Evaluation of salivary function by sialoscintigraphy in locally advanced nasopharyngeal cancer patients after intensity modulated radiotherapy.

PURPOSE: This study aimed to evaluate the salivary gland function changes by sialoscintigraphy in locally advanced nasopharyngeal cancer (NPC) after intensity modulated radiotherapy (IMRT). MATERIALS AND METHODS: Salivary function was assessed by sialoscintigraphy. Quantitative sialoscintigraphy was performed in 24 NPC patients prior to and after IMRT. Results were categorized in four groups according to the duration of treatment. The sialoscintigraphy parameters were examined. RESULTS: Sialoscintigraphy showed a significant difference in the secretion of each interval groups. The parameters of scintigraphy, except maximum accumulation (MA) of submandibular glands, decreased first after radiotherapy, and then recovered. However, the MA of submandibular glands was continuously downhill after radiation. CONCLUSIONS: The sialoscintigraphy parameters of each gland changed with the different radiation dose and follow-up intervals. The salivary function was influenced after radiotherapy in locally advanced NPC, especially, in the submandibular gland. Strategies to improve the salivary function should be assessed.

Protease inhibitor 10 inhibits tumor necrosis factor alpha -induced cell death. Evidence for the formation of intracellular high M(r) protease inhibitor 10-containing complexes.

Protease inhibitor 10 (PI10) is a member of the ovalbumin family of serine protease inhibitors (ov-serpin) that is expressed at elevated levels in patients with acute myeloid leukemia and chronic myelomonocytic leukemia. Based upon the ability of the related serpin plasminogen activator inhibitor 2 (PAI-2) to protect cells against tumor necrosis factor alpha (TNFalpha)-induced cell death, this study was initiated to investigate the potential cytoprotective activity of PI10. Two different expression systems (i.e. plasmids encoding either PI10 alone or PI10 fused to the tag: enhanced green fluorescent protein, EGFP) were utilized to stably transfect an eukaryotic model cell system (i.e. HeLa cells) that neither expresses PAI-2 nor PI10. The level of PI10 expression in the stable transfectants was found to correlate with their resistance to TNFalpha-induced cell death. Immunoprecipitation/immunoblotting experiments demonstrated that PI10 is able to form SDS-stable complexes (i.e. M(r) >100,000) with a cytosolic protein(s). Increased levels of the PI10-containing complexes can be detected by TNFalpha treatment by preventing intracellular degradative activities with the proteasome inhibitor N-carbobenzyloxy-leucine-leucine-norvalinal. PI10-containing complexes are dissociated with conditions known to separate classical protease-serpin complexes (i.e., 1.5 m ammonium hydroxide in the presence of SDS). These data support a role for the regulation of intracellular protease activities by ov-serpins.

Identification of a nuclear targeting domain in the insertion between helices C and D in protease inhibitor-10.

Protease inhibitor 10 (PI-10), an intracellular ovalbumin-serpin, contains a series of basic amino acids in the loop between helices C and D that exhibit homology to known nuclear targeting signals. Transfection of HeLa cells with plasmids encoding enhanced green fluorescent protein (EGFP) coupled to PI-10 revealed an intense fluorescence of the nucleus. Immunoblotting demonstrated a single Mr 80,000 EGFP.PI-10 complex in isolated nuclei. Mutation of four basic amino acids in the interhelical loop to alanines (i.e. K74A, K75A, R76A, K77A) resulted in the fluorescent complex being confined to the cytoplasm. Further evidence for a nuclear targeting signal in this region was provided by localization of the fluorescent label to the nucleus in cells transfected with a plasmid encoding EGFP fused to the 25 amino acids comprising the interhelical loop of PI-10 (i.e. Arg-63 to Glu-87), whereas a cytoplasmic distribution was noted for the construct encoding EGFP coupled to the mutated interhelical loop. These data raise the possibility that PI-10 may play a role in regulating protease activity within the nucleus, a property unique in the field of serpin biology.

Purification of storage granule protein-23. A novel protein identified by phage display technology and interaction with type I plasminogen activator inhibitor.

Type 1 plasminogen activator inhibitor (PAI-1) is a key regulator of the fibrinolytic cascade that is stored in a rapidly releasable form within platelet alpha-granules. To identify proteins that may participate in the targeting or storage of this potent inhibitor, this report investigates the applicability of utilizing filamentous bacteriophages to display proteins expressed by cells containing a regulated secretory pathway and their enrichment based upon an interaction with PAI-1. For this purpose, RNA was extracted from AtT-20 cells (i.e. a classical model cell system for intracellular protein sorting), reverse transcribed, amplified using polymerase chain reaction primers containing internal restriction sites, and cloned into the phagemid pCOMB3H for expression as fusion constructs with the bacteriophage gene III protein. Escherichia coli was transformed with the phagemids and infected with VCSM13 helper phage, and the resulting AtT-20 cDNA-bacteriophage library was enriched by panning against solid- and solution-phase PAI-1. The enriched cDNA library was subcloned into a prokaryotic expression vector system that replaces the gene III protein with a decapeptide tag for immunologic quantitation. One novel cDNA clone (i.e. A-61), which preferentially recognized solution-phase PAI-1 and reacted positively with antibodies derived from a rabbit immunized with alpha-granules, was subcloned into the prokaryotic expression vector pTrcHis to create a construct containing an N-terminal six-histidine purification tag. This construct was expressed in E. coli, purified by nickel-chelate chromatography followed by preparative SDS-polyacrylamide gel electrophoresis, and utilized for the generation of polyclonal antibodies. Immunoblotting analysis employing antibodies against the purified A-61 construct revealed a 23-kDa protein present in the regulated secretory pathway of AtT-20 cells. The 23-kDa molecule was purified from media conditioned by AtT-20 cells by ion exchange chromatography on DEAE-Sephacel, molecular sieve chromatography on Sephacryl S-100, chromatofocusing on Polybuffer exchanger 94, and affinity chromatography on PAI-1-Sepharose. N-terminal amino acid sequencing of a 16-kDa Lys-C proteolytic fragment of the 23-kDa storage granule protein was employed to confirm its identity with the cDNA sequence of clone A-61. These data indicate that phage display of cDNA libraries fused to the C-terminal region of the gene III protein and their enrichment via an interaction with a target molecule can be utilized to define other proteins present within a particular cellular pathway.