l-Proline transport in Saccharomyces cerevisiae

Transport of l-proline into Saccharomyces cerevisiae K is mediated by two systems, one with a K_T of 31 μM and J_max of 40 nmol · s^?1 · (g dry wt.)^?1, the other with K_T > 2.5 mM and J_max of 150–165 nmol · s^?1 · (g dry wt.)^?1, The kinetic properties of the high-affinity system were studied in detail. It proved to be highly specific, the only potent competitive inhibitors being (i) l-proline and its analogs l-azetidine-2-carboxylic acid, sarcosine, d-proline and 3,4-dehydro-dl-proline, and (ii) l-alanine. The other amino acids tested behaved as noncompetitive inhibitors. The high-affinity system is active, has a sharp pH optimum at 5.8–5.9 and, in an Arrhenius plot, exhibits two inflection points at 15°C and 20–21°C. It is trans-inhibited by most amino acids (but probably only the natural substrates act in a trans-noncompetitive manner) and its activity depends to a considerable extent on growth conditions. In cells grown in a rich medium with yeast extract maximum activity is attained during the stationary phase, on a poor medium it is maximal during the early exponential phase. Some 50–60% of accumulated l-proline can leave cells in 90 min (and more if washing is done repeatedly), the efflux being insensitive to 0.5 mM 2,4-dinitrophenol and uranyl ions, to pH between 3 and 7.3, as well as to the presence of 10–100 mM unlabeled l-proline in the outside medium. Its rate and extent are increased by 1% d-glucose and by 10 μg nystatin per ml.     

Localisation and distance between ABL and BCR genes in interphase nuclei of bone marrow cells of control donors and patients with chronic myeloid leukaemia

Quantitative measurements of the nuclear localisation of the ABL and BCR genes and the distance between them were performed in randomly oriented bone marrow cells of control donors and patients with chronic myeloid leukaemia (CML). Most ABL and BCR genes (75%) are located at a distance of 20–65% of the local radius from the nuclear centre to the nuclear membrane. A chimeric BCR-ABL gene located on a derivative chromosome 22 resulting from t(9;22)(q34;q11) [the so-called Philadelphia (Ph) chromosome] as well as the intact ABL and BCR genes of patients suffering from chronic myeloid leukaemia are also located mostly in this region, which has a mean thickness of 2 μm in bone marrow cells. We have not found any significant differences in the location of the two genes in the G₁ and G₂ phases of the cell cycle, nor between bone marrow cells and stimulated lymphocytes. Irradiation of lymphocytes with a dose of 5 Gy of γ-rays results in a shift of both genes to the central region of the nucleus (0–20% of the radius distant from the nuclear centre) in about 15% of the cells. The minimum distance between one ABL and one BCR gene is less than 1 μm in 47.5% of bone marrow cells of control donors. Such a small distance is found between homologous ABL and between homologous BCR genes in only 8.1% and 8.4% of cells, respectively. It is possible that the relative closeness of nonhomologous ABL and BCR genes in interphase nuclei of bone marrow cells could facilitate translocation between these genes. In 16.4% of bone marrow cells one ABL and one BCR gene are juxtaposed (the distance between them varies from 0–0.5 μm) and simulate the Ph chromosome. This juxtaposition is the result of the projection of two genes located one above another into a plane, as follows from the probability calculation. Received: 5 September 1996 / Accepted: 15 April 1997     

Right ventricular function in acute myocardial ischaemia

A haemodynamic examination of 10 dogs was carried out at rest, during volume loading and after ligation of the right coronary artery in the presence of a closed pericardium. Ligation of the right coronary artery led to haemodynamic signs of depression of right ventricular function--a drop in systolic pressure and an increase in end diastolic pressure, together with a shift of the functional curve to the right and downwards. Overall performance of the heart (cardiac output and the mean systemic pressure, also fell. Our results show that the depression of the systolic function of the myocardium in the presence of right ventricular infarction can be an important factor in the genesis of low cardiac output syndrome observed in clinical situations. Its pathophysiological mechanisms and some of the clinical consequences are discussed.     

Mitochondrial and Nucleolar Localization of Cysteine Desulfurase Nfs and the Scaffold Protein Isu in Trypanosoma brucei

Trypanosoma brucei has a complex life cycle during which its single mitochondrion is subjected to major metabolic and morphological changes. While the procyclic stage (PS) of the insect vector contains a large and reticulated mitochondrion, its counterpart in the bloodstream stage (BS) parasitizing mammals is highly reduced and seems to be devoid of most functions. We show here that key Fe-S cluster assembly proteins are still present and active in this organelle and that produced clusters are incorporated into overexpressed enzymes. Importantly, the cysteine desulfurase Nfs, equipped with the nuclear localization signal, was detected in the nucleolus of both T. brucei life stages. The scaffold protein Isu, an interacting partner of Nfs, was also found to have a dual localization in the mitochondrion and the nucleolus, while frataxin and both ferredoxins are confined to the mitochondrion. Moreover, upon depletion of Isu, cytosolic tRNA thiolation dropped in the PS but not BS parasites.     

Assessing Niche Separation among Coexisting Limnohabitans Strains through Interactions with a Competitor,Viruses, and a Bacterivore

We investigated potential niche separation in two closely related (99.1% 16S rRNA gene sequence similarity) syntopic bacterial strains affiliated with the R-BT065 cluster, which represents a subgroup of the genus Limnohabitans. The two strains, designated B4 and D5, were isolated concurrently from a freshwater reservoir. Differences between the strains were examined through monitoring interactions with a bacterial competitor, Flectobacillus sp. (FL), and virus- and predator-induced mortality. Batch-type cocultures, designated B4+FL and D5+FL, were initiated with a similar biomass ratio among the strains. The proportion of each cell type present in the cocultures was monitored based on clear differences in cell sizes. Following exponential growth for 28 h, the cocultures were amended by the addition of two different concentrations of live or heat-inactivated viruses concentrated from the reservoir. Half of virus-amended treatments were inoculated immediately with an axenic flagellate predator, Poterioochromonas sp. The presence of the predator, of live viruses, and of competition between the strains significantly affected their population dynamics in the experimentally manipulated treatments. While strains B4 and FL appeared vulnerable to environmental viruses, strain D5 did not. Predator-induced mortality had the greatest impact on FL, followed by that on D5 and then B4. The virus-vulnerable B4 strain had smaller cells and lower biomass yield, but it was less subject to grazing. In contrast, the seemingly virus-resistant D5, with slightly larger grazing-vulnerable cells, was competitive with FL. Overall, our data suggest contrasting ecophysiological capabilities and partial niche separation in two coexisting Limnohabitans strains.Bacterioplankton communities in freshwater systems frequently are characterized by the dominance of a relatively small number of clades as defined by 16S rRNA gene phylogenies (4, 50). These genus-like phylogenetic groups usually contribute large fractions to total prokaryotic numbers in typical stagnant freshwater systems (1, 34, 37, 39). While determining ecological differences between the organisms of these rather distantly related 16S rRNA clades is of enormous importance for understanding the mechanisms and factors shaping the diversity and composition of freshwater bacterioplankton (21), it is important to know if such clades actually represent ecologically coherent taxa, or if there is significant ecological variation within a single clade. Recent studies have uncovered ecological differences between closely related organisms within the same 16S rRNA clade (7, 13, 16, 18, 20, 26, 35). Several of these studies compared organisms affiliated with the same clade but originating from ecologically different habitats. To date only a few investigations have examined ecological differences between closely related and coexisting (syntopic) freshwater bacteria (7, 18, 35). For example, niche separation among Vibrionaceae strains coexisting in coastal bacterioplankton was demonstrated (16). Among a broad diversity of sympatric strains, Hunt and coworkers (16) identified phylogenetic subgroups differing in lifestyle (free living or association with different particle size classes) and seasonal preferences.Here, we investigate if two closely related strains coexisting in the same freshwater habitat differ significantly in their interactions with biological factors. We examined vulnerability to predation by a bacterivorous flagellate, sensitivity to virus assemblages recovered from their native habitat, and the interaction with a competitor of high growth potential. The two strains both were isolated from the same water sample taken from the meso-eutrophic Římov reservoir and belong to the recently proposed betaproteobacterial genus Limnohabitans (11). Within Limnohabitans, these two strains belong to a monophyletic group known as the R-BT065 cluster targeted by a homonymous fluorescent in situ hybridization (FISH) probe (41), which enabled intensive investigations of the ecology of this abundant bacterioplankton group in several different habitats (1, 15, 28, 29, 34, 37, 38). A recent survey on the distribution of this taxon revealed that it is a ubiquitous member of bacterioplankton communities in a broad variety of pH-neutral freshwater systems (102 systems inspected; see reference 39) that typically comprises ∼5 to 30% of total bacterial cell numbers. Previous investigations indicated a rather homogenous ecology of the R-BT065 group, characterized by the potential for a rapid response to changes of environmental conditions (34, 37, 38, 41) and a high sensitivity to flagellate predation (19, 42).We hypothesized that the two sympatric strains affiliated with the R-BT065 group would show ecological differences. These strains, designated B4 and D5, represent the most distantly (99.1% 16S rRNA gene sequence similarity) related pairing among the R-BT065 strains isolated so far (V. Kasalický, K. Šimek, J. Jezbera, and M. W. Hahn, unpublished data). To reveal ecological differences, the two strains were exposed to different manipulations in a laboratory experiment: (i) each of the R-BT065 strains were always separately cocultured with a fast-growing competitor of the genus Flectobacillus (strain MWH38; see reference 12), (ii) the cocultures were amended by additions of different amounts of a live or heat-killed virus concentrate collected from their home environment, and (iii) in parallel, these cocultures received an axenic bacterivore, Poterioochromonas sp., that was able to feed on all three bacterial strains involved in the study.     

The double-blind sham-controlled study of high-frequency rTMS (20 Hz) for negative symptoms in schizophrenia: negative results

The high-frequency repetitive transcranial magnetic stimulation (HF-rTMS) over the prefrontal cortex is a promising method for the treatment of negative symptoms of schizophrenia. Using double-blind sham-controlled parallel design, we evaluated the effect of HF-rTMS over the left dorsolateral prefrontal cortex (DLPFC) on negative symptoms in patients with schizophrenia. Sixteen schizophrenia patients with predominantly negative symptoms on stable antipsychotic medication were treated with 20 Hz rTMS (90% of motor threshold, 2000 stimuli per session) over ten days within 2 weeks with six weeks follow-up. The effect was assessed using PANSS, CGI, MADRS and neuropsychological tests. We failed to find any significant effect of active rTMS. Sham rTMS showed a trend for improvement over time on positive and negative subscales of PANSS and MADRS. Between-group comparisons failed to reveal any significant differences on any rating scales except a positive subscale of PANSS after 8 weeks. Results from our study did not confirm that HF-rTMS over the left DLPCF affects the negative symptoms of schizophrenia and alternative rTMS approaches are discussed.