Differences in the expression of caveolin-1 isoforms in cancer-associated and normal fibroblasts of patients with oral squamous cell carcinoma.

OBJECTIVES: For many years, tumor development has been viewed as a cell-autonomous process; however, today we know that the tumor microenvironment (TME) and especially cancer-associated fibroblasts (CAFs) significantly contribute to tumor progression. Caveolin-1 (Cav-1) is a scaffolding protein which is involved in several cancer-associated processes as important component of the caveolae. Our goal was to shed light on the expression of the two different isoforms of Cav-1 in normal fibroblasts (NFs) and CAFs of patients with oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: Fibroblasts from normal mucosa and CAFs were isolated and propagated in vitro. Gene expression of the different Cav-1 isoforms was assessed via quantitative real-time PCR (qPCR) and supplemented by protein expression analysis. RESULTS: We could show that the Cav-1ß isoform is more highly expressed in NFs and CAFs compared to Cav-1α. Furthermore, the different Cav-1 isoforms tended to be differently expressed in different tumor stages. However, this trend could not be seen consistently, which is in line with the ambiguous role of Cav-1 in tumor progression described in literature. Western blotting furthermore revealed that NFs and CAFs might differ in the oligomerization profile of the Cav-1 protein. CONCLUSION: These differences in expression of Cav-1 between NFs and CAFs of patients with OSCC confirm that the protein might play a role in tumor progression and is of interest for further analyses. CLINICAL RELEVANCE: Our findings support a possible role of the two isoforms of Cav-1 in the malignant transformation of OSCC.

Adaptations to a Loss-of-Function Mutation in the Betaproteobacterium Aromatoleum aromaticum: Recruitment of Alternative Enzymes for Anaerobic Phenylalanine Degradation.

Anaerobic phenylalanine (Phe) degradation in the betaproteobacterium Aromatoleum aromaticum involves transamination and decarboxylation to phenylacetaldehyde, followed by oxidation to phenylacetate. The latter reaction is catalyzed simultaneously by two enzymes, a highly specific phenylacetaldehyde dehydrogenase (PDH) and a rather unspecific tungsten-dependent aldehyde oxidoreductase (AOR). Attempting to establish increased synthesis of AOR, we constructed a mutant lacking the gene for PDH. This mutant still grew on phenylalanine, exhibiting increased AOR activities on medium containing tungstate. In the absence of tungstate, the mutant showed initially severe growth deficiency, but it resumed growth on Phe after longer incubation times. Moreover, the growth rates of the mutant increased during several reinoculation cycles on either tungstate-proficient or -deficient media, reaching the same values as recorded in wild-type strains. We confirmed AOR as the major alternative enzyme serving Phe degradation under tungstate-supplied conditions and identified and characterized the alternative NAD-dependent aldehyde dehydrogenase AldB taking over the function under tungstate-deficient conditions. Sequence analysis of the respective genes from adapted cultures under either growth condition revealed a mutation in the upstream region of the aor operon and a mutation within the coding region of aldB, which are likely involved in the observed adaptation of the deletion mutant to regain fast growth on Phe.IMPORTANCE The betaproteobacterium Aromatoleum aromaticum degrades many aromatic compounds under denitrifying conditions. One of the steps of phenylalanine degradation is catalyzed by two simultaneously induced enzymes, a NAD(P)-dependent phenylacetaldehyde dehydrogenase and a W-containing aldehyde oxidoreductase. We report here that the latter fully complements a constructed deletion mutant lacking the gene for phenylacetaldehyde dehydrogenase and is overproduced after several reinoculations. Moreover, an alternative NAD-dependent dehydrogenase is recruited to resume growth in tungstate-free medium, which does not allow the production of aldehyde oxidoreductase. This alternative enzyme is overproduced and seems to have acquired a point mutation in the active center. Our research illustrates the flexibility of environmentally important bacteria in adapting their metabolic pathways to new challenges within only a few generations.

Asymmetric reduction of ketones and ß-keto esters by (S)-1-phenylethanol dehydrogenase from denitrifying bacterium Aromatoleum aromaticum.

Enzyme-catalyzed enantioselective reductions of ketones and keto esters have become popular for the production of homochiral building blocks which are valuable synthons for the preparation of biologically active compounds at industrial scale. Among many kinds of biocatalysts, dehydrogenases/reductases from various microorganisms have been used to prepare optically pure enantiomers from carbonyl compounds. (S)-1-phenylethanol dehydrogenase (PEDH) was found in the denitrifying bacterium Aromatoleum aromaticum (strain EbN1) and belongs to the short-chain dehydrogenase/reductase family. It catalyzes the stereospecific oxidation of (S)-1-phenylethanol to acetophenone during anaerobic ethylbenzene mineralization, but also the reverse reaction, i.e., NADH-dependent enantioselective reduction of acetophenone to (S)-1-phenylethanol. In this work, we present the application of PEDH for asymmetric reduction of 42 prochiral ketones and 11 ß-keto esters to enantiopure secondary alcohols. The high enantioselectivity of the reaction is explained by docking experiments and analysis of the interaction and binding energies of the theoretical enzyme-substrate complexes leading to the respective (S)- or (R)-alcohols. The conversions were carried out in a batch reactor using Escherichia coli cells with heterologously produced PEDH as whole-cell catalysts and isopropanol as reaction solvent and cosubstrate for NADH recovery. Ketones were converted to the respective secondary alcohols with excellent enantiomeric excesses and high productivities. Moreover, the progress of product formation was studied for nine para-substituted acetophenone derivatives and described by neural network models, which allow to predict reactor behavior and provides insight on enzyme reactivity. Finally, equilibrium constants for conversion of these substrates were derived from the progress curves of the reactions. The obtained values matched very well with theoretical predictions.

Prevalence of suicidal ideation in German psychotherapy outpatients: A large multicenter assessment.

BACKGROUND: Suicidal ideation is a major concern in clinical practice. Yet, little is known about prevalence rates of suicidal ideation in patients undergoing outpatient psychotherapeutic treatment. Therefore, the aim of the current study is to assess the prevalence of suicidal ideation in a large sample of psychotherapy outpatients in Germany. The data analyzed in this study is taken from the KODAP-project on the coordination of data collection and analysis at German university-based research and training outpatient clinics for psychotherapy. METHODS: A total of N = 10,357 adult outpatients (64.4 % female; age: M(SD) = 35.94 (13.54), range: 18-92 years of age) starting cognitive-behavioral therapy at one of 27 outpatient clinics in Germany were included in the current study. Prevalence of suicidal ideation was assessed with the Suicide Item (Item 9) of the Beck-Depression Inventory II. RESULTS: Suicidal ideation was reported by 36.7 % (n = 3795) of the participants. Borderline Personality Disorder, Posttraumatic Stress Disorder, and recurrent Major Depression were the diagnoses most strongly associated with the presence and severity of suicidal ideation. LIMITATION: Suicide ideation was assessed only with the respective item of the Beck Depression Inventory II. CONCLUSION: Suicidal ideation is very common among adult patients who start psychotherapy in Germany. A well-founded knowledge of risk assessment in suicidal patients and suicide-specific treatment options is therefore highly relevant.

[Excisional surgery of orbital tumors]. / Exzisionale Chirurgie orbitaler Tumoren.

The indications for orbital tumor surgery are an incisional biopsy to confirm the diagnosis or in malignant operable tumors a complete excision or a debulking to avoid complications in large invasively infiltrating tumors. In the case of benign tumors, the indications for surgery depend mostly on the clinical symptoms and cosmetic esthetic disfigurement. In the present article the preoperative examinations as well as surgical access approaches to different orbital regions, endoscopic procedures and methods of intraoperative navigation are presented. Magnetic resonance imaging is the instrument of choice, whereby in many cases computed tomography (CT) adds further information. Depending on the indications, diffusion-weighted sequences, CT angiography and digital subtraction angiography (DSA, catheter angiography) are added to the preoperative diagnostics. For space-occupying lesions located anterior to the bulbar equator, an anterior orbitotomy can be performed transconjunctivally or transpalpebrally. A lateral orbitotomy is used to reach lateral, laterocranial, and lateroinferior orbital segments, whereas transcranial approaches are suitable for processes located far posterior and for those with retro-orbital intracranial extension as well as for processes in the optic foramen/superior orbital fissure. The indications for an endonasal access approach are processes medial to the bulb or optic nerve and up to the orbital apex. A transantral access can be chosen for caudal, mediolateral, and medioinferior space-occupying lesions. Modern orbital surgery is complemented by endoscopic procedures and intraoperative navigation. Orbital tumors belong to the interdisciplinary relevant diseases. Therefore, an optimal management takes place at specialized multidisciplinary centers.

Selenoprotein synthesis: an expansion of the genetic code.

A number of enzymes employ the unusual amino acid selenocysteine as part of their active site because of its high chemical reactivity. Selenocysteine is incorporated into these proteins co-translationally: biosynthesis occurs on a specific tRNA and insertion into a growing polypeptide is directed by a UGA codon in the mRNA. In E. coli, this requires a specific translation factor. Selenocysteine thus represents a unique expansion of the genetic code.

Investigation of toxicity and mutagenicity of cold atmospheric argon plasma.

Cold atmospheric argon plasma is recognized as a new contact free approach for the decrease of bacterial load on chronic wounds in patients. So far very limited data are available on its toxicity and mutagenicity on eukaryotic cells. Thus, the toxic/mutagenic potential of cold atmospheric argon plasma using the MicroPlaSter ß® , which has been used efficiently in humans treating chronic and acute wounds, was investigated using the XTT assay in keratinocytes and fibroblasts and the HGPRT (hypoxanthine guanine phosphoribosyl transferase) assay with V79 Chinese hamster cells. The tested clinical parameter of a 2 min cold atmospheric argon plasma treatment revealed no relevant toxicity on keratinocytes (viability: 76% ± 0.17%) and on fibroblasts (viability: 81.8 ± 0.10) after 72 hr as compared to the untreated controls. No mutagenicity was detected in the HGPRT assay with V79 cells even after repetitive CAP treatments of 2-10 min every 24 hr for up to 5 days. In contrast, UV-C irradiation of V79 cells, used as a positive control in the HGPRT test, led to DNA damage and mutagenic effects. Our findings indicate that cold atmospheric plasma using the MicroPlaSter ß® shows negligible effects on keratinocytes and fibroblasts but no mutagenic potential in the HGPRT assay, indicating a new contact free safe technology. Environ. Mol. Mutagen. 58:172-177, 2017. © 2017 Wiley Periodicals, Inc.

A new family of CoA-transferases.

CoA-transferases are found in organisms from all lines of descent. Most of these enzymes belong to two well-known enzyme families, but recent work on unusual biochemical pathways of anaerobic bacteria has revealed the existence of a third family of CoA-transferases. The members of this enzyme family differ in sequence and reaction mechanism from CoA-transferases of the other families. Currently known enzymes of the new family are a formyl-CoA: oxalate CoA-transferase, a succinyl-CoA: (R)-benzylsuccinate CoA-transferase, an (E)-cinnamoyl-CoA: (R)-phenyllactate CoA-transferase, and a butyrobetainyl-CoA: (R)-carnitine CoA-transferase. In addition, a large number of proteins of unknown or differently annotated function from Bacteria, Archaea and Eukarya apparently belong to this enzyme family. Properties and reaction mechanisms of the CoA-transferases of family III are described and compared to those of the previously known CoA-transferases.

Antiatherogenic properties of calcium antagonists. State of the art.

Atherosclerosis is an arterial disease characterized by localized accumulation of collagen, elastin, lipids, and calcium at sites associated with macrophage infiltration and altered smooth muscle metabolism. Studies in several types of animal models, especially cholesterol-fed rabbits, have shown that calcium competitors, calcium chelators, anticalcifying agents, and calcium antagonists can reduce the accumulation of atherogenic lesion components and decrease the progression of lesions. Although there are some conflicting data in the animal model studies, it is now apparent that several classes of calcium antagonists inhibit the progression of early arterial lesions induced by cholesterol-feeding in animals. The dihydropyridine class of calcium antagonists may be more potent as anti-atherosclerotic agents than the other classes. Mechanisms involving regulation of endothelial cell, smooth muscle cell, and macrophage metabolism may be responsible for the effects of calcium antagonists on early lesion progression. Recent studies in cell culture-model systems suggest that calcium antagonists may significantly alter activities that regulate lipoprotein-derived cholesterol accumulation by arterial wall cells. Some of these activities are independent of calcium flux across voltage-operated calcium channels. Thus, calcium antagonists may reduce the progression of atherogenic lesions by a combination of decreasing calcium accumulation within arterial wall cells and by altering calcium channel-independent metabolic activities, which affect lesion development.

Specific bradycardic agents. 2. Heteroaromatic modifications in the side chain of specific bradycardic benzazepinones: chemistry, pharmacology, and structure-activity relationships.

Compound 1 (UL-FS 49) has recently been described as the representative of a novel class of antiischemic compounds termed "specific bradycardic agents". In search of specific bradycardic agents with different pharmacokinetic profiles, heteroaromatic analogues of 1 have been synthesized and evaluated for their bradycardic activity, selectivity, and duration of action. The chain length n and the nature of the heteroaromatic system of compounds 2 strongly determine the biological activities. Unsubstituted benzothiophenes and benzofurans in combination with a chain length of n = 2 give the most active bradycardic compounds. Some of the new compounds combine high bradycardic potency and selectivity with a short duration of action and may thus be useful for the development of short-acting specific bradycardic drugs.

Specific bradycardic agents. 1. Chemistry, pharmacology, and structure-activity relationships of substituted benzazepinones, a-new class of compounds exerting antiischemic properties.

Structural modification of the calcium-antagonist verapamil (1) by replacement of the lipophilic alpha-isopropylacetonitrile moiety by various heterocyclic ring systems has led to a new class of cardiovascular compounds which are characterized by a specific bradycardic activity. These agents reduce heart rate without binding to classical calcium channels or beta-adrenoceptors, interacting instead specifically with structures at the sino atrial node. Therefore they have also been termed sinus node inhibitors. The prototype falipamil (2) has been submitted to further optimization mainly by manipulation of the phthalmidine moiety. This has resulted in a second generation of specific bradycardic agents with increased potency and selectively and prolonged duration of action represented by the benzazepinone-derivative UL-FS 49 (4). Structure-activity relationships within this novel class of compounds have revealed a marked dependence of activity on the substitution pattern of the aromatic rings, the nature of the central nitrogen atom, and the length of the connecting alkyl chains. The crucial role of the benzazepinone ring for bradycardic activity can be best explained by its special impact on the overall molecular conformation.

Antiatherogenic properties of calcium antagonists.

A generalized accumulation of cholesterol, calcium and matrix materials (collagen, elastin and proteoglycans) occurs in an age-dependent manner in major arteries. Human atherogenesis is a disease of arteries characterized by a focal accumulation of fibrous matrix elements, lipids and calcium at lesion sites. Studies in cholesterol-fed animal models have indicated that calcium competitors and chelating agents can reduce calcium, lipid and matrix accumulation in arterial lesions and reduce the extent of lesion formation. These agents generally alter soft and hard tissue calcium pools or have deleterious side-effect profiles. Antiatherogenic studies with calcium antagonists (which have been shown to be safe in human clinical studies) have created confusion because of conflicting results. It is apparent, however, that high doses of calcium antagonists can significantly decrease atherogenic lesion development in cholesterol-fed rabbits. The antiatherogenic effects of calcium antagonists may be the result of changes in intracellular calcium pools within smooth muscle cells, which may lead to alterations in cellular metabolic activity or may be due to activities not related to calcium channel effects. Several mechanisms involving regulation of lipoprotein receptor synthesis, lipoprotein uptake or degradation, cholesterol ester hydrolytic activity and arterial matrix synthesis are discussed as potential sites of activity for calcium antagonists. A dihydropyridine channel antagonist, PN 200-110 (isradipine), has been shown to be a very potent antiatherogenic agent in the rabbit and also to be a potent inhibitor of smooth muscle cell matrix synthesis.

Protective action of calcium channel antagonists in atherogenesis and experimental vascular injury.

Experimental research using in vitro and in vivo models of vascular injury have delineated several common mechanisms that characterize the arterial damage in diseases such as atherosclerosis and hypertension. Changes in endothelial permeability, smooth muscle cell proliferation, and accumulation of connective tissue matrix are major common mechanisms. Chronic hyperlipidemia is a major determinant of the proliferative arterial lesions in atherogenic models. Calcium antagonists of very diverse structure and function have been shown to have antiatherogenic potential in several animal model systems of arterial injury. Calcium channel-blockers of several chemical classes have been demonstrated to alter endothelial function, intimal smooth muscle proliferation, and lipid accumulation in the arterial wall. Cell culture model systems have elucidated several potential mechanisms that may contribute to the antiatherogenic potential of the calcium channel-blockers. These activities may in part involve protection of arterial cells from calcium overload via inhibition of calcium flux across voltage-regulated ion channels. However, other activities of these drugs, such as inhibition of cholesterol esterification and matrix protein formation, appear to function independently of calcium flux. A hypothesis is presented that lipophilic calcium channel-blockers are accumulated in cell membranes and perturb metabolic function as a result of altering local membrane structure.

A two-component system involved in regulation of anaerobic toluene metabolism in Thauera aromatica.

The genes for a two-component regulatory system of the denitrifying toluene-degrading bacterium Thauera aromatica were identified immediately upstream of the genes for benzylsuccinate synthase (bssDCAB), the first enzyme involved in anaerobic toluene metabolism. The genes apparently encode the regulators of toluene catabolic enzymes and were therefore termed tdiSR (for toluene degradation including sensor and regulator). The tdiR gene product was overproduced in Escherichia coli and assayed for binding to a DNA fragment containing the 5' region of the bss operon. We observed specific DNA binding with cell extracts containing overproduced TdiR, but not with control extracts. The tdiSR genes are almost identical to two genes of Thauera strain T1, which have not been assigned a function so far. In addition, the derived gene products share similarity with regulators of toluene and styrene catabolic pathways in aerobic Pseudomonas species, and with the tutCB gene products of Thauera strain T1. The latter have previously been implicated in regulating anaerobic toluene metabolism. Our data suggest that toluene catabolism under aerobic and anaerobic conditions is regulated by similar, but distinct two-component systems.

Phototrophic utilization of toluene under anoxic conditions by a new strain of blastochloris sulfoviridis

The capacity of anoxygenic phototrophic bacteria to utilize aromatic hydrocarbons was investigated in enrichment cultures with toluene. When mineral medium with toluene (provided in an inert carrier phase) was inoculated with activated sludge and incubated under infrared illumination (> 750 nm), a red-to-brownish culture developed. Agar dilution series indicated the dominance of two types of phototrophic bacteria. One type formed red colonies, had rod-shaped cells with budding division, and grew on benzoate but not on toluene. The other type formed yellow-to-brown colonies, had oval cells, and utilized toluene and benzoate. One strain of the latter type, ToP1, was studied in detail. Sequence analysis of the 16S rRNA gene and DNA-DNA hybridization indicated an affiliation of strain ToP1 with the species Blastochloris sulfoviridis, a member of the alpha-subclass of Proteobacteria. However, the type strain (DSM 729) of Blc. sulfoviridis grew neither on toluene nor on benzoate. Light-dependent consumption of toluene in the presence of carbon dioxide and formation of cell mass by strain ToP1 were demonstrated in quantitative growth experiments. Strain ToP1 is the first phototrophic bacterium shown to utilize an aromatic hydrocarbon. In the supernatant of toluene-grown cultures and in cell-free extracts incubated with toluene and fumarate, the formation of benzylsuccinate was detected. These findings indicate that the phototrophic bacterium activates toluene anaerobically by the same mechanism that has been reported for denitrifying and sulfate-reducing bacteria. The natural abundance of phototrophic bacteria with the capacity for toluene utilization was examined in freshwater habitats. Counting series revealed that up to around 1% (1.8 x 10(5) cells per gram dry mass of sample) of the photoheterotrophic population cultivable with acetate grew on toluene.

Suitability of the hydrocarbon-hydroxylating molybdenum-enzyme ethylbenzene dehydrogenase for industrial chiral alcohol production.

The molybdenum/iron-sulfur/heme protein ethylbenzene dehydrogenase (EbDH) was successfully applied to catalyze enantiospecific hydroxylation of alkylaromatic and alkylheterocyclic compounds. The optimization of the synthetic procedure involves use of the enzyme in a crude purification state that saves significant preparation effort and is more stable than purified EbDH without exhibiting unwanted side reactions. Moreover, immobilization of the enzyme on a crystalline cellulose support and changes in reaction conditions were introduced in order to increase the amounts of product formed (anaerobic atmosphere, electrochemical electron acceptor recycling or utilization of ferricyanide as alternative electron acceptor in high concentrations). We report here on an extension of effective enzyme activity from 4h to more than 10 days and final product yields of up to 0.4-0.5g/l, which represent a decent starting point for further optimization. Therefore, we expect that the hydrocarbon-hydroxylation capabilities of EbDH may be developed into a new process of industrial production of chiral alcohols.

Anaerobic toluene catabolism of Thauera aromatica: the bbs operon codes for enzymes of beta oxidation of the intermediate benzylsuccinate.

The pathway of anaerobic toluene oxidation to benzoyl coenzyme A (benzoyl-CoA) consists of an initial reaction catalyzed by benzylsuccinate synthase, a glycyl radical enzyme adding the methyl group of toluene to the double bond of a fumarate cosubstrate, and a subsequent beta-oxidation pathway of benzylsuccinate. Benzylsuccinate synthase has been studied in some detail, whereas the enzymes participating in beta oxidation of benzylsuccinate are unknown. We have investigated these enzymes by analyzing substrate-induced proteins in toluene-grown cells. Toluene-induced proteins were identified and N-terminally sequenced. Nine of these proteins are encoded by an 8.5-kb operon consisting of bbs (beta-oxidation of benzylsuccinate) genes whose products are apparently involved in the beta-oxidation pathway of benzylsuccinate. Two of the genes, bbsE and bbsF, code for the subunits of a succinyl-CoA:benzylsuccinate CoA-transferase whose activity was previously detected in toluene-grown Thauera aromatica. The bbsG gene codes for a specific benzylsuccinyl-CoA dehydrogenase, as confirmed by overexpression of the gene in Escherichia coli and detection of enzyme activity. The further enzymes of the pathway are probably encoded by bbsH (enoyl-CoA hydratase), bbsCD (3-hydroxyacyl-CoA dehydrogenase), and bbsB (3-oxoacyl-CoA thiolase). The operon contains two additional genes, bbsA and bbsI, for which no obvious function could be derived. The bbs operon is expressed only in toluene-grown cells and is regulated at the transcriptional level. Promoter mapping revealed a transcription start site upstream of the bbsA gene. This represents the first known promoter site in Thauera spp.