BMAL1 links the circadian clock to viral airway pathology and asthma phenotypes.

Patients with asthma experience circadian variations in their symptoms. However it remains unclear how specific aspects of this common airway disease relate to clock genes, which are critical to the generation of circadian rhythms in mammals. Here, we used a viral model of acute and chronic airway disease to examine how circadian clock disruption affects asthmatic lung phenotypes. Deletion of the core clock gene bmal1 or environmental disruption of circadian function by jet lag exacerbated acute viral bronchiolitis caused by Sendai virus (SeV) and influenza A virus in mice. Post-natal deletion of bmal1 was sufficient to trigger increased SeV susceptibility and correlated with impaired control of viral replication. Importantly, bmal1-/- mice developed much more extensive asthma-like airway changes post infection, including mucus production and increased airway resistance. In human airway samples from two asthma cohorts, we observed altered expression patterns of multiple clock genes. Our results suggest a role for bmal1 in the development of asthmatic airway disease via the regulation of lung antiviral responses to common viral triggers of asthma.

Early detection of life-threatening intracranial haemorrhage using a portable near-infrared spectroscopy device.

OBJECTIVE: To determine whether infrared spectroscopy allows early recognition of epidural and subdural haematomas among trauma patients. METHODS: Injured people admitted to two trauma units were enrolled in a prospective multicentre observational study, and infrared spectroscopy was performed before computed tomography of the head as a part of their initial evaluation. Subsequent CT findings suggestive of epidural or subdural haematoma served as controls. RESULTS: Over 12 months, 110 patients were enrolled; 64 (58.1%) were men and 46 (41.9%) were women. Mean age was 56.2 years, and mean Glasgow Coma Scale on admission was 12.6. Infrared spectroscopy was 90.5% sensitive and 95.5% specific for epidural and subdural haematoma. Positive and negative predictive values were 82.6% and 97.7%, respectively. CONCLUSIONS: Infrared spectroscopy allows early recognition of epidural and subdural haematomas in trauma cases. Further studies are needed to evaluate whether immediate confirmation or exclusion of epidural and subdural haematomas with portable near-infrared spectroscopy devices improves the decision-making process in the treatment of severely injured people.

Developing and implementing a model for changing physicians' prescribing habits-- the role of clinical pharmacy in leading the change.

BACKGROUND AND OBJECTIVE: Budgetary constraints led the Israeli Hillel Yaffe Medical Center management to implement policies for reducing expenditure while maintaining the quality of care. For this purpose, the pharmacy services management developed and implemented a three-tier intervention feedback model for changing physicians' prescribing habits, and achieving cost-effective changes in antibiotic utilization. METHODS: A prospective drug utilization evaluation was conducted to profile antibiotic utilization. The results established a base from which a three-tier feedback, evidence-based intervention model was built. This model corresponds to the following three hierarchical levels: Level 1 activities involved management actions that influenced all levels of staff and concentrated mainly on the creation of guidelines. Level 2 activities involved the reorganization of the restricted antibiotics prescription authorization system, through the co-operation of the clinical pharmacy unit and the hospital infection control specialist. Level 3 focussed on clinical pharmacist activities on the wards. The model was implemented and assessed in the hospital from June 2002 until December 2004. RESULTS AND DISCUSSION: Implementation of the model resulted in a cumulative decrease of 6,473 i.v. antibiotics daily defined doses (DDDs) and a parallel increase in total oral antibiotic DDDs (Table 1). These changes were especially notable with high-bioavailability antibiotics and co-amoxiclav, where over 2.5 years there was a reduction of 2,472 and 4,752 i.v. DDDs, respectively (P < 0.000). The successful implementation of the model resulted in a reduction of 375,000 NIS ( approximately 66,190 euro) in pharmacy antibiotic costs, equivalent to 10 i.v. DDDs or 570 NIS ( approximately 102 euro) saved per clinical pharmacist working day. CONCLUSIONS: Our study demonstrates the successful implementation of a three-tier model for changing physicians' antibiotic prescribing.

Disulfide-mediated dimerization of L1 Ig domains.

The neural cell adhesion molecule L1 contains immunoglobulin-like (Ig) domains in its extracellular region that mediate homophilic binding, neurite outgrowth and other activities relevant to CNS development. To correlate conformations of these domains to biological function, several L1-Fc fusion proteins whose bioactivities were previously characterized were analyzed by rotary shadowing electron microscopy. We found that bioactive L1-Fcs containing Ig domains 1-4 or 1-6 exhibited extended, branched structures. In contrast, inactive L1-Fcs containing only the first two or three Ig domains assumed compact shapes that suggested interactions between the L1 arms of these proteins. Analysis of an untagged L1 fragment composed of Ig domains 1-3 demonstrated a mixture of monomeric and dimeric forms. Surprisingly, these dimers were stabilized by intermolecular disulfide bonds. Finally, cell surface L1-GFP fusion proteins containing only the first two or three Ig domains in the extracellular region also engaged in disulfide-mediated dimerization. These results suggest a novel mechanism by which mutations in L1 could interfere with its biological functioning.

Cell adhesion molecule L1 in folded (horseshoe) and extended conformations.

We have investigated the structure of the cell adhesion molecule L1 by electron microscopy. We were particularly interested in the conformation of the four N-terminal immunoglobulin domains, because x-ray diffraction showed that these domains are bent into a horseshoe shape in the related molecules hemolin and axonin-1. Surprisingly, rotary-shadowed specimens showed the molecules to be elongated, with no indication of the horseshoe shape. However, sedimentation data suggested that these domains of L1 were folded into a compact shape in solution; therefore, this prompted us to look at the molecules by an alternative technique, negative stain. The negative stain images showed a compact shape consistent with the expected horseshoe conformation. We speculate that in rotary shadowing the contact with the mica caused a distortion of the protein, weakening the bonds forming the horseshoe and permitting the molecule to extend. We have thus confirmed that the L1 molecule is primarily in the horseshoe conformation in solution, and we have visualized for the first time its opening into an extended conformation. Our study resolves conflicting interpretations from previous electron microscopy studies of L1.

System for cleavable Fc fusion proteins using tobacco etch virus (TEV) protease.

We describe a novel Fc fusion protein system that can be cleaved by tobacco etch virus (TEV) protease. This system is desirable because it takes advantage of the high specificity of TEV protease and its activity at 4 degrees C. We produced two TEV-Fc fusion proteins that contain the first three Ig domains and all six Ig domains of the cell adhesion molecule L1. Both proteins were efficiently cleaved by TEV protease at 4 degrees C. Functional analysis of the cleavage products in neurite outgrowth assays showed they had similar activities to their parental Fc fusion proteins. Therefore, TEV-Fc fusion proteins may increase the utility and flexibility of the Fc fusion protein system.

Critical and optimal Ig domains for promotion of neurite outgrowth by L1/Ng-CAM.

Mammalian L1 and avian Ng-CAM are homologous neural cell adhesion molecules (CAMs) that promote neurite outgrowth and cell adhesion in most neurons. Previous attempts to map these activities to discrete regions in the CAMs have suggested the involvement of a variety of different domains. However, these studies mainly used bacterially expressed proteins that were much less active on a molar basis than the native molecules. To define regions that are critical for maximal neurite outgrowth, we constructed and tested a panel of eukaryotically expressed proteins containing various extracellular segments of human L1 (hL1) or Ng-CAM. Our results indicate that Ig domains 1-4 of hL1 are critical for homophilic binding and neurite outgrowth; however this segment is less potent than the entire extracellular region. Optimal neurite outgrowth activity was seen with proteins containing all six Ig domains of hL1 or Ng-CAM. The adhesive properties of hL1 fragments correlated tightly with their neurite outgrowth activities, suggesting that these two processes are closely linked. These results suggest that Ig domains 1-4 form a structural cassette responsible for hL1 homophilic binding, while Ig domains 1-6 represent a functional region for optimal promotion of neurite outgrowth in vitro and possibly in vivo.

A nickel-binding serpin, pNiXa, induces maturation of Xenopus oocytes and shows synergism with oncogenic ras-p21 protein.

A nickel-binding serine proteinase inhibitor, pNiXa (43 kDa), was isolated from Xenopus ovary and assayed for effects on oocyte maturation. Microinjection of pNiXa (0.12 pmol/50 nl) induced maturation in 60% of Xenopus oocytes, beginning at 4 hours and reaching completion by 9 hours. Microinjection of oncogenic ras-p21 protein (0.12 pmol/50 nl) induced maturation in 79% of oocytes, beginning at 6 hours and reaching completion by 12 hours. Microinjection of pNiXa in combination with ras-p21 protein had a synergistic effect on maturation, which occurred in 92% of oocytes, beginning at 4 hours and reaching completion by 9 hours. Oocyte maturation did not occur in control oocytes, which received a microinjection of bovine serum albumin. In oocytes exposed to a combination of pNiXa (0.12 pmol/50 nl, by microinjection) and progesterone (10 micrograms/ml, in the medium), maturation was intermediate (68% at 9 hours) between that induced by pNiXa (60%) or progesterone (85%) alone. This study shows (a) that pNiXa is a potent inducer of oocyte maturation, (b) that pNiXa's effect is synergistic with that of oncogenic ras-p21 protein, and (c) that pNiXa partially antagonizes progesterone induction of oocyte maturation.

Antibacterial activity of the pancreatic fluid.

The antibacterial activity of canine pancreatic fluid was investigated in an attempt to understand the resistance of this organ, when intact, to ascending bacterial infections. The pancreatic fluid demonstrated bactericidal activity against Escherichia coli, Shigella species, Salmonella species, and Klebsiella pneumoniae; bacteriostatic activity against coagulase-positive and coagulase-negative staphylococci and Pseudomonas aeruginosa; and fungistatic activity against Candida albicans. There was no demonstrable antibacterial activity against Bacteroides fragilis and Streptococcus faecalis. The antibacterial activity was dialyzable and pH dependent, but independent of heat, the activity of several digestive pancreatic enzymes, and the bacterial inoculum. Electron micrographs of Escherichia coli exposed to pancreatic fluid did not demonstrate changes in the bacterial cell wall. Tracer studies of susceptible bacteria demonstrated decreased leucine uptake when briefly exposed to pancreatic fluid. The antibacterial activity was found by column chromatography to be a small molecular peptide. It is likely that pancreatic antibacterial factors protect the pancreas from ascending bacterial infections and operate along with other factors in the homeostasis of the upper small bowel flora.

Effects of parathyroid hormone on exocrine pancreatic secretion in parathyroidectomized dogs.

The effects of intravenous parathyroid hormone (PTH) on steady state Secretin-induced pancreatic secretion were studied in seven dogs before and after parathyroidectomy. Free flow of pancreatic juice was obtained by direct cannulation of the main pancreatic duct (the minor duct being ligated) : a gastric fistula prevented the entry of gastric acid into the duodenum. In the normal dog PTH caused a significant increase in volume and bicarbonate concentration, reciprocal change in chloride and no change in total protein concentration. The stimulatory effect of PTH was dose-dependent. In the parathyroidectomized dog, the basic Secretin-induced secretion was lower than the preoperative values, but PTH infusion caused a significant increase in volume of fluids and bicarbonate concentration, reciprocal change in chloride and no change in protein concentration. These results were not dependent on calcium blood level, and did not change after calcium injection to the hypocalcemic parathyroidectomized dog. It is suggested, that PTH may have a direct effect on pancreatic exocrine secretion.

Effect of pancreatitis on ampicillin excretion in pancreatic fluids of dogs.

Pancreatic excretion of ampicillin was evaluated in normal dogs and in dogs with induced pancreatis. A 100-mg/kg ampicillin dose administered intravenously induced mean peak serum levels of 100 micrograms/ml, and a 200-mg/kg intravenous dose induced a mean peak serum level of 273 microgram/ml. Ampicillin serum levels did not differ between the group of normal dogs and those with pancreatitis. In normal dogs, the peak pancreatic fluid ampicillin concentration after the 100-mg/kg dose was 0.4 microgram/ml, and that after the 200-mg/kg dose was 2.7 micrograms/ml. In dogs with pancreatitis, the mean peak ampicillin concentration in the pancreatic fluid after the 100 mg/kg dose was 19 micrograms/ml, and that after the 200-mg/kg dose was 38.5 micrograms/ml. Pancreatic fluid ampicillin concentrations were therapeutic in dogs with pancreatitis and subtherapeutic in normal dogs.

Clindamycin excretion in the pancreatic fluid of dogs.

Clindamycin phosphate in dosage of 15 mg/kg was administered intravenously to dogs. Serum and pancreatic juice clindamycin concentrations were assayed. Pancreatic juice clindamycin concentrations varied between 1.32--5.5 micrograms/ml. Clindamycin was still detectable in the pancreatic juice 4 1/2 h after its administration. Clindamycin, in combination with other antimicrobials, may be potentially effective in the prophylaxis and therapy of some of the infectious complications associated with pancreatitis.