Ultrastructure of Acaryochloris marina, an oxyphotobacterium containing mainly chlorophyll d

We present a detailed investigation of the ultrastructure of the chlorophyll a/d-containing unicellular oxyphotobacterium Acaryochloris marina, combining light and transmission electron microscopy and showing freeze fractures of this organism for the first time. The cells were 1.8-2.1 microm x 1.5-1.7 microm in size. The cell envelope consisted of a peptidoglycan layer of approximately 10 nm thickness combined with an outer membrane. Cell division was intermediate between the constrictive and the septum type. The nucleoplasm, which contained several carboxysomes, was surrounded by 7-11 concentrically arranged thylakoids, which were predominantly stacked, with the exception of distinct areas where phycobiliproteins were located. The thylakoids were perforated by channel-like structures connecting the central and peripheral portions of the cytoplasm and not yet observed in other organisms. In freeze fractures, the protoplasmic fracture faces of thylakoid membranes were densely covered with particles of inhomogenous size. The particle size histogram peaked at 10-11, 13 and 18 nm. The 18-nm particles are assumed to represent photosystem I trimers. The particles on exoplasmic fracture faces, proposed to represent photosystem II complexes, were significantly larger than the corresponding particles of cyanobacteria and clustered to form large aggregates. This kind of arrangement is unique among photosynthetic organisms.     

Expression and purification of dynamin II domains and initial studies on structure and function

Dynamin II, a large GTP-binding protein, is involved in endocytosis and in vesicle formation at the trans-Golgi network. To further elucidate functions of dynamin II, the pleckstrin homology domain (PHD), the proline-rich domain (PRD), and the C-terminal part of dynamin II (dynamin(500-870)) were expressed in Escherichia coli. The PHD, tagged C-terminally by a (His)(6) peptide, was expressed to 15% of cellular proteins and could be purified on nickel-chelating agarose. On the contrary, the PRD and dynamin(500-870) had to be tagged with a (His)(6) peptide at the N-terminus to bind to nickel-chelating agarose. Additional tagging with the S-peptide, which forms a stable complex with immobilized S-protein, allowed removal of strongly interacting E. coli proteins. Circular dichroic spectra indicate a structured recombinant PHD with a secondary structure content similar to that of the known PHD from dynamin I. The N-terminally tagged, recombinant PRD is unfolded but nevertheless binds specifically to the SH3 domain of amphiphysin II as well as to proteins extracted from rat brain. The described methods are suitable to isolate functionally active domains of dynamin II in sufficient amount and purity for further studies.     

Classification and diagnostic prediction of cancers using gene expression profiling and artificial neural networks

The purpose of this study was to develop a method of classifying cancers to specific diagnostic categories based on their gene expression signatures using artificial neural networks (ANNs). We trained the ANNs using the small, round blue-cell tumors (SRBCTs) as a model. These cancers belong to four distinct diagnostic categories and often present diagnostic dilemmas in clinical practice. The ANNs correctly classified all samples and identified the genes most relevant to the classification. Expression of several of these genes has been reported in SRBCTs, but most have not been associated with these cancers. To test the ability of the trained ANN models to recognize SRBCTs, we analyzed additional blinded samples that were not previously used for the training procedure, and correctly classified them in all cases. This study demonstrates the potential applications of these methods for tumor diagnosis and the identification of candidate targets for therapy.     

Shell growth and chamber formation of aquarium-reared Nautilus pompilius (Mollusca, Cephalopoda) by X-ray analysis

Observations on the growth rate of aquarium maintained Nautilus pompilius in different developmental stages, i.e. juveniles (shell length about 8.75 cm), late juveniles (approximately 10 cm), and early adolescent (approximately 13.5 cm), indicate that this species is fully grown at an age of 7.3-8 years. The age calculations are based on two different computations: (1) the measurement of the increase of the shell length per day and (2) the formation of new septa in time intervals of 150+/-5 days, as demonstrated by X-ray analyses. After N. pompilius hatches, its shell grows about 139 mm to reach full growth and approximately 28 septa are formed. With an increase of the shell length of 0.052 mm per day, it takes about 2,673 days (7.3 years) to reach maturity. Provided that the process of chamber formation follows an exponential function, these computations result in approximately 2,925 days (8 years) to reach full maturity. Supposing that N. pompilius may live for several years after onset of maturity like Nautilus belauensis, the total life span for this species may exceed 11-12 years.     

Granulocyte/macrophage-colony-stimulating-factor plus interleukin-2 plus interferon α in the treatment of metastatic renal cell carcinoma: a pilot study

 Granulocyte/macrophage-colony-stimulating factor (GM-CSF) plays a central role in the differentiation and function of dendritic cells, which are crucial for the elicitation of MHC-restricted T cell responses. Preclinical and the first clinical data provide a rationale for the application of GM-CSF in immunotherapy of cancer. Ten patients with renal cell carcinoma stage IV (Holland/Robson) were treated in this pilot study. Therapy was started with GM-CSF alone (2 weeks). Interleukin (IL-2) and interferon α (IFNα) were added sequentially (3 weeks GM-CSF plus IL-2 or IFNα, 3 weeks GM-CSF plus IL-2 plus IFNα). Therapy was performed on an outpatient basis. The cytokine regimen was evaluated for toxicity, clinical response and immunomodulatory effects [fluorescence-activated cell sorting analysis of peripheral blood mononuclear cells (PBMC), mixed-lymphocyte reaction and cytotoxicity of PBMC]. GM-CSF treatment caused a significant increase in the number of PBMC expressing costimulatory molecules. Addition of IL-2 and IFNα led to an increase in CD3⁺, CD4⁺, CD8⁺ and CD56⁺ PBMC in week 9. In an autologous mixed-lymphocyte reaction a 2.1-fold increase in T cell proliferation was observed after 2 weeks of GM-CSF treatment, and cytotoxicity assays showed changes in natural-killer- (NK)- and non-NK-mediated cytotoxicity in some patients. Two patients achieved partial remission, one patient had a mixed response. The toxicity of the regimen was mild to moderate with fever, flu-like symptoms and nausea being observed in most patients. Severe organ toxicity was not observed. We conclude that GM-CSF might be useful for immunotherapy of renal cell carcinoma, especially in combination with T-cell-active cytokines. Further studies are warranted. Received: 16 March 2000 / Accepted: 10 August 2000     

Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, reduction of NAD(+) to NADH occurs in dissimilatory as well as in assimilatory reactions. This review discusses mechanisms for reoxidation of NADH in this yeast, with special emphasis on the metabolic compartmentation that occurs as a consequence of the impermeability of the mitochondrial inner membrane for NADH and NAD(+). At least five mechanisms of NADH reoxidation exist in S. cerevisiae. These are: (1) alcoholic fermentation; (2) glycerol production; (3) respiration of cytosolic NADH via external mitochondrial NADH dehydrogenases; (4) respiration of cytosolic NADH via the glycerol-3-phosphate shuttle; and (5) oxidation of intramitochondrial NADH via a mitochondrial 'internal' NADH dehydrogenase. Furthermore, in vivo evidence indicates that NADH redox equivalents can be shuttled across the mitochondrial inner membrane by an ethanol-acetaldehyde shuttle. Several other redox-shuttle mechanisms might occur in S. cerevisiae, including a malate-oxaloacetate shuttle, a malate-aspartate shuttle and a malate-pyruvate shuttle. Although key enzymes and transporters for these shuttles are present, there is as yet no consistent evidence for their in vivo activity. Activity of several other shuttles, including the malate-citrate and fatty acid shuttles, can be ruled out based on the absence of key enzymes or transporters. Quantitative physiological analysis of defined mutants has been important in identifying several parallel pathways for reoxidation of cytosolic and intramitochondrial NADH. The major challenge that lies ahead is to elucidate the physiological function of parallel pathways for NADH oxidation in wild-type cells, both under steady-state and transient-state conditions. This requires the development of techniques for accurate measurement of intracellular metabolite concentrations in separate metabolic compartments.     

A Phytase-Based Reporter System for Identification of Functional Secretion Signals in Bifidobacteria

Health-promoting effects have been attributed to a number of Bifidobacterium sp. strains. These effects as well as the ability to colonise the host depend on secreted proteins. Moreover, rational design of protein secretion systems bears the potential for the generation of novel probiotic bifidobacteria with improved health-promoting or therapeutic properties. To date, there is only very limited data on secretion signals of bifidobacteria available. Using in silico analysis, we demonstrate that all bifidobacteria encode the major components of Sec-dependent secretion machineries but only B. longum strains harbour Tat protein translocation systems. A reporter plasmid for secretion signals in bifidobacteria was established by fusing the coding sequence of the signal peptide of a sialidase of Bifidobacterium bifidum S17 to the phytase gene appA of E. coli. The recombinant strain showed increased phytase activity in spent culture supernatants and reduced phytase levels in crude extracts compared to the control indicating efficient phytase secretion. The reporter plasmid was used to screen seven predicted signal peptides in B. bifidum S17 and B. longum E18. The tested signal peptides differed substantially in their efficacy to mediate protein secretion in different host strains. An efficient signal peptide was used for expression and secretion of a therapeutically relevant protein in B. bifidum S17. Expression of a secreted cytosine deaminase led to a 100-fold reduced sensitivity of B. bifidum S17 to 5-fluorocytosine compared to the non-secreted cytosine deaminase suggesting efficient conversion of 5-fluorocytosine to the cytotoxic cancer drug 5-fluorouracil by cytosine deaminase occurred outside the bacterial cell. Selection of appropriate signal peptides for defined protein secretion might improve therapeutic efficacy as well as probiotic properties of bifidobacteria.     

The Association between Mycobacterium Tuberculosis Genotype and Drug Resistance in Peru

BackgroundThe comparison of Mycobacterium tuberculosis bacterial genotypes with phenotypic, demographic, geospatial and clinical data improves our understanding of how strain lineage influences the development of drug-resistance and the spread of tuberculosis.MethodsTo investigate the association of Mycobacterium tuberculosis bacterial genotype with drug-resistance. Drug susceptibility testing together with genotyping using both 15-loci MIRU-typing and spoligotyping, was performed on 2,139 culture positive isolates, each from a different patient in Lima, Peru. Demographic, geospatial and socio-economic data were collected using questionnaires, global positioning equipment and the latest national census.ResultsThe Latin American Mediterranean (LAM) clade (OR 2.4, p<0.001) was significantly associated with drug-resistance and alone accounted for more than half of all drug resistance in the region. Previously treated patients, prisoners and genetically clustered cases were also significantly associated with drug-resistance (OR''s 2.5, 2.4 and 1.8, p<0.001, p<0.05, p<0.001 respectively).ConclusionsTuberculosis disease caused by the LAM clade was more likely to be drug resistant independent of important clinical, genetic and socio-economic confounding factors. Explanations for this include; the preferential co-evolution of LAM strains in a Latin American population, a LAM strain bacterial genetic background that favors drug-resistance or the "founder effect" from pre-existing LAM strains disproportionately exposed to drugs.     

Virulence determinants of the human pathogenic fungus Aspergillus fumigatus protect against soil amoeba predation

Filamentous fungi represent classical examples for environmentally acquired human pathogens whose major virulence mechanisms are likely to have emerged long before the appearance of innate immune systems. In natural habitats, amoeba predation could impose a major selection pressure towards the acquisition of virulence attributes. To test this hypothesis, we exploited the amoeba Dictyostelium discoideum to study its interaction with Aspergillus fumigatus, two abundant soil inhabitants for which we found co‐occurrence in various sites. Fungal conidia were efficiently taken up by D. discoideum, but ingestion was higher when conidia were devoid of the green fungal spore pigment dihydroxynaphtalene melanin, in line with earlier results obtained for immune cells. Conidia were able to survive phagocytic processing, and intracellular germination was initiated only after several hours of co‐incubation which eventually led to a lethal disruption of the host cell. Besides phagocytic interactions, both amoeba and fungus secreted cross inhibitory factors which suppressed fungal growth or induced amoeba aggregation with subsequent cell lysis, respectively. On the fungal side, we identified gliotoxin as the major fungal factor killing Dictyostelium, supporting the idea that major virulence attributes, such as escape from phagocytosis and the secretion of mycotoxins are beneficial to escape from environmental predators.