Vitamin K-dependent carboxylase: utilization of decarboxylated bone Gla protein and matrix Gla protein as substrates.

The ability of des-gamma-carboxy bone Gla protein (dBGP) and des-gamma-carboxy matrix Gla protein (dMGP) to act as substrates for the rat liver vitamin K-dependent carboxylase has been investigated. An amino-terminal 'propeptide' is present on the intracellular form of BGP and is thought to interact with a recognition site on the enzyme. dBGP, lacking this extension, is a poor, high apparent Km, carboxylase substrate, but is a much better substrate when free propeptide is added. MGP lacks an amino-terminal propeptide, but contains a a homologous region in the mature protein. dMGP is an excellent substrate for the carboxylase with a low apparent Km and its carboxylation is inhibited by free propeptide.

Purification and characterization of secreted human leptin produced in baculovirus-infected insect cells.

The product of the human ob (obesity) gene, leptin, appears to function in the maintenance of body weight in vivo. When injected into mice, this hormone reduces food consumption and causes weight loss. This work has been done with recombinant leptin (re-leptin) purified and renatured from inclusion bodies in Escherichia coli. We have expressed the human obesity gene encoding the predicted full-length leptin in Spodoptera frugiperda (Sf-9) cells by infection with the recombinant baculovirus system. Protein corresponding to re-leptin was secreted into the culture medium and purified in sufficient quantity for testing biological activity. The secreted re-protein was characterized and found to be unmodified except for correct cleavage of the signal peptide during export from the cells. The resulting molecule is expected to be properly folded and has been purified to a high level of homogeneity. The re-leptin secreted from Sf-9 cells should be an appropriate source of protein for study of the native structure.

Leptin is a four-helix bundle: secondary structure by NMR.

Leptin is a signaling protein that in its mutant forms has been associated with obesity and Type II diabetes. The lack of sequence similarity has precluded analogies based on structural resemblance to known systems. Backbone NMR signals for mouse leptin (13C/15N -labeled) have been assigned and its secondary structure reveals it to be a four-helix bundle cytokine. Helix lengths and disulfide pattern are in agreement with leptin as a member of the short-helix cytokine family. A three-dimensional model was built verifying the mechanical consistency of the identified elements with a short-helix cytokine core.

Suppression of an amyloid beta peptide-mediated calcium channel response by a secreted beta-amyloid precursor protein.

Secreted isoforms of the beta-amyloid precursor protein potently enhance neuronal survival in cell cultures exposed to toxic amyloid beta peptide. Lowering of intracellular calcium levels to offset the increases in intraneuronal calcium caused by amyloid beta peptide is thought to underly this neuroprotection. Because we have shown previously that an amyloid beta peptide-mediated potentiation of calcium channel currents may contribute to this cytosolic calcium overload, the present study examined the effects of a secreted beta-amyloid precursor protein on the calcium channel response to amyloid beta peptide. When compared with untreated cultured rat hippocampal neurons, cells that underwent a 24 h preincubation with beta-amyloid precursor protein 751 displayed decreases in the relative size of the calcium channel response to amyloid beta peptide. A membrane-permeable analog of cyclic GMP, a second messenger believed to be involved in the calcium regulation process mediated by beta-amyloid precursor proteins, also attenuated the modulatory calcium channel response. Co-application of beta-amyloid precursor protein 751 with amyloid beta peptide did not alter calcium channel response to amyloid beta peptide. Taken together, these findings suggest that secreted beta-amyloid precursor proteins can suppress a calcium channel response to amyloid beta peptide that is potentially injurious to the cell, and as such, may define a neuroprotective mechanism that is specific for amyloid beta toxicity.

Effects of osteochondral defect size on cartilage contact stress.

Contact stress distributions were studied in vitro for 13 dog knees, with full-thickness osteochondral defects drilled in the weight-bearing area of both femoral condyles. Diameters of the circular defects were concentrically enlarged from 1 to 7 mm. Digitally-imaged Fuji film was used to record cartilage contact stress distribution on femoral condyles for each increment of defect diameter. All specimens showed at least some tendency for contact stress concentration at the rim of the defects. However, detailed distributions had large interspecimen variability and, within a given specimen, contact stress distributions became progressively more nonuniform around the defect rim as the diameter was enlarged. Averaged over the full series of 26 condyles, circumferential mean cartilage contact stress around the defect rim was only moderately higher (by 10-30%) than intact surface's peak local contact stress [series average = 6.2 mega pascals (MPa)]. Maximal rim stress concentration occurred for 2 mm defects, there being a consistent trend toward mild rim stress decrease with further defect enlargement. Such modest contact stress elevations, per se, are probably insufficient to inhibit defect repair or to cause degeneration of surrounding cartilage. However, near the defect rim (for all diameters), the radial component of the gradient of contact stress (i.e., radial-direction variation of contact stress) was consistently elevated by an order of magnitude above that for intact, condyle articular cartilage.

Biomechanical evaluation of early fracture healing in normal and diabetic rats.

Diabetes mellitus has been shown to alter the properties of bone and impair fracture healing in both humans and animals. The objective of this study was to document changes in the structural and material properties of intact bone and bone with healed fractures in diabetic rats compared with nondiabetic controls after 3 and 4 weeks of healing. Rods were inserted in the right femurs of control rats and rats with streptozotocin-induced diabetes, and the femurs were fractured in a standardized procedure and then allowed to heal for 3 and 4 weeks. After death, all femurs were mechanically tested to failure in torsion. The degree of healing was quantified for each animal by normalizing mechanical parameters for the femur with a healed fracture with those for the intact contralateral femur. At both time points of healing, diabetic rats exhibited inferior healing compared with that of control animals in terms of failure torque, failure stress, structural stiffness, and material stiffness of the femur with the healed fracture relative to the intact contralateral femur (p < 0.05). Our results demonstrate that the recovery of structural and material strength in femurs with healed fractures in diabetic rats is delayed by at least 1 week compared with that in controls.

Sub-Tenon's local anaesthesia: the effect of hyaluronidase.

AIMS: A prospective, randomised, double blind study was used to investigate the effect of hyaluronidase on the quality of block achieved with sub-Tenon's local anaesthesia. METHODS: 150 patients scheduled for elective cataract surgery were randomly allocated to either sub-Tenon's block with 3 ml lignocaine 2%/adrenaline 1:200 000 alone or with the addition of 30 IU/ml of hyaluronidase. The blocks were assessed for degree of akinesia and reduction of eyelid movement, and also post-injection and postoperative pain scores. RESULTS: Akinesia and reduction of eyelid movement measured 10 minutes after injection were significantly better in the group with hyaluronidase added to the anaesthetic solution. Postoperative pain scores were not significantly different between the two groups but the post-injection pain score was greater (marginally significant) in the group with hyaluronidase added. CONCLUSION: The addition of hyaluronidase significantly improves the quality of the motor blockade achieved with sub-Tenon's local anaesthesia, but has no effect on the sensory blockade.

Indentation assessment of biphasic mechanical property deficits in size-dependent osteochondral defect repair.

The apparent biphasic material properties of 10-month osteochondral defect repair tissue were determined for a series of full thickness defects of 1, 3, or 5 mm diameter, created in weight-bearing regions of 48 canine femoral condyles. Load cell recordings from indentation tests were compared with resultant contact forces computed using a corresponding linear biphasic finite element model. The spread of cartilage engagement by a spherical ended indentor was modeled by successively imposing an impenetrability kinematic boundary condition at cartilage surface nodes for which incipient indentor surface penetration was detected. For each indentation test, a least-squares-error curve fitting procedure was used to identify a set of biphasic coefficients (aggregate modulus, permeability, Poisson ratio) that closely modeled experimental behavior. In the near neighborhood of best-fit, the finite element solutions were found to be much more sensitive to aggregate modulus perturbations than to permeability permutations, suggesting that perceived permeability increase may be of lesser value as a discriminant of repair tissue inadequacy. Compared to surrounding cartilage, the repair tissue for all defect sizes had statistically significant decreases in aggregate modulus and in Poisson ratio (much more so for 3 and 5 mm defects than for 1 mm defects). The two larger defect diameters had significant increases in permeability, whereas the 1 mm defects did not. While the material property deficits were consistent, substantial and comparable to those in other recent animal models of osteochondral defect repair, the size-dependence per se of the observed constitutive differences was only modest.

An algorithm for approximate crinkle artifact compensation in pressure-sensitive film recordings.

An objective, empirically based image-processing technique was devised to compensate for the presence of crinkle artifact in Pressensor pressure-sensitive film recordings. A spherical indentor was used to produce film stains which deliberately included radially directed artifact streaks, superimposed upon otherwise smooth, nearly axisymmetric stain recordings. An interactive, threshold-based search algorithm was developed to delineate explicitly the perimeters of specific artifacts present within manually (cursor) circumscribed regions where crinkle features were visually apparent. Three mathematical artifact transformation operators were parametrically evaluated in terms of their ability to approximate objectively the corresponding artifact-free axisymmetric pressure fields. All three operators were found to reduce substantially the quantitative deviation from the idealized distributions. When appropriately tuned transformation operators were applied to typical in vitro intraarticular contact stains, the visual prominence of crinkle artifact features was markedly reduced.

Rate-independent characteristics of an arthroscopically implantable force probe in the human achilles tendon.

The effect of loading rate on specimen calibration was investigated for an implantable force sensor of the two-point loading variety. This variety of sensor incorporates a strain gage to measure the compressive load applied to the sensor due to tensile loading in a soft tissue specimen. The Achilles tendon in each of four human cadaveric lower extremities was instrumented with a force sensor and then loaded in tension using a materials testing machine. Each specimen was tensile tested at three different displacement rates, 0.25, 2.5 and 12.7 cm s(-1), corresponding with mean loading rates of 33.8, 513.2, and 2838.6 N s(-1), respectively. A calibration curve relating the force sensor signal and applied tendon tension was generated for each specimen/ displacement rate combination. For each specimen, calibration curves were compared by calculating an RMS error for the entire data set (eRMS = 1.6% of the full load value) and a coefficient of determination, R2, of a curve fit through all of the data (R2 = 99.6%). Over the range of rates tested, no measurable change in sensor sensitivity due to loading rate was observed. Hysteresis for all displacement rates was on the order of 2.4%.

Effects of imposed hindfoot constraint on ankle contact mechanics for displaced lateral malleolar fractures.

Contact stress distributions on the tibial plafond were mapped in a series of eight fresh-frozen cadaver specimens in which displaced lateral malleolar fractures were studied. These included gripping (a) by snugly lacing the foot in an athletic shoe, (b) by polymethylmethacrylate potting of the calcaneus alone, and (c) by potting of the calcaneus plus talus. Each of these three gripping conditions was tested both for rigid and for nearly frictionless transverse external constraint conditions. Across the series, the grip-dependent changes in contact stress distributions were found to be very minor compared with the wide interspecimen variability that was consistently present. Moreover, although contact stresses generally increased with progressive lateral fibular fragment offsets of up to 5 mm, such an effect was far more modest than that seen in previous cadaver work. The present laboratory cadaver findings suggest that the contact stress elevations occurring clinically for displaced lateral malleolar fractures are probably relatively mild and likely not directly responsible for late secondary degeneration.

Some problems in using a polyvinylidene fluoride transducer for the intra-articular determination of joint contact stress.

A piezoelectric transducer and associated instrumentation were developed and evaluated as a means of experimentally determining joint contact stress. Each transducer, fabricated from a polyvinylidene fluoride film, comprised four discrete sensing elements. Following dynamic calibration of all sensing elements, in vitro evaluations were performed with transducers positioned in canine tibio-femoral joints. Quantitative measurements of contact stress as a function of time were obtained using these transducers, the magnitudes of which ranged between 0.01 and 7.99 MPa. Limitations associated with the transducer material and its use in this specific application included calibration variability and temporal phase shift of the transducer output signal relative to the applied load.

Irreversible, oriented immobilization of antibodies to cobalt-iminodiacetate resin for use as immunoaffinity media.

A method for the construction of antibody affinity columns has been developed which utilizes the innate affinity of antibodies for metal. Antibodies that are reversibly bound to a cobalt-iminodiacetate resin are attached in an "exchange inert" fashion when the cobalt is oxidized from the 2+ to 3+ state. Antibodies bound in this fashion are not removed by metal chelating reagents, high salt, detergents, or chaotropic agents. Only reagents which reduce the metal are capable of removing the antibody. Since the metal binding site in antibodies is in the C-terminus of the heavy chain, antibodies bound to the resin are oriented with the combining site directed away from the resin thus allowing maximal antigen binding. The oxidation conditions used are mild and do not subject the antibody to harsh immobilization chemistry. This technique may be used to construct affinity media using multiple subclasses of antibodies since the metal binding site is conserved. This immobilization strategy may be a general technique for the oriented immobilization of antibody to surfaces.

Identification of cysteine residues alkylated with 3-bromopropylamine by protein sequence analysis.

A new reagent for the routine identification of cysteine residues during protein sequencing is described. This method employs 3-bromopropylamine to alkylate cysteines in proteins after reduction with dithiothreitol. Upon sequencing of the protein on an Applied Biosystems 477A protein sequencer, the aminopropylcysteine residue yields a phenylthiohydantoin (PTH) derivative which elutes reproducibly at a unique position immediately after PTH-leucine; baseline resolution is achieved without modification of the PTH analyzer gradient. Unlike PTH-pyridylethylcysteine, the relative elution position of the aminopropylcysteine PTH derivative is not affected by changes in the ionic strength of the analyzer solvents. In addition, the previewing of the next amino acid which is observed in proteins modified with 4-vinylpyridine does not occur in aminopropylated proteins. Preparation of alkylated proteins for electroblotting onto polyvinylidene difluoride (PVDF) membranes and methods for desalting alkylated proteins immobilized on precoated glass fiber filters or PVDF membranes are also described.

On-line procedures for alkylation of cysteine residues with 3-bromopropylamine prior to protein sequence analysis.

We have previously shown that 3-bromopropylamine offers several advantages over other alkylating reagents in the modification and subsequent identification of cysteine residues by protein sequencing. We describe here simple on-sequencer procedures for alkylating cysteines in proteins which employ the reduction of cystines in proteins with tri-n-butylphosphine and concomitant alkylation of the resulting cysteines with 3-bromopropylamine. Addition of an aqueous acetone wash to a modified reaction cycle on the Applied Biosystems 477A sequencer removes excess 3-bromopropylamine. As a result, very little background in the first step of the sequence analysis is seen. Under these conditions, cysteines are readily modified and identified during sequencing. Moreover, very little preview of the next amino acid is observed, which indicates that the N-terminal amino acid is not appreciably alkylated by 3-bromopropylamine. On-sequencer methods have been developed for proteins spotted onto glass fiber filters and proteins electroblotted onto polyvinylidene difluoride membranes.