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.

Transport of endosomal early antigen I in the rat sciatic nerve and location in cultured neurons

Early endosomal antigen I (EEAI) is known to be a marker of early endosomes and in cultured hippocampal neurons it preferentially localizes to the dendritic but not the axonal compartment. We show in cultured dorsal root ganglia and superior cervical ganglia neurons that EEAI localizes to the cell bodies and the neurites of both sensory and sympathetic neurons. We then show in vivo using a ligated rat sciatic nerve that EEAI significantly accumulates on the proximal side and not on the distal side of the ligation. This suggests that EEAI is transported in the anterograde direction in axons either as part of the homeostatic process or to the nerve ligation site in response to nerve injury. NeuroReport 12:281-284 (C) 2001 Lippincott Williams & Wilkins.

A common inhibitory binding site for zinc and odorants at the voltage-gated K+ channel of rat olfactory receptor neurons

This study compared the effects of zinc and odorants on the voltage-gated K+ channel of rat olfactory neurons. Zinc reduced current magnitude, depolarized the voltage activation curve and slowed activation kinetics without affecting inactivation or deactivation kinetics. Zinc inhibition was potentiated by the NO compound, S-nitroso-cysteine. The pH- and diethylpyrocarbonate-dependence of zinc inhibition suggested that zinc acted by binding to histidine residues. Cysteine residues were eliminated as contributing to the zinc-binding site. The odorants, acetophenone and amyl acetate, also reduced current magnitude, depolarized the voltage activation curve and selectively slowed activation kinetics. Furthermore, the diethylpyrocarbonate- and pH-dependence of odorant inhibition implied that the odorants also bind to histidine residues. Zinc inhibitory potency was dramatically diminished in the presence of odorants, implying competition for a common binding site. These observations indicate that the odorants and zinc share a common inhibitory binding site on the external surface of the voltage-gated K+ channel.

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.

Developmental changes in hyperpolarization-activated currents I-h and I-K(IR) in isolated rat intracardiac neurons

The hyperpolarization-activated nonselective cation current, I-h, was investigated in neonatal and adult rat intracardiac neurons. I-h was observed in all neurons studied and displayed slow time-dependent rectification. I-h was isolated by blockade with external Cs+ (2 mM) and was inhibited irreversibly by the bradycardic agent, ZD 7288. Current density of I-h was approximately twofold greater in neurons from neonatal (-4.1 pA/pF at -130 mV) as compared with adult (-2.3 pA/pF) rats; however, the reversal potential and activation parameters were unchanged. The reversal potential and amplitude of I-h was sensitive to changes in external Na+ and K+ concentrations. An inwardly rectifying K+ current, I-K(IR), was also present in intracardiac neurons from adult but not neonatal rats and was blocked by extracellular Ba2+. I-K(IR) was present in approximately one-third of the adult intracardiac neurons studied, with a current density of -0.6 pA/pF at -130 mV. I-K(IR) displayed rapid activation kinetics and no time-dependent rectification consistent with the rapidly activating, inward K+ rectifier described in other mammalian autonomic neurons. I-K(IR) was sensitive to changes in external K+, whereby raising the external K+ concentration from 3 to 15 mM shifted the reversal potential by approximately +36 mV. Substitution of external Na+ had no effect on the reversal potential or amplitude of I-K(IR). I-K(IR) density increases as a function of postnatal development in a population of rat intracardiac neurons, which together with a concomitant decrease in I-h may contribute to changes in the modulation of neuronal excitability in adult versus neonatal rat intracardiac ganglia.

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.

Temperature dependence of polaronic transport through single molecules and quantum dots

Motivated by recent experiments on electric transport through single molecules and quantum dots, we investigate a model for transport that allows for significant coupling between the electrons and a boson mode isolated on the molecule or dot. We focus our attention on the temperature-dependent properties of the transport. In the Holstein picture for polaronic transport in molecular crystals the temperature dependence of the conductivity exhibits a crossover from coherent (band) to incoherent (hopping) transport. Here, the temperature dependence of the differential conductance on resonance does not show such a crossover, but is mostly determined by the lifetime of the resonant level on the molecule or dot.

Nondeterministic gates for photonic single-rail quantum logic

We discuss techniques for producing, manipulating, and measuring qubits encoded optically as vacuum- and single-photon states. We show that a universal set of nondeterministic gates can be constructed using linear optics and photon counting. We investigate the efficacy of a test gate given realistic detector efficiencies.