Subcellular localization of EGFR in esophageal carcinoma cell lines

Background: The EGF receptor is a therapeutic target in cancer cells, whereby mutations of EGFR and/or signalling members act as predictive markers. EGFR however also exhibits dynamic changes of subcellular localization, leading to STAT5 complex formation, nuclear translocation and induction of Aurora-A expression in squamous cancer cells. We previously described high EGFR and Aurora-A expression in esophageal cancer cells. Here, we investigated subcellular localization of EGFR and STAT5 in esophageal cancer cells. Results: Quantitative immunofluorescence analyses of four esophageal cancer cell lines reflecting esophageal squamous cell carcinomas (ESCC) and esophageal adenocarcinomas (EAC) revealed that the subcellular localization of EGFR was shifted from a membranous to cytoplasmic localization upon EGF-stimulation in OE21 (ESCC) cells. Thereby, EGFR in part co-localized with E-Cadherin. In parallel, phosphorylated STAT5-Tyr694 appeared to increase in the nucleus and to decrease at the cell membrane. In three additional cell lines, EGFR was only marginally (Kyse-410/ESCC; OE19/EAC) and weakly (OE33, EAC) detectable at the cell membrane. Partial co-localization of EGFR and E-Cadherin occurred in OE33 cells. Post EGF-stimulation, EGFR was detected in the cytoplasm, resembling endosomal compartments. Furthermore, OE19 and OE33 exhibited nuclear STAT5-Tyr694 phosphorylation upon EGF-stimulation. None of the four cell lines showed nuclear EGFR expression and localization. Conclusion: In contrast to other (squamous) cancer cells, activation of EGFR in esophageal squamous cancer cells does not result in nuclear translocation of EGFR. Still, the subcellular localization of EGFR may influence STAT5-associated signaling pathways in esophageal cancer cells and hence possibly also the responses to ErbB, respective EGFR-targeted therapies.     

Enzymatic Dehalogenation of Pentachlorophenol by <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> of the Microbial Community from Tannery Effluent

Four different bacterial isolates obtained from a stable bacterial consortium were capable of utilizing pentachlorophenol (PCP) as sole carbon and energy source. The consortium was developed by continuous enrichment in the chemostat. The degradation of PCP by bacterial strain was preceded through an oxidative route as indicated by accumulation of tetrachloro-ρ-hydroquinone and dichlorohydroquinone as determined by high performance liquid chromatography (HPLC). Among the four isolates, Pseudomonas fluorescens exhibited maximum degradation capability and enzyme production. PCP-monooxygenase enzyme was extracted from culture extract and fractionated by DEAE-cellulose ion exchange chromatography. The molecular weight of the enzyme, purified from Pseudomonas fluorescens, determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography was found to be 24,000 Da. Received: 22 July 2002 / Accepted: 23 September 2002     

Multi-Stability and Multi-Instability Phenomena in a Mathematical Model of Tumor-Immune-Virus Interactions

Recent advances in virology, gene therapy, and molecular and cell biology have provided insight into the mechanisms through which viruses can boost the anti-tumor immune response, or can infect and directly kill tumor cells. A recent experimental report (Bridle et al. in Molec. Ther. 18(8):1430–1439, 2010) showed that a sequential treatment approach that involves two viruses that carry the same tumor antigen leads to an improved anti-tumor response compared to the effect of each virus alone. In this article, we derive a mathematical model to investigate the anti-tumor effect of two viruses, and their interactions with the immune cells. We discuss the conditions necessary for permanent tumor elimination and, in this context, we stress the importance of investigating the long-term effect of non-linear interactions. In particular, we discuss multi-stability and multi-instability, two complex phenomena that can cause abrupt transitions between different states in biological and physical systems. In the context of cancer immunotherapies, the transitions between a tumor-free and a tumor-present state have so far been associated with the multi-stability phenomenon. Here, we show that multi-instability can also cause the system to switch from one state to the other. In addition, we show that the multi-stability is driven by the immune response, while the multi-instability is driven by the presence of the virus.     

Associations between primates and other mammals in a central Amazonian forest landscape

Little information exists on mixed-species groups between primates and other mammals in Neotropical forests. In this paper, we describe three such associations observed during an extensive large-vertebrate survey in central Amazonia, Brazil. Mixed-species groups between a primate species and another mammal were observed on seven occasions between squirrel monkeys (Saimiri cf. ustus) and either South American coatis (Nasua nasua) or tayras (Eira barbara) and between brown capuchins (Cebus apella) and coatis. All associations were restricted to floodplain forest during its dry stage. We suggest that the associations involving the coatis are connected to foraging and vigilance but may be induced by a common alternative food resource at a time of food shortage.     

The chimeric cytokine Hyper-IL-6 enhances the efficiency of lentiviral gene transfer in hepatocytes both in vitro and in vivo

Lentiviral vectors have been used for gene transfer into the liver but their ability to efficiently transduce quiescent hepatocytes remains controversial. Lentivirus-mediated gene transfer is more efficient in cycling cells. We determine the effect of H-IL6 in the lentiviral transduction. The lentiviral vector was used to transduce HepG2 cells and mice liver cells, previously treated with H-IL6. The highest transduction level was observed in HepG2 cells treated with 30 ng/mL H-IL6 and in the mice that received 4 μg H-IL6. Our results suggest that H-IL6 is an inducer of lentiviral gene transfer into the liver cells without any toxicity.     

Sortase-mediated protein ligation: an emerging biotechnology tool for protein modification and immobilisation

Sortases are transpeptidases produced by Gram-positive bacteria to anchor cell surface proteins covalently to the cell wall. The Staphylococcus aureus sortase A (SrtA) cleaves a short C-terminal recognition motif (LPXTG) on the target protein followed by the formation of an amide bond with the pentaglycine cross-bridge in the cell wall. Over recent years, several researchers have exploited this specific reaction for a range of biotechnology applications, including the incorporation of non-native peptides and non-peptidic molecules into proteins, the generation of nucleic acid–peptide conjugates and neoglycoconjugates, protein circularisation, and labelling of cell surface proteins on living cells.     

The effect of jasmonic acid on the accumulation of ABA,proline and spermidine and its influence on membrane injury under water deficit in two barley genotypes

Seedlings of two barley genotypes (‘Maresi’ and wild form of Hordeum spontaneum) were treated with jasmonic acid (JA 5 μM and 15 μM) for 24 h, and then subjected to water stress (PEG 6000 solution of − 1.5 MPa). JA caused an increase in the content of ABA but not in that of proline and spermidine in the two studied genotypes. The effect of the treatment did not depend on the applied JA concentration. The pre-stress treatment with JA changed plant response to water deficit with regard to membrane injury. Treatment with a lower JA concentration (5 μM) caused a substantial reduction of the stress-induced membrane damage in the both genotypes. A higher JA concentration (15 μM) caused the reduction of membrane injury only in H. spontaneum and was ineffective in ‘Maresi’. JA had no influence on the leaf water status in water-stressed plants. A possible role of JA in leaf ABA accumulation and alleviation of cell membrane injury under water deficit is discussed. The work was partly supported by the Polish Committee For Scientific Research, grant No 5 PO6A 036 18     

Molecular diversity of 1-aminocyclopropane-1-carboxylate (ACC) deaminase producing PGPR from wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) rhizosphere

Aims

The present study was planned to investigate the diversity of 1-aminocyclopropane-1-carboxylate (ACC) deaminase producing bacteria from the rhizosphere of wheat plants and subsequent evaluation of selected PGPR on growth enhancement of wheat seedlings under drought and saline conditions.

Methods

ACC deaminase producing plant growth promoting rhizobacteria (PGPR) were isolated from the rhizosphere of wheat and identified using 16S rRNA gene sequence analysis. Isolates were evaluated for various direct and indirect plant growth promoting (PGP) traits. Plant inoculation experiment was conducted using isolates IG 19 and IG 22 in wheat to assess their plant growth promotion potential under salinity and drought stress.

Results

Thirty-eight ACC deaminase producing PGPR were isolated which belonged to 12 distinct genera and falling into four phyla γ-proteobacteria, β-proteobacteria, Flavobacteria and Firmicutes. Klebsiella sp. was the most abundant genera and followed by Enterobacter sp. The isolates exhibited ACC deaminase activities ranging from 0.106–0.980 μM α- ketobutyrate μg protein^?1 h^?1. The isolates showed multiple PGP traits such as IAA production, phosphate, zinc, potassium solubilization and siderophore production. Enterobacter cloacae (IG 19) and Citrobacter sp. (IG 22) inoculated wheat seedlings showed notable increases in fresh and dry biomass under non-stress as well as under stressed condition.

Conclusion

To the best of our knowledge this is the first report of presence of ACC deaminase activity and other PGP traits from the genus Citrobacter and Empedobacter. Our finding revealed that the γ-proteobacteria group dominated the wheat rhizosphere. Plant inoculation with PGPR could be a sustainable approach to alleviate abiotic stresses in wheat plants. These native PGPR isolates could be used as potential biofertilizers for sustainable agriculture.

     

Comparison of three ELISA kits for the differentiation of foot-and-mouth disease virus-infected from vaccinated animals

A study was performed to validate 3 FMDV 3ABC-I-ELISA kits developed in China for the differentiation of FMDV infected and vaccinated animals. Sets of sera from naive and vaccinated cattle as well as from cattle that had been infected were tested for antibodies against nonstructural proteins (NSPs) of FMDV by commercial diagnosis kits, Ceditest® FMDV-NS (Ceditest® kit), UBI® FMDV NONSTRUCTURAL PROTEIN ELISA DIRECTION INSERT (UBI® kit) and a FMDV 3ABC-I-ELISA kit developed at the Lanzhou Veterinary Research Institute. The test parameters (sensitivity and specificity) of the three kits were determined, and the result obtained from FMD 3ABC-I-ELISA kit was compared with that obtained from two foreign kits. The results indicated that the coincidence rate between the FMDV 3ABC-I-ELISA and Ceditest® kits was 98.05%, and the coincidence rate between the FMDV 3ABC-I-ELISA and UBI® kits was 94.4%; the sensitivity of both Ceditest® and FMDV 3ABC-I-ELISA kit was 100%. However, the sensitivity of the UBI® kit was only 81.8%. With sera from naive or vaccinated non-infected animals, the specificity of all tests exceeded 90%.     

A modified Coomassie Brilliant Blue staining method at nanogram sensitivity compatible with proteomic analysis

A more sensitive and convenient Coomassie Brilliant Blue (CBB) staining method for visualizing proteins was developed. Compared with the modifications include the supplement of 10% (v/v) methanol into the fixing solution, an increase of an additional sensitization step and CBB raised from 0.1 to 0.125%. The improved method can detect proteins at nanogram level. The improved method is more sensitive than Blue Silver and more convenient than the Silver protocol. Mass spectrometry results confirmed that it is suitable for subsequent proteomic research.