Endoscopic carpal tunnel release surgery: report of patient satisfaction.

Recently, a trend has developed to use an endoscope to achieve carpal tunnel release. Proponents of the endoscopic technique believe it has benefits to patients that include minimal incision, minimal pain and scarring, a shortened recovery period and a high level of patient satisfaction. To test these beliefs, a retrospective analysis of the first 42 cases that were done between May 1997 and June 1998 was completed. Endoscopic carpal tunnel release surgery was performed on patients with the classical clinical and neurophysiological findings of carpal tunnel syndrome. The procedure was performed in an outpatient surgery center under primarily local anesthesia and by the same neurosurgeon (RG), who was blind to data analysis. The biportal technique (Instratek Inc., Houston, TX, USA) was used as described by Brown. The first 42 patients (n = 35, seven patients had bilateral surgeries) were sent a survey (modified Health Outcomes Carpal Tunnel Questionnaire, Health Outcomes, Bloomington, MN, USA) that measured a wide spectrum of variables, with a year follow-up. Patient demography indicated wide patient selection. All subjects (100%) had claimed work-related injury. Patient satisfaction was reported in 86%. No or mild incisional pain, night pain, absent tingling, and improved grip strengthening were reported in 100%, 95%, 81%, and 85% respectively. The mean for return to daily activity and work was 14 and 25 days respectively. No recurrent hematoma, infection, or structure injury was reported. Endoscopic carpal tunnel release can be done safely and effectively with excellent self-reports of patient satisfaction. Reduced recovery period and hospitalization with minimal tissue violation and incisional pain can be expected.

Small heat-shock protein structures reveal a continuum from symmetric to variable assemblies.

The small heat-shock proteins (sHSPs) form a diverse family of proteins that are produced in all organisms. They function as chaperone-like proteins in that they bind unfolded polypeptides and prevent uncontrolled protein aggregation. Here, we present parallel cryo-electron microscopy studies of five different sHSP assemblies: Methanococcus jannaschii HSP16.5, human alphaB-crystallin, human HSP27, bovine native alpha-crystallin, and the complex of alphaB-crystallin and unfolded alpha-lactalbumin. Gel-filtration chromatography indicated that HSP16.5 is the most monodisperse, while HSP27 and the alpha-crystallin assemblies are more polydisperse. Particle images revealed a similar trend showing mostly regular and symmetric assemblies for HSP16.5 particles and the most irregular assemblies with a wide range of diameters for HSP27. A symmetry test on the particle images indicated stronger octahedral symmetry for HSP16.5 than for HSP27 or the alpha-crystallin assemblies. A single particle reconstruction of HSP16.5, based on 5772 particle images with imposed octahedral symmetry, resulted in a structure that closely matched the crystal structure. In addition, the cryo-EM reconstruction revealed internal density presumably corresponding to the flexible 32 N-terminal residues that were not observed in the crystal structure. The N termini were found to partially fill the central cavity making it unlikely that HSP16.5 sequesters denatured proteins in the cavity. A reconstruction calculated without imposed symmetry confirmed the presence of at least loose octahedral symmetry for HSP16.5 in contrast to the other sHSPs examined, which displayed no clear overall symmetry. Asymmetric reconstructions for the alpha-crystallin assemblies, with an additional mass selection step during image processing, resulted in lower resolution structures. We interpret the alpha-crystallin reconstructions to be average representations of variable assemblies and suggest that the resolutions achieved indicate the degree of variability. Quaternary structural information derived from cryo-electron microscopy is related to recent EPR studies of the alpha-crystallin domain fold and dimer interface of alphaA-crystallin.

Review: resolution issues in single-particle reconstruction.

Specific factors that affect the resolution of single-particle reconstructions are discussed. We present reconstructions of six particles (DNA-dependent protein kinase catalytic subunit, alphaB-crystallin, the ribonucleoprotein vault, hepatitis A virus, adenovirus type 2, and the adenovirus type 12/alpha(v)beta5 integrin complex), which have a variety of symmetries (asymmetric to 60-fold) and a wide range of molecular masses (470 kDa to 150 MDa). In the case of icosahedral viruses, we have found that applying a "soft" mask to remove regions of disordered density improves the resolution given by the Fourier shell correlation 0.5 criterion. This masking procedure is also useful during refinement to improve the quality of the reference model and thus aid in precise alignment of the particle images. For asymmetric particles, we note that image classification, although often a necessary step to generate a first reconstruction, can limit the achievable resolution. The diameter of the particle and the available computational power can also affect the resolution, as can structural variability within the particle.

Mutation R120G in alphaB-crystallin, which is linked to a desmin-related myopathy, results in an irregular structure and defective chaperone-like function.

alphaB-crystallin, a member of the small heat shock protein family, possesses chaperone-like function. Recently, it has been shown that a missense mutation in alphaB-crystallin, R120G, is genetically linked to a desmin-related myopathy as well as to cataracts [Vicart, P., Caron, A., Guicheney, P., Li, A., Prevost, M.-C., Faure, A., Chateau, D., Chapon, F., Tome, F., Dupret, J.-M., et al. (1998) Nat. Genet. 20, 92-95]. By using alpha-lactalbumin, alcohol dehydrogenase, and insulin as target proteins, in vitro assays indicated that R120G alphaB-crystallin had reduced or completely lost chaperone-like function. The addition of R120G alphaB-crystallin to unfolding alpha-lactalbumin enhanced the kinetics and extent of its aggregation. R120G alphaB-crystallin became entangled with unfolding alpha-lactalbumin and was a major portion of the resulting insoluble pellet. Similarly, incubation of R120G alphaB-crystallin with alcohol dehydrogenase and insulin also resulted in the presence of R120G alphaB-crystallin in the insoluble pellets. Far and near UV CD indicate that R120G alphaB-crystallin has decreased beta-sheet secondary structure and an altered aromatic residue environment compared with wild-type alphaB-crystallin. The apparent molecular mass of R120G alphaB-crystallin, as determined by gel filtration chromatography, is 1.4 MDa, which is more than twice the molecular mass of wild-type alphaB-crystallin (650 kDa). Images obtained from cryoelectron microscopy indicate that R120G alphaB-crystallin possesses an irregular quaternary structure with an absence of a clear central cavity. The results of this study show, through biochemical analysis, that an altered structure and defective chaperone-like function of alphaB-crystallin are associated with a point mutation that leads to a desmin-related myopathy and cataracts.

Lens alpha-crystallin: function and structure.

alpha-Crystallin is a major lens protein, comprising up to 40% of total lens proteins, where its structural function is to assist in maintaining the proper refractive index in the lens. In addition to its structural role, it has been shown to function in a chaperone-like manner. The chaperone-like function of alpha-crystallin will help prevent the formation of large light-scattering aggregates and possibly cataract. In the lens, alpha-crystallin is a polydisperse molecule consisting of a 3:1 ratio of alpha A to alpha B subunits. In this study, we expressed recombinant alpha A- and alpha B-crystallin in E. coli and compared the polydispersity, structure and aggregation state between each other and native bovine lens alpha-crystallin. Using gel permeation chromatography to assay for polydispersity, we found native alpha-crystallin to be significantly more polydisperse than either recombinant alpha A- or alpha B-crystallin, with alpha B-crystallin having the most homogeneous structure of the three. Reconstructed images of alpha B-crystallin obtained with cryo-electron microscopy support the concept that alpha B-crystallin is an extremely dynamic molecule and demonstrated that it has a hollow interior. Interestingly, we present evidence that native alpha-crystallin is significantly more thermally stable than either alpha A- or alpha B-crystallin alone. In fact, our experiments suggest that a 3:1 ratio of alpha A to alpha B subunit composition in an alpha-crystallin molecule is optimal in terms of thermal stability. This fascinating result explains the stoichiometric ratios of alpha A- and alpha B-crystallin subunits in the mammalian lens.

The small heat-shock protein, alphaB-crystallin, has a variable quaternary structure.

alphaB-crystallin is a major structural protein in the lens that is found in a variety of other tissues and is associated with numerous neurological disorders. It is a member of the small heat-shock protein family and possesses chaperone-like properties. Cryo-electron microscopy has been applied to analyze the quaternary structure of human recombinant alphaB-crystallin, which spontaneously forms roughly spherical multimers 8 to 18 nm in diameter. Class-sum images based on nearly 5000 alphaB-crystallin particles reveal the presence of a large central cavity, weak regions of density within the protein shell, and an asymmetric quaternary structure. The class-sum images are variable in size and shape, and are suggestive of snapshots of a conformationally flexible assembly. As gel-filtration chromatography reveals a range of molecular masses (650 (+/-140) kDa) for the assembly, the class-sum images were further classified on the basis of total molecular mass. A reconstruction at approximately 4 nm resolution was calculated from the images assigned to 32 subunit (approximately 645 kDa) assemblies. Comparison of class-sum images with reprojections of the reconstruction indicates that the resolution is limited by the variable nature of the assembly. A three-dimensional variance map indicates significant structural divergence within the protein shell and on the outer surface of the particle. Some of the strong variance may correspond to the flexible, exposed C-terminal residues of the alphaB-crystallin monomers. The variable quaternary structure of alphaB-crystallin is consistent with the polydisperse size of the assembly and the previously observed subunit exchange between multimers. Thus, we propose that the monomer packing is variable, and that the quaternary structure of the assembly is not completely defined. A variable alphaB-crystallin quaternary structure may facilitate binding of target proteins in up to stoichiometric ratios.

Pharmacological analysis of the magnocellular red nucleus during classical conditioning of the rabbit nictitating membrane response.

Previous experiments have suggested that the red nucleus is an essential structure in the neural pathways subserving the conditioned responses (CRs) elicited in several simple associative learning paradigms. The present investigation confirms the involvement of the magnocellular red nucleus in production of the classically conditioned nictitating membrane response in the rabbit and suggests that gamma-aminobutyric acid (GABA) processes within this structure are involved in expression of the CR. Specifically, these studies demonstrate that microinfusion of a GABA antagonist (either picrotoxin or bicuculline methiodide) into the magnocellular red nucleus can selectively and reversibly reduce or abolish retention of the CR, without altering the unconditioned reflex response. Furthermore, these pharmacological manipulations that disrupt the CR are both anatomically and pharmacologically specific, and demonstrate a predictable dose-dependent function. These findings suggest that GABAergic processes within the magnocellular red nucleus are part of the critical circuitry subserving the CR.