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The uropathogenic Escherichia coli wiJd..:type strain 536 produces S-fimbriae, P-related fimbriae and type I fimbriae. Using immuno-colony dot and ELISA techniques, variants were detected showing an increased degree of S-fimbrial production. It was demonstrated by itrtmunofluorescence microscopy that in noimal (wild-type) and hyperS- fimbriated E. coli populaiions non-fimbriated cells also · exist, and that the percentage of Sfinibrlated and non-fimbriated bacteria was roughly identica1 in either population. Hyper-Sfimbriated variants could be stably maintained. The transition from wild-type to hyper-S-fimbriation, which occurs spontaneously, is markedly higher than vice versa. Southern blot analysis of the S fimbrial adhesin (sfa) determinants of normal and hyper-fimbriated strains revealed no marked difference in the gene structure.
Das Enzym alpha-Dioxygenase (alpha-DOX) aus Erbsen (Pisum sativum) wurde mit folgenden Zielsetzungen untersucht: Isolierung und Charakterisierung der für die P. sativum alpha-DOX codierenden cDNA, Überproduktion der P. sativum alpha-DOX in Escherichia coli und nachfolgende Isolierung, Untersuchung der Interaktion der P. sativum alpha-DOX mit Fettsäuresubstraten sowie systematische Studie der Expression der P. sativum alpha-DOX während der Keimung und Entwicklung von Erbsenpflanzen. alpha-Dioxygenasen katalysieren in Pflanzen den Initialschritt der alpha-Oxidation von langkettigen Fettsäuren, die über die intermediäre Bildung von (R)-2-Hydroperoxyfettsäuren führt. Folgeprodukte dieser Reaktion sind die entsprechende (R)-2-Hydroxysäure sowie der um ein C-Atom kettenverkürzte Aldehyd. Es wurde die für die alpha-Dioxygenase aus Erbsen codierende cDNA mit einer Gesamtlänge von 2132 bp isoliert, die ein offenes Leseraster von 1929 bp beinhaltet. Sie codiert für ein Protein mit 643 Aminosäuren und einem errechneten Molekulargewicht von ca. 73 kD. Die Pisum sativum alpha-Dioxygenase wurde in E. coli als Fusionsprotein mit einem 6 x His-tag überproduziert und mittels Metallaffinitätschromatographie an Ni-NTA-Agarose isoliert. Studien zur Interaktion der P. sativum alpha-Dioxygenase mit Fettsäuresubstraten umfassten sowohl Versuche zu Anforderungen auf Seiten des Substrats als auch zu potentiellen Interaktionspartnern auf Seiten des Enzym. Es wurde gezeigt, dass für die Reaktion von alpha-Dioxygenasen mit Fettsäuren die freie Carboxylgruppe des Substrats unerlässlich ist. Aufgrund eines Aminosäuresequenzvergleichs zwischen der alpha-Dioxygenase aus Erbsen und PGHS-1 aus O. aries wurden vier Aminosäuren als potentielle Interaktionspartner auf Seiten der alpha-Dioxygenase aus Erbsen ausgewählt. Es handelte sich um die Arginin-Reste Arg-87, Arg-391, Arg-569 und Arg-570. Mit Hilfe der ortsspezifischen Mutagenese wurde gezeigt, dass der Aminosäurerest Arg-570 für die katalytische Aktivität unerlässlich ist. Die Expression der P. sativum alpha-Dioxygenase in keimenden Erbsen und jungen Erbsenpflanzen wurde sowohl in ihrem zeitlichen Verlauf als auch hinsichtlich der Gewebespezifität betrachtet. Die Ergebnisse zeigten, dass Keimung zu einer deutlichen Akkumulation von alpha-Dioxygenase mRNA in Erbsen führte. Auch alpha-Dioxygenase Protein war in großer Menge in keimenden und jungen Erbsenpflanzen vorhanden. Ausgeprägte Gewebespezifität war festzustellen: alpha-DOX mRNA fand sich fast ausschließlich in Wurzeln von Erbsenpflanzen, in Sprossgewebe dagegen war sie kaum vorhanden. Im Gegensatz dazu lag alpha-DOX Protein gleichermaßen in Spross- und in Wurzelgewebe vor. Parallel zur Reifung der Pflanzen nahm die Menge an alpha-DOX mRNA und Protein ab. Alpha-Dioxygenase-Aktivität war bereits in trockenen Samen detektierbar, während der Keimung nahm sie deutlich zu. Im Vergleich von Spross- und Wurzelgewebe war die Aktivität in Wurzeln höher, bezogen sowohl auf das Frischgewicht der Pflanzen als auch auf die Menge an Gesamtprotein (spezifische Aktivität). Die Untersuchungen an Wurzeln zeigten, dass die Aktivität bezogen auf das Frischgewicht der Pflanzen über den betrachteten Zeitraum kaum variierte, während die spezifische Aktivität mit zunehmendem Alter der Pflanzen kontinuierlich zunahm. Dieses Ergebnis deutet darauf hin, dass in Erbsen mehrere alpha-Dioxygenase-Isoenzyme vorhanden sind, so wie man dies für andere höhere Pflanzen bereits postuliert hat. Ein zellprotektiver Effekt von alpha-Dioxygenasen auf Pflanzen während der Interaktion mit Pathogenen ist bekannt. Möglicherweise ist dies auch der Grund für eine verstärkte Expression während der Keimung von Pflanzen. Die bevorzugte Expression in Wurzeln könnte auf eine Funktion als permanentes Schutzsystem gegen Infektion hindeuten.
Der transkriptionelle Repressor Blimp-1 wurde ursprünglich als essentiell für die terminale Differenzierung von B-Zellen zu Antikörper-produzierenden Plasmazellen beschrieben. Im Rahmen dieser Arbeit wurde die Expression von Blimp-1 in humanen T-Zellen untersucht. Die Versuchsergebnisse zeigen, dass Blimp-1 auch in humanen T-Zellen sowohl auf mRNA- als auch auf Proteinebene exprimiert wird. Es ist deshalb anzunehmen, dass Blimp-1 auch für die terminale Differenzierung von T-Zellen eine wichtige Rolle spie
Single-cell time-lapse analysis of depletion of the universally conserved essential protein YgjD
(2011)
Background:
The essential Escherichia coli gene ygjD belongs to a universally conserved group of genes whose function has been the focus of a number of recent studies. Here, we put ygjD under control of an inducible promoter, and used time-lapse microscopy and single cell analysis to investigate the phenotypic consequences of the depletion of YgjD protein from growing cells.
Results:
We show that loss of YgjD leads to a marked decrease in cell size and termination of cell division. The transition towards smaller size occurs in a controlled manner: cell elongation and cell division remain coupled, but cell size at division decreases. We also find evidence that depletion of YgjD leads to the synthesis of the intracellular signaling molecule (p) ppGpp, inducing a cellular reaction resembling the stringent response. Concomitant deletion of the relA and spoT genes - leading to a strain that is uncapable of synthesizing (p) ppGpp abrogates the decrease in cell size, but does not prevent termination of cell division upon YgjD depletion.
Conclusions:
Depletion of YgjD protein from growing cells leads to a decrease in cell size that is contingent on (p) ppGpp, and to a termination of cell division. The combination of single-cell time-lapse microscopy and statistical analysis can give detailed insights into the phenotypic consequences of the loss of essential genes, and can thus serve as a new tool to study the function of essential genes.
Background:
Single drug use has not achieved satisfactory results in the treatment of prostate cancer, despite application of increasingly widespread targeted therapeutics. In the present study, the combined impact of the mammalian target of rapamycin (mTOR)-inhibitor RAD001, the dual EGFr and VGEFr tyrosine kinase inhibitor AEE788 and the histone deacetylase (HDAC)-inhibitor valproic acid (VPA) on prostate cancer growth and adhesion in vitro was investigated.
Methods:
PC-3, DU-145 and LNCaP cells were treated with RAD001, AEE788 or VPA or with a RAD-AEE-VPA combination. Tumor cell growth, cell cycle progression and cell cycle regulating proteins were then investigated by MTT-assay, flow cytometry and western blotting, respectively. Furthermore, tumor cell adhesion to vascular endothelium or to immobilized extracellular matrix proteins as well as migratory properties of the cells was evaluated, and integrin alpha and beta subtypes were analyzed. Finally, effects of drug treatment on cell signaling pathways were determined.
Results:
All drugs, separately applied, reduced tumor cell adhesion, migration and growth. A much stronger anticancer effect was evoked by the triple drug combination. Particularly, cdk1, 2 and 4 and cyclin B were reduced, whereas p27 was elevated. In addition, simultaneous application of RAD001, AEE788 and VPA altered the membranous, cytoplasmic and gene expression pattern of various integrin alpha and beta subtypes, reduced integrin-linked kinase (ILK) and deactivated focal adhesion kinase (FAK). Signaling analysis revealed that EGFr and the downstream target Akt, as well as p70S6k was distinctly modified in the presence of the drug combination.
Conclusions:
Simultaneous targeting of several key proteins in prostate cancer cells provides an advantage over targeting a single pathway. Since strong anti-tumor properties became evident with respect to cell growth and adhesion dynamics, the triple drug combination might provide progress in the treatment of advanced prostate cancer.
Background:
Retinitis pigmentosa (RP) is an inherited eye disease characterized by the progressive degeneration of rod photoreceptor cells. Mutations in pre-mRNA splicing factors including PRPF31 have been identified as cause for RP, raising the question how mutations in general factors lead to tissue specific defects.
Results:
We have recently shown that the zebrafish serves as an excellent model allowing the recapitulation of key events of RP. Here we use this model to investigate two pathogenic mutations in PRPF31, SP117 and AD5, causing the autosomal dominant form of RP. We show that SP117 leads to an unstable protein that is mislocalized to the rod cytoplasm. Importantly, its overexpression does not result in photoreceptor degeneration suggesting haploinsufficiency as the underlying cause in human RP patients carrying SP117. In contrast, overexpression of AD5 results in embryonic lethality, which can be rescued by wild-type Prpf31. Transgenic retina-specific expression of AD5 reveals that stable AD5 protein is initially localized in the nucleus but later found in the cytoplasm concurrent with progressing rod outer segment degeneration and apoptosis. Importantly, we show for the first time in vivo that retinal transcripts are wrongly spliced in adult transgenic retinas expressing AD5 and exhibiting increased apoptosis in rod photoreceptors.
Conclusion:
Our data suggest that distinct mutations in Prpf31 can lead to photoreceptor degeneration through different mechanisms, by haploinsufficiency or dominant-negative effects. Analyzing the AD5 effects in our animal model in vivo, our data imply that aberrant splicing of distinct retinal transcripts contributes to the observed retina defects.
Transient receptor potential channels are important mediators of thermal and mechanical stimuli and play an important role in neuropathic pain. The contribution of hereditary variants in the genes of transient receptor potential channels to neuropathic pain is unknown. We investigated the frequency of transient receptor potential ankyrin 1, transient receptor potential melastin 8 and transient receptor potential vanilloid 1 single nucleotide polymorphisms and their impact on somatosensory abnormalities in neuropathic pain patients. Within the German Research Network on Neuropathic Pain (Deutscher Forscbungsverbund Neuropathischer Schmerz) 371 neuropathic pain patients were phenotypically characterized using standardized quantitative sensory testing. Pyrosequencing was employed to determine a total of eleven single nucleotide polymorphisms in transient receptor potential channel genes of the neuropathic pain patients and a cohort of 253 German healthy volunteers. Associations of quantitative sensory testing parameters and single nucleotide polymorphisms between and within groups and subgroups, based on sensory phenotypes, were analyzed. Single nucleotide polymorphisms frequencies did not differ between both the cohorts. However, in neuropathic pain patients transient receptor potential ankyrin 1 710G>A (rs920829, E179K) was associated with the presence of paradoxical heat sensation (p=0.03), and transient receptor potential vanilloid 1 1911A>G (rs8065080, I585V) with cold hypoalgesia (p=0.0035). Two main subgroups characterized by preserved (1) and impaired (2) sensory function were identified. In subgroup 1 transient receptor potential vanilloid 1 1911A>G led to significantly less heat hyperalgesia, pinprick hyperalgesia and mechanical hypaesthesia (p=0.006, p=0.005 and p<0.001) and transient receptor potential vanilloid 1 1103C>G (rs222747, M315I) to cold hypaesthesia (p=0.002), but there was absence of associations in subgroup 2. In this study we found no evidence that genetic variants of transient receptor potential channels are involved in the expression of neuropathic pain, but transient receptor potential channel polymorphisms contributed significantly to the somatosensory abnormalities of neuropathic pain patients.
Introduction:
Oncolytic viruses show promise for treating cancer. However, to assess therapeutic efficacy and potential toxicity, a noninvasive imaging modality is needed. This study aimed to determine if insertion of the human sodium iodide symporter (hNIS) cDNA as a marker for non-invasive imaging of virotherapy alters the replication and oncolytic capability of a novel vaccinia virus, GLV-1h153.
Methods:
GLV-1h153 was modified from parental vaccinia virus GLV-1h68 to carry hNIS via homologous recombination. GLV-1h153 was tested against human pancreatic cancer cell line PANC-1 for replication via viral plaque assays and flow cytometry. Expression and transportation of hNIS in infected cells was evaluated using Westernblot and immunofluorescence. Intracellular uptake of radioiodide was assessed using radiouptake assays. Viral cytotoxicity and tumor regression of treated PANC-1tumor xenografts in nude mice was also determined. Finally, tumor radiouptake in xenografts was assessed via positron emission tomography (PET) utilizing carrier-free (124)I radiotracer.
Results:
GLV-1h153 infected, replicated within, and killed PANC-1 cells as efficiently as GLV-1h68. GLV-1h153 provided dose-dependent levels of hNIS expression in infected cells. Immunofluorescence detected transport of the protein to the cell membrane prior to cell lysis, enhancing hNIS-specific radiouptake (P < 0.001). In vivo, GLV-1h153 was as safe and effective as GLV-1h68 in regressing pancreatic cancer xenografts (P < 0.001). Finally, intratumoral injection of GLV-1h153 facilitated imaging of virus replication in tumors via (124)I-PET.
Conclusion:
Insertion of the hNIS gene does not hinder replication or oncolytic capability of GLV-1h153, rendering this novel virus a promising new candidate for the noninvasive imaging and tracking of oncolytic viral therapy.
Background:
Mantle cell lymphoma (MCL) is genetically characterized by the t(11; 14)(q13; q32) translocation and a high number of secondary chromosomal alterations. The contribution of DNA methylation to MCL lymphomagenesis is not well known. We sought to identify epigenetically silenced genes in these tumours that might have clinical relevance.
Methodology/Principal Findings:
To identify potential methylated genes in MCL we initially investigated seven MCL cell lines treated with epigenetic drugs and gene expression microarray profiling. The methylation status of selected candidate genes was validated by a quantitative assay and subsequently analyzed in a series of primary MCL (n = 38). After pharmacological reversion we identified 252 potentially methylated genes. The methylation analysis of a subset of these genes (n = 25) in the MCL cell lines and normal B lymphocytes confirmed that 80% of them were methylated in the cell lines but not in normal lymphocytes. The subsequent analysis in primary MCL identified five genes (SOX9, HOXA9, AHR, NR2F2, and ROBO1) frequently methylated in these tumours. The gene methylation events tended to occur in the same primary neoplasms and correlated with higher proliferation, increased number of chromosomal abnormalities, and shorter survival of the patients.
Conclusions:
We have identified a set of genes whose methylation degree and gene expression levels correlate with aggressive clinicopathological features of MCL. Our findings also suggest that a subset of MCL might show a CpG island methylator phenotype (CIMP) that may influence the behaviour of the tumours.