3- Instead of 4-helix formation in a de novo designed protein in solution revealed by small-angle X-ray scattering

De novo design and chemical synthesis of proteins and their mimics are central approaches for understanding protein folding and accessing proteins with novel functions. We have previously described carbohydrates as templates for the assembly of artificial proteins, so-called carboproteins. Here, we describe the preparation and structural studies of three alpha-helical bundle carboproteins, which were assembled from three different carbohydrate templates and one amphiphilic hexadecapeptide sequence. This heptad repeat peptide sequence has been reported to lead to 4-alpha-helix formation. The low resolution solution structures of the three carboproteins were analyzed by means of small-angle X-ray scattering (SAXS) and synchrotron radiation circular dichroism (SRCD). The ab initio SAXS data analysis revealed that all three carboproteins adopted an unexpected 3+1-helix folding topology in solution, while the free peptide formed a 3-helix bundle. This finding is consistent with the calculated alpha-helicities based on the SRCD data, which are 72 and 68 % for two of the carboproteins. The choice of template did not affect the overall folding topology (that is for the 3+1 helix bundle) the template did have a noticeable impact on the solution structure. This was particularly evident when comparing 4-helix carboprotein monomers with the 2x2-helix carboprotein dimer as the latter adopted a more compact conformation. Furthermore, the clear conformational differences observed between the two 4-helix (3+1) carboproteins based on D-altropyranoside and D-galactopyranoside support the notion that folding is affected by the template, and subtle variations in template distance-geometry design may be exploited to control the solution fold. In addition, the SRCD data show that template assembly significantly increases thermostability.     

Expression of genes encoding the E2 and E3 components of the Bacillus stearothermophilus pyruvate dehydrogenase complex and the stoichiometry of subunit interaction in assembly in vitro

Genes encoding the dihydrolipoyl acetyltransferase (E2) and dihydrolipoyl dehydrogenase (E3) components of the pyruvate dehydrogenase (PDH) multienzyme complex from Bacillus stearothermophilus were overexpressed in Escherichia coli. The E2 component was purified as a large soluble aggregate (molecular mass > 1 x 10(6) Da) with the characteristic 532 symmetry of an icosahedral (60-mer) structure, and the E3 as a homodimer with a molecular mass of 110 kDa. The recombinant E2 component in vitro was capable of binding either 60 E3(alpha2) dimers or 60 heterotetramers (alpha2beta2) of the pyruvate decarboxylase (E1) component (also the product of B. stearothermophilus genes overexpressed in E. coli). Assembling the E2 polypeptide chain into the icosahedral E2 core did not impose any restriction on the binding of E1 or E3 to the peripheral subunit-binding domain in each E2 chain. This has important consequences for the stoichiometry of the assembled complex in vivo. The lipoyl domain of the recombinant E2 protein was found to be unlipoylated, but it could be correctly post-translationally modified in vitro using a recombinant lipoate protein ligase from E. coli. The lipoylated E2 component was able to bind recombinant E1 and E3 components in vitro to generate a PDH complex with a catalytic activity comparable with that of the wild-type enzyme. Reversible unfolding of the recombinant E2 and E3 components in 6 M guanidine hydrochloride was possible in the absence of chaperonins, with recoveries of enzymic activities of 95% and 85%, respectively. However, only 26% of the E1 enzyme activity was recovered under the same conditions as a result of irreversible denaturation of both E1alpha and E1beta. This represents the first complete post-translational modification and assembly of a fully active PDH complex from recombinant proteins in vitro.     

Change in optic disk topography after trabeculectomy

PURPOSE: To investigate the relationship between optic disk topography and intraocular pressure before and after trabeculectomy with confocal scanning laser ophthalmoscopy. METHODS: The eyes of 49 consecutive patients undergoing trabeculectomy at a university-based glaucoma practice underwent preoperative and postoperative imaging using a confocal scanning laser ophthalmoscope (Heidelberg Retina Tomograph). Three images of one eye of each patient were obtained with a 15-degree field of view. Preoperative images were obtained approximately 2 months before surgery (mean +/- SD, 2.4 +/- 1.6 months). Postoperative images were obtained at least 3 months after surgery (mean, 4.5 +/- 2.6 months). RESULTS: Mean preoperative intraocular pressure, postoperative intraocular pressure, and percent change in intraocular pressure respectively were 23.1 +/- 6.8 mm Hg, 12.7 +/- 7.1 mm Hg, and 43.8% +/- 29.9%. A significant association (P < .01) was found between percent decrease in intraocular pressure and decreases in cup area, cup volume, and cup/disk area ratio as well as between percent decrease in intraocular pressure and increases in rim area, rim volume, mean height contour, retinal cross-section area, and height in contour. Between 11.7% and 31.2% of the variability (R2) in these parameters was explained by the percent change in intraocular pressure. Topography changes were more strongly associated with percent change than with mean change in intraocular pressure. We found no association between percent decrease in intraocular pressure and reference plane height or maximum cup depth. CONCLUSIONS: Changes in optic nerve topography were associated with reduction in intraocular pressure after trabeculectomy.     

State laws on youth access to tobacco in the United States: measuring their extensiveness with a new rating system

OBJECTIVE: To develop and implement a rating system evaluating the extensiveness of state laws restricting youth access to tobacco. DESIGN: State laws on youth access to tobacco were analysed and assigned ratings on nine items. Six items addressed specific tobacco-control provisions, and three related to enforcement provisions. For each item, a target was specified reflecting public health objectives. Achieving the target resulted in a rating of +4 points; for three items, a rating of +5 was possible if the target was exceeded. Criteria for lower ratings were established for situations when the target was not met. SETTING: United States. RESULTS: State scores (sum of the ratings across all nine items) ranged from 0-18 in 1993, 2-21 in 1994, and 1-21 in 1995 and 1996, out of a possible total of 39. The average score across states was 7.2 in 1993, 7.9 in 1994, 8.2 in 1995, and 9.0 in 1996. The overall mean rating (per item) was 0.80 in 1993, 0.88 in 1994, 0.91 in 1995, and 1.00 in 1996, on a scale where 4.0 indicates that the target goals (per item) were met. From 1993 to 1996, scores increased for 20 states, decreased for one state, and remained unchanged for the others. The number of states for which state preemption of local tobacco regulation was a factor doubled from 10 states in 1993 to 20 states in 1996. CONCLUSIONS: Although all states have laws addressing youth access to tobacco, this analysis reveals that, as of the end of 1996, the progress towards meeting health policy targets is slow, and state legislation that preempts local tobacco regulation is becoming more common.     

Technology adoption as process: a case of integrating an information-intensive website into a patient education helpline

This study followed the introduction of the Arthritis Source website into the existing teaching practices of Arthritis Foundation Helpline volunteers. The goal was to examine what factors may affect a particular group of educators adopt a potentially valuable Internet tool into an existing instructional environment. Defining the possible uses of the website in reference to the volunteers' actual job duties helped provide a clearer understanding of how the volunteers might use this new technology. The researchers used qualitative techniques to focus on three volunteers who experienced different physical, environmental and cognitive means that impacted their use of the new tool. Each volunteer experienced varying levels of motivation in areas of learning, satisfaction and responses to outside influences. Each volunteer also had varying amounts of opportunity prompts in which to interact or refer the website. Consequently, Helpline volunteers experienced different rates of adopting the information-intensive website into their traditional work system.     

Range of motion and complications after postburn heterotopic bone excision about the elbow

OBJECTIVE: To review the results of surgical management of heterotopic ossification about the elbow in burned patients. DESIGN: Retrospective analysis with long-term patient follow-up. MATERIALS AND METHODS: Eleven patients with 16 elbows requiring surgery were admitted between January 1, 1982 and December 31, 1993. A posterior approach to the elbow with release of the encased ulnar nerve +/- anterior transposition and transolecranon osteotomy to access extensive bone formation in the olecranon fossa was employed. Eight patients (11 elbows) were available for long-term follow-up conducted at mean 50 +/- 13 months after surgery. Long-term follow-up consisted of measurement of range of elbow motion, as well as clinical assessment of ulnar nerve function. MAIN RESULTS: For the 11 elbows examined postoperatively, the mean range of motion preoperatively in flexion-extension was 11 degrees +/- 5 degrees compared to 89 degrees +/- 12 degrees postoperatively (p < 0.001). Three patients with poor long-term results had ankylosis of the joint preoperatively. Of four patients with ulnar nerve paresis preoperatively, none had ulnar nerve dysfunction at follow-up. Of 16 elbows operated on, four (25%) had postoperative complications. Two suffered soft-tissue breakdown with hardware exposure requiring abdominal flap closure, one early failure of olecranon fixation, and one late infected hardware. CONCLUSIONS: Surgery for both limited range of motion as well as ulnar nerve compression is effective in cases of heterotopic ossification about the elbows of burned patients. Early operative intervention is indicated in progressive disease, particularly ulnar nerve palsy, if soft-tissue quality is adequate. Complications with 25% of elbows suggest that use of olecranon osteotomy for joint access may warrant review.     

The Imbrication Model for the integration of social services in a community

An innovative model for organising social services in a community, the Imbrication Model, is contrasted with two traditional models, the Entrepreneurial and the Umbrella Agency. The structural characteristics and dynamics of the three models are illustrated with actual case histories. Imbrication Model calls for the interlocking of personnel from several agencies, with the purpose of redirecting the dysfunctional interagency rivalry prevalent in the traditional models. Imbrications at all organisational levels--Board of Directors, Administrators and Staff--facilitate adoption of the superordinate goal of providing clients with the best services available, regardless of which particular agency delivers the service. Few observers of the current social service scene would challenge the statement that needs for service are unlimited and resources limited. In the USA the imbalance between needs and resources persists despite a decade of massive governmental programmes intended to alleviate social ills. Recent substantial cutbacks in federal funds, moreover are not likely to improve the situation. The resource shortage involves more than a limitation of funds. Deliverers of service and competent programme administrators are also on critically short supply. These shortages are more often than not exacerbated by a chronic spirit of competition among agencies and programmes at the local level. Three organizational models for the delivery and administration of social services, two conventional and one of more recent date, are examined in this article. The innovative model, which has been named the Imbrication Model, explicitly calls for redirecting interagency rivalry and competition. Its ambitious goal is to integrate the efforts of those attempting to meet a community's social service needs.