Desulfoferrodoxin of Clostridium acetobutylicum functions as a superoxide reductase

Desulfoferrodoxin (cac2450) of Clostridium acetobutylicum was purified after overexpression in E. coli. In an in vitro assay the enzyme exhibited superoxide reductase activity with rubredoxin (cac2778) of C. acetobutylicum as the proximal electron donor. Rubredoxin was reduced by ferredoxin:NADP(+) reductase from spinach and NADPH. The superoxide anions, generated from dissolved oxygen using Xanthine and Xanthine oxidase, were reduced to hydrogen peroxide. Thus, we assume that desulfoferrodoxin is the key factor in the superoxide reductase dependent part of an alternative pathway for detoxification of reactive oxygen species in this obligate anaerobic bacterium.     

DNA sequences from Arabidopsis, which encode protein kinases and function as upstream regulators of Snf1 in yeast

Sucrose nonfermenting-1 (Snf1)-related protein kinase-1 (SnRK1) of plants is a global regulator of carbon metabolism through the modulation of enzyme activity and gene expression. It is structurally and functionally related to the yeast protein kinase, Snf1, and to mammalian AMP-activated protein kinase. Two DNA sequences from Arabidopsis thaliana, previously known only by their data base accession numbers of NM_ 125448.3 (protein ID NP_200863) and NM_114393.3 (protein ID NP_566876) each functionally complemented a Saccharomyces cerevisiae elm1 sak1 tos3 triple mutant. This indicates that the Arabidopsis proteins are able to substitute for one of the missing yeast upstream kinases, which are required for activity of Snf1. Both plant proteins were shown to phosphorylate a peptide with the amino acid sequence of the phosphorylation site in the T-loop of SnRK1 and by inference SnRK1 in Arabidopsis. The proteins encoded by NM_125448.3 and NM_114393.3 have been named AtSnAK1 and AtSnAK2 (Arabidopsis thaliana SnRK1-activating kinase), respectively. We believe this is the first time that upstream activators of SnRK1 have been described in any plant species.     

Differential expression of sarcoplasmic proteins in four heterogeneous ovine skeletal muscles

Fiber-type distribution is known to vary widely within and between muscles according to differences in muscle functions. 2-DE and MALDI-MS were used to investigate the molecular basis of muscle fiber type-related variability. We compared four lamb skeletal muscles with heterogeneous fiber-type composition that are relatively rich in fast-twitch fiber types, i.e., the semimembranosus, vastus medialis, longissimus dorsi, and tensor fasciae latae (TL). Our results clearly showed that none of the glycolytic metabolism enzymes detected, including TL which was most strongly glycolytic, made intermuscular differentiation possible. Muscle differentiation was based on the differential expression of proteins involved in oxidative metabolism, including not only citric acid cycle enzymes but also other classes of proteins with functions related to oxidative metabolism, oxidative stress, and probably to higher protein turnover. Detected proteins were involved in transport (carbonate dehydratase, myoglobin, fatty acid-binding protein), repair of misfolding damage (heat shock protein (HSP) 60 kDa, HSP-27 kDa, alpha-crystallin beta subunit, DJ1, stress-induced phosphoprotein), detoxification or degradation of impaired proteins (GST-Pi, aldehyde dehydrogenase, peroxiredoxin, ubiquitin), and protein synthesis (tRNA-synthetase). The fractionating method led to the detection of proteins involved in different functions related to oxidative metabolism that have not previously been shown concomitancy.     

Proteomics demonstration that normal breast epithelial cells can induce apoptosis of breast cancer cells through insulin-like growth factor-binding protein-3 and maspin

Normal breast epithelial cells are known to exert an apoptotic effect on breast cancer cells, resulting in a potential paracrine inhibition of breast tumor development. In this study we purified and characterized the apoptosis-inducing factors secreted by normal breast epithelial cells. Conditioned medium was concentrated by ultrafiltration and separated on reverse phase Sep-Pak C18 and HPLC. The proapoptotic activity of eluted fractions was tested on MCF-7 breast cancer cells, and nano-LC-nano-ESI-MS/MS allowed the identification of insulin-like growth factor-binding protein-3 (IGFBP-3) and maspin as the proapoptotic factors produced by normal breast epithelial cells. Western blot analysis of conditioned media confirmed the specific secretion of IGFBP-3 and maspin by normal cells but not by breast cancer cells. Immunodepletion of IGFBP-3 and maspin completely abolished the normal cell-induced apoptosis of cancer cells, and recombinant proteins reproduced the effect of normal cell-conditioned medium on apoptosis of breast cancer cells. Together our results indicated that normal breast epithelial cells can induce apoptosis of breast cancer cells through IGFBP-3 and maspin. These findings provide a molecular hypothesis for the long observed inhibitory effect of normal surrounding cells on breast cancer development.     

Glutamine regulates the human epithelial intestinal HCT-8 cell proteome under apoptotic conditions

Glutamine plays a key role in the metabolism of rapidly dividing cells, including enterocytes and lymphocytes, which may contribute to its beneficial clinical effects. Gut mucosal homeostasis is achieved through a balance between cell proliferation and apoptosis. In T cells, glutamine up-regulates antiapoptotic proteins and down-regulates proapoptotic proteins. In gut mucosa, glutamine prevents apoptosis in rat epithelial cell lines, whereas glutamine starvation induces apoptosis through caspase activation. Finally glutamine specifically prevents tumor necrosis factor-alpha-related apoptosis in the human intestinal cell line HT-29. Comparative functional proteomics enables the characterization of each differentially expressed protein in intestinal cells in response to modifications of nutritional environment. The influence of glutamine on intestinal proteome expression in apoptotic conditions has not been studied and evaluated. This comparative proteomics study was performed in the human epithelial intestinal cell line HCT-8 under experimental apoptotic conditions to investigate the influence of glutamine on protein expression during apoptosis. The pharmaconutritional effects of glutamine were determined under 2 mm (physiological concentration) and 10 mm (pharmaconutritional concentration) conditions. About 1,800 protein spots were revealed in both conditions. Comparative assessments indicated that 28 proteins were differentially expressed significantly (i.e. at least 2-fold modulated and Student's t test with p 相似文献   

Specific on-plate enrichment of phosphorylated peptides for direct MALDI-TOF MS analysis

An on-plate specific enrichment method is presented for the direct analysis of peptides phosphorylation. An array of sintered TiO 2 nanoparticle spots was prepared on a stainless steel plate to provide porous substrate with a very large specific surface and durable functions. These spots were used to selectively capture phosphorylated peptides from peptide mixtures, and the immobilized phosphopeptides could then be analyzed directly by MALDI MS after washing away the nonphosphorylated peptides. beta-Casein and protein mixtures were employed as model samples to investigate the selection efficiency. In this strategy, the steps of phosphopeptide capture, purification, and subsequent mass spectrometry analysis are all successfully accomplished on a single target plate, which greatly reduces sample loss and simplifies analytical procedures. The low detection limit, small sample size, and rapid selective entrapment show that this on-plate strategy is promising for online enrichment of phosphopeptides, which is essential for the analysis of minute amount of samples in high-throughput proteome research.     

Über festlegung und einhaltung der mittleren dichte von insektenpopulationen

Zusammenfassung Die Arbeit verzichtet bewußt auf empirische Beweise jeder Art, weil sie sich mit Vorgängen befaßt, die sich über lange Zeit erstrecken und deshalb der Beobachtung und dem Experiment einstweilen nicht zugänglich sind. Alle derartigen Versuche konnen bisher nicht überzeugen.Regulation der Populationsdichte liegt vor, wenn es irgendwelche endlichen Grenzen gibt, die vor ihr auf die Dauer nicht über- bzw. unterschritten werden. Da die Populationsdichte die auf sie ausgeübten Einflüsse im Laufe der Zeit summiert bzw. multipliziert, vermögen Zufallsfaktoren die Einhaltung solcher Grenzen nicht zu garantieren. Die Wetterereignisse konnen in ihrem zeitlichen Ablauf in periodischer und eventuell auch in aperiodischer Art von der Zufallserwartung abweichen, ohne dadurch aber die Überschreitung der Grenzen durch wetterbedingten Massenwechsel zu verhindern oder wesentlich zu verzögern. Auch das Zusammenw-irken mehrerer dichteunabhängiger Faktoren führt nicht zur Regulation.Wanderungen von Insekten können nur darn regulierend wirken, wenn sie in Abhängigkeit vor der Populationsdichte auftreten. Der regulierende Einfluß schützender Stellen im Biotop ist eine Folge intraspezifischer Konkurrenz. Änderungen in der Häufigkeit vor Genen oder Genanordnungen, welche eventuell mit dem Massenwechsel verbunden sind, können ebenfalls höchstens dann eine regulierende Wirkung haben, wenn sie dichteabhängig erfolgen.Alle dichteabhängigen Faktoren werden nicht nur vor der Populationsdichte, sondern auch vor anderen (diehteunabhängigen) Umweltkomponenten beeinflußt. Trotzdem ist die Dichteabhängigkeit das einzig mögliche regulierende Prinzip. Das Begriffssystem der Kybernetik gestattet eine eindeutige Beschreibung der Regulationsvorgänge. Dabei zeigt es sich, daß die Einhaltung irgendwelcher endlichen Dichtegrenzen und die Bestimmung der mittleren Populationsdichte zwei zwar zusammengehörige, aber wesensmäßig verschiedene Prozesse sind. Dichteunabhängige Faktoren (vor allem das Wetter) wirken einerseits als Führungsgröße, andererseits als kausales Agens für die Störgröße, dichteabhängige, vor allem die intraspezifische Konkurrenz, als Regler.Zur Beseitigung der bestehenden begrifflichen Schwierigkeiten wird vorgeschlagen, die Bezeichnung Regulation auf den Vorgang im Regelkreis (Populationsdichte — dichteabhängiger Faktor — dichteabhängige Sterblichkeit und Fruchtbarkeit - Populationsdichte) zu beschränken und die Festlegung der mittleren Populationsdichte als Determination zu bezeichnen. Das Wetter ist kein Regulations-, sondern ein Massenwechselfaktor, das Klima dagegen ist ein Determinationsfaktor.Die bisherigen Meinungsverschiedenheiten haben außer Mißverständnissen hauptsächlich drei Ursachen: 1. die ungenügende begriffliche Trennung zwischen Massenwechsel und Regulation, 2. ungenaue Vorstellungen über die Auswirkung dichteunabhängiger Faktoren, 3. fehlende Unterscheidung zwischen Regulation und Determination.     

Activities of the EM10 protein from Echinococcus multilocularis in cultured mammalian cells demonstrate functional relationships to ERM family members

The ezrin-radixin-moesin (ERM) homolog EM10 is expressed by the larval stage of the parasite E. multilocularis and shows 46.9% overall identity in the primary structure with human ezrin. To determine whether EM10 has similar activities to ERM proteins, we investigated properties of the protein expressed in mammalian cells. In particular, we transiently expressed haemagglutinin-tagged (HA-tagged) versions of the full-length EM10 as well as the amino- and the carboxy-terminal halves of EM10 in HtTA-1 cells. In addition we stably transfected NIH-3T3 cells with untagged full-length EM10. The data demonstrate that EM10 polypeptides behave like their corresponding portions of radixin when transiently expressed in mammalian cells. The full-length and amino-terminal EM10 polypeptides were localized to cortical structures. Cells expressing the carboxy-terminal polypeptide of EM10 showed long actin-filled protrusions. Cells expressing full-length EM10 showed a reduction in endogenous moesin-staining at cortical structures. In stably transfected NIH-3T3 cells EM10 was not crisply localized but rather was diffuse throughout the cytoplasm. These cells showed a conspicuous loss of stress-fibers, a phenotype that was not seen in analogous experiments with ERM proteins. The results demonstrate both similarities and differences between the functional properties of EM10 and ERM proteins expressed in vertebrate cells.     

Switch from an aquaporin to a glycerol channel by two amino acids substitution

The MIP (major intrinsic protein) proteins constitute a channel family of currently 150 members that have been identified in cell membranes of organisms ranging from bacteria to man. Among these proteins, two functionally distinct subgroups are characterized: aquaporins that allow specific water transfer and glycerol channels that are involved in glycerol and small neutral solutes transport. Since the flow of small molecules across cell membranes is vital for every living organism, the study of such proteins is of particular interest. For instance, aquaporins located in kidney cell membranes are responsible for reabsorption of 150 liters of water/day in adult human. To understand the molecular mechanisms of solute transport specificity, we analyzed mutant aquaporins in which highly conserved residues have been substituted by amino acids located at the same positions in glycerol channels. Here, we show that substitution of a tyrosine and a tryptophan by a proline and a leucine, respectively, in the sixth transmembrane helix of an aquaporin leads to a switch in the selectivity of the channel, from water to glycerol.