Quantitative X-ray fluorescence analysis of an Egyptian faience pendant and comparison with PIXE.

The X-ray fluorescence (XRF) technique is a common choice in the archaeometric field for in situ investigations with portable instruments. This work shows that XRF portable systems can be used for quantitative analyses using appropriate software, obtaining a similar accuracy to that provided with other techniques such as particle-induced X-ray emission (PIXE), as shown for an Egyptian faience pendant and for two glass standards.

Distinct cellular locations of the syntaxin family of proteins in rat pancreatic acinar cells.

Syntaxins are cytoplasmically oriented integral membrane soluble NEM-sensitive factor receptors (SNAREs; soluble NEM-sensitive factor attachment protein receptors) thought to serve as targets for the assembly of protein complexes important in regulating membrane fusion. The SNARE hypothesis predicts that the fidelity of vesicle traffic is controlled in part by the correct recognition of vesicle SNAREs with their cognate target SNARE partner. Here, we show that in the exocrine acinar cell of the pancreas, multiple syntaxin isoforms are expressed and that they appear to reside in distinct membrane compartments. Syntaxin 2 is restricted to the apical plasma membrane whereas syntaxin 4 is found most abundantly on the basolateral membranes. Surprisingly, syntaxin 3 was found to be localized to a vesicular compartment, the zymogen granule membrane. In addition, we show that these proteins are capable of specific interaction with vesicle SNARE proteins. Their nonoverlapping locations support the general principle of the SNARE hypothesis and provide new insights into the mechanisms of polarized secretion in epithelial cells.

The vesicle-associated membrane protein family of proteins in rat pancreatic and parotid acinar cells.

BACKGROUND & AIMS: The vesicle-associated membrane protein (VAMP) family of proteins may play an important role in regulating enzyme secretion from pancreatic and parotid acini. The purpose of this study was to characterize the isoforms produced in pancreatic and parotid acini and determine their subcellular locations. METHODS: Using a battery of specific antisera and recombinant tetanus toxin light chain (which cleaves VAMP-2 and cellubrevin), the presence of each VAMP molecule in the acini was determined by immunoblotting of subcellular membrane fractions; their localization was determined by confocal immunofluorescence microscopy and immunogold electron microscopy. RESULTS: Both VAMP-2 and cellubrevin were present on both the zymogen granule membrane and plasma membrane. VAMP-1 was not present in the acinar cell but was found in the nerve endings innervating the acini. As expected, pancreatic acinar VAMP-2 and cellubrevin were sensitive to cleavage by recombinant tetanus toxin. CONCLUSIONS: VAMP-2 and cellubrevin may play integral roles in exocytosis of the pancreatic and parotid acinar cells, whereas VAMP-1 is restricted to nerves that innervate the acini and may function to modulate exocrine activity.

Characterization of SNARE protein expression in beta cell lines and pancreatic islets.

Pancreatic beta cells and cell lines were used in the present study to test the hypothesis that the molecular mechanisms controlling exocytosis from neuronal cells may be used by the beta cell to regulate insulin secretion. Using specific antisera raised against an array of synaptic proteins (SNAREs) implicated in the control of synaptic vesicle fusion and exocytosis, we have identified the expression of several SNAREs in the islet beta cell lines, beta TC6-f7 and HIT-T15, as well as in pancreatic islets. The v-SNARE vesicle-associated membrane protein (VAMP)-2 but not VAMP-1 immunoreactive proteins were detected in beta TC6-f7 and HIT-T15 cells and pancreatic islets. In these islet-derived cell lines, this 18-kDa protein comigrated with rat brain synaptic vesicle VAMP-2, which was cleaved by Tetanus toxin (TeTx). Immunofluorescence confocal microscopy and electron microscopy localized the VAMP-2 to the cytoplasmic side of insulin containing secretory granule membrane. In streptolysin O permeabilized HIT-T15 cells, TeTx inhibited Ca2+-evoked insulin release by 83 +/- 4.3%, which correlated well to the cleavage of VAMP-2. The beta cell lines were also shown to express a second vesicle (v)-SNARE, cellubrevin. The proposed neuronal target (t)-membrane SNAREs, SNAP-25, and syntaxin isoforms 1-4 were also detected by Western blotting. The beta cell 25-kDa SNAP-25 protein and syntaxin isoforms 1-3 were specifically cleaved by botulinum A and C toxins, respectively, as observed with the brain isoforms. These potential t-SNARES were localized by immunofluorescence microscopy primarily to the plasma membrane in beta cell lines as well as in islet beta cells. To determine the specific identity of the immunoreactive syntaxin-2 and -3 isoforms and to explore the possibility that these beta cells express the putative Ca2+-sensing molecule synaptotagmin III, RT-PCR was performed on the beta cell lines. These studies confirmed that betaTC6-F7 cells express syntaxin-2 isoforms, 2 and 2', but not 2'' and express syntaxin-3. They further demonstrate the expression of synaptotagmin III. DNA sequence analysis revealed that rat and mouse beta cell syntaxins 2, 2' and synaptotagmin III are highly conserved at the nucleotide and predicted amino acid levels (95-98%). The presence of VAMP-2, nSec/Munc-18, SNAP-25 and syntaxin family of proteins, along with synaptotagmin III in the islet cells and in beta cell lines provide evidence that neurons and beta cells share similar molecular mechanisms for Ca2+-regulated exocytosis. The inhibition of Ca2+-evoked insulin secretion by the proteolytic cleavage of HIT-T15 cell VAMP-2 supports the hypothesis that these proteins play an integral role in the control of insulin exocytosis.

Simultaneous solid-phase extraction and chromatographic analysis of morphine and hydromorphone in plasma by high-performance liquid chromatography with electrochemical detection.

High-performance liquid chromatography has become an important analytical tool for the quantitation of opioid drugs. Using solid-phase extraction and coulometric electrochemical detection, we have developed a chromatographic method for the simultaneous measurement of morphine and hydromorphone which is both sensitive and specific. Using 1 ml of plasma, intra-assay and inter-assay data show that the detection limit for accurate quantitation of these compounds is about 1.2 ng/ml (coefficient of variation 11.6%) for morphine and 2.5 ng/ml (coefficient of variation 10.5%) for hydromorphone. The method is simple and readily adaptable to most pharmacokinetic studies and toxic screens involving these drugs.

Comparison of continuous subcutaneous and intravenous hydromorphone infusions for management of cancer pain.

To compare the safety and efficacy of subcutaneous and intravenous infusion of opioid analgesics, a randomised, double-blind, crossover trial was carried out in inpatients. 15 patients with severe cancer pain received two 48 h infusions of hydromorphone--one subcutaneously and one intravenously in randomly allocated order. The study was made double-blind by the use of two infusion pumps throughout; during the active subcutaneous infusion the intravenous pump delivered saline and vice versa. Serial measurements of pain intensity, pain relief, mood, and sedation by means of visual analogue scales showed no clinically or statistically significant difference between the two infusion routes. Side-effects were slight, and the mean number of morphine injections for breakthrough pain did not differ significantly between the routes (4.8 [SD 4.5] for intravenous vs 5.3 [5.6] for subcutaneous). Plasma hydromorphone concentrations measured at 24 h and 48 h of infusion showed stable steady-state pharmacokinetics; the mean bioavailability from subcutaneous infusion was 78% of that with intravenous infusion. Because of the simplicity, technical advantages, and cost-effectiveness of continuous subcutaneous opioid infusion into the chest wall or trunk, intravenous opioid infusion for the management of severe cancer pain should be abandoned.