Microdensitometric analysis for study of skeletal growth and aging in the living subject

Microdensitometric analysis of a radiographic image of mineralized tissue offers information on the morphologic characteristics of the specimen examined. The use of microstructure (such as individual trabecula of bone) and macrostructure (such as Harris lines of arrest) as in vivo bone markers allows for assessment of gross skeletal changes in nondestructive fashion. This technique is also applicable to the study of the interrelationship of tooth and bone. In addition, microdensitometry offers precise information on the fine structure of bone as regards the trabecular elements. This technique is applicable in determining the size and quantity of trabeculae, their persistence over long periods of time, and the factors responsible for change or lack of change in trabeculae during growth and aging.     

Importance of the interaction protein–protein of the CaM–PDE1A and CaM–MLCK complexes in the development of new anti‐CaM drugs

Protein–protein interactions play central roles in physiological and pathological processes. The bases of the mechanisms of drug action are relevant to the discovery of new therapeutic targets. This work focuses on understanding the interactions in protein–protein–ligands complexes, using proteins calmodulin (CaM), human calcium/calmodulin‐dependent 3′,5′‐cyclic nucleotide phosphodiesterase 1A active human (PDE1A), and myosin light chain kinase (MLCK) and ligands αII–spectrin peptide (αII–spec), and two inhibitors of CaM (chlorpromazine (CPZ) and malbrancheamide (MBC)). The interaction was monitored with a fluorescent biosensor of CaM (hCaM M124C–mBBr). The results showed changes in the affinity of CPZ and MBC depending on the CaM–protein complex under analysis. For the Ca²⁺–CaM, Ca²⁺–CaM–PDE1A, and Ca²⁺–CaM–MLCK complexes, CPZ apparent dissociation constants (K_ds) were 1.11, 0.28, and 0.55 μM, respectively; and for MBC K_ds were 1.43, 1.10, and 0.61 μM, respectively. In competition experiments the addition of calmodulin binding peptide 1 (αII–spec) to Ca²⁺–hCaM M124C–mBBr quenched the fluorescence (K_d = 2.55 ± 1.75 pM) and the later addition of MBC (up to 16 μM) did not affect the fluorescent signal. Instead, the additions of αII–spec to a preformed Ca²⁺–hCaM M124C–mBBr–MBC complex modified the fluorescent signal. However, MBC was able to displace the PDE1A and MLCK from its complex with Ca²⁺–CaM. In addition, docking studies were performed for all complexes with both ligands showing an excellent correlation with experimental data. These experiments may help to explain why in vivo many CaM drugs target prefer only a subset of the Ca²⁺–CaM regulated proteins and adds to the understanding of molecular interactions between protein complexes and small ligands. Copyright © 2013 John Wiley & Sons, Ltd.     

Intra- and interobserver concordance in scoring Harris lines: A test on bone sections and radiographs

Little attention has been devoted to assessing the reproducibility of (paleo)pathological observations. Harris lines (HL) are among the markers most used to determine chronology of stresses suffered during growth. Nevertheless, their scoring entails remarkable methodological difficulty. Bone sections (S) and radiographs (R) of 29 adult tibiae of archeological provenance (medieval) were scored for HL by five observers. At regular intervals of time, each observer gave two independent counts on both series. Results show a) a substantial interobserver disagreement of HL estimates for both sectional and radiographic records, and b) a high level of intraobserver error. © 1994 Wiley-Liss, Inc.     

Divergent selection for skeletal malformations in chickens alters polymorphism at microsatellite loci

The objective of this study was to determine microsatellite polymorphism in Rhode Island Red (RIR) and Sussex (SX) chickens, divergently selected over six generations for high (H) or low (L) incidence of skeletal defects in embryos (30.7% for H lines, 3.7% for L lines). The polymorphism analysis covered 15 microsatellite markers within four lines (a total of 60 individuals). Eight alleles were identified as specific to H lines and six alleles as specific to L lines. The selection for skeletal malformation appears to have affected the frequency of microsatellite alleles. The experimental material examined constitutes a valuable source for identification of real genes causing skeletal defects.     

Spectroscopic studies of bovine serum albumin adsorbed onto magnetic–thermosensitive carbon microspheres

To investigate the influence of magnetic–thermosensitive carbon microspheres (MTCMSs) as a targeting drug carrier on serum albumins in vitro, in this study, bovine serum albumin (BSA) was chosen as a template protein to explore the interaction between serum proteins and MTCMSs. Fluorescence spectrophotometry, ultraviolet–visible absorbance (UV–vis) spectrophotometry and circular dichroism spectrometry were used to investigate the interaction between MTCMSs and BSA. Results indicate that BSA interacts with MTCMSs and the fluorescence intensity of BSA is quenched by 50% in a static quenching at 310 K when the concentration of MTCMSs reaches 30 mg/L. Thermodynamic parameters including free energy change (△G^θ), enthalpy change (△H^θ) and entropy change (△S^θ) were calculated. The results (△G^θ < 0, △H^θ < 0 and △S^θ > 0) suggest a spontaneous process and the formation of a hydrogen bond between MTCMSs and BSA. UV–vis measurements reveal that the micro‐environment of an amino acid residue is altered in the presence of MTCMSs. The α‐helix content of BSA decreases by 4% and the β‐sheet content increases by 3.2% with increasing concentrations of MTCMSs to 30 mg/L, illustrating a change in the skeletal structure of BSA. These results demonstrate that MTCMSs as a targeting drug carrier impact the structure of serum albumins. This work provides not only a theoretical basis of BSA adsorption onto MTCMSs, but also an understanding of safe drug carriers in biomedicine. Copyright © 2016 John Wiley & Sons, Ltd.     

The Occurrence and Age Distribution of Harris Lines in Turkey

Harris lines are widely accepted as indicators of physiological stress and provide valuable data for determining the extent and nature of the physiological stress factors acting on a human community. Traditionally, Harris lines are studied in skeletal populations. In the study reported here, data were collected on living children to eventually clarify if stress is basically chronic or acute in nature, if it has a greater impact on children or adults, and if it is correlated with increased rates of mortality. The existence of Harris lines was determined in a sample of 400 children, 210 males and 190 females, randomly selected from those under examination in the radiology services of hospitals. Radiological analysis was used to analyze Harris lines. The age of Harris line formation and variations in the number of lines with age were established to determine at which age the densest line population was present. For this sample, the formation of Harris lines is around 2–3 years of age, in agreement with published literature. It should be taken into consideration that Harris lines are the end result of multiple factors, rather than a single stress factor, and are influenced by an individual’s immune system and resistance to stress.     

Typing of Listeria monocytogenes strains isolated in Italy by inlA gene characterization and evaluation of a new cost‐effective approach to antisera selection for serotyping

Aims: In this study, 105 Listeria monocytogenes strains isolated from humans, foods and environmental samples were characterized using several typing methods. Moreover, serotyping procedure was evaluated, and a cost‐effective methodological approach based on preliminary PCRs screening was proposed. Methods and Results: The isolates were analysed by conventional serotyping, multiplex‐PCRs for serogroup and lineage identification and PCR–RFLP of inlA gene to identify potentially noninvasive L. monocytogenes. Among the strains, only the serotypes 1/2a, 1/2c, 1/2b, 4b and 3a were identified. The isolates were classified into serogroups I (58·10%), II (22·85%), III (12·38%) and IV (6·67%). Among clinical strains, lineage I was more represented (68·75%) than lineage II; whereas, lineage II was more associated with food (90·24%) and environmental (85·72%) isolates. Most of food (89·02%) and environmental (85·71%) isolates were classified into truncated InlA profiles, whereas the 93·75% of clinical strains were associated with a complete form of the protein. Conclusion: Molecular techniques were sensitive and specific for classifying strains into serogroup and lineage and in agreement with the serotyping. Moreover, a preliminary PCRs‐based screening was proposed to select only the necessary antisera by a flow chart; this methodological approach allows cost saving up to 42%. Our results further suggest the role of InlA protein in human listeriosis, particularly in immunocompetent individuals, and a correlation between truncated protein and serotype. Significance and Impact of the Study: This study further validates molecular methods for L. monocytogenes analysis and proposed a new cost‐effective approach for serotyping. It could help to improve a national surveillance network for L. monocytogenes infections in Italy.     

Overcoming climate change adaptation barriers: A study on food–energy–water impacts of the average American diet by demographic group

Effectively adapting to climate change involves overcoming social and ecological system barriers. The present study uses a three‐phase adaptation framework to propose adaptation strategies aimed at overcoming socioecological barriers of the food–energy–water (FEW) nexus. Cradle‐to‐farm‐gate land, greenhouse gas (GHG), and water impacts—that derive from food consumption in the United States—are analyzed and differentiated by major demographic groups (Black, Latinx, and White). Results indicate that the White demographic yields the highest per capita GHG (680 kg of CO₂ eq?year^?1) and water impacts (328,600 L?year^?1) from food consumption, whereas the Black demographic yields the highest per capita land impacts (1,770 m²?year^?1) from food consumption. Our findings suggest that obtaining data with the intention of building consensus across sociodemographic lines overcomes barriers in the understanding phase, leading to increased social receptivity for many planning and managing phase processes. Specifically, we find that identifying and developing leaders who possess the cognitive and interpersonal capacity to manage many variables and stakeholders is key to assessing and selecting adaptation options in the planning phase. We also propose using government programming to encourage environmentally friendly food purchasing behavior. Then, we discuss how our proposals could be used in adaptation feasibility and evaluation activities in the managing phase. In all, these findings facilitate the development of improved climate change adaptation and policy by satisfying the understanding phase of the climate change adaptation framework, establishing a cross‐disciplinary methodological approach to addressing socioecological problems, and providing useful FEW impact data for FEW nexus and climate change researchers.     

GENETIC VARIATION AMONG STRAINS OF PSEUDOPFIESTERIA SHUMWAYAE AND PFIESTERIA PISCICIDA (DINOPHYCEAE)1

The putatively toxic dinoflagellates Pseudopfiesteria shumwayae (Glasgow et J. M. Burkh.) Litaker, Steid., P. L. Mason, Shields et P. A. Tester and Pfiesteria piscicida Steid. et J. M. Burkh. have been implicated in massive fish kills and of having negative impacts on human health along the mid‐Atlantic seaboard of the USA. Considerable debate still remains as to the mechanisms responsible for fish mortality (toxicity vs. micropredation) caused by these dinoflagellates. Genetic differences among these cultures have not been adequately investigated and may account for or correlate with phenotypic variability among strains within each species. Genetic variation among strains of Ps. shumwayae and P. piscicida was examined by PCR–RFLP analysis using cultures obtained from the Provasoli‐Guillard National Center for Culture of Marine Phytoplankton (CCMP), as well as those from our own and other colleagues’ collection efforts. Examination of restriction digest banding profiles for 22 strains of Ps. shumwayae revealed the presence of 10 polymorphic restriction endonuclease sites within the first and second internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene of the rDNA complex, and the cytochrome oxidase subunit I (COI) gene. Three compound genotypes were represented within the 22 Ps. shumwayae strains. Conversely, PCR–RFLP examination of 14 strains of P. piscicida at the same ITS1, 5.8S, and ITS2 regions revealed only one variable restriction endonuclease site, located in the ITS1 region. In addition, a dinoflagellate culture listed as P. piscicida (CCMP 1928) and analyzed as part of this study was identified as closely related to Luciella masanensis P. L. Mason, H. J. Jeong, Litaker, Reece et Steid.     

An in vitro system for the rapid functional characterization of genes involved in carotenoid biosynthesis and accumulation

We have developed an assay based on rice embryogenic callus for rapid functional characterization of metabolic genes. We validated the assay using a selection of well‐characterized genes with known functions in the carotenoid biosynthesis pathway, allowing rapid visual screening of callus phenotypes based on tissue color. We then used the system to identify the functions of two uncharacterized genes: a chemically synthesized β–carotene ketolase gene optimized for maize codon usage, and a wild‐type Arabidopsis thaliana ortholog of the cauliflower Orange gene. In contrast to previous reports (Lopez, A.B., Van Eck, J., Conlin, B.J., Paolillo, D.J., O'Neill, J. and Li, L. ( 2008 ) J. Exp. Bot. 59, 213–223; Lu, S., Van Eck, J., Zhou, X., Lopez, A.B., O'Halloran, D.M., Cosman, K.M., Conlin, B.J., Paolillo, D.J., Garvin, D.F., Vrebalov, J., Kochian, L.V., Küpper, H., Earle, E.D., Cao, J. and Li, L. ( 2006 ) Plant Cell 18, 3594–3605), we found that the wild‐type Orange allele was sufficient to induce chromoplast differentiation. We also found that chromoplast differentiation was induced by increasing the availability of precursors and thus driving flux through the pathway, even in the absence of Orange. Remarkably, we found that diverse endosperm‐specific promoters were highly active in rice callus despite their restricted activity in mature plants. Our callus system provides a unique opportunity to predict the effect of metabolic engineering in complex pathways, and provides a starting point for quantitative modeling and the rational design of engineering strategies using synthetic biology. We discuss the impact of our data on analysis and engineering of the carotenoid biosynthesis pathway.     

Structural basis for type I and type II deficiencies of antithrombotic plasma protein C: Patterns revealed by three-dimensional molecular modelling of mutations of the protease domain

Familial deficiency of protein C is associated with inherited thrombophilia. To explore how specific missense mutations might cause observed clinical phenotypes, known protein C missense mutations were mapped onto three-dimensional homology models of the protein C protease domain, and the implications for domain folding and structure were evaluated. Most Type I missense mutations either replaced internal hydrophobic residues (I201T, L223F, A259V, A267T, A346T, A346V, G376D) or nearby interacting residues (I403M, T298M, Q184H), thus disrupting the packing of internal hydrophobic side chains, or changed hydrophilic residues, thus disrupting ion pairs (N256D, R178W). Mutations (P168L, R169W) at the activation site destabilized the region containing the activation peptide structure. Most Type II mutations involved solvent-exposed residues and were clustered either in a positively charged region (R147W, R157Q, R229Q, R352W) or were located in or near the active site region (S252N, D359N, G381S, G391S, H211Q). The cluster of arginines 147, 157, 229, and 352 may identify a functionally important exosite. Identification of the spatial relationships of natural mutations in the protein C model is helpful for understanding manifestations of protein C deficiency and for identification of novel, functionally important molecular features and exosites. © 1994 John Wiley & Sons, Inc.     

Epitope mapping of a monoclonal antibody directed against the α‐subunit of phosphofructokinase‐1 from Saccharomyces cerevisiae by screening phage display libraries

Phosphofructokinase‐1 from Saccharomyces cerevisiae is composed of two types of subunits, α and β. Subunit‐specific monoclonal antibodies were raised to elucidate structural and functional properties of both subunits. One monoclonal antibody, α‐F3, binds to an epitope either at the C‐terminal or at the N‐terminal part of the α‐polypeptide chain. By screening a heptapeptide library with this monoclonal antibody, a set of heptapeptides was selected, which contained the consensus sequences D–A–F and D–S–F. Two heptapeptides with these motifs were synthesized in order assess their capacity to inhibit the binding of antibody α‐F3 to native phosphofructokinase‐1. The peptide G–I–K–D–A–F–L inhibited the binding more strongly (IC₅₀ = 1.5 µM) than the peptide A–P–W–H–D–S–F (IC₅₀ = 33.3 µM). Sequence matching revealed the presence of the D–A–F motif in the polypeptide chain of phosphofructokinase‐1 at amino acid position 172–174. As a control, the nonapeptide A–P–T–S–K–D–A–F–L which corresponds to the sequence of the putative epitope was tested in the inhibition assay. In view of the high inhibitory capacity (IC₅₀ = 0.3 µM) it was concluded that this nonapeptide represents the continuous epitope of phosphofructokinase‐1 that is recognized by antibody α‐F3. Copyright © 1999 John Wiley & Sons, Ltd.     

Drimane Sesquiterpenoids from the Aspergillus oryzae QXPC‐4

Three new drimane sesquiterpenoids, astellolides C–E ( 1 – 3 , resp.), four new drimane sesquiterpenoid p‐hydroxybenzoates, astellolides F–I ( 4 – 7 , resp.), together with two known compounds astellolides A and B ( 8 and 9 , resp.), have been isolated from the liquid culture of Aspergillus oryzae (strain No. QXPC‐4). Their structures were established by comprehensive analysis of spectroscopic data. The relative and absolute configurations were determined on the basis of NOESY and CD data, together with single‐crystal X‐ray diffraction analyses of compounds 1 – 3 . The metabolites were evaluated for their cytotoxic activities, however, no compounds showed a significant cytotoxicity against the tested cell lines at a concentration of 20 μM .     

Skeletal manifestations of stress in child victims of the Great Irish Famine (1845–1852): Prevalence of enamel hypoplasia,Harris lines,and growth retardation

The Great Irish Famine of 1845–1852 is among the worst food crises in human history. While numerous aspects of this period have been studied by generations of scholars, relatively little attention has so far been given to the physiological impact it is likely to have had on the people who suffered and succumbed to it. This study examines the prevalence of enamel hypoplasia, Harris lines, and growth retardation in the nonadult proportion of a skeletal population comprising victims of the Famine who died in the workhouse in the city of Kilkenny between 1847 and 1851. The frequency of enamel hypoplasia in these children does not appear to have increased as a consequence of famine, although this fact is likely to be a reflection of the osteological paradox. Harris lines and growth retardation; however, were very prevalent, and the manifestation and age‐specific distribution of these may be indicators of the Famine experience. While there was no clear correlation in the occurrence of the assessed markers, the presence of cribra orbitalia displayed a significant relationship to enamel hypoplasia in 1‐ to 5‐year‐old children. While starvation, metabolic disorders and infectious diseases are likely to have greatly contributed to the manifestation of the markers, the psychosocial stress relating to institutionalization in the workhouse should not be underestimated as a substantial causative factor for skeletal stress in this population. Am J Phys Anthropol 155:149–161, 2014. © 2014 Wiley Periodicals, Inc.     

Interleukin-1 receptor antagonist competitively inhibits the binding of interleukin-1 to the type II interleukin-1 receptor.

The interleukin-1 receptor antagonist (IL-1ra) inhibits the binding of interleukin-1 (IL-1) to T-cell lines possessing the type I IL-1 receptor; evidence has been published (Carter, D. B., Deibel, M. R. J., Dunn, C. J., Tomich, C. S., Laborde, A. L., Slightom, J. L., Berger, A. E., Bienkowski, M. J., Sun, F. F., McEwan, R. N., Harris, P. K. W., Yem, A. W., Waszak, G. A., Chosay, J. G., Sieu, L. C., Hardee, M. M., Zurcher-Neely, H. A., Reardon, I. M., Heinrickson, R. L., Truesdell, S. E., Shelly, J. A., Eessalu, T. E., Taylor, B. M., and Tracey, D. E. (1990) Nature 344, 633-638; Hannum, C. H., Wilcox, C. J., Arend, W. P., Joslin, F. G., Dripps, D. J., Heimdal, P. L., Armes, L. G., Sommer, A., Eisenberg, S. P., and Thompson, R. C. (1990) Nature 343, 336-340) that IL-Ira does not bind to the type II IL-1 receptor (IL-1RtII). In this study we examined the ability of human recombinant IL-1ra to block the binding of IL-1 to the IL-1RtII on human polymorphonuclear leukocytes (PMN) and Raji human B-lymphoma cells. The binding of 125I-IL-1 beta to PMN was competively inhibited by IL-1ra. IL-1 beta was more potent in inhibiting the binding of 125I-IL-1 beta than IL-1ra. Incubating PMN with 125I-IL-1ra in the presence of increasing concentrations of IL-1 beta or IL-1ra showed that IL-1 beta was an approximately 40-fold more potent inhibitor of binding of 125I-IL-1ra than unlabeled IL-1ra. The IL-1ra was approximately 500-fold less potent in inhibiting the binding of 125I-IL-1 alpha than IL-1 alpha. IL-1ra was also able to competitively inhibit binding of 125I-IL-1 beta to Raji cells. PMN or Raji cells were also incubated with 125I-IL-1 in the absence or presence of IL-1 or IL-1ra. After cross-linking of IL-1 to cells followed by specific immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a band at 85 kDa corresponding to the 68-kDa IL-1RtII. However, in the presence of an excess of either unlabeled IL-1 or IL-1ra, the 85-kDa IL-1.IL-1RtII complex was not present. These findings demonstrate that the IL-1ra recognizes and blocks IL-1 binding to the IL-1RtII.     

Gender-related differences in carnosine,anserine and lysine content of murine skeletal muscle

Summary. The aminoacyl-imidazole dipeptides carnosine (-alanyl-L-histidine) and anserine (-alanyl-1-methyl-histidine) are present in relatively high concentrations in excitable tissues, such as muscle and nervous tissue. In the present study we describe the existence of a marked sexual dimorphism of carnosine and anserine in skeletal muscles of CD1 mice. In adult animals the concentrations of anserine were higher than those of carnosine in all skeletal muscles studied, and the content of aminoacyl-imidazole dipeptides was remarkably higher in males than in females. Postnatal ontogenic studies and hormonal manipulations indicated that carnosine synthesis was up-regulated by testosterone whereas anserine synthesis increased with age. Regional variations in the concentrations of the dipeptides were observed in both sexes, skeletal muscles from hind legs having higher amounts of carnosine and anserine than those present in fore legs or in the pectoral region. The concentration of L-lysine in skeletal muscles also showed regional variations and a sexual dimorphic pattern with females having higher levels than males in all muscles studied. The results suggest that these differences may be related with the anabolic action of androgens on skeletal muscle.     

Determination of trace α‐fetoprotein variant by affinity adsorption solid substrate‐room temperature phosphorimetry

The 3.5‐generation dendrimers (3.5G‐D)–porphyrin (P) dual luminescent molecule (3.5G‐D–P) was used to label concanavalin agglutinin (Con A); the product of the reaction is 3.5G‐D–P–Con A. A new method for the determination of trace AFP‐V by affinity adsorption solid substrate–room temperature phosphorimetry (AA‐SS–RTP) was established, based on the room temperature phosphorescence (RTP) property of the product on polyamide membrane (PAM) substrate and the specific affinity adsorption (AA) reaction between 3.5G‐D–P–Con A and α‐fetoprotein variant (AFP‐V), which caused the RTP of the system to be sharply enhanced, the ΔIp was linearly correlated to the content of AFP‐V. The sensitivity of the method was obviously high. It could accurately detect the content of AFP‐V in serum. The results were tallied well with those obtained by the ELISA method. Copyright © 2008 John Wiley & Sons, Ltd.     

Skeletal development and abnormalities of the vertebral column and of the fins in hatchery-reared turbot Scophthalmus maximus

To describe the skeletal development and abnormalities in turbot Scophthalmus maximus, samples were collected every day from hatching to 60 days after hatching (DAH). A whole-mount cartilage and bone-staining technique was used. Vertebral ontogeny started with the formation of anterior haemal arches at 5·1 mm standard length (L(S) ) c. 11 DAH, and was completed by the full attainment of parapophyses at 16·9 mm L(S) c. 31 DAH. Vertebral centra started to develop at 6·3 mm L(S) c. 16 DAH and ossification in all centra was visible at 11·0 mm L(S) c. 25 DAH. The caudal fin appeared at 5·1 mm L(S) c. 11 DAH and ossification was visible at 20·6 mm L(S) c. 37 DAH. The onset of dorsal and anal fin elements appeared at 5·8 mm L(S) c. 15 DAH and 6·3 mm L(S) c. 16 DAH, respectively. Ossifications of both dorsal fin and anal fin were visible at 20·6 mm L(S) c. 37 DAH. The pectorals were the only fins present before first feeding, their ossifications were completed at 23·5 mm L(S) c. 48 DAH. Pelvic fins began forming at 7·2 mm L(S) c. 19 DAH and calcification of the whole structure was visible at 19·8 mm L(S) c. 36 DAH. In the present study, 24 types of skeletal abnormalities were observed. About 51% of individuals presented skeletal abnormalities, and the highest occurrence was found in the haemal region of the vertebral column. As for each developmental stage, the most common abnormalities were in the dorsal fin during early metamorphic period (stage 2), vertebral fusion during climax metamorphosis (stage 3) and caudal fin abnormality during both late-metamorphic period (stage 4) and post-metamorphic period (stage 5). Such research will be useful for early detection of skeletal malformations during different growth periods of reared S. maximus.