Structural essentials for ß-N-acetylhexosaminidase inhibition by amides of prolines, pipecolic and azetidine carboxylic acids.

This paper explores the computer modelling aided design and synthesis of ß-N-acetylhexosaminidase inhibitors along with their applicability to human disease treatment through biological evaluation in both an enzymatic and cellular setting. We investigated the importance of individual stereocenters, variations in structure-activity relationships along with factors influencing cell penetration. To achieve these goals we modified nitrogen heterocycles in terms of ring size, side chains present and ring nitrogen derivatization. By reducing the inhibitor interactions with the active site down to the essentials we were able to determine that besides the established 2S,3R trans-relationship, the presence and stereochemistry of the CH2OH side chain is of crucial importance for activity. In terms of cellular penetration, N-butyl side chains favour cellar uptake, while hydroxy- and carboxy-group bearing sidechains on the ring nitrogen retarded cellular penetration. Furthermore we show an early proof of principle study that ß-N-acetylhexosaminidase inhibitors can be applicable to use in a potential anti-invasive anti-cancer strategy.

Health-related quality of life of women with menopausal hot flushes and night sweats.

OBJECTIVES: Despite a large body of research on menopause, there is little definitive evidence of the impact of vasomotor symptoms on health-related quality of life (HrQoL). Therefore, this study describes the HrQoL of menopausal women with hot flushes and night sweats and examines predictors of HrQoL. METHODS: A total of 140 women reporting at least ten hot flushes/night sweats (vasomotor symptoms) a week for at least a month completed an assessment interview (including medical history, past and current physical and mental health and menopausal status) and questionnaires eliciting sociodemographic and help-seeking information, HrQoL and the Hot Flush Rating Scale. RESULTS: Women with vasomotor symptoms reported somewhat reduced HrQoL compared to SF-36 US norms and a general sample of UK menopausal women; 53% reported comorbid physical illness and 66% had current psychosocial concerns; 77% had visited their doctor about menopausal symptoms; 28% were past and 3% current users of hormone therapy. Overall, poor HrQoL was associated with having problematic hot flushes, current psychosocial concerns, (younger) age, (higher) body mass index and poor general health. CONCLUSIONS: This sample of relatively healthy mid-aged women with vasomotor symptoms reported reduced HrQoL compared to age-matched norms and a general sample of menopausal women. Problem rating, rather than frequency, of hot flushes was associated with reduced HrQoL, as were health and psychosocial factors.

Setting performance standards for medical practice: a theoretical framework.

BACKGROUND: The assessment of performance in the real world of medical practice is now widely accepted as the goal of assessment at the postgraduate level. This is largely a validity issue, as it is recognised that tests of knowledge and in clinical simulations cannot on their own really measure how medical practitioners function in the broader health care system. However, the development of standards for performance-based assessment is not as well understood as in competency assessment, where simulations can more readily reflect narrower issues of knowledge and skills. This paper proposes a theoretical framework for the development of standards that reflect the more complex world in which experienced medical practitioners work. METHODS: The paper reflects the combined experiences of a group of education researchers and the results of literature searches that included identifying current health system data sources that might contribute information to the measurement of standards. CONCLUSION: Standards that reflect the complexity of medical practice may best be developed through an "expert systems" analysis of clinical conditions for which desired health care outcomes reflect the contribution of several health professionals within a complex, three-dimensional, contextual model. Examples of the model are provided, but further work is needed to test validity and measurability.

The reproducibility of assessing radiological reporting: studies from the development of the General Medical Council's Performance Procedures.

OBJECTIVES: To investigate the reproducibility of peer ratings of consultant radiologists' reports, as part of the new General Medical Council (GMC) Performance Procedures. DESIGN: An evaluation protocol was piloted, used in a blocked, balanced, randomized generalizability analysis with three blocks of three judges (raters), each rating 30 reports from 10 radiologists, and re-rated to estimate intrarater reliability with conventional statistics (kappa). SETTING: Rating was performed at the Royal College of Radiologists. Volunteers were sampled from 23 departments of radiology in university teaching and district general hospitals. PARTICIPANTS: A nationally drawn non-random sample of 30 consultant radiologists contributing a total of 900 reports. Three trained and six non-trained judges were used in the rating analysis. RESULTS: A protocol was generated that was usable by judges. Generalizable results would be obtained with not less than three judges all rating the same 60 reports from a radiologist. CONCLUSIONS: Any assessment of performance of technical abilities in this field will need to use multiple assessors, basing judgements on an adequate sample of reports.

Iliac to popliteal artery bypass through the iliac wing: An alternative extracavitary route for management of complex groin injuries.

Extracavitary bypass through the iliac wing allows placement of the grafts into the posterior thigh and is another alternative route when an obturator bypass is not possible, or an axillary-popliteal bypass is to be avoided. The transiliac wing bypass is relatively simple and easy to perform. The bypass route is short and direct, has excellent inflow, and is accompanied by minimal neurological or bleeding risks. An illustrative case is presented with a complete description of the operative technique. Review of the literature is also included.

Chemical ligation of folded recombinant proteins: segmental isotopic labeling of domains for NMR studies.

A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N. Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.

Introduction of unnatural amino acids into proteins using expressed protein ligation.

Here we describe the results of studies designed to explore the scope and limitations of expressed protein ligation (EPL), a protein semisynthesis approach that allows unnatural amino acids to be site specifically introduced into large proteins. Using Src homology 3 domains from the proteins c-Abl and c-Crk as model systems, we show here that EPL can be performed in the presence of moderate concentrations of the chemical denaturant, guanidine hydrochloride, and the organic solvent dimethylsulfoxide. Use of these solubilizing agents allowed the successful preparation of two semisynthetic proteins, 10 and 12, both of which could not be prepared using standard procedures due to the low solubility of the synthetic peptide reactants in aqueous buffers. We also report the results of thiolysis and kinetic studies which indicate that stable alkyl thioester derivatives of recombinant proteins can be generated for storage and purification purposes, and that 2-mercaptoethanesulfonic acid compares favorably with thiophenol as the thiol cofactor for EPL reactions, while having superior handling properties. Finally, we describe the semisynthesis of the fluorescein/rhodamine-containing construct (12) and the ketone-containing construct (14). The efficiency of these two syntheses indicates that EPL offers a facile way of incorporating these important types of biophysical and biochemical probes into proteins.

Studying receptor-ligand interactions using encoded amino acid scanning.

A novel technique is described that allows the synthesis, functional analysis, and quantitative readout of defined arrays of polypeptide analogues in aqueous solution. Key to this approach is the use of a simple encoding-decoding system in which a unique Fmoc-amino acid tag is covalently attached to the C terminus of each member of a molecular array through a selectively cleavable bond. These tags can be cleanly removed from the molecules they encode, allowing single-step characterization and quantification of the entire mixture by HPLC. The utility of this technique is illustrated through the preparation of an array of proline-rich sequences based on the exchange factor C3G, one of the natural ligands of the N-terminal SH3 domain from the proto-oncogene, c-Crk. The array was designed to systematically modify those residues within the C3G peptide ligand thought to make key interactions with the c-Crk SH3 domain. Using competition binding experiments, it was possible to determine the relative ED50 values for the entire array of molecules simultaneously. These studies revealed that in order to maintain optimal binding to the SH3 domain, the P-3 side chain of the ligand must be positively charged and the P-0 side chain must be hydrophobic and extend beyond the gamma-carbon. The excellent correlation between these relative ED50 values and a series of relative Kd values determined from individual peptides suggests that this approach may be useful in determining, in a parallel fashion, the relative biological activities of arrays of polypeptides.

Constricted flux through the branched-chain amino acid biosynthetic enzyme acetolactate synthase triggers elevated expression of genes regulated by rpoS and internal acidification.

The first common enzyme of isoleucine and valine biosynthesis, acetolactate synthase (ALS), is specifically inhibited by the herbicide sulfometuron methyl (SM). To further understand the physiological consequences of flux alterations at this point in metabolism, Escherichia coli genes whose expression was induced by partial inhibition of ALS were sought. Plasmid-based fusions of random E. coli DNA fragments to Photorhabdus luminescens luxCDABE were screened for bioluminescent increases in actively growing liquid cultures slowed 25% by the addition of SM. From more than 8,000 transformants, 12 unique SM-inducible promoter-lux fusions were identified. The lux reporter genes were joined to seven uncharacterized open reading frames, f253a, f415, frvX, o513, o521, yciG, and yohF, and five known genes, inaA, IdcC, osmY, poxB, and sohA. Inactivation of the rpoS-encoded sigma factor, sigmaS, reduced basal expression levels of six of these fusions 10- to 200-fold. These six genes defined four new members of the sigmaS regulon, f253a, IdcC, yciG, and yohF, and included two known members, osmY and poxB. Furthermore, the weak acid salicylate, which causes cytoplasmic acidification, also induced increased bioluminescence from seven SM-inducible promoter-lux fusion-containing strains, namely, those with fusions of the sigmaS-controlled genes and inaA. The pattern of gene expression changes suggested that restricted ALS activity may result in intracellular acidification and induction of the sigmaS-dependent stress response.

Aortic valve replacement with the Jyros bileaflet prosthetic valve; early echocardiographic and radiological evaluation.

The Jyros bileaflet prosthetic heart valve has a unique hinge mechanism permitting rotation of the leaflets within the valve ring. Thirty patients undergoing aortic valve replacement with a Jyros valve were assessed clinically, echocardiographically and radiologically. Rotation of the valve in vivo was assessed, as well as the hemodynamic characteristics of the valve at rest and after exercise, and functional capacity of the valve recipients. Measured transvalvar gradients were comparable with other bileaflet valves at rest and after exercise, as were calculated effective orifice areas. Initially only four prostheses (13%) showed any evidence of rotating, but this improved to 14 of 23 investigated (60.9%) after exercise; rotation appears to be more common in smaller valves. There were no operative or early deaths, but one patient died at 11 months from a type A ascending aortic dissection. During a total of 28.5 patient-years of follow up (range 2-24 months) valve related complication has not been reported. All survivors are in good clinical condition. Preliminary results with the Jyros valve compare well with other bileaflet devices for aortic valve replacement; the hemodynamic performance of the valve does not appear to be affected by rotation or non-rotation.

Improved MR venography: use of fast short inversion time inversion-recovery technique in evaluation of venous angiomas.

Eleven patients (five female and six male, aged 6-67 years) with venous angiomas underwent axial and coronal T1-weighted and fast short inversion time inversion-recovery (STIR) magnetic resonance (MR) imaging to assess the full extent of the malformations. Images were assessed subjectively by the authors in a nonblinded study. In all patients, fast STIR images depicted more anatomic detail than did T1-weighted images and depicted venous angiomas more clearly than did contrast material-enhanced CT scans but did not help discrimination of arteries and veins.

The use of ultrasound in the venous assessment of lower limb Klippel-Trenaunay syndrome.

Colour Doppler ultrasound was used to study the affected lower limbs of a group of patients with the Klippel-Trenaunay syndrome. The abnormal lateral venous channel characteristic of this syndrome was examined, together with its deep venous connections and deep venous patency established if present. Seventeen patients (18 limbs) were studied. Ultrasound demonstrated calf vein patency in 15 out of 17 limbs and deep venous patency from the popliteal vein to the inferior vena cava in 17 out of 17 limbs. Ultrasound was also effective in demarcation of the abnormal lateral venous channel, together with its deep connections (13 out of 16 limbs). We conclude that ultrasound is superior to the more established venographic techniques in the investigations of patients with the Klippel-Trenaunay syndrome. From this study, the deep venous aplasia said to occur in the condition would appear to be less common than previously thought.

Easing transitions in care delivery with a core training group.

A collaborative approach was used by the nursing staff of a state psychiatric hospital to make a transition from traditional nursing services to a self-care model on one ward. The core training group, including representatives from all levels of nursing staff, developed and evaluated a personal hygiene protocol for 30 patients with chronic mental illness. Underwood's adaptation of Orem's self-care model of nursing was modified to provide the framework for this protocol. Evaluation of the project demonstrated both improved personal hygiene for the patients and greater job satisfaction for the nursing staff. The collaborative core training group approach was effective in facilitating the transition to the self-care nursing model.

Resolution of hypergastrinaemia after parathyroidectomy in multiple endocrine neoplasia syndrome type I (MEN type I).

The treatment of choice for gastric acid hypersecretion in MEN type I has now changed from total gastrectomy to the long-term administration of H2 receptor blockers or similar agents. However, the importance of parathyroidectomy for the concomitant hypercalcaemia is not fully realized. A case is reported of a subject with MEN type I, who was initially treated with parathyroidectomy and H2 receptor blockade. Following parathyroidectomy, there was a rapid fall of the markedly elevated gastrin levels to the upper limit of the normal range. During follow-up of 2.5 years, there has been no increase in serum gastrin, and the subject's gastric symptoms have resolved, despite the cessation of H2 blockade. Infusion of calcium to pre-operative levels and superimposed secretin stimulation after this period of time closely reproduced the pre-operative hypergastrinaemia. On screening, the subject's only child was found to have hypercalcaemia but normal serum gastrin levels and pituitary function; parathyroidectomy has been carried out. No abnormality of gastrin secretion has been found during follow-up. This case highlights the importance of early parathyroidectomy in this syndrome.