DJ-1 interactions with α-synuclein attenuate aggregation and cellular toxicity in models of Parkinson's disease.

Parkinson's disease (PD) is a devastating neurodegenerative disorder characterized by the loss of neurons in the substantia nigra pars compacta and the presence of Lewy bodies in surviving neurons. These intracellular protein inclusions are primarily composed of misfolded α-synuclein (aSyn), which has also been genetically linked to familial and sporadic forms of PD. DJ-1 is a small ubiquitously expressed protein implicated in several pathways associated with PD pathogenesis. Although mutations in the gene encoding DJ-1 lead to familial early-onset PD, the exact mechanisms responsible for its role in PD pathogenesis are still elusive. Previous work has found that DJ-1--which has protein chaperone-like activity--modulates aSyn aggregation. Here, we investigated possible physical interactions between aSyn and DJ-1 and any consequent functional and pathological relevance. We found that DJ-1 interacts directly with aSyn monomers and oligomers in vitro, and that this also occurs in living cells. Notably, several PD-causing mutations in DJ-1 constrain this interaction. In addition, we found that overexpression of DJ-1 reduces aSyn dimerization, whereas mutant forms of DJ-1 impair this process. Finally, we found that human DJ-1 as well as yeast orthologs of DJ-1 reversed aSyn-dependent cellular toxicity in Saccharomyces cerevisiae. Taken together, these data suggest that direct interactions between DJ-1 and aSyn constitute the basis for a neuroprotective mechanism and that familial mutations in DJ-1 may contribute to PD by disrupting these interactions.

Resistance and adaptation to quinidine in Saccharomyces cerevisiae: role of QDR1 (YIL120w), encoding a plasma membrane transporter of the major facilitator superfamily required for multidrug resistance.

As predicted based on structural considerations, we show results indicating that the member of the major facilitator superfamily encoded by Saccharomyces cerevisiae open reading frame YIL120w is a multidrug resistance determinant. Yil120wp was implicated in yeast resistance to ketoconazole and quinidine, but not to the stereoisomer quinine; the gene was thus named QDR1. Qdr1p was proved to alleviate the deleterious effects of quinidine, revealed by the loss of cell viability following sudden exposure of the unadapted yeast population to the drug, and to allow the earlier eventual resumption of exponential growth under quinidine stress. However, QDR1 gene expression had no detectable effect on the susceptibility of yeast cells previously adapted to quinidine. Fluorescence microscopy observation of the distribution of the Qdr1-green fluorescent protein fusion protein in living yeast cells indicated that Qdr1p is a plasma membrane protein. We also show experimental evidence indicating that yeast adaptation to growth with quinidine involves the induction of active expulsion of the drug from preloaded cells, despite the fact that this antiarrhythmic and antimalarial quinoline ring-containing drug is not present in the yeast natural environment. However, we were not able to prove that Qdr1p is directly implicated in this export. Results clearly suggest that there are other unidentified quinidine resistance mechanisms that can be used in the absence of QDR1.

Transcriptional activation of FLR1 gene during Saccharomyces cerevisiae adaptation to growth with benomyl: role of Yap1p and Pdr3p.

The adaptation of Saccharomyces cerevisiae to growth in the presence of the antimitotic fungicide benomyl involves the dramatic activation of FLR1 transcription, taking place during benomyl-induced latency following sudden exposure to the fungicide. FLR1 gene encodes a plasma membrane transporter of the major facilitator superfamily (MFS) conferring resistance to multiple drugs, in particular to benomyl. FLR1 activation is completely abolished in a mutant devoided of YAP1 gene being exerted by Yap1p either directly or via Pdr3p. YAP1 gene was proved to be a determinant of benomyl resistance; the duration of the adaptation period preceding cell division under benomyl stress was longer for the Deltayap1 population, presumably due to the abolishment of FLR1 activation during latency. Although benomyl resistance mediated by Yap1p is reduced in a FLR1 deletion mutant, results also indicate that Yap1p may have other target genes that confer benomyl resistance in yeast.

Expression of the AZR1 gene (ORF YGR224w), encoding a plasma membrane transporter of the major facilitator superfamily, is required for adaptation to acetic acid and resistance to azoles in Saccharomyces cerevisiae.

In this work, we report results on the functional analysis of Saccharomyces cerevisiae ORF YGR224w, predicted to code for an integral membrane protein, with 14 potential transmembrane segments, belonging to the major facilitator superfamily (MFS) of transporters which are required for multiple-drug resistance (MDR). This MFS-MDR homologue is required for yeast adaptation to high stress imposed by low-chain organic acids, in particular by acetic acid, and for resistance to azoles, especially to ketoconazole and fluconazole; the encoding gene was thus named the AZR1 gene. These conclusions were based on the higher susceptibility to these compounds of an azr1Delta deletion mutant strain compared with the wild-type and on the increased resistance of both azr1Delta and wild-type strains upon increased expression of the AZR1 gene from a centromeric plasmid clone. AZR1 gene expression reduces the duration of acetic acid-induced latency, although the growth kinetics of adapted cells under acetic acid stress is apparently independent of AZR1 expression level. Fluorescence microscopy observation of the distribution of the Azr1-GFP fusion protein in yeast living cells indicated that Azr1 is a plasma membrane protein. Studies carried out to gain some understanding of how this plasma membrane putative transporter facilitates yeast adaptation to acetic acid did not implicate Azr1p in the alteration of acetic acid accumulation into the cell through the active efflux of acetate.

FLR1 gene (ORF YBR008c) is required for benomyl and methotrexate resistance in Saccharomyces cerevisiae and its benomyl-induced expression is dependent on pdr3 transcriptional regulator.

In this work we report the disruption of a Saccharomyces cerevisiae ORF YBR008c (FLR1 gene) within the context of EUROFAN (EUROpean Functional Analysis Network) six-pack programme, using a PCR-mediated gene replacement protocol as well as the results of the basic phenotypic analysis of a deletant strain and the construction of a disruption cassette for inactivation of this gene in any yeast strain. We also show results extending the knowledge of the range of compounds to which FLR1 gene confers resistance to the antimitotic systemic benzimidazole fungicide benomyl and the antitumor agent methotrexate, reinforcing the concept that the FLR1 gene is a multidrug resistance (MDR) determinant. Our conclusions were based on the higher susceptibility to these compounds of flr1Delta compared with wild-type and on the increased resistance of both flr1Delta and wild-type strains upon increased expression of FLR1 gene from a centromeric plasmid clone. The present study also provides, for the first time, evidence that the adaptation of yeast cells to growth in the presence of benomyl involves the dramatic activation of FLR1 gene expression during benomyl-induced latency (up to 400-fold). Results obtained using a FLR1-lacZ fusion in a plasmid indicate that the activation of FLR1 expression in benomyl-stressed cells is under the control of the transcriptional regulator Pdr3p. Indeed, PDR3 deletion severely reduces benomyl-induced activation of FLR1 gene expression (by 85%), while the homologous Pdr1p transcription factor is apparently not involved in this activation.

Thermonema rossianum sp. nov., a new thermophilic and slightly halophilic species from saline hot springs in Naples, Italy.

Six slightly halophilic, thermophilic bacterial strains were isolated from saline hot springs along the Bay of Naples, Italy. These strains produce bright yellow colonies and have a filamentous morphology and an optimum growth temperature of about 60 degrees C. Lipid composition and 16S ribosomal DNA sequence analyses showed that these strains belong to the genus Thermonema, a member of the cytophaga-flavobacter-bacteroides phylum. Growth was observed in medium containing 1 to 3% NaCl. The DNA G + C content was 50.9 mol%. DNA-DNA hybridization studies showed that these strains represent a new species of the genus Thermonema. We propose that strain NR-27T (T = type strain) and the other strains represent a new species, Thermonema rossianum. Strain NR-27 (= DSM 10300) is the type strain of this species.

Thermus silvanus sp. nov. and Thermus chliarophilus sp. nov., two new species related to thermus ruber but with lower growth temperatures.

Strains of Thermus silvanus sp. nov. and strains of Thermus chliarophilus sp. nov. were isolated from the hot spring at Vizela in northern Portugal and the hot spring at Alcafache in central Portugal, respectively. The strains of T. silvanus produce orange-red-pigmented colonies and have an optimum growth temperature of about 55 degrees C, while the strains of T. chliarophilus produce yellow-pigmented colonies and have an optimum growth temperature of about 50 degrees C. The strains of both species are catalase negative. These species can be distinguished from each other and from Thermus ruber by biochemical characteristics, fatty acid composition data, and 16S rRNA gene sequence data. Our phylogenetic analysis showed that strains VI-R2T (T = type strain) and ALT-8T belong to the T. ruber line of descent. The type strain of T. silvanus is strain VI-R2 (= DSM 9946), and the type strain of T. chliarophilus is strain ALT-8 (= DSM 9957).

DNA:DNA hybridization and chemotaxonomic studies of Thermus scotoductus.

The species Thermus scotoductus was recently described as containing several non-pigmented isolates from Selfoss, Iceland, and the X-1 strain from the USA (Kristjansson et al., 1994). In this study, we performed DNA:DNA hybridizations and chemotaxonomic studies on several non-pigmented Thermus isolates from other geographical areas to assess their relationship to the strains originally assigned to this species. The results of DNA:DNA hybridizations showed that strains NH and Dl from London and strains Vl-7a and Vl-13 from Vizela, Portugal, belonged to T. scotoductus. T. scotoductus X-1 (ATCC 27978) was composed of two stable colony types, one of which had a major glycolipid different from the one present in the other colony type and from all other Thermus strains examined as well. The fatty acid composition of the isolates from Selfoss and London were practically identical. However, the fatty acid composition of strain X-1, the individual colony types of this strain and the Vizela strains were different from the Selfoss-London isolates and from each other. Another non-pigmented strain, designated SPS-11, belonged to a different DNA homology group.

Polar lipids and fatty acid composition of Thermus strains from New Zealand.

The polar lipids and fatty acid composition of Thermus aquaticus YT-1 and YS 041, T. filiformis Wai33 A1 and eighteen isolates from New Zealand, several of which are attributed to T. filiformis, were compared to complement the taxonomy of these organisms. The polar lipid patterns were essentially similar in all strains and consisted of one major phospholipid and one major glycolipid. The fatty acid analysis produced three basic groups corresponding to T. filiformis Wai33 A1, T. aquaticus and the third to the other New Zealand strains. The presence of hydroxy fatty acids is reported in Thermus spp. for the first time.

Skin temperature and heart rate rhythms in infants of extreme prematurity.

Nine preterm infants of 26 to 29 weeks' gestational age and 792 to 1200 g birth weight spent six to 17 weeks in our neonatal medical unit. Hourly recordings of skin temperature and heart rate were carried out. The first five to 15 weeks were spent in the intensive care ward, in continuous light, due to various medical conditions. After recovery they were moved to a nursery for one to nine weeks, with 12 hourly periods of light and darkness. Four infants developed circadian rhythms in temperature and three in heart rate in light-dark periods, the remainder failing to do so. Some infants take longer than others to develop circadian rhythms but the reasons for this are not clear. It is suggested that earlier exposure to a light-dark environment may synchronize the 'body clock' to a 24 hour period in more preterm infants.

The development of ultradian and circadian rhythms in premature babies maintained in constant conditions.

Twenty very premature babies, born at 24-29 weeks gestation, have been studied while they were maintained in intensive care with continuous intravenous feeding and constant ambient lighting and temperature. Hourly records of insulated skin temperature and heart rate were made for a continuous period of 6-17 weeks, always starting the recording within 24 h of birth. The development of rhythms within the ultradian, circadian and infradian domains was sought by methods including maximum entropy spectral analysis and autocorrelation. Circadian and ultradian rhythms were present, but not regularly so; rather they appeared and disappeared erratically in successive weeks. As a consequence, the group as a whole did not show an increasing rhythmicity with chronological age. In some cases, babies were later placed in a ward in which the lighting was dimmed at night, and feeding by mouth at regular intervals was instituted. There was some evidence for increases in circadian and ultradian rhythmicity after these changes. These results enable inferences to be drawn as to the origin of fetal rhythms in the third trimester of pregnancy, as well as speculation to be made on the ontogeny of ultradian and circadian rhythms in the neonate.