In vivo and in vitro liver cancer metabolism observed with hyperpolarized [5-(13)C]glutamine.

Glutamine metabolism is, with its many links to oncogene expression, considered a crucial step in cancer metabolism and it is thereby a key target for alteration in cancer development. In particular, strong correlations have been reported between oncogene expression and expression and activity of the enzyme glutaminase. This mitochondrial enzyme, which is responsible for the deamidation of glutamine to form glutamate, is overexpressed in many tumour tissues. In animal models, glutaminase expression is correlated with tumour growth rate and it is readily possible to limit tumour growth by suppression of glutaminase activity. In principle, hyperpolarized (13)C MR spectroscopy can provide insight to glutamine metabolism and should hence be a valuable tool to study changes in glutaminase activity as tumours progress. However, no such successful in vivo studies have been reported, even though several good biological models have been tested. This may, at least partly, be due to problems in preparing glutamine for hyperpolarization. This paper reports a new and improved preparation of hyperpolarized [5-(13)C]glutamine, which provides a highly sensitive (13)C MR marker. With this preparation of hyperpolarized [5-(13)C]glutamine, glutaminase activity in vivo in a rat liver tumour was investigated. Moreover, this marker was also used to measure response to drug treatment in vitro in cancer cells. These examples of [5-(13)C]glutamine used in tumour models warrant the new preparation to allow metabolic studies with this conditionally essential amino acid.

Marizomib, a proteasome inhibitor for all seasons: preclinical profile and a framework for clinical trials.

The proteasome has emerged as an important clinically relevant target for the treatment of hematologic malignancies. Since the Food and Drug Administration approved the first-in-class proteasome inhibitor bortezomib (Velcade) for the treatment of relapsed/refractory multiple myeloma (MM) and mantle cell lymphoma, it has become clear that new inhibitors are needed that have a better therapeutic ratio, can overcome inherent and acquired bortezomib resistance and exhibit broader anti-cancer activities. Marizomib (NPI-0052; salinosporamide A) is a structurally and pharmacologically unique ß-lactone-γ-lactam proteasome inhibitor that may fulfill these unmet needs. The potent and sustained inhibition of all three proteolytic activities of the proteasome by marizomib has inspired extensive preclinical evaluation in a variety of hematologic and solid tumor models, where it is efficacious as a single agent and in combination with biologics, chemotherapeutics and targeted therapeutic agents. Specifically, marizomib has been evaluated in models for multiple myeloma, mantle cell lymphoma, Waldenstrom's macroglobulinemia, chronic and acute lymphocytic leukemia, as well as glioma, colorectal and pancreatic cancer models, and has exhibited synergistic activities in tumor models in combination with bortezomib, the immunomodulatory agent lenalidomide (Revlimid), and various histone deacetylase inhibitors. These and other studies provided the framework for ongoing clinical trials in patients with MM, lymphomas, leukemias and solid tumors, including those who have failed bortezomib treatment, as well as in patients with diagnoses where other proteasome inhibitors have not demonstrated significant efficacy. This review captures the remarkable translational studies and contributions from many collaborators that have advanced marizomib from seabed to bench to bedside.

Pestalone, a new antibiotic produced by a marine fungus in response to bacterial challenge.

The isolation and structure determination of a new chlorinated benzophenone antibiotic, pestalone (1), is described. The new compound was produced by a cultured marine fungus only when a unicellular marine bacterium, strain CNJ-328, was co-cultured in the fungal fermentation. The fungus, isolated from the surface of the brown alga Rosenvingea sp. collected in the Bahamas Islands, was identified as an undescribed member of the genus Pestalotia. The structure of 1, initially assigned with only modest confidence by combined spectral and chemical data, was confirmed by single-crystal X-ray analysis. Pestalone (1) exhibits moderate in vitro cytotoxicity in the National Cancer Institute's 60 human tumor cell line screen (mean GI(50) = 6.0 microM). More importantly, pestalone shows potent antibiotic activity against methicillin-resistant Staphylococcus aureus (MIC = 37 ng/mL) and vancomycin-resistant Enterococcus faecium (MIC = 78 ng/mL), indicating that pestalone should be evaluated in advanced models of infectious disease.

Hemin reconstitutes proton extrusion in an H(+)-ATPase-negative mutant of Lactococcus lactis.

H(+)-ATPase is considered essential for growth of Lactococcus lactis. However, media containing hemin restored the aerobic growth of an H(+)-ATPase-negative mutant, suggesting that hemin complements proton extrusion. We show that inverted membrane vesicles prepared from hemin-grown L. lactis cells are capable of coupling NADH oxidation to proton translocation.

The membrane-bound H(+)-ATPase complex is essential for growth of Lactococcus lactis.

The eight genes which encode the (F(1)F(o)) H(+)-ATPase in Lactococcus lactis subsp. cremoris MG1363 were cloned and sequenced. The genes were organized in an operon with the gene order atpEBFHAGDC; i.e., the order of atpE and atpB is reversed with respect to the more typical bacterial organization. The deduced amino acid sequences of the corresponding H(+)-ATPase subunits showed significant homology with the subunits from other organisms. Results of Northern blot analysis showed a transcript at approximately 7 kb, which corresponds to the size of the atp operon. The transcription initiation site was mapped by primer extension and coincided with a standard promoter sequence. In order to analyze the importance of the H(+)-ATPase for L. lactis physiology, a mutant strain was constructed in which the original atp promoter on the chromosome was replaced with an inducible nisin promoter. When grown on GM17 plates the resulting strain was completely dependent on the presence of nisin for growth. These data demonstrate that the H(+)-ATPase is essential for growth of L. lactis under these conditions.

Mangicols: structures and biosynthesis of A new class of sesterterpene polyols from a marine fungus of the genus Fusarium.

A marine fungal isolate, tentatively identified as Fusarium heterosporum, has been found to produce a series of structurally novel sesterterpene polyols, the mangicols A-G (4-10). The structures of the new compounds, including the stereochemistry of mangicol A, were assigned by interpretation of spectral data derived from both natural products and synthetic derivatives. The mangicols, which possess unprecedented spirotricyclic skeletal components, show only weak to modest cytotoxicities toward a variety of cancer cell lines in in vitro testing. Mangicols A and B, however, showed significant antiinflammatory activity in the PMA (phorbol myristate acetate)-induced mouse ear edema model. A biosynthetic pathway for the neomangicol and mangicol carbon skeletons is proposed on the basis of the incorporation of appropriate radiolabeled precursors.

The frequency of mutators in populations of Escherichia coli.

Owing to occasional spontaneous mutations in genes encoding DNA repair, any population of a reasonable size is expected to harbor a sub-population of genetic mutators. Using a genetically modified strain of Escherichia coli K-12, we have estimated the frequency of mutators to be about 3x10(-5). By and large, this corresponds to a mutation rate from non-mutators to mutators of 5x10(-6) per bacterium per generation. Using a mutS∷Tn10 derivative as representative for mutators, we estimated the increase in mutation rates in mutators to be 19- to 82-fold, depending on the test-mutation under consideration. The load associated with this increase in mutation rate resulted in a growth inhibition of 1%. From these data, we estimated that the rate of detrimental mutations in the non-mutators to be 2x10(-4)-8x10(-4). The situations where adaptive mutations may result in an increase in the frequency of mutators are discussed.

N-Methylsansalvamide, a cytotoxic cyclic depsipeptide from a marine fungus of the genus fusarium.

N-Methylsansalvamide (1), a new cyclic depsipeptide, was isolated from extracts of a cultured marine fungus, strain CNL-619, identified as a member of the genus Fusarium. N-Methylsansalvamide exhibits weak in vitro cytotoxicity in the NCI human tumor cell line screen (GI50 8.3 microM). The structure of 1 was determined by combined spectral and chemical methods.

Extensive regulation compromises the extent to which DNA gyrase controls DNA supercoiling and growth rate of Escherichia coli.

As DNA gyrase is the only enzyme to supercoil DNA actively, we address here the question of whether it does play the expected dominant role in controlling the level of DNA supercoiling and growth rate in Escherichia coli. We modulated the expression of DNA gyrase around its wild-type level, and measured the effect on plasmid supercoiling and growth rate. As both the activity and the transcription rate of DNA gyrase are sensitive to DNA supercoiling we distinguish two types of control (with control defined as the percentage change observed on a 1% modulation of a parameter). The first type of control, here named inherent control, quantifies the effect of a sustained modulation of the transcription rate of gyrase. At its wild-type expression level this inherent control exerted by DNA gyrase on growth rate was very low, i.e. c mu/gyrase = 0.05 - 0.00, as was the inherent control on DNA supercoiling, c aLK/gyrase = 0.2. The second type of control, here named global control, quantifies the effect of a change in gyrase activity whilst allowing the cell to respond by readjusting gyrase transcription. Both types of control are linked via the sensitivity of gyrase transcription to DNA supercoiling, as determined from the inherent control by gyrase of the gyrase promoter activity using a chromosomal gyrB:lacZ fusion. As expected, the latter control was negative, but small, i.e. c gyr promoter/gyrase=-0.3. The global control by gyrase of active linking number was 0.1. These results show that although gyrase is an essential enzyme it does not have a high control, on either growth rate or DNA supercoiling. Homeostatic regulation of physiological DNA structure appears to dominate. At low degrees of DNA supercoiling, the control by DNA gyrase and by the other topoisomerases is much stronger.

atp Mutants of Escherichia coli fail to grow on succinate due to a transport deficiency.

Escherichia coli atp mutants, which lack a functional H+-ATPase complex, are capable of growth on glucose but not on succinate or other C4-dicarboxylates (Suc- phenotype). Suc+ revertants of an atp deletion strain were isolated which were capable of growth on succinate even though they lack the entire H+-ATPase complex. Complementation in trans with the yhiF gene suppressed the growth of the Suc+ mutants on succinate, which implicates the yhiF gene product in the regulation of C4-dicarboxylate metabolism. Indeed, when the E. coli C4-dicarboxylate transporter (encoded by the dctA gene) was expressed in trans, the Suc- phenotype of the atp deletion strain reverted to Suc+, which shows that the reason why the E. coli atp mutant is unable to grow aerobically on C4-dicarboxylates is insufficient transport capacity for these substrates.

Eleutherobin, a novel cytotoxic agent that induces tubulin polymerization, is similar to paclitaxel (Taxol).

Eleutherobin is a novel natural product isolated from a marine soft coral that is extremely potent for inducing tubulin polymerization in vitro and is cytotoxic for cancer cells with an IC50 similar to that of paclitaxel. This compound is cross-resistant along with other multidrug-resistant agents against P-glycoprotein-expressing cells and is cross-resistant with paclitaxel against a cell line that has altered tubulin. In mechanistic studies, eleutherobin shares with paclitaxel the ability to induce tubulin polymerization in vitro and is most likely cytotoxic by virtue of this mechanism. Human colon carcinoma cells exposed to eleutherobin contain multiple micronuclei and microtubule bundles, and they arrest in mitosis, depending on concentration, cell line, and length of exposure. These morphological abnormalities appearing in cultured cells are indistinguishable from those induced by paclitaxel. Electron microscopy reveals that eleutherobin induces homogeneous populations of long, rigid microtubules similar to those formed by paclitaxel. Thus, eleutherobin is a new chemotype with a mechanism of action similar to that of paclitaxel and, as such, has promising potential as a new anticancer agent.

DNA supercoiling depends on the phosphorylation potential in Escherichia coli.

ATP/ADP ratios were varied in different ways and the degree of negative supercoiling was determined in Escherichia coli. Independent of whether the ATP/ ADP ratio was reduced by a shift to anaerobic conditions, by addition of a protonophore (dinitrophenol) or by potassium cyanide addition, DNA supercoiling decreased similarly with the ATP/ADP ratio. The experiments were performed under well-defined conditions, where oxidative phosphorylation was the dominant route for ATP synthesis, i.e. using a minimal salts medium with succinate as the sole free-energy and carbon source, and in the presence or absence of ammonia as the nitrogen source. The results of the different experiments were consistent with a single linear relationship between the log(ATP/ADP) and the change in linking number. The dependence of DNA supercoiling on the ATP/ADP ratio was not influenced by inhibitors of transcription or translation. Because the ATP/ADP ratio was modulated in different ways, the unique relationship suggests coupling between the phosphorylation potential and DNA supercoiling. This was most probably mediated by the DNA gyrase, independent of topoisomerase I or transcription.

Changes in the cellular energy state affect the activity of the bacterial phosphotransferase system.

The effect of different cellular free-energy states on the uptake of methyl alpha-D-glucopyranoside, an analogue of glucose, by the Escherichia coli phosphoenolpyruvate:carbohydrate phosphotransferase system was investigated. The intracellular [ATP]/[ADP] ratio was varied by changing the expression of the atp operon, which codes for the H+-ATPase, or by adding an uncoupler of oxidative phosphorylation or an inhibitor of respiration. Corresponding initial phosphotransferase uptake rates were determined using an improved uptake assay that works with growing cells in steady state. The results show that the initial uptake rate was decreased under conditions of lowered intracellular [ATP]/[ADP] ratios, irrespective of which method was used to change the cellular energy state. When either the expression of the atp operon was changed or 2,4-dinitrophenol was added to wild-type cells, the relationship between initial phosphotransferase uptake rate and the logarithm of the [ATP]/[ADP] ratio was approximately linear. These results suggest that the cellular free-energy state, as reflected in the intracellular [ATPI]/[ADP] ratio, plays an important role in regulating the activity of the phosphotransferase system.

Experimental determination of control by the H(+)-ATPase in Escherichia coli.

Strains carrying deletions in the atp genes, encoding the H(+)-ATPase, were unable to grow on nonfermentable substrates such as succinate, whereas with glucose as the substrate the growth rate of an atp deletion mutant was surprisingly high (some 75-80% of wild-type growth rate). The rate of glucose and oxygen consumption of these mutants was increased compared to the wild-type rates. In order to analyze the importance of the H(+)-ATPase at its physiological level, the cellular concentration of H(+)-ATPase was modulated around the wild-type level, using genetically manipulated strains. The control coefficient by the H(+)-ATPase with respect to growth rate and catabolic fluxes was measured. Control on growth rate was absent at the wild-type concentration of H(+)-ATPase, independent of whether the substrate for growth was glucose or succinate. Control by the H(+)-ATPase on the catabolic fluxes, including respiration, was negative at the wild-type H(+)-ATPase level. Moreover, the turnover number of the individual H(+)-ATPase enzymes increased as the H(+)-ATPase concentration was lowered. The negative control by the H(+)-ATPase on catabolism may thus be involved in a homeostatic control of ATP synthesis and, to some extent, explain the zero control by the H(+)-ATPase on E. coli growth rate.

Energy, control and DNA structure in the living cell.

Maintenance (let alone growth) of the highly ordered living cell is only possible through the continuous input of free energy. Coupling of energetically downhill processes (such as catabolic reactions) to uphill processes is essential to provide this free energy and is catalyzed by enzymes either directly or via "storage" in an intermediate high energy form, i.e., high ATP/ADP ratio or H+ ion gradient. Although maintenance of a sufficiently high ATP/ADP ratio is essential to overcome the thermodynamic burden of uphill processes, it is not clear to what degree enzymes that control this ratio also control cell physiology. Indeed, in the living cell homeostatic control mechanisms might exist for the free-energy transduction pathways so as to prevent perturbation of cellular function when the Gibbs energy supply is compromised. This presentation addresses the extent to which the intracellular ATP level is involved in the control of cell physiology, how the elaborate control of cell function may be analyzed theoretically and quantitatively, and if this can be utilized selectively to affect certain cell types.

Energy buffering of DNA structure fails when Escherichia coli runs out of substrate.

To study how changes in the [ATP]/[ADP] ratio affect the level of DNA supercoiling in Escherichia coli, the cellular content of H(+)-ATPase was modulated around the wild-type level. A relatively large drop in the [ATP]/[ADP] ratio from the normal ratio resulted in a small increase in the linking number of our reporter plasmid (corresponding to a small decrease in negative supercoiling). However, when cells depleted their carbon and energy source, the ensuing drop in energy state was accompanied by a strong increase in linking number. This increase was not due to reduced transcription of the DNA in the absence of growth substrate, since rifampin had virtually no effect on the plasmid linking number. To examine whether DNA supercoiling depends more strongly on the cellular energy state at low [ATP]/[ADP] ratios than at high ratios, we used cells that were already at a low energy state after substrate depletion; after the addition of an uncoupler to these cells, the [ATP]/[ADP] ratio decreased further, which resulted in a strong increase in plasmid linking number. Our results suggest that the strong thermodynamic control of DNA supercoiling takes over at low [ATP]/[ADP] ratios, whereas at high ratios homeostatic control mechanisms attenuate thermodynamic control.

[Penile vein surgery. A follow-up study of erectile dysfunction of presumed venous origin]. / Penil venekirurgi. En efterundersøgelse ved formodet venøst betinget erektiv dysfunktion.

Forty patients who underwent penile venous surgery (PVS) because of suspected veno-occlusive dysfunction, were evaluated approximately 15 months (median) postoperatively as to their sexual capability. Although 47.5% were able to obtain penile rigidity, this was sufficient for intercourse in only 35%. Short-term subjective improvement was noted in 55% as opposed to long-term improvement in 35%. Analysis for predictive factors was negative. In conclusion, the indication for PVS should be reconsidered, as an insufficient theoretical concept might explain the low long-term success rate.

Control analysis of the dependence of Escherichia coli physiology on the H(+)-ATPase.

The H(+)-ATPase plays a central role in Escherichia coli free-energy transduction and hence in E. coli physiology. We here investigate the extent to which this enzyme also controls the growth rate, growth yield, and respiratory rate of E. coli. We modulate the expression of the atp operon and determine the effect on said properties. When quantified in terms of control coefficients, we find that, in the wild-type cell growing on glucose in minimal medium, this key enzyme (H(+)-ATPase) exerts virtually no control on growth rate (magnitude of C < 0.01), a minor positive control on growth yield (C = 0.15), and a small but negative control on respiration rate (C = -0.25). The control the enzyme exerts on the consumption rate of the carbon and free-energy substrate is negative (C = -0.15). We also studied how the control coefficients themselves vary with the expression of the atp operon. As the level of expression of the atp operon was reduced, the control exerted by the H(+)-ATPase on growth rate and growth yield increased slightly; the control on growth rate passed through a maximum (C = 0.1) and disappeared when the atp operon was not expressed at all, reflecting that with this substrate there are alternative routes for ATP synthesis. At elevated levels of the H(+)-ATPase compared to the wild type, the control exerted by the enzyme on growth rate became negative. The evolutionary context of the absence of control by the atp operon on growth rate is discussed.

Excess capacity of H(+)-ATPase and inverse respiratory control in Escherichia coli.

With succinate as free-energy source, Escherichia coli generating virtually all ATP by oxidative phosphorylation might be expected heavily to tax its ATP generating capacity. To examine this the H(+)-ATPase (ATP synthase) was modulated over a 30-fold range. Decreasing the amount of H(+)-ATPase reduced the growth rate much less than proportionally; the H(+)-ATPase controlled growth rate by < 10%. This lack of control reflected excess capacity: the rate of ATP synthesis per H(+)-ATPase (the turnover number) increased by 60% when the number of enzymes was decreased by 40%. At 15% H(+)-ATPase, the enzyme became limiting and its turnover was increased even further, due to an increased driving force caused by a reduction in the total flux through the enzymes. At smaller reductions of [H(+)-ATPase] the total flux was not reduced, revealing a second cause for increased turnover number through increased membrane potential: respiration was increased, showing that in E.coli, respiration and ATP synthesis are, in part, inversely coupled. Indeed, growth yield per O2 decreased, suggesting significant leakage or slip at the high respiration rates and membrane potential found at low H(+)-ATPase concentrations, and explaining that growth yield may be increased by activating the H(+)-ATPase.