Synthesis and properties of anionic cellulose ethers: influence of functional groups and molecular weight on flowability of concrete

The suitability of anionic cellulose ethers as superplasticizers and the effect of chemical structure on the fluidity of cement mixtures was investigated. To elucidate the influence of molecular weight and degree of cellulose backbone substitution, cellulose and hydroxyethyl cellulose with molecular weights <50,000 g/mol were synthesized by acid-catalyzed and oxidative degradation. Commercial as well as degraded samples were functionalized by carboxymethylation and sulfobutylation, controlling the degree of substitution (DS) by the molar ratio of reactants and by taking advantage of the high reactivity of sultones towards salts of carboxylic acids, even in aqueous solutions. The fluidizing effect of the cellulose ethers with anionic ‘cement-anchoring‘ groups was prescreened, measuring the static flow of cement pastes. The results indicated a high potential of sulfobutylated cellulose mixed ethers as dispersing agents for concrete. The fluidizing action increased with increasing DS and an optimum range of molecular weight between 100,000 and 150,000 g/mol was found.     

Molecular and Functional Characterization of the Four-Transmembrane Molecule L6 in Epidermal Keratinocytes

In normal human epidermal keratinocytes (NHEK) proteolytic detachment from the substrate induces a complex activation cascade including expression of new proteins, morphological alterations, and the onset of migration for epidermal regeneration. By subtractive cloning we have shown that L6, a four-transmembrane protein, is newly expressed after proteolytic keratinocyte detachment. In this study, we have generated a novel anti-L6 antibody (clone HD-pKe#104-1.1) and investigated L6 expression regulation in vitro and in vivo as well as L6 function in keratinocyte migration. Dispase-mediated detachment induced L6 expression in NHEK at the mRNA and protein level. Immunohistology of skin biopsies displayed a strong expression of L6 in follicular epidermis and epidermolytic lesions of autoimmune bullous dermatoses (bullous pemphigoid, pemphigus vulgaris), but not in normal interfollicular epidermis. In contrast to normal keratinocytes, HaCaT cells showed constitutive L6 expression, indicating a constitutively active phenotype. After artificial wounding of confluent HaCaT cultures, anti-L6 antibody strongly impaired cell migration velocity and migratory reepithelization of the defect, indicating L6 involvement in keratinocyte migration. These findings suggest that L6 is an important activation-dependent regulator of keratinocyte function and epidermal tissue regeneration.     

Enterotoxigenic Potential of Staphylococcus intermedius

Staphylococcal food poisoning (SFP) caused by enterotoxigenic staphylococci is one of the main food-borne diseases. In contrast to Staphylococcus aureus, a systematic screening for the enterotoxins has not yet been performed on the genomic level for the coagulase-positive species S. intermedius. Therefore, the enterotoxigenic potential of 281 different veterinary (canine, n = 247; equine, n = 23; feline, n = 9; other, n = 2) and 11 human isolates of S. intermedius was tested by using a multiplex PCR DNA-enzyme immunoassay system targeting the staphylococcal enterotoxin genes sea, seb, sec, sed, and see. Molecular results were compared by in vitro testing of enterotoxin production by two immunoassays. A total of 33 (11.3%) S. intermedius isolates, including 31 (12.6%) canine isolates, 1 equine isolate, and 1 human isolate, tested positive for the sec gene. In vitro production of the respective enterotoxins was detected in 30 (90.9%) of these isolates by using immunological tests. In contrast, none of 65 veterinary specimen-derived isolates additionally tested and comprising 13 (sub)species of coagulase-negative staphylococci were found to be enterotoxigenic. This study shows on both molecular and immunological levels that a substantial number of S. intermedius isolates harbor the potential for enterotoxin production. Since evidence for noninvasive zoonotic transmission of S. intermedius from animal hosts to humans has been documented, an enterotoxigenic role of this microorganism in SFP via contamination of food products may be assumed.     

Characterization of the gltF gene product of Escherichia coli

The glt operon of Escherichia coli comprises the structural genes for the glutamate synthase subunits (gltB and gltD) and gltF, whose product was previously suggested to have regulatory functions. The A/T-rich region between gltD and gltF contains a weak promoter and a translation initiation site for gltF. The GltF protein is preceded by a signal peptide, which is cleaved off during export into the periplasmic space. A gltF::Km(R) insertion mutant was constructed and shown here to have no detectable phenotype with respect to amino acid utilization or ammonium transport. Thus, GltF is apparently not involved in regulation of nitrogen catabolism.     

Acrylyl-Coenzyme A Reductase,an Enzyme Involved in the Assimilation of 3-Hydroxypropionate by Rhodobacter sphaeroides

The anoxygenic phototroph Rhodobacter sphaeroides uses 3-hydroxypropionate as a sole carbon source for growth. Previously, we showed that the gene (RSP_1434) known as acuI, which encodes a protein of the medium-chain dehydrogenase/reductase (MDR) superfamily, was involved in 3-hydroxypropionate assimilation via the reductive conversion to propionyl-coenzyme A (CoA). Based on these results, we speculated that acuI encoded acrylyl-CoA reductase. In this work, we characterize the in vitro enzyme activity of purified, recombinant AcuI using a coupled spectrophotometric assay. AcuI from R. sphaeroides catalyzes the NADPH-dependent acrylyl-CoA reduction to produce propionyl-CoA. Two other members of the MDR012 family within the MDR superfamily, the products of SPO_1914 from Ruegeria pomeroyi and yhdH from Escherichia coli, were shown to also be part of this new class of NADPH-dependent acrylyl-CoA reductases. The activities of the three enzymes were characterized by an extremely low K_m for acrylyl-CoA (<3 μM) and turnover numbers of 45 to 80 s⁻¹. These homodimeric enzymes were highly specific for NADPH (K_m = 18 to 33 μM), with catalytic efficiencies of more than 10-fold higher for NADPH than for NADH. The introduction of codon-optimized SPO_1914 or yhdH into a ΔacuI::kan mutant of R. sphaeroides on a plasmid complemented 3-hydroxypropionate-dependent growth. However, in their native hosts, SPO_1914 and yhdH are believed to function in the metabolism of substrates other than 3-hydroxypropionate, where acrylyl-CoA is an intermediate. Complementation of the ΔacuI::kan mutant phenotype by crotonyl-CoA carboxylase/reductase from R. sphaeroides was attributed to the fact that the enzyme also uses acrylyl-CoA as a substrate.     

Serratia odorifera: analysis of volatile emission and biological impact of volatile compounds on Arabidopsis thaliana

Bacteria emit a wealth of volatiles. The combination of coupled gas chromatography/mass spectrometry (GC/MS) and proton-transfer-reaction mass spectrometry (PTR-MS) analyses provided a most comprehensive profile of volatiles of the rhizobacterium Serratia odorifera 4Rx13. An array of compounds, highly dominated by sodorifen (approximately 50%), a bicyclic oligomethyl octadiene, could be detected. Other volatiles included components of the biogeochemical sulfur cycle such as dimethyl disulfide (DMDS), dimethyl trisulfide and methanethiol, terpenoids, 2-phenylethanol, and other aromatic compounds. The composition of the bouquet of S. odorifera did not change significantly during the different growth intervals. At the beginning of the stationary phase, 60 μg of volatiles per 24 h and 60 easily detectable components were released. Ammonia was also released by S. odorifera, while ethylene, nitric oxide (NO) and hydrogen cyanide (HCN) could not be detected. Dual culture assays proved that 20 μmol DMDS and 2.5 μmol ammonia, individually applied, represent the IC₅₀ concentrations that cause negative effects on Arabidopsis thaliana.     

Raman microspectroscopic discrimination of TCam-2 cultures reveals the presence of two sub-populations of cells

TCam-2 cells are the main in vitro model for investigations into seminomatous tumors. However, despite their widespread use, questions remain regarding the cells’ homogeneity and consequently how representative they are of seminomas. We assess the TCam-2 cell line using routine and novel authentication methods to determine its homogeneity, identify any cellular sub-populations and resolve whether any changes could be due to generational differentiation. TCam-2, embryonal carcinoma cells (2102EP) and breast cancer cell (MCF7) lines were assessed using qRT-PCR, immunocytochemistry, flow cytometry and short tandem repeat analyses. Raman maps of individual cells (minimum of 10) and single scan spectra from 200 cells per culture were obtained. TCam-2s displayed the characteristic marker gene expression pattern for seminoma, were uniform in size and granularity and short tandem repeat analysis showed no contamination. However, based only on physical parameters, flowcytometry was unable to differentiate between TCam-2 and 2102EPs. Raman maps of TCam-2s comprised three equally distributed, distinct spectral patterns displaying large intercellular single spectral variation. All other cells showed little variation. Principal component, cluster and local spectral angle analyses indicated that the TCam-2s contained two different types of cells, one of which comprised two subgroups and was similar to some 2102EP cells. Protein expression corroborated the presence of different cells and generational differences. The detailed characterization provided by the Raman spectra, augmented by the routine methods, provide substantiation to the long-held suspicion that TCam-2 are not homogeneous but comprise differing cell populations, one of which may be embryonal carcinoma in origin.     

Alpha-Catulin Contributes to Drug-Resistance of Melanoma by Activating NF-κB and AP-1

Melanoma is the most dangerous type of skin cancer accounting for 48,000 deaths worldwide each year and an average survival rate of about 6-10 months with conventional treatment. Tumor metastasis and chemoresistance of melanoma cells are reported as the main reasons for the insufficiency of currently available treatments for late stage melanoma. The cytoskeletal linker protein α-catulin (CTNNAL1) has been shown to be important in inflammation, apoptosis and cytoskeletal reorganization. Recently, we found an elevated expression of α-catulin in melanoma cells. Ectopic expression of α-catulin promoted melanoma progression and occurred concomitantly with the downregulation of E-cadherin and the upregulation of mesenchymal genes such as N-cadherin, Snail/Slug and the matrix metalloproteinases 2 and 9. In the current study we showed that α-catulin knockdown reduced NF-κB and AP-1 activity in malignant melanoma cells. Further, downregulation of α-catulin diminished ERK phosphorylation in malignant melanoma cells and sensitized them to treatment with chemotherapeutic drugs. In particular, cisplatin treatment led to decreased ERK-, JNK- and c-Jun phosphorylation in α-catulin knockdown melanoma cells, which was accompanied by enhanced apoptosis compared to control cells. Altogether, these results suggest that targeted inhibition of α-catulin may be used as a viable therapeutic strategy to chemosensitize melanoma cells to cisplatin by down-regulation of NF-κB and MAPK pathways.