High-level expression of recombinant Fc chimeric proteins in suspension cultures of stably transfected J558L cells.

Recombinant Fc chimeric proteins are useful tools for studying protein function, including the analysis of molecular interactions by techniques such as expression cloning. Here we describe a method we have used to express the IgLON family proteins, CEPU1 and OBCAM, as recombinant Fc chimeric proteins in stably transfected mouse J558L myeloma cells. The use of this cell line provided the opportunity to maximize protein production, as it secretes antibodies in large quantities and can be grown to high density in small volumes of culture medium. Isolation of recombinant OBCAMFc from the adherent COS7 cell line suggested a minimum level of expression of 0.07 mg OBCAMFc/100 mL culture medium, while the J558L cell line expressed OBCAMFc at approximately 11.4 mg/100 mL culture medium. Purification of IgLON-Fc expressed by J558L cells was simpler than purification from COS7 cells because of the lower volume of culture medium generated. Furthermore, contamination of J558L expressed IgLONFc with bovine IgG from the culture medium was negligible. The method presented, which utilizes a commercially available small-scale bioreactor, provides the nonspecialist protein expression laboratory with the means to produce recombinant proteins quickly and easily in milligram quantities.

Identification and characterization of CEPU-Se-A secreted isoform of the IgLON family protein, CEPU-1.

CEPU-1/Neurotrimin is a neuronal glycoprotein thought to play a role in axon guidance and cell-cell recognition. It is a member of the IgLON family, has three C2 domains, and is attached to the plasma membrane by a GPI-anchor. We report here the characterisation of an alternatively-spliced isoform of CEPU-1 that is secreted. This isoform, termed CEPU-Se, is coexpressed with CEPU-1 in retina, cerebellum, and DRG neurons. In the cerebellum CEPU-1/CEPU-Se is expressed predominantly on granule cells and in the molecular layer. Divalent but not monovalent CEPU-Se interacts with CEPU-1 and other IgLONs, suggesting that the ability of CEPU-Se to modify the activity of the IgLON family may require an additional cofactor. CEPU-Se does not support the outgrowth of DRG neurons or the extension of established growth cones; however, neurite outgrowth on laminin is unaffected by CEPU-Se. Our data suggest that CEPU-Se may act to modulate the ability of CEPU-1, LAMP, and OBCAM to influence neurite outgrowth.

Co-localisation, heterophilic interactions and regulated expression of IgLON family proteins in the chick nervous system.

The chick glycoprotein GP55 has been shown to inhibit the growth and adhesion of DRG and forebrain neurons. GP55 consists of several members of the IgLON family, a group of glycoproteins including LAMP, OBCAM, CEPU-1 (chick)/neurotrimin (rat) and neurotractin (chick)/kilon (rat) thought to play a role in the guidance of growing axons. IgLONs belong to the Ig superfamily and have three C2 domains and a glycosyl phosphatidylinositol anchor which tethers them to the neuronal plasma membrane. We have now completed the deduced amino acid sequence for two isoforms of chicken OBCAM and used recombinant LAMP, OBCAM and CEPU-1 to raise antisera specific to these three IgLONs. LAMP and CEPU-1 are co-expressed on DRG and sympathetic neurons, while both overlapping and distinct expression patterns for LAMP, OBCAM and CEPU-1 are observed in retina. Analysis of IgLON mRNA expression reveals that alternatively spliced forms of LAMP and CEPU-1 are developmentally regulated. In an attempt to understand how the IgLONs function, we have begun to characterise their molecular interactions. LAMP and CEPU-1 have already been shown to interact homophilically. We now confirm that OBCAM will bind homophilically and also that LAMP, OBCAM and CEPU-1 will interact heterophilically with each other. We propose that IgLON activity will depend on the complement of IgLONs expressed by each neuron.

Treatment of chronic contained spontaneous esophageal perforations.

Spontaneous esophageal perforations are associated with a high mortality and morbidity without surgery. The treatment mortality for early (<24) and late (>24 h) spontaneous esophageal perforations is reviewed as well as all recent cases of chronic spontaneous esophageal perforations. Chronic esophageal perforations with mediastinal cavities may be best treated by internal drainage of the cavity into the esophagus in order to convert the transmural perforation into an intramural esophageal dissection.

Identification of chURP, a nuclear calmodulin-binding protein related to hnRNP-U.

In a screen for myosin-like proteins in embryonic chicken brain, we have identified a novel nuclear protein structurally related to hnRNP-U (heterogeneous nuclear ribonuclear protein U). We have called this protein chURP, for chicken U-related protein. In this screen, chURP was immunoreactive with two myosin antibodies and, in common with the unconventional myosins, bound calmodulin in vitro in both the presence and absence of calcium ions. Determination of 757 amino acids of the chURP sequence revealed that it shares 41% amino acid identity with human and rat hnRNP-U, although chURP and hnRNP-U appear not to be orthologous proteins. ChURP is ubiquitously expressed in the nuclei of all chick tissues and, as one of a growing number of calmodulin-binding proteins to be identified in the nucleus, further highlights the potential of calmodulin as a regulator of nuclear metabolism.

Purulent pericarditis secondary to Candida parapsilosis and Peptostreptococcus species.

Purulent pericarditis secondary to Candida pericarditis is a rare but lethal infection. The first case of pericardial infection by Candida parapsilosis in combination with Peptostreptococcus species in an immunocompetent nondebilitated individual is reported. Urgent pericardiectomy was required after pericardial drainage failed to stop symptoms of tamponade. The etiology, microbiology and treatment of this unusual condition are reviewed.

Octogenarian with a congenital bronchoesophageal fistula.

Bronchoesophageal fistula are commonly caused by a lung or esophageal malignancy eroding into the neighboring structure. Benign forms of bronchoesophageal fistula are less common and may have a congenital nature. Congenital bronchoesophageal fistula usually present in adult life with chronic symptoms of lung suppuration. We present a case of congenital bronchoesophageal fistula in an octogenarian and review the literature on this subject. We also suggest an extrapleural approach to the fistula to lessen the possibility of postoperative empyema.

Cell cycle-dependent morphological changes in the actin cytoskeleton induced by agents which elevate cyclic AMP.

Agents which increase the intracellular concentration of cyclic adenosine-5'-monophosphate, induce a highly arborised morphology in a proportion of sub-confluent Swiss 3T3 fibroblasts. During this process the organisation of actin filaments progressively changes from a characteristic stress fibre pattern to leave a network of actin filaments within each and every arborisation. Despite this massive reorganisation of the actin cytoskeleton no changes are observed in the extent of polymerisation of actin during arborisation. The proportion of cells in asynchronous cultures undergoing arborisation at maximal concentrations of agents reaches a maximum of 30%; suggesting that the effect might be mediated only in cells during a restricted period of the cell cycle. More than 80% of serum-starved cells responded to these agents between 1 and 8 hours after readdition of serum, but not at other times, suggesting that the arborisation response can occur only in the G1 phase of the cell cycle.

Defective endothelium-dependent relaxation in the JCR:LA-corpulent rat.

Endothelium-dependent relaxation of the aorta was assessed in JCR:LA-corpulent rats, which are hyperphagous, hyperlipidemic, hyperinsulinemic, and obese and spontaneously develop atherosclerotic disease and myocardial lesions. The findings in corpulent rats (6 months of age) were compared with those in age-and sex-matched lean rats. Aortic rings were prepared and mounted in Krebs-Henseleit buffer in a conventional organ bath. The tissue was contracted with norepinephrine (10(-6) mol/L), and relaxation was induced using acetylcholine, the calcium ionophore A23187, or bradykinin. The maximum relaxation to acetylcholine was impaired in corpulent male rats compared with lean rats, whereas relaxation in response to the calcium ionophore was similar in the corpulent and lean animals. Aortic rings from corpulent and lean female rats showed no differences in response to acetylcholine or to the calcium ionophore. Removal of endothelium resulted in the loss of relaxant response to acetylcholine and the calcium ionophore. The relaxant responses to sodium nitrite were not significantly different in the corpulent and lean male rats when deendothelialized tissues were examined, but the sensitivity to sodium nitrite was significantly lower in rings from corpulent male rats with intact endothelium. There were no differences in the response to bradykinin between corpulent and lean rats. These findings suggest that there is a specific impairment of endothelium-dependent relaxation in the corpulent male rat that is limited to that mediated by muscarinic receptors. The possibility that endothelium-derived contractile agents are secreted in the vessels of corpulent male rats cannot be excluded.

Blunt traumatic avulsion of an intercostal artery: an unusual case of thoracic aortic injury.

The aortic isthmus is the most commonly injured part of the thoracic aorta in patients who survive blunt deceleration injury to that vessel long enough to reach a hospital. Occasionally, avulsion of the brachiocephalic arteries from the aortic arch is seen. The authors describe an unusual form of intrathoracic vascular injury in which mediastinal hemorrhage occurred secondary to avulsion of an intercostal artery from the descending thoracic aorta as a result of a vertical deceleration injury.

Bronchogenic cyst masquerading as a chronic post-traumatic pseudoaneurysm of the aortic isthmus.

The serious nature of false aneurysms that develop in the aortic isthmus after blunt chest trauma is well known. The authors describe the case of a 33-year-old woman who presented with symptoms of chronic post-traumatic pseudoaneurysm of the aorta 3 months after blunt chest trauma. Radiologic investigations could not substantiate an aortic disruption. A bronchogenic cyst masquerading as a false aneurysm of the aorta was identified at thoracotomy. Bronchogenic cysts are one of the most common causes of primary mid-mediastinal masses and should be considered as potential causes of mid-mediastinal enlargement. However, this consideration should not delay urgent surgery if vascular damage cannot be ruled out.

New method for dealing with late-presenting spontaneous esophageal ruptures.

A new technique is described for dealing with late-presenting spontaneous esophageal ruptures. This method requires only a short period of general anesthesia to drain the periesophageal abscess by a drainage tube inserted into the abscess cavity from the esophagus with the aid of a gastroscope and fluoroscopy. Gastric fluids are diverted from the esophageal rupture with a gastrostomy, and a jejunostomy is used for enteral feeding. The esophagus is retained, and closure of the fistula with resumption of normal swallowing is documented with serial sinograms.

Flexible bronchoscopy via the laryngeal mask: a new technique.

Malignant tracheal tumours often cause airway obstruction and this may be aggravated by vocal cord paralysis due to invasion of the recurrent laryngeal nerve. Conventional endoscopic techniques performed under general anaesthesia do not give a simultaneous view of vocal cord function and the distal airways. The technique of bronchoscopy via the laryngeal mask allowed full assessment of the cause of stridor in a patient with a malignant tracheal tumour that was causing airways obstruction and vocal cord paralysis.

Multiple small-bowel carcinoids presenting with gastrointestinal hemorrhage: a case report.

Gastrointestinal hemorrhage from a small-bowel lesion can be a problem diagnostically and therapeutically. The authors present a case of multiple small-bowel carcinoids, which were the cause of multiple upper gastrointestinal hemorrhages over a 15-year period. Despite advances in endoscopy and diagnostic imaging, accurate localization and definitive diagnosis remain elusive in such cases. Optimal treatment depends on careful clinical evaluation and timely laparotomy.