Laboratory gloves as a source of trace element contamination

Contamination in a trace element laboratory can come from a variety of sources, including laboratory gloves. Therefore, vinyl and latex gloves were obtained from as many manufacturers as would supply gloves. These gloves were either prepared for acid-washing and subsequent soaking in an acid solution, or immersed in an acid solution for a duration of either 1 min or 1 h. Incubation washes were analyzed for a variety of trace elements by flame atomic abosrption spectroscopy (AAS) or inductively coupled mass spectrometry (ICP-MS). Results indicated that only three brands of vinyl gloves were acceptable for use in a trace element laboratory, whereas others had contamination of different elements. Latex gloves contained such high levels of biologically important elements that they were not considered suitable for routine trace element work. Vinyl gloves of choice should be routinely acid-washed before use in a trace element laboratory.     

Simultaneous rapid high-performance liquid chromatographic determination of phenytoin and its prodrug, fosphenytoin in human plasma and ultrafiltrate

A reversed-phase high-performance liquid chromatographic assay for the simultaneous determination of phenytoin and fosphenytoin, a prodrug for phenytoin, in human plasma and plasma ultrafiltrate is described. For plasma, the method involves simple extraction of drugs with diethyl ether and evaporation of solvent, followed by injection of the reconstituted sample onto a reversed-phase C₁₈ column. Plasma ultrafiltrate is injected directly into the HPLC column. Compounds are eluted using an ion-pair mobile phase containing 20% acetonitrile. The eluent is monitored by UV absorbance at 210 nm. The fosphenytoin standard curves are linear in the concentration range 0.4 to 400 μg/ml for plasma and 0.03 to 80 μg/ml for ultrafiltrate. Phenytoin standard curves are linear from 0.08 to 40 μg/ml for plasma and from 0.02 to 5.0 μg/ml for ultrafiltrate. No interferences with the assay procedure were found in drug-free blank plasma or plasma ultrafiltrate. Relative standard deviation for replicate plasma or ultrafiltrate samples was less than 5% at concentrations above the limit of quantitation for both within- and between-run calculations.     

Performance of Multiplex Cytokine Assays in Serum and Saliva among Community-Dwelling Postmenopausal Women

Multiplexing arrays increase the throughput and decrease sample requirements for studies employing multiple biomarkers. The goal of this project was to examine the performance of Multiplex arrays for measuring multiple protein biomarkers in saliva and serum. Specimens from the OsteoPerio ancillary study of the Women’s Health Initiative Observational Study were used. Participants required the presence of at least 6 teeth and were excluded based on active cancer and certain bone issues but were not selected on any specific condition. Quality control (QC) samples were created from pooled serum and saliva. Twenty protein markers were measured on five multiplexing array panels. Sample pretreatment conditions were optimized for each panel. Recovery, lower limit of quantification (LLOQ) and imprecision were determined for each analyte. Statistical adjustment at the plate level was used to reduce imprecision estimates and increase the number of usable observations. Sample pre-treatment improved recovery estimates for many analytes. The LLOQ for each analyte agreed with manufacturer specifications except for MMP-1 and MMP-2 which were significantly higher than reported. Following batch adjustment, 17 of 20 biomarkers in serum and 9 of 20 biomarkers in saliva demonstrated acceptable precision, defined as <20% coefficient of variation (<25% at LLOQ). The percentage of cohort samples having levels within the reportable range for each analyte varied from 10% to 100%. The ratio of levels in saliva to serum varied from 1∶100 to 28∶1. Correlations between saliva and serum were of moderate positive magnitude and significant for CRP, MMP-2, insulin, adiponectin, GM-CSF and IL-5. Multiplex arrays exhibit high levels of analytical imprecision, particularly at the batch level. Careful sample pre-treatment can enhance recovery and reduce imprecision. Following statistical adjustments to reduce batch effects, we identified biomarkers that are of acceptable quality in serum and to a lesser degree in saliva using Multiplex arrays.     

Evidence for growth ofSporothrix schenckii on dead but not on living sphagnum moss

When clinical isolates ofSporothrix schenckii were inoculated onto the apices of living or dead sphagnum moss plants maintained under growth chamber conditions, populations of the fungus, assessed by standard dilution plate methods, increased swiftly up to about 70-fold on moist, dead plants but did not increase on the live moss. Light and scanning electron microscopy revealed fungal growth and sporulation on and within dead plants, but no evidence of either on live plants. These data provide indirect support for the contention thatS. schenckii does not grow on living sphagnum in bogs, but rather that sporotrichosis epidemics associated with sphagnum moss are likely to result from contamination of the dead plants at some point(s) in the chain of events during or after harvest. One practical implication of our results is that precautions should be taken to insure that sphagnum moss is stored dry and that it is not wetted any sooner than necessary before use. We also report here improvement of the Mycose isolation medium by an increase in cycloheximide from 400 to 800 mg/l, chloramphenicol from 50 to 250 mg/l, and the addition of rifampicin at 20 mg/l.     

The australopithecines in review

Over the past 75 years since the discovery of the first australopithecine at Taung in southern Africa there has been a growing realisation that there is no simple, linear ancestor-descendant relationship connecting the australopithecines to laterHomo. There are currently at least ten recognised species of australopithecine, including two species of earlyHomo, that have been recently transferred to the genusAustralopithecus. These known species span the period between about 4.2-1.2 Ma and throughout the majority of this period there are multiple contemporaneous hominin species in eastern and southern Africa. This contribution reviews current knowledge about the australopithecine species and their inferred relationships to each other and to the genusHomo. At present it is impossible to resolve the phylogenetic relationships of the australopithecines with any degree of confidence. There is a growing realisation of the ‘bushy’ nature of hominin evolution throughout the australopithecine period and also of the inevitability that additional early hominin species remain to be discovered. Paper submitted for inclusion in the Proceedings of the International Symposium of the Ramón Areces Foundation “Evolution of the Human Family: State of the Art” held in Madrid on the 11–13 March, 1998     

Sources of Variability in the Measurement of Fungal Spore Yields

Variability in the production of fungal spores and in the measurement of spore yields was investigated in four species of fungi: Colletotrichum gloeosporioides, Colletotrichum coccodes, Colletotrichum phomoides, and Acremonium strictum. When the fungi were grown on solid medium in microplates and spore yields were measured by counting the subsamples with a hemacytometer, the variability among hemacytometer squares was always the largest source of variation, accounting for 51 to 91% of the total variation. Variability among replicate cultures and results of repeat experiments were generally also significant. The effect of square-to-square variability on the precision of spore yield measurement was minimized by counting a moderate number (ca. 30) of squares per culture. Culture-to-culture variability limited the practical precision of spore production measurements to a 95% confidence interval of approximately the mean ± 25%. We provide guidelines for determining the number of replicate cultures required to attain this or other degrees of precision. Particle counter-derived spore counts and counts based on spore weights were much less variable than were hemacytometer counts, but they did not improve spore production estimates very much because of culture-to-culture variability. Results obtained by both of these methods differed from those obtained with a hemacytometer; particle counter measurements required a correction for spore pairs, while the relationship between spore weights and spore counts changed as the cultures aged.     

Identification of cellular prosomatostatin and nonsomatostatin peptides derived from its amino terminus

Rat prosomatostatin was isolated from a somatostatin-producing cell line and was partially microsequenced. This indicated the amino terminal structure of cellular prosomatostatin and implied a 92-amino acid sequence for the somatostatin precursor. Based on the structure for cellular prosomatostatin, a peptide was synthesized and used to develop a radioimmunoassay directed toward the amino terminal portion of prosomatostatin. This assay has revealed two peptides containing the amino-terminal portion of prosomatostatin in a somatostatin-secreting CA-77 rat medullary thyroid carcinoma cell line. These two peptides - MW 4000 and 8000 daltons - lack somatostatin immunoreactivity. Thus, processing of prosomatostatin occurs both at the amino and carboxyl regions. These results open the way for elucidation of the structure, function and metabolism of non-somatostatin peptides derived from the amino terminus of prosomatostatin.     

There is more to life and death than mitochondria: Bcl-2 proteins at the endoplasmic reticulum

Proteins of the Bcl-2 family are important regulators of cell fate. The role of these proteins in controlling mitochondrial apoptotic processes has been extensively investigated, although exact molecular mechanisms are incompletely understood. However, mounting evidence indicates that these proteins also function at the endoplasmic reticulum and other locations within the cell. Both pro- and anti-apoptotic Bcl-2 family members can regulate endoplasmic reticulum calcium, cellular pH and endoplasmic reticulum resident proteins. In this review, we discuss the activities and potential targets of Bcl-2 family members at the endoplasmic reticulum and other cellular locations.