Isolation and identification of the cyanotoxin cylindrospermopsin and deoxy-cylindrospermopsin from a Thailand strain of Cylindrospermopsis raciborskii (Cyanobacteria)

A strain of Cylindrospermopsis (Cyanobacteria) isolated from a fishpond in Thailand was examined for its taxonomy based upon morphology and 16S rRNA gene sequence. It was also examined for production of the hepatotoxic cyanotoxin called cylindrospermopsin (CYN) and deoxycylindrospermopsin (deoxy-CYN). The strain (CY-Thai) was identified as C. raciborskii (Woloszynska) Seenaya and Subba Raju based upon morphological examination which was confirmed by 16S rRNA gene sequences and phylogenetic comparisons based upon its 16S rRNA gene. The alkaloid heptatotoxin CYN was confirmed using mouse bioassay, HPLC and HPLC-MS/MS while deoxy-CYN was confirmed using HPLC-MS/MS. The mouse bioassay gave a minimum lethal dose at 250 mg dry weight cells/kg body weight within 24 h and 125 mg/kg at 72 h, with signs of poisoning the same as in literature reports for CYN. HPLC chromatographic comparison of the CY-Thai toxin with standard CYN gave the same retention time and an absorbance maximum at 262 nm. HPLC-MS/MS confirmed the presence of CYN (M + H 416) and deoxy-CYN (M + H 400). The CYN content in strain CY-Thai was estimated at 1.02 mg/g and approximately 1/10 of this amount for deoxy-CYN. This is the first report from Asia of a CYN, deoxy-CYN producing Cylindrospermopsis raciborskii. (C) 2001 Elsevier Science Ltd. All rights reserved.

Single-electron measurements with a micromechanical resonator

A mechanical electroscope based on a change in the resonant frequency of a cantilever one micron in size in the presence of charge has recently been fabricated. We derive the decoherence rate of a charge superposition during measurement with such a device using a master equation theory adapted from quantum optics. We also investigate the information produced by such a measurement, using a quantum trajectory approach. Such instruments could be used in mesoscopic electronic systems, and future solid-state quantum computers, so it is useful to know how they behave when used to measure quantum superpositions of charge.

Single institution outcomes of treatment of severe aplastic anaemia

Background: In severe aplastic anaemia, the treatment of choice for young patients with a human leucocyte antigen-matched sibling is now established as allogeneic bone marrow transplantation (BMT). In older patients and in those without a matched sibling donor, immunosuppressive therapy is the usual first option. 'Alternative' marrow donors are emerging as an option for those without a matched sibling donor. Aims: To review 10 years of local experience in treating severe aplastic anaemia with BMT and immunosuppressive therapy with emphasis on long-term outcomes. Methods: A retrospective analysis was performed of all patients with severe aplastic anaemia presenting to the Royal Brisbane and Royal Children's Hos- pitals between 1989 and 1999. Data were abstracted regarding patient demographics, pretreatment characteristics and outcome measures, including response rates, overall survival and long-term complications. Results: Twenty-seven consecutive patients were identified, 12 treated with immunosuppression alone and 15 with BMT. In these two groups, transfusion independence was attained in 25% and 100%, respectively, with overall survival being 36% and 100%, respectively. Those treated with immunosuppression were significantly older (median 41.5 versus 22 years, P = 0.008). Long-term survivors of either treatment had extremely low morbidity. Three patients carried pregnancies to term post-transplant. Three patients received alternative donor BMT with correspondingly excellent survival. Conclusions: Patients treated with allogeneic BMT for severe aplastic anaemia enjoyed extremely good long-term survival and minimal morbidity. Patients treated with immunosuppressive therapy had a poorer outcome reflecting their older age and different usage of therapies over the past decade. Optimal treatment strategies for severe aplastic anaemia remain to be determined.

Sexual recombination in Phytophthora cinnamomi in vitro and aggressiveness of single-oospore progeny to Eucalyptus

Fifty single oospore progeny were established from an in vitro mating of A1 and A2 mating type isolates of Phytophthora cinnamomi from South Africa. Forty-nine progeny were identified as F-1 hybrids using seven random amplified polymorphic DNA (RAPD) primers, and one was a selfed isolate of the A1 mating type parent. Among the hybrid progeny, 24 and 25 were A1 and A2 mating type, respectively. Aggressiveness of progeny and parental isolates was assessed on 1-year-old seedlings of Eucalyptus smithii. The mean aggressiveness of hybrid oosporic isolates, expressed as lesion length, was significantly (P = 0.0001) lower than that of the parental isolates. No significant difference in aggressiveness of A1 and A2 mating type F-1 hybrid isolates was observed. This is the first report demonstrating sexual recombination in vitro in P. cinnamomi.

A single beta subunit M2 domain residue controls the picrotoxin sensitivity of alpha beta heteromeric glycine receptor chloride channels

This study investigated the residues responsible for the reduced picrotoxin sensitivity of the alpha beta heteromeric glycine receptor relative to the alpha homomeric receptor. By analogy with structurally related receptors, the beta subunit M2 domain residues P278 and F282 were considered the most likely candidates for mediating this effect. These residues align with G254 and T258 of the alpha subunit. The T258A, T258C and T258F mutations dramatically reduced the picrotoxin sensitivity of the alpha homomeric receptor. Furthermore, the converse F282T mutation in the beta subunit increased the picrotoxin sensitivity of the alpha beta heteromeric receptor. The P278G mutation in the beta subunit did not affect the picrotoxin sensitivity of the alpha beta heteromer. Thus, a ring of five threonines at the M2 domain depth corresponding to alpha subunit T258 is specifically required for picrotoxin sensitivity. Mutations to alpha subunit T258 also profoundly influenced the apparent glycine affinity. A substituted cysteine accessibility analysis revealed that the T258C sidechain increases its pore exposure in the channel open state. This provides further evidence for an allosteric mechanism of picrotoxin inhibition, but renders it unlikely that picrotoxin las an allosterically acting 'competitive' antagonist) binds to this residue.

Isolation of microsatellite loci from spotted gum (Corymbia variegata), and cross-species amplification in Corymbia and Eucalyptus

Corymbia variegata (spotted gum) is an important commercial hardwood timber species in Australia. Fourteen polymorphic microsatellite loci were isolated from C. variegata, with 3-5 alleles amplified in three individuals examined. Cross-species amplification in Corymbia was successful for all primer pairs, while 10 loci (71%) were successfully transferred to at least one species in the closely related genus Eucalyptus.

Using MF-PCR to diagnose multiple defects from single cells: implications for PGD

Single cell genetic analysis is generally performed using PCR and FISH. Until recently, FISH has been the method of choice. FISH however is expensive, has significant misdiagnosis rates, can result in interpretation difficulties and is labour intensive making it unsuitable for high throughput processing. Recently fluorescent PCR reliability has increased to levels at or surpassing FISH whilst maintaining low cost. However, PCR accuracy has been a concern due to allelic dropout. Multiplex PCR can now increase accuracy by using multiple markers for each chromosome to firstly provide diagnosis if markers fail and,or secondly confirm diagnosis. We compare a variety of diagnostic methods and demonstrate for the first time a multiplex PCR system providing simultaneous diagnosis and confirmation of the major aneuploidy chromosomes (21, 18, 13) and sex as well as DNA fingerprint in single cells. We also discuss the implications of using PCR for aneuploidy screening in preimplantation genetic diagnosis. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

Identifying the origin of single corneal cells by DNA fingerprinting - Part I - Implications for corneal limbal allografting

Purpose. To demonstrate that the combination of impression cytology and single cell DNA fingerprinting represents a powerful tool that is suitable for detecting transplanted cells after corneal limbal allografting. Methods, Fifty single cells were obtained by corneal impression cytology from 12 patients undergoing cataract surgery. Individual cells were isolated from samples by micromanipulation. Polymerase chain reaction and short tandem repeat profiling was used to obtain forensic standard DNA fingerprints from single cells. Blood samples taken at the time of impression cytology provided control fingerprints. Results, informative DNA fingerprints were obtained from all corneal samples and 66% (33 of 50 cells) of isolated single cells, Of all fingerprints obtained, most (91%, 30 of 33 fingerprints) corneal fingerprints matched corresponding blond sample fingerprints. At least one corneal fingerprint matched the corresponding blood sample fingerprint in 83% (10 of 12 patients) of the patients in the study, Conclusions. This extremely specific single cell DNA fingerprinting system permits accurate identification of individual corneal epithelial cells, allowing very reliable determination of their origin, which will enable host and donor cells to be distinguished from each other after keratolimbal allografting procedures. even if the host and donor are the same sex or siblings. These DNA fingerprinting methods allow assessment of quality and quantity of donor cell survival, as well as survival time. The extreme sensitivity and accuracy of the technique means that should contamination occur, it would be identified, thus ensuring meaningful results.

A spongin-boring alpha-proteobacterium is the etiological agent of disease in the Great Barrier Reef sponge Rhopaloeides odorabile

High levels of mortality in the Mediterranean bath sponge industry have raised concerns for the future of sponge farms. Healthy sponges feed predominantly on bacteria, and many harbour a wide diversity of inter- and extra-cellular symbiotic bacteria. Here we describe the first isolation and description of a pathogenic bacterium from an infected marine sponge. Microbiological examination of tissue necrosis in the Great Barrier Reef sponge Rhopaloeides odorabile resulted in isolation of the bacterial strain NW4327. Sponges infected with strain NW4327 exhibited high levels of external tissue necrosis, and the strain was re-isolated from infected sponges. A single morphotype, which had burrowed through the collagenous spongin fibres causing severe necrosis, was observed microscopically. Strain NW4327 was capable of degrading commercial preparations of azo-collagen, providing further evidence of its involvement in spongin fibre necrosis, Strain NW4327 disrupted the microbial community associated with R. odorabile and was able to infect and kill healthy sponge tissue. 16S rRNA sequence analysis revealed that strain NW4327 is a novel member of the alpha-proteobacteria.

Isolation (from a basal cell carcinoma) of a functionally distinct fibroblast-like cell type that overexpresses Ptch

In this study we report on the isolation and characterization of a nonepithelial, nontumorigenic cell type (BCC1) derived from a basal cell carcinoma from a patient. The BCC1 cells share many characteristics with dermal fibroblasts, such as the expression of vimentin, lack of expression of cytokeratins, and insensitivity to agents that cause growth inhibition and differentiation of epithelial cells; however, significant differences between BCC1 cells and fibroblasts also exist. For example, BCC1 cells are stimulated to undergo DNA synthesis in response to interferon-gamma, whereas dermal fibroblasts are not. More over, BCC1 cells overexpress the basal cell carcinoma-specific genes ptch and ptch2 . These data indicate that basal cell carcinomas are associated with a functionally distinct population of fibroblast-like cells that overexpress known tumor-specific markers (ptch and ptch2 ).