bluuebunny/crossref_metadata_2025 huggingface dataset:
The genome of Rhizobium leguminosarum has recognizable core and accessory components
Authors: Young J Peter W , Crossman Lisa C , Johnston Andrew WB , Thomson Nicholas R , Ghazoui Zara F , Hull Katherine H , Wexler Margaret , Curson Andrew RJ , Todd Jonathan D , Poole Philip S , Mauchline Tim H , East Alison K , Quail Michael A , Churcher Carol , Arrowsmith Claire , Cherevach Inna , Chillingworth Tracey , Clarke Kay , Cronin Ann , Davis Paul , Fraser Audrey , Hance Zahra , Hauser Heidi , Jagels Kay , Moule Sharon , Mungall Karen , Norbertczak Halina , Rabbinowitsch Ester , Sanders Mandy , Simmonds Mark , Whitehead Sally , Parkhill Julian
Published: April 2006

Background

Rhizobium leguminosarum is an α-proteobacterial N2-fixing symbiont of legumes that has been the subject of more than a thousand publications. Genes for the symbiotic interaction with plants are well studied, but the adaptations that allow survival and growth in the soil environment are poorly understood. We have sequenced the genome of R. leguminosarum biovar viciae strain 3841.

Results

The 7.75 Mb genome comprises a circular chromosome and six circular plasmids, with 61% G+C overall. All three rRNA operons and 52 tRNA genes are on the chromosome; essential protein-encoding genes are largely chromosomal, but most functional classes occur on plasmids as well. Of the 7,263 protein-encoding genes, 2,056 had orthologs in each of three related genomes (Agrobacterium tumefaciens, Sinorhizobium meliloti, and Mesorhizobium loti), and these genes were over-represented in the chromosome and had above average G+C. Most supported the rRNA-based phylogeny, confirming A. tumefaciens to be the closest among these relatives, but 347 genes were incompatible with this phylogeny; these were scattered throughout the genome but were over-represented on the plasmids. An unexpectedly large number of genes were shared by all three rhizobia but were missing from A. tumefaciens.

Conclusion

Overall, the genome can be considered to have two main components: a 'core', which is higher in G+C, is mostly chromosomal, is shared with related organisms, and has a consistent phylogeny; and an 'accessory' component, which is sporadic in distribution, lower in G+C, and located on the plasmids and chromosomal islands. The accessory genome has a different nucleotide composition from the core despite a long history of coexistence.
Protective effect of fasudil, a Rho-kinase inhibitor, on chemokine expression, leukocyte recruitment, and hepatocellular apoptosis in septic liver injury
Authors: Thorlacius Karin , Slotta Jan E , Laschke Matthias W , Wang Yusheng , Menger Michael D , Jeppsson Bengt , Thorlacius Henrik
Published: April 2006

Rho-kinase signaling regulates important features of inflammatory reactions. Herein, we investigated the effect and mechanisms of action of the Rho-kinase inhibitor fasudil in endotoxemic liver injury. C57/BL/6 mice were challenged with lipopolysaccharide (LPS) and D-galactosamine, with or without pretreatment with the Rho-kinase inhibitor fasudil. Six hours after endotoxin challenge, leukocyte-endothelium interactions in the hepatic microvasculature were studied by use of intravital fluorescence microscopy and tumor necrosis factor α (TNF-α); CXC chemokines as well as liver enzymes and apoptosis were determined. Administration of fasudil reduced LPS-induced leukocyte adhesion in postsinusoidal venules and sequestration in sinusoids. Moreover, we found that fasudil abolished extravascular infiltration of leukocytes as well as production of TNF-α and CXC chemokines in the liver of endotoxemic mice. Liver enzymes and hepatocellular apoptosis were markedly reduced, and sinusoidal perfusion was improved significantly in endotoxemic mice pretreated with fasudil. Our novel data document that fasudil is a potent inhibitor of endotoxin-induced expression of TNF-α and CXC chemokines as well as leukocyte infiltration and hepatocellular apoptosis in the liver. Based on the present findings, it is suggested that inhibition of the Rho-kinase signaling pathway may be a useful target in the treatment of septic liver injury.
Cardiopulmonary bypass in the immature fetus through novel use of a mini-centrifugal pump
Authors: Lombardi John , Sedgwick Joseph , Schenbeck Jennifer , Lubbers Walter , Ferguson Robert E , Gardner Aimee , McNamara Jerri L , Eghtesady Pirooz
Published: April 2006

The concept that the natural history of certain heart defects could be positively modified through in utero intervention has stimulated extensive research in fetal cardiac intervention and surgery since the early 1980s. Since the management of certain defects would require the use of cardiopulmonary support, extensive studies have been directed toward the application of a variety of perfusion circuits. The unique features of the fetal patient have directed the focus of many of these designs toward miniaturization of components and minimization of prime volume. Large extracorporeal surface contact areas and prime volumes have been identified as potential contributors to a frequently observed placental dysfunction following fetal cardiopulmonary bypass (CPB). We set out to develop means of CPB using a centrifugal micro-system that would not require supplemental prime volume. We describe the unique application of an adult right heart assist device, primarily used for ‘off-pump’ coronary revascularization for fetal cardiopulmonary support. Finally, while previous fetal experiments have used late-gestation mature fetuses, we studied more immature fetuses of mid-gestation, relevant to current clinical attempts in fetal therapy.
Direct evidence of endothelial injury during cardiopulmonary bypass by demonstration of circulating endothelial cells
Authors: Schmid Franz-Xaver , Floerchinger Bernhard , Vudattu Nalini Kumar , Eissner Günther , Haubitz Marion , Holler Ernst , Andreesen Reinhard , Birnbaum Dietrich E
Published: April 2006

Endothelial activation is considered a key process in the development of a whole body inflammatory response secondary to cardiopulmonary bypass (CPB). Increased levels of a multitude of soluble mediators have been described as being released during and after cardiac surgery. Circulating endothelial cells have recently been established as a novel marker of endothelial damage in a variety of vascular disorders.

Blood samples from 20 patients undergoing elective coronary artery bypass surgery were obtained preoperatively and 1, 6, 12, 24, and 48 h after termination of CPB. Control samples were obtained from ten healthy volunteers. Circulating endothelial cells (CEC) were isolated with immunomagnetic anti-CD146-coated Dynabeads, and counted in a Nageotte chamber.

Low numbers of CEC were observed in healthy control volunteers (12±6 cells/mL; median: 9 cells/mL). CEC numbers were already significantly elevated in all patients before CPB, and there was a further significant increase after weaning from CPB (maximum increase at 6 h after CPB: 73±30 cells/mL; range: 30-153 cells/mL, p < 0.001).

The number of CEC provides further and direct evidence that CPB is associated with a pronounced endothelial injury and/or damage. CEC appear to be most useful markers for vascular endothelial activation because they are specific, stable, and circulating components of injured vessel wall.
Influence of hematocrit and pump prime on cerebral oxygen saturation in on-pump coronary revascularization
Authors: McCusker Kevin , Chalafant Anthony , de Foe Gordon , Gunaydin Serdar , Vijay Venkataramana
Published: April 2006

Background: The couplings between cerebral oxygenation (rSO2), on-pump hematocrit and circuit prime are explored in this study.

Methods: Thirty-eight consecutive patients undergoing coronary revascularization with cardiopulmonary bypass (CPB) were matched on preoperative hematocrit < 40% and >40% ( n=16). Similarly, six blood prime patients were matched with six crystalloid prime patients. Hematocrit and rSO2 levels were then compared on CPB.

Results: The pre-operative hematocrit >40% group retained higher levels on pump run ( p < 0.01) and significantly higher rSO2 prior to CPB (64.8±9.6 versus 73.2±7.3), and on and off CPB (61.1±8.8 versus 67.4±6.4). Blood priming increased absolute rSO2 (2.3± 6.3 versus − 10.9±5.9) and% rSO2 (4.7±11.8 versus −14.2±7.4%) in the low hematocrit group.

Conclusion: Blood primes are instrumental in high-risk and low preoperative hematocrit patients in preventing cerebral oxygen desaturation during initiation and maintenance of CPB.
The junctional SR protein JP-45 affects the functional expression of the voltage-dependent Ca2+ channel Cav1.1
Authors: Anderson Ayuk A. , Altafaj Xavier , Zheng Zhenlin , Wang Zhong-Min , Delbono Osvaldo , Ronjat Michel , Treves Susan , Zorzato Francesco
Published: April 2006

JP-45, an integral protein of the junctional face membrane of the skeletal muscle sarcoplasmic reticulum (SR), colocalizes with its Ca2+-release channel (the ryanodine receptor), and interacts with calsequestrin and the skeletal-muscle dihydropyridine receptor Cav1. We have identified the domains of JP-45 and the Cav1.1 involved in this interaction, and investigated the functional effect of JP-45. The cytoplasmic domain of JP-45, comprising residues 1-80, interacts with Cav1.1. JP-45 interacts with two distinct and functionally relevant domains of Cav1.1, the I-II loop and the C-terminal region. Interaction between JP-45 and the I-II loop occurs through the α-interacting domain in the I-II loop. β1a, a Cav1 subunit, also interacts with the cytosolic domain of JP-45, and its presence drastically reduces the interaction between JP-45 and the I-II loop. The functional effect of JP-45 on Cav1.1 activity was assessed by investigating charge movement in differentiated C2C12 myotubes after overexpression or depletion of JP-45. Overexpression of JP-45 decreased peak charge-movement and shifted VQ1/2 to a more negative potential (-10 mV). JP-45 depletion decreased both the content of Cav1.1 and peak charge-movements. Our data demonstrate that JP-45 is an important protein for functional expression of voltage-dependent Ca2+ channels.
High Luminance in Organic Electroluminescent Devices with Bis(10-hydroxybenzo[h]quinolinato)beryllium as an Emitter
Authors: Hamada Yuji , Sano Takeshi , Fujita Masayuki , Fujii Takanon , Nishio Yoshitaka , Shibata Kenichi
Published: April 2006

An organic electroluminescent (EL) devices with bis(10-hydroxybenzo[h]quinolinate) beryllium (Bebq2) as an emitter was fabricated. A device structure of [ITO/hole transport layer/emitting layer/MgIn] was employed. Its color was green, and its emission peak was 516 nm. This device offered high performance with a luminance of 1.9 × 104 cd/m2 and a luminous efficiency of 3.5 lm/W.
Synthesis of Novel π-Conjugated Polymer Containing Porphyrins
Authors: Aota Hiroyuki , Itai Yasuyuki , Matsumoto Akira , Kamachi Mikiharu
Published: April 2006

Polymers containing porphyrins were synthesized from pyrrole, 1-dodecanal, and (2-formyl-5,10,15,20-tetraphenylporphyrinato)oxovanadium(IV) or 2,8,12,18-tetraethyl-3,7,13,17-tetramethyl-5,10-dipyrrylporphine. The number-average molecular weights of the resulting polymers were about 3000. NMR and absorption spectral data show the formation of the π-conjugated polymer.
Synthesis, Molecular Structure and Reactions of a Novel μ-σ,π-Cyclopentadienediyl Platinum-Manganese Complex
Authors: Onitsuka Kiyotaka , Hosokawa Naoki , Adachi Tomohiro , Yoshida Toshikatsu , Sonogashira Kenkichi
Published: April 2006

A novel σ,π-cyclopentadienediyl-bridged dinuclear complex {μ-η1,η5-C5H4Pt(COD)Cl}Mn(CO)3 (COD = 1,5-cyclooctadiene) (3), of which structure was determined by X-ray crystallography, was prepared by the reaction between (η5-C5H4SnMe3)Mn(CO)3 and Pt(COD)Cl2. The reaction of 3 with phosphine caused the ligand exchange reaction to give {μ-η1,η5-C5H4Pt(PR3)Cl}Mn(CO)3 (4). On treatment of complex 4b (R = Et) with carbon monoxide acyl complex {η5-C5H4COPt(PR3)Cl}Mn(CO)3 was obtained.
Layer-by-Layer Assembly of Alternate Protein/Polyion Ultrathin Films
Authors: Lvov Yuri , Ariga Katsuhiko , Kunitake Toyoki
Published: April 2006

Layer-by-layer assembly of multilayer films was realized by means of alternate adsorption of positively charged globular proteins (myoglobin or lysozyme) and anionic poly(styrenesulfonate). Regular growth during at least 20 alternate adsorption cycles was proved by means of UV spectroscopy and quartz crystal microbalance. Superlattices containing alternate layers of both myoglobin and lysozyme were similarly prepared.
Total Synthesis of (−)-Cotylenol, a Fungal Metabolite Having a Leaf Growth Activity
Authors: Okamoto Hiroaki , Arita Hiroaki , Kato Nobuo , Takeshita Hitoshi
Published: April 2006

Cotylenol, one of the representative fusicoccane diterpenoids having 5-8-5-membered tricyclic carbon framework, is now synthesized for the first time via an eight-membered ring formation by an intramolecular ene reaction followed by an oxidative formation of the 8β,9α-glycol function on the central eight-membered ring.
The effect of alkaline phosphatase inhibitors on intracellular lipid accumulation in preadipocytes isolated from human mammary tissue
Authors: Ali Aus T , Penny Clem B , Paiker Janice E , Psaras George , Ikram Faisel , Crowther Nigel J
Published: April 2006

Background: A previous study has demonstrated that alkaline phosphatase (AP) may play a role in the control of intracellular lipid accumulation in the rodent preadipocyte cell line, 3T3-L1. The present study investigated whether AP may have a similar function in preadipocytes isolated from human mammary gland tissue.

Methods: Preadipocyte maturation was induced in the presence or absence of the tissue non-specific AP inhibitors levamisole and histidine, and the tissue-specific AP inhibitor PheGlyGly. Cellular AP activity and adipogenesis were both assessed at 0 and 12 days post-induction of differentiation.

Results: After differentiation, AP activity increased 5.1±1.3-fold in the absence and 8.9±2.8-fold ( P<0.05) in the presence of levamisole. However, adipogenesis increased 1.95±0.11-fold in the absence but only 1.36±0.06-fold ( P<0.001) in the presence of levamisole. There was a 4.2±2.2-fold increase in AP activity in the absence and a 0.51±0.46-fold ( P<0.05) decrease in the presence of histidine. Adipogenesis increased 2.09±0.35-fold in the absence of histidine but only 1.22±0.30-fold ( P<0.05) in the presence of histidine. PheGlyGly had no effects. Fluorescent microscopy showed AP activity was localized to the triglyceride-containing droplets of the cell.

Conclusion: This is the first study to show that tissue non-specific AP inhibitors can block adipogenesis in human preadipocytes.
Hyperhomocysteinaemia, methylenetetrahydrofolate reductase polymorphism and risk of coronary artery disease
Authors: Kerkeni Mohsen , Addad Faouzi , Chauffert Maryline , Myara Anne , Gerhardt Marie , Chevenne Didier , Trivin François , Farhat Mohamed Ben , Miled Abdelhedi , Maaroufi Khira
Published: April 2006

Background: Hyperhomocysteinaemia is an independent, graded risk factor for coronary artery disease (CAD). The methylenetetrahydrofolate reductase (MTHFR) polymorphism is associated with hyperhomcysteinaemia and may therefore influence individual susceptibility to CAD. We have investigated this risk factor in a Tunisian Arab population.

Methods: Polymerase chain reaction-restriction fragment length polymorphism analysis was used to detect the C677T and A1298C variants of the MTHFR gene in 100 patients with CAD and 120 healthy controls. The severity of CAD was expressed as the number of affected vessels. Plasma total homocysteine (tHcy) concentration was determined using a direct chemiluminescence assay.

Results: MTHFR CC, CT and TT genotype frequencies in the CAD group were significantly different from those observed in the control group (49%, 35% and 16% versus 48.3%, 45.8% and 5.8%, respectively; P=0.031). However, MTHFR AA, AC and CC genotypes frequencies in the CAD group were not significantly different from the control group ( P = 0.568). Patients with CAD showed higher plasma tHcy concentrations than patients without CAD (15.86±8.63 μmol/L versus 11.90 ± 3.25 μmol/L, P<0.001). There was no association between the MTHFR polymorphisms and the number of stenosed vessels. Patients with the MTHFR TT genotype had higher plasma tHcy, serum creatinine, cholesterol and triglyceride concentrations than patients with the MTHFR CC genotype.

Conclusions: The C677T polymorphism of the MTHFR gene is associated with hyperhomocysteinaemia, lipid dysregulation and the presence of CAD in this Tunisian Arab population.
A case of low cortisol-binding globulin: use of plasma free cortisol in interpretation of hypothalamic-pituitary-adrenal axis tests
Authors: Davidson James S , Bolland Mark J , Croxson Michael S , Chiu Weldon , Lewis John G
Published: April 2006

We investigated a patient with suspected hypopituitarism who showed subnormal cortisol responses to stimulation tests with adrenocorticotrophic hormone (ACTH) and hypoglycaemia, but normal ACTH and 11-deoxycortisol responses in the metyrapone test. Cortisol-binding globulin (CBG) was measured by enzyme-linked immunosorbent assay (ELISA), and was used to calculate plasma free cortisol and free cortisol index. The patient had a low plasma CBG concentration. Reinterpreted in terms of free cortisol and free cortisol index, his responses to ACTH were normal. We conclude that despite subnormal total cortisol responses to ACTH and hypoglycaemia, the patient had a normal hypothalamic-pituitary-adrenal (HPA) axis. Measurement of CBG concentration and calculation of free cortisol or free cortisol index can help avoid false interpretations of dynamic tests of the HPA axis based on plasma total cortisol.
Nonsteroidal Antiinflammatory Drugs During Third Trimester and the Risk of Premature Closure of the Ductus Arteriosus: A Meta-Analysis
Authors: Koren Gideon , Florescu Ana , Costei Adriana Moldovan , Boskovic Radinka , Moretti Myla E
Published: April 2006

Background:

Nonsteroidal antiinflammatory drugs (NSAIDs) are increasingly being used during pregnancy to treat a variety of conditions. An evaluation of the risk of premature closure of the ductus arteriosus is useful in determining the safety of NSAIDs at different stages of pregnancy.

Objective:

To determine whether NSAID use during the third trimester of pregnancy is associated with an increased risk of premature constriction of the ductus arteriosus.

Methods:

A systematic review was conducted of MEDLINE (1966–2004), Embase (1980–2004), and the Cochrane Database of Systematic Reviews (1991–2004). Summary estimates of the odds ratios, comparing ductal outcomes in exposed and unexposed fetuses, and their 95% confidence intervals were calculated assuming a random effects model.

Results:

Based on 217 patients exposed to indomethacin and 221 to placebo, the risk of ductal closure was 15-fold higher in the group of women exposed to NSAIDs compared with those receiving either placebo or other NSAIDs (8 studies; OR = 15.04, 95% CI 3.29 to 68.68). There was no significant increased risk of ductal closure in the infants of women treated with indomethacin compared with those receiving other drugs (4 studies; OR = 2.12, 95% CI 0.48 to 9.25). Similar results were found when calculating rate differences.

Conclusions:

Short-term use of NSAIDs in late pregnancy is associated with a significant increase in the risk of premature ductal closure.
Propofol-Induced Priapism, a Case Confirmed with Rechallenge
Authors: Vesta Kimi S , Martina Shaunta' D , Kozlowski Ellen A
Published: April 2006

Objective:

To report a case of propofol-induced priapism.

Case Summary:

A 17-year-old male receiving propofol for induction of anesthesia during a cardiac ablation procedure developed priapism that required medical intervention and discontinuation of propofol. The priapism developed after a total propofol dose of 550 mg, lasted for 2 hours, and resolved immediately following medical intervention. The following day, the patient underwent a transesophageal echocardiogram and received a total propofol dose of 40 mg throughout the procedure. He again developed priapism, which resolved within one hour without medical intervention.

Discussion:

Priapism experienced by this patient is considered to be drug-induced because of the temporal relationship, recurrence with rechallenge, and dose–response relationship. Using the Naranjo probability scale, we determined that propofol was a highly probable causative agent of priapism in this patient.

Conclusions:

Although, as of April 4, 2006, this adverse effect has not been previously reported, there is a strong correlation of propofol to priapism in this patient. Clinicians should be aware of this adverse effect.
Absolute versus relative difference measures of priming: Which is appropriate when baseline scores change with age?
Authors: Murphy Kristina , McKone Elinor , Slee Judith
Published: April 2006

It is often of theoretical interest to know if implicit memory (repetition priming) develops across childhood under a given circumstance. Methodologically, however, it is difficult to determine whether development is present when baseline performance for unstudied items improves with age. Calculation of priming in absolute (priming=studied – unstudied) or relative‐to‐baseline terms can lead to different conclusions. In first noting this problem, Parkin (1993) suggested using the Snodgrass (1989a) calculation of relative priming [priming=(studied – unstudied)/(maximum – unstudied)], and most developmental studies have since adopted this procedure. Here, we question the Snodgrass method because the Snodgrass method's results are not replicated in the picture identification task when baselines are equated experimentally across age groups. Instead, results support an absolute measure of priming. Theoretically, we argue against its core assumption; namely, that children and adults always lie on the same learning curve, with an equal maximum performance level and equal rate of learning.
Drawing boundaries: From individual to common region – the development of spatial region attribution in children
Authors: Lange‐Küttner C.
Published: April 2006

The study investigated at what age children draw boundaries around pairs of objects that share either similarity or proximity. In two studies (N=132 and N=252) using a Wertheimer array, a clear age trend between 4 and 8 years showed that while young children were more likely to code objects into individual regions, older children were more likely to attribute common regions to pairs of dots. Unsystematic coding occurred as a transitional pattern until age 6, but except for the youngest group, at all ages a proportion of children drew boundaries of common region around arbitrary numbers of stimuli. Future research might show whether constructing exact, stimulus‐matched boundaries of common region is a performance predictor in other cognitive domains, such as visual memory.
Growth Hormone Attenuates the Transcriptional Activity of Runx2 by Facilitating Its Physical Association With Stat3β
Authors: Ziros Panos G , Georgakopoulos Tassos , Habeos Ioannis , Basdra Efthimia K , Papavassiliou Athanasios G
Published: April 2006

We document that GH controls osteoblast function by modulating the biological activity of the osteospecific transcription factor Runx2. Evidence is provided for a physical interaction between Runx2 and Stat3β, which is enhanced by GH and downregulates the transcriptional properties of this key osteogenic regulator.

Introduction: Growth hormone (GH) signals to bone either through insulin-like growth factor-1 or directly by influencing the function of osteoblasts, the bone-forming cells. This study aimed at exploring the molecular events that underlie the direct biological action of GH on osteoblastic cells, and specifically, the effects that it might exert on the function of the bone-specific transcriptional regulator Runx2.

Materials and Methods: The GH-responsive human osteoblastic cell line Saos-2 was used as our experimental system. Western blot analyses were used to monitor the presence of several parameters known to be affected by GH in these cells (i.e., downregulation of GH receptor, induction of STATs, and extracellular signal-regulated kinase [ERK] mitogen-activated protein kinase [MAPK] pathways). Electrophoretic mobility shift assays were used to assess Runx2 and Stat3 binding activity on an osteoblast-specific element (OSE2) after GH treatment. A combination of yeast two-hybrid and co-immunoprecipitation assays were performed to test for the existence of a physical Runx2·Stat3β association. Finally, co-transfection experiments were used to investigate the interplay of the two transcription factors on the activity of a p6OSE2-Luc promoter after GH stimulation.

Results: We show that GH signaling through Stat3/ERK MAPK potentiates the DNA binding activity of Runx2 but, at the same time, restrains its transcriptional potential. Moreover, a novel physical interaction of Runx2 with transcription factor Stat3β, which is enhanced by GH stimulation, was documented both in vitro and in vivo. Importantly, this interaction impairs the transcriptional activity of Runx2 without affecting its DNA binding capacity.

Conclusion: Our data provide the first evidence that GH modulates the transcriptional function of Runx2 in osteoblastic cells by promoting its inhibitory interaction with Stat3β. Shedding light on such mechanisms will contribute to a better understanding of GH effects on skeletal homeostasis that may impact on decisions at the clinical level, especially in diseases affecting bone quantity and quality (e.g., osteoporosis).
Interleukin-1β and TNF-α Act in Synergy to Inhibit Longitudinal Growth in Fetal Rat Metatarsal Bones
Authors: Mårtensson Katarina , Chrysis Dionisios , Sävendahl Lars
Published: April 2006

We hypothesized that pro-inflammatory cytokines can act locally in the growth plate to impair longitudinal growth. In a model of cultured fetal rat metatarsal bones, we found that IL-1β and TNF-α act in synergy to inhibit longitudinal growth, an effect linked to decreased proliferation and increased apoptosis of growth plate chondrocytes. IGF-I could partially reverse all these effects.

Introduction: Children with chronic inflammatory conditions, such as Crohn's disease or rheumatoid arthritis, experience impaired longitudinal growth. The inflammatory process itself, which includes upregulation of the pro-inflammatory cytokines interleukin (IL)-1β, IL-6, and TNF-α, is believed to be at least partly responsible for the poor growth in these patients. This study aimed to clarify whether these cytokines can act locally in the growth plate to suppress longitudinal growth and whether any negative effects can be reversed by insulin-like growth factor-I (IGF-I).

Materials and Methods: The effects of cytokines on longitudinal bone growth were studied in fetal (day E20) rat metatarsal bones kept in culture. After a 7-day culture, the bones were sectioned, and chondrocyte proliferation was assessed by bromodeoxyuridine (BrdU) incorporation and apoptosis by TUNEL.

Results: When added separately, IL-1β and TNF-α impaired longitudinal bone growth only at a high concentration (100 ng/ml each; p < 0.05 versus control). In contrast, when added in combination, IL-1β and TNF-α potently inhibited growth at far lower concentrations (from 3 ng/ml each; p < 0.001 versus control) and also decreased chondrocyte proliferation and increased apoptosis. Growth failure induced by the combination of IL-1β and TNF-α (10 ng/ml each) could be counteracted by anti-IL-1β (100 ng/ml; p < 0.001), anti-TNF-α (100 ng/ml; p < 0.001), or IGF-I (100 ng/ml; p < 0.01). IL-6 did not affect longitudinal growth even when added in combination with IL-1β or TNF-α (10 ng/ml each).

Conclusions: We show that IL-1β and TNF-α act in synergy to locally suppress longitudinal growth, an effect that can be partially reversed by IGF-I. Although growth hormone (GH)/IGF-I may improve longitudinal growth in children with chronic inflammatory diseases, our results suggest that the inflammatory process itself must be targeted to achieve normal growth.
Downregulation of Osteoblast Markers and Induction of the Glial Fibrillary Acidic Protein by Oncostatin M in Osteosarcoma Cells Require PKCδ and STAT3
Authors: Chipoy Céline , Berreur Martine , Couillaud Séverine , Pradal Gilbert , Vallette François , Colombeix Caroline , Rédini Françoise , Heymann Dominique , Blanchard Frédéric
Published: April 2006

The effects of OSM on proliferation and differentiation of osteosarcoma and nontransformed osteoblasts were analyzed. OSM downregulates osteoblast markers but induces the glial fibrillary acidic protein by the combined activation of PKCδ and STAT3, offering new lines of therapeutic investigations.

Introduction: Oncostatin M (OSM) is a multifunctional cytokine of the interleukin-6 family implicated in embryonic development, differentiation, inflammation, and regeneration of various tissues, mainly the liver, bone, and the central nervous and hematopoietic systems. One particularity of OSM relies on its growth inhibitory and pro-differentiating effects on a variety of tumor cell lines such as melanoma, providing arguments for a therapeutic application of OSM. The objective of this study was to analyze the effects of OSM on osteosarcoma cell lines proliferation and differentiation.

Materials and Methods: Proliferation was analyzed by3H thymidine incorporation. Differentiation was analyzed by semiquantitative RT-PCR and immunocytochemistry for various markers. Alizarin red S staining was used to evaluate bone nodule formation. Morphological changes were studied by confocal and electron microscopy. Western blotting, kinases inhibitors, and dominant negative STAT3 were used to identified the signaling pathways implicated.

Results: OSM inhibits the growth of rat osteosarcoma cell lines as well as normal osteoblasts, in correlation with induction of the cyclin-dependent kinases inhibitor p21WAF1. However, OSM reduces osteoblast markers such as alkaline phosphatase, osteocalcin, and bone sialoprotein, leading to strong inhibition of mineralized nodule formation. This inhibitory effect is restricted to mature osteoblasts and differentiated osteosarcoma because OSM effectively stimulates osteoblast markers and bone nodule formation in early, but not late, bone marrow mesenchymal stem cell (BMSC) cultures. In osteosarcoma cells or BMSC, OSM induces expression of the glial fibrillary acidic protein (GFAP) as well as morphological and ultrastructural changes, for example, elongated shape and bundles of microfilaments in cell processes. Rottlerin (PKCδ inhibitor), and to a lesser degree UO126 (MEK/ERK inhibitor), prevents the loss of osteoblastic markers by OSM, whereas dominant negative STAT3 prevents GFAP induction.

Conclusions: These results highlight the particular gene expression profile of OSM-treated osteosarcoma cells and BMSCs, suggesting either a osteocytic or a glial-like phenotype. Together with the implication of PKCδ, ERK1/2, and STAT3, these results offer new lines of investigations for neural cell transplantation and osteosarcoma therapy.
Quantitative Trait Loci That Determine BMD in C57BL/6J and 129S1/SvImJ Inbred Mice
Authors: Ishimori Naoki , Li Renhua , Walsh Kenneth A , Korstanje Ron , Rollins Jarod A , Petkov Petko , Pletcher Mathew T , Wiltshire Tim , Donahue Leah Rae , Rosen Clifford J , Beamer Wesley G , Churchill Gary A , Paigen Beverly
Published: April 2006

BMD is highly heritable; however, little is known about the genes. To identify loci controlling BMD, we conducted a QTL analysis in a (B6 × 129) F2 population of mice. We report on additional QTLs and also narrow one QTL by combining the data from multiple crosses and through haplotype analysis.

Introduction: Previous studies have identified quantitative trait loci (QTL) that determine BMD in mice; however, identification of genes underlying QTLs is impeded by the large size of QTL regions.

Materials and Methods: To identify loci controlling BMD, we performed a QTL analysis of 291 (B6 × 129) F2 females. Total body and vertebral areal BMD (aBMD) were determined by peripheral DXA when mice were 20 weeks old and had consumed a high-fat diet for 14 weeks.

Results and Conclusions: Two QTLs were common for both total body and vertebral aBMD: Bmd20 on chromosome (Chr) 6 (total aBMD; peak cM 26, logarithm of odds {LOD} 3.8, and vertebral aBMD; cM 32, LOD 3.6) and Bmd22 on Chr 1 (total aBMD; cM 104, LOD 2.5, and vertebral aBMD; cM 98, LOD 2.6). A QTL on Chr 10 (Bmd21, cM 68, LOD 3.0) affected total body aBMD and a QTL on Chr 7 (Bmd9, cM 44, LOD 2.7) affected vertebral aBMD. A pairwise genome-wide search did not reveal significant gene-gene interactions. Collectively, the QTLs accounted for 21.6% of total aBMD and 17.3% of vertebral aBMD of the F2 population variances. Bmd9 was previously identified in a cross between C57BL/6J and C3H/HeJ mice, and we narrowed this QTL from 34 to 22 cM by combining the data from these crosses. By examining the Bmd9 region for conservation of ancestral alleles among the low allele strains (129S1/SvImJ and C3H/HeJ) that differed from the high allele strain (C57BL/6J), we further narrowed the region to ∼9.9 cM, where the low allele strains share a common haplotype. Identifying the genes for these QTLs will enhance our understanding of skeletal biology.
Four and Half Lim Protein 2 (FHL2) Stimulates Osteoblast Differentiation
Authors: Lai Chung-Fang , Bai Shuting , Uthgenannt Brian A , Halstead Linda R , McLoughlin Patricia , Schafer Beat W , Chu Po-Hsien , Chen Ju , Otey Carol A , Cao Xu , Cheng Su-Li
Published: April 2006

FHL2, a molecule that interacts with many integrins and transcription factors, was found to play an important role in osteoblast differentiation. Overexpression of FHL2 increases the accumulation of osteoblast differentiation markers and matrix mineralization, whereas FHL2 deficiency results in inhibition of osteoblast differentiation and decreased bone formation.

Introduction: Integrin-matrix interaction plays a critical role in osteoblast function. It has been shown that the cytoplasmic domains of integrin β subunits mediate signal transduction induced by integrin-matrix interaction. We reasoned that the identification of proteins interacting with β-cytoplasmic tails followed by analysis of the function of these proteins would enhance our understanding on integrin signaling and the roles of these proteins in osteoblast activities.

Materials and Methods: Yeast two hybrid assay was used to identify proteins interacting with the cytoplasmic domain of integrin β5 subunit. The association of these proteins with integrin αvβ5 was confirmed by confocal analysis and co-immunoprecipitation. A stable MC3T3-E1 cells line overexpressing Four and Half Lim Protein 2 (FHL2) and mouse osteoblasts deficient in FHL2 were used to study the roles of FHL2 in osteoblast differentiation and bone formation. Matrix protein expression was determined by mRNA analysis and Western blotting. Matrix mineralization was detected by Alizarin red staining. Alkaline phosphatase activity was also measured. μCT was used to determine bone histomorphometry.

Results and Conclusions: FHL2 and actin-binding proteins, palladin and filamin A, were identified as proteins interacting with β5 cytoplasmic domain. FHL2 co-localized with αvβ5 at the focal adhesion sites in association with palladin and filamin A. FHL2 was also present in nuclei. Osteoblasts overexpressing FHL2 exhibited increased adhesion to and migration on matrix proteins. Conversely, FHL2 stimulation of CREB activity was dependent on integrin function because it was inhibited by Gly-Arg-Gly-Asp-Ser (GRGDS) peptide. The expression of osteoblast differentiation markers and Msx2 was upregulated, and bone matrix mineralization was increased in FHL2 overexpressing cells. In contrast, FHL2-deficient bone marrow cells and osteoblasts displayed decreased osteoblast colony formation and differentiation, respectively, compared with wildtype cells. Moreover, FHL2-deficient female mice exhibited greater bone loss than the wildtype littermates after ovariectomy. Thus, FHL2 plays an important role in osteoblast differentiation and bone formation.
TREM2, a DAP12-Associated Receptor, Regulates Osteoclast Differentiation and Function
Authors: Humphrey Mary Beth , Daws Michael R , Spusta Steve C , Niemi Eréne C , Torchia James A , Lanier Lewis L , Seaman William E , Nakamura Mary C
Published: April 2006

Deficiency of the signaling adapter protein DAP12 or its associated receptor TREM2 is associated with abnormal OC development in humans. Here we examine the role of TREM2 in mouse OC development and function, including migration and resorption in vitro. These results provide new evidence that TREM2 regulates OC function independent of its effects on multinucleated OC differentiation.

Introduction: TREM2 (triggering receptor expressed in myeloid cells-2) associates with the signaling adapter DAP12 in osteoclasts (OCs). Genetic mutation or deletion of either the TYROBP (DAP12) or TREM2 gene is associated with the human disorder of brain and bone, Nasu-Hakola disease. We and others recently showed the critical requirement for immunoreceptor tyrosine-based activation motif (ITAM) signals through DAP12 and the Fc Receptor γ chain (FcRγ) during OC development. Here, we further define the role of TREM2 in OC differentiation and describe a role for TREM2 in OC migration and bone resorption.

Materials and Methods: We generated monoclonal anti-mouse TREM2 antibodies (mAb), analyzed pre-osteoclasts and mature OCs for TREM2 surface expression, and determined the effect of antibody ligation on in vitro OC differentiation, resorption, and migration. TREM2 RNA interference (RNAi) was used to disrupt expression of TREM2 in pre-osteoclasts.

Results: Using flow cytometry, our studies reveal that TREM2 is weakly expressed on C57BL/6 bone marrow macrophages (BMMs) and is upregulated during culture with RANKL and macrophage-colony stimulating factor (M-CSF). The expression of TREM2 is unaltered in DAP12-deficient OCs. Using C57BL/6 BMMs or RAW264.7 precursors, anti-TREM2 mAb treatment with RANKL and M-CSF enhances the formation of multinuclear TRACP+ OCs compared with control mAb treatment. In contrast, these agents have no effect on DAP12-deficient precursors. Monoclonal Ab blockade of TREM2 on OCs generated from C57BL/6 BMMs results in decreased resorption of artificial calcium-phosphate substrate and dentine. Reduction of TREM2 expression in RAW264.7 cells by RNAi results in loss of OC formation in response to RANKL and M-CSF. Anti-TREM2 cross-linking enhances migration of C57BL/6 OCs and RAW246.7 OCs in response to M-CSF.

Conclusions: Our studies indicate that the TREM2 receptor regulates OC multinucleation as well as resorption and migration of mature OCs. Thus, TREM2-DAP12 signals regulate both OC formation and function.
Gorham-Stout Syndrome: A Monocyte-Mediated Cytokine Propelled Disease
Authors: Colucci Silvia , Taraboletti Giulia , Primo Luca , Viale Andrea , Roca Cristina , Valdembri Donatella , Geuna Massimo , Pagano Marco , Grano Maria , Pogrel Anthony M , Harris Adrian L , Athanasou Nicholas N , Mantovani Alberto , Zallone Alberta , Bussolino Federico
Published: April 2006

We studied the biological features and the immunophenotype of a cell culture established from the lesion of soft tissues of a woman affected by Gorham-Stout syndrome. We found that these cells belonged to a monocytic lineage with some characteristics of immature osteoclasts and were able to release large amounts of osteoclastogenic and angiogenic molecules that may contribute to disease progression.

Introduction: Gorham-Stout syndrome is a rare disease characterized by osteolysis and proliferation of vascular or lymphatic vessels, with a severe outcome. Its etiology and the identification of the cell types involved are completely unknown.

Materials and Methods: A cell culture from a lesion of soft tissues was established, and its behavior in vitro and in immunodeficient mice was studied. We analyzed (1) the cell phenotype by flow cytometry; (2) the adhesive and migratory properties on different substrates; (3) the ability to differentiate into mature osteoclasts; (4) the production of osteclastogenic and angiogenic molecules; (5) the in vivo angiogenic activity of the cells subcutaneously implanted in mouse in a Matrigel plug; and (6) the ability to recapitulate the disease when transplanted in nude mice.

Results and Conclusions: The established culture consisted of a morphologically homogeneous cell population belonging to a monocytic lineage having some features of an osteoclast-like cell type. Cells had an invasive phenotype, were angiogenic, and produced osteoclastogenic (IL-6, TGF-β1, IL-1β) and angiogenic (vascular endothelial growth factor-A {VEGF-A}, CXCL-8) molecules when challenged with inflammatory cytokines. Immunodeficient mice injected with these cells did not show any bone lesions or vascular alteration, but had high amounts of circulating human IL-6 and VEGF-A. Cells isolated from a cutaneous lymphangiomatosis did not show any of these findings. These data suggest that cells of monocyte-macrophage lineage play an essential role in the pathogenesis of Gorham-Stout disease, whose progression is propelled by cytokine circuits that accelerate angiogenesis and osteoclastogenesis.
Effect of Teriparatide {rhPTH(1-34)} on BMD When Given to Postmenopausal Women Receiving Hormone Replacement Therapy
Authors: Ste-Marie Louis G , Schwartz Sherwyn L , Hossain Anwar , Desaiah Durisala , Gaich Gregory A
Published: April 2006

The effects of teriparatide when given in combination with HRT were studied in postmenopausal women with low bone mass or osteoporosis. The data provide evidence that the adverse event profile for combination therapy with teriparatide + HRT together is consistent with that expected for each treatment alone and that the BMD response is greater than for HRT alone.

Introduction: Teriparatide {rhPTH(1-34)}, given as a once-daily injection, activates new bone formation in patients with osteoporosis. Hormone replacement therapy (HRT) prevents osteoporosis by reducing bone resorption and formation. Combination therapy with these two compounds, in small clinical trials, increased BMD and reduced vertebral fracture burden. The purpose of this study was to determine whether teriparatide provided additional effect on BMD when given in combination with HRT.

Materials and Methods: A randomized, double-blind, placebo-controlled study was conducted in postmenopausal women with either low bone mass or osteoporosis. Patients were randomized to placebo subcutaneous plus HRT (n = 125) or teriparatide 40 μg/day (SC) plus HRT (TPTD40 + HRT; n = 122) for a median treatment exposure of 13.8 months. Approximately one-half of the patients in each group were pretreated with HRT for at least 12 months before randomization. Patients received 1000 mg calcium and 400–1200 IU of vitamin D daily as oral supplementation. BMD was measured by DXA.

Results: Compared with HRT alone, TPTD40 + HRT produced significant (p < 0.001) increases in spine BMD (14% versus 3%), total hip (5.2% versus 1.6%), and femoral neck (5.2% versus 2%) at study endpoint. BMD, in whole body and ultradistal radius, was higher, and in the one-third distal radius was lower, in the combination therapy but not in the HRT group. Serum bone-specific alkaline phosphatase and urinary N-telopeptide/Cr were increased significantly (p < 0.01) in the women receiving TPTD40 + HRT compared with HRT. A similar profile of BMD and bone markers was evident in both randomized patients as well as in subgroups of patients not pretreated or pretreated with HRT. Patients tolerated both the treatments well. Nausea and leg cramps were more frequently reported in the TPTD40 + HRT group.

Conclusions: Adding teriparatide, a bone formation agent, to HRT, an antiresorptive agent, provides additional increases in BMD beyond that provided by HRT alone. The adverse effects of teriparatide when added to HRT were similar to the adverse effects described for teriparatide administered alone. Whether teriparatide was initiated at the same time as HRT or after at least 1 year on HRT, the incremental increases over HRT alone were similar.
Human Microvascular Endothelial Cell Activation by IL-1 and TNF-α Stimulates the Adhesion and Transendothelial Migration of Circulating Human CD14+ Monocytes That Develop With RANKL Into Functional Osteoclasts
Authors: Kindle Libby , Rothe Linda , Kriss Michael , Osdoby Philip , Collin-Osdoby Patricia
Published: April 2006

Circulating pre-OCs may be recruited to locally inflamed sites through specific interactions with activated microvasculature. We found that HMVECs stimulated the adhesion and TEM of circulating pre-OCs, in an ICAM-1- and CD44-dependent manner, leading to greater RANKL-induced OC formation and bone pit resorption.

Introduction: Inflammation is critical for healing processes but causes severe tissue destruction when chronic. Local osteoclast (OC) formation and bone resorption may increase at inflammatory sites through multiple mechanisms, including direct stimulation by inflamed microvasculature of circulating OC precursor (pre-OC) migration through a blood vessel barrier into bone or joint tissue. How this might occur is not yet well understood.

Materials and Methods: Cytokine-activated human microvascular endothelial cell (HMVEC) monolayers, with or without IL-1 and TNF-α preactivation (24 h), were incubated in adhesion (1-3 h) or porous transwell transendothelial migration (TEM; 3 h) assays with human peripheral blood mononuclear cells (hPBMCs) or CD14+ monocyte or CD14− lymphocyte subsets. The number of cells that adhered or transmigrated, and their ability to thereafter develop with macrophage-colony stimulating factor (M-CSF) + RANKL into bone pit-resorbing OCs, were analyzed. Immunostaining and neutralizing antibodies to key cell adhesion molecules were used to determine their potential involvement in stimulated CD14+ monocyte TEM.

Results: M-CSF + RANKL caused OC and bone pit formation only from hPBMCs and CD14+ cells but not CD14− cells. Adhesion of hPBMCs or CD14+ cells but not CD14− cells was stimulated by cytokine preactivation of HMVECs and led to the full capture of all circulating pre-OCs capable of developing into OCs. Cytokine-preactivated HMVECs also promoted the postadhesion TEM of hPBMCs and CD14+ populations, resulting in markedly greater OC formation and bone pit resorption by transmigrated cells. Immunodetectable vascular cell adhesion molecule (VCAM-1), intercellular adhesion molecule (ICAM-1), and CD44 levels increased on cytokine-treated HMVEC surfaces, and neutralizing antibodies to ICAM-1 or CD44, but not VCAM-1 or platelet endothelial cell adhesion molecule (PECAM-1), inhibited stimulated CD14+ cell TEM through activated HMVECs.

Conclusions: This is the first demonstration that cytokine-activated HMVECs efficiently capture and promote the TEM of circulating pre-OCs capable of differentiating into bone-resorbing OCs. Thus, direct pre-OC recruitment by activated microvasculature at inflammatory sites may significantly contribute to normal OC bone remodeling during fracture healing or exacerbate pathological bone loss in various chronic inflammatory disorders.