 10 mM in standard conditions, which shifts to 5 mM when the MG²⁺/EDTA ratio is increased to 10; Mn²⁺ and Co²⁺ are slightly active. EDTA enhances FDPase activity slightly, with an optimum at 0·4–0·8 mM. Cysteine has no activating effect, and acts as an inhibitor above 10 mM. Both I and II have an optimum substrate concentration of 4 mM, and the substrate inhibits at concns above this value. Kinetic velocity curves are sigmoidal, with the concave zone located in the range of physiological substrate concns. (Hill coefficient 1·75 for both). This suggests a strong regulatory role of fructose-1,6-diphosphate. K_m values are 1·4 × 10⁻³ M (fraction I) and 1·1 × 10⁻³ M (fraction II). The highest activity rate occurs at 60°, in accordance with the high thermostability of both fractions; the activation energies are 14·3 kcal/mol (fraction I) and 13·0 kcal/mol (fraction II).     

Ultrahigh resolution drug design. II. Atomic resolution structures of human aldose reductase holoenzyme complexed with Fidarestat and Minalrestat: implications for the binding of cyclic imide inhibitors

The X-ray structures of human aldose reductase holoenzyme in complex with the inhibitors Fidarestat (SNK-860) and Minalrestat (WAY-509) were determined at atomic resolutions of 0.92 A and 1.1 A, respectively. The hydantoin and succinimide moieties of the inhibitors interacted with the conserved anion-binding site located between the nicotinamide ring of the coenzyme and active site residues Tyr48, His110, and Trp111. Minalrestat's hydrophobic isoquinoline ring was bound in an adjacent pocket lined by residues Trp20, Phe122, and Trp219, with the bromo-fluorobenzyl group inside the "specificity" pocket. The interactions between Minalrestat's bromo-fluorobenzyl group and the enzyme include the stacking against the side-chain of Trp111 as well as hydrogen bonding distances with residues Leu300 and Thr113. The carbamoyl group in Fidarestat formed a hydrogen bond with the main-chain nitrogen atom of Leu300. The atomic resolution refinement allowed the positioning of hydrogen atoms and accurate determination of bond lengths of the inhibitors, coenzyme NADP+ and active-site residue His110. The 1'-position nitrogen atom in the hydantoin and succinimide moieties of Fidarestat and Minalrestat, respectively, form a hydrogen bond with the Nepsilon2 atom of His 110. For Fidarestat, the electron density indicated two possible positions for the H-atom in this bond. Furthermore, both native and anomalous difference maps indicated the replacement of a water molecule linked to His110 by a Cl-ion. These observations suggest a mechanism in which Fidarestat is bound protonated and becomes negatively charged by donating the proton to His110, which may have important implications on drug design.     

Homeostasis of cell composition during prolonged darkness

The chemical composition of organisms in relation to their environmental resource availability is an area of intense research activity. We studied the changes in cell composition of the cyanobacterium Phormidium autumnale in response to prolonged darkness. Cells allocated their internal resources in a homeostatic manner, oxidizing all the three major cellular constituents in a proportional way. This resulted in constant C/N and carbohydrates, lipids and proteins ratios that remained unaltered throughout the whole incubation period. We propose the maintenance of balanced cell composition (homeostasis) as an evolutionary strategy related to environmental transitory changes.     

A BASIC microcomputer program to calculate the secondary structure of proteins from their circular dichroism spectrum

A BASIC program (CDPROT) has been developed to calculate thesecondary structure of proteins from their far UV circular dichroismspectrum. This implementation can use different reference spectra,calculated either from model polypeptides or proteins of knowntertiary structure. Apart from obtaining the a-helical, ß-structure,ß-turns or random percentages which would generatethe spectrum of best fit with respect to the experimental measures,CDPROT represents on screen both theoretical and experimentalspectra indicating the root-mean-square error. The provisionof additional reference spectra by the user is also considered,and another program (STOREREF) performs the editing in an adequateformat for CDPROT. Received on March 8, 1988; accepted on June 3, 1988