Preparing recombinant "Split AEP" for protein labeling.

A variant originated from Oldenlandia affinis asparaginyl ligase, OaAEP1-C247A, has emerged as an ideal tool for protein labeling. However, its preparation was laborious and time-consuming. It is recombinantly produced as a zymogen, requiring acid activation and four chromatographic steps; despite these extensive steps, the catalytically active enzyme exhibited only moderate purity. Here, we report a novel preparation protocol, in which the cap and catalytically active core domains are produced as separate entities. The active enzyme can be obtained in two chromatographic steps, immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC), with no acid activation required, thereby shortening the purification procedure from at least 2 days to less than 6 h. In addition to the original C247A mutation which enhanced reaction with various amino nucleophiles, an extra D29E mutation was introduced to prevent self-cleavage, which led to noticeable improvements in homogeneity and activity of the enzyme. Indeed, the resulting "split AEP" (i.e., core domain of OaAEP1-D29E/C247A) exhibited improved catalytic efficiency constant (kcat/KM) that was found to be ∼3-fold higher than that of the original acid-activated counterpart (OaAEP1-C247A). Furthermore, we described a protein labeling protocol that couples the enzymatic reaction with an irreversible chemical transformation, thereby enabling high conversion of labeled protein with a lowered amount of reagent. Precisely, an alternative Asn-Cys-Leu (NCL) recognition sequence was used for substrate recognition. As the byproduct contains an N-terminal cysteine, it can be transformed into an inert 1,2 aminothiol motif by reacting with formylphenyl boronic acid (FPBA). Finally, the opportunities and challenges associated with the use of asparaginyl ligase are discussed.

The differential mediating effects of pain and depression on the physical and mental dimension of quality of life in Hong Kong Chinese adults.

OBJECTIVE: The impact of pain and depression on health-related quality of life (QoL) is widely investigated, yet the pain-depression interactions on QoL remain unclear. This study aims to examine the pain-depression-QoL mediation link. METHODS: Pain severity were assessed in a sample of Chinese professional teachers (n = 385). The subjects were also assessed on depressive symptoms and QoL. Regression models were fitted to evaluate the pain-depression-QoL relationships. RESULTS: About 44% of the sample had 3-5 painful areas in the past 3 months. Shoulder pain (60%) and headache (53%) were common painful areas. The results of regression analyses showed that pain mediated the effects of depression on the mental aspect of QoL (standardized beta = -0.111; Sobel test: z = -3.124, p < 0.005) whereas depression mediated the effects of pain on the physical aspect of QoL (standardized beta = -0.026; Sobel test: z = -4.045, p < 0.001). CONCLUSIONS: Our study offered tentative evidence that pain and depression impacted differently on the mental and physical aspect of QoL. As these findings were based on a Chinese teacher sample, future studies should employ more representative samples across cultures to verify the present data.

[Bioluminescent reporter genes]. / Systemy genów reporterowych opartych na zjawisku bioluminescencji.

Reporter genes typically are used to monitor changes in transcriptional rate, which can vary quickly in response to a specific cellular event. Here we give background on bioluminescent reporters and assays and their uses in research.

Intra-articular distribution and residence time of Hylan A and B: a study in the goat knee.

OBJECTIVE: This study evaluates the viscosupplementation material Hylan A and B in relation to its: (1) joint distribution, residence time and mechanism of removal and/or degradation, and (2) associated synovial fluid leukocyte response, in a goat model. METHOD: One green fluorophore was covalently bound to the Hylan A low molecular weight (MW) molecule (viscous fluid fraction) and a second red fluorophore was covalently bound to Hylan B high MW molecule (globule gel-like fraction). Goats were anesthetized and the right knee received 0.5 ml of test material or unbound fluorophore dyes. Gross and histological serial evaluations were performed over an 8-week period. RESULTS: By 24 h, the non-covalently linked control labels were not present in the tissues. For the covalently linked labels, the green fluorophore Hylan A diminished rapidly in intensity grossly but persisted to 28 days within the superficial synovial and articular cartilage layers in histologic sections. The red fluorophore linked Hylan B was seen only as globules in the synovial fluid. Mononuclear cells remained attached to these globules for 28 days and showed phagocytosis of the globules as well as the green fluorophore Hylan A. The globules were absent at 56 days after injection. The synovial fluid leukocyte count peaked at 24h (mean 9767 cells/mm(3) +/- 8574 S.D.) and declined by 7 days. CONCLUSIONS: The smaller MW Hylan was removed more rapidly than the higher MW Hylan. The globules were degraded by a different mechanism involving monocytes/macrophages on the surface of the higher MW globules.

The structure at 1.6 Angstroms resolution of the protein product of the At4g34215 gene from Arabidopsis thaliana.

The crystal structure of the At4g34215 protein of Arabidopsis thaliana was determined by molecular replacement and refined to an R factor of 14.6% (R(free) = 18.3%) at 1.6 Angstroms resolution. The crystal structure confirms that At4g34215 belongs to the SGNH-hydrolase superfamily of enzymes. The catalytic triad of the enzyme comprises residues Ser31, His238 and Asp235. In this structure the catalytic serine residue was found to be covalently modified, possibly by phenylmethylsulfonyl fluoride. The structure also reveals a previously undescribed variation within the active site. The conserved asparagine from block III, which provides a hydrogen bond for an oxyanion hole in the SGNH-hydrolase superfamily enzymes, is missing in At4g34215 and is functionally replaced by Gln30 from block I. This residue is positioned in a catalytically competent conformation by nearby residues, including Gln159, Gly160 and Glu161, which are fully conserved in the carbohydrate esterase family 6 enzymes.

The structure at 2.4 A resolution of the protein from gene locus At3g21360, a putative Fe(II)/2-oxoglutarate-dependent enzyme from Arabidopsis thaliana.

The crystal structure of the gene product of At3g21360 from Arabidopsis thaliana was determined by the single-wavelength anomalous dispersion method and refined to an R factor of 19.3% (Rfree = 24.1%) at 2.4 A resolution. The crystal structure includes two monomers in the asymmetric unit that differ in the conformation of a flexible domain that spans residues 178-230. The crystal structure confirmed that At3g21360 encodes a protein belonging to the clavaminate synthase-like superfamily of iron(II) and 2-oxoglutarate-dependent enzymes. The metal-binding site was defined and is similar to the iron(II) binding sites found in other members of the superfamily.

The structure at 1.7 A resolution of the protein product of the At2g17340 gene from Arabidopsis thaliana.

The crystal structure of the At2g17340 protein from A. thaliana was determined by the multiple-wavelength anomalous diffraction method and was refined to an R factor of 16.9% (Rfree = 22.1%) at 1.7 A resolution. At2g17340 is a member of the Pfam01937.11 protein family and its structure provides the first insight into the structural organization of this family. A number of fully and highly conserved residues defined by multiple sequence alignment of members of the Pfam01937.11 family were mapped onto the structure of At2g17340. The fully conserved residues are involved in the coordination of a metal ion and in the stabilization of loops surrounding the metal site. Several additional highly conserved residues also map into the vicinity of the metal-binding site, while others are clearly involved in stabilizing the hydrophobic core of the protein. The structure of At2g17340 represents a new fold in protein conformational space.

Structure at 1.6 A resolution of the protein from gene locus At3g22680 from Arabidopsis thaliana.

The gene product of At3g22680 from Arabidopsis thaliana codes for a protein of unknown function. The crystal structure of the At3g22680 gene product was determined by multiple-wavelength anomalous diffraction and refined to an R factor of 16.0% (Rfree = 18.4%) at 1.60 A resolution. The refined structure shows one monomer in the asymmetric unit, with one molecule of the non-denaturing detergent CHAPS {3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate} tightly bound. Protein At3g22680 shows no structural homology to any other known proteins and represents a new fold in protein conformation space.

The structure at 2.5 A resolution of human basophilic leukemia-expressed protein BLES03.

The crystal structure of the human basophilic leukemia-expressed protein (BLES03, p5326, Hs.433573) was determined by single-wavelength anomalous diffraction and refined to an R factor of 18.8% (Rfree = 24.5%) at 2.5 A resolution. BLES03 shows no detectable sequence similarity to any functionally characterized proteins using state-of-the-art sequence-comparison tools. The structure of BLES03 adopts a fold similar to that of eukaryotic transcription initiation factor 4E (eIF4E), a protein involved in the recognition of the cap structure of eukaryotic mRNA. In addition to fold similarity, the electrostatic surface potentials of BLES03 and eIF4E show a clear conservation of basic and acidic patches. In the crystal lattice, the acidic amino-terminal helices of BLES03 monomers are bound within the basic cavity of symmetry-related monomers in a manner analogous to the binding of mRNA by eIF4E. Interestingly, the gene locus encoding BLES03 is located between genes encoding the proteins DRAP1 and FOSL1, both of which are involved in transcription initiation. It is hypothesized that BLES03 itself may be involved in a biochemical process that requires recognition of nucleic acids.

High resolution X-ray structure of dTDP-glucose 4,6-dehydratase from Streptomyces venezuelae.

Desosamine is a 3-(dimethylamino)-3,4,6-trideoxyhexose found in some macrolide antibiotics. In Streptomyces venezuelae, there are seven genes required for the biosynthesis of this unusual sugar. One of the genes, desIV, codes for a dTDP-glucose 4,6-dehydratase, which is referred to as DesIV. The reaction mechanisms for these types of dehydratases are quite complicated with proton abstraction from the sugar 4'-hydroxyl group and hydride transfer to NAD+, proton abstraction at C-5, and elimination of the hydroxyl group at C-6 of the sugar, and finally return of a proton to C-5 and a hydride from NADH to C-6. Here we describe the cloning, overexpression, and purification, and high resolution x-ray crystallographic analysis to 1.44 A of wild-type DesIV complexed with dTDP. Additionally, for this study, a double site-directed mutant protein (D128N/E129Q) was prepared, crystallized as a complex with NAD+ and the substrate dTDP-glucose and its structure determined to 1.35 A resolution. In DesIV, the phenolate group of Tyr(151) and O(gamma) of Thr(127) lie at 2.7 and 2.6 A, respectively from the 4'-hydroxyl group of the dTDP-glucose substrate. The side chain of Asp(128) is in the correct position to function as a general acid for proton donation to the 6'-hydroxyl group while the side chain of Glu(129) is ideally situated to serve as the general base for proton abstraction at C-5. This investigation provides further detailed information for understanding the exquisite chemistry that occurs in these remarkable enzymes.

The structure of NADH in the enzyme dTDP-d-glucose dehydratase (RmlB).

The structure of Streptococcus suis serotype type 2 dTDP-d-glucose 4,6-dehydratase (RmlB) has been determined to 1.5 A resolution with its nicotinamide coenzyme and substrate analogue dTDP-xylose bound in an abortive complex. During enzyme turnover, NAD(+) abstracts a hydride from the C4' atom of dTDP-glucose-forming NADH. After elimination of water, hydride is then transferred back to the C6' atom of dTDP-4-keto-5,6-glucosene-regenerating NAD(+). Single-crystal spectroscopic studies unambiguously show that the coenzyme has been trapped as NADH in the crystal. Electron density clearly demonstrates that in contrast to native structures of RmlB where a flat nicotinamide ring is observed, the dihydropyridine ring of the reduced cofactor in this complex is found as a boat. The si face, from which the pro-S hydride is transferred, has a concave surface. Ab initio electronic structure calculations demonstrate that the presence of an internal hydrogen bond, between the amide NH on the nicotinamide ring and one of the oxygen atoms on a phosphate group, stabilizes this distorted conformation. Additionally, calculations show that the hydride donor ability of NADH is influenced by the degree of bending in the ring and may be influenced by an active-site tyrosine residue (Tyr 161). These results demonstrate the ability of dehydratase enzymes to fine-tune the redox potential of NADH through conformational changes in the nicotinamide ring.

Toward a structural understanding of the dehydratase mechanism.

dTDP-D-glucose 4,6-dehydratase (RmlB) was first identified in the L-rhamnose biosynthetic pathway, where it catalyzes the conversion of dTDP-D-glucose into dTDP-4-keto-6-deoxy-D-glucose. The structures of RmlB from Salmonella enterica serovar Typhimurium in complex with substrate deoxythymidine 5'-diphospho-D-glucose (dTDP-D-glucose) and deoxythymidine 5'-diphosphate (dTDP), and RmlB from Streptococcus suis serotype 2 in complex with dTDP-D-glucose, dTDP, and deoxythymidine 5'-diphospho-D-pyrano-xylose (dTDP-xylose) have all been solved at resolutions between 1.8 A and 2.4 A. The structures show that the active sites are highly conserved. Importantly, the structures show that the active site tyrosine functions directly as the active site base, and an aspartic and glutamic acid pairing accomplishes the dehydration step of the enzyme mechanism. We conclude that the substrate is required to move within the active site to complete the catalytic cycle and that this movement is driven by the elimination of water. The results provide insight into members of the SDR superfamily.

Symptomatic articular cartilage degeneration: the impact in the new millennium.

The symptomatic degeneration of articular cartilage and associated arthritis is among the most prevalent chronic conditions in the United States and the population most at risk is increasing. It is the leading cause of limitations in activities of daily living and is second to heart disease in causing work disability. The current and future socioeconomic impact of chronic articular cartilage disease on the healthcare system will be magnified by increasing numbers of patients who will seek relief of their symptoms and their disability to remain active. Because these individuals live longer and remain active, the proportion of their life living with symptoms and disability from articular cartilage degeneration increases. The economic, psychologic, and social impact of degenerative articular cartilage can be enormous for these individuals but it also impacts their family and society. The direct traditional medical costs and indirect economic and wage loss from arthritis in individuals the United States has reached in excess of $65 billion annually and is expected to increase as the population ages. In addition, the expenditures for complementary and alternative professional services and therapies for arthritis is increasing and is also in the billions of dollars annually. Because of these escalating costs, documenting the value of the patient and cost effectiveness to society of prevention and treatment programs for symptomatic articular cartilage degeneration will be required.

Cartilage substitutes: overview of basic science and treatment options.

Articular cartilage defects that are symptomatic and refractory to nonoperative treatment represent a clinical management challenge. Although there have been important advances in stimulating intrinsic repair mechanisms, cartilage regeneration, and other substitution techniques, to date none has unlocked the understanding necessary to duplicate normal articular cartilage. The objectives of treatment of cartilage lesions are to obtain pain relief, reduce effusions and inflammation, restore function, reduce disability, and postpone or alleviate the need for prosthetic replacement. As the field of articular cartilage repair continues to evolve rapidly, the most appropriate treatment option for an individual patient should be based on the pathologic characteristics of the lesion and the patient's symptoms and expectations. The orthopaedic surgeon needs to be familiar with both the existing and the newly emerging cartilage treatment techniques in order to best educate patients and meet their expectations for long-term benefits.

Spontaneous repair of full-thickness defects of articular cartilage in a goat model. A preliminary study.

BACKGROUND: Full-thickness defects measuring 3 mm in diameter have been commonly used in studies of rabbits to evaluate new procedures designed to improve the quality of articular cartilage repair. These defects initially heal spontaneously. However, little information is available on the characteristics of repair of larger defects. The objective of the present study was to define the characteristics of repair of 6-mm full-thickness osteochondral defects in the adult Spanish goat. METHODS: Full-thickness osteochondral defects measuring 6 x 6 mm were created in the medial femoral condyle of the knee joint of adult female Spanish goats. The untreated defects were allowed to heal spontaneously. The knee joints were removed, and the defects were examined at ten time-intervals, ranging from time zero (immediately after creation of the defect) to one year postoperatively. The defects were examined grossly, microradiographically, histologically, and with magnetic resonance imaging and computed tomography. RESULTS: The 6-mm osteochondral defects did not heal. Moreover, heretofore undescribed progressive, deleterious changes occurred in the osseous walls of the defect and the articular cartilage surrounding the defect. These changes resulted in a progressive increase in the size of the defect, the formation of a large cavitary lesion, and the collapse of both the surrounding subchondral bone and the articular cartilage into the periphery of the defect. Resorption of the osseous walls of the defect was first noted by one week, and it was associated with extensive osteoclastic activity in the trabecular bone of the walls of the defect. Flattening and deformation of the articular cartilage at the edges of the defect was also observed at this time. By twelve weeks, bone resorption had transformed the surgically created defect into a larger cavitary lesion, and the articular cartilage and subchondral bone surrounding the defect had collapsed into the periphery of the defect. By twenty-six weeks, bone resorption had ceased and the osseous walls of the lesion had become sclerotic. The cavitary lesion did not become filled in with fibrocartilage. Instead, a cystic lesion was found in the center of most of the cavitary lesions. Only a thin layer of fibrocartilage was present on the sclerotic osseous walls of the defect. Specimens examined at one year postoperatively showed similar characteristics. CONCLUSIONS: Full-thickness osteochondral defects, measuring 6 mm in both diameter and depth, that are created in the medial femoral condyle of the knee joint of adult Spanish goats do not heal spontaneously. Instead, they undergo progressive changes resulting in resorption of the osseous walls of the defect, the formation of a large cavitary lesion, and the collapse of the surrounding articular cartilage and subchondral bone. CLINICAL RELEVANCE: As surgeons apply new reparative procedures to larger areas of full-thickness articular cartilage loss, we believe that it is important to consider the potential deleterious effects of a "zone of influence" secondary to the creation of a large defect in the subchondral bone. When biologic and synthetic matrices with or without cells or bioactive factors are placed into surgically created osseous defects, the osseous walls serve as shoulders to protect and stabilize the preliminary repair process. It is important to protect the repair process until biologic incorporation occurs and the chondrogenic switch turns the cells on to synthesize an articular-cartilage-like matrix. It takes a varying period of time to fill a large, surgically created bone defect underlying a chondral surface. The repair of such a defect requires bone synthesis and the reestablishment of a subchondral plate with a tidemark transition to the new overlying articular surface. The prevention of secondary changes in the surrounding bone and articular cartilage and the durability of the new reparative tissue making up the articulating surface are issues that must be addressed in future studies.

Tissue engineering principles in orthopaedic surgery.

Advances in the fields of biotechnology and biomaterials offer the orthopaedic surgeon the exciting possibility of repair or regeneration of tissue lost to injury, disease, or aging. The promising area of tissue engineering represents a multidisciplinary approach aimed at solving some of the most perplexing biologic problems, namely, the creation of new tissues and organs similar to the original tissues and organs. In addition, strategies using new synthetic polymer formulations can facilitate tissue replacement and represent alternatives to tissue regeneration in certain conditions. Although biotechnology has broad application over many medical specialties, orthopaedics is receiving a large focus of the research effort devoted to techniques for developing bone, articular cartilage, ligaments, and tendons. Because bioengineered tissue and/or techniques to stimulate tissue regeneration soon may be used clinically, orthopaedic surgeons should have a working knowledge of the basic concepts involved. Terms, such as biomaterial, biotechnology, matrices of synthetic or biologic polymers or both, adhesion, cohesion, porosity, induction, conduction, stem cell, progenitor cell, mesenchymal cell, tissue growth factor, bone morphogenetic protein, mitogenic and chemotactic factors, and numerous other terms will become part of the working language of clinicians of the twenty-first century.

Reduced anterior tibial translation associated with adaptive changes in the anterior cruciate ligament-deficient joint: goat model.

Adaptive changes in the menisci and adjacent posterior capsule were documented within anterior cruciate ligament-deficient knee (stifle) joints in the goat model. These physical changes in the menisci and capsule developed over time and were associated with reduction in the initial (time zero) abnormal anterior tibial translation following transection of the anterior cruciate ligament. At 50 N of applied force, the normal goat knee joint has a total anterior-posterior translation of 0.6+/-0.1 mm (+/- SEM) at 45 degrees of flexion and 0.3+/-0.1 mm at 90 degrees. The translation immediately after transection (time zero) with 50 N of force was 8.2+/-0.5 mm at 45 degrees and 4.9+/-0.9 mm at 90 degrees. Within 8 months after transection and at 50 N of force, the treated knees had reduced translation values of 5.3+/-0.6 mm at 45 degrees of flexion and 2.9+/-0.5 mm at 90 degrees, or 35 (p<0.001) and 40% reductions, respectively, compared with the values at time zero. Magnetic resonance images of the ligament-deficient stifle joints, as well as gross measurements and image analysis after dissection, consistently demonstrated increases in cross-sectional area and volume of the menisci compared with the contralateral controls. These secondary changes were most pronounced in the posterior portion of the medial menisci, and histologic evaluation demonstrated hypercellularity with the accumulation of poorly organized collagen, reduced safranin O staining (proteoglycan matrix synthesis), a thickened capsule and capsule attachment, and increased vascularity at the meniscal capsule interface.

Characterization of the repair tissue after removal of the central one-third of the patellar ligament. An experimental study in a goat model.

UNLABELLED: The purpose of this study was to characterize the repair tissue that develops after removal of a portion of the patellar ligament for use as a graft. A six-millimeter-wide strip was obtained from the central portion of the patellar ligament with tibial and patellar bone plugs from one knee (stifle joint) of eight goats. The repair tissue that formed in the defect was characterized in terms of its structural, material, histological, and ultrastructural properties twenty-one months after the operation. The contralateral patellar ligament served as a control. Representative specimens were taken from the proximal, middle, and distal portions of the repair tissue and the control tissue for histological study and examination with transmission electron microscopy. The six-millimeter-long defect filled with repair tissue that increased the cross-sectional area by a mean of 42 per cent compared with the control values (p < 0.05). The maximum force to failure and the ultimate stress of the repair tissue were significantly decreased (by a mean of 51 and 65 per cent, respectively) compared with those of the controls (p < 0.001 for both). The stiffness also was reduced, by a mean of 27 per cent, but this was not significant (p > 0.05). Magnetic resonance imaging of the donor site showed slightly increased signal intensity compared with the intensity on the control side. Histological sections from the donor site contained collagenous (scar) tissue that was less organized, more cellular, and more vascular than the control tissue. Evaluation of the ultrastructure revealed that the repair tissue was composed primarily of collagen fibrils with a small diameter (range, fifty to 100 nanometers). CLINICAL RELEVANCE: The results of the present study suggest that the repair tissue that develops after removal of a strip of the patellar ligament for use as a graft is not comparable with normal tissue in terms of its structural, material, histological, and ultrastructural properties by twenty-one months. This should be kept in mind when this repair tissue is considered for use as a graft for revision of a reconstruction of the anterior cruciate ligament.

Chondrocyte transplantation.

The transplantation of chondrocytes as a treatment to repair defects and degeneration in hyaline articular cartilage is being tested in numerous laboratory and clinical settings. This has included transplanting chondrocytes grown in tissue culture that were procured from non-weight-bearing areas of the affected joint to transplanting allografts with living chondrocytes in their intact cartilaginous matrix. Reported success with transplanting host and donor chondrocytes has varied and widespread application of these techniques still awaits more definitive studies. The clinician needs more evidence that the transplanted chondrocytes maintain their viability and that they synthesize the appropriate extracellular matrix. This new matrix needs to reproduce the functional, mechanical, and long-term wear properties of the native articular cartilage. Chondrocyte transplantation also merits further monitoring for possible delayed immunogenicity or for any signs of neoplastic potential. This exciting technology and its potential application to damaged and degenerated articular cartilage remains a stimulus to encourage further scientific work. Duplicating the unique and complex interrelations of the chondrocytes, matrix, and various bioactive factors is still some years away from general patient care.