Dictyostelium, a microbial model for brain disease.

BACKGROUND: Most neurodegenerative diseases are associated with mitochondrial dysfunction. In humans, mutations in mitochondrial genes result in a range of phenotypic outcomes which do not correlate well with the underlying genetic cause. Other neurodegenerative diseases are caused by mutations that affect the function and trafficking of lysosomes, endosomes and autophagosomes. Many of the complexities of these human diseases can be avoided by studying them in the simple eukaryotic model Dictyostelium discoideum. SCOPE OF REVIEW: This review describes research using Dictyostelium to study cytopathological pathways underlying a variety of neurodegenerative diseases including mitochondrial, lysosomal and vesicle trafficking disorders. MAJOR CONCLUSIONS: Generalised mitochondrial respiratory deficiencies in Dictyostelium produce a consistent pattern of defective phenotypes that are caused by chronic activation of a cellular energy sensor AMPK (AMP-activated protein kinase) and not ATP deficiency per se. Surprisingly, when individual subunits of Complex I are knocked out, both AMPK-dependent and AMPK-independent, subunit-specific phenotypes are observed. Many nonmitochondrial proteins associated with neurological disorders have homologues in Dictyostelium and are associated with the function and trafficking of lysosomes and endosomes. Conversely, some genes associated with neurodegenerative disorders do not have homologues in Dictyostelium and this provides a unique avenue for studying these mutated proteins in the absence of endogeneous protein. GENERAL SIGNIFICANCE: Using the Dictyostelium model we have gained insights into the sublethal cytopathological pathways whose dysregulation contributes to phenotypic outcomes in neurodegenerative disease. This work is beginning to distinguish correlation, cause and effect in the complex network of cross talk between the various organelles involved. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.

Quantitative analysis of cell motility and chemotaxis in Dictyostelium discoideum by using an image processing system and a novel chemotaxis chamber providing stationary chemical gradients.

An image processing system was programmed to automatically track and digitize the movement of amebae under phase-contrast microscopy. The amebae moved in a novel chemotaxis chamber designed to provide stable linear attractant gradients in a thin agarose gel. The gradients were established by pumping attractant and buffer solutions through semipermeable hollow fibers embedded in the agarose gel. Gradients were established within 30 min and shown to be stable for at least a further 90 min. By using this system it is possible to collect detailed data on the movement of large numbers of individual amebae in defined attractant gradients. We used the system to study motility and chemotaxis by a score of Dictyostelium discoideum wild-type and mutant strains, including "streamer" mutants which are generally regarded as being altered in chemotaxis. None of the mutants were altered in chemotaxis in the optimal cAMP gradient of 25 nM/mm, with a midpoint of 25 nM. The dependence of chemotaxis on cAMP concentration, gradient steepness, and temporal changes in the gradient were investigated. We also analyzed the relationship between turning behavior and the direction of travel during chemotaxis in stable gradients. The results suggest that during chemotaxis D. discoideum amebae spatially integrate information about local increases in cAMP concentration at various points on the cell surface.

Selection of chemotaxis mutants of Dictyostelium discoideum.

A method has been developed for the efficient selection of chemotaxis mutants of Dictyostelium discoideum. Mutants defective in the chemotactic response to folate could be enriched up to 30-fold in one round of selection using a chamber in which a compartment that contained the chemoattractant was separated by a sandwich of four nitrocellulose filters from a compartment that contained buffer. Mutagenized cells were placed in the center of the filter layer and exposed to the attractant gradient built up between the compartments for a period of 3-4 h. While wild-type cells moved through the filters in a wave towards the compartment that contained attractant, mutant cells remained in the filter to which they were applied. After several repetitions of the selection procedure, mutants defective in chemotaxis made up 10% of the total cell population retained in that filter. Mutants exhibiting three types of alterations were collected: motility mutants with either reduced speed of movement, or altered rates of turning; a single mutant defective in production of the attractant-degrading enzyme, folate deaminase; and mutants with normal motility but reduced chemotactic responsiveness. One mutant showed drastically reduced sensitivity in folate-induced cGMP production. Morphogenetic alterations of mutants defective in folate chemotaxis are described.

Photosensory and thermosensory responses in Dictyostelium slugs are specifically impaired by absence of the F-actin cross-linking gelation factor (ABP-120).

Chemotactic aggregation of starving amoebae of Dictyostelium discoideum leads to formation of a motile, multicellular organism - the slug - whose anterior tip controls its phototactic and thermotactic behaviour. To determine whether proteins that regulate the in vitro assembly of actin are involved in these responses, we tested phototaxis and thermotaxis in mutant slugs in which the gene encoding one of five actin-binding proteins had been disrupted. Of the proteins tested - severin, alpha-actinin, fimbrin, the 34 kD actin-bundling protein and the F-actin cross-linking gelation factor (ABP-120) - only ABP-120 proved essential for normal phototaxis and thermotaxis in the multicellular slugs. The related human protein ABP-280 is required for protein phosphorylation cascades initiated by lysophosphatidic acid and tumor necrosis factor alpha. The repeating segments constituting the rod domains of ABP-120 and ABP-280 may be crucial for the function of both proteins in specific signal transduction pathways by mediating interactions with regulatory proteins.

Genetic analysis of Dictyostelium slug phototaxis mutants.

Mapping and complementation analysis with 17 phototaxis mutations has established 11 complementation groups phoA-phoK distributed over six linkage groups. Statistical calculations from the complementation data yielded 17 as the maximum likelihood estimate of the number of pho genes assuming all loci are equally mutable. Most of the phototaxis mutants were found to exhibit bimodal phototaxis and all were found to be impaired in positive thermotaxis supporting convergence of the photosensory and thermosensory pathways. The thermotaxis mutant HPF228 was unaltered in phototaxis suggesting that the mutation in this strain affects a gene product whose site of action is before the convergence of the two pathways. Other phenotypes such as multiple tip formation by aggregates, stumpy fruiting bodies with short or absent stalks and short migration were associated with some pho alleles suggesting multiple biological roles for some gene products important in phototransduction.

Lack of correlation between streptococcal Fc receptors and symptomatic pharyngitis.

To determine whether bacterial Fc(gamma) surface receptors are associated with symptomatic streptococcal pharyngitis, throat cultures were obtained from 264 children (grouped according to symptoms of pharyngeal infection) and from 328 who were asymptomatic. Group A beta-hemolytic streptococci which were recovered were tested for the presence of Fc(gamma) surface receptors. There were no statistically significant differences in the incidence of Fc(gamma) surface receptors on strains from the various patient groups.

Phototaxis genes on linkage group V in Dictyostelium discoideum.

During the course of mapping and complementation analysis of phototaxis (pho) mutations in Dictyostelium discoideum we have assigned to linkage group V three mutant pho alleles belonging to complementation groups phoG and phoK. These are the first genetic markers with an easily recognizable phenotype to be found on this linkage group.

Exploring subjective vs. objective issues in the validation of computer-based critiquing advice.

Evaluation is an important part of the development of computer-based medical expert systems. Such evaluation may be particularly difficult when judging a critiquing system which responds to a proposed management strategy with a discussion of the advisability of that approach. DxCON is an expert system which produces a prose critique discussing the radiologic workup of obstructive jaundice. This paper describes DxCON, and its experimental validation by three independent judges. A central component of the validation involved allowing the judges to react to the system's advice in a quite flexible, unstructured fashion. This project provides a case study of how subjective issues impact both the design and implementation of a validation of a medical expert system whose output is explanatory prose.

Identifiable semen donors--attitudes of donors and recipient couples.

Fifty-three couples embarking on donor insemination (DI) replied to an anonymous questionnaire asking what they wanted to know about the donor should they become pregnant. Fifty-one percent were definitely and 32% probably going to tell a child of its donor origins. Items most frequently listed for themselves and on behalf of a child were--interests/sports (57%), physical attributes (41%), occupation (37%) and family background (26%). Forty-two percent of the women and 28% of the men thought the child should have access to the identity of the donor eventually. Thirty-eight donors were surveyed by a questionnaire; 68% were agreeable to their identity being available to a donor insemination child when the child reached maturity. When the category of identifiable donor was created, 20 of 36 (56%) new donors and donors still donating chose this option.

Genetics of phototaxis in a model eukaryote, Dictyostelium discoideum.

The life cycle of Dictyostelium discoideum offers a unique opportunity to study signal transduction in eukaryotic cells at both the unicellular and multicellular levels of organization. Adding to the already extensive knowledge of the unicellular stages, classical and molecular genetics have begun to unravel transduction of signals controlling morphogenesis and behaviour (phototaxis and thermotaxis) in the multicellular 'slug' stage of the life cycle. Distributed over all seven genetic linkage groups are probably about 20, but possibly as many as 55, genes of importance for slug behaviour. The encoded proteins appear from pharmacological studies and mutant phenotypes to govern transduction pathways involving the intracellular second messengers cyclic AMP, cyclic GMP, IP3 and Ca2+. Pathways from the photo- and thermoreceptors converge first with each other and thence, at the level of the second messengers, with those from extracellular tip activation (cyclic AMP) and inhibition (Slug Turning Factor and/or ammonia and/or adenosine) signals that control slug movement and morphogenesis.

Pseudopodium activation and inhibition signals in chemotaxis by Dictyostelium discoideum amoebae.

The behaviour of Dictyostelium discoideum amoebae has been studied in natural cAMP waves and in controlled spatial and temporal gradients. Chemoattractant gradients induce responses which indicate that amoebae spatially compare concentration increases at different points on the cell surface. This allows them to respond to the relative spatial and temporal gradients in a manner that is little affected by the absolute attractant concentration over several orders of magnitude. The changes in turning behaviour, motility and morphology that are induced by attractant gradients are consistent with transduction of stimuli into two intracellular signals - one activating and the other inhibiting pseudopodium formation. The former measures the present attractant concentration at particular points on the cell surface - the local, current signal. The latter measures the average attractant concentration over the whole cell surface during the recent past - the global, past signal. Both signals may be part of a normal pseudopodium autoactivation and inhibition system responsible for amoeboid morphology and motility. Attractants could modulate this system to generate the complex behavioural responses observed.

Analysis of a complex plasmid insertion in a phototaxis-deficient transformant of Dictyostelium discoideum selected on a Micrococcus luteus lawn.

A novel method for clonal selection of G418-resistant Dictyostelium discoideum transformants on lawns of Micrococcus luteus was developed. The procedure was used to isolate transformants deficient in phototaxis after nontargeted insertion of shuttle vector DNA. Southern blot analysis as well as restriction, T-tracking, and sequencing analysis of plasmids rescued from the genomic DNA of one of the putative phototaxis gene disruptants showed that it contained a complex multicopy insertion of the vector. While insertions of such plasmid vectors might typically be in tandem multicopy format, they can be much more complex, containing, as in this case, inverse as well as tandemly duplicated copies and various deletions.

Intracellular Ca2+ signals in Dictyostelium chemotaxis are mediated exclusively by Ca2+ influx.

We measured folate- and cAMP-induced changes in cytoplasmic free calcium concentration ([Ca2+]i) using recombinant aequorin reconstituted in living Dictyostelium cells with coelenterazine-h. The resulting semi-synthetic protein displayed increased sensitivity to Ca2+ allowing accurate measurement of chemoattractant-induced transients at low resting levels. Both folate- and cAMP-induced Ca2+ responses were developmentally regulated, exhibited remarkably similar kinetics and were dependent on the relative rather than the absolute magnitude of increases in attractant concentration. They began after a short delay of 5-10 seconds, leading to a maximum increase in cytosolic calcium concentration after approximately 25 seconds and a return to basal level within approximately 60 seconds after stimulation. Responses elicited by the two chemoattractants were dose-dependent and saturated between 4 and 20 microM. They depended on the presence of free extracellular calcium ions and were inhibited in a concentration-dependent manner between 10(-4) and 10(-5) M. In accordance with 45Ca2+-uptake measurements by Milne and Coukell (J. Cell Biol. (1991) 112, 103-110), both responses were also completely inhibited by 15 microM Ruthenium Red, 15 microM carbonylcyanide m-chlorophenyl-hydrazone (CCCP) and 500 microM gadolinium ions. Under conditions that prohibited influx of Ca2+ from the extracellular medium there were no detectable changes in [Ca2+]i that could be related to a separate release of the ion from intracellular stores. Together, these results show that the Ca2+ signals involved in chemotaxis correlate temporally with actin depolymerization (not polymerization) and are mediated by Ca2+ influx, not IP3-mediated intracellular release.

The birthing chair in the second stage of labour.

A prospective study of 56 primigravidas was performed to assess the advantages, disadvantages and acceptability of the upright posture during the second stage of labour. Twenty-seven patients laboured in the second stage in a birthing chair, in an upright position. Twenty-one patients laboured in bed in the recumbent position and acted as controls. No difference could be found in the length of second stage, ease or type of delivery between the 2 groups. No differences were detected in the condition of the neonates between the 2 groups. This birthing chair was found to be an acceptable mode of delivery to most of those patients using it.

Controlled study of 16,16-dimethyl-trans-delta 2 prostaglandin E1 methyl ester vaginal pessaries prior to suction termination of first trimester pregnancies.

A pessary containing the prostaglandin analogue, 16,16-dimethyl-trans-delta 2 prostaglandin E1 methyl ester (Cervagem) was inserted into the vagina of primigravid women 2 h before suction termination of pregnancy. The cervix was softer, easier to dilate and more widely dilated at the start of the procedure than in a control group of women who received placebo pessaries containing the vehicle alone. Intra-operative blood loss was diminished. The incidence of pre- and post-operative abdominal cramps was increased in the Cervagem-treated group. No gastrointestinal side-effects were noted.

PIP: A case control study to assess the effect on the cervix of inserting a pessary containing the prostaglandin analogue, 16,16-dimethyl-trans-delta 2 prostaglandin E1 methyl ester (Cervagem), into the vagina 2 hours prior to suction curettage was undertaken at the National Woem's Hospital in Uckland, New Zealand. Previous investigaors found that pretreatment of the cervix with Cervagem 3 hours prior to termination reduced cervical damage by making it easier to dilate the cervix. In order to facilitate termination procedures in a busy outpatient clinic, the pesent investigation was undertaken to determine if the treatment time could be reduced to 2 hours and still be effective. The study group consisted of 110 consecutively admitted primigravid patients, aged 14-35, at 6-12 weeks gestation. A randomized supply of 55 placebo pessaries and 55 active pessaries containing 1 mg of Cervagem were inserted, and temperature, pluse, and blood pressure measurements were taken just prior to insertion and then hourly. Prior to termination the patients received an injection of 50-100 ug fentanyl and paracervical block of 10-20 ml of 1% lignocaine was used as a local anesthesia. An assessment of the ease of dilation was made just prior to the operation, and blood loss during the procedure was measured and recored. Chi-square and t-test techniques were used to anaylyze the data. Results indicated that the Cervagem treatment significantly increased the ease of dilation but that the degree proceeded the operation by 3 hours. Spkecific findings were: 1) the mean dilation just prior to termination was 5.8 mm in the Cervagem treated group and 4.9 mm in the control group; 2) mechanical dilation was requred for 49 of the treated women and for 54 of the controls, and the difference was significant (P0.05); 3) dilation was difficult or moderately difficult in 17 of the treated women and in 28 of the placebo patients, and this difference was significant (P0.02); and 4) blood loss during the operation was significantly less for the treated group than for the untreated group (P0.02). Preoperative side effects in the Cervagem treated group were more common than in the control group, but none of the effects were serious. Postoperative complaints of abdominal cramping were significantly more common among the treated patients than among the control (P0.001); however, there were no differences between the 2 groups in regard to the incidence of postoperative pain, nausea, or diarrhea.

Isolation and characterization of the pesticide-degrading plasmid pJP1 from Alcaligenes paradoxus.

A strain of Alcaligenes paradoxus, unable to degrade phenoxyacetic acid, was shown to degrade two synthetic derivatives of this molecule, the herbicides 2,4-dichlorophenoxyacetic acid and 2-methyl-4-chlorophenoxyacetic acid. The ability to degrade these pesticides is encoded by a 58-megadalton conjugal plasmid, pJP1.

The role of cGMP in photosensory and thermosensory transduction in Dictyostelium discoideum.

Weak and strong light/heat stimuli induced changes in cyclic guanosine monophosphate (cGMP) levels in vegetative and aggregation competent amoebae and in slug cells of the Dictyostelium discoideum strain X22. Mutant strains derived from X22 with mutations in the phototaxis loci phoA-phoK fell into four phenotypic classes with respect to cGMP responses to weak and strong light/heat stimuli. These results suggest an intermediary role for cGMP in photosensory and thermosensory processing in slugs and amoebae. The streamer F mutant NP368 which has previously been shown to exhibit a prolonged cGMP response to cAMP, showed a wild-type cGMP response to light and heat. All phototaxis mutant strains with altered cGMP responses to light and heat were unaltered in their cGMP response to cAMP. These results suggest that cAMP and light/heat regulate cGMP via independent pathways. The thermotaxis mutant HPF228 showed altered cGMP responses to heat but not to light stimuli. This suggests that the mutation in HPF228 affects thermosensory transduction before convergence with the phototaxis pathway and the subsequent cGMP response.

Polycistronic transcription and editing of the mitochondrial small subunit (SSU ) ribosomal RNA in Dictyostelium discoideum.

Northern analyses and reverse transcription-polymerase chain reaction (RT-PCR) experiments were performed on total RNA of Dictyostelium discoideum. The mitochondrial genes encoding the small subunit ribosomal RNA (SSU), cytochrome b (CYTB) and subunit 3 of the NADH dehydrogenase (ND3) were found to be co-transcribed. Further post-transcriptional processing resulted in a dicistronic transcript for CYTB and ND3, and a monocistronic SSU transcript. Markedly higher steady state transcript levels were detected for the mature SSU ribosomal RNA. A comparison of the SSU cDNA sequence with the mitochondrial DNA sequence of the SSU gene revealed C-to-U substitutional editing of the SSU ribosomal RNA at a single site, as a consequence of which the cDNA contained a PvuII site not present in the genomic DNA. The editing was shown to be highly efficient and to occur in the primary transcript before the release of the mature mRNA, rRNA and tRNAs. It is suggested that the editing may be required for normal pseudoknot formation in the 530 loop of the RNA and thus is important for efficient, accurate translation in the mitochondria.