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Biomedical subjects

J S Metcalf

Publications and source records attributed to J S Metcalf.

At least 19 recordsLinked to original sources
PubMed2006

Protection against the toxicity of microcystin-LR and cylindrospermopsin in Artemia salina and Daphnia spp. by pre-treatment with cyanobacterial lipopolysaccharide (LPS).

Purified cyanobacterial lipopolysaccharide (LPS) was not acutely toxic to three aquatic invertebrates (Artemia salina, Daphnia magna and Daphnia galeata) in immersion trials. However, pre-exposure (24 h) to 2 ngmL(-1) LPS increased the LC(50) of microcystin-LR significantly in all 3 species. Similar results were observed with A. salina pre-treated with the same concentration of cyanobacterial LPS and subsequently exposed to cylindrospermopsin, increasing the LC(50) by 8. The findings indicate the need to include exposures to defined combinations of cyanotoxins, and in defined sequences, to understand the contributions of individual cyanotoxins in accounting for cyanobacterial toxicity to invertebrates in natural aquatic environments.

Alkaloids↗
PubMed2006

Legal and security requirements for the air transportation of cyanotoxins and toxigenic cyanobacterial cells for legitimate research and analytical purposes.

Cyanotoxins are now recognised by international and national health and environment agencies as significant health hazards. These toxins, and the cells which produce them, are also vulnerable to exploitation for illegitimate purposes. Cyanotoxins are increasingly being subjected to national and international guidelines and regulations governing their production, storage, packaging and transportation. In all of these respects, cyanotoxins are coming under the types of controls imposed on a wide range of chemicals and other biotoxins of microbial, plant and animal origin. These controls apply whether cyanotoxins are supplied on a commercial basis, or stored and transported in non-commercial research collaborations and programmes. Included are requirements concerning the transportation of these toxins as documented by the United Nations, the International Air Transport Association (IATA) and national government regulations. The transportation regulations for "dangerous goods", which by definition include cyanotoxins, cover air mail, air freight, and goods checked in and carried on flights. Substances include those of determined toxicity and others of suspected or undetermined toxicity, covering purified cyanotoxins, cyanotoxin-producing laboratory strains and environmental samples of cyanobacteria. Implications of the regulations for the packaging and air-transport of dangerous goods, as they apply to cyanotoxins and toxigenic cyanobacteria, are discussed.

Aviation↗
PubMed2002

Effects of organic solvents on the high performance liquid chromatographic analysis of the cyanobacterial toxin cylindrospermopsin and its recovery from environmental eutrophic waters by solid phase extraction.

The effect of organic solvents on the high performance liquid chromatography (HPLC) analysis of cylindrospermopsin using photodiode array detection was examined since organic solvents are commonly used to extract this toxin from cyanobacteria and in the mobile phase compositions used in HPLC. Increasing concentrations of methanol resulted in an increase in the UV absorbance of purified cylindrospermopsin according to spectrometry, but to a marked decrease during HPLC analysis when the concentration of this solvent was greater than 50% methanol, or when acetonitrile concentrations exceeded 30% (v/v). Precipitation of cylindrospermopsin at these high concentrations of organic solvents was not observed. Solid phase extraction methods were developed to recover the toxin from spent extracellular growth medium after laboratory culture of Cylindrospermopsis raciborskii strain CR3 as an aid to toxin purification and from spiked environmental water samples. Using C18 and polygraphite carbon cartridges in series, 100% recoveries of cylindrospermopsin were achieved for lake waters spiked at 1 micro g l(-1).

Alkaloids↗
PubMed2002

Effects of enteric bacterial and cyanobacterial lipopolysaccharides, and of microcystin-LR, on glutathione S-transferase activities in zebra fish (Danio rerio).

Cyanobacteria (blue-green algae) can produce a variety of toxins including hepatotoxins e.g. microcystins, and endotoxins such as lipopolysaccharides (LPS). The combined effects of such toxins on fish are little known. This study examines the activities of microsomal (m) and soluble (s) glutathione S-transferases (GST) from embryos of the zebra fish, Danio rerio at the prim six embryo stage, which had been exposed since fertilisation to LPS from different sources. A further aim was to see how activity was affected by co-exposure to LPS and microcystin-LR (MC-LR). LPS were obtained from Salmonella typhimurium, Escherichia coli, a laboratory culture of Microcystis CYA 43 and natural cyanobacterial blooms of Microcystis and Gloeotrichia. Following in vivo exposure of embryos to each of the LPS preparations, mGST activity was significantly reduced (from 0.50 to between 0.06 and 0.32 nanokatals per milligram (nkat mg(-1)) protein). sGST activity in vivo was significantly reduced (from 1.05 to between 0.19 and 0.22 nkat mg(-1) protein) after exposure of embryos to each of the cyanobacterial LPS preparations, but not in response to S. typhimurium or E. coli LPS. Activities of both m- and sGSTs were reduced after co-exposure to MC-LR and cyanobacterial LPS, but only mGST activity was reduced in the S. typhimurium and E. coli LPS-treated embryos. In vitro preparations of GST from adult and prim six embryo D. rerio showed no significant changes in enzyme activity in response to the LPS preparations with the exception of Gloeotrichia bloom LPS, where mGST was reduced in adult and embryo preparations. The present study represents the first investigations into the effects of cyanobacterial LPS on the phase-II microcystin detoxication mechanism. LPS preparations, whether from axenic cyanobacteria or cyanobacterial blooms, are potentially capable of significantly reducing activity of both the s- and mGSTs, so reducing the capacity of D. rerio to detoxicate microcystins. The results presented here have wide ranging implications for both animal and human health.

Animals↗
PubMed2002

Toxicity of cylindrospermopsin to the brine shrimp Artemia salina: comparisons with protein synthesis inhibitors and microcystins.

The Artemia salina bioassay was successfully applied to the analysis of the hepatotoxic cyanobacterial alkaloid and protein synthesis inhibitor, cylindrospermopsin. A dose-dependent response in mortality was observed for purified cylindrospermopsin and LC(50) values decreased with time from 8.1 to 0.71 microg/ml(-1), between 24 and 72 h, respectively. Cylindrospermopsin was slightly less potent than micro cystin-LR, with similar LC(50) values on a gravimetric basis, but was more toxic to A.salina than the protein synthesis inhibitors, cycloheximide, chloramphenicol and tetracycline. Cylindrospermopsin-containing strains of the cyanobacterium Cylindrospermopsis raciborskii were found to be toxic to A.salina and the LC(50) concentration for these strains over time was greater than the LC(50) for purified cylindrospermopsin, with the exception of C. raciborskii strain CR1.

Alkaloids↗
PubMed2001

Quantitative real-time RT-PCR detection of breast cancer micrometastasis using a multigene marker panel.

Real-time RT-PCR is a relatively new technology that uses an online fluorescence detection system to determine gene expression levels. It has the potential to significantly improve detection of breast cancer metastasis by virtue of its exquisite sensitivity, high throughput capacity and quantitative readout system. To assess the utility of this technology in breast cancer staging, we determined the relative expression levels of 12 cancer-associated genes (mam, PIP, mamB, CEA, CK19, VEGF, erbB2, muc1, c-myc, p97, vim and Ki67) in 51 negative-control normal lymph nodes and in 17 histopathology-positive ALNs. We then performed a receiver operating characteristic (ROC) curve analysis to determine the sensitivity and specificity levels of each gene. Areas under the ROC curve indicated that the most accurate diagnostic markers were mam (99.6%), PIP (93.3%), CK19 (91.0%), mamB (87.9%), muc1 (81.5%) and CEA (79.4.0%). mam was overexpressed in 16 of 17 lymph nodes known to contain metastatic breast cancer at levels ranging from 22- to 2.8 x 10(5)-fold above normal mean expression, whereas PIP was overexpressed from 30- to 2.2 x 10(6)-fold above normal in 13 lymph nodes. Real-time RT-PCR analysis of pathology-negative LN from breast cancer patients revealed evidence of overexpression of PIP (6 nodes), mam (3 nodes) and CEA (1 node) in 8 of 21 nodes (38%). Our results provide evidence that mam, PIP, CK19, mamB, muc1 and CEA can be applied as a panel for detection of metastatic and occult micrometastatic disease.

Base Sequence↗
PubMed2001

Melanoma thickness and histology predict sentinel lymph node status.

BACKGROUND: It remains unclear which patients with melanoma will benefit most from lymphatic mapping and sentinel lymphadenectomy. The purpose of this study is to determine whether primary melanoma histopathologic features could be applied to predict sentinel node status. METHODS: One hundred twelve patients underwent sentinel node biopsy between May 1995 and August 1999. Reported histologic features were assessed for predictive value by univariate and multivariate logistic regression. RESULTS: The sentinel node was located successfully in 105 of the 112 patients (94%). Twenty-one of these 105 patients (20%) had sentinel nodes that were positive for metastatic disease. Multivariate analyses revealed that tumor thickness greater than 1.5 mm (P = 0.01), ulceration (P <0.01), and lymphovascular invasion (P = 0.05) predicted the presence of micrometastases. CONCLUSIONS: The presence of unfavorable histopathology such as ulceration and lymphovascular invasion may identify a group of patients with thin melanomas who would benefit from sentinel lymphadenectomy.

Female↗
PubMed2001

Losses of the cyanobacterial toxin microcystin-LR from aqueous solution by adsorption during laboratory manipulations.

The effect of plastic and methanol on the loss of microcystin-LR from solution was analysed by HPLC with photodiode array detection (HPLC-PDA). With plastic disposable pipette tips, the loss from an aqueous microcystin-LR (MC-LR) solution was 4.2% per tip operation. Using the same pipette tip, four operations were required to completely saturate a single tip with toxin. MC-LR attached to plastic pipette tips could subsequently be eluted by methanol and detected by HPLC-PDA. At methanol concentrations below 25% (v/v), recovered concentrations of MC-LR decreased significantly. Differences in MC-LR concentration were also noted by performing 50% dilution with Milli-Q water or methanol. The results are discussed in relation to the hydrophobicity of MC-LR, analytical procedures and the avoidance of toxin losses from solution during laboratory manipulations.

Adsorption↗
PubMed2001

Effects of adsorption to plastics and solvent conditions in the analysis of the cyanobacterial toxin microcystin-LR by high performance liquid chromatography.

Effects of adsorption to plastics and solvent conditions in the high performance liquid chromatographic analysis of the cyanobacterial toxin microcystin-LR were investigated. Aqueous microcystin-LR readily adsorbed to the disposable polypropylene pipette tips commonly used in laboratory manipulations. This was not affected by the pH or salinity of the solution. Furthermore, dilutions of microcystin-LR in varying concentrations of methanol and acetonitrile influenced the quantification of the microcystin-LR concentration by high performance liquid chromatography.

Adsorption↗
PubMed2001

Colorimetric immuno-protein phosphatase inhibition assay for specific detection of microcystins and nodularins of cyanobacteria.

A novel immunoassay was developed for specific detection of cyanobacterial cyclic peptide hepatotoxins which inhibit protein phosphatases. Immunoassay methods currently used for microcystin and nodularin detection and analysis do not provide information on the toxicity of microcystin and/or nodularin variants. Furthermore, protein phosphatase inhibition-based assays for these toxins are not specific and respond to other environmental protein phosphatase inhibitors, such as okadaic acid, calyculin A, and tautomycin. We addressed the problem of specificity in the analysis of protein phosphatase inhibitors by combining immunoassay-based detection of the toxins with a colorimetric protein phosphatase inhibition system in a single assay, designated the colorimetric immuno-protein phosphatase inhibition assay (CIPPIA). Polyclonal antibodies against microcystin-LR were used in conjunction with protein phosphatase inhibition, which enabled seven purified microcystin variants (microcystin-LR, -D-Asp3-RR, -LA, -LF, -LY, -LW, and -YR) and nodularin to be distinguished from okadaic acid, calyculin A, and tautomycin. A range of microcystin- and nodularin-containing laboratory strains and environmental samples of cyanobacteria were assayed by CIPPIA, and the results showed good correlation (R2 = 0.94, P < 0.00001) with the results of high-performance liquid chromatography with diode array detection for toxin analysis. The CIPPIA procedure combines ease of use and detection of low concentrations with toxicity assessment and specificity for analysis of microcystins and nodularins.

Bacterial Toxins↗
PubMed2000

Immuno-crossreactivity and toxicity assessment of conjugation products of the cyanobacterial toxin, microcystin-LR.

Immunoassays are increasingly used to investigate the production, properties and fates of the cyanobacterial hepatotoxic microcystins in vitro and in vivo. Responses of an ELISA immunoassay to microcystins have been determined using the authentic toxin antigen, microcystin-LR, and conjugation products between the toxin and glutathione, cysteine-glycine and cysteine. The antibodies against microcystin-LR crossreacted with the toxin conjugation products with similar affinities (96-112%) to that of microcystin-LR, when assayed at a concentration of 1 microg l(-1). Toxicity assessment of the conjugates, in comparison to microcystin-LR, indicated a reduction according to mouse bioassay. In vitro protein phosphatase inhibition assay indicated that the conjugates possessed approximately 3-9-fold lower toxicity than microcystin-LR.

Animals↗
PubMed2000

Microwave oven and boiling waterbath extraction of hepatotoxins from cyanobacterial cells.

Low-cost, straightforward methods for the extraction of microcystins and nodularins from cyanobacterial cells were developed using a microwave oven and boiling waterbath. The use of organic solvents, such as methanol, which can interfere with sensitive analytical procedures, e.g. immunoassays, can thus be avoided. Analysis by protein phosphatase inhibition assay and high performance liquid chromatography indicated that purified microcystin-LR was unaffected by the microwave oven and boiling waterbath treatments. Four microcystins of differing hydrophobicities were successfully extracted from Microcystis PCC 7813 by both treatments at yields equivalent to those obtained by longer protocols using methanol. Assessment of the microwave oven and boiling waterbath extraction methods with laboratory strains and environmental samples of cyanobacteria showed good correlation with results from lyophilisation and methanol extraction, when extracts were analysed by high performance liquid chromatography with diode array detection (R(2)>/=0.92). The microwave and boiling waterbath extraction methods also sterilised the environmental bloom samples, as evidenced by the abolition of heterotrophic bacterial growth.

Bacterial Toxins↗
PubMed2000

Effects of physicochemical variables and cyanobacterial extracts on the immunoassay of microcystin-LR by two ELISA kits.

Two types of commercially available ELISA kits for the immunoassay of cyanobacterial microcystins were evaluated for potential interference effects due to methanol, salinity, pH, plasticware and cyanobacterial extract. Of the treatments examined, methanol had the greatest effect, giving false positive microcystin concentrations with increasing methanol concentrations up to 30% (v/v) compared with the negative calibrators of each kit. False positive microcystin results were also produced with increasing salinity up to full strength seawater. Decreases in microcystin-LR equivalents were observed when assaying purified microcystin-LR at pH values between 6.25 and 10. Aqueous microcystin-LR solutions in plastic microcentrifuge tubes after pipetting with disposable plastic tips had lower toxin concentrations than expected when analysed by ELISA. Indicated microcystin concentrations in cyanobacterial extracts varied between kit types and the choice of blanks used. Although ELISAs can be useful tools for the screening of water and cyanobacterial blooms for microcystins and nodularins, users should be aware that commercial kits can be susceptible to interference by commonly encountered environmental and laboratory conditions and materials.

Cyanobacteria↗
PubMed2000

The effect of tamoxifen and cisplatin on the disease-free and overall survival of patients with high risk malignant melanoma.

The adjuvant treatment of high-risk malignant melanoma remains problematic. Previously we reported moderate success in the treatment of metastatic disease using tamoxifen, cisplatin, dacarbazine and carmustine. Based upon data that suggested tamoxifen and cisplatin were the active agents in this regimen, we initiated a phase II trial of this combination in the adjuvant setting. We treated 153 patients with 4 cycles of tamoxifen (160 mg day(-1), days 1-7) and cisplatin (100 mg m(-2), day 2) for 28-day intervals. Patients received an anti-nausea regimen of dexamethasone with ondansetron or granisetron. During the first 2 years of follow-up, patients were evaluated every 2 months with a history, physical exam, laboratory work and computed tomography scans of the chest, abdomen and pelvis every 4 months. Thereafter, patients were evaluated every 3 months and radiographic studies were performed if necessary. Currently, with a median follow-up of 36 months, the disease-free survival (DFS) is 68.4% and overall survival (OS) is 84.5%. Kaplan-Meier analysis predicts a 5-year DFS of 62% with an OS of 79%. Relapses after 20 months have been rare. No effect of gender or number of positive lymph nodes was noted, however, stage of disease prior treatment was a factor. The major toxicity proved to be gastrointestinal in nature with nausea the most prevalent symptom. Minimal renal, haematologic and neurologic toxicity occurred. These preliminary results suggest that there is a positive impact of tamoxifen and cisplatin on both the DFS and OS of high-risk malignant melanoma patients. The 5-year projected DFS and OS compare favourably with those reported for the ECOG 1684 trial and warrant confirmation in a prospective randomized trial.

Adult↗
PubMed1999

Retention of Microcystis aeruginosa and microcystin by salad lettuce (Lactuca sativa) after spray irrigation with water containing cyanobacteria.

Colonies and single cells of Microcystis aeruginosa and the hepatotoxin microcystin were retained by salad lettuce after growth with spray irrigation water containing the microcystin-producing cyanobacteria. These findings are discussed in terms of crop spray irrigation with water containing cyanobacteria and potential human exposure to cyanobacterial toxins via plant foods grown in such circumstances.

Carcinogens↗