Therapeutic efficacy of melanoma-reactive TIL injected in stage III melanoma patients

Adoptive therapy for cancer using tumor-infiltrating lymphocytes (TIL) has mainly been investigated in cancer patients with advanced stage disease. The limited clinical success has not been encouraging, although this might be explained by poor TIL specificity and/or high tumor burden. To re-evaluate the effectiveness of adoptive therapy, we analyzed the capacity of tumor-reactive TIL injection in preventing the further development of disease in stage III melanoma patients after complete tumor resection. A phase II/III randomized trial was performed on 88 melanoma patients, who received autologous TIL plus interleukin-2 (IL-2) or IL-2 only. The duration of relapse-free survival was analyzed, taking into account the immunological specificity of injected TIL and the number of metastatic lymph nodes removed before treatment. Kaplan-Meyer analysis revealed that the injection of tumor-reactive TIL was statistically correlated with prolonged relapse-free survival in patients with only one metastatic lymph node. Therefore, improved clinical outcome could be obtained after adoptive therapy by selecting appropriate groups of patients and monitoring the specificity of the injected TIL populations.     

Identification and properties of type I-signal peptidases of Bacillus amyloliquefaciens.

The use of Bacillus amyloliquefaciens for enzyme production and its exceptional high protein export capacity initiated this study where the presence and function of multiple type I signal peptidase isoforms was investigated. In addition to type I signal peptidases SipS(ba) [Meijer, W.J.J., de Jong, A., Bea, G., Wisman, A., Tjalsma, H., Venema, G., Bron, S. & van Dijl, J.M. (1995) Mol. Microbiol. 17, 621-631] and SipT(ba) [Hoang, V. & Hofemeister, J. (1995) Biochim. Biophys. Acta 1269, 64-68] which were previously identified, here we present evidence for two other Sip-like genes in B. amyloliquefaciens. Same map positions as well as sequence motifs verified that these genes encode homologues of Bacillus subtilis SipV and SipW. SipU-encoding DNA was not found in B. amyloliquefaciens. SipW-encoding DNA was also found for other Bacillus strains representing different phylogenetic groups, but not for Bacillus stearothermophilus and Thermoactinomyces vulgaris. The absence of these genes, however, could have been overlooked due to sequence diversity. Sequence alignments of 23 known Sip-like proteins from Bacillus origin indicated further branching of the P-group signal peptidases into clusters represented by B. subtilis SipV, SipS-SipT-SipU and B. anthracis Sip3-Sip5 proteins, respectively. Each B. amyloliquefaciens sip(ba) gene was expressed in an Escherichia coli LepBts mutant and tested for genetic complementation of the temperature sensitive (TS) phenotype as well as pre-OmpA processing. Although SipS(ba) as well as SipT(ba) efficiently restored processing of pre-OmpA in E. coli, only SipS(ba) supported growth at TS conditions, indicating functional diversity. Changed properties of the sip(ba) gene disruption mutants, including cell autolysis, motility, sporulation, and nuclease activities, seemed to correlate with specificities and/or localization of B. amyloliquefaciens SipS, SipT and SipV isoforms.     

Questa (Annelida, Polychaeta, Orbiniidae) from Pacific regions — new species and reassessment of the genus Periquesta

On the basis of new material from various Pacific regions we critically evaluate the characters for the taxonomy of the genus Questa and the former Questidae, a taxon now concluded to be a lineage within the family Orbiniidae. Two new species, Questa retrospermatica sp. n. from Hawaii (with similar forms also from New Caledonia and China), and Questa fijiensis sp. n. from Fiji, are described, and the morphological character patterns and phylogeny of the genus are revisited on the basis of a cladistic analysis. The finding of Hawaiian material of a new species, Levinsenia hawaiiensis sp. n., similar to Periquesta canariensis Brito & Nunez, 2002 (=Levinsenia canariensis comb. n.), challenges the view that Periquesta Brito & Nunez, 2002 is closely related to Questa; rather, it belongs to Paraonidae as a junior synonym of Levinsenia Mesnil, 1897.     

Knocking out the regulatory beta subunit of protein kinase CK2 in mice: Gene dosage effects in ES cells and embryos

Knocking out the regulatory β subunit of protein kinase CK2 in mice leads to early embryonic lethality. Heterozygous CK2β (CK2β^+/−) knockout mice do not show an obvious phenotype. However, the number of heterozygous offsprings from CK2β^+/− inter-crossings is lower than expected, meaning that some heterozygous embryos do not survive. Interestingly, CK2β^+/− ES (Embryonic Stem) cells express a considerably lower level of CK2β than wild-type ES cells, whereas the level of CK2β in organs from heterozygous adult mice does not significantly differ from those of wild-type mice. The data suggest a compensatory mechanism that adjusts CK2β levels during development in the majority of, but not in all, cases (Mol Cell Biol {23:} 908–915, 2003).In order to find an explanation for the gene dosage effect observed for heterozygous offsprings, we analysed embryos at mid-gestation (E10.5) as well as wild-type and CK2β^+/− ES cells for differences in growth rate and response to different stress agents. Analysis of E10.5 embryos generated from heterozygous matings revealed about 20% of smaller retarded CK2β^+/− embryos. No correlation between CK2β levels in normal looking and retarded CK2β^+/− embryos were found. However, a different post-translational form of CK2β protein has been detected in these retarded embryos. Cellular parameters such as growth rate and G1-, G2-checkpoints in ES cells were identical in both wild-type and CK2β^+/− cells. When ES cells were injected to induce differentiated teratocarcinoma in syngenic mice, the size of the tumours correlated with the level of CK2β.     

Characterization of myo-inositol utilization by Corynebacterium glutamicum: the stimulon, identification of transporters, and influence on L-lysine formation

Although numerous bacteria possess genes annotated iol in their genomes, there have been very few studies on the possibly associated myo-inositol metabolism and its significance for the cell. We found that Corynebacterium glutamicum utilizes myo-inositol as a carbon and energy source, enabling proliferation with a high maximum rate of 0.35 h-1. Whole-genome DNA microarray analysis revealed that 31 genes respond to myo-inositol utilization, with 21 of them being localized in two clusters of >14 kb. A set of genomic mutations and functional studies yielded the result that some genes in the two clusters are redundant, and only cluster I is necessary for catabolizing the polyol. There are three genes which encode carriers belonging to the major facilitator superfamily and which exhibit a >12-fold increased mRNA level on myo-inositol. As revealed by mutant characterizations, one carrier is not involved in myo-inositol uptake whereas the other two are active and can completely replace each other with apparent Kms for myo-inositol as a substrate of 0.20 mM and 0.45 mM, respectively. Interestingly, upon utilization of myo-inositol, the L-lysine yield is 0.10 mol/mol, as opposed to 0.30 mol/mol, with glucose as the substrate. This is probably not only due to myo-inositol metabolism alone since a mixture of 187 mM glucose and 17 mM myo-inositol, where the polyol only contributes 8% of the total carbon, reduced the L-lysine yield by 29%. Moreover, genome comparisons with other bacteria highlight the core genes required for growth on myo-inositol, whose metabolism is still weakly defined.     

Frequent Long-Range Epigenetic Silencing of Protocadherin Gene Clusters on Chromosome 5q31 in Wilms' Tumor

Wilms'' tumour (WT) is a pediatric tumor of the kidney that arises via failure of the fetal developmental program. The absence of identifiable mutations in the majority of WTs suggests the frequent involvement of epigenetic aberrations in WT. We therefore conducted a genome-wide analysis of promoter hypermethylation in WTs and identified hypermethylation at chromosome 5q31 spanning 800 kilobases (kb) and more than 50 genes. The methylated genes all belong to α-, β-, and γ-protocadherin (PCDH) gene clusters (Human Genome Organization nomenclature PCDHA@, PCDHB@, and PCDHG@, respectively). This demonstrates that long-range epigenetic silencing (LRES) occurs in developmental tumors as well as in adult tumors. Bisulfite polymerase chain reaction analysis showed that PCDH hypermethylation is a frequent event found in all Wilms'' tumor subtypes. Hypermethylation is concordant with reduced PCDH expression in tumors. WT precursor lesions showed no PCDH hypermethylation, suggesting that de novo PCDH hypermethylation occurs during malignant progression. Discrete boundaries of the PCDH domain are delimited by abrupt changes in histone modifications; unmethylated genes flanking the LRES are associated with permissive marks which are absent from methylated genes within the domain. Silenced genes are marked with non-permissive histone 3 lysine 9 dimethylation. Expression analysis of embryonic murine kidney and differentiating rat metanephric mesenchymal cells demonstrates that Pcdh expression is developmentally regulated and that Pcdhg@ genes are expressed in blastemal cells. Importantly, we show that PCDHs negatively regulate canonical Wnt signalling, as short-interfering RNA–induced reduction of PCDHG@ encoded proteins leads to elevated β-catenin protein, increased β-catenin/T-cell factor (TCF) reporter activity, and induction of Wnt target genes. Conversely, over-expression of PCDHs suppresses β-catenin/TCF-reporter activity and also inhibits colony formation and growth of cancer cells in soft agar. Thus PCDHs are candidate tumor suppressors that modulate regulatory pathways critical in development and disease, such as canonical Wnt signaling.     

Asymmetric lipid bilayer formation stabilized by DNA at the air/water interface

The lipid bis(guanidinium)-tren-cholesterol (BGTC) is a cationic cholesterol derivative bearing guanidinium polar headgroups used for gene transfection either alone or formulated as liposomes with the zwitterionic lipid 1,2-di-[cis-9-octadecenoyl]-sn-glycero-3-phosphoethanolamine (DOPE). Previous investigations have shown its ability to strongly interact with DNA and form asymmetric lipid bilayers at the air/water interface when mixed with DOPE. Here, with a view to further investigate its physicochemical behavior, we studied the interactions of mixtures of BGTC with another zwitterionic lipid, 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine, (DMPC), with DNA at the air/water interface by using the Langmuir monolayer technique coupled with Brewster Angle Microscopy (BAM) and Polarization Modulation Infra Red Reflexion Absorption (PMIRRAS) spectroscopy and we investigate DNA–BGTC/DMPC interactions. We demonstrate that when DNA is injected into the subphase in excess compared to the positive charges of BGTC, it adsorbs to BGTC/DMPC monolayers at 20 mN/m whatever the lipid monolayer composition (1/5, 2/3 or 3/2 BGTC/DMPC molar ratio) and forms an incomplete monolayer of either isotropic or anisotropic double strands depending on the BGTC content in the monolayer. Compression beyond the collapse of some mixed DNA–BGTC/DMPC (2/3 and 3/2 molar ratio) systems leads to the formation of DNA monolayers underneath asymmetric lipid bilayers characterized by a bottom layer of BGTC in contact with DNA and a top layer mainly constituted of DMPC.     

Activity of the Enterococcus faecalis EIIA PTS component and its strong interaction with EIIB

Eubacteria can import and simultaneously phosphorylate a range of different carbohydrates by means of sugar specific phosphoenolpyruvate (PEP) dependent sugar phosphotransferase systems (PTSs). Here, we report the biochemical characterization of the gluconate specific PTS component EIIA^gnt from Enterococcus faecalis and its unexpectedly strong complex with EIIB^gnt. We analyze the activity of the complex regarding phosphoryl transfer using kinetic measurements and demonstrate by mutagenesis that His-9 of EIIA^gnt is essential for this process and represents most likely the phosphoryl group carrier of EIIA^gnt. With a combination of isothermal titration calorimetry (ITC), analytical ultracentrifugation (AUC), native gel electrophoresis and chemical crosslinking experiments we show that EIIA^gnt and EIIB^gnt form a strong 2:2 heterotetrameric complex, which seems to be destabilized upon phosphorylation of EIIB^gnt.     

Données nouvelles sur les mammifères et les nummulites de l'éocène supérieur subalpin (synclinaux du Charbon et d'entrevernes,Bauges, Savoie et Haute-Savoie)

Vertebrate remains and nummulites have been investigated from the Late Eocene—Early Oligocene transgressive sequence of the «Chaînes subalpines south of the lake Annecy. The sedimentary environments range from fluvio-lacustrine shales and conglomerates at the base to hemipelagic Globigerina shales at the top. The fluvio-lacustrine white marls contain mammals of the upper, probably terminal Ludian, with affinities to the assemblage of Saint-Capraise. The brackish Cerithium beds have furnished charophytes of the Bembridge zone. The nummulite limestones are not dated precisely, but isolated, probably redeposited nummulites from the base of the Globigerina shales are of terminal Eocene or basal Oligocene age. The Globigerina shales themselves belong to the Oligocene.