Characterization of Brain-Penetrant Pyrimidine-Containing Molecules with Differential Microtubule-Stabilizing Activities Developed as Potential Therapeutic Agents for Alzheimer's Disease and Related Tauopathies.

The microtubule (MT)-stabilizing protein tau disengages from MTs and forms intracellular inclusions known as neurofibrillary tangles in Alzheimer's disease and related tauopathies. Reduced tau binding to MTs in tauopathies may contribute to neuronal dysfunction through decreased MT stabilization and disrupted axonal transport. Thus, the introduction of brain-penetrant MT-stabilizing compounds might normalize MT dynamics and axonal deficits in these disorders. We previously described a number of phenylpyrimidines and triazolopyrimidines (TPDs) that induce tubulin post-translational modifications indicative of MT stabilization. We now further characterize the biologic properties of these small molecules, and our results reveal that these compounds can be divided into two general classes based on the cellular response they evoke. One group composed of the phenylpyrimidines and several TPD examples showed a bell-shaped concentration-response effect on markers of MT stabilization in cellular assays. Moreover, these compounds induced proteasome-dependent degradation of α- and ß-tubulin and caused altered MT morphology in both dividing cells and neuron cultures. In contrast, a second group comprising a subset of TPD molecules (TPD+) increased markers of stable MTs in a concentration-dependent manner in dividing cells and in neurons without affecting total tubulin levels or disrupting MT architecture. Moreover, an example TPD+ compound was shown to increase MTs in a neuron culture model with induced tau hyperphosphorylation and associated MT deficits. Several TPD+ compounds were shown to be both brain penetrant and orally bioavailable, and a TPD+ example increased MT stabilization in the mouse brain, making these compounds potential candidate therapeutics for neurodegenerative tauopathies such as Alzheimer's disease.

Survival as medical registrar on call: remember the doughnut.

Life as a medical registrar presents many challenges and concern is growing that a downwards trend in recruitment to General Medicine is the direct result of a perception by junior doctors that the role of the medical registrar is excessively demanding and results in poor work-life balance. A solution-focused approach (SFA) can be used successfully to find a satisfying outcome for both registrar and patient in many of the situations encountered during an on-call, as well as carrying over benefits into life outside of work. We explore the origins of SFA and the ways in which it can be successfully applied to clinical medicine, providing case studies from the author's own experience to illustrate the principles of this way of thinking.

Inserting tunnelled hemodialysis catheters using elective guidewire exchange from nontunnelled catheters: is there a greater risk of infection when compared with new-site replacement?

The objective is to evaluate bacteremia outcomes and survival rates when using guidewire exchange to place tunnelled hemodialysis catheter (THDC) compared with a new-site replacement. Retrospectively, all patients were identified who received a THDC between January 1, 2000 and January 1, 2007. Any THDC having received antibiotic line locks or tunnel-to-tunnel exchange were excluded. This left 408 THDC placed in 329 patients: 46 guidewire exchange, 362 new-site replacement. Bacteremia rate from the new-site insertion group was 3.0 per 1000 catheter days, the guidewire exchange group demonstrated a rate of 2.8 per 1000 catheter days. Local infection rates did not differ between the groups at 1.2 per 1000 catheters days. The actuarial catheter survival rates using Kaplan-Meier survival analysis demonstrated no difference between the 2 groups. The placing of tunnelled cuffed hemodialysis catheters to replace temporary catheters using a guidewire exchange did not contribute to further episodes of sepsis and has the advantage of preserving venous access and minimizing invasive procedures for the patient.

Markers of oxidative stress in the skeletal muscle of patients on haemodialysis.

BACKGROUND: Increased oxidative stress may play a role in morbidity and mortality of patients with renal failure. Most studies have examined serum markers of oxidation, but it is unclear whether oxidative stress is involved in skeletal muscle atrophy. METHODS: This study examined markers of oxidative stress in the skeletal muscle of 10 haemodialysed patients and 10 control subjects. Biopsies from the quadriceps femoris were analysed for reduced and oxidized glutathione, protein thiols, malonaldehyde and heat shock proteins (HSP27, HSP60 and HSP70), superoxide dismutase and catalase activities. A novel microdialysis procedure was used to examine hydroxyl radical activity in the interstitial fluid of the tibialis anterior. RESULTS: Patients had muscle atrophy with a reduced diameter of both type I and II fibres (by 15 and 20%, respectively). Muscle microdialysates contained 2,3- and 2,5-dihydroxybenzoates formed from salicylate indicating hydroxyl radical activity, with no differences between patients and control subjects. Muscle protein thiol and oxidized glutathione contents were unchanged in patients, but malonaldehyde content was reduced. In contrast, total muscle glutathione and heat shock protein contents were increased. Muscle superoxide dismutase activity was unchanged, but catalase activity was reduced in patients. CONCLUSIONS: The muscle of patients undergoing haemodialysis undergoes some adaptive responses in total glutathione content, heat shock protein content and catalase activity that are potentially related to chronic oxidative stress. However, there is no evidence of gross oxidation, nor any clear relationship between oxidative stress and muscle fibre atrophy, arguing against a direct role of oxidants in the degenerative processes.

Abnormal mitochondrial function and muscle wasting, but normal contractile efficiency, in haemodialysed patients studied non-invasively in vivo.

BACKGROUND: Muscle dysfunction, which contributes to morbidity in patients on haemodialysis, has several manifestations and a number of possible causes. We applied the non-invasive techniques of (31)P-magnetic resonance spectroscopy ((31)P-MRS), magnetic resonance imaging (MRI) and near-infrared spectroscopy (NIRS) to calf muscle of dialysed patients to define the abnormalities in muscle cross-sectional area (CSA), contractile efficiency, mitochondrial function and vascular O(2) supply. METHODS: We performed (31)P-MRS/NIRS/MRI studies on the lateral gastrocnemius during isometric plantarflexion and recovery in 23 male patients on haemodialysis (age 24-71 years; haemoglobin 9.9-14.2 g/dl; bicarbonate 17-30 mmol/l; urea reduction ratio 53-77%; parathyroid hormone 1-95 U/l) and 15 male controls (age 29-71 years). To understand the relationships between calf CSA and body mass we also performed MRI only in a further six male patients and 18 male controls. RESULTS: In patients, exercise duration was 30+/-11% lower than in controls. Muscle CSA was lower by 26+/-5%, but contractile efficiency (force/CSA/ATP turnover) was normal. Slowing of post-exercise phosphocreatine (PCr) recovery implied a 22+/-5% defect in effective 'mitochondrial capacity'. That PCr recovery was slow relative to NIRS recovery suggests that this is largely an intrinsic mitochondrial problem (not the result of impaired O(2) supply), one which, furthermore, correlated with CSA. Urea reduction ratio showed a negative correlation with body mass and CSA, but none with PCr rate constant. CONCLUSIONS: The relationships to urea reduction ratio reflect the effect of muscle mass on dialysis efficiency, rather than direct effects on muscle CSA or metabolism. The relationship between PCr recovery and calf CSA suggests a role for the mitochondrial defect, whatever its cause, in the development of muscle wasting, although a common cause (e.g. physical inactivity) for both abnormalities cannot be ruled out.

The antiproteinuric effect of losartan is systemic blood pressure dependent.

BACKGROUND: It has been suggested that high doses of angiotensin II receptor antagonists (AIIAs) may reduce proteinuria by a non-haemodynamic action additional to their effect on systemic blood pressure. METHODS: We tested this for the AIIA losartan using a prospective single-blind randomized design in patients with proteinuria (>1 g/24 h) due to non-diabetic chronic renal failure (stable creatinine clearance >20 ml/min) and mild to moderate hypertension (130/80 < blood pressure < 160/110 mmHg). Twenty-one patients were randomized into two groups: group A received losartan 50 mg daily for 4 weeks, then 100 mg daily for 4 weeks; group B received losartan 50 mg daily for 8 weeks. Twenty-four hour ambulatory blood pressure and renal parameters were measured at baseline and at 4 and 8 weeks of treatment. RESULTS: Overall there was a 7 +/- 2 mmHg fall (mean +/- SEM) in mean daytime systolic blood pressure at 4 weeks, and a 22 +/- 7% fall in protein/creatinine ratio (both P < 0.05), with no difference between groups A and B or between 4 and 8 weeks. These two changes were highly correlated (r = 0.64, P = 0.006, taking both groups together). Changes in diastolic pressure and in night-time systolic pressure did not reach statistical significance. Changes in renal plasma flow (measured by Tc 99m MAGIII), glomerular filtration rate and filtration fraction (measured by 51Cr EDTA) did not reach statistical significance, did not differ between the two groups and did not correlate with effects on proteinuria. CONCLUSION: This study provides no evidence that the effect of losartan on proteinuria has a non-haemodynamic component.

A comparison of amyloid fibrillogenesis using the novel fluorescent compound K114.

Proteinaceous inclusions with amyloidogenic properties are a common link between many neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Histological and in vitro studies of amyloid fibrils have advanced the understanding of protein aggregation, and provided important insights into pathogenic mechanisms of these neurodegenerative brain amyloidoses. The classical amyloid dyes Congo Red (CR) and thioflavin T and S, have been used extensively to detect amyloid inclusions in situ. These dyes have also been utilized to monitor the maturation of amyloid fibrils assembled from monomer subunits in vitro. Recently, the compound (trans,trans)-1-bromo-2,5-bis-(3- hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB), derived from the structure of CR, was shown to bind to a wide range of amyloid inclusions in situ. More importantly it was also used to label brain amyloids in live animals. Herein, we show that an analogue of BSB, (trans,trans)-1-bromo-2,5-bis-(4-hydroxy)styrylbenzene (K114), recognizes amyloid lesions, and has distinctive properties which allowed the quantitative monitoring of the formation of amyloid fibrils assembled from the amyloid-beta peptide, alpha-synuclein, and tau.