The effects of parathyroid hormone (PTH), prostaglandins (PGE1, PGE2, PGF2a), cAMP, cAMP-analogues, phosphodiesterase (PDE) inhibitors and la (OH) D3 on bone resorption and associated cellular process have been studied in a bone organ culture system using half- calvaria from 6-7 day-old mice. Bone resorption was assessed by determining the release of calcium (Ca2+), inorganic phosphate (Pi) and 45Ca from the calvarial bones to the culture media. The release of lysosomal enzymes was studied by analysing the activities of β-glucuronidase, β-N-acetyl- glucosaminidase, β-galactosidase and p-nitrophenyl phosphatase in bone expiants and culture media. The release of non-lysosomal enzymes was followed by assaying the activities of lactate dehydrogenase (LDH), aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) in the expiants as well as the media. In addition glucose consumption and lactate production was registered. The findings may be summarized as follows: 1. cAMP and PDE-inhibitors have the capacity to inhibit the initial stages of spontaneous as well as PTH- PGE1- and PGE2-stimulated bone resorption. 2. cAMP and PDE-inhibitors produce after a lag period, or a period of reduced bone resorption, a stimulatory effect on bone resorption. 3. There is a significant correlation between bone resorption and lysosomal enzyme release both as regards the inhibitory and stimulatory effect of cAMP. 4. PGE2 and la (OH) D3 increase the release of lysosomal enzymes in parallel with bone resorption. 5. Bone resorption stimulated by cAMP and PGE2 is associated with increased glucose consumption and lactate production, while la (OH) D3 promotes bone resorption without any change with regard to these parameters of glucose metabolism. It is concluded that the initial stages of bone resorption stimulated by PTH, PGE1 and PGE2 is medited by cAMP-independent mechanisms, but that this nucleotide may be an intracellular mediator of these hormones of later stages of bone resorption. It is suggested that the role played by cAMP may be related to the capacity of PTH and PGE2 to develop new osteoclasts. The observations further support the concept that lysosomal enzyme release is intimately associated with bone resorption. Finally it is concluded that increased lactate production seems to be related to bone resorption stimulated by agents which increase the level of cAMP (PTH, PGE2, dbcAMP), but that it is not an indispensible part of the mechanism by which the osteoclasts solubilize bone mineral. / digitalisering@umu.se

Human α-N-Acetylglucosaminidase (Naglu) is a lysosomal acid hydrolase implicated in tthe rare metabolic storage disorder known as mucopolysaccharidosis type IIIB (MPS IIIB; also Sanfilippo syndrome B). Absence of this enzyme results in cytotoxic accumulation of heparan sulphate in the central nervous system, causing mental retardation and a shortened lifespan. Enzyme replacement therapy is not currently effective to treat neurological symptoms due to the inability of exogenous Naglu to access the brain. This laboratory uses a Spodoptera frugiperda (Sf9) insect cell system to express Naglu fused to a synthetic protein transduction domain with the intent to facilitate delivery of Naglu across the blood-brain barrier. The project described herein may be broken down into three main sections. Firstly, the impact of two cryptic splice sites on Naglu expression levels was analyzed in both transiently expressing Sf9 cultures and stably selected cell lines. Secondly, the effectiveness of the native Naglu secretion-signaling peptide in the Sf9 system was examined. Finally, purification of a Naglu fusion protein from suspension culture medium was performed using hydrophobic interaction chromatographic techniques. The ultimate goal of this research is to develop an efficient system for economical, large-scale production of a human recombinant Naglu fusion protein that has the potential to be successfully used for enzyme replacement therapy to treat MPS IIIB. / Graduate