Bending Assay Research Articles

Z-Scan Analysis: a New Method to Determine the Oxidative State of Low-Density Lipoprotein and Its Association with Multiple Cardiometabolic Biomarkers

The great atherogenic potential of oxidized low- density lipoprotein has been widely described in the literature. The objective ofthisstudy was to investigate whether the state of oxidized low-density lipoprotein in human plasma mea- sured by the Z-scan technique has an association with differ- ent cardiometabolic biomarkers. Total cholesterol, high- density lipoprotein cholesterol, triacylglycerols, apolipopro- tein A-I and apolipoprotein B, paraoxonase-1, and glucose were analyzed using standard commercial kits, and low- density lipoprotein cholesterol was estimated using the Friedewald equation. A sandwichenzyme-linked immunosor- bent assay was used to detect electronegative low-density li- poprotein. Low-density lipoprotein and high-density lipopro- tein sizes were determined by Lipoprint ® system. The Z-scan technique was usedto measure the non-linear optical response oflow-densitylipoproteinsolution.Principalcomponentanal- ysis and correlations were used respectively to resize the data from the sample and test association between the θ parameter, measured with the Z-scan technique, and the principal com- ponent. A total of 63 individuals, from both sexes, with mean age 52 years (±11), being overweight and having high levels of total cholesterol and low levels of high-density lipoprotein cholesterol, were enrolled in this study. A positive correlation betweenthe θparameter and moreanti-atherogenicpattern for cardiometabolic biomarkers together with a negative correla- tion for an atherogenic pattern was found. Regarding the pa- rameters related with an atherogenic low-density lipoprotein profile, the θ parameter was negatively correlated with a more atherogenic pattern. By using Z-scan measurements, we were able to find an association between oxidized low-density lipo- protein state and multiple cardiometabolic biomarkers in sam- ples from individuals with different cardiovascular risk factors.

Lentivirus-TAZ Administration Alleviates Osteoporotic Phenotypes in the Femoral Neck of Ovariectomized Rats

Background: Osteoporosis is characterized by impairment of bone mass, strength, and microarchitecture, leading to the susceptibility to fragility fractures, especially in femoral neck region. Transcriptional coactivator with PDZ-binding motif (TAZ) facilitates osteogenesis while suppressing adipogenesis via regulation of transcriptional activities of runt-related transcription factor 2 and peroxisome proliferator-activated receptor γ. Here, we validated the role of TAZ in vivo using an ovariectomized (OVX) rat model of osteoporosis. Methods: Serum alkaline phosphatase, triglyceride, cholesterol and urinary hydroxyproline were measured on an automatic analyzer using diagnostic reagent kits. Serum OCN and C-terminal cross-linked telopeptides of type I collagen were measured using ELISA. Bone mineral density was measured using dual-energy X-ray scanner. Mechanical parameters were detected by three-point bending assays. Bone volume per tissue volume (BV/TV), trabecular thickness (Tb. Th), trabecular number (Tb. No), and trabecular separation (Tb. Sp) were measured by MicroCT. The mRNA and protein levels were quantified by Realtime PCR and Western Blotting respectively. Results: After injections of lentivirus overexpressing TAZ into the femoral neck region, bone mineral density, ultimate force, stiffness, BV/TV, Tb. Th, and Tb. No were significantly increased, whereas Tb. Sp was dramatically decreased. In the TAZ-overexpression region in the femoral neck of OVX rats, the mRNA levels of Runx2 and osteocalcin were obviously elevated, whereas that of PPARγ and adipocyte protein 2 were downregulated. Conclusion: Lentivirus-mediated TAZ gene therapy alleviated the osteoporotic phenotypes in the femoral neck of OVX rats, providing an alternative strategy for the treatment of postmenopausal osteoporosis and prevention of osteoporotic fracture.

Water Potential and Its Components in Developing Sweet Cherry

Susceptibility of sweet cherry (Prunus avium L.) fruit to rain cracking increases toward maturity and is thought to be related to increases in both tissue pressure () and cell pressure (). Furthermore, at a given water potential (), one might expect the increase in and the to balance the decrease in osmotic potential (). The objectives of our study were to quantify and in developing sweet cherry using vapor pressure osmometry (VPO), compression plate (CP), and the cell pressure probe (CPP). In addition, the tissue water potential was determined by quantifying the bending of strips of fruit skin and the change in projected area of discs excised from the flesh when incubated in a range of sucrose solutions of varying osmotic potentials (). Fruit growth followed a sigmoid pattern with time with the Stage II/Stage III transition occurring at ≈55 days after full bloom (DAFB). The and the were constant up to ≈55 DAFB but decreased to –2.8 MPa at maturity. The calculated by subtracting the from averaged ≈350 kPa up to 48 DAFB and then decreased at a decreasing rate to ≈21 kPa toward maturity. The determined from bending assays using excised skin strips or from water uptake of excised flesh discs was essentially constant up to ≈48 DAFB, then decreased until ≈75 DAFB and remained constant thereafter. These values were in good agreement with those determined by VPO. The as determined by CP passed through a transient peak at ≈41 DAFB, then decreased until ≈63 DAFB and remained constant and low until maturity. Similarly, by CPP increased from 27 to 48 DAFB, remained constant until ≈55 DAFB, and then decreased until maturity. Our data demonstrate a consistent decrease in and that coincides with a decrease in of sweet cherry during Stage III. Because and are low relative to , the change in parallels that in . The reason for the low turgor most likely lies in the accumulation of apoplastic solutes. These prevent a catastrophic increase in pressure that would otherwise lead to the bursting of individual cells and the cracking of entire fruit.

Stabilization of dentin matrix after cross-linking treatments, in vitro

ObjectivesTo evaluate the effect of EDC on elastic modulus (E), MMPs activity, hydroxyproline (HYP) release and thermal denaturation temperature of demineralized dentin collagen. MethodsDentin beams were obtained from human molars and completely demineralized in 10wt% H3PO4 for 18h. The initial E and MMP activity were determined with three-point bending and microcolorimetric assay, respectively. Extra demineralized beams were dehydrated and the initial dry mass (DM) was determined. All the beams were distributed into groups (n=10) and treated for 30s or 60s with: water, 0.5M, 1M or 2M EDC or 10% glutaraldehyde (GA). After treatment, the new E and MMP activity were redetermined. The beams submitted to DM measurements were storage for 1 week in artificial saliva, after that the mass loss and HYP release were evaluated. The collagen thermal denaturation temperature (TDT) was determined by DSC analysis. Data for E, MMP activity and HYP release were submitted to Wilcoxon and Kruskal–Wallis or Mann–Whitney tests. Mass loss and TDT data were submitted to ANOVA and Tukey tests at the 5% of significance. ResultsEDC was able to significantly increase collagen stiffness in 60s. 10% GA groups obtained the highest E values after both 30 and 60s. All cross-linking agents decreased MMP activity and HYP release and increased TDT temperature. Significant differences were identified among EDC groups after 30 or 60s of cross-linking, 1M or 2M EDC showed the lowest MMP activity. SignificanceCross-linking agents are capable of preventing dentin collagen degradation. EDC treatment may be clinically useful to increase resin-dentin stability.

The inhibitory effect of a polymerisable cationic monomer on functional matrix metalloproteinases

ObjectivesThis study examined the use of methacryloxylethyl cetyl dimethyl ammonium chloride (DMAE-CB) as a potential matrix metalloproteinases (MMPs) inhibitor on both soluble recombinant and dentine matrix-bound endogenous MMPs, meanwhile attempted to determine the effective anti-MMP group of quaternary ammonium methacrylates (QAMs). MethodsThe possible inhibitory effects of five DMAE-CB mass concentrations (0.1%, 0.5%, 1%, 3%, 5%) on soluble rhMMP-9 were measured using a colorimetic assay kit. Methyl methacrylate (MMA) and [2-(methacryloyloxy)ethyl] trimethylammonium chloride (METMAC) were also screened against rhMMP-9 to compare the inhibitory effect with DMAE-CB. Matrix-bound endogenous MMP-activity was evaluated in completely demineralized dentine beams. Thirty beams were randomly divided into three groups (N=10) and respectively placed into 500μL of calcium- and zinc-containing media (CM; control), 0.2% chlorhexidine or 3% DMAE-CB in CM aged for 30 days. The changes in modulus of elasticity, loss of dry mass and solubilization of collagen peptides were measured via three-point bending, precision weighing and hydroxyproline assay, respectively. Results0.5–5% mass concentrations of DMAE-CB were highly effective (P<0.05) in inhibiting rhMMP-9 varied between 76.56±6.44% and 97.06±3.24%, the inhibitory effect of MMA was much lower than that of METMAC and DMAE-CB at the same concentration (P<0.05). Dentine beams incubated in 3% DMAE-CB showed only a 26.34% decrease in the modulus of elasticity (75.74% decrease in control), a 1.72±1.56% loss of dry mass (29.70±9.12% loss in control), and significantly less solubilized hydroxyproline when compared with the control (P<0.05). ConclusionsDMAE-CB is effective in inhibiting both soluble recombinant MMPs and matrix-bound dentine MMPs. Quaternary ammonium group is the effective anti-MMP group of QAMs. Clinical significanceThe incorporation of DMAE-CB into dental adhesives has the potential to enhance the durability of dentine bonding and thus increases the longevity of restorations.

The Tail That Wags the Dog: Topoisomerase IV ParC C-Terminal Domain Controls Strand Passage Activity through Multipartite Topology-Dependent Interactions with DNA

The Tail That Wags the Dog: Topoisomerase IV ParC C-Terminal Domain Controls Strand Passage Activity through Multipartite Topology-Dependent Interactions with DNA

CK2 Phosphorylation of Schistosoma mansoni HMGB1 Protein Regulates Its Cellular Traffic and Secretion but Not Its DNA Transactions

BackgroundThe helminth Schistosoma mansoni parasite resides in mesenteric veins where fecundated female worms lay hundred of eggs daily. Some of the egg antigens are trapped in the liver and induce a vigorous granulomatous response. High Mobility Group Box 1 (HMGB1), a nuclear factor, can also be secreted and act as a cytokine. Schistosome HMGB1 (SmHMGB1) is secreted by the eggs and stimulate the production of key cytokines involved in the pathology of schistosomiasis. Thus, understanding the mechanism of SmHMGB1 release becomes mandatory. Here, we addressed the question of how the nuclear SmHMGB1 can reach the extracellular space.Principal FindingsWe showed in vitro and in vivo that CK2 phosphorylation was involved in the nucleocytoplasmic shuttling of SmHMGB1. By site-directed mutagenesis we mapped the two serine residues of SmHMGB1 that were phosphorylated by CK2. By DNA bending and supercoiling assays we showed that CK2 phosphorylation of SmHMGB1 had no effect in the DNA binding activities of the protein. We showed by electron microscopy, as well as by cell transfection and fluorescence microscopy that SmHMGB1 was present in the nucleus and cytoplasm of adult schistosomes and mammalian cells. In addition, we showed that treatments of the cells with either a phosphatase or a CK2 inhibitor were able to enhance or block, respectively, the cellular traffic of SmHMGB1. Importantly, we showed by confocal microscopy and biochemically that SmHMGB1 is significantly secreted by S. mansoni eggs of infected animals and that SmHMGB1 that were localized in the periovular schistosomotic granuloma were phosphorylated.ConclusionsWe showed that secretion of SmHMGB1 is regulated by phosphorylation. Moreover, our results suggest that egg-secreted SmHMGB1 may represent a new egg antigen. Therefore, the identification of drugs that specifically target phosphorylation of SmHMGB1 might block its secretion and interfere with the pathogenesis of schistosomiasis.

Biomechanical Evaluation for Mechanisms of Periprosthetic Femoral Fractures

There are a number of biomechanical tests for various treatment options of periprosthetic femoral fractures, but different loading modalities prelude their direct comparison. This study was designed to develop an experimental model of osteoporotic bone fractures near the femoral stem that is based on a simple testing protocol to increase the reproducibility. In addition, we wanted to clarify whether a femoral prosthetic stem reduces the femoral fracture strength. Twenty human cadaveric femurs were harvested, and five groups were randomized on the basis of the bone mineral density using a pQCT device. The specimens of three groups were provided with a cemented Exeter V40 stem and loaded to failure with torsion (I), anterior (II), and lateral load (III). The femurs of groups IV and V remained uninstrumented and were tested in a four-point bending assay similar to groups II and III. All biomechanical testings were realized with a servohydraulic testing machine (MTS). There was no significant difference regarding bone mineral density of all groups. Torsional testing generated proximal intertrochanteric fractures and anterior loading resulted exclusively in supracondylar fractures. Introducing the force from the lateral side, all fracture lines occurred close to the tip of the stem, similar to a Vancouver-B fracture. Assuming that lateral load application is a main responsible mechanism of periprosthetic femoral fracture near the tip of the stem, the fracture strength of instrumented femurs was significantly reduced (group III: 4,692 N vs. group V: 6,931 N; p < 0.05). Prosthetic stems reduce femoral fracture strength significantly. In an osteoporotic bone model, a four-point bending test with lateral load application seems to be a suitable approach.

Corrigendum to “Functional characterization of three water deficit stress-induced genes in tobacco and Arabidopsis: An approach based on gene down regulation” [Plant Physiol. Biochem. 48 (2010) 35–44

The authors regret that in the above published article, the figure captions of Figs. 1e5 were incomplete, which have been corrected to read as follows: Fig. 1. Antibiotic selection and confirmation of transgenic RNAi lines. Seeds from T0 generation RNAi lines were germinated on MS medium supplemented with kanamycin. A representative plates for wild-type and each transgenic tobacco (Nicotiana tabacum) lines are shown here (A). PCR amplificationwas performed in the leaf samples collected from T1 generation RNAi lines using attB1-attB2 primers and amplification data for two lines are shown here (B). Arrow marks in the panel A show representative white (dead) seedlings. Fig. 2. Real time PCR (qRT-PCR) analysis showing the down regulation of ADH or CcoAOMTor F3OGTgene transcripts in respective tobacco RNAi lines. The transcript levels were studied by qRT-PCR in non-stressed and water deficit stressed plants of RNAi lines of ADH (A), CcoAOMT (B) and F3OGT (C) at T1 generation. Plants from two independent lines were analyzed and relative expression ratio values over their corresponding wild-type are represented in the graph. In all the experiments vector control plants were also analyzed and they did not show silencing. Relative amounts of transcripts were calculated using method described in Pfaffl (2001). The data was normalized to the expression of elongation factor 1-a (EF1a) as the internal housekeeping gene and the wild-type (WT) plants of stress or non-stress mRNA levels of respective genes as an expression reference (WT stress1⁄4 1; WT non-stress1⁄4 1). Silenced lines are shown by filled bars and theWT control(s) is shown by empty bar. Bars represent standard deviation values. Fig. 3. Phenotype of the RNAi transgenic tobacco plants down regulated with ADH or CcoAOMT or F3OGT under water deficit stress. Fortyday-old wild-type and RNAi plants (RNAi line 1) were subjected to water deficit stress by gradually with-holding water for 7 d and later maintained at 40% FC for 7 d. The photographs of representative plants from each gene silenced lines were taken at the end of stress. Values indicate leaf relative water content (%) at the end of 7 d severe stress. Fig. 4. Performance of the transgenic RNAi plants down regulated for ADH or CcoAOMT or F3OGT under water deficit stress. RNAi transgenic lines were initially exposed to acclimation treatment by gradually with-holding the irrigation and later exposed to severe stress (40% FC-7 d). Data from a selected representative line (RNAi line 1) is presented here. Cellular level tolerance was assessed by estimating chlorophyll reduction (A), cell viability by TTC assay (B) and cell membrane stability (C). Percent reduction values under stress in (A) and (B) are calculated over corresponding well watered (100% FC) plants. Bars represent standard error values. The same letters are not significantly different at 5% level by Duncan’s Multiple Range Test. WT, wild-type; FC, field capacity. Fig. 5. Response of Arabidopsis mutant plants to salinity and water deficit stress. The f3ogt, ccoaomt and adh mutants seedlings were initially grown in MS medium and transferred to 100 mM NaCl medium for root bending assay. Root growth was studied at the end of 7 d (A, B). To assess the desiccation response, seedling rosettes were detached and left on the lab bench for drying. Fresh weight loss was measured at the indicated time intervals and water lose was assessed (C). Water deficit stress was imposed by gradually with-holding water from pottingmix (acclimation) and exposing to severe stress. The cell membrane stability was assessed (D) in the stressed seedlings at 14.5% soil water content as measured by soil moisture monitor (Decagon devices Pullman WA USA; probe EC-5). The non-stress plants were maintained at 46% soil water content. Bars represent standard deviation values.

The inhibitory effect of polyvinylphosphonic acid on functional matrix metalloproteinase activities in human demineralized dentin.

This study has examined the use of polyvinylphosphonic acid (PVPA) as a potential matrix metalloproteinase (MMP) inhibitor and how brief cross-linking of demineralized dentin matrix that did not affect its mechanical properties enhanced the anti-MMP activity of PVPA. The anti-MMP potential of five PVPA concentrations (100-3000 microgml(-1)) was initially screened using a rhMMP-9 colorimetic assay. Demineralized dentin beams were treated with the same five concentrations of PVPA to collagen and then aged for 30 days in a calcium- and zinc-containing medium. The changes in modulus of elasticity, loss of dry mass and dissolution of collagen peptides were measured via three-point bending, precision weighing and hydroxyproline assay, respectively. All tested PVPA concentrations were highly effective (P<0.05) in inhibiting MMP-9. Ageing in the incubation medium did not significantly alter the modulus of elasticity of the five PVPA treatment groups. Conversely, aged dentin beams from the control group exhibited a significant decline in their modulus of elasticity (P<0.05) over time. Mass loss from the dentin beams and the corresponding increase in hydroxyproline in the medium in the five PVPA treatment groups were significantly lower than for the control (P<0.05). PVPA is a potent inhibitor of endogenous MMP activities in demineralized dentin. It may be used as an alternative to chlorhexidine to prevent collagen degradation within hybrid layers to extend the longevity of resin-dentin bonds.

DWARF AND LOW‐TILLERING, a new member of the GRAS family, plays positive roles in brassinosteroid signaling in rice

Rapid progress has been made regarding the understanding of brassinosteroid (BR) signaling in Arabidopsis. However, little is known about BR signaling in monotyledons. Here, we characterized a rice dwarf and low-tillering (dlt) mutant and cloned the corresponding gene via map-based cloning. DLT encodes a new member of the plant-specific GRAS family. The dwarf phenotype of dlt is similar to BR-deficient or signaling mutants in rice. In addition, both lamina bending and coleoptile elongation assays show that dlt is insensitive or much less responsive to brassinolide (BL), the most active BR, suggesting that DLT is involved in BR signaling. Consistent with this conclusion, the accumulation of transcripts of BR biosynthesis genes in the dlt mutant indicated that DLT is involved in feedback inhibition of BR biosynthesis genes. In addition, transcription of several other BR-regulated genes is altered in the dlt mutant. Finally, consistent with the fact that DLT is also negatively feedback-regulated by BR treatment, a gel mobility shift assay showed that OsBZR1 can bind to the DLT promoter through the BR-response element. Taken together, these studies have enabled us to identify a new signaling component that is involved in several specific BR responses in rice.

Purification and Characterization of Bacteriophage P22 Xis Protein

The temperate bacteriophages lambda and P22 share similarities in their site-specific recombination reactions. Both require phage-encoded integrase (Int) proteins for integrative recombination and excisionase (Xis) proteins for excision. These proteins bind to core-type, arm-type, and Xis binding sites to facilitate the reaction. lambda and P22 Xis proteins are both small proteins (lambda Xis, 72 amino acids; P22 Xis, 116 amino acids) and have basic isoelectric points (for P22 Xis, 9.42; for lambda Xis, 11.16). However, the P22 Xis and lambda Xis primary sequences lack significant similarity at the amino acid level, and the linear organizations of the P22 phage attachment site DNA-binding sites have differences that could be important in quaternary intasome structure. We purified P22 Xis and studied the protein in vitro by means of electrophoretic mobility shift assays and footprinting, cross-linking, gel filtration stoichiometry, and DNA bending assays. We identified one protected site that is bent approximately 137 degrees when bound by P22 Xis. The protein binds cooperatively and at high protein concentrations protects secondary sites that may be important for function. Finally, we aligned the attP arms containing the major Xis binding sites from bacteriophages lambda, P22, L5, HP1, and P2 and the conjugative transposon Tn916. The similarity in alignments among the sites suggests that Xis-containing bacteriophage arms may form similar structures.

The Caenorhabditis elegans AMP-activated Protein Kinase AAK-2 Is Phosphorylated by LKB1 and Is Required for Resistance to Oxidative Stress and for Normal Motility and Foraging Behavior

AAK-2 is one of two alpha isoforms of the AMP-activated protein kinase in Caenorhabditis elegans and is involved in life span maintenance, stress responses, and germ cell cycle arrest upon dauer entry. We found that AAK-2 was phosphorylated at threonine 243 in response to paraquat treatment and that this phosphorylation depends on PAR-4, the C. elegans LKB1 homologue. Both aak-2 mutation and par-4 knockdown increased the sensitivity of C. elegans worms to paraquat, and the double deficiency did not further increase sensitivity, indicating that aak-2 and par-4 act in a linear pathway. Both mutations also slowed body bending during locomotion and failed to reduce head oscillation in response to anterior touch. Consistent with this abnormal motility and behavioral response, expression of the AAK-2::green fluorescent protein fusion protein was observed in the ventral cord, some neurons, body wall muscle, pharynx, vulva, somatic gonad, and excretory cell. Our study suggests that AMPK can influence the behavior of C. elegans worms in addition to its well known function in metabolic control.

Bioorganic/inorganic hybrid composition of sponge spicules: Matrix of the giant spicules and of the comitalia of the deep sea hexactinellid Monorhaphis

The giant basal spicules of the siliceous sponges Monorhaphis chuni and Monorhaphis intermedia (Hexactinellida) represent the largest biosilica structures on earth (up to 3m long). Here we describe the construction (lamellar organization) of these spicules and of the comitalia and highlight their organic matrix in order to understand their mechanical properties. The spicules display three distinct regions built of biosilica: (i) the outer lamellar zone (radius: >300 microm), (ii) the bulky axial cylinder (radius: <75 microm), and (iii) the central axial canal (diameter: <2 microm) with its organic axial filament. The spicules are loosely covered with a collagen net which is regularly perforated by 7-10 microm large holes; the net can be silicified. The silica layers forming the lamellar zone are approximately 5 microm thick; the central axial cylinder appears to be composed of almost solid silica which becomes porous after etching with hydrofluoric acid (HF). Dissolution of a complete spicule discloses its complex structure with distinct lamellae in the outer zone (lamellar coating) and a more resistant central part (axial barrel). Rapidly after the release of the organic coating from the lamellar zone the protein layers disintegrate to form irregular clumps/aggregates. In contrast, the proteinaceous axial barrel, hidden in the siliceous axial cylinder, is set up by rope-like filaments. Biochemical analysis revealed that the (dominant) molecule of the lamellar coating is a 27-kDa protein which displays catalytic, proteolytic activity. High resolution electron microscopic analysis showed that this protein is arranged within the lamellae and stabilizes these surfaces by palisade-like pillars. The mechanical behavior of the spicules was analyzed by a 3-point bending assay, coupled with scanning electron microscopy. The load-extension curve of the spicule shows a biphasic breakage/cracking pattern. The outer lamellar zone cracks in several distinct steps showing high resistance in concert with comparably low elasticity, while the axial cylinder breaks with high elasticity and lower stiffness. The complex bioorganic/inorganic hybrid composition and structure of the Monorhaphis spicules might provide the blueprint for the synthesis of bio-inspired material, with unusual mechanical properties (strength, stiffness) without losing the exceptional properties of optical transmission.

Co-Treatment of PTH With Osteoprotegerin or Alendronate Increases Its Anabolic Effect on the Skeleton of Oophorectomized Mice

We examined the effects of 60 days of co-treatment of PTH with either OPG or alendronate in oophorectomized mice. Compared with PTH alone, co-treatment of PTH with either of these two mechanistically distinct anti-catabolics improved bone volume, mechanical strength, and appendicular and axial mineralization and prolonged the beneficial effect of PTH on BMD. Conflicting evidence exists as to whether the anabolic effect of PTH is inhibited by the action of anti-catabolics. To examine this issue, we assessed the effects of alendronate and osteoprotegerin (OPG), two anti-catabolics with different modes of action, on the anabolic activity of PTH(1-34) in the skeleton of 4-month-old oophorectomized mice. Mice treated with vehicle alone (PBS), alendronate alone (100 microg/kg/week), OPG alone (10 mg/kg twice a week), or PTH alone (80 microg/kg/day) were compared with each other and with animals administered PTH plus alendronate or PTH plus OPG. We assessed lumbar spine and femoral BMD at 0, 30, and 60 days. Contact radiography, histology, and histomorphometry, three-point bending assay of the femur, and serum osteocalcin and TRACP5b assays were performed at 2 months. Although alendronate and OPG each suppressed bone turnover, at the doses used, this was more profound with OPG. Increases in lumbar spine and femoral BMD and in trabecular bone volume were at least as great with OPG as with alendronate, and mechanical indices of femoral bone strength improved only with OPG. Both produced a plateau in spine and femoral BMD increases by 30 days. Co-treatment of PTH with each anti-catabolic produced additive increases in BMD in the femur and supra-additive increases in the lumbar spine with no plateau effects. Neither anti-catabolic impeded the PTH-induced increase in bone volume or the increase in mechanical strength of the femur. These studies show that the highly potent anti-catabolic OPG can produce dramatic increases in BMD and bone strength; that the temporal pattern of activity of bone formation and resorption modulators may have major influence on net skeletal accrual; and that, depending on timing, inhibition of osteoclastic activity may markedly augment the anabolic action of PTH.

Long‐term fluoxetine administration does not result in major changes in bone architecture and strength in growing rats

Many studies have indicated that serotonin and its transporter play a role in bone metabolism. In this study we investigated the effect of selective serotonin re-uptake inhibitor (SSRI), fluoxetine (Prozac) on bone architecture and quality in growing female rats. We therefore administrated rats with clinically relevant doses of fluoxetine for a period of 6 months. DXA scans were performed during the treatment period in order to follow parameters as body weight, fat percentage and BMD. After 6 months of treatment, femurs were used to analyze bone architecture and bone strength, by means of microCT scans and three-point bending assays, respectively. We found a slightly diminished bone quality, reflected in a lower bone tissue strength, which was compensated by changes in bone geometry. As leptin and adiponectin could be possible factors in the serotonergic regulation of bone metabolism, we also determined the levels of these factors in plasma samples of all animals. Leptin and adiponectin levels were not different between the control group and fluoxetine-treated group, indicating that these factors were not involved in the observed changes in bone geometry and quality.

Interaction between fructooligosaccharides (FOS) and dietary lipids: Effects on mineral absorption and balance, bone mass and biomechanical properties in rats

Fructooligosaccharides (FOS) stimulate the mineral absorption in the large intestine, which increases bone mineral retention. Changes in lipid composition have been related to changes on fluidity of enterocytes brush border membranes. Our hypothesis was that dietary lipid composition might influence the effects of FOS on the intestinal absorption of Ca, Mg, Fe and Cu, and some bone characteristics. Male rats (n=24) were fed a 15% lipid diet (soy oil or a 1:0.3 mix of fish and soy oils) or diets containing the same lipid sources supplemented with 10% FOS (Raftilose® Synergy 1) for 15 days. Feces and urine were collected for the evaluation of mineral absorption and balance. At sacrifice, the cecum contents and walls were weighed and the pH was determined in situ. Femurs were collected for Ca analyses (AAS) and bone strength evaluation (3-point bending assay). FOS consumption resulted in an increase (p<0.05) in cecal wall and contents, as well as a decrease in the luminal pH. These effects probably contributed to the higher intestinal mineral absorption (p<0.05) which, except for Mg, was increased by the fish oil (p<0.001). A higher Ca balance and bone retention (p<0.05) was observed in the FOS + fish-oil group with a significant increase in the peak and yield load bone strength variables. The results confirm the positive influence of FOS on mineral absorption and bone parameters which were potentialized by the fish oil.

Biochemical Screening of Stable Dinucleosomes Using DNA Fragments from a Dinucleosome DNA Library

The dinucleosome is an informative unit for analysis of the higher-order chromatin structure. DNA fragments forming stable dinucleosomes were screened from a dinucleosome DNA library after the reconstitution of nucleosomes in vitro and digestion with micrococcal nuclease. Reconstituted dinucleosomes showed a diversity of sensitivity to micrococcal nuclease, suggesting that the biochemical stability of a dinucleosome depends, in part, on the DNA fragments. The DNA fragments after the screening were classified into three groups represented by clones bf10, af14 and af32 according to the sensitivity to micrococcal nuclease. Mapping of the nucleosome boundaries by Southern blotting of the DNA after restriction digestion and by primer extension analysis showed that each nucleosome position of clone af32 was fixed. Analysis of reconstituted dinucleosomes using mutant DNA fragments of clone af32 revealed a unique property characteristic of a key nucleosome, given that the replacement of a DNA fragment corresponding to the right nucleosome position resulted in marked sensitivity to micrococcal nuclease, whereas the replacement of the other nucleosome fragment had almost no effect on sensitivity as compared to the original af32 construct. The mutant construct in which the right nucleosome was removed showed multiple nucleosome phases, suggesting that the right nucleosome stabilized first each mononucleosome and then the dinucleosome. An oligonucleotide bending assay revealed that the DNA fragment in the right nucleosome included curved DNA, suggesting that the positioning activity of the nucleosome was attributed to its DNA structure. These results suggest that information for forming stable dinucleosome is embedded in the genomic DNA and that a further characterization of the key nucleosome is useful for understanding the building up of the chromatin structure.

A Novel Cytochrome P450 Is Implicated in Brassinosteroid Biosynthesis via the Characterization of a Rice Dwarf Mutant, dwarf11, with Reduced Seed Length

We have characterized a rice (Oryza sativa) dwarf mutant, dwarf11 (d11), that bears seeds of reduced length. To understand the mechanism by which seed length is regulated, the D11 gene was isolated by a map-based cloning method. The gene was found to encode a novel cytochrome P450 (CYP724B1), which showed homology to enzymes involved in brassinosteroid (BR) biosynthesis. The dwarf phenotype of d11 mutants was restored by the application of the brassinolide (BL). Compared with wild-type plants, the aberrant D11 mRNA accumulated at higher levels in d11 mutants and was dramatically reduced by treatment with BL, implying that the gene is feedback-regulated by BL. Precise determination of the defective step(s) in BR synthesis in d11 mutants proved intractable because of tissue specificity and the complex control of BR accumulation in plants. However, 6-deoxotyphasterol (6-DeoxoTY) and typhasterol (TY), but not any upstream intermediates before these compounds, effectively restored BR response in d11 mutants in a lamina joint bending assay. Multiple lines of evidence together suggest that the D11/CYP724B1 gene plays a role in BR synthesis and may be involved in the supply of 6-DeoxoTY and TY in the BR biosynthesis network in rice.

AtTBP2 and AtTRP2 in Arabidopsis encode proteins that bind plant telomeric DNA and induce DNA bending in vitro

Telomeric DNA-binding proteins (TBPs) are crucial components that regulate the structure and function of eukaryotic telomeres and are evolutionarily conserved. We have identified two homologues of AtTBP1 (for Arabidopsis thaliana telomeric DNA binding protein 1), designated as AtTBP2 and AtTRP2, which encode proteins that specifically bind to the telomeric DNA of this plant. These proteins show extensive homology with other known plant TBPs. The isolated C-terminal segments of these proteins were capable of sequence-specific binding to duplex telomeric plant DNA in vitro. DNA bending assays using the Arabidopsis TBPs revealed that AtTBP1 and AtTBP2 have DNA-bending abilities comparable to that of the human homologue hTRF1, and higher than those of AtTRP1 and AtTRP2.