Normal Skeletal Development of Mice Lacking Matrilin 1: Redundant Function of Matrilins in Cartilage?

Matrilin 1, or cartilage matrix protein, is a member of a novel family of extracellular matrix proteins. To date, four members of the family have been identified, but their biological role is unknown. Matrilin 1 and matrilin 3 are expressed in cartilage, while matrilin 2 and matrilin 4 are present in many tissues. Here we describe the generation and analysis of mice carrying a null mutation in the Crtm gene encoding matrilin 1. Anatomical and histological studies demonstrated normal development of homozygous mutant mice. Northern blot and biochemical analyses show no compensatory up-regulation of matrilin 2 or 3 in the cartilage of knockout mice. Although matrilin 1 interacts with the collagen II and aggrecan networks of cartilage, suggesting that it may play a role in cartilage tissue organization, studies of collagen extractability indicated that collagen fibril maturation and covalent cross-linking were unaffected by the absence of matrilin 1. Ultrastructural analysis did not reveal any abnormalities of matrix organization. These data suggest that matrilin 1 is not critically required for cartilage structure and function and that matrilin 1 and matrilin 3 may have functionally redundant roles.     

Regeneration of autotransplanted lymph node fragments

Summary In normal young minipigs thin slices of autologous mesenteric or superficial inguinal lymph nodes were implanted either in the greater omentum or subcutaneously in the groin region. The regeneration was studied histologically and connections between the afferent lymphatics and the regenerated tissue were checked. In the greater omentum, no regenerated lymph node tissue was found. In the inguinal region, lymphoid tissue with all the typical lymph node compartments was identified following antigenic stimulation in the draining area. Sinuses, germinal centres with a lymphatic corona, and a paracortex with typical high endothelial venules were seen. There was evidence of afferent lymphatics, e.g., macroscopically visible lymphatics, the occurrence of a subcutaneously injected dye, the effect of antigenic stimulation and a normal lymph node structure. Avascular transplants of autologous lymph node fragments regenerate subcutaneously, possibly providing a future technique for treating lymphoedema after radical excision or irradiation of lymph nodes.     

Bacterial mutagenicity investigation of epoxides: drugs,drug metabolites,steroids and pesticides

Although it has been observed that many epoxides are ultimate mutagens, surprisingly little is known about epoxides to which man may be extensively exposed, e.g., physiological compounds, drugs, drug metabolites and pesticides. We have now investigated 35 such and related epoxides for mutagenicity, using reversion of his^?Salmonella typhimurium TA98 and TA100 as biological end-point. None of the tested steroids (12 compounds), vitamin K epoxides (3 compounds) and pesticides (dieldrin, endrin, HEOM (1,2,3,4,9,9-hexachloro-6,7-epoxy-1,4,4a5,6,7,8,8a-octahydro-1,4-methanonaphthalene), heptachlor epoxide) showed any mutagenic activity. Negative results were also obtained with the antibiotics oleandomycin, anti-capsin and asperlin, the cardiotonic drug resibufogenin, the widely used parasympatholytic drugs butylscopolamine and scopolamine, the sedatives valtratum, didovaltratum and acevaltratum, the tranquilizer oxanamide as well as with the drug metabolites carbamazepine 10,11-oxide and diethylstilbestrol α,β-oxide. Three barbiturate epoxides, formed by metabolism of allobarbital, alphenal and secobarbital, caused weak but reproducible mutagenic effects at high concentrations. The cytostatic agent ethoglucide was the only drug having substantial mutagenic activity. Its mutagenic potency was similar to those of the control epoxides styrene 7,8-oxide, p-bromostyrene 7,8-oxide and m-bromostyrene 7,8-oxide, but much lower than those of benzo[a]pyrene 4,5-oxide, benzo[e]pyrene 4,5-oxide and 7,12-dimethylbenz[a]-anthracene 5,6-oxide.Some epoxides were also tested in other Salmonella typhimurium strains or in the presence of rat-liver S9 mix. Positive results were only obtained with compounds that had already been detected as mutagens in the direct test with strain TA100.     

Effects of Multi-Deficiencies-Diet on Bone Parameters of Peripheral Bone in Ovariectomized Mature Rat

Many postmenopausal women have vitamin D and calcium deficiency. Therefore, vitamin D and calcium supplementation is recommended for all patients with osteopenia and osteoporosis. We used an experimental rat model to test the hypothesis that induction of osteoporosis is more efficiently achieved in peripheral bone through combining ovariectomy with a unique multi-deficiencies diet (vitamin D depletion and deficient calcium, vitamin K and phosphorus). 14-week-old Sprague-Dawley rats served as controls to examine the initial bone status. 11 rats were bilaterally ovariectomized (OVX) and fed with multi-deficiencies diet. Three months later the treated group and the Sham group (n = 8) were euthanized. Bone biomechanical competence of the diaphyseal bone was examined on both, tibia and femur. Image analysis was performed on tibia via µCT, and on femur via histological analysis. Lower torsional stiffness indicated inferior mechanical competence of the tibia in 3 month OVX+Diet. Proximal metaphyseal region of the tibia showed a diminished bone tissue portion to total tissue in the µCT despite the increased total area as evaluated in both µCT and histology. Cortical bone showed higher porosity and smaller cross sectional thickness of the tibial diaphysis in the OVX+Diet rats. A lower ALP positive area and elevated serum level of RANKL exhibited the unbalanced cellular interaction in bone remodeling in the OVX+Diet rat after 3 month of treatment. Interestingly, more adipose tissue area in bone marrow indicated an effect of bone loss similar to that observed in osteoporotic patients. Nonetheless, the presence of osteoid and elevated serum level of PTH, BGP and Opn suggest the development of osteomalacia rather than an osteoporosis. As the treatment and fracture management of both osteoporotic and osteomalacia patients are clinically overlapping, this study provides a preclinical animal model to be utilized in local supplementation of minerals, drugs and growth factors in future fracture healing studies.     

Nonlinearities make a difference: comparison of two common Hill-type models with real muscle

Compared to complex structural Huxley-type models, Hill-type models phenomenologically describe muscle contraction using only few state variables. The Hill-type models dominate in the ever expanding field of musculoskeletal simulations for simplicity and low computational cost. Reasonable parameters are required to gain insight into mechanics of movement. The two most common Hill-type muscle models used contain three components. The series elastic component is connected in series to the contractile component. A parallel elastic component is either connected in parallel to both the contractile and the series elastic component (model [CC+SEC]), or is connected in parallel only with the contractile component (model [CC]). As soon as at least one of the components exhibits substantial nonlinearities, as, e.g., the contractile component by the ability to turn on and off, the two models are mechanically different. We tested which model ([CC+SEC] or [CC]) represents the cat soleus better. Ramp experiments consisting of an isometric and an isokinetic part were performed with an in situ cat soleus preparation using supramaximal nerve stimulation. Hill-type models containing force–length and force–velocity relationship, excitation–contraction coupling and series and parallel elastic force–elongation relations were fitted to the data. To test which model might represent the muscle better, the obtained parameters were compared with experimentally determined parameters. Determined in situations with negligible passive force, the force–velocity relation and the series elastic component relation are independent of the chosen model. In contrast to model [CC+SEC], these relations predicted by model [CC] were in accordance with experimental relations. In conclusion model [CC] seemed to better represent the cat soleus contraction dynamics and should be preferred in the nonlinear regression of muscle parameters and in musculoskeletal modeling.     

Ena/VASP Is Required for neuritogenesis in the developing cortex

Mammalian cortical development involves neuronal migration and neuritogenesis; this latter process forms the structural precursors to axons and dendrites. Elucidating the pathways that regulate the cytoskeleton to drive these processes is fundamental to our understanding of cortical development. Here we show that loss of all three murine Ena/VASP proteins, a family of actin regulatory proteins, causes neuronal ectopias, alters intralayer positioning in the cortical plate, and, surprisingly, blocks axon fiber tract formation during corticogenesis. Cortical fiber tract defects in the absence of Ena/VASP arise from a failure in neurite initiation, a prerequisite for axon formation. Neurite initiation defects in Ena/VASP-deficient neurons are preceded by a failure to form bundled actin filaments and filopodia. These findings provide insight into the regulation of neurite formation and the role of the actin cytoskeleton during cortical development.     

Modulation of the CD95-induced apoptosis: the role of CD95 N-glycosylation

Protein modifications of death receptor pathways play a central role in the regulation of apoptosis. It has been demonstrated that O-glycosylation of TRAIL-receptor (R) is essential for sensitivity and resistance towards TRAIL-mediated apoptosis. In this study we ask whether and how glycosylation of CD95 (Fas/APO-1), another death receptor, influences DISC formation and procaspase-8 activation at the CD95 DISC and thereby the onset of apoptosis. We concentrated on N-glycostructure since O-glycosylation of CD95 was not found. We applied different approaches to analyze the role of CD95 N-glycosylation on the signal transduction: in silico modeling of CD95 DISC, generation of CD95 glycosylation mutants (at N136 and N118), modulation of N-glycosylation by deoxymannojirimycin (DMM) and sialidase from Vibrio cholerae (VCN). We demonstrate that N-deglycosylation of CD95 does not block DISC formation and results only in the reduction of the procaspase-8 activation at the DISC. These findings are important for the better understanding of CD95 apoptosis regulation and reveal differences between apoptotic signaling pathways of the TRAIL and CD95 systems.     

The Aspergillus nidulans CENP-E kinesin motor KipA interacts with the fungal homologue of the centromere-associated protein CENP-H at the kinetochore

Chromosome segregation is an essential process for nuclear and cell division. The microtubule cytoskeleton, molecular motors and protein complexes at the microtubule plus ends and at kinetochores play crucial roles in the segregation process. Here we identified KatA (KipAtarget protein, homologue of CENP-H) as a kinesin-7 (KipA, homologue of human CENP-E) interacting protein in Aspergillus nidulans. KatA located at the kinetochore during the whole cell cycle and colocalized with KipA and partially with the putative microtubule polymerase AlpA (XMAP215) during mitosis. Deletion of katA was lethal at 37°C and caused severe growth and morphology defects at room temperature. KipA was shown before to play an important role in growth directionality determination and our new results suggest a second function of KipA in the interaction between the microtubule plus ends and the kinetochores during mitosis.