The role of calcium signalling in the chondrogenic response of mesenchymal stem cells to hydrostatic pressure.

The object of this study was to elucidate the role of Ca++ signalling in the chondrogenic response of mesenchymal stem cells (MSCs) to hydrostatic pressure (HP). MSCs were seeded into agarose hydrogels, subjected to HP or kept in free swelling conditions, and were cultured either with or without pharmacological inhibitors of Ca++ mobility and downstream targets. Chelating free Ca++, inhibiting voltage-gated calcium channels, and depleting intracellular calcium storessuppressed the beneficial effect of HP on chondrogenesis, indicating that Ca++ mobility may play an important role in the mechanotransduction of HP. However, inhibition of stretch-activated calcium channels in the current experiment yielded similar results to the control group, suggesting that mechanotransduction of HP is distinct from loads that generate cell deformations. Inhibition of the downstream targets calmodulin, calmodulin kinase II, and calcineurin all knocked down the effect of HP on chondrogenesis, implicating these targets in MSCs response to HP. All of the pharmacological inhibitors that abolished the chondrogenic response to HP also maintained a punctate vimentin organisation in the presence of HP, as opposed to the mechanoresponsive groups where the vimentin structure became more diffuse. These results suggest that Ca++ signalling may transduce HP via vimentin adaptation to loading.

The pericellular environment regulates cytoskeletal development and the differentiation of mesenchymal stem cells and determines their response to hydrostatic pressure.

The objective of this study was to examine the interplay between matrix stiffness and hydrostatic pressure (HP) in regulating chondrogenesis of mesenchymal stem cells (MSCs) and to further elucidate the mechanotransductive roles of integrins and the cytoskeleton. MSCs were seeded into 1 %, 2 % or 4 % agarose hydrogels and exposed to cyclic hydrostatic pressure. In a permissive media, the stiffer hydrogels supported an osteogenic phenotype, with little evidence of chondrogenesis observed regardless of the matrix stiffness. In a chondrogenic media, the stiffer gels suppressed cartilage matrix production and gene expression, with the addition of RGDS (an integrin blocker) found to return matrix synthesis to similar levels as in the softer gels. Vinculin, actin and vimentin organisation all adapted within stiffer hydrogels, with the addition of RGDS again preventing these changes. While the stiffer gels inhibited chondrogenesis, they enhanced mechanotransduction of HP. RGDS suppressed the mechanotransduction of HP, suggesting a role for integrin binding as a regulator of both matrix stiffness and HP. Intermediate filaments also appear to play a role in the mechanotransduction of HP, as only vimentin organisation adapted in response to this mechanical stimulus. To conclude, the results of this study demonstrate that matrix density and/or stiffness modulates the development of the pericellular matrix and consequently integrin binding and cytoskeletal structure. The study further suggests that physiological cues such as HP enhance chondrogenesis of MSCs as the pericellular environment matures and the cytoskeleton adapts, and points to a novel role for vimentin in the transduction of HP.

Cell-matrix interactions regulate mesenchymal stem cell response to hydrostatic pressure.

Both hydrostatic pressure (HP) and cell-matrix interactions have independently been shown to regulate the chondrogenic differentiation of mesenchymal stem cells (MSCs). The objective of this study was to test the hypothesis that the response of MSCs to hydrostatic pressure will depend on the biomaterial within which the cells are encapsulated. Bone-marrow-derived MSCs were seeded into either agarose or fibrin hydrogels and exposed to 10 MPa of cyclic HP (1 Hz, 4 h per day, 5 days per week for 3 weeks) in the presence of either 1 or 10 ng ml(-1) of TGF-ß3. Agarose hydrogels were found to support a spherical cellular morphology, while MSCs seeded into fibrin hydrogels attached and spread, with clear stress fiber formation. Hydrogel contraction was also observed in MSC-fibrin constructs. While agarose hydrogels better supported chondrogenesis of MSCs, HP only enhanced sulfated glycosaminoglycan (sGAG) accumulation in fibrin hydrogels, which correlated with a reduction in fibrin contraction. HP also reduced alkaline phosphatase activity in the media for both agarose and fibrin constructs, suggesting that this stimulus plays a role in the maintenance of the chondrogenic phenotype. This study demonstrates that a complex relationship exists between cell-matrix interactions and hydrostatic pressure, which plays a key role in regulating the chondrogenic differentiation of MSCs.

The effect of cyclic hydrostatic pressure on the functional development of cartilaginous tissues engineered using bone marrow derived mesenchymal stem cells.

Mechanical signals can play a key role in regulating the chondrogenic differentiation of mesenchymal stem cells (MSCs). The objective of this study was to determine if the long-term application of cyclic hydrostatic pressure could be used to improve the functional properties of cartilaginous tissues engineered using bone marrow derived MSCs. MSCs were isolated from the femora of two porcine donors, expanded separately under identical conditions, and then suspended in cylindrical agarose hydrogels. Constructs from both donors were maintained in a chemically defined media supplemented with TGF-ß3 for 42 days. TGF-ß3 was removed from a subset of constructs from day 21 to 42. Loaded groups were subjected to 10 MPa of cyclic hydrostatic pressurisation at 1 Hz for one hour/day, five days/week. Loading consisted either of continuous hydrostatic pressure (CHP) initiated at day 0, or delayed hydrostatic pressure (DHP) initiated at day 21. Free swelling (FS) constructs were cultured in parallel as controls. Constructs were assessed at days 0, 21 and 42. MSCs isolated from both donors were morphologically similar, demonstrated comparable colony forming unit-fibroblast (CFU-F) numbers, and accumulated near identical levels of collagen and GAG following 42 days of free swelling culture. Somewhat unexpectedly the two donors displayed a differential response to hydrostatic pressure. For one donor the application of CHP resulted in increased collagen and GAG accumulation by day 42, resulting in an increased dynamic modulus compared to FS controls. In contrast, CHP had no effect on matrix accumulation for the other donor. The application of DHP had no effect on either matrix accumulation or construct mechanical properties for both donors. Variability in the response to hydrostatic pressure was also observed for three further donors. In conclusion, this study demonstrates that the application of long-term hydrostatic pressure can be used to improve the functional properties of cartilaginous tissues engineered using bone marrow derived MSCs by enhancing collagen and GAG accumulation. The response to such loading however is donor dependent, which has implications for the clinical utilisation of such a stimulus when engineering cartilaginous grafts using autologous MSCs.

Can non-mechanical proteins withstand force? Stretching barnase by atomic force microscopy and molecular dynamics simulation.

Atomic force microscopy (AFM) experiments have provided intriguing insights into the mechanical unfolding of proteins such as titin I27 from muscle, but will the same be possible for proteins that are not physiologically required to resist force? We report the results of AFM experiments on the forced unfolding of barnase in a chimeric construct with I27. Both modules are independently folded and stable in this construct and have the same thermodynamic and kinetic properties as the isolated proteins. I27 can be identified in the AFM traces based on its previous characterization, and distinct, irregular low-force peaks are observed for barnase. Molecular dynamics simulations of barnase unfolding also show that it unfolds at lower forces than proteins with mechanical function. The unfolding pathway involves the unraveling of the protein from the termini, with much more native-like secondary and tertiary structure being retained in the transition state than is observed in simulations of thermal unfolding or experimentally, using chemical denaturant. Our results suggest that proteins that are not selected for tensile strength may not resist force in the same way as those that are, and that proteins with similar unfolding rates in solution need not have comparable unfolding properties under force.

The folding nucleus of a fibronectin type III domain is composed of core residues of the immunoglobulin-like fold.

To identify the contacts that stabilise the rate-limiting transition state for folding of FNfn10 (the tenth fnIII domain of human fibronectin), 42 mutants have been analysed at 29 positions across this domain. An anomalous response to mutation means that structure formation in the A, B and G strands cannot be evaluated by this method. In all the residues analysed, phi-values are fractional and no completely structured region is observed. The analysis reveals that hydrophobic residues from the central strands of the beta-sandwich form a large core of interactions in the transition state. Brønsted analysis shows that the stabilisation energy from the amino acid side-chains in the transition state is approximately 40 % of that in the native state. The protein folds by a nucleation-condensation mechanism, and tertiary interactions within the core make up the folding nucleus. Local interactions, in turns and loops, are apparently much less significant. Comparison with an homologous domain from human tenascin (TNfn3), shows that FNfn10 has a more extended, structured transition state spanning three different "layers" of the beta-sandwich. The results support the hypothesis that interactions in the common structural core guide the folding of these domains.

The folding of an immunoglobulin-like Greek key protein is defined by a common-core nucleus and regions constrained by topology.

TNfn3, the third fibronectin type III domain of human tenascin, is an immunoglobulin-like protein that is a good model for experimental and theoretical analyses of Greek key folding. The third fibronectin type III domain of human tenascin folds and unfolds in a two-state fashion over a range of temperature and pH values, and in the presence of stabilising salts. Here, we present a high resolution protein engineering analysis of the single rate determining transition state. The 48 mutations report on the contribution of side-chains at 32 sites in the core and loop regions. Three areas in the protein exhibit high Phi-values, indicating that they are partially structured in the transition state. First, a common-core ring of four positions in the central strands B, C, E and F, that are in close contact, form a nucleus of tertiary interactions. The two other regions that appear well-formed are the C' region and the E-F loop. The Phi-values gradually decrease away from these regions such that the very ends of the two terminal strands A and G, have Phi-values of zero. We propose a model for the folding of immunoglobulin-like proteins in which the common-core "ring" forms the nucleus for folding, whilst the C' and E-F regions are constrained by topology to pack early. Folding characteristics of a group of structurally related proteins appear to support this model.

CYP2D6 is associated with Parkinson's disease but not with dementia with Lewy Bodies or Alzheimer's disease.

The similarities between the clinical and pathological findings of dementia with Lewy Bodies (DLB) with Alzheimer's disease and Parkinson's disease are complex, and their significance for pathogenesis is unresolved. It is likely that DLB shares common disease determinants with both Alzheimer's disease and Parkinson's disease. Clinically DLB shows the presence of dementia similar, though not identical, to that found in Alzheimer's disease. A parkinsonian movement disorder is present in a proportion of DLB cases. Pathologically DLB shows senile plaques, as with Alzheimer's disease, and also substantia nigra neurone loss and Lewy bodies, as with Parkinson's disease. At a genetic level, DLB shows an elevated Apolipoprotein E epsilon4 frequency as described in Alzheimer's disease, but this is absent in Parkinson's disease. An elevated frequency of the CYP2D6*4 allele has been found in Parkinson's disease and we have therefore genotyped a large series of clinically and neuropathologically confirmed cases of DLB, Alzheimer's disease, Parkinson's disease and age-matched control individuals for the CYP2D6*4 allele. Whilst an elevated frequency of the CYP2D6*4 allele was found in Parkinson's disease, no such elevations were found in DLB or Alzheimer's disease. Stratification of the CYP2D6*4 allele with respect to the Apolipoprotein E epsilon4 also did not show any significant associations with the CYP2D6*4 allele. The CYP2D6*4 allele is not a major genetic determinant of DLB and the results place DLB with Alzheimer's disease rather than Parkinson's disease on a genetic level.

Co-administration of polyanions with a phosphorothioate oligodeoxynucleotide (CGP 69846A): a role for the scavenger receptor in its in vivo disposition.

The effects of co-administering polyanions on the pharmacokinetics of a 20-mer phosphorothioate oligodeoxynucleotide (CGP 69846A), and the role of scavenger receptors in its in vivo disposition, have been investigated. Following i.v. administration, CGP 69846A was rapidly cleared from the plasma and distributed amongst high (e.g. kidney, liver, spleen), low (e.g. skeletal muscle) and negligible (e.g. brain) accumulating tissues. In addition it was shown that: 1) dextran sulphate co-administration has a dose-dependent effect on the disposition of CGP 69846A; 2) CGP 69846A undergoes renal filtration and renal accumulation largely results from tubular reabsorption; 3) cross-inhibition studies are consistent with CGP 69846A being recognized by scavenger receptors in vitro and in vivo; and 4) the scavenger receptor may be an important determinant for the in vivo disposition of CGP 69846A in mice. These studies contribute toward an increased understanding of the mechanism underlying the pharmacokinetic behaviour of phosphorothioate oligodeoxynucleotides.

Short tau inversion recovery magnetic resonance imaging in occult scaphoid injuries: effect on management.

Over a 21-month period, 16 patients with possible scaphoid fractures, where conventional radiographs were essentially normal on the day of injury and 10 days later, were examined by coronal short tau inversion recovery (STIR) and T1 weighted sequences on a 0.2 Tesla scanner. The magnetic resonance (MR) examinations were done in between electively booked patients and management was altered in 50% of patients. It was expedient to do MR as radio-isotope facilities were not available at the hospital. In four patients, the examination was entirely normal. In another four patients, intercarpal fluid collections only were demonstrated (two generalized and two localized). In these patients a plaster cast was not re-applied. In two patients, bony injuries of the distal radius were demonstrated instead, with the scaphoid being normal. In the remaining six patients, variable appearances were shown, ranging from an actual fracture line demonstrated on both STIR and T1-weighted images with the remainder of the scaphoid being hyperintense on STIR and hypointense on T1, to non-visualization of the actual fracture line, which appeared to be represented by a much more hyperintense band through the waist of an otherwise hyperintense scaphoid on the STIR sequence. In all these patients a plaster cast was re-applied empirically, including those patients with a definite fracture and those patients with apparent bone contusion only. Although the T1-weighted images were anatomically superior, their addition did not alter management and it is suggested that coronal STIR images should suffice to demonstrate occult fractures of the scaphoid.

CYP2D6 phenotype-genotype relationships in African-Americans and Caucasians in Los Angeles.

CYP2D6 genotyping (CYP2D6*3, CYP2D6*4, CYP2D6*5, CYP2D6*13, CYP2D6*16 alleles and gene duplications) was previously performed on 1053 Caucasian and African-American lung cancer cases and control individuals and no significant difference in allele frequencies between cases and control individuals detected. We have carried out additional genotyping (CYP2D6*6, CYP2D6*7, CYP2D6*8, CYP2D6*9, CYP2D6*10, CYP2D6*17 alleles) and debrisoquine phenotyping on subgroups from this study to assess phenotype-genotype relationships. African-Americans showed significant differences from Caucasians with respect to frequency of defective CYP2D6 alleles, particularly CYP2D6*4 and CYP2D6*5. The CYP2D6*17 allele occurred at a frequency of 0.26 among 87 African-Americans and appeared to explain higher average metabolic ratios among African-Americans compared with Caucasians. CYP2D6*6, CYP2D6*8, CYP2D6*9 and CYP2D6*10 were rare in both ethnic groups but explained approximately 40% of higher than expected metabolic ratios among extensive metabolizers. Among individuals phenotyped with debrisoquine, 32 out of 359 were in the poor metabolizer range with 24 of these (75%) also showing two defective CYP2D6 alleles. Additional single strand conformational polymorphism analysis screening of samples showing large phenotype-genotype discrepancies resulted in the detection of three novel polymorphisms. If subjects taking potentially interfering drugs were excluded, this additional screening enabled the positive identification of 88% of phenotypic poor metabolizers by genotyping. This sensitivity was comparable with that of phenotyping, which identified 90% of those with two defective alleles as poor metabolizers.

Significance of short tau inversion recovery magnetic resonance sequence in the management of skeletal injuries.

The conventional radiographs and urgent short tau inversion recovery (STIR) magnetic resonance image (MRI) examinations of 27 consecutive patients with occult bony injuries were prospectively analysed over a 12 month period. A STIR MRI study was undertaken where the plain films were normal (n = 15) or inconclusive (n = 12) and where the patients' clinical setting was highly suggestive of an underlying bony injury. In six patients, MRI only revealed soft-tissue injuries or joint effusions and did not demonstrate any bony injury but in the remainder fractures or bone contusions were shown to be present. The MRI studies were performed on a 0.2 Tesla lower field strength unit and the examinations were expeditiously performed, inexpensive, and done on a priority basis between electively booked patients. Radio-isotope studies were not available and hence were not included in the study protocol. Apart from demonstrating the value of STIR MRI (without additional T1-weighted sequences in most patients), the purpose of this study was to highlight the alteration in management in 18/27 patients (66%) and the significant alteration in management in six of these patients.

An improved animal model for studying desferrioxamine.

A hamster model has been developed for studying desferrioxamine. The hamster shows many similarities to man in terms of plasma stability and metabolites formed from desferrioxamine. The [59Fe]ferritin derived from rats has been shown to be sequestered into liver parenchymal cells when injected intravenously into hamsters. The technique has proved sufficiently sensitive to enable detection of differences of < 1% of the radioactivity administered in the elimination of iron. Alterations in iron excretion were seen when dosing desferrioxamine via different routes. The principal route of iron excretion was into the intestines. The effectiveness of the dosing routes for desferrioxamine in removing iron were subcutaneous (10.5%) > intravenous (6.25%) > oral (3.66%) > control (2.19%). A dose-response relationship was demonstrated using the intravenous dose route. The model offers a simple method for comparing the efficacy of administration routes for determining the optimal use of desferrioxamine.

Disposition and metabolism of 2,3,7,8-tetrachlorodibenzofuran by rainbow trout (Oncorhynchus mykiss).

The disposition and metabolism of 2,3,7,8-tetrachlorodibenzofuran (TCDF) was investigated in rainbow trout (Oncorhynchus mykiss) in order to better understand the metabolic and physiological factors that modulate the fate of this extremely toxic compound in rainbow trout compared to other species. The fish were dosed orally with [3H]TCDF (1 microgram/kg); fish were terminated at 1-19 days for the determination of whole body half-life or at 0.3-28 days for determination of tissue distribution. Unassimilated TCDF (51.5% of the dose) was eliminated with a half-life of 0.84 days. The assimilated body burden of TCDF equivalents decreased with a half-life of 14.8 days (determined between 3 and 19 days). Trout muscle showed a relatively high capacity to accumulate and retain (unmetabolized) TCDF, accounting, at 3 days, for 32% of the body burden of TCDF equivalents (half-life in muscle, 15.2 days). Trout liver, on the other hand, showed a relatively low capacity to accumulate and metabolize TCDF. At 3 days, the concentrations of TCDF equivalents in liver and bile were, respectively, 0.37 ng/g liver (0.88% of the body burden) and 4.8 ng/ml bile. The data suggest that the relatively high affinity of lipid-rich trout muscle for TCDF limits the ability of the liver to accumulate and metabolize TCDF. The major TCDF metabolites found in trout liver and bile were, respectively, 4-OH-TCDF and TCDF-4-O-glucuronide.

Disposition and metabolism of 2,3,7,8-tetrachlorodibenzofuran by channel catfish (Ictalurus punctatus).

The disposition and metabolism of 2,3,7,8-tetrachlorodibenzofuran (TCDF) was was investigated in channel catfish (Ictalurus punctatus) in order to better understand the metabolic and physiological factors that modulate the fate of this extremely toxic compound in channel catfish compared to other species. The fish were dosed orally with [3H]TCDF (1 microgram/kg); tissue were harvested at 3, 7, and 14 days for radioassay. The body burden of TCDF equivalents in catfish at 3 days was 0.36 microgram/kg, which was decreasing with a half-life of 3.6 days. Catfish muscle showed a relatively low capacity to accumulate and retain TCDF, accounting, at 3 days, for only 19.0% of the body burden of TCDF equivalents (half-life in muscle, 5.0 days). Catfish liver, on the other hand, showed a high capacity to accumulate and metabolize TCDF and to secrete TCDF metabolites into the bile. At 3 days, the concentrations of TCDF equivalents in liver and bile were, respectively, 5.7 ng/g liver (19% of the body burden) and 129 ng/ml bile. However, the concentration of TCDF equivalents in liver decreased with a half-life of 1.8 days to 0.04 ng/g (2.0% of the body burden) at 14 days. Thus, the capacity of catfish liver to retain TCDF decreased dramatically as the body burden decreased. The data suggest that the low affinity of lipid poor catfish muscle for TCDF may allow catfish liver to accumulate a concentration of TCDF sufficient to induce the metabolism of this compound by liver monooxygenases. The major TCDF metabolites found in catfish liver and bile were, respectively, 4-OH-TCDF and TCDF-4-O-glucuronide.

The transmucosal absorption of recombinant human interferon-alpha B/D hybrid in the rat and rabbit.

For therapeutic proteins such as interferon-alpha B/D, a non-parenteral route of delivery is desirable. Possible sites of administration include the various regions of the gastrointestinal tract and airways, and this paper reports the bioavailability of interferon-alpha B/D via these routes in the rat and rabbit. Apart from the stomach, detectable levels of interferon-alpha B/D in the serum were achieved via all routes. Bioavailabilities were less than 1%, except from the lung (6.8% in the rat) and nasal cavity (2.9% in the rabbit). Absorption from the gastrointestinal tract was similar for both species, but in the nasal cavity of the rabbit was sixfold that of the rat, and in the lung of the rat was tenfold that in the rabbit. Absorption from all routes, except the buccal cavity, resulted in detectable biochemical changes in the liver of the rabbit. Comparison with reports from other groups show differences in the extent of absorption of interferon-alpha B/D and of natural or homologous recombinant interferon-alpha. The non-parenteral delivery of biochemically active amounts of interferon-alpha B/D is thus demonstrated.

Metabolism of 2-acetylaminofluorene by hepatocytes isolated from rainbow trout.

The metabolism of 2-acetyl-[9-14C]aminofluorene (AAF) by hepatocytes isolated from rainbow trout (Oncorhynchus mykiss), Shasta strain, was investigated in order to assess the competing activation and detoxification pathways which may explain the resistance of this species and strain to the initiation of carcinogenesis by this model carcinogenic aromatic amide. Freshly isolated hepatocytes (per milliliter: 1.0 mg dry wt; 1.5 (10(6)) hepatocytes) incubated with 65 microM AAF for 4 hr converted 15.4 nmol AAF to metabolites, including 7.8 nmol of water-soluble compounds. AAF-derived radioactivity extracted from the incubation mixtures, before and after hydrolysis by beta-glucuronidase and arylsulfatase, was analyzed by reversed-phase HPLC. The metabolite profile following incubation of hepatocytes with 6.5 microM AAF for 4 hr included (as percentage of total metabolites); 7-OH-AAF, 5-/8-/9-OH-AAF and 2-aminofluorene (AF) (17, 2.4, and 2.7%, respectively); conjugates of these respective primary metabolites (39, 9, and 4%, respectively). Glucuronides amounted to 49% of the total metabolites. N-OH-AAF and its conjugates always amounted to < 1% of total metabolites. The relative amount of (unconjugated) AF increased considerably (to 26%) following incubation of hepatocytes with 65 microM AAF, with a corresponding decrease in the total amount of glucuronides formed. Following incubation with 65 microM AAF, 1.6% of AAF metabolites was covalently bound to macromolecules, giving a ratio of covalently bound derivatives to detoxification products of 0.028. These data are consistent with the hypothesis that rainbow trout are resistant to AAF-induced hepatocarcinogenesis, in part, because trout liver efficiently detoxifies AAF and forms only relatively small amounts of active intermediates capable of binding to macromolecules, including DNA.