Distribution of cartilage molecules in the developing mouse joint.

This study describes the precise spatial and temporal patterns of protein distribution for aggrecan, fibromodulin, cartilage oligomeric matrix protein (COMP) and cartilage matrix protein (CMP) in the developing mouse limb with particular attention to those cells destined to form articular chondrocytes in comparison to those cells destined to form a mineralized tissue and become replaced by bone. Mouse glenohumeral joints from fetal mice (12-18 days post coitus (dpc) to the young adult (37 days after birth) were immunostained with antibodies specific for these molecules. Aggrecan staining defined the general chondrocytic phenotype, whether articular or transient. Fibromodulin was associated with prechondrocytic mesenchymal cells in the interzone prior to joint cavitation and with the mesenchymal cells of the perichondrium or the periosteum encapsulating the joint elements of the maturing and young adult limb. Staining was most intense around developing articular chondrocytes and much less abundant or absent in those differentiating cells along the anlage. CMP showed an almost reciprocal staining pattern to fibromodulin and was not detected in the matrix surrounding articular chondrocytes. COMP was not detected in the cells at the articular surface prior to cavitation but by 18 dpc, as coordinated movement of the mouse forelimb intensifies, staining for COMP was most intense around the maturing articular chondrocytes. These results show that the cells that differentiate into articular chondrocytes elaborate an extracellular matrix distinct from those cells that are destined to form bone. Fibromodulin may function in the early genesis of articular cartilage and COMP may be associated with elaboration of a weight-bearing chondrocyte matrix.     

Diets of giants: the nutritional value of sauropod diet during the Mesozoic

A major uncertainty in estimating energy budgets and population densities of extinct animals is the carrying capacity of their ecosystems, constrained by net primary productivity (NPP) and its digestible energy content. The hypothesis that increases in NPP due to elevated atmospheric CO₂ contributed to the unparalleled size of the sauropods has recently been rejected, based on modern studies on herbivorous insects that imply a general, negative correlation of diet quality and increasing CO₂. However, the nutritional value of plants grown under elevated CO₂ levels might be very different for vertebrate megaherbivores than for insects. Here we show plant species‐specific responses in metabolizable energy and nitrogen content, equivalent to a two‐fold variation in daily food intake estimates for a typical sauropod, for dinosaur food plant analogues grown under CO₂ concentrations spanning estimates for Mesozoic atmospheric concentrations. Our results potentially rebut the hypothesis that constraints on sauropod diet quality were driven by Mesozoic CO₂ concentration.     

Direct radioimmunoassay of estradiol-17β in defatted bovine milk

A radioimmunoassay was developed for rapid determination of estradiol-17β concentrations in unextracted defatted bovine milk. The assay was dependent on the use of a highly specific anti-estradiol-17β antiserum. Application of a formula to correct for the interference associated with individual milk samples and use of appropriate assay blanks facilitated interpolation on a buffer standard curve. The assay offered a high degree of sensitivity (0.6pg/ml milk) and a precision (within-assay coefficient of variation: 0.196; between-assay CV:0.191) comparable with contemporary extraction methods.     

A family-based approach to preventing excessive weight gain

Objective: Preventing weight gain in adults and excessive weight gain in children is a high priority. We evaluated the ability of a family‐based program aimed at increasing steps and cereal consumption (for breakfast and snacks) to reduce weight gain in children and adults. Research Methods and Procedures: Families (n = 105) with at least one 8‐ to 12‐year‐old child who was at‐risk‐for‐overweight or overweight (designated as the target child) were recruited for the study. Eighty‐two families were randomly assigned to receive the family‐based intervention and 23 families to the control condition. The 13‐week intervention consisted of specific increases in daily steps (an additional 2000 steps/d) and consumption of 2 servings/d of ready‐to‐eat cereal. Results: The intervention was successful in increasing walking (steps) and cereal consumption. The intervention had positive, significant effects on percentage BMI‐for‐age and percentage body fat for target children and weight, BMI, and percentage body fat for parents. On further analysis, the positive effects of the intervention were seen largely in target girls and moms, rather than in target boys and dads. Discussion: This family‐based weight gain prevention program based on small changes holds promise for reducing excessive weight gain in families and especially in growing overweight children.     

Ku and DNA-dependent Protein Kinase Dynamic Conformations and Assembly Regulate DNA Binding and the Initial Non-homologous End Joining Complex

DNA double strand break (DSB) repair by non-homologous end joining (NHEJ) is initiated by DSB detection by Ku70/80 (Ku) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) recruitment, which promotes pathway progression through poorly defined mechanisms. Here, Ku and DNA-PKcs solution structures alone and in complex with DNA, defined by x-ray scattering, reveal major structural reorganizations that choreograph NHEJ initiation. The Ku80 C-terminal region forms a flexible arm that extends from the DNA-binding core to recruit and retain DNA-PKcs at DSBs. Furthermore, Ku- and DNA-promoted assembly of a DNA-PKcs dimer facilitates trans-autophosphorylation at the DSB. The resulting site-specific autophosphorylation induces a large conformational change that opens DNA-PKcs and promotes its release from DNA ends. These results show how protein and DNA interactions initiate large Ku and DNA-PKcs rearrangements to control DNA-PK biological functions as a macromolecular machine orchestrating assembly and disassembly of the initial NHEJ complex on DNA.     

Versatile High Resolution Oligosaccharide Microarrays for Plant Glycobiology and Cell Wall Research

Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.     

PURIFICATION AND CHARACTERIZATION OF A HOMODIMERIC ENOLASE FROM SYNECHOCOCCUS PCC 6301 (CYANOPHYCEAE)1

Enolase from Synechococcus PCC 6301 was purified 1450‐fold to electrophoretic homogeneity and a final specific activity of 68 μmol of phosphoenolpyruvate produced·min^?1·mg protein^?1. Analytical gel filtration and nondenaturing and SDS‐gel electrophoresis demonstrated that this enolase exists as a 118‐kDa homodimer composed of 56‐kDa subunits. The purified enzyme displayed 1) a broad pH‐activity profile with maximal activity occurring at pH 8.0 and 7.5 for the forward and reverse reactions, respectively, 2) a forward‐to‐reverse maximal activity ratio of about 1.6, 3) a K_m (2‐phosphoglycerate) of 0.28 mM, and 4) an absolute requirement for a divalent metal cation cofactor that was best satisfied by Mg²⁺ (K_m=0.62 mM). Enolase activity increased by about 200% after the first purification step (60° C heat treatment), whereas addition of increasing amounts of a clarified extract led to a progressive 70% inhibition in the activity of the purified enzyme. This was reflected by a reduction in enolase's V_max from 73 to 22 U·mg^?1 and forward‐to‐reverse activity ratio from 1.6 to 1.3. This inhibition was negated when the clarified extract was either preincubated with trypsin or warmed to approximately 40° for 5 min. Results are indicative of a heat‐labile enolase inhibitor protein in Synechococcus PCC 6301. By contrast, the purified enolase lost no activity when incubated at 70° C for up to 5 min. This study represents the first purification of enolase from the Cyanophyceae. Characterization of the purified enzyme's physical and kinetic features has provided insights into the structural and functional properties of cyanobacterial enolase.     

The Wnt antagonist secreted frizzled-related protein-1 is a negative regulator of trabecular bone formation in adult mice

Previous studies have associated activation of canonical Wnt signaling in osteoblasts with elevated bone formation. Here we report that deletion of the murine Wnt antagonist, secreted frizzled-related protein (sFRP)-1, prolongs and enhances trabecular bone accrual in adult animals. sFRP-1 mRNA was expressed in bones and other tissues of +/+ mice but was not observed in -/- animals. Despite its broad tissue distribution, ablation of sFRP-1 did not affect blood and urine chemistries, most nonskeletal organs, or cortical bone. However, sFRP-1-/- mice exhibited increased trabecular bone mineral density, volume, and mineral apposition rate when compared with +/+ controls. The heightened trabecular bone mass of sFRP-1-/- mice was observed in adult animals between the ages of 13-52 wk, occurred in multiple skeletal sites, and was seen in both sexes. Mechanistically, loss of sFRP-1 reduced osteoblast and osteocyte apoptosis in vivo. In addition, deletion of sFRP-1 inhibited osteoblast lineage cell apoptosis while enhancing the proliferation and differentiation of these cells in vitro. Ablation of sFRP-1 also increased osteoclastogenesis in vitro, although changes in bone resorption were not observed in intact animals in vivo. Our findings demonstrate that deletion of sFRP-1 preferentially activates Wnt signaling in osteoblasts, leading to enhanced trabecular bone formation in adults.