Comparative immunological characterization of various photosynthetic cytochromes c from pro- and eukaryotic algae

Photosynthetic c-type cytochromes isolated from various pro- and eukaryotic algae have been compared by an immunochemical method. Thereby the extent of cross-reactivity of several cytochromes with antisera to cytochrome c from Spirulina platensis, Bumilleriopsis filiformis, and Scenedesmus acutus was quantitatively determined by antigen-binding tests. When immunological relationship is taken as a measure of structural relationship, the following conclusions can be drawn: (1) c-type cytochromes from Anabaena variabilis, Nostoc muscorum, Calothrix membranacea, and Spirulina platensis show large differences in cross-reactivity. (2) The acidic Spirulina cytochrome c is fairly closely related to the two eukaryotic cytochromes assayed here.Abbreviations SAUG Sammlung von Algenkulturen am Pflanzenphysiologischen Institut der Universität Göttingen, FRG - PCC Pasteur Culture Collection     

Overexpression of GLUT1 in colorectal cancer is independently associated with poor prognosis

Background: To investigate the expression of glucose transporter 1 (GLUT1) in colorectal cancer (CRC) and its relationship to clinicopathological variables. Methods: The expression of GLUT1 in 163 primary tumors together with the corresponding normal mucosa, and 36 liver metastases was examined using real-time PCR. Results: The mean value of GLUT1 was higher in primary tumors (50.390 ± 68.648) than in the corresponding normal mucosa (20.437 ± 28.703, p<0.0001), while there was no significant difference in GLUT1 expression between CRC and liver metastasis (50.390 ± 68.648 vs 52.277 ± 52.482, p=0.190). In CRCs, GLUT1 expression was higher in poorly differentiated than in well and moderately differentiated tumors (p=0.022), and higher in stage III + IV than in stage I + II tumors (p=0.035). The patients with high-expressed GLUT1 had a worse prognosis than those with low-expressed GLUT1 independently of gender, age, tumor site, stage and differentiation (p=0.026, RR 2.737, 95% CI 1.126-6.651) in stage I-III CRCs. In liver metastasis, GLUT1 expression was higher in larger tumors than in smaller ones (p=0.025). Conclusions: Overexpression of GLUT1 in stage I-III CRCs was independently associated with poor prognosis.     

Strontium-Induced Repetitive Calcium Spikes in a Unicellular Green Alga

The divalent cation Sr²⁺ induced repetitive transient spikes of the cytosolic Ca²⁺ activity [Ca²⁺]_cy and parallel repetitive transient hyperpolarizations of the plasma membrane in the unicellular green alga Eremosphaera viridis. [Ca²⁺]_cy measurements, membrane potential measurements, and cation analysis of the cells were used to elucidate the mechanism of Sr²⁺-induced [Ca²⁺]_cy oscillations. Sr²⁺ was effectively and rapidly compartmentalized within the cell, probably into the vacuole. The [Ca²⁺]_cy oscillations cause membrane potential oscillations, and not the reverse. The endoplasmic reticulum (ER) Ca²⁺-ATPase blockers 2,5-di-tert-butylhydroquinone and cyclopiazonic acid inhibited Sr²⁺-induced repetitive [Ca²⁺]_cy spikes, whereas the compartmentalization of Sr²⁺ was not influenced. A repetitive Ca²⁺ release and Ca²⁺ re-uptake by the ER probably generated repetitive [Ca²⁺]_cy spikes in E. viridis in the presence of Sr²⁺. The inhibitory effect of ruthenium red and ryanodine indicated that the Sr²⁺-induced Ca²⁺ release from the ER was mediated by a ryanodine/cyclic ADP-ribose type of Ca²⁺ channel. The blockage of Sr²⁺-induced repetitive [Ca²⁺]_cy spikes by La³⁺ or Gd³⁺ indicated the necessity of a certain influx of divalent cations for sustained [Ca²⁺]_cy oscillations. Based on these data we present a mathematical model that describes the baseline spiking [Ca²⁺]_cy oscillations in E. viridis.     

Soluble and membrane-bound ferrisiderophore reductases of Escherichia coli K-12

After uptake of microbial ferrisiderophores, iron is assumed to be released by reduction. Two ferrisiderophore-reductase activities were identified in Escherichia coli K-12. They differed in cellular location, susceptibility to amytal, and competition between oxygen and ferrichrome-iron(III) reduction. The ferrisiderophore reductase associated with the 40,000×g sediment (membrane-bound enzyme) was inhibited by 10 mM amytal in contrast to the ferrisiderophore reductase present in the 100,000×g supernatant (soluble enzyme). Reduction by the membrane-bound enzyme followed sigmoid kinetics, but was biphasic in the case of the soluble enzyme. The soluble reductase could be assigned to a protein consisting of a single polypeptide of M _r 26000. Reduction of iron(III) by the purified enzyme depended on the addition of NADH or NADPH which were equally active reductants. The cofactor FMN and to a lesser degree FAD stimulated the reaction. Substrate specificity of the soluble reductase was low. In addition to the hydroxamate siderophores arthrobactin, schizokinen, fusigen, aerobactin, ferrichrome, ferrioxamine B, coprogen, and ferrichrome A, the iron(III) complexes of synthetic catecholates, dihydroxy benzoic acid, and dicitrate, as well as carrier-free iron(III) were accepted as substrates. Both ferrisiderophore reductases were not controlled by the fur regulatory system and were not suppressed by anaerobic growth.Abbreviations DHB dihydroxybenzoic acid - MECAM 1,3,5-N,N,N-tris-(2,3-dihydroxybenzoyl)-triamino-methylbenzene - MECAMS 2,3-dihydroxy-5-sulfonyl-derivative of MECAM     

Purification and characterization of the beta-trefoil fold protein barley alpha-amylase/subtilisin inhibitor overexpressed in Escherichia coli

Barley alpha-amylase/subtilisin inhibitor (BASI) is a beta-trefoil fold protein related to soybean trypsin inhibitor (Kunitz) and inhibits barley alpha-amylase isozyme 2 (AMY2), which is de novo synthesized in the seed during germination. Recombinant BASI was produced in Escherichia coli in an untagged form (untagged rBASI), in two His(6)-tag forms (His(6)-rBASI and His(6)-Xa-rBASI), and in an intein-CBD-tagged form (rBASI (intein)). The yields per liter culture after purification were (i) 25 mgl(-1) His(6)-rBASI; (ii) 6 mgl(-1) rBASI purified after cleavage of His(6)-Xa-rBASI by Factor Xa; (iii) 3 mgl(-1) untagged rBASI; and (iv) 0.2 mgl(-1) rBASI after a chitin-column and autohydrolysis of the rBASI-intein-CBD. In Pichia pastoris, rBASI was secreted at 0.1 mgl(-1). The recombinant BASI forms and natural seed BASI (sBASI) all had an identical isoelectric point of 7.2 and a mass of 19,879 Da, as determined by mass spectrometry. The fold of rBASI from the different preparations was confirmed by circular dichroism spectroscopy and rBASI (intein), His(6)-rBASI, and sBASI inhibited AMY2 catalyzed starch hydrolysis with K(i) of 0.10, 0.06, and 0.09 nM, respectively. Surface plasmon resonance analysis of the formation of AMY2/rBASI (intein) gave k(on)=1.3x10(5)M(-1)s(-1), k(off)=1.4x10(-4)s(-1), and K(D)=1.1 nM, and of the savinase-His(6)-rBASI complex k(on)=21.0x10(4)M(-1)s(-1), k(off)=53.0x10(-4)s(-1), and K(D)=25.0 nM, in agreement with sBASI values. K(i) was 77 and 65 nM for inhibition of savinase activity by His(6)-rBASI and sBASI, respectively.     

Lactate-Modulated Induction of THBS-1 Activates Transforming Growth Factor (TGF)-beta2 and Migration of Glioma Cells In Vitro

Background

An important phenomenon observed in glioma metabolism is increased aerobic glycolysis in tumor cells, which is generally referred to as the Warburg effect. Transforming growth factor (TGF)-beta2, which we previously showed to be induced by lactic acid, is a key pathophysiological factor in glioblastoma, leading to increased invasion and severe local immunosuppression after proteolytic cleavage from its latency associated peptide. In this study we tested the hypothesis, that lactate regulates TGF-beta2 expression and glioma cell migration via induction of Thrombospondin-1 (THBS-1), a TGF-beta activating protein.

Methods

Lactate levels were reduced by knockdown of LDH-A using specific small interfering RNA (siRNA) and competitive inhibition of LDH-A by sodium oxamate. Knockdown of THBS-1 was performed using specific siRNA. Western Blot, qRT-PCR, and ELISA were used to investigate expression levels of LDH-A, LDH-B, TGF-beta2 and THBS-1. Migration of cells was examined by Spheroid, Scratch and Boyden Chamber assays.

Results

Knockdown of LDH-A with subsequent decrease of lactate concentration leads to reduced levels of THBS-1 and TGF-beta2 in glioma cells. Lactate addition increases THBS-1 protein, leading to increased activation of TGF-beta2. Inhibition of THBS-1 reduces TGF-beta2 protein and migration of glioma cells. Addition of synthetic THBS-1 can rescue reduced TGF-beta2 protein levels and glioma cell migration in siLDH-A treated cells.

Conclusion

We define a regulatory cascade between lactate, THBS-1 and TGF-beta2, leading to enhanced migration of glioma cells. Our results demonstrate a specific interaction between tumor metabolism and migration and provide a better understanding of the mechanisms underlying glioma cell invasion.     

A distinctive fungal community inhabiting cryoconite holes on glaciers in Svalbard

Cryoconite holes on glacier surfaces are ice-cold hot spots of microbial diversity and activity but still little is known about their fungal inhabitants. We provide the first report of distinctive fungal communities in cryoconite debris from three valley glaciers at Kongsfjorden, Svalbard. Multivariate analysis of terminal-restriction fragment length polymorphism (T-RFLP) profiles of rRNA ITS amplicons revealed that quite distinct fungal communities were found in cryoconite holes compared with soils from adjacent moraine and tundra sites, and that communities on glaciers with contrasting ice-surface hydrology also differed. Most of the fungi cultured from cryoconite sediment were basidiomycetous yeasts or filamentous Ascomycota (Helotiales/Pleosporales). The latter included aeroaquatic fungi, such as Articulospora and Varicosporium, implying a role for these important freshwater decomposers in the carbon dynamics of cryoconite holes. Matching of the dominant peaks from T-RFLP analysis to predicted peaks of cultured isolates confirmed the abundance of these aeroaquatic fungi but also revealed that most of the dominant T-RFLP peaks did not match any cultured isolates. Considering the prevalence and endangerment of glacial environments worldwide, these findings would suggest that their potential as reservoirs of fungal diversity should not be overlooked.     

Evidence that acetyl phosphate functions as a global signal during biofilm development

We used DNA macroarray analysis to identify genes that respond to the status of the intracellular acetyl phosphate (acP) pool. Genes whose expression correlated negatively with the ability to synthesize acP (i.e. negatively regulated genes) function primarily in flagella biosynthesis, a result consistent with observations that we published previously (Prüss and Wolfe, 1994, Mol Microbiol 12: 973-984). In contrast, genes whose expression correlated positively with the ability to synthesize acP (i.e. positively regulated genes) include those for type 1 pilus assembly, colanic acid (capsule) biosynthesis and certain stress effectors. To our knowledge, this constitutes the first report that these genes may respond to the status of the intracellular acP pool. Previously, other researchers have implicated flagella, type 1 pili, capsule and diverse stress effectors in the formation of biofilms. We therefore tested whether cells altered in their ability to metabolize acP could construct normal biofilms, and found that they could not. Cells defective for the production of acP and cells defective for the degradation of acP could both form biofilms, but these biofilms exhibited characteristics substantially different from each other and from biofilms formed by their wild-type parent. We confirmed the role of individual cell surface structures, the expression of which appears to correlate with acP levels, in fim or fli mutants that cannot assemble type 1 pili or flagella respectively. Thus, the information gained by expression profiling of cells with altered acP metabolism indicates that acP may help to co-ordinate the expression of surface structures and cellular processes involved in the initial stages of wild-type biofilm development.     

Chromoplast differentiation in bell pepper (Capsicum annuum) fruits

We report here a detailed analysis of the proteome adjustments that accompany chromoplast differentiation from chloroplasts during bell pepper (Capsicum annuum) fruit ripening. While the two photosystems are disassembled and their constituents degraded, the cytochrome b₆f complex, the ATPase complex, and Calvin cycle enzymes are maintained at high levels up to fully mature chromoplasts. This is also true for ferredoxin (Fd) and Fd-dependent NADP reductase, suggesting that ferredoxin retains a central role in the chromoplasts’ redox metabolism. There is a significant increase in the amount of enzymes of the typical metabolism of heterotrophic plastids, such as the oxidative pentose phosphate pathway (OPPP) and amino acid and fatty acid biosynthesis. Enzymes of chlorophyll catabolism and carotenoid biosynthesis increase in abundance, supporting the pigment reorganization that goes together with chromoplast differentiation. The majority of plastid encoded proteins decline but constituents of the plastid ribosome and AccD increase in abundance. Furthermore, the amount of plastid terminal oxidase (PTOX) remains unchanged despite a significant increase in phytoene desaturase (PDS) levels, suggesting that the electrons from phytoene desaturation are consumed by another oxidase. This may be a particularity of non-climacteric fruits such as bell pepper that lack a respiratory burst at the onset of fruit ripening.     

Range‐wide patterns of migratory connectivity in the western sandpiper Calidris mauri

Understanding the population dynamics of migratory animals and predicting the consequences of environmental change requires knowing how populations are spatially connected between different periods of the annual cycle. We used stable isotopes to examine patterns of migratory connectivity across the range of the western sandpiper Calidris mauri. First, we developed a winter isotope basemap from stable‐hydrogen (δD), ‐carbon (δ¹³C), and ‐nitrogen (δ¹⁵N) isotopes of feathers grown in wintering areas. δD and δ¹⁵N values from wintering individuals varied with the latitude and longitude of capture location, while δ¹³C varied with longitude only. We then tested the ability of the basemap to assign known‐origin individuals. Sixty percent of wintering individuals were correctly assigned to their region of origin out of seven possible regions. Finally, we estimated the winter origins of breeding and migrant individuals and compared the resulting empirical distribution against the distribution that would be expected based on patterns of winter relative abundance. For breeding birds, the distribution of winter origins differed from expected only among males in the Yukon‐Kuskokwim (Y‐K) Delta and Nome, Alaska. Males in the Y‐K Delta originated overwhelmingly from western Mexico, while in Nome, there were fewer males from western North America and more from the Baja Peninsula than expected. An unexpectedly high proportion of migrants captured at a stopover site in the interior United States originated from eastern and southern wintering areas, while none originated from western North America. In general, we document substantial mixing between the breeding and wintering populations of both sexes, which will buffer the global population of western sandpipers from the effects of local habitat loss on both breeding and wintering grounds.