The biocatalysis of a crude chlorophyllase extract, obtained from the biomass culture of Phaeodactylum tricornutum, in neat organic solvent media was investigated. The addition of selected excipients, including crown ether (enzyme:crown ether, 135:1--2.7:1, w/w), dextran (enzyme:dextran, 1:2--1:0.25, w/w), Span 40 and Span 60 (enzyme:Span, 1:2, w/w) and sodium bis (2-ethylhexyl) sulfosuccinate (enzyme:AOT, 1:12.6, w/w), to the crude solid enzyme preparation decreased the chlorophyllase activity. The effects of selected parameters, including solvent hydrophobicity (Log P, 2.40--4.45), initial water activity (aw, 0.44--0.97), agitation speed (0--200 rpm), reaction temperature (25--45°C) and enzyme concentration (1.67--5.3 mg solid enzyme/mL) on chlorophyllase activity, were investigated using a crude solid enzyme. The experimental findings showed that the highest chlorophyllase specific activity of 362.4 nmol hydrolyzed chlorophyll/g solid enzyme/min and bioconversion yield of 90.7% were obtained with the hexane/octanone mixture (98.7:1.3, v/v), aw of 0.90, agitation speed of 200 rpm, reaction temperature of 35°C and enzyme concentration of 3.33 mg solid enzyme/mL.

As nascent secretory and membrane proteins are inserted into the endoplasmic reticulum (ER), they are maintained in folding and/or assembly competent states by molecular chaperones including the Hsp70 and Hsp90 homologues, BiP and GRP94, and the lectin-like chaperones calnexin (CNX) and calreticulin (CRT). Folding is catalyzed by protein disulfide isomerase (PDI), its CNX (and CRT) associated homologue, ERp57, and protein prolyl isomerase (PPI). Moreover, N-linked glycoproteins benefit from a lectin-based "quality control apparatus" that ensures their correct folding or oligomeric assembly. Binding to these lectins occurs through oligosaccharide trimming from Glc 3Man9GlcNAc2 to the monoglucosylated form (Glc 1Man9GlcNAc2). Release and subsequent rebinding occurs though the hydrolysis and reglucosylation of the innermost glucose by glucosidase II and UDP-glucose glycoprotein:glucosyltransferase, respectively. This cyclical process, termed the "Calnexin Cycle", continues until their correct conformation is achieved. / The cloning and characterization of human glucosidase II is reported here. cDNAs for two splice variants of the catalytic alpha subunit and the beta subunit were isolated. Expression of the beta subunit was shown to be required for enzymatic activity, solubility and/or stability, and ER retention of the enzyme. Detailed kinetic analysis on recombinant alpha1/beta and alpha2/beta isoforms, using p-nitrophenyl alpha-D-glucopyranoside as a substrate, reveals that both exhibit kinetic profiles of a two binding site model, and share properties of catalysis and inhibition on this substrate. Moreover, similar rates of hydrolysis of the oligosaccharide substrates rules out the possibility that the two binding site kinetic model, first proposed by Alonso et al. (1999, Biochem J. 278:721--7), is the result of co-purified isoforms of glucosidase II that have different substrate specificities. / Also, an ER protein two-hybrid system, based on Ire1p and the unfolded protein response (UPR) pathway in Saccharomyces cerevisiae, was developed to examine and map the interactions between CNX/CRT and ERp57. Ire1p fusions with CNX and CRT were shown to interact specifically with ERp57, and as expected, PDI did not. Through deletion analysis, new roles were assigned to the proline-rich loop domains of CNX and CRT, and the non-catalytic B thioredoxin domain of ERp57 in mediating their heterodimerization.

A partially purified chlorophyllase, obtained from alga Phaeodactylum tricornutum, was assayed for its hydrolytic activity towards the pheophytin in ternary micellar systems of hexane/Tris-HCl/surfactant. A wide range of surfactants, sorbitans (Span 20, 40, 60, 80 and 85) and polysorbates (Tween 20, 40, 60, 80 and 85), was used. The use of either 50 $ mu$M of Span 85 or 1 $ mu$M of Tween 80 increased the hydrolytic activity of chlorophyllase by 110 and 23%, respectively. The optimum values of pH, enzyme content, incubation time and temperature for the hydrolytic activity of chlorophyllase were determined as 8.25, 8 $ mu$g protein/ml, 60 min and 27.5$ sp circ$C, respectively. The enzyme was assayed for its hydrolytic activity in the most appropriate ternary system containing Span 85 with purified pheophytin, as well as chlorophyll derivatives, as substrates. Moreover, the values of $V sb{ rm max}/K sb{ rm m}$ ratio for chlorophyllase, using the partially purified pheophytin as substrate, in ternary systems with Span 85 and Tween 80 as surfactants, were 0.15 and 0.08, respectively; however, the value of $V sb{ rm max}/K sb{ rm m}$ ratio for the enzyme, in the ternary system with Span 85, using purified pheophytin as substrate was 0.07. The addition of optimized amounts of individual membrane lipids, L-$ alpha$-phosphatidylcholine, L-$ alpha$-phosphatidyl-DL-glycerol and $ beta$-carotene increased the hydrolytic activity of chlorophyllase, using partially purified pheophytin as substrate, by 50, 36 and 10%, respectively, for Span 85, and 30, 48 and 15%, respectively, for Tween 80; in addition, these lipids increased the enzyme activity by 6, 23 and 31%, respectively, in the Span 85 media, using purified pheophytin as substrate. Phytol showed a competitive inhibitory effect on chlorophyllase activity in both Span and Tween systems containing partially purified pheophytin substrate; however, phytol had an uncompetitive inhibitory effect on the enzyme activity in the S

Polymorphisms of platelet glycoproteins (GPs) are frequently targets for anti-platelet antibodies. At least 19 antigenic polymorphisms have been identified on platelet GPs. Antibodies against the HPA-lb polymorphism (a Leu to Pro switch at amino acid residue 33 of the IIIa sub-unit of GP IIb/Illa) have been attributed to as much as 90% of all cases of neonatal alloimmune thrombocytopenic purpura and posttransfusion purpura in caucasians. The HPA-lb polymorphism has also been equivocally associated with coronary artery disease, particularly early onset (<60 years of age) myocardial infarction. Current technology for identifying individuals with the HPA-lb phenotype is limited to the labor-intensive, highly technical and expensive process of DNA amplification by polymerase chain reaction and restriction fragment length polymorphism (PCR/RFLP) analysis.This study proposes an alternative method for phenotyping individuals for the HPA-1 polymorphism using the Biocytex Platelet HPA-1 kit. The kit identifies the HPA-1 polymorphism utilizing two monoclonal CD61 (platelet glycoprotein IIb/IIIa) antibodies, one of which has a lowered affinity for GP llb/Illa possessing the HPA-lb polymorphism. Fluorescent labeling of bound antibody allows for flow cytometric quantitation of antibody binding capacity (ABC) for both monoclonal antibodies, and ratios derived from the ABC can be used to phenotype previously unknown samples.The Biocytex HPA-1 kit identified 73 of 74 (98.6%) individuals possessing the HPA1 a/HPA-1 a phenotype, 22 of 22 (100%) HPA-1 a/HPA-1 b individuals and 4 of 4 (100%) HPAIb/HPA-Ib individuals. All HPA-lb phenotypes were confirmed by PCR/RFLP. Total accuracy of the test system was 99%. / Department of Biology

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), also called human herpesvirus 8 (HHV8), has been been identified in all epidemiological forms of KS as well as in tissue obtained from primary effusion lymphoma (PEL) and Multicentric Castleman's disease (MCD). The KSHV genome contains several putative oncogenes, suggesting that viral infection may induce cellular transformation and tumorgenesis. Herpesviruses encode a number of different surface glycoproteins, which are involved in virus-host interactions. Studies have shown that the viral glycoproteins H and L form a complex that plays an essential role in viral attachment and cell to cell fusion. Both glycoproteins have been identified in KSHV and were expressed in mammalian cells. Expression studies revealed that KSHV gH and gL exhibit similar features to those seen in other herpesviruses. However, KSI-IV gL appears to traffic independently and may function in cell to cell fusion processes even when expressed alone. KSI-IV de novo infections are rare and the lack of a reliable cell culture system has delayed pathogenesis studies. As part of this thesis the hepatoma cell line HepG2 has been shown to allow limited KSHV infection, as judged by nested PCR. Studies have shown that infection leads to increased apoptosis, although viral replication could not be detected. Furthermore, Epstein Barr Virus (EBV) appeared to modulate the ability of KSHV to infect HepG2 cells. Finally, a microtitre plate assay has been established for the quantification of the KSHV genome. A comparison of plasma and serum samples obtained at the same time point showed that plasma is more reliable in testing for KSHV, the DNA copy number in serum samples being reduced up to 10 fold. In conclusion, this new assay is a potentially useful tool for both diagnostic proposes and research studies.

We investigated whether human lysosomal hydrolases, in common with secretory and plasma membrane glycoproteins, associate with the ER chaperone calnexin. Neither $ alpha$- or $ beta$-chains of $ beta$-hexosaminidase A, cathepsin D, nor the endogenous proteases cathepsins B or L associated with calnexin in COS-I cells. Hex $ alpha$-chains misfolded due to either the incorporation of azetidine-2-carboxylic acid, treatment with dithiothreitol, or the presence of a Tay-Sachs Disease mutation (leading to retention of Hex A $ alpha$-chains in the ER) also did not associate with calnexin. Chemical-crosslinking reagents or long-term labeling also failed to show a Hex A $ alpha$-chain association with calnexin. Lysosomal hydrolases also did not associate with the ER chaperone calreticulin. Surprisingly, $ alpha$-L-iduronidase and Hex A $ alpha$-chains associated with calnexin when overexpressed using a CMV promoter. The segregation of lysosomal hydrolases from secretory proteins thus occurs at an earlier stage than predicted. Hydrolase folding appears to be controlled by a pathway different from that used by secretory and plasma membrane glycoproteins.

Thesis (Ph. D.)--University of Adelaide, Dept. of Pathology, 1985. / Includes bibliographical references (leaves 266-303).

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 5 uppsatser.

Diss. (sammanfattning) Göteborg : Univ., 2007. / Härtill 5 uppsatser.

Thesis (Ph. D.)--Auburn University. / Abstract. Vita. Includes bibliographical references (p. 76-102).