bluuebunny/crossref_metadata_2025 huggingface dataset:
RANKL is an essential cytokine mediator of polymethylmethacrylate particle‐induced osteoclastogenesis
Authors: Clohisy John C. , Frazier Elfaridah , Hirayama Teruhisa , Abu‐Amer Yousef
Published: February 2003

RANKL is a TNF superfamily member and an essential cytokine mediator of developmental osteoclastogenesis. We examined the role of RANKL in PMMA particle‐induced osteoclastogenesis in vitro. In murine whole bone marrow cultures, PMMA particles stimulate a 2.5 fold increase in secreted RANKL, a 5–8 fold increase in osteoclast number and induce the formation of giant multinuclear osteoclasts. RANKL and TNF, potential cytokine mediators of PMMA, had similar osteoclastogenic effects. The RANKL inhibitor OPG was utilized to define the role of RANKL in mediating the PMMA response and was found to inhibit basal and PMMA particle‐induced osteoclastogenesis. Additionally, particles stimulate osteoclast formation in RANKL‐primed osteoclast precursor cells (devoid of supporting stromal cells) while RANKL untreated osteoclast precursors demonstrate no osteoclastogenic response to particles. Since TNF can potentiate RANKL action and is thought to mediate implant osteolysis we analyzed TNF−/− whole bone marrow cultures to elucidate the role of this cytokine. In TNF‐/‐ cultures basal osteoclastogenesis remains intact, yet the PMMA effect is blunted. Finally, we show that PMMA, RANKL and TNF all activate the NF‐kB and c‐jun/AP‐1 signaling pathways which are both fundamental to osteoclast formation and are potential sites of signal convergence in RANKL‐mediated particle osteoclastogenesis.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Type II collagen modulates the composition of extracellular matrix synthesized by articular chondrocytes
Authors: Qi Wen‐Ning , Scully Sean P.
Published: February 2003

The articular cartilage extracellular matrix (ECM) interfaces with chondrocytes and influences many biological processes important to cartilage homeostasis and repair. The alginate bead culture system can be viewed as a model of cartilage repair in which the chondrocyte attempts to recreate the pericellular matrix while maintaining a differentiated phenotype. The purpose of this study was to evaluate the alteration in epitopes of proteoglycan and tenascin synthesized by chondrocytes in the presence of exogenous extracellular type II collagen. We evaluated the effects on four biomarkers associated with the creation of the denovo matrix using ELISA and immunohistochemistry: keratan sulfate epitope (5D4), 3B3(−) neoepitope of chondroitin‐6‐ sulfate, 3B3(+) chondroitinase‐generatedepitope of chondroitin‐6‐sulfate, and tenascin‐C expression. TGF‐β1 stimulated the production of 3B3(+), 5D4, and tenascin‐C in a dose‐dependent manner and decreased 3B3(−) levels. Following the addition of exogenous type II collagen, 3B3(−) increased and tenascin‐C decreased but did not change the direction of TGF‐β1 effects. In contrast, 5D4 expression decreased in the presence of collagen II as TGF‐β1 increased to 10 ng/ml. Interestingly, the amount of 3B3(+) epitope was not affected by the incorporation of type II collagen. Immunohistochemistry found there was no significant difference in distribution of these biomarkers in the presence and absence of extracellular type II collagen incorporation. These results elucidate the subtle biochemical differences in ECM synthesized by chondrocytes in the presence of type II collagen and further characterize the role played by ECM in the TGF‐β1 regulation of the articular cartilage physiology.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Pulsed electromagnetic fields affect phenotype and connexin 43 protein expression in MLO‐Y4 osteocyte‐like cells and ROS 17/2.8 osteoblast‐like cells
Authors: Lohmann C. H. , Schwartz Z. , Liu Y. , Li Z. , Simon B. J. , Sylvia V. L. , Dean D. D. , Bonewald L. F. , Donahue H. J. , Boyan B. D.
Published: February 2003

Osteocytes, the predominant cells in bone, are postulated to be responsible for sensing mechanical and electrical stimuli, transducing signals via gap junctions. Osteocytes respond to induced shear by increasing connexin 43 (Cx43) levels, suggesting that they might be sensitive to physical stimuli like low‐frequency electromagnetic fields (EMF). Immature osteoblasts exhibit decreased intercellular communication in response to EMF but no change in Cx43. Here, we examined long term effects of pulsed EMF (PEMF) on MLO‐Y4 osteocyte‐like cells and ROS 17/2.8 osteoblast‐like cells. In MLO‐Y4 cell cultures, PEMF for 8 h/day for one, two or four days increased alkaline phosphatase activity but had no effect on cell number or osteocalcin. Transforming growth factor beta‐1 (TGF‐β1) and prostaglandin E2 were increased, and NO2‐ was altered. PEMFs effect on TGF‐β1 was via a prostaglandin‐dependent mechanism involving Cox‐1 but not Cox‐2. In ROS 17/2.8 cells, PEMF for 24, 48 or 72 h did not affect cell number, osteocalcin mRNA or osteocalcin protein. PEMF reduced Cx43 protein in both cells. Longer exposures decreased Cx43 mRNA. This indicates that cells in the osteoblast lineage, including well‐differentiated osteoblast‐like ROS 17/2.8 cells and terminally differentiated osteocyte‐like MLO‐Y4 cells, respond to PEMF with changes in local factor production and reduced Cx43, suggesting decreased gap junctional signaling.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Autologous osteoblasts enhance osseointegration of porous titanium implants
Authors: Frosch K.‐H. , Sondergeld I. , Dresing K. , Rudy T. , Lohmann C. H. , Rabba J. , Schild D. , Breme J. , Stuermer K. M.
Published: February 2003

The goal of this study was to assess the osseointegration of porous titanium implants by means of coating with autologous osteoblasts.

Titanium implants (8 × 5 × 4 mm) having drill channels with diameters of 400, 500, and 600 μm were coated with autologous osteoblasts obtained from spongiosa chips. The implants were inserted into the distal femora of 17 adult Chinchilla Bastard rabbits (group I). Uncoated implants were inserted as controls in the contralateral femur (group II). The animals were sacrificed after 5, 11, and 42 days. Intravital fluorochrome labeling and microradiography were used for the assessment of bone ingrowth into the titanium channels.

In both groups, no bone tissue was formed in the channels up to day 5. On day 11, group I exhibited significantly more (p<0.05) bone tissue (19.8 ± 14.0% vs. 5.8 ± 9.1%) with greater bone‐implant contact (13.3 ± 15.1% vs. 5.7 ± 5.3%, p<0.05) at the channel mouths than group II. Bone tissue was formed mainly between day 15 and 30 in group I, in group II between day 25 and 40.

Six weeks after implantation, bone tissue filled on an average 68.8 ± 15.1% of the mouths of the drill channels in implants in group I, the filling for group II was 49.8 ± 18.1% (p<0.05). The average bone‐implant contact at the channel mouths after six weeks was 56.5 ± 13.5% in group I, 40.2 ± 21.9% in group II (p<0.05). 600‐μm channels showed at this time point the best osseous integration (p<0.05).

Coating with autologous osteoblasts accelerates and enhances the osseointegration of titanium implants and could be a successful biotechnology for future clinical applications.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Localization of insulin‐like growth factor binding protein‐2 in chondrocytes of bovine articular cartilage
Authors: Morales Teresa I. , Hunziker Ernst B.
Published: February 2003

Purpose: Previous work indicated that transforming growth factor (TGF‐β) treatment of bovine articular cartilage resulted in an accumulation of insulin‐like growth factor binding protein‐2 (IGF‐BP‐2). The purpose of the work presented in this paper was to define the localization of the IGF‐BP‐2 in freshly excised articular cartilage and in slices cultured in the presence and absence of TGF‐β.

Method: Newborn calf articular cartilage was dissected and immediately fixed or maintained in organ culture for five days under basal conditions (media without added serum or growth factors) or with basal media containing 15 ng/ml of TGF‐β1. Frozen or paraffin embedded sections were prepared, and immunohistochemistry using anti‐IGF‐BP‐2 performed.

Results: The paraffin sections provided the best preservation of morphology and consistency of immunohistochemical staining patterns. In fresh cartilage slices, IGF‐BP‐2 was associated with most of the chondrocytes. The basal cultured cartilage showed positive immunostaining in some areas, but not others: the most consistently stained area was the upper radial zone. In all cases where a positive reaction was observed, it was associated mostly with chondrocytes. On the other hand, all the TGF‐β treated samples that were examined in this study were evenly stained, and most chondrocytes were positive in all areas from superficial to deep zones, thus closely resembling the pattern of fresh tissue.

Conclusions: It is concluded that IGF‐BP‐2 is closely cell associated in bovine articular cartilage. Following culture of cartilage slices, TGF‐β increases the number of cells with positive immunostaining. These data help to support the postulate that TGF‐β exerts at least some of its actions in articular cartilage via cross‐talk mechanisms involving the IGF‐BP‐2 system.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Neurohistological examination of the inferior glenohumeral ligament of the shoulder
Authors: Steinbeck Jörn , Brüntrup Jens , Greshake Oliver , Pötzl Wolfgang , Filler Timm , Liljenqvist Ulf
Published: February 2003

The neural histology of the anterior band of the inferior glenohumeral ligament (IGHL) was studied in 11 fresh shoulder specimen using a special silver impregnation technique. Between the collagen fibers small myelinated and unmyelinated dendrites could be detected. The appearance of neurovascular structures in the adjacent synovial layer clearly exceeded the typical supply to soft tissues. Analysing about 11,000 sections Ruffini mechanoreceptors that are known to be slow adapting were found on the humeral insertion of the band. The sections containing these neural end organs were identified by means of transillumination and reflection‐contrast microscopy and reconstructed using three‐dimensional image processing. The presence of neural structures including Ruffini corpuscles in these most important passive stabilizers of the shoulder joint shows that these ligaments function also as an active safety device. There slow adaption is a prerequisite for muscular reflexes counteracting the tensile stresses to which the passive stabilizing structures of the shoulder are exposed. A disruption of the continuity of these structures by mechanical forces or surgery can reduce the biofeedback and proprioceptive quality and thus lead to a decrease of shoulder function and/or stability. These observations should be taken into account when planning surgical interventions involving the IGHL. Procedures like capsule shifts or plications may affect mechanoreceptor orientation and concentrations, thereby affecting the interaction between these structures and the synergistic muscles. When possible, these intervention should avoid receptor‐dense regions while attempting to restore normal anatomical orientation and tissue tension.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Determination of cortical bone porosity and pore size distribution using a low field pulsed NMR approach
Authors: Wang Xiaodu , Ni Qingwen
Published: February 2003

The objective of this study was first to prove the concept of a low field pulsed nuclear magnetic resonance (NMR) process for assessing the cortical porosity and pore size distribution of human bone in vitro, and then to apply the technique to detect age‐related changes of bone in these parameters. The Carr‐Purcell‐Meiboom‐Gill NMR spin echo train method is used to determine the porosity, and an inversion NMR spin‐spin relaxation (T2) spectrum is used to assess the pore size distribution in cortical bone. Using these techniques, cortical porosity and pore size distribution of 19 specimens of human cadaveric bone, ranging from 16 to 89 years of age, were assessed. The NMR results were compared with the histomorphometric data of the same bone samples to verify the efficacy of the NMR approach. Moreover, a coefficient (surface relaxivity) relating the pore size to the T2 relaxation time was determined empirically for the Haversian canals and the osteocytic lacunae. The results of this study demonstrate that the in vitro NMR approach using T2 relaxation techniques can directly assess the porosity and pore size distribution (Haversian canals and osteocytic lacunae) in human cortical bone. In addition, this study indicates that the age‐related changes in cortical porosity relate predominantly to Haversian canals, whereas the porosity of osteocytic lacunae appears to be independent of age.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Induction of CD‐RAP mRNA during periosteal chondrogenesis
Authors: Sanyal Arunik , Clemens Victoria , Fitzsimmons James S. , Reinholz Gregory G. , Sarkar Gobinda , Mukherjee Nilay , O'Driscoll Shawn W.
Published: February 2003

Induction of chondrogenesis and maintenance of the chondrocyte phenotype are critical events for autologous periosteal transplantation, which is a viable approach for cartilage repair. Cartilage‐derived retinoic acid‐sensitive protein (CD‐RAP) is a recently discovered protein that is mainly produced in cartilage. During development, CD‐RAP expression starts at the beginning of chondrogenesis and continues throughout cartilage maturation. In order to investigate the involvement of CD‐RAP during periosteal chondrogenesis we have determined the nucleotide sequence of the rabbit CD‐RAP mRNA and utilized this information to evaluate the temporal and spatial expression pattern of CD‐RAP at the mRNA level during chondrogenesis. When the periosteal explants were cultured under chondrogenic conditions, the expression of CD‐RAP was induced, as shown by a 40‐fold increase in CD‐RAP mRNA between days 7 and 10. The temporal expression pattern of CD‐RAP closely mimicked that of collagen type IIB mRNA. Also, the CD‐RAP mRNA was localized to the matrix forming chondrocytes in the cambium layer of the periosteum by in situ hybridization as indicated by colocalization with collagen type II mRNA and positive safranin O staining. These data suggest a regulatory role of CD‐RAP in periosteal chondrogenesis, which is potentially important for both cartilage repair and fracture healing via callus formation.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Effects of locally applied vascular endothelial growth factor (VEGF) and VEGF‐inhibitor to the rabbit tibia during distraction osteogenesis
Authors: Eckardt Henrik , Bundgaard Kristian G. , Christensen Knud S. , Lind Martin , Hansen Ebbe S. , Hvid Ivan
Published: February 2003

Introduction: Therapeutic angiogenesis, a novel concept in tissue engineering, is neo‐formation of blood vessels in a tissue upon delivery of an angiogenic growth factor to the tissue. We hypothesised that therapeutic angiogenesis could enhance bone formation and challenged the hypothesis in an experimental model of distraction osteogenesis.

Methods: Rabbits, divided into three equal groups of 12, had their right tibia lengthened by distraction osteogenesis. A miniosmotic pump delivered to the osteotomy gap either recombinant human vascular endothelial growth factor (VEGF), VEGF‐inhibitor, or vehicle alone during the latency and distraction phase. After consolidation, we assessed bone blood flow by radioactive microsphere entrapment, measured torsional stiffness and bone mineral content, and did histomorphometry.

Results: VEGF and VEGF‐inhibitor treatment failed to influence bone blood flow, torsional stiffness, bone mineral content and histomorphometric indices of the bone regenerate. However, VEGF treatment increased the blood flow in bone of the distracted limb and VEGF‐inhibitor treatment decreased bone blood flow.

Conclusion: The regenerate was unresponsive to VEGF and VEGF‐inhibitor treatment in contrast to the neighbouring bone, which implies different biological properties of the vasculature in native and regenerating bone. VEGF is not recommended for enhancement of bone formation in this setting.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
The relationship between knee strength and functional stability before and after anterior cruciate ligament reconstruction
Authors: Keays S. L. , Bullock‐Saxton J. E. , Newcombe P. , Keays A. C.
Published: February 2003

Functional stability of the knee is dependent on an intact ligamentous system and the timely and efficient contraction of supporting musculature. The aim of this study was to assess the relationship between muscle strength and functional stability in 31 patients pre‐ and post‐operatively, following a unilateral anterior cruciate ligament rupture. All subjects underwent reconstructive surgery using semitendonosis and gracilis tendons. Isokinetic strength assessment of quadriceps and hamstring muscles was performed at a rate of movement of 60°/s and 120°/s. Functional stability was determined by performance during five functional stability tests that included the shuttle run, side step, carioca, single and triple hop tests. Pearson's correlation coefficient statistics were applied to pre‐operative and post‐operative data respectively. These analyses demonstrated a significant positive correlation between quadriceps strength indices at both testing speeds and the two hop tests pre‐operatively (p's<0.007) and between quadriceps strength indices at both speeds and all five functional tests post‐operatively (p's<0.01). Assessed using Steiger's formula, there was a significant increase in the correlation between quadriceps strength indices and three functional tests post‐operatively compared to pre‐operatively (p<0.05). No significant correlation between hamstring strength indices and functional scores existed pre‐ or post‐operatively. This study has shown a significant correlation exists between quadriceps strength indices and functional stability both before and after surgery, this relationship does not reach significance between hamstring strength indices and functional stability.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Characterization of proliferation and differentiation of EGF‐responsive striatal and septal precursor cells
Authors: Castillo Lazara , Martı́nez Lisis , Grygar Elizabeth , Hutter‐Paier Birgit
Published: February 2003

The epigenetic manipulation of precursors may provide data to elucidate the potential interactions among these cells in different brain regions. However, the response to epigenetic signals is modulated by the environment in which the cells are manipulated. Therefore, data regarding the action of a particular factor must be considered in the light of a specific system. To compare septal and striatal precursors, we have tested the effect of nerve growth factor (NGF) on the proliferation and neuronal differentiation of epidermal growth factor (EGF)‐responsive cells from these brain regions. Precursors were cultivated as ‘neuropheres’ in serum free medium (SFM) to which NGF was added. NGF did not support the proliferation of EGF‐generated precursors so that no differences in the cell magnitude with respect to control cultures were observed. Differentiation of precursors in SFM plus 1% fetal bovine serum (FBS) on poly‐d‐lysine showed that the neuron number was increased two‐fold in septal cultures treated with NGF but not in those from striatum. A quantitative evaluation of the soma surface and the number of primary neurites showed differences between both populations of precursor‐generated neurons. In addition, we also observed no influence of NGF on these parameters of cellular morphology. Thus, taken together these results seem to indicate that at this developmental stage in which these populations of precursors were isolated, heterogeneities exist between them, which is probably related to their origin and/or functional roles in vivo.
GABAAβ2–3 immunoreactive cells in the developing chick retina
Authors: Barros Pedro Henrique Oliveira Cavalcanti , Calaza Karin da Costa , Gardino Patrı́cia Franca
Published: February 2003

Gama‐aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system (CNS). It has been shown that GABA is an important factor for CNS maturation and that its functions are mainly mediated by GABAA receptors. Thus, in order to fully comprehend the role of GABA during development, it is essential to establish the developmental features of the catalytic subunits (β) of GABAA receptor. Here, we determine the ontogenesis and neurogenesis of cells expressing β2–3 subunits of GABAA receptor (GABAAβ2–3) in the chick retina. In the ontogenetic experiments, only the immunohistochemistry for GABAAβ2–3 approach was employed. For neurogenesis a double‐labeling method (autoradiography and immunohistochemistry) was applied. [H3]‐thymidine was injected into eggs (2–11 days) and the embryos were sacrificed at embryonic day 19 (E19). GABAAβ2–3 immunohistochemistry was processed and then autoradiography was performed. We used a cumulative counting method to quantify the autoradiographic grains. The ontogenesis study revealed that at E9, GABAAβ2–3 immunoreactivity was restricted to the inner plexiform layer and the first cell bodies immunoreactive to GABAAβ2–3 were seen at E14. Thereafter, the number of cell bodies and the intensity of GABAAβ2–3 immunoreactivity increased until the adult pattern was established. The neurogenesis study showed that cells that will express GABAAβ2–3 were generated between E6 and E9. In addition, from E7 to E9 the rate of neurogenesis of GABAAβ2–3 immunoreactive cells quickly increases. Therefore, the detection of GABAAβ2–3 occurred only after the end of generation period of this cell population.
Corn feeding during development induces changes in the number of serotonergic neurons in the raphe nuclei
Authors: Orozco‐Suárez Sandra , Del Angel Alma Rosa , Beas‐Zárate Carlos , Manjarrrez Gabriel , Feria‐Velasco Alfredo
Published: February 2003

Serotonin (5‐HT) plays a trophic role during brain development; chronic changes in cerebral concentration of this neurotransmitter during the critical stage of development can produce severe damage in the formation of the neural circuits. For the present work a hypoproteic (HYP) diet based on corn (CORN) meal which is deficient in tryptophan (TRY) was given to rats before and during pregnancy, which continued to the offspring until they reached 60 days of age. An isocaloric but hypoproteic diet containing normal amount of TRY, and normal chow (Ch) Purina were given with the same scheme to two groups of rats considered as controls. 5‐HT immunohistochemistry was revealed by avidin–biotin complex (ABC) method to quantify serotonergic nerve cells in the nine raphe nuclei. The number of cells immunoreactive to 5‐HT immunoreactive (5‐HTir) were quantified by means of stereological analysis. Results demonstrated a significant variation in 5‐HT expression in the raphe nuclei. Thus, a significant reduction in the number of 5‐HTir cells in the rostral raphe nuclei was seen at all ages studied in the animals fed the corn diet, compared to data obtained from the control groups. This decrease was more evident between the postnatal ages of 30 and 60 days. It is concluded that the variations in the available TRY affect the brain cells producing 5‐HT and the innervation of their target areas.
NEURORECEPTOR mRNA EXPRESSION IN THE RAT MESENTERIC ARTERY DEVELOPS INDEPENDENTLY OF INNERVATION
Authors: Phillips Jacqueline K. , Hill Caryl E.
Published: February 2003

Studies in autonomic targets have shown that nerves may be required for the development and maintenance of postsynaptic receptor populations. We have examined this relationship in the rat mesenteric artery, assessing mRNA expression levels for a range of neuroreceptors after neonatal sympathectomy, using 6‐hydroxydopamine or antisera directed against nerve growth factor, and sensory denervation, using capsaicin. Total RNA was extracted from 28 day old rats and reverse transcription‐polymerase chain reaction was performed, using primers specific for the α1(A,B,D)‐ and α2(A,B,C)‐adrenergic, neurokinin (NK1–NK3), muscarinic (M1–M5) and P2X purinergic (P2X1–7) receptor families. Results showed no decreases in mRNA expression of any of the specific receptor subtypes after either sympathetic or sensory denervation. Small increases in mRNA expression were detected following sensory denervation for some of the receptor subtypes. We conclude that neither sympathetic nor sensory nerves are mandatory for the expression of mRNA of a range of neuroreceptors in the mesenteric vascular bed of the rat.
Fetal human cortical neurons grown in culture: morphological differentiation, biochemical correlates and development of electrical activity
Authors: Kerkovich Danielle M. , Sapp Douglas , Weidenheim Karen , Brosnan Celia F. , Pfeiffer Steven E. , Yeh Hermes H. , Busciglio Jorge
Published: February 2003

Cultured fetal human cortical neurons derived from second trimester human fetal cortex were analyzed with regard to their morphological differentiation and expression of cell‐specific markers. The culture method was adapted from standardized protocols originally developed for the isolation and culture of rodent oligodendrocytes and astrocytes. This technique takes advantage of the different adhesive properties and stratification of central nervous system cells in vitro. Under these culture conditions fetal human cortical neurons underwent morphological differentiation, expressed neuron‐specific markers and voltage‐ and ligand‐gated ion channels. Highly enriched cultures of microglia and astrocytes generated from the same starting material also expressed cell‐type specific markers. These cultures serve as a valuable tool for the establishment of normative data and as experimental models for neurodevelopmental and neurodegenerative studies.
Development and testing of the European Heart Failure Self‐Care Behaviour Scale
Authors: Jaarsma T. , Strömberg A. , Mårtensson J. , Dracup K.
Published: February 2003

Background:

Improvement of self‐care behaviour is an aim of several non‐pharmacological nurse‐led management programmes for patients with heart failure. These programmes are often evaluated based on their effects on readmission, costs and quality of life. It is, however, also important to know how patients changed their self‐care behaviour as a result of such a programme. Therefore a comprehensive, reliable and valid measure of the self‐care behaviour of HF patients is needed.

Objectives:

To develop a scale measuring the behaviour that heart failure patients perform to maintain life, healthy functioning, and well‐being.

Method:

The European Heart Failure Self‐Care Behaviour Scale (EHFScBS) was developed in three phases: (1) concept analysis and first construction; (2) revision of items and response and scoring format; and (3) testing of the new scale for validity and reliability.

Results:

The European Heart Failure Self‐Care Behaviour Scale is a 12‐item, self‐administered questionnaire that covers items concerning self‐care behaviour of patients with heart failure. Face‐validity and concurrent validity was established and the internal consistency of the scale was tested using pooled data of 442 patients from two centres in Sweden, three in the Netherlands and one in Italy. Cronbachs's alpha was 0.81.

Conclusion:

The instrument is a valid, reliable and practical scale to measure the self‐reported self‐care behaviour of heart failure patients. It is ready to use by investigators evaluating the outcome of heart failure management programmes that target changes in patients’ self‐care practices.
Structure of the large FK506-binding protein FKBP51, an Hsp90-binding protein and a component of steroid receptor complexes
Authors: Sinars Cindy R. , Cheung-Flynn Joyce , Rimerman Ronald A. , Scammell Jonathan G. , Smith David F. , Clardy Jon
Published: February 2003

The ability to bind immunosuppressive drugs such as cyclosporin and FK506 defines the immunophilin family of proteins, and the FK506-binding proteins form the FKBP subfamily of immunophilins. Some FKBPs, notably FKBP12 (the 12-kDa FK506-binding protein), have defined roles in regulating ion channels or cell signaling, and well established structures. Other FKBPs, especially the larger ones, participate in important biological processes, but their exact roles and the structural bases for these roles are poorly defined. FKBP51 (the 51-kDa FKBP) associates with heat shock protein 90 (Hsp90) and appears in functionally mature steroid receptor complexes. In New World monkeys, FKBP51 has been implicated in cortisol resistance. We report here the x-ray structures of human FKBP51, to 2.7 Å, and squirrel monkey FKBP51, to 2.8 Å, by using multiwavelength anomalous dispersion phasing. FKBP51 is composed of three domains: two consecutive FKBP domains and a three-unit repeat of the TPR (tetratricopeptide repeat) domain. This structure of a multi-FKBP domain protein clarifies the arrangement of these domains and their possible interactions with other proteins. The two FKBP domains differ by an insertion in the second that affects the formation of the progesterone receptor complex.
Stabilization of perfect and imperfect tandem repeats by single-strand DNA exonucleases
Authors: Feschenko Vladimir V. , Rajman Luis A. , Lovett Susan T.
Published: February 2003

Rearrangements between tandemly repeated DNA sequences are a common source of genetic instability. Such rearrangements underlie several human genetic diseases. In many organisms, the mismatch-repair (MMR) system functions to stabilize repeats when the repeat unit is short or when sequence imperfections are present between the repeats. We show here that the action of single-stranded DNA (ssDNA) exonucleases plays an additional, important role in stabilizing tandem repeats, independent of their role in MMR. For perfect repeats of ≈100 bp in
Escherichia coli
that are not susceptible to MMR, exonuclease (Exo)-I, ExoX, and RecJ exonuclease redundantly inhibit deletion. Our data suggest that >90% of potential deletion events are avoided by the combined action of these three exonucleases. Imperfect tandem repeats, less prone to rearrangements, are stabilized by both the MMR-pathway and ssDNA-specific exonucleases. For 100-bp repeats containing four mispairs, ExoI alone aborts most deletion events, even in the presence of a functional MMR system. By genetic analysis, we show that the inhibitory effect of ssDNA exonucleases on deletion formation is independent of the MutS and UvrD proteins. Exonuclease degradation of DNA displaced during the deletion process may abort slipped misalignment. Exonuclease action is therefore a significant force in genetic stabilization of many forms of repetitive DNA.
Genetic diversity, asymmetrical aggression, and recognition in a widespread invasive species
Authors: Tsutsui Neil D. , Suarez Andrew V. , Grosberg Richard K.
Published: February 2003

The evolution and persistence of cooperative social units depends on the ability to distinguish group members from nonmembers. The precision of discrimination, in turn, relies on variation in the labels that individuals use to recognize group members. However, this same variation can be selected against if individuals that are rejected as nonmembers incur a high cost. Here we provide evidence that selection against individuals from genetically diverse groups has contributed to the formation of the unicolonial colony structure that characterizes introduced populations of the invasive Argentine ant (Linepithema humile). Studies in both the laboratory and the field showed that individuals from less genetically diverse colonies attack individuals from more diverse colonies and that attackers survived agonistic encounters more than six times as often as recipients of aggression. This selection, in concert with reductions in genetic diversity after a founder event, likely creates a barrier to the establishment of new, genetically diverse introductions from the native range and may reduce genetic diversity within established populations in the introduced range.
Hysteresis drives cell-cycle transitions in
Xenopus laevis
egg extracts
Authors: Sha Wei , Moore Jonathan , Chen Katherine , Lassaletta Antonio D. , Yi Chung-Seon , Tyson John J. , Sible Jill C.
Published: February 2003

Cells progressing through the cell cycle must commit irreversibly to mitosis without slipping back to interphase before properly segregating their chromosomes. A mathematical model of cell-cycle progression in cell-free egg extracts from frog predicts that irreversible transitions into and out of mitosis are driven by hysteresis in the molecular control system. Hysteresis refers to toggle-like switching behavior in a dynamical system. In the mathematical model, the toggle switch is created by positive feedback in the phosphorylation reactions controlling the activity of Cdc2, a protein kinase bound to its regulatory subunit, cyclin B. To determine whether hysteresis underlies entry into and exit from mitosis in cell-free egg extracts, we tested three predictions of the Novak–Tyson model. (
i
) The minimal concentration of cyclin B necessary to drive an interphase extract into mitosis is distinctly higher than the minimal concentration necessary to hold a mitotic extract in mitosis, evidence for hysteresis. (
ii
) Unreplicated DNA elevates the cyclin threshold for Cdc2 activation, indication that checkpoints operate by enlarging the hysteresis loop. (
iii
) A dramatic “slowing down” in the rate of Cdc2 activation is detected at concentrations of cyclin B marginally above the activation threshold. All three predictions were validated. These observations confirm hysteresis as the driving force for cell-cycle transitions into and out of mitosis.
Structural evidence for substrate strain in antibody catalysis
Authors: Yin Jun , Andryski Scott E. , Beuscher Albert E. , Stevens Raymond C. , Schultz Peter G.
Published: February 2003

The crystal structure of the Michaelis complex between the Fab fragment of ferrochelatase antibody 7G12 and its substrate mesoporphyrin has been solved to 2.6-Å resolution. The antibody-bound mesoporphyrin clearly adopts a nonplanar conformation and reveals that the antibody catalyzes the porphyrin metallation reaction by straining/distorting the bound substrate toward the transition-state configuration. The crystal structures of the Fab fragment of the germ-line precursor antibody to 7G12 and its complex with the hapten
N
-methylmesoporphyrin have also been solved. A comparison of these structures with the corresponding structures of the affinity-matured antibody 7G12 reveals the molecular mechanism by which the immune system evolves binding energy to catalyze this reaction.
Sex pheromone biosynthetic pathway for disparlure in the gypsy moth,
Lymantria dispar
Authors: Jurenka Russell A. , Subchev Mitko , Abad José-Luis , Choi Man-Yeon , Fabriàs Gemma
Published: February 2003

The pheromone biosynthetic pathway for production of the sex pheromone disparlure, 2-methyl-7
R
,8
S
-epoxy-octadecane, was determined for the gypsy moth. Each step in the pathway was followed by using deuterium-labeled compounds that could be identified by using GC/MS. This approach provides unequivocal determination of specific reactions in the pathway. It was shown that the alkene precursor, 2-methyl-
Z
7-octadecene, is most likely made in oenocyte cells associated with abdominal epidermal cells. The pathway begins with valine contributing carbons for chain initiation, including the methyl-branched carbon, followed by chain elongation to 19 carbons. The double bond is introduced with an unusual Δ12 desaturase that utilizes a methyl-branched substrate. The resulting 18-methyl-
Z
12-nonadecenoate is decarboxylated to the hydrocarbon, 2-methyl-
Z
7-octadecene. The alkene is then transported to the pheromone gland through the hemolymph, most probably by lipophorin. At the pheromone gland, the alkene is unloaded and transformed into the epoxide disparlure for release into the environment. A chiral HPLC column was used to demonstrate that the (
R
,
S
)-stereoisomer of the epoxide, (+)-disparlure is found in pheromone glands.
A photochemical approach to the lipid accessibility of engineered cysteinyl residues
Authors: Li Jing , Shi Lei , Karlin Arthur
Published: February 2003

Ordinary electrophilic reagents react too slowly in a nonpolar environment to be useful for the determination of the accessibility to lipid of continuous stretches of residues mutated to cysteine. By contrast, photoactivated 5-iodonaphthyl-1-azide (INA) reacted readily with 2-mercaptoethanol and dodecanethiol in nonpolar solvents and in liposomes. Continuous stretches of residues in the amphipathic N-terminal helix and first transmembrane helix of the bacterial potassium channel KcsA were replaced with cysteine, and the mutants were expressed in
Escherichia coli
and isolated in inner membranes. These membranes were dissolved in detergent and reconstituted into asolectin liposomes incorporating INA. The extent of light-induced reaction of INA with each cysteine was assayed by subsequent reaction with the gel-shifting, SH-specific methoxy-polyethylene glycol-2-pyridine disulfide. The pattern of apparent second-order rate constants for the photoreactions of eight substituted cysteines in the N-terminal helix conformed to other measures of lipid exposure. The pattern of the rate constants for the photoreactions of 15 cysteines in the first transmembrane helix had peaks every third residue, which partly conformed to other measures of lipid exposure.
Hox gene clusters in the Indonesian coelacanth,
Latimeria
menadoensis
Authors: Koh Esther G. L. , Lam Kevin , Christoffels Alan , Erdmann Mark V. , Brenner Sydney , Venkatesh Byrappa
Published: February 2003

The Hox genes encode transcription factors that play a key role in specifying body plans of metazoans. They are organized into clusters that contain up to 13 paralogue group members. The complex morphology of vertebrates has been attributed to the duplication of Hox clusters during vertebrate evolution. In contrast to the single Hox cluster in the amphioxus (
Branchiostoma floridae
), an invertebrate-chordate, mammals have four clusters containing 39 Hox genes. Ray-finned fishes (Actinopterygii) such as zebrafish and fugu possess more than four Hox clusters. The coelacanth occupies a basal phylogenetic position among lobe-finned fishes (Sarcopterygii), which gave rise to the tetrapod lineage. The lobe fins of sarcopterygians are considered to be the evolutionary precursors of tetrapod limbs. Thus, the characterization of Hox genes in the coelacanth should provide insights into the origin of tetrapod limbs. We have cloned the complete second exon of 33 Hox genes from the Indonesian coelacanth,
Latimeria menadoensis
, by extensive PCR survey and genome walking. Phylogenetic analysis shows that 32 of these genes have orthologs in the four mammalian HOX clusters, including three genes (
HoxA6
,
D1
, and
D8
) that are absent in ray-finned fishes. The remaining coelacanth gene is an ortholog of
hoxc1
found in zebrafish but absent in mammals. Our results suggest that coelacanths have four Hox clusters bearing a gene complement more similar to mammals than to ray-finned fishes, but with an additional gene,
HoxC1
, which has been lost during the evolution of mammals from lobe-finned fishes.
Optimized expression and specific activity of IL-12 by directed molecular evolution
Authors: Leong Steven R. , Chang Jean C. C. , Ong Randal , Dawes Glenn , Stemmer Willem P. C. , Punnonen Juha
Published: February 2003

DNA delivery of IL-12 has shown promise in reducing the toxic side effects associated with administration of recombinant human (h)IL-12 protein while maintaining the ability to inhibit tumor growth and abolish tumor metastases in animal models. We have developed a more potent version of IL-12 by using DNA shuffling and screening to improve its expression in human cells and specific activity on human T cells. The most improved evolved IL-12 (EvIL-12) derived from seven mammalian genes encoding both the p35 and p40 subunits of IL-12 showed a 128-fold improvement in human T cell proliferation compared with native hIL-12 during the initial screening of supernatants from transected cells. When purified hIL-12 and EvIL-12 proteins were comparedin vitroin human T cell proliferation and Th1 differentiation assays, it was demonstrated that EvIL-12 exhibited a concomitant 10-fold increase in the specific activity of the protein compared with hIL-12. Furthermore, DNA shuffling improved the level of expression and homogeneity of the heterodimer synthesized by 293 human embryonic kidney cells transfected with EvIL-12 by at least 10-fold. Molecular analysis of the variant revealed strategic placement of amino acid substitutions that potentially may facilitate heterodimer formation and product expression. The enhanced expression and biological activity of EvIL-12 may improve the effectiveness of IL-12 gene-based vaccines and therapeutics without the toxic side effects sometimes associated with hIL-12 protein administration.
Modular organization of cellular networks
Authors: Rives Alexander W. , Galitski Timothy
Published: February 2003

We investigated the organization of interacting proteins and protein complexes into networks of modules. A network-clustering method was developed to identify modules. This method of network-structure determination was validated by clustering known signaling-protein modules and by identifying module rudiments in exclusively high-throughput protein-interaction data with high error frequencies and low coverage. The signaling network controlling the yeast developmental transition to a filamentous form was clustered. Abstraction of a modular network-structure model identified module-organizer proteins and module-connector proteins. The functions of these proteins suggest that they are important for module function and intermodule communication.
Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells
Authors: Blyszczuk Przemyslaw , Czyz Jaroslaw , Kania Gabriela , Wagner Martin , Roll Ursula , St-Onge Luc , Wobus Anna M.
Published: February 2003

Mouse embryonic stem (ES) cells differentiate into cells of all three primary germ layers including endodermal cells that produce insulin
in vitro
. We show that constitutive expression of Pax4 (Pax4
+
), and to a lesser extent Pdx1 (Pdx1
+
), affects the differentiation of ES cells and significantly promote the development of insulin-producing cells. In Pax4 overexpressing R1 ES cells, isl-1, ngn3, insulin, islet amyloid polypeptide, and glucose transporter 2 (Glut-2) mRNA levels increase significantly. The number of nestin-expressing (nestin+) cells also increases. Constitutive Pax4 expression combined with selection of nestin+ cells and histotypic culture conditions give rise to spheroids containing insulin-positive granules typical of embryonal and adult β cells. In response to glucose, Pax4
+
and wild-type ES-derived cells release insulin. Transplantation of these cells into streptozotocin-treated diabetic mice results in a normalization of blood glucose levels. We conclude that constitutive expression of Pax4 in combination with histotypic cultivation facilitates ES cell differentiation into the pancreatic lineage, which leads to the formation of islet-like spheroid structures that produce increased levels of insulin.
CTXΦ-independent production of the RS1 satellite phage by
Vibrio
cholerae
Authors: Faruque Shah M. , Kamruzzaman M. , Asadulghani  , Sack David A. , Mekalanos John J. , Nair G. Balakrish
Published: February 2003

The cholera toxin genes of
Vibrio cholerae
are encoded by the filamentous phage, CTXΦ. Chromosomal CTXΦ prophage DNA is often found flanked by copies of a related genetic element designated RS1, and RS1 DNA can be packaged into filamentous phage particles (designated RS1Φ) by using the CTXΦ morphogenesis genes. RS1Φ is a satellite phage that further controls expression and dissemination of CTXΦ. Here we describe a CTXΦ-independent mechanism for production of RS1Φ. A nontoxigenic environmental
V. cholerae
strain (55V71) was identified that supports production of RS1Φ. However, newly infected CTX-negative strains did not produce RS1Φ, indicating that additional 55V71 genes were involved in production of RS1Φ. Analysis of nucleic acids from phage preparations of 55V71 revealed a 7.5-kb single-stranded DNA, whose corresponding replicative form was found in plasmid preparations. This DNA likely corresponds to the genome of a new filamentous phage, which we have designated KSF-1Φ. The replicative form DNA of KSF-1Φ was cloned into pUC18, and the resulting construct pKSF-1.1 supported the production of RS1Φ particles by CTX-negative
V. cholerae
strains. RS1Φ particles produced in this way infect recipient
V. cholerae
strains by a mechanism that is independent of the CTXΦ receptor, the toxin-coregulated pilus. Thus, KSF-1Φ is capable of facilitating the transfer of the RS1 element to strains that do not express toxin coregulated pilus. Given that RS1Φ can enhance coproduction of CTXΦ particles, KSF-1Φ-mediated dissemination of RS1 may indirectly promote the spread of toxin genes among
V. cholerae
strains. This study also shows that filamentous phages can package diverse DNA elements and thus may play a role in horizontal transfer of more genes than previously appreciated.
Bcl-x
L
overexpression blocks bax-mediated mitochondrial contact site formation and apoptosis in rod photoreceptors of lead-exposed mice
Authors: He Lihua , Perkins Guy A. , Poblenz Ann T. , Harris Jeffrey B. , Hung Michael , Ellisman Mark H. , Fox Donald A.
Published: February 2003

Photoreceptor apoptosis and resultant visual deficits occur in humans and animals with inherited and disease-, injury-, and chemical-induced retinal degeneration. A clinically relevant mouse model of progressive rod photoreceptor-selective apoptosis was produced by low-level developmental lead exposure and studied in combination with transgenic mice overexpressing Bcl-x
L
only in the photoreceptors. A multiparametric analysis of rod apoptosis and mitochondrial structure-function was performed. Mitochondrial cristae topography and connectivity, matrix volume, and contact sites were examined by using 3D electron tomography. Lead-induced rod-selective apoptosis was accompanied by rod Ca
2+
overload, rhodopsin loss, translocation of Bax from the cytosol to the mitochondria, decreased rod mitochondrial respiration and membrane potential, mitochondrial cytochrome
c
release, caspase-3 activation, and an increase in the number of mitochondrial contact sites. These effects occurred without mitochondrial matrix swelling, outer membrane rupture, caspase-8 activation, or Bid cleavage. Bcl-x
L
overexpression completely blocked all apoptotic events, except Ca
2+
overload, and maintained normal rod mitochondrial function throughout adulthood. This study presents images of mitochondrial contact sites in an
in vivo
apoptosis model and shows that Bcl-x
L
overexpression blocks increased contact sites and apoptosis. These findings extend our
in vitro
retinal studies with Pb
2+
and Ca
2+
and suggest that developmental lead exposure produced rod-selective apoptosis without mitochondrial swelling by translocating cytosolic Bax to the mitochondria, which likely sensitized the Pb
2+
and Ca
2+
overloaded rod mitochondria to release cytochrome
c
. These results have relevance for therapies in a wide variety of progressive retinal and neuronal degenerations where Ca
2+
overload, lead exposure, and/or mitochondrial dysfunction occur.