Determination of selenium in human blood by high-performance liquid chromatography with fluorescence detection

Selenium was determined in human whole blood or red cells by high-performance liquid chromatography (HPLC) using a new internal standard. The method provides a 0.15-ng detection limit, a between-day standard deviation of 1% at the 20-ng level, and a 3% within-day standard deviation at the 1-ng level. Samples were wet-ashed, and a selenium-diaminonaphthalene derivative was formed, followed by addition of tetraphenylnaphthacene internal standard, reverse-phase HPLC separation (10 min/run), and fluorescence detection. The detection limit was reduced by discrimination of fluorescence excitation of the selenium complex from the background using long-wavelength excitation (480 nm), removal of stray light in the excitation beam, and other optimizations described. Representative aliquots of frozen and thawed whole blood samples were obtained by using a nitric acid predigestion at ambient temperature. Procedures to validate the method included standard addition and neutron activation analysis.     

The extravesicular domain of synaptotagmin-1 is released with the latent fibroblast growth factor-1 homodimer in response to heat shock

The heparin-binding fibroblast growth factor (FGF) prototypes lack a classical signal sequence, yet their presence is required in the extracellular compartment for the activation of cell-surface receptor-dependent signaling. Early studies with FGF-1 demonstrated its presence in bovine brain as a novel high molecular weight complex, and subsequent studies identified a second heparin-binding protein that co-purified with FGF-1. Polypeptide sequence analysis revealed that this heparin-binding protein corresponded to the extravesicular domain of bovine synaptotagmin (Syn)-1, a transmembrane component of synaptic vesicles involved in the regulation of organelle traffic. Since FGF-1 is released in response to heat shock as a mitogenically inactive Cys-30 homodimer, we sought to determine whether this heparin-binding protein was involved in the release of FGF-1. We report that a proteolytic fragment of the extravesicular domain of Syn-1 is associated with FGF-1 in the extracellular compartment of FGF-1-transfected NIH 3T3 cells following temperature stress. By using heparin-Sepharose affinity to discriminate between the monomer and homodimer forms of FGF-1 and resolution by conventional and limited denaturant gel shift immunoblot analysis, it was possible to identify FGF-1 and Syn-1 as potential components of a denaturant- and reducing agent-sensitive extracellular complex. It was also possible to demonstrate that the expression of an antisense-Syn-1 gene represses the release of FGF-1 in response to heat shock. These data indicate that FGF-1 may be able to utilize the cytosolic face of conventional exocytotic vesicles to traffic to the inner surface of the plasma membrane where it may gain access to the extracellular compartment as a complex with Syn-1.     

Rapid diagnosis of tuberculous meningitis by polymerase chain reaction assay of cerebrospinal fluid

A polymerase chain reaction (PCR) method for the rapid diagnosis of tuberculous meningitis (TBM) was used to study prospectively 47 cerebrospinal fluid (CSF) samples from 45 patients. Twenty CSF samples were from patients with clinically suspected TBM and another 27 samples came from patients without clinically suspected TBM. Mycobacterial DNA was detected in 15 CSF samples (14 from patients with clinically suspected TBM and 1 from a patient not suspected of having TBM). Of the PCR-positive samples, 4 were also positive for mycobacterial culture. However, 32 PCR-negative samples were all culture-negative. All samples were negative for the acid-fast bacillus by direct smear. The single PCR-positive patient in the clinically unsuspected TBM group was initially diagnosed as suffering from aseptic meningitis on the basis of his clinical features. The mycobacterial culture of his CSF specimen was also positive and a revised diagnosis of an aseptic type of TBM was made. The estimations of specificity and sensitivity in this study were 100% and 70% respectively. The results showed that using a PCR to detect mycobacterial DNA in CSF for the early diagnosis of TBM is not only a rapid but also an accurate method.     

Structure-activity analysis of the interaction of curacin A, the potent colchicine site antimitotic agent, with tubulin and effects of analogs on the growth of MCF-7 breast cancer cells

Originally purified as a major lipid component of a strain of the cyanobacterium Lyngbya majuscula isolated in Cura?ao, curacin A is a potent inhibitor of cell growth and mitosis, binding rapidly and tightly at the colchicine site of tubulin. Because its molecular structure differs so greatly from that of colchicine and other colchicine site inhibitors, we prepared a series of curacin A analogs to determine the important structural features of the molecule. These modifications include reduction and E-to-Z transitions of the olefinic bonds in the 14-carbon side chain of the molecule; disruption of and configurational changes in the cyclopropyl moiety; disruption, oxidation, and configurational reversal in the thiazoline moiety; configurational reversal and substituent modifications at C13; and demethylation at C10. Inhibitory effects on tubulin assembly, the binding of colchicine to tubulin, and the growth of MCF-7 human breast carcinoma cells were examined. The most important portions of curacin A required for its interaction with tubulin seem to be the thiazoline ring and the side chain at least through C4, the portion of the side chain including the C9-C10 olefinic bond, and the C10 methyl group. Only two modifications totally eliminated the tubulin-drug interaction. The inactive compounds were a segment containing most of the side chain, including its two substituents, and analogs in which the methyl group at the C13 oxygen atom was replaced by a benzoate residue. Antiproliferative activity comparable with that observed with curacin A was only reproduced in compounds that were potent inhibitors of the binding of colchicine to tubulin. Molecular modeling and quantitative structure-activity relationship studies demonstrated that most active analogs overlapped extensively with curacin A but failed to provide an explanation for the apparent structural analogy between curacin A and colchicine.     

Palliation of symptomatic adrenal gland metastases by radiotherapy

Whilst adrenal metastases are common in patients with metastatic disease, they are rarely symptomatic. We present a series of seven patients with painful adrenal metastases who were seen in one centre over an 18-month period. All responded to palliative radiotherapy with a reduction in pain.     

Visual field defects after macular hole surgery. A new finding

PURPOSE: The purpose of the study is to report the problem of a temporal visual field defect occurring after macular hole surgery. METHODS: The authors reviewed the records of 13 patients found to have visual field defects after vitrectomy for macular holes. Fluorescein angiograms (13 patients), optic nerve photographs (13 patients), focal electroretinograms (3 patients), and nerve fiber analyses (8 patients) were performed in patients with visual field defects. RESULTS: An absolute, temporal, usually inferior field defect was noted in 13 patients. In eight patients, the defect was detected because of specific reports or retrospective field examination results. Five patients examined in a prospective manner were found to have field defects. No history of abnormal intraocular pressure or direct trauma to the optic nerve or retinal vessels was identified. Four patients showed optic nerve pallor and three had an anomalous-appearing disc. Focal electroretinograms were of similar amplitude in the involved retina compared to corresponding areas in the healthy fellow eye. Nerve fiber analysis showed a reduction in nerve fiber layer thickness correlating to the visual field defect in those eight patients in which this test was used. CONCLUSION: A significant temporal field defect may occur in patients after otherwise uncomplicated surgery for macular holes. The cause is unclear; however, reductions in nerve fiber layer thickness from the superior and nasal peripapillary area suggest that acute surgical release of the posterior hyaloid and the use of long-acting intraocular gas may in certain patients result in visual field defects.     

Biomarkers in Barrett''s esophagus (review)

Lens major intrinsic protein (MIP) is the founding member of the MIP family of membrane channel proteins. Its isolation from ovine lens fibre cell membranes and its two-dimensional crystallization are described. Membranes were solubilized with N-octyl-beta-D-glucoside and proteins fractionated by sucrose gradient centrifugation containing decyl-beta-D-maltoside. MIP was purified by cation exchange chromatography, and homogeneity was assessed by mass analysis in the scanning transmission electron microscope. Purified MIP reconstituted into a lipid bilayer at a low lipid-to-protein ratio formed highly ordered tetragonal two-dimensional crystals. The square unit cell had a side length of 6.4 nm, and exhibited in negative stain four stain-excluding elongated domains surrounding a central stain-filled depression. Projection maps of freeze-dried crystals exhibited a resolution of 9 A, and revealed a monomer structure of MIP consisting of distinct densities. Despite significant differences in the packing of tetramers in the crystals, the projection map of the MIP monomer was similar to that of aquaporin-1 (AQP1), the first member of the MIP family which had its structure resolved to 6 A. Our protocols for the purification and reconstitution of MIP establish the feasibility for future work to visualize structure elements which determine the diverse functional properties of the MIP family members.