Membrane Proteins in Human Neutrophils - Identification and Characterization of Lipid Rafts in Subcellular Organelles

The human neutrophil is an important effector cell in acute inflammation and in the innate immune response against bacteria and fungi. When immune reactions occur in the tissue in response to antigen challenge, neutrophils are the first cells to enter the site of inflammation. The neutrophil is equipped with a vast amount of receptors that both interact with inflammatory mediators and host tissue as well as with the prey. These receptors are found on the cell surface but are also stored in different types of granules and vesicles in the cell. By mobilizing the granules and vesicles to various extents and thereby upregulate receptors to the plasma membrane, the mature neutrophil can modulate its communication with the environment. One granule type, the azurophil granules, primarily delivers a killing machinery to intracellular organelles containing a prey that has been engulfed. These granules have traditionally been regarded as classical lysosomes, but their membrane is so far largely uncharacterized. The aim of this thesis was to elucidate details regarding azurophil granule membrane composition in order to further understand their role in neutrophil function. The studies led to identification of so-called lipid rafts in the azurophil and other granule membranes, and a detailed characterization of the azurophil granule lipid rafts with regard to protein composition was thus performed. One of the proteins identified in azurophil granule membranes was stomatin. This protein was present also in other granule/vesicle membranes and the plasma membrane. Furthermore, the protein was localized to lipid rafts. Apart from stomatin, the azurophil granule membrane rafts contained a vast number of proteins, of possible importance for membrane structure/integrity and fusion. The fact that several cytoskeletal proteins also were identified, suggests that the granule membrane is organized in much the same way as the plasma membrane. The thesis also includes studies on the neutrophil receptors for galectin-3, a potent activator of extravasated human neutrophils. Since granule mobilization is a prerequisite for galectin-3-induced activation of the cells, the receptors have been suggested to be granule localized. Here, galectin-3-binding proteins from specific/gelatinase granules were isolated, and among these, CD66a and CD66b were identified as the most plausible receptor candidates. The CD66b is a glycophosphatidyl inositol (GPI)-linked protein that was found to be localized to lipid rafts, suggesting that raft-associated signaling may be of importance for the galectin-3-induced neutrophil responses

RNA silencing of lactate dehydrogenase gene in Rhizopus oryzae

Rhizopus oryzae is a filamentous fungus, belonging to the order Mucorales. It can ferment a wide range of carbohydrates hydrolyzed from lignocellulosic materials and even cellobiose to produce ethanol. However, R. oryzae also produces lactic acid as a major metabolite, which reduces the yield of ethanol. In this study, we show that significant reduction of lactic acid production could be achieved by short (25nt) synthetic siRNAs targeting the ldhA gene. The average yield of lactic acid production by R. oryzae during the batch fermentation process, where glucose had been used as a sole carbon source, diminished from 0.07gm/gm in wild type to 0.01gm/gm in silenced samples. In contrast, the average yield of ethanol production increased from 0.39gm/gm in wild type to 0.45gm/gm in silenced samples. These results show 85.7% (gm/gm) reduction in lactic acid production as compared with the wild type R. oryzae, while an increase of 15.4% (gm/gm) in ethanol yield

Biological treatment of chicken feather waste for improved biogas production

A two-stage system was developed which combines the biological degradation of keratin-rich waste with the production of biogas. Chicken feather waste was treated biologically with a recombinant Bacillus megaterium strain showing keratinase activity prior to biogas production. Chopped, autoclaved chicken feathers (4%, W/V) were completely degraded, resulting in a yellowish fermentation broth with a level of 0.51 mg/mL soluble proteins after 8 days of cultivation of the recombinant strain. During the subsequent anaerobic batch digestion experiments, methane production of 0.35 Nm(3)/kg dry feathers (i.e., 0.4 Nm(3)/kg volatile solids of feathers), corresponding to 80% of the theoretical value on proteins, was achieved from the feather hydrolyzates, independently of the pre-hydrolysis time period of 1, 2 or 8 days. Cultivation with a native keratinase producing strain, Bacillus licheniformis resulted in only 0.25 mg/mL soluble proteins in the feather hydrolyzate, which then was digested achieving a maximum accumulated methane production of 0.31 Nm(3)/kg dry feathers. Feather hydrolyzates treated with the wild type B. megaterium produced 0.21 Nm(3) CH(4)/kg dry feathers as maximum yield

Major histocompatibility complex class II (DR) antigen and costimulatory molecules on in vitro and in vivo activated human polymorphonuclear neutrophils

We have previously shown that normal human peripheral blood polymorphonuclear neutrophils (PMNs) contain cytoplasmic ‘stores’ of three key molecules normally associated with antigen presentation and T-cell costimulation, i.e. major histocompatibility complex class II (DR) antigen, CD80 (B7-1) and CD86 (B7-2). These cytoplasmic molecules were found to translocate to the cell surface within a few minutes following cross-linking (X-L) of Mac-1: an early neutrophil activation signal. In this study we have compared X-L of Mac −1 in parallel with four other well documented in vitro neutrophil activators: phorbol myristate acetate, N-formyl methionyl leucyl phenylalanine, lipopolysaccharide, and phagocytosis of immunoglobulin G–Latex particles. In addition, we have used paired samples of neutrophils obtained from peripheral blood (as a control) and synovial fluid from patients with rheumatoid arthritis as a source of in vivo activated cells. With the exception of phagocytosis, all activators resulted in the rapid (within 30 min) generation of two populations of activated neutrophils (designated P1 and P2) based on flow-cytometry measurements of size, granularity and phenotype. Significant up-regulation of DR and costimulatory molecules was observed, predominantly on P2 cells, with all activators except phagocytosis. CD80 and CD86 were noted to respond to the various activation signals in a different pattern suggesting that their intracellular granule location may be different. Dual-staining confocal laser microscopy studies showed that CD80 is largely confined to secretory vesicles (SVs) while CD86 appears to have a much wider distribution being found in SVs and within secondary (specific) and primary (azurophilic) granules. Increased surface expression of these antigens was also observed on P2 synovial fluid neutrophils appearing as large heterogeneous clusters on the cell surface when visualized by confocal laser microscopy