Estimating nanoscale deformation in bone by X-ray diffraction imaging method

Knowledge of internal stress-strain in bone tissue is important for clinical diagnosis and remedies. The inorganic mineral phase of apatite crystals in bone composite, because of its crystalline nature, provides a reliable way of measurement through X-ray diffraction system. Use of two-dimensional detector, imaging plate (IP), is considered to expedite the process with much more information, hence, is widely applied in the study of organization, stress, strain, etc. for crystalline substance. The distortion of Debye rings in the image obtained by IP can be directly related to the deformation in lattice plane of the crystals. Since X-ray diffraction method involves measurement at nano-level, proper focus on the extraction of data and corresponding analysis is needed. In the current work, we considered weighted average value of intensity to locate radius vectors along azimuthal direction in the diffracted rings from the primary array of digital data in steps of pixels. The widely applied approaches for profile shift measurement--peak shift and full width at half maximum (FWHM) of a peak, and shift of center of gravity of profile--were compared with a new concept of segmental shift (SS) proposed previously by the authors. We observed reliable and effective outcomes with higher precision in the consideration of SS while using IP as a detector. Our approach in this work for intensity integration and radius vector positioning especially add precision in such applications.     

Understanding site-specific residual strain and architecture in bovine cortical bone

Living bone is considered as adaptive material to the mechanical functions, which continually undergoes change in its histological arrangement with respect to external prolonged loading. Such remodeling phenomena within bone depend on the degree of stimuli caused by the mechanical loading being experienced, and therefore, are specific to the sites. In the attempts of understanding strain adaptive phenomena within bones, different theoretical models have been proposed. Also, the existing literatures mostly follow the measurement of surface strains using strain gauges to experimentally quantify the strains experienced in the functional environment. In this work, we propose a novel idea of understanding site-specific functional adaptation to the prolonged load in bone on the basis of inherited residual strains and structural organization. We quantified the residual strains and amount of apatite crystals distribution, i.e., the degree of orientation, using X-ray diffraction procedures. The sites of naturally existing hole in bone, called foramen, are considered from bovine femur and metacarpal samples. Significant values of residual strains are found to exist in the specimens. Trends of residual strains noted in the specimens are mostly consistent with the degree of orientation of the crystallites. These features explain the response behavior of bone to the mechanical loading history near the foramen sites. Preferential orientation of crystals mapped around a femoral foramen specimen showed furnished tailored arrangement of the crystals around the hole. Effect of external loading at the femoral foramen site is also explained by the tensile loading experiment.     

Discovery of pyridone-containing imidazolines as potent and selective inhibitors of neuropeptide Y Y5 receptor

A series of 2-pyridone-containing imidazoline derivatives was synthesized and evaluated as neuropeptide Y Y5 receptor antagonists. Optimization of the 2-pyridone structure on the 2-position of the imidazoline ring led to identification of 1-(difluoromethyl)-5-[(4S,5S)-4-(4-fluorophenyl)-4-(6-fluoropyridin-3-yl)-5-methyl-4,5-dihydro-1H-imidazol-2-yl]pyridin-2(1H)-one (7m). Compound 7m displayed statistically significant inhibition of food intake in an agonist-induced food intake model in SD rats and no adverse cardiovascular effects in anesthetized dogs. In addition, markedly higher brain penetrability and a lower plasma Occ90 value were observed in P-gp-deficient mdr1a (?/?) mice compared to mdr1a (+/+) mice after oral administration of 7m.     

Inhibition of tissue-bound semicarbazide-sensitive amine oxidase by two haloamines, 2-bromoethylamine and 3-bromopropylamine

Various mammalian tissues contain membrane-bound amine oxidase termed semicarbazide-sensitive amine oxidase (SSAO). A variety of compounds has been identified as relatively selective SSAO inhibitors, but those inhibitors currently available also inhibit monoamine oxidase (MAO). In the present study, inhibitory properties of 2-bromoethylamine (2-BEA) and 3-bromopropylamine (3-BPA) toward rat lung-bound SSAO have been studied. Regardless of preincubation, 2-BEA could not appreciably inhibit MAO-A and MAO-B activity, but 3-BPA at relatively high concentrations inhibited only MAO-B activity. 3-BPA was a competitive and reversible SSAO inhibitor with a Ki value of 17 microM regardless of preincubation. In contrast, without preincubation, 2-BEA competitively inhibited SSAO activity with the Ki value of 2.5 microM and after preincubation, the mode of inhibition changed to be noncompetitive, indicating irreversible inhibition after the preincubation. Dialysis experiments with 2-BEA-pretreated homogenate resulted in no recovery of SSAO activity even after overnight dialysis. A decreased rate of SSAO inhibition under N2 atmosphere to that obtained under O2 was produced upon preincubation of enzyme with 2-BEA, suggesting that oxidized intermediate was necessary for its inhibitory activity. Thus, 2-BEA first interacts with SSAO to form a reversible complex with a subsequent reaction, leading this complex to the covalently bound enzyme-inhibitor adduct. The data analyzed by the plot of 1/k' vs 1/2-BEA concentrations intersected on the y-axis indicate that the inhibition by 2-BEA is not mediated by a bimolecular reaction; thus it is not an affinity-labeling agent, but a suicide SSAO inhibitor. 2-BEA may be employed as a useful compound in the studying SSAO.     

Genes from Pseudomonas sp. strain BS involved in the conversion of L-2-amino-Delta(2)-thiazolin-4-carbonic acid to L-cysteine

DL-2-amino-Delta(2)-thiazolin-4-carbonic acid (DL-ATC) is a substrate for cysteine synthesis in some bacteria, and this bioconversion has been utilized for cysteine production in industry. We cloned a DNA fragment containing the genes involved in the conversion of L-ATC to L-cysteine from Pseudomonas sp. strain BS. The introduction of this DNA fragment into Escherichia coli cells enabled them to convert L-ATC to cysteine via N-carbamyl-L-cysteine (L-NCC) as an intermediate. The smallest recombinant plasmid, designated pTK10, contained a 2.6-kb insert DNA fragment that has L-cysteine synthetic activity. The nucleotide sequence of the insert DNA revealed that two open reading frames (ORFs) encoding proteins with molecular masses of 19.5 and 44.7 kDa were involved in the L-cysteine synthesis from DL-ATC. These ORFs were designated atcB and atcC, respectively, and their gene products were identified by overproduction of proteins encoded in each ORF and by the maxicell method. The functions of these gene products were examined using extracts of E. coli cells carrying deletion derivatives of pTK10. The results indicate that atcB and atcC are involved in the conversion of L-ATC to L-NCC and the conversion of L-NCC to cysteine, respectively. atcB was first identified as a gene encoding an enzyme that catalyzes thiazolin ring opening. AtcC is highly homologous with L-N-carbamoylases. Since both enzymes can only catalyze the L-specific conversion from L-ATC to L-NCC or L-NCC to L-cysteine, it is thought that atcB and atcC encode L-ATC hydrolase and N-carbamyl-L-cysteine amidohydrolase, respectively.     

Common reed produces starch granules at the shoot base in response to salt stress

Common reed (Phragmites australis) is a well known salt-tolerant plant and it is suggested that reeds recover Na(+) in the xylem sap of the shoot base (basal part of the shoot), store it temporarily in the shoot base, release it into the phloem sap, and then retranslocate it to the roots. To investigate whether Na(+) is retained in the shoot base of reeds, confocal laser scanning microscope (CLSM) observations were conducted using an intracellular Na(+)-specific fluorescent probe. The CLSM observations revealed that reeds produced a large number of the starch granules at the shoot base when salt-stressed, and that the fluorescence indicating the location of intracellular free Na(+) was observed in the same position as the starch granules. The Na content of starch granules was considerably greater than that of the shoot base, whereas the potassium (K) contents of the granules was only slightly greater than that of the shoot base. Reeds produced Na(+)-binding starch granules in the parenchyma cells of the shoot base when salt-stressed; these starch granules may decrease intracellular free Na(+). It is proposed that the site-specific production of Na(+)-binding starch granules constitutes a novel salt tolerance mechanism.     

Health effects of particulate matter formation in Life Cycle Impact Assessment: critical review and recommendation of models for Brazil

The International Journal of Life Cycle Assessment - The impact of particulate matter (PM) formation on human health in Life Cycle Impact Assessment (LCIA) can be characterized through combining...     

Induction of nociceptive responses by intrathecal injection of interleukin-1 in mice.

Intrathecal (i.t.) injection (between lumbar vertebrae 5 and 6) into mice of a markedly low dose of IL-1alpha (3x10(-4) fmol or 5.4 fg in 5 microl per mouse) induced behaviors involving scratching, biting, and licking of non-stimulated hindpaws. The IL-1-induced behaviors appeared within 10 min of the injection of IL-1alpha, peaked at 20-40 min, and had disappeared 60 min after the injection. The IL-1-induced behaviors were similar to the nociceptive responses induced in mice by i.t. injection of substance P (SP) or subcutaneous (s.c.) injection of formalin into the footpad. The IL-1-induced behaviors were suppressed by intraperitoneal morphine, indicating that they are nociceptive responses. The nociceptive responses induced by 3x10(-4) (5.4 fg) of IL-1alpha were almost completely suppressed by co-injection of 0.3 fmol (7.2 pg) of an IL-1 receptor antagonist (IL-1ra). An antiserum against substance P, but not an antiserum against somatostatin, suppressed the IL-1-induced nociceptive responses. The nociceptive responses induced by s.c. injection of 2% formalin into the footpad were also inhibited by i.t. injection of 30 pmol (720 ng) of IL-1ra. These results suggest that IL-1 may play a role in hyperalgesia in mice by acting as a factor augmenting pain transmission in the spinal cord at least in part by either directly or indirectly releasing substance P.     

Site-directed mutagenesis of restriction endonuclease HindIII

Site-directed mutagenesis by inverse PCR was done on the HindIII gene. Target residues to be mutated were chosen according to (i) the fact that a mutant obtained by sodium nitrite treatment showed almost no HindIII activity, where Asp-123 was replaced with Asn, and (ii) the model proposed by Stahl et al. (Stahl, F., Wende, W., Jeltsch, A. and Pingoud, A. Biol. Chem. 379, 467-473 (1998)). Seven kinds of mutants were obtained by the PCR, and their enzymatic and biochemical properties were examined. Three mutants, P50S, D108L, and D123N, showed fairly low HindIII activity. On the other hand, the other four, P84Q, E86K, V106E, and K125N, retained the activity. In particular, E86K showed higher activity than the wild enzyme. This fact was confirmed when activities of the purified wild and E86K enzymes were assayed. These results coincided fairly well with data using E. coli strains that carry the respective mutant plasmids, on their resistance to phage T7 and on growth rate. We conclude that the PE motif at residues 50 and 51, and DXK motif at residues 108-110, are responsible for the enzymic reaction of HindIII.