Functional analysis of phosphorylation of the mitotic centromere-associated kinesin by Aurora B kinase in human tumor cells

Mitotic centromere-associated kinesin (MCAK) is the best characterized member of the kinesin-13 family and plays important roles in microtubule dynamics during mitosis. Its activity and subcellular localization is tightly regulated by an orchestra of mitotic kinases, such as Aurora B. It is well known that serine 196 of MCAK is the major phosphorylation site of Aurora B in Xenopus leavis extracts and that this phosphorylation regulates its catalytic activity and subcellular localization. In the current study, we have addressed the conserved phosphorylation site serine 192 in human MCAK to characterize its function in more depth in human cancer cells. Our data confirm that S192 is the major phosphorylation site of Aurora B in human MCAK and that this phosphorylation has crucial roles in regulating its catalytic activity and localization at the kinetochore/centromere region in mitosis. Interfering with this phosphorylation leads to a delayed progression through prometa- and metaphase associated with mitotic defects in chromosome alignment and segregation. We show further that MCAK is involved in directional migration and invasion of tumor cells, and interestingly, interference with the S192 phosphorylation affects this capability of MCAK. These data provide the first molecular explanation for clinical observation, where an overexpression of MCAK was associated with lymphatic invasion and lymph node metastasis in gastric and colorectal cancer patients.     

Effect of bioadctive glass templates on osteoblast proliferation and in vitro synthesis of bone-like tissue

Using in vitro synthesifzed bone tissue with cells aspirated fpom the patient's marrow is an appealing idea to avoid the profound limitations of biological of biologiaal and synthetic grafts. Procedures to synthesize bone tiqsue on vitro primapily relied on seeding various subqtpates with cellq that have osteogenia capacity in culture. It should be noted that in an in vitro system, msteoppogenitor cells, as well as bone themselves an papidiy change their phenotype, hence the substrate needs to promote the expression or the bone cell Phenotype. Furthermore, it needs to provide a template for bone deposition while gradually resorbing once bone tissue has been laid down. This paper presents initial evidence that optimally combines the requirements of the ideal template for in vitro synthesis of bone tissue. When made in popous dorm, and conditioned to detelop a bone-like surface prior to being seeded with pluripoteltial cells capable of expressing the osteoblastic phenotype, these templates lead to expeditious and a undalt in vitro synthesis of extracellular matrix with most important characteristics of bone tissue.     

Brain-derived neurotrophic factor (BDNF) mediates bone morphogenetic protein-2 (BMP-2) effects on cultured striatal neurones

Bone morphogenetic proteins are members of the transforming growth factor-beta superfamily that have multiple functions in the developing nervous system. One of them, bone morphogenetic protein-2 (BMP-2), promotes the differentiation of cultured striatal neurones, enhancing dendrite growth and calbindin-positive phenotype. Bone morphogenetic proteins have been implicated in cooperative interactions with other neurotrophic factors. Here we examined whether the effects of BMP-2 on cultured striatal neurones are mediated or enhanced by other neurotrophic factors. BMP-2 had a cooperative effect with low doses of brain-derived neurotrophic factor or neurotrophin-3 (but not with other neurotrophic factors such as glial cell line-derived neurotrophic factor, neurturin or transforming growth factor-beta 2) on the number of calbindin-positive striatal neurones. Moreover, BMP-2 induced phosphorylated Trk immunoreactivity in cultured striatal neurones, suggesting that neurotrophins are involved in BMP-2 neurotrophic effects. The addition of TrkB-IgG or antibodies against brain-derived neurotrophic factor abolished the effects of BMP-2 on the number and degree of differentiation of calbindin-positive striatal neurones. Indeed, BMP-2 treatment increased brain-derived neurotrophic factor protein levels in treated cultures media and BDNF immunocytochemistry revealed that this neurotrophin was produced by neuronal cells. Taken together, these results indicate that brain-derived neurotrophic factor mediates the effects of BMP-2 on striatal neurones.     

Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification,Distraction Osteogenesis,or Healing of Critical Sized Defects

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens⁴. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice ^5-7, after application of bioactive molecules ^8,9, after altering physiological parameters of healing ¹⁰, after modifying the amount or time of stabilization ¹¹, after distraction osteogenesis ¹², after creation of a non-union ¹³, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice.     

Quantitative Analysis of the Sex Pheromone of Several Phycitid Moths by Electron-capture Gas Chromatography

β-Phenetyl alcohol and procaine hydrochloride are known to alter membrane structure. Their effects on the syntheses of tyramine oxidase and arylsulfatase were studied in Klebsiella aerogenes. β-Phenetyl alcohol inhibited the syntheses of membrane-bound tyramine oxidase and arylsulfatase, located in the periplasm, under non-repressing and derepressing conditions, but did not affect the syntheses of β-galactosidase and histidase, which are located internally. In contrast, procaine hydrochloride stimulated the synthesis of tyramine oxidase and derepressed the synthesis of arylsulfatase, but inhibited non-repressed synthesis of arylsulfatase. Thus, derepressed synthesis of cellular arylsulfatase was affected by the level of tyramine oxidase synthesis. Structural alterations in the cell membrane seem to impair the formation of active-arylsulfatase protein in the periplasmic space.     

Validation of density–elasticity relationships for finite element modeling of human pelvic bone by modal analysis

In total hip arthroplasty and particularly in revision surgery, computer assisted pre-operative prediction of the best possible anchorage strategy for implant fixation would be a great help to the surgeon. Computer simulation relies on validated numerical models. In the current study, three density–elasticity relationships (No. 1–3) from the literature for inhomogeneous material parameter assignment from CT data in automated finite element (FE) modeling of long bones were evaluated for their suitability for FE modeling of human pelvic bone. Numerical modal analysis was conducted on 10 FE models of hemipelvic bone specimens and compared to the gold standard provided by experimental modal analysis results from a previous in-vitro study on the same specimens. Overall, calculated resonance frequencies came out lower than measured values. Magnitude of mean relative deviation of numerical resonance frequencies with regard to measured values is lowest for the density–elasticity relationship No. 3 (−15.9%) and considerably higher for both density–elasticity relationships No. 1 (−41.1%) and No. 2 (−45.0%). Mean MAC values over all specimens amount to 77.8% (No. 1), 78.5% (No. 2), and 83.0% (No. 3). MAC results show, that mode shapes are only slightly influenced by material distribution. Calculated resonance frequencies are generally lower than measured values, which indicates, that numerical models lack stiffness. Even when using the best suited (No. 3) out of three investigated density–elasticity relationships, in FE modeling of pelvic bone a considerable underestimation of model stiffness has to be taken into account.     

Osteoblasts and collagen orientation

Summary Bone was removed from the calvaria of anaesthetized 70 g rats or freshly killed young monkeys and the fibrous periosteum dissected off the inner, formative surface under 0.15 M cacodylate buffer. The bone and undisturbed osteoblasts were fixed in 3% glutaraldehyde in the same buffer for 24 to 48 hours, critical point dried and coated with evaporated carbon and gold for scanning electron microscopy (SEM). Fields of osteoblasts were photographed and chosen cells dissected off the osteoid using a tungsten needle. The control of the dissection was made possible by the use of a system of real-time stereo TV-speed SEM. The fields were rephotographed and the orientations of the osteoblasts were compared with that of the underlying collagen fibres. 62% of all osteoblasts lay with their long axes within 15° of the collagen fibre orientation below and 80% within 30°. Montages of large areas of osteoblasts were also made, and then compared with ones of the same area after the cells had been stripped off on adhesive tape. In general, the orientation of the collagen tended to be the same as the cell that formed it. Collagen fibres below cells at the periphery of a domain sometimes had the orientation of the cells in the adjacent patch. It is not possible to determine whether the cells controlled the orientation of the collagen, or vice versa, from this experiment, but other SEM evidence suggests that the collagen orientation in hard tissue matrices depends on the freedom of cells to move with respect to the matrix surface. Acknowledgements. This work has been supported by generous grants from the Medical Research Council and the Science Research Council. We are grateful to Elaine Bailey and Mr. P. Reynolds for technical assistance.     

Monostotic fibrous dysplasia in the temporal bone: A late prehistoric occurrence

Fibrous dysplasia, characterized by benign osteolytic and osteoblastic lesions may involve one or several bones. Recent investigators have suggested that it may be merely a phase of what have previously been thought to be several different bone disease. Isolated fibrous dysplasia in the temporal bone is infrequent. Several reports of this disease have appeared in the literature of paleopathology, but none involved only the temporal bone. Monostotic involvement of the right temporal bone was discovered in the skull of an adult male recovered from an archeological site dating from the Late Mississippian period (A. D. 1,350–A. D. 1,650). It will provide an opportunity for preliminary documentation of the antiquity of this disease in the southeastern portion of the United States.     

Suspension osteopenia in mice: Whole body electromagnetic field effects

Whole-body fields were tested for their efficacy in preventing the osteopenia caused by tail suspension in mice. The fields had fundamental frequencies corresponding to the upper range of predicted endogenous impact-generated frequencies (0.25–2.0 kHz) in the long bones. Three distinct whole-body EMFs were applied for 2 weeks on growing mice. Structural, geometric, and material properties of the femora, tibiae, and humeri of suspended mice were altered compared to controls. Comparison of suspended mice and mice subjected to caloric restriction indicates that the changes in caloric intake do not explain either the suspension or the field-induced effects. In agreement with past studies, rather, unloading appears to cause the suspension effects and to be addressed by the EMFs. The EMF effects on bone properties were apparently frequency dependent, with the lower two fundamental frequencies (260 and 910 Hz) altering, albeit slightly, the suspension-induced bone effects. The fields are not apparently optimized for frequency, etc., with respect to therapeutic potential; however, suspension provides a model system for further study of the in vivo effects of EMFs. © 1995 Wiley-Liss, Inc.     

Aquatic Habits and Niche Partitioning in the Extraordinarily Long-Necked Triassic Reptile Tanystropheus

1. Download : Download high-res image (278KB)
2. Download : Download full-size image