New and fast method to quantify respiration rates of bacterial and plankton communities in freshwater ecosystems by using optical oxygen sensor spots

A new method of respiration rate measurement based on oxygen luminescence quenching in sensor spots was evaluated for the first time for aquatic bacterial communities. The commonly used Winkler and Clark electrode methods to quantify oxygen concentration both require long incubation times, and the latter additionally causes signal drift due to oxygen consumption at the cathode. The sensor spots proved to be advantageous over those methods in terms of precise and quick oxygen measurements in natural bacterial communities, guaranteeing a respiration rate estimate during a time interval short enough to neglect variations in organism composition, abundance, and activity. Furthermore, no signal drift occurs during measurements, and respiration rate measurements are reliable even at low temperatures and low oxygen consumption rates. Both a natural bacterioplankton sample and a bacterial isolate from a eutrophic river were evaluated in order to optimize the new method for aquatic microorganisms. A minimum abundance of 2.2 x 10(6) respiring cells ml(-1) of a bacterial isolate was sufficient to obtain a distinct oxygen depletion signal within 20 min at 20 degrees C with the new oxygen sensor spot method. Thus, a culture of a bacterial isolate from a eutrophic river (OW 144; 20 x 10(6) respiring bacteria ml(-1)) decreased the oxygen saturation about 8% within 20 min. The natural bacterioplankton sample respired 2.8% from initially 94% oxygen-saturated water in 30 min. During the growth season in 2005, the planktonic community of a eutrophic river consumed between 0.7 and 15.6 micromol O(2) liter(-1) h(-1). The contribution of bacterial respiration to the total plankton community oxygen consumption varied seasonally between 11 and 100%.     

Construction of a thermostable cytochrome P450 chimera derived from self-sufficient mesophilic parents

The P450 monooxygenases CYP102A1 from Bacillus megaterium and CYP102A3 from Bacillus subtilis are fusion flavocytochromes comprising of a P450 heme domain and a FAD/FMN reductase domain. This protein organization is responsible for the extraordinary catalytic activities making both monooxygenases promising enzymes for biocatalysis. CYP102A1 and CYP102A3 are fatty acid hydroxylases that share 65% identity, and their mutants are able to oxidize a wide range of substrates. In an attempt to increase the process stability of CYP102A1, we exchanged the more unstable reductase domain of CYP102A1 with the more stable reductase domain of CYP102A3. Stability of the chimeric fusion protein was determined spectrophotometrically as well as by measuring the hydroxylation activity towards 12-para-nitrophenoxydodecanoic acid (12-pNCA) after incubation at elevated temperatures. In the reaction with 12-pNCA, the new chimeric protein exhibited 88 and 38% of the activity of CYP102A3 and CYP102A1, respectively, but was able to hydroxylate substrates within a wider temperature range compared with the parental enzymes. Maximum activity was obtained at 51°C, and the half-life at 50°C was with 100 min more than ten times longer than that of CYP102A1 (8 min).     

Formation of cyclotides and variations in cyclotide expression in <Emphasis Type="Italic">Oldenlandia affinis</Emphasis> suspension cultures

Cyclotides, a family of disulfide-rich mini-proteins, show a wide range of biological activities, making them interesting targets for pharmaceutical and agrochemical applications, but little is known about their natural function and the events that trigger their expression. An investigation of nutritional variations and irradiation during a batch process involving plant cell cultures has been performed, using the native African medical herb, Oldenlandia affinis, as a model plant. The results demonstrated the biosynthesis of kalata B1, the main cyclotide in O. affinis, in a combined growth/nongrowth-associated pattern. The highest concentration, 0.37 mg g⁻¹ dry weight, was accumulated in irradiated cells at 35 μmol m⁻² s⁻¹. Furthermore, 12 novel cyclotides were identified and the expression of various cyclotides compared in irradiated vs non-irradiated cultures. The results indicate that cyclotide expression varies greatly depending on physiological conditions and environmental stress. Kalata B1 is the most abundant cyclotide in plant suspension cultures, which underlies its importance as a natural defense molecule. The identification of novel cyclotides in suspension cultures, compared to whole plants, indicates that there may be more novel cyclotides to be discovered and that the genetic network regulating cyclotide expression is a very sensitive system, ready to adapt to the current environmental growth condition.     

The heat shock protein HSP70 promotes mouse NK cell activity against tumors that express inducible NKG2D ligands

The stress-inducible heat shock protein (HSP) 70 is known to function as an endogenous danger signal that can increase the immunogenicity of tumors and induce CTL responses. We show in this study that HSP70 also activates mouse NK cells that recognize stress-inducible NKG2D ligands on tumor cells. Tumor size and the rate of metastases derived from HSP70-overexpressing human melanoma cells were found to be reduced in T and B cell-deficient SCID mice, but not in SCID/beige mice that lack additionally functional NK cells. In the SCID mice with HSP70-overexpressing tumors, NK cells were activated so that they killed ex vivo tumor cells that expressed NKG2D ligands. In the tumors, the MHC class I chain-related (MIC) A and B molecules were found to be expressed. Interestingly, a counter selection was observed against the expression of MICA/B in HSP70-overexpressing tumors compared with control tumors in SCID, but not in SCID/beige mice, suggesting a functional relevance of MICA/B expression. The melanoma cells were found to release exosomes. HSP70-positive exosomes from the HSP70-overexpressing cells, in contrast to HSP70-negative exosomes from the control cells, were able to activate mouse NK cells in vitro to kill YAC-1 cells, which express NKG2D ligands constitutively, or the human melanoma cells, in which MICA/B expression was induced. Thus, HSP70 and inducible NKG2D ligands synergistically promote the activation of mouse NK cells resulting in a reduced tumor growth and suppression of metastatic disease.     

Intimate cell conjugate formation and exchange of membrane lipids precede apoptosis induction in target cells during antibody-dependent, granulocyte-mediated cytotoxicity

Ab-dependent polymorphonuclear granulocyte (PMN)-mediated cytotoxicity may play an important role in the control of malignant diseases. However, little is known as to which particular pathways are used for the killing of malignant cells by PMN. The production of reactive oxygen intermediates (ROI) has been observed to occur during Ab-dependent, cell-mediated cytotoxicity (ADCC). However, PMN from a patient with chronic granulomatous disease demonstrated strong ADCC against malignant lymphoma cells. Furthermore, the inhibition of ROI production in PMN from healthy donors had no significant effect on ADCC. Therefore, ROI production by the NADPH oxidase of PMN does not appear to be mandatory for PMN-mediated ADCC. Recent data suggest a role for perforins in PMN-mediated cytotoxicity. However, in our assays concanamycin A, an inhibitor of perforin-mediated ADCC by mononuclear cells, had no inhibitory effect on PMN-mediated ADCC. Using electron microscopy we observed that PMN and their target cells intimately interact with the formation of interdigitating membrane protrusions. During PMN and target cell contact there was a mutual exchange of fluorescent membrane lipid dyes that was strongly increased in the presence of tumor-targeting Abs. This observation may be closely related to the recently described process of trogocytosis by lymphocytes. The presence of transient PMN-tumor cell aggregates and the accumulation of PMN with tumor cell-derived membrane lipids and vice versa were associated with effective ADCC as measured by chromium-release or apoptosis induction.     

v-SNARE cellubrevin is required for basolateral sorting of AP-1B-dependent cargo in polarized epithelial cells

The epithelial cell-specific adaptor complex AP-1B is crucial for correct delivery of many transmembrane proteins from recycling endosomes to the basolateral plasma membrane. Subsequently, membrane fusion is dependent on the formation of complexes between SNARE proteins located at the target membrane and on transport vesicles. Although the t-SNARE syntaxin 4 has been localized to the basolateral membrane, the v-SNARE operative in the AP-1B pathway remained unknown. We show that the ubiquitously expressed v-SNARE cellubrevin localizes to the basolateral membrane and to recycling endosomes, where it colocalizes with AP-1B. Furthermore, we demonstrate that cellubrevin coimmunoprecipitates preferentially with syntaxin 4, implicating this v-SNARE in basolateral fusion events. Cleavage of cellubrevin with tetanus neurotoxin (TeNT) results in scattering of AP-1B localization and missorting of AP-1B-dependent cargos, such as transferrin receptor and a truncated low-density lipoprotein receptor, LDLR-CT27. These data suggest that cellubrevin and AP-1B cooperate in basolateral membrane trafficking.     

BimS-induced apoptosis requires mitochondrial localization but not interaction with anti-apoptotic Bcl-2 proteins

Release of apoptogenic proteins such as cytochrome c from mitochondria is regulated by pro- and anti-apoptotic Bcl-2 family proteins, with pro-apoptotic BH3-only proteins activating Bax and Bak. Current models assume that apoptosis induction occurs via the binding and inactivation of anti-apoptotic Bcl-2 proteins by BH3-only proteins or by direct binding to Bax. Here, we analyze apoptosis induction by the BH3-only protein Bim(S). Regulated expression of Bim(S) in epithelial cells was followed by its rapid mitochondrial translocation and mitochondrial membrane insertion in the absence of detectable binding to anti-apoptotic Bcl-2 proteins. This caused mitochondrial recruitment and activation of Bax and apoptosis. Mutational analysis of Bim(S) showed that mitochondrial targeting, but not binding to Bcl-2 or Mcl-1, was required for apoptosis induction. In yeast, Bim(S) enhanced the killing activity of Bax in the absence of anti-apoptotic Bcl-2 proteins. Thus, cell death induction by a BH3-only protein can occur through a process that is independent of anti-apoptotic Bcl-2 proteins but requires mitochondrial targeting.     

Redundant role of DEAD box proteins p68 (Ddx5) and p72/p82 (Ddx17) in ribosome biogenesis and cell proliferation

The DEAD box proteins encoded by the genes ddx5 (p68) and ddx17 (isoforms p72 and p82) are more closely related to each other than to any other member of their family. We found that p68 negatively controls p72/p82 gene expression but not vice versa. Knocking down of either gene does not affect cell proliferation, in case of p68 suppression, however, only on condition that p72/p82 overexpression was granted. In contrast, co-silencing of both genes causes perturbation of nucleolar structure and cell death. In mutant studies, the apparently redundant role(s) of p68 and p72/p82 correspond to their ability to catalyze RNA rearrangement rather than RNA unwinding reactions. In search for possible physiological targets of this RNA rearrangement activity it is shown that the nucleolytic cleavage of 32S pre-rRNA is reduced after p68 subfamily knock-down, most probably due to a failure in the structural rearrangement process within the pre-60S ribosomal subunit preceding the processing of 32S pre-rRNA.