Acute lung injury refers to a non-specific response of the lung to a plethora of precipitating events and factors. Despite intensive research for decades, many of the fundamental mechanisms in the initiation and progression of acute lung injury are poorly understood. Abnormal mechanical forces due to mechanical ventilation and pathological changes in pulmonary function and structure have been hypothesized to influence acute lung injury. The main objective of the research described in this dissertation was to investigate the responses of pulmonary epithelial cells to mechanical stimulation in vitro in relationship to acute lung injury. This dissertation presents the first in vitro model of airway reopening that incorporates living pulmonary epithelial cells. In the model, the progression of a semi-infinite bubble in a narrow channel lined with pulmonary epithelial cells inflicts significant injury to epithelial cell population. The presence of pulmonary surfactant (Infasurf, ONY, Inc., Buffalo, NY) in sufficient quantity (1 mg/mL) effectively abates this injury, verifying its protective role in airway collapse and reopening. The complex hydrodynamics of the bubble progression in this model influence the degree of injury. A computational analysis demonstrated that the most mechanically-damaging element of the stress cycle associated with bubble progression was the steep pressure gradient near the perimeter of the bubble cap. The cellular injury that was observable immediately following 'reopening' was found to be lethal for individual cells, but survivable for the population as whole. The tissues of the lung are exposed to a variety of physical forces. Motivated by the distension of the airways and alveoli during normal breathing and mechanical ventilation, cellular stretch has been assumed to be the predominant physical force that influences pulmonary epithelial function. This dissertation explores influences of mechanical stimuli other than substrate stretch on the biological function of pulmonary epithelial cells, namely the production of pulmonary surfactant, cytokines, and nitric oxide. Taken as a group, the findings suggest that physical stresses, such as transmural pressure and fluid shear stress, may impact acute lung injury by altering the function of the pulmonary epithelium / acase@tulane.edu

acase@tulane.edu

While studying the sialosyl linkages in glycoconjugates, we found that commercially available sialidases prepared from Clostridium perfringens ATCC 10543 were contaminated with an unusual endo-beta-galactosidase (Endo-beta-GalAB) capable of liberating the blood group A- and B-trisaccharides from human ovarian cyst glycoproteins. We have isolated Endo-beta-GalAB in pure form from the culture supernatant of Clostridium perfringens and cloned the gene encoding this enzyme. The gene (eabC) consists of a 2400 by open reading frame, including a 35-residue signal peptide at the N-terminus. The mature protein (765 amino acids) has a predicted molecular mass of 87 kDa. Sequence comparisons of Endo-beta-GalAB to other microbial endo-beta-galactosidases revealed no significant sequence homology. Although a BLAST search did reveal a modest homology (34%) to a 'fucolectin-related protein of unknown function' from Streptococcus pneumoniae, this protein did not cleave blood group A- and B-glycoconjugates. Endo-beta-Gal AB has a very broad optimal pH (5.5--8.0) and is stable with little loss of activity upon storage at -20°C for up to 2 years. We have established the structures of the liberated trisaccharides from A+ porcine gastric mucin and B+ human ovarian cyst glycoprotein by NMR spectroscopy. Endo-beta-GalAB was found to effectively liberate the blood group A- and B-trisaccharide epitopes, GalNAc/Galalpha1 → 3(Fucalpha1 → 2) Gal, from these glycoconjugates. Also, Endo-beta-Gal AB was able to completely abolish the blood group B antigenicity of type B human red blood cells. Due to its unique specificity, Endo-beta-Gal AB should prove useful in studying the structure and function of blood group A- and B-containing glycoconjugates / acase@tulane.edu

acase@tulane.edu

acase@tulane.edu

acase@tulane.edu

Ceramide glycanase is an enzyme which hydrolyzes the linkage between the oligosaccharide chains and lipid moieties of glycolipids. The intact glycan chains are released from various glycolipids in which the glycan chains are attached to the lipid moieties through a $\beta$-glucosyl linkage. This dissertation includes isolation and characterization of ceramide glycanase from leeches and earthworms and brief examination of the distribution of this unique lipid splitting enzyme in certain living organisms and tissues Ceramide glycanase was isolated from the leech, Macrobdella decora. The general properties of the purified enzyme and its relative activities on various natural glycosphingolipids were carefully characterized. The rate of the hydrolysis by this enzyme on various glycolipids either having the same oligosaccharide chains but differing in the lipid moieties or having the same lipid portions but differing in the sugar chains was compared. The results indicated that both the hydrophilic and hydrophobic moieties in glycolipids affect the action of ceramide glycanase Additionally, ceramide glycanase was purified from the earthworm, Lumbricus terrestris and its properties were compared with those of the leech enzyme. Although they differ somewhat in molecular weight, optimal pH, sensitivity to metal ions, detergent requirement and substrate specificities, both enzymes hydrolyze various glycolipids including ceramide glycans (glycosphingolipids), sphingosine glycans (lyso-glycosphingolipids), alkyl-glycans and dialkylglycerol which have oligosaccharide chains linked to the lipid moieties through a $\beta$-glucosyl linkage Among all the plants, animals and mammalian tissues and organs examined, ceramide glycanase was found only in annelids. No ceramide glycanase activity has been detected in any tissues or organs from mammalian sources / acase@tulane.edu

acase@tulane.edu

acase@tulane.edu

acase@tulane.edu