The P2Y4 receptor forms homo-oligomeric complexes in several CNS and PNS neuronal cells

It is well established that several cell surface receptors interact with each other to form dimers and oligomers, which are essential for their activation. Since little is known about the quaternary structure of P2Y receptors, in the present work, we investigated the expression of the G-protein-coupled P2Y₄ subunit as monomeric or higher-order complex protein. We examined both endogenously expressed P2Y₄ subtype with the aid of specific anti-P2Y₄ antiserum, and heterologously transfected P2Y₄-tagged receptors with the use of antitag antibodies. In both cases, we found the P2Y₄ receptor displaying molecular masses corresponding to monomeric, dimeric and oligomeric structures. Experiments performed in the absence of reducing agents demonstrated that there is a strict correlation among the multiple protein bands and that the multimeric forms are at least partially assembled by disulphide bonds. The direct demonstration of P2Y₄ homodimerisation comes instead from co–transfection and differential co–immunoprecipitation experiments, with the use of differently tagged P2Y₄ receptors and antitag antibodies. The structural propensity of the P2Y₄ protein to form homo-oligomers may open the possibility of a novel regulatory mechanism of physiopathological functions for this and additional P2Y receptors.     

Biotransformation of Flurbiprofen by Cunninghamella Species

The biotransformation of the fluorinated anti-inflammatory drug flurbiprofen was investigated in Cunninghamella spp. Mono- and dihydroxylated metabolites were detected using gas chromatography-mass spectrometry and fluorine-19 nuclear magnetic resonance spectroscopy, and the major metabolite 4′-hydroxyflurbiprofen was isolated by preparative high-pressure liquid chromatography (HPLC). Cunninghamella elegans DSM 1908 and C. blakesleeana DSM 1906 also produced a phase II (conjugated) metabolite, which was identified as the sulfated drug via deconjugation experiments.One of the objectives of the recent European Union legislation governing the testing and evaluation of chemicals, REACH (Regulation, Evaluation, Authorisation and Restriction of Chemicals), is to further reduce the need for animals in the testing process. Some microorganisms, such as the zygomycete fungus Cunninghamella and actinomycetes bacteria, have been shown to metabolize xenobiotic compounds in a fashion analogous to that of mammals (3, 5, 11, 17). It was suggested over 3 decades ago that microorganisms had potential as models of mammalian metabolism (16), although there are concerns about their predictive value (8). Nevertheless, certain microorganisms can be applied to the generation of useful quantities of drug metabolic intermediates (13), which is more desirable than isolation of these compounds from dosed animals, and avoids the concerns often associated with chemical synthesis, such as the use of toxic reagents and harsh reaction conditions.Owing to the desirable physicochemical properties of the fluorine atom (small Van der Waals radius, electronegativity, and strength of the carbon-fluorine bond), approximately 25% of drugs either currently on the market or in the pipeline are fluorinated (12). One such example is flurbiprofen [(RS)-2-(2-fluoro-4-biphenyl)propionic acid], which is a nonsteroidal anti-inflammatory drug (NSAID) used in the treatment of inflammation caused by arthritis. In humans it is transformed to the phase I (oxidative) metabolites 4′-hydroxyflurbiprofen, 3′,4′-dihydroxyflurbiprofen, and 3′-hydroxy,4′-methoxyflurbiprofen; glucuronide and sulfate conjugates (phase II metabolites) have also been detected (9, 15). In equine urine additional hydroxylated and methoxylated metabolites were detected (20). Tracy et al. (18) demonstrated that only one cytochrome P450 isoform (2C9) is involved in the oxidation of flurbiprofen, which makes the drug a potentially useful in vivo probe for this particular isoform. Despite the prevalence of fluorinated drugs, only a few investigations have been undertaken to determine the microbial biotransformation of these compounds (7, 21). Here we describe the biotransformation of flurbiprofen by Cunninghamella species and the determination of the metabolites by nuclear magnetic resonance (NMR) spectroscopy (¹H and ¹⁹F), gas chromatography-mass spectrometry (GC-MS), and high-pressure liquid chromatography (HPLC).Three species of Cunninghamella were selected for the biotransformation experiments: C. elegans (strains DSM 1908, DSM 8217, and DSM 63299), C. echinulata DSM 1905, and C. blakesleeana DSM 1906. The fungi were grown on Sabouraud dextrose agar plates (Sigma) for 5 days at 26°C before being homogenized in 100 ml of sterile saline solution. The homogenate (10%, vol/vol) was used to inoculate 50 ml of fresh Sabouraud dextrose broth in 250-ml Erlenmeyer flasks, which were incubated at 28°C with shaking at 150 rpm. Following previously established procedures (2), 5 mg of flurbiprofen (Sigma) dissolved in dimethyl formamide (20 μl) was added to the cultures after 72 h, and the incubation was continued up to a further 120 h. Control experiments were conducted in the absence of either flurbiprofen or fungus. The cultures (supernatant and cells) were sonicated on ice (Sonicator U200S control; IKA Labortechnik) for 5 min at 50% amplitude, with intervals of 30 s after each minute to prevent overheating. The sonicates were centrifuged, the supernatant was extracted with 50 ml of ethyl acetate, and the extracts were evaporated to dryness.     

Temporal response of canine flexor tendon to limb suspension

Tendon disuse, or stress deprivation, frequently accompanies clinical disorders and treatments, yet the metabolism of tendons subject to stress deprivation has rarely been investigated systematically. The effects of stress deprivation on canine flexor tendon were investigated in this study. One adult canine forepaw was suspended for 21 or 42 days. Control forepaws were collected from dogs that had no intervention on their limbs and paws. The expression of collagen I and III was not significantly altered in the tendons disused for 21 days but was significantly decreased at 42 days (P < 0.03). The expression of collagen II, aggrecan, decorin, and fibronectin was significantly decreased in the tendons in the suspended limbs at 21 days (P < 0.002) and further reduced at 42 days. With stress deprivation, the expression of matrix metalloproteinase 2 (MMP2) was significantly increased (P < 0.004) at 21 and 42 days. The expression of MMP3 was significantly decreased at 21 and 42 days (P < 0.03). The expression of MMP13 was not altered with stress deprivation at 21 and 42 days. The expression of MMP14 was significantly increased at 21 days (P = 0.0015) and returned to the control level at 42 days. Tissue inhibitor of metalloproteinase 1 (TIMP1) expression was decreased after the limbs were suspended for 42 days (P = 0.0043), but not 21 days. However, TIMP2 expression was not significantly different from control at 21 or 42 days. Furthermore, the cross-sectional area of the stress-deprived tendons at 42 days was decreased compared with the control group (P < 0.01). The intervention method in this study did not result in any alteration of stiffness of the tendon. Our study demonstrated that stress deprivation decreases the anabolic process and increases the catabolic process of extracellular matrix in flexor tendon.     

Electrospun micro- and nanofiber tubes for functional nervous regeneration in sciatic nerve transections

Background

Rhipicephalus (Boophilus) spp. ticks economically impact on cattle production in Africa and other tropical and subtropical regions of the world. Tick vaccines constitute a cost-effective and environmentally friendly alternative to tick control. The R. microplus Bm86 protective antigen has been produced by recombinant DNA technology and shown to protect cattle against tick infestations.

Results

In this study, the genes for Bm86 (R. microplus), Ba86 (R. annulatus) and Bd86 (R. decoloratus) were cloned and characterized from African or Asian tick strains and the recombinant proteins were secreted and purified from P. pastoris. The secretion of recombinant Bm86 ortholog proteins in P. pastoris allowed for a simple purification process rendering a final product with high recovery (35–42%) and purity (80–85%) and likely to result in a more reproducible conformation closely resembling the native protein. Rabbit immunization experiments with recombinant proteins showed immune cross-reactivity between Bm86 ortholog proteins.

Conclusion

These experiments support the development and testing of vaccines containing recombinant Bm86, Ba86 and Bd86 secreted in P. pastoris for the control of tick infestations in Africa.     

Analysis of the gliding pattern of the canine flexor digitorum profundus tendon through the A2 pulley

Friction between a tendon and its pulley was first quantified using the concept of the arc of contact. Studies of human tendons conformed closely to a theoretical nylon cable/nylon rod model. However, we observed differences in measured friction that depended on the direction of motion in the canine model. We hypothesized that fibrocartilaginous nodules in the tendon affected the measurements and attempted to develop a theoretical model to explain the observations we made. Two force transducers were connected to each end of the canine flexor digitorum profundus tendon and the forces were recorded when it was moved through the A2 pulley toward a direction of flexion by an actuator and then reversed a direction toward extension. The changes of a force as a function of tendon excursion were evaluated in 20 canine paws. A bead cable/rod model was developed to simulate the canine tendon-pulley complex. To interpret the results, a free-body diagram was developed. The two prominent fibrocartilaginous nodules in the tendon were found to be responsible for deviation from a theoretical nylon cable gliding around the rod model, in a fashion analogous to the effect of the patella on the quadriceps mechanism. A bead cable/rod model qualitatively reproduced the findings observed in the canine tendon-pulley complex. Frictional coefficient of the canine flexor tendon-pulley was 0.016+/-0.005. After accounting for the effect created by the geometry of two fibrocartilaginous nodules within the tendon, calculation of frictional force in the canine tendon was possible.     

Hyaluronic acid diminishes the resistance to excursion after flexor tendon repair: an in vitro biomechanical study

Adhesion between the tendon and tendon sheath after primary flexor tendon repair is seen frequently, and postoperative finger function is occasionally unsatisfactory. A reduction of the friction may facilitate tendon mobilization, which in turn may reduce the risk of the adhesion and restriction of range of motion. We considered the possibility of utilizing the hyaluronic acid (HA) as a lubricant. To evaluate the effect of HA, the gliding resistance between the canine flexor digitorum profundus tendon repaired by a modified Kessler suture technique with running epitendinous suture and the annular pulley located on the proximal phalanx (corresponding to the A2 pulley in humans) was evaluated and compared before and after administration of HA. The HA solution measurement groups were identified as follows; intact tendon as a control; repaired tendon; tendon soaked in 0.1, 1, and 10 mg/ml HA. The resistance increased after repairing, then it decreased after soaking in 10 mg/ml HA solution. The results of this study revealed that HA diminishes the excursion resistance after flexor tendon repair. We believe that some style of administration of the HA might reduce the excursion resistance and prevent adhesion until the synovial surface is fully developed.     

Human fibroblasts from normal and malignant breast tissue grown in vitro show a distinct senescence profile and telomerase activity

The telomerase activity and the senescence profile of cultured breast fibroblasts from normal human interstitial and malignant stromal tissue were studied in comparison with their proliferation and differentiation pattern. Fibroblasts were grown either in the presence or absence of a conditioned medium (CM) obtained from cultures of the oestrogen receptor-positive breast cancer MCF-7 cell line. At different passages (from the 2nd up to the 48th), fibroblasts were examined for the telomerase activity by the Telomerase Repeats Amplification Protocol (TRAP) assay, for proliferation profile by Ki-67 antigen expression, and the myofibroblast or smooth muscle cell-like differentiation pattern by immunofluorescence with monoclonal antibodies specific for smooth muscle markers. Serial passages of fibroblasts from normal or tumour breast reveal that the relationship between the levels of telomerase activity and phenotypic/proliferation profile changes with cell subcultivation in a different manner in the two cell populations. The fibroblasts from normal tissue completed 12 passages in a CM-independent way prior to senescence whereas fibroblasts from tumour stroma senescence were attained after 48 passages. These cells showed a marked decrease of telomerase activity, growth rate and smooth muscle -actin expressing myofibroblasts after the 32nd passage. CM treatment of this fibroblast population induces a decline in the myofibroblast content, which precedes the changes in telomerase activity. Passaged fibroblasts from normal breast tissue can be converted to myofibroblasts upon CM treatment whereas those from tumour stroma were CM-insensitive. Taken together our data suggest that a heterogeneous fibroblast population with different life span is activated/recruited in the breast interstitium and poses the problem of a unique activation/recruitment of fibroblasts in neoplastic conditions.     

Age-dependent changes in the mechanical properties of tail tendons in TGF-beta inducible early gene-1 knockout mice.

The purpose of this study is to investigate age-dependent changes in the architecture and mechanical properties of tendon in TGF-beta inducible early gene-1 (TIEG) knockout mice. Wild-type and TIEG knockout mice, aged 1, 2, and 15 mo, were used. The mechanical properties of tail tendons isolated from these mice were determined using uniaxial tensile ramp (0.05 mm/s) and relaxation (5 mm/s) tests, with a strain of 10%. Mechanical parameters (Young's modulus from the ramp test; fast and static stresses from the relaxation test) were measured and recorded. The structure of the tail tendon fascicle was characterized by transmission electron microscopy. The results of the mechanical testing revealed no significant difference between the knockout and wild-type groups at 1 or 15 mo of age. However, the fascicles of the knockout mice at 3 mo of age exhibited decreased fast and static stresses compared with those of the wild-type mice. Electron microscopy revealed an increase in fibril size in the knockout mouse tendons relative to wild-type controls at 1 and 3 mo of age. These data indicate an important role for TIEG in tendon microarchitecture and strength in adult mice.