Parasexuality and Ploidy Change in Candida tropicalis

Candida species exhibit a variety of ploidy states and modes of sexual reproduction. Most species possess the requisite genes for sexual reproduction, recombination, and meiosis, yet only a few have been reported to undergo a complete sexual cycle including mating and sporulation. Candida albicans, the most studied Candida species and a prevalent human fungal pathogen, completes its sexual cycle via a parasexual process of concerted chromosome loss rather than a conventional meiosis. In this study, we examine ploidy changes in Candida tropicalis, a closely related species to C. albicans that was recently revealed to undergo sexual mating. C. tropicalis diploid cells mate to form tetraploid cells, and we show that these can be induced to undergo chromosome loss to regenerate diploid forms by growth on sorbose medium. The diploid products are themselves mating competent, thereby establishing a parasexual cycle in this species for the first time. Extended incubation (>120 generations) of C. tropicalis tetraploid cells under rich culture conditions also resulted in instability of the tetraploid form and a gradual reduction in ploidy back to the diploid state. The fitness levels of C. tropicalis diploid and tetraploid cells were compared, and diploid cells exhibited increased fitness relative to tetraploid cells in vitro, despite diploid and tetraploid cells having similar doubling times. Collectively, these experiments demonstrate distinct pathways by which a parasexual cycle can occur in C. tropicalis and indicate that nonmeiotic mechanisms drive ploidy changes in this prevalent human pathogen.     

Is Rapoport's rule a recent phenomenon? A deep time perspective on potential causal mechanisms

Macroecology strives to identify ecological patterns on broad spatial and temporal scales. One such pattern, Rapoport''s rule, describes the tendency of species'' latitudinal ranges to increase with increasing latitude. Several mechanisms have been proposed to explain this rule. Some invoke climate, either through glaciation driving differential extinction of northern species or through increased seasonal variability at higher latitudes causing higher thermal tolerances and subsequently larger ranges. Alternatively, continental tapering or higher interspecific competition at lower latitudes may be responsible. Assessing the incidence of Rapoport''s rule through deep time can help to distinguish between competing explanations. Using fossil occurrence data from the Palaeobiology Database, we test these hypotheses by evaluating mammalian compliance with the rule throughout the Caenozoic of North America. Adherence to Rapoport''s rule primarily coincides with periods of intense cooling and increased seasonality, suggesting that extinctions caused by changing climate may have played an important role in erecting the latitudinal gradients in range sizes seen today.     

Antibodies to Heteromeric Glycolipid Complexes in Guillain-Barré Syndrome

Autoantibodies are infrequently detected in the sera of patients with the demyelinating form of Guillain-Barré syndrome most commonly encountered in the Western world, despite abundant circumstantial evidence suggesting their existence. We hypothesised that antibody specificities reliant on the cis interactions of neighbouring membrane glycolipids could explain this discrepancy, and would not have been detected by traditional serological assays using highly purified preparations of single gangliosides. To assess the frequency of glycolipid complex antibodies in a Western European cohort of patients GBS we used a newly developed combinatorial glycoarray methodology to screen against large range of antigens (11 gangliosides, 8 other single glycolipids and 162 heterodimeric glycolipid complexes). Serum samples of 181 patients from a geographically defined, Western European cohort of GBS cases were analysed, along with 161 control sera. Serum IgG binding to single gangliosides was observed in 80.0% of axonal GBS cases, but in only 11.8% of cases with demyelinating electrophysiology. The inclusion of glycolipid complexes increased the positivity rate in demyelinating disease to 62.4%. There were 40 antigens with statistically significantly increased binding intensities in GBS as compared to healthy control sera. Of these, 7 complex antigens and 1 single ganglioside also produced statistically significantly increased binding intensities in GBS versus neurological disease controls. The detection of antibodies against specific complexes was associated with particular clinical features including disease severity, requirement for mechanical ventilation, and axonal electrophysiology. This study demonstrates that while antibodies against single gangliosides are often found in cases with axonal-type electrophysiology, antibodies against glycolipid complexes predominate in cases with demyelinating electrophysiology, providing a more robust serum biomarker than has ever been previously available for such cases. This work confirms the activation of the humoral immune system in the dysimmune disease process in GBS, and correlates patterns of antigen recognition with different clinical features.     

Searching for the bull's eye: agents and targets of selection vary among geographically disparate cyanogenesis clines in white clover (Trifolium repens L.)

The recurrent evolution of adaptive clines within a species can be used to elucidate theselective factors and genetic responses that underlie adaptation. White clover ispolymorphic for cyanogenesis (HCN release with tissue damage), and climate-associatedcyanogenesis clines have evolved throughout the native and introduced species range. Thispolymorphism arises through two independently segregating Mendelian polymorphisms for thepresence/absence of two required components: cyanogenic glucosides and theirhydrolyzing enzyme linamarase. Cyanogenesis is commonly thought to function in herbivoredefense; however, the individual cyanogenic components may also serve other physiologicalfunctions. To test whether cyanogenesis clines have evolved in response to the sameselective pressures acting on the same genetic targets, we examined cyanogenesis clineshape and its environmental correlates in three world regions: southern New Zealand, thecentral United States and the US Pacific Northwest. For some regional comparisons, clineshapes are remarkably similar despite large differences in the spatial scales over whichclines occur (40–1600 km). However, we also find evidence for majordifferences in both the agents and targets of selection among the sampled clines.Variation in cyanogenesis frequency is best predicted using a combination of minimumwinter temperature and aridity variables. Together, our results provide evidence thatrecurrent adaptive clines do not necessarily reflect shared adaptive processes.     

Detection of circulating antigen of Aspergillus fumigatus in sera of mice and rabbits by enzyme-linked immunosorbent assay

Circulating antigen of Aspergillus fumigatus was demonstrated in the sera of experimentally infected, cortisone-treated mice and rabbits by enzyme-linked immunosorbent assay (micro-ELISA), confirming earlier results where fungal antigen had been detected by counter-immunoelectrophoresis (CIE). Peaks of detection of circulating antigen by CIE and micro-ELISA in mice were not simultaneous suggesting that the nature of the predominant antigens may have altered during the course of infection. CIE failed to detect fungal antigen in infected rabbits whereas micro-ELISA monitored antigenemia until death. Both CIE and micro-ELISA demonstrated the rapid clearance of intravenously inoculated Aspergillus-antigen from the rabbit circulation.     

Functional morphology of synostosial articulations in the crinoid column

The rarest articulation found in the crinoid column is the synostosis, in which adjacent articular facets are essentially planar. 'Synostoses' in the columns of post-Palaeozoic isocrinids are more correctly termed cryptosymplexies. comprising symplectial articulations which have become infilled by secondary calcite. Cryptosymplexial joints are totally inflexible and are the preferred sites of stem autotomy. Synostoses sensu stricto appear to have been limited to early Palaeozoic taxa, but this form of articulation was soon succeeded by the symplexy. Synostoses were probably commonest in meric columns which evolved from hohlwurzels and in which the main flexibility was along meric sutures, rather than between columnals. With the evolution of the holomeric columnal. a more flexible articulation between columnals was a functional necessity in order that the stem did not develop into a stiffened rod. The solution was the evolution of the symplexy. *** Crinoids, columnals. synostosis, functional morphology evolution.     

Elimination and reconstitution of the requirement for hormone in promoting temperature-dependent transformation of cytosolic glucocorticoid receptors to the DNA-binding state

Cytosols contain a heat-stable, chelatable, anionic, molybdate-like factor that stabilizes glucocorticoid receptors in a heteromeric complex with hsp90 (refers to the 90-kDa heat shock protein) and inhibits their transformation to the DNA-binding state (Meshinchi, S., Grippo, J.F., Sanchez, E.R., Bresnick, E.H., and Pratt, W.B. (1988) J. Biol. Chem. 263, 16809-16817). In this work, we demonstrate that removal of this factor by passage of L cell cytosol through the metal-chelating resin Chelex-100 makes the glucocorticoid receptor unstable, thus markedly facilitating both its dissociation from hsp90 and its transformation to the DNA-binding state. In normal cytosol, both temperature-mediated dissociation of hsp90 and temperature-mediated receptor transformation are hormone-dependent events. In the Chelex-treated, metal-depleted cytosol, however, temperature-mediated dissociation of hsp90 and receptor transformation occur very rapidly in a manner that is no longer hormone-dependent. When boiled L cell cytosol is added to the metal-depleted receptor system, the hormone dependence of both temperature-mediated dissociation of receptor from hsp90 and receptor transformation to the DNA-binding state is reconstituted. Like boiled cytosol, molybdate stabilizes the receptor complex and inhibits its transformation in metal-depleted cytosol, but it does not reconstitute the hormone dependence of the system. These results support the proposal that an endogenous metal anion interacts with the glucocorticoid receptor to stabilize it in the heteromeric, inactive, non-DNA-binding state in cytosol and that binding of the hormone promotes conversion of the receptor to the DNA-binding state through an effect on this metal anion center.     

Blood group A-active glycosphingolipids analysis by the combination of TLC-immunostaining assay and TLC/SIMS mass spectrometry

Blood group A-active glycosphingolipids from human erythrocyte membranes were identified by the combination of thin-layer chromatography and matrix-assisted secondary ion mass spectrometry (TLC/SIMS). Partially purified lipid extracts were chromatographed by TLC and then blood group A-active glycolipids were detected by TLC-immunostaining assay using anti-A antibody. The parts of the plates which contained the same Rf area as anti-A positive spots were cut out and subjected to direct SIMS analysis. The TLC/SIMS spectra were quite similar to those obtained by ordinary SIMS. Detailed information, such as molecular weight, molecular species, ceramide portion, and oligosaccharide sequence, was obtained. Also, peracetylated blood group A-active glycolipids were analyzed in a similar manner. After the position of A-active glycolipids on a TLC plate was confirmed by in situ deacetylation and TLC-immunostaining, acetylated A-active glycolipids were also analyzed by the TLC/SIMS. Enhanced sensitivity was obtained with peracetylated glycolipids. Consequently, small amounts of unpurified bioactive glycolipids can be readily analyzed by TLC/SIMS.     

Effect of LH-RH on the release of sulfated lutropin: a potential in vitro bioassay for LH-RH

Sheep pituitary cells prelabelled with radioactive [35S] sulfate (35SO4(2-)) were incubated with different concentrations of LH-RH and the release of LH (lutropin) into the medium was monitored in terms of immunoprecipitable [35S] sulfated LH radioactivity and estimation of LH in the same sample by radioimmunoassay. A dose dependent response was obtained with a maximum of a 16 fold increase in immunoprecipitable 35SO4(2-) -labelled LH radioactivity in the medium which was confirmed by radioimmunoassay. Similar results were also obtained for Buserelin, a well known superactive analogue of LH-RH. However, the half maximal response for Buserelin was obtained at 3-5 nM in comparison to 80.5 nM for LH-RH. After the maximal response to LH-RH as well as Buserelin, a further increase in the concentrations caused a decrease in the release of immunoprecipitable [35S]-sulfate labelled LH into the medium. Differential labelling of stored and newly synthesized LH with radioactive [35S] sulfate and [3H]-labelled leucine revealed that there was a dose dependent increase in the [35S] sulfate labelled LH into the medium whereas the release of [3H]-leucine labelled newly synthesized LH did not show a parallel increase either at different concentrations of LH-RH or at different time intervals. The above observations strongly suggest the possibility of sulfation of LH being the potential signal indicating the storage of LH in sheep pituitary cells. Another important observation in our study was that the dose dependent response of LH-RH in the form of release of [35S]-sulfate labelled LH, which was monitored by immunoprecipitation with specific LH antiserum, can be used in an in vitro bioassay for LH-RH. We believe that a new cheap and sensitive in vitro bioassay could be developed on the basis of this observation.     

Stereospecific production of the herbicide phosphinothricin (glufosinate) by transamination: cloning, characterization, and overexpression of the gene encoding a phosphinothricin-specific transaminase from Escherichia coli

We have cloned the gene encoding a 43-kilodalton transaminase from Escherichia coli K-12 with a specificity for L-phosphinothricin [L-homoalanine-4-yl-(methyl)phosphinic acid], the active ingredient of the herbicide Basta (Hoechst AG). The structural gene was isolated, together with its own promoter, and shown to be localized on a 1.6-kilobase DraI-BamHI fragment. The gene is subject to catabolite repression by glucose; however, repression could be relieved completely when 4-aminobutyrate (GABA) served as the sole nitrogen source. The regulation pattern obtained and a comparison of the restriction map of the initially cloned 15-kilobase SalI fragment with the physical map of the E. coli K-12 genome suggest that the cloned gene is identical with gabT, a locus on the gab gene cluster of E. coli K-12 which codes for the GABA:2-ketoglutartate transaminase (EC 2.6.1.19). A number of expression plasmids carrying the isolated transaminase gene were constructed. With these constructs, the transaminase expression in transformants of E. coli could be increased up to 80-fold compared with that in a wild-type control, and the transaminase constituted up to 20% of the total soluble protein of the bacteria. Thus, the protein crude extracts of the transformants could be used, after a simple heat precipitation step, for the biotechnological production of L-phosphinothricin in an enzyme reactor.