31Phospho-NMR Demonstration of Phosphocysteine as a Catalytic Intermediate on the Escherichia coli Phosphotransferase System EIIMtl

The mannitol-specific phosphotransferase system transport protein, Enzyme IIMtl, contains two catalytically important phosphorylated amino acid residues, both present on the cytoplasmic part of the enzyme. Recently, this portion has been subcloned, purified, and shown to be an enzymatically active domain. The N-terminal half has also been subcloned and shown to be the mannitol-binding domain. When combined the two domains catalyze mannitol phosphorylation at the expense of phospho-HPr. The phospho-NMR spectrum of the purified phosphorylated cytoplasmic domain, taken at pH 8.0, shows two signals, one at -6.9 ppm compared with inorganic phosphate resulting from phosphohistidine and one at +11.9 ppm originating from phosphocysteine. Addition of mannitol plus membranes containing the N-terminal mannitol-binding domain results in the formation of mannitol 1-phosphate and the disappearance of the two signals at -6.9 and +11.9 ppm.

Preoperative patient and injury factors of successful rehabilitation after anterior cruciate ligament reconstruction with single-bundle techniques

The aim of this systematic review was to determine which patient determinants and injury factors, before anterior cruciate ligament reconstruction by arthroscopic single-bundle techniques, affect postoperative rehabilitation. A search of PubMed, Embase, and the Cochrane Database of Clinical Trials was performed up to February 2013. After application of our inclusion criteria, a final selection was made based on studies' methodologic score assessed with the Newcastle-Ottawa Scale. Meta-analysis was planned for each prognostic factor when data were considered clinically and statistically homogeneous. Meta-analysis showed that male patients have better functional outcomes. Qualitative synthesis from 18 high-quality studies showed that patients operated on before 30 years of age reach higher activity levels. Patients with high baseline body mass index have lower activity levels after surgery. Smoking results in more symptoms and lower activity levels and subjective scores. Reconstruction before 3 months results in higher activity levels. Preoperatively, a less than 20% quadriceps strength difference, 50° of tibial external rotation or less, absence of flexion deficits, low knee influence on the patient's activity level, and less anterior knee pain result in higher functional scores. Preoperative anterior laxity difference does not predict functional scores. The prognostic value of preoperative activity and competition level for postoperative functional outcome is controversial. Patients with concomitant meniscal injuries have worse functional outcomes. The prognostic value of concomitant chondral pathology for postoperative functional outcome is controversial. Collateral ligament injury could predict functional scores or activity level. Male gender, patient age younger than 30 years, reconstruction before 3 months, and high baseline activity level contribute to better functional outcomes. Smoking, high body mass index, quadriceps strength, and range-of-motion deficits affect rehabilitation negatively. Preoperative anterior laxity does not influence rehabilitation. The role of preoperative prognostic injury factors remains unclear because of limited evidence. Level III, systematic review of Level II and III studie

Serial CSF sampling over a period of 30 h via an indwelling spinal catheter in healthy volunteers:headache, back pain, tolerability and measured acetylcholine profile

<p>Timed interval cerebrospinal fluid (CSF) sampling by indwelling catheterization can be a valuable corroborative tool for the pharmacokinetic and pharmacodynamic assessment of drugs. CSF sampling in studies on drug candidates for Alzheimer's disease have been conducted in evaluations of the biomarkers acetylcholine (ACh), tau proteins, amyloid precursor protein and beta-amyloid fragments. The primary aim of this study was to study the feasibility and the burden on the healthy volunteers of serial CSF sampling within the contract research organization environment in order to establish a standardized research tool for future drug development studies.</p><p>This study is a validation study in healthy subjects: eight healthy male subjects aged 55-75 years were enrolled. After eligibility had been confirmed, the subjects were admitted to the clinical pharmacology unit 2 days before starting the CSF sampling procedure. Hydration by drip infusion of 2 L saline was performed for 24 h before starting the CSF sampling procedure, and for antithrombotic purposes, Fraxiparine (nadroparine calcium) was given 12 and 36 h after intradural catheterization. CSF catheterization was performed by board-certified anesthesiologists with experience in inserting indwelling intrathecal catheters. Subjects only required to remain in a horizontal position for the first 24 h after removal of the catheter. CSF and blood samples were collected by interval sampling over a 30-h period.</p><p>The study was completed by seven of the eight subjects. Six subjects who completed the study reported adverse effects (AEs) which were all mild and from which they recovered during their stay in the clinic. A total of 25 AEs were reported of which 13 were considered to be procedure-related. The procedure was well tolerated by all participating subjects, and the VAS scale scores for headache and back pain were low. CSF samples were analyzed for ACh. All values were above the lowest limit of quantification. On average, the ACh concentration started at a low level but rose between 1 and 2 h after insertion of the catheter and then remained high during the whole sampling period up to 30 h.</p><p>Serial sampling of CSF in seven healthy volunteers up to 30 h occurred without serious complications and was well tolerated. The CSF collected was of good quality and facilitated the assessment of an Alzheimer's disease-sensitive biomarker. We conclude that this validation study can form the basis for future patient studies aimed at elucidating disease mechanisms and the pharmacodynamics of drugs in the developmental stage.</p>