Dipeptidyl aminopeptidase yspI mutants of Schizosaccharomyces pombe: Genetic mapping of dpa1+ on chromosome III

Abstract A mutant strain of Schizosaccharomyces pombe lacking dipeptidyl aminopeptidase yspI was isolated from a strain already defective in aminopeptidase activity by means of a staining technique with the chromogenic substrate ala-pro-4-methoxy-β-naphthylamide to screen colonies for the absence of the enzyme. The defect segregated 2⁺ :2⁻ in meiotic tetrads, indicating a single chromosomal gene mutation, which was shown to be recessive. Gene dosage experiments indicated that the mutation resides in the structural gene of dipeptidyl aminopeptidase yspI, dpa 1⁺. The dpa 1⁺ gene was located on chromosome III by using l m- fluorophen-ylalanine-induced haploidization and mitotic analysis. dpa1 mutants did not show any obvious phenotype under a variety of conditions tested.     

Isolation of fungi from bats of the Amazon basin

A total of 2,886 bats captured in the Amazon Basin of Brazil were processed for the isolation of fungi. From the livers, spleens, and lungs of 155 bats (5.4%), 186 fungal isolates of the genera Candida (123 isolates), Trichosporon (26 isolates), Torulopsis (25 isolates), Kluyveromyces (11 isolates), and Geotrichum (1 isolate) were recovered. Seven known pathogenic species were present: Candida parapsilosis, C. guilliermondii, C. albicans, C. stellatoidea, C. pseudotropicalis, Trichosporon beigelii, and Torulopsis glabrata. Twenty-three culture-positive bats showed identical fungal colonization in multiple organs or mixed colonization in a single organ. The fungal isolation rates for individual bat species varied from 1 fungus per 87 bats to 3 fungi per 13 bats, and the mycoflora diversity for members of an individual fungus-bearing bat species varied from 16 fungi per 40 bats to 7 fungi per 6 bats. Of the 38 fungal species isolated, 36 had not been previously described as in vivo bat isolates. Of the 27 culture-positive bat species, 21 had not been previously described as mammalian hosts for medically or nonmedically important fungi.     

Characterisation of Trypanosoma cruzi populations by dna polymorphism of the cruzipain gene detected by single-stranded dna conformation polymorphism (SSCP) and direct sequencing

Fifty fresh isolates of Trypanosoma cruzi from Triatoma dimidiata vectors and 31 from patients with Chagas disease were analysed for DNA polymorphisms within the 432-bp core region of the cruzipain gene which encodes the active site of cathepsin L-like cystein proteinase. The cruzipain gene showed signs of polymorphism consisting of four different DNA sequences in Central and South American isolates of T. cruzi. The PCR fragments of Guatemalan isolates could be divided into three groups, Groups 1, 2 and 3, based on different patterns of single-stranded DNA conformation polymorphism. All of the strains isolated from Brazil, Chile, and Paraguay, except for the CL strain, showed a Group 4 pattern. Two to four isolates from each group were analysed by cloning and sequencing. A silent mutation occurred between Groups 1 and 2, and five nucleotides and two aa substitutions were detected between Groups 1 and 3. The DNA sequence of Group 4 contained five nucleotides and one aa substitution from Group 1. All of the DNA sequences corresponded well with the single-stranded DNA conformation polymorphism. The Group 1 isolates, the majority in the Guatemalan population (70/81, 86.4%), were isolated from both triatomines and humans, but Group 3 were isolated only from humans. Moreover, the Group 2 isolates were detected only in triatomine vectors (9/50; 18%), but never in humans (0/32, P<0.05) suggesting that this group has an independent life-cycle in sylvatic animals and is maintained by reservoir hosts other than humans.     

A predictive model for freshwater bioassessment (Mondego River,Portugal)

We sampled macroinvertebrates at 75 locations in the Mondego river catchment, Central Portugal, and developed a predictive model for water quality assessment of this basin, based on the Reference Condition Approach. Sampling was done from June to September 2001. Fifty-five sites were identified as “Reference sites” and 20 sites were used as “Test sites” to test the model. At each site we also measured 40 habitat variables to characterize water physics and chemistry, habitat type, land use, stream hydrology and geographic location. Macroinvertebrates were generally identified to species or genus level; a total of 207 taxa were found. By Unweighted Pair Group Method with Arithmetic mean (UPGMA) clustering and analysis of species contribution to similarities percentage (SIMPER), two groups of reference sites were established. Using Discriminant Analysis (stepwise forward), four variables correctly predicted 78% of the reference sites to the appropriate group: stream order, pool quality, substrate quality and current velocity. Test sites’ environmental quality was established from their relative distance to reference sites, in MDS ordination space, using a series of bands (BEAST methodology). The model performed well at upstream sites, but at downstream sites it was compromised by the lack of reference sites. As with the English RIVPACS predictive model, the Mondego model should be continually improved with the addition of new reference sites. The adaptation of the Mondego model methodology to the Water Framework Directive is possible and would consist mainly of the integration of the WFD typology and increasing the number of ellipses that define quality bands. Handling editor: K. Martens     

Promotion by phloroglucinol of micropropagation of Minuartia valentina, an endangered and endemic Spanish plant

Tissue culture techniques for the propagation and conservation of endemic or threatened plants can be used to complement the methods usually applied in ex situ conservation. Thus, Minuartia valentina (Caryophyllaceae), an endangered plant species endemic to the Valencia Community (Eastern Spain), was successfully regenerated through shoot proliferation from wild plants growing in their natural area. Nodal segments, 10~mm long, were cut from rametes of adult material, sterilised and established in vitro. Equally successful shoot multiplication was achieved on Murashige and Skoog (MS) medium with 80 mg l-1 phloroglucinol in combination with either 1 mg l-1 6-benzylaminopurine or 1 mg l-1 kinetin. Excised shoots were rooted in MS medium supplemented with an auxin (indole acetic acid, indole-3-butyric acid, or napththalene acetic acid). Shoots rooted well (96–100%) within three weeks in all auxin treatments. However, the use of napththalene acetic acid was discarded because this auxin delayed root differentiation, and induced adventitious root malformation. Rooted plantlets were transferred to pots and 85% of them acclimatized successfully four weeks after transfer to greenhouse conditions, where they exhibited normal morphology and growth.     

The Peculiar Facets of Nitric Oxide as a Cellular Messenger: From Disease-Associated Signaling to the Regulation of Brain Bioenergetics and Neurovascular Coupling

In this review, we address the regulatory and toxic role of ^·NO along several pathways, from the gut to the brain. Initially, we address the role on ^·NO in the regulation of mitochondrial respiration with emphasis on the possible contribution to Parkinson’s disease via mechanisms that involve its interaction with a major dopamine metabolite, DOPAC. In parallel with initial discoveries of the inhibition of mitochondrial respiration by ^·NO, it became clear the potential for toxic ^·NO-mediated mechanisms involving the production of more reactive species and the post-translational modification of mitochondrial proteins. Accordingly, we have proposed a novel mechanism potentially leading to dopaminergic cell death, providing evidence that NO synergistically interact with DOPAC in promoting cell death via mechanisms that involve GSH depletion. The modulatory role of NO will be then briefly discussed as a master regulator on brain energy metabolism. The energy metabolism in the brain is central to the understanding of brain function and disease. The core role of ^·NO in the regulation of brain metabolism and vascular responses is further substantiated by discussing its role as a mediator of neurovascular coupling, the increase in local microvessels blood flow in response to spatially restricted increase of neuronal activity. The many facets of NO as intracellular and intercellular messenger, conveying information associated with its spatial and temporal concentration dynamics, involve not only the discussion of its reactions and potential targets on a defined biological environment but also the regulation of its synthesis by the family of nitric oxide synthases. More recently, a novel pathway, out of control of NOS, has been the subject of a great deal of controversy, the nitrate:nitrite:NO pathway, adding new perspectives to ^·NO biology. Thus, finally, this novel pathway will be addressed in connection with nitrate consumption in the diet and the beneficial effects of protein nitration by reactive nitrogen species.

     

Dual Cyclin-Binding Domains Are Required for p107 To Function as a Kinase Inhibitor

The retinoblastoma (pRB) family of proteins includes three proteins known to suppress growth of mammalian cells. Previously we had found that growth suppression by two of these proteins, p107 and p130, could result from the inhibition of associated cyclin-dependent kinases (cdks). One important unresolved issue, however, is the mechanism through which inhibition occurs. Here we present in vivo and in vitro evidence to suggest that p107 is a bona fide inhibitor of both cyclin A-cdk2 and cyclin E-cdk2 that exhibits an inhibitory constant (K_i) comparable to that of the cdk inhibitor p21/WAF1. In contrast, pRB is unable to inhibit cdks. Further reminiscent of p21, a second cyclin-binding site was mapped to the amino-terminal portions of p107 and p130. This amino-terminal domain is capable of inhibiting cyclin-cdk2 complexes, although it is not a potent substrate for these kinases. In contrast, a carboxy-terminal fragment of p107 that contains the previously identified cyclin-binding domain serves as an excellent kinase substrate although it is unable to inhibit either kinase. Clustered point mutations suggest that the amino-terminal domain is functionally important for cyclin binding and growth suppression. Moreover, peptides spanning the cyclin-binding region are capable of interfering with p107 binding to cyclin-cdk2 complexes and kinase inhibition. Our ability to distinguish between p107 and p130 as inhibitors rather than simple substrates suggests that these proteins may represent true inhibitors of cdks.