Mediation of pinocytosis in cultured arterial smooth muscle and endothelial cells by platelet-derived growth factor

Pinocytosis was measured in monkey aortic smooth muscle cells (SMC), bovine aortic endothelial cells, and Swiss 3T3 cells in culture as cellular uptake of [U-(14)C]sucrose and horseradish peroxidase (HRP) from the tissue culture medium. Monkey arterial SMC and Swiss 3T3 cells were maintained in a quiescent state of growth at low cells density in medium containing 5 percent monkey plasma-derived serum (PDS). Replacement of PDS with 5 percent monkey whole blood serum (WBS) from the same donor, or addition to PDS of partially purified platelet-derived growth factor(s) (PF), resulted in a marked stimulation of pinocytosis as well as of cellular proliferation. In SMC, enhancement of the rate of pinocytosis occurred 4-6 h after exposure to WBS or PF, and the rate was up to twofold higher than the rate in medium containing PDS. In contrast, [(3)H]thymidine uptake by SMC did not increase until 12-16 h after exposure to PF. In endothelial cells the presence of PF or WBS did not enhance either the rate of pinocytosis or the rate of proliferation over that in PDS. Thus, endothelial cells did not become quiescent at subconfluent densities in PDS but maintained rates of proliferation and pinocytosis that were equivalent to those in WBS. By autoradiography, the fraction of labeled nuclei in SMC cultures 24 h after change of medium increased from 0.061 +/- 0.004 in quiescent cultures to 0.313 +/- 0.028 after exposure to WBS or PF. In contrast, labeling indices of endothelial cells were similar for cultures grown in PDS, WBS, or PF at any single time point after change of medium. These findings suggest that the rate of pinocytosis maybe be coupled in some fashion to growth regulation, which may be mediated in part by specific growth factors, such as that derived from the thrombocyte.     

Degradation Pathway of Bisphenol A: Does ipso Substitution Apply to Phenols Containing a Quaternary α-Carbon Structure in the para Position?

The degradation of bisphenol A and nonylphenol involves the unusual rearrangement of stable carbon-carbon bonds. Some nonylphenol isomers and bisphenol A possess a quaternary α-carbon atom as a common structural feature. The degradation of nonylphenol in Sphingomonas sp. strain TTNP3 occurs via a type II ipso substitution with the presence of a quaternary α-carbon as a prerequisite. We report here a new degradation pathway of bisphenol A. Consequent to the hydroxylation at position C-4, according to a type II ipso substitution mechanism, the C-C bond between the phenolic moiety and the isopropyl group of bisphenol A is broken. Besides the formation of hydroquinone and 4-(2-hydroxypropan-2-yl)phenol as the main metabolites, further compounds resulting from molecular rearrangements consistent with a carbocationic intermediate were identified. Assays with resting cells or cell extracts of Sphingomonas sp. strain TTNP3 under an ¹⁸O₂ atmosphere were performed. One atom of ¹⁸O₂ was present in hydroquinone, resulting from the monooxygenation of bisphenol A and nonylphenol. The monooxygenase activity was dependent on both NADPH and flavin adenine dinucleotide. Various cytochrome P450 inhibitors had identical inhibition effects on the conversion of both xenobiotics. Using a mutant of Sphingomonas sp. strain TTNP3, which is defective for growth on nonylphenol, we demonstrated that the reaction is catalyzed by the same enzymatic system. In conclusion, the degradation of bisphenol A and nonylphenol is initiated by the same monooxygenase, which may also lead to ipso substitution in other xenobiotics containing phenol with a quaternary α-carbon.     

Degradation of a Nonylphenol Single Isomer by Sphingomonas sp. Strain TTNP3 Leads to a Hydroxylation-Induced Migration Product

Sphingomonas sp. strain TTNP3 degrades 4(3′,5′-dimethyl-3′-heptyl)-phenol and unidentified metabolites that were described previously. The chromatographic analyses of the synthesized reference compound and the metabolites led to their identification as 2(3′,5′-dimethyl-3′-heptyl)-1,4-benzenediol. This finding indicates that the nonylphenol metabolism of this bacterium involves unconventional degradation pathways where an NIH shift mechanism occurs.     

Genetic covariance between indices of body condition and immunocompetence in a passerine bird

Background  

Condition-dependence is a ubiquitous feature of animal life histories and has important implications for both natural and sexual selection. Mate choice, for instance, is typically based on condition-dependent signals. Theory predicts that one reason why condition-dependent signals may be special is that they allow females to scan for genes that confer high parasite resistance. Such explanations require a genetic link between immunocompetence and body condition, but existing evidence is limited to phenotypic associations. It remains unknown, therefore, whether females selecting males with good body condition simply obtain a healthy mate, or if they acquire genes for their offspring that confer high immunocompetence.     

Control of stem cell fate by engineering their micro and nanoenvironment

Stem cells are capable of long-term self-renewal and differentiation into specialised cell types, making them an ideal candidate for a cell source for regenerative medicine. The control of stem cell fate has become a major area of interest in the field of regenerative medicine and therapeutic intervention. Conventional methods of chemically inducing stem cells into specific lineages is being challenged by the advances in biomaterial technology, with evidence highlighting that material properties are capable of driving stem cell fate. Materials are being designed to mimic the clues stem cells receive in their in vivo stem cell niche including topographical and chemical instructions. Nanotopographical clues that mimic the extracellular matrix(ECM) in vivo have shown to regulate stem cell differentiation. The delivery of ECM components on biomaterials in the form of short peptides sequences has also proved successful in directing stem cell lineage. Growth factors responsible for controlling stem cell fate in vivo have also been delivered via biomaterials to provide clues to determine stem cell differentiation. An alternative approach to guide stem cells fate is to provide genetic clues including delivering DNA plasmids and small interfering RNAs via scaffolds. This review, aims to provide an overview of the topographical, chemical and molecular clues that biomaterials can provide to guide stem cell fate. The promising features and challenges of such approaches will be highlighted, to provide directions for future advancements in this exciting area of stem cell translation for regenerative medicine.     

Biodegradation of sulfamethoxazole by a bacterial consortium of Achromobacter denitrificans PR1 and Leucobacter sp. GP

In the last decade, biological degradation and mineralization of antibiotics have been increasingly reported feats of environmental bacteria. The most extensively described example is that of sulfonamides that can be degraded by several members of Actinobacteria and Proteobacteria. Previously, we reported sulfamethoxazole (SMX) degradation and partial mineralization by Achromobacter denitrificans strain PR1, isolated from activated sludge. However, further studies revealed an apparent instability of this metabolic trait in this strain. Here, we investigated this instability and describe the finding of a low-abundance and slow-growing actinobacterium, thriving only in co-culture with strain PR1. This organism, named GP, shared highest 16S rRNA gene sequence similarity (94.6–96.9%) with the type strains of validly described species of the genus Leucobacter. This microbial consortium was found to harbor a homolog to the sulfonamide monooxygenase gene (sadA) also found in other sulfonamide-degrading bacteria. This gene is overexpressed in the presence of the antibiotic, and evidence suggests that it codes for a group D flavin monooxygenase responsible for the ipso-hydroxylation of SMX. Additional side reactions were also detected comprising an NIH shift and a Baeyer–Villiger rearrangement, which indicate an inefficient biological transformation of these antibiotics in the environment. This work contributes to further our knowledge in the degradation of this ubiquitous micropollutant by environmental bacteria.

     

Quantification of the Influence of Extracellular Laccase and Intracellular Reactions on the Isomer-Specific Biotransformation of the Xenoestrogen Technical Nonylphenol by the Aquatic Hyphomycete Clavariopsis aquatica

The aquatic hyphomycete Clavariopsis aquatica was used to quantify the effects of extracellular laccase and intracellular reactions on the isomer-specific biotransformation of technical nonylphenol (t-NP). In laccase-producing cultures, maximal removal rates of t-NP and the isomer 4-(1-ethyl-1,4-dimethylpentyl)phenol (NP112) were about 1.6- and 2.4-fold higher, respectively, than in laccase-lacking cultures. The selective suppression of either laccase or intracellular reactions resulted in essentially comparable maximal removal rates for both compounds. Evidence for an unspecific oxidation of t-NP isomers was consistently obtained from laccase-expressing fungal cultures when intracellular biotransformation was suppressed and from reaction mixtures containing isolated laccase. This observation contrasts with the selective degradation of t-NP isomers by bacteria and should prevent the enrichment of highly estrogenic isomers in remaining t-NP. In contrast with laccase reactions, intracellular fungal biotransformation caused a significant shift in the isomeric composition of remaining t-NP. As a result, certain t-NP constituents related to more estrogenic isomers were less efficiently degraded than others. In contrast to bacterial degradation via ipso-hydroxylation, the substitution pattern of the quaternary α-carbon of t-NP isomers does not seem to be very important for intracellular transformation in C. aquatica. As-yet-unknown intracellular enzymes are obviously induced by nonylphenols. Mass spectral data of the metabolites resulting from the intracellular oxidation of t-NP, NP112, and 4-(1-ethyl-1,3-dimethylpentyl)phenol indicate nonyl chain hydroxylation, further oxidation into keto or aldehyde compounds, and the subsequent formation of carboxylic acid derivatives. Further metabolites suggest nonyl chain desaturation and methylation of carboxylic acids. The phenolic moieties of the nonylphenols remained unchanged.Nonylphenol ethoxylates (NPEOs) represent a major group of industrial nonionic surfactants. Technical nonylphenol (t-NP), used for the production of NPEOs, is synthesized by Friedel-Crafts alkylation of phenol with a mixture of differently branched nonenes. It therefore comprises a great variety of mainly para-substituted isomers, with variously branched nonyl chains. About 50 to 80 t-NP isomers were estimated to occur in environmentally relevant matrices (19). The incomplete bioconversion of NPEOs in wastewater treatment plants results in the formation of the less biodegradable t-NP and is considered a major source of this contaminant in the aquatic environment (57). The recalcitrance of t-NP to biodegradation is partly due to the presence in more than 85% of the t-NP isomers of a quaternary α-carbon in the branched nonyl chain. Such structural characteristics are considered to limit biological nonyl chain oxidation (11, 53, 55). Nonylphenols are known to disrupt normal endocrine functions in vertebrates (57). Certain isomers contained in t-NP have been reported to possess a considerably higher estrogenic activity than the t-NP mixture (15). Due to increasing concerns with respect to their largely unknown environmental fate and potentially adverse environmental and human health effects, nonylphenols have been listed as priority hazardous substances in the EU water framework directive.In light of the concerns above, microbial reactions with the potential to reduce nonylphenol concentrations in the environment but also offering new possibilities for applications such as effluent treatment have received increasing attention (11). Among environmental microorganisms, both aquatic and terrestrial fungi, as well as bacteria, have been shown to degrade t-NP (11). Fungal attack on nonylphenols differs from bacterial nonylphenol degradation. In the case of intracellular nonylphenol biotransformation reactions catalyzed by fungi, only metabolites modified in the alkyl chain have been described (23, 52). Metabolites indicative of oxidation of the phenolic ring have not been described to date. Bacterial degradation pathways have only been documented in the genera Sphingomonas and Sphingobium. Bacterial mineralization of the aromatic moiety of t-NP isomers to CO₂ and H₂O is initiated via ring hydroxylation at the ipso (C-4) position of the phenolic ring, and nonanols are produced from the nonyl chains (10, 11, 15, 16). Bacteria have been shown to utilize branched-chain nonylphenols as growth substrates (11, 12, 17, 43). In contrast, only one report describes the growth of a fungus, the yeast Candida aquaetextoris, on nonylphenol (the isomer 4-n-NP containing a linear nonyl chain) (52). With respect to fungal attack on t-NP, cometabolism seems to be the dominating process (11). Recent literature data indicate that certain t-NP isomers with an estrogenic potency higher than those of the original t-NP mixture can be enriched in remaining t-NP. This results from the selective removal of individual isomers upon bacterial ipso-substitution degradation mechanisms (15). However, the effects of fungal biotransformation reactions on the isomeric profile of t-NP have not yet been quantified.Laccases are extracellular multicopper oxidases. These have most frequently been described in white-rot basidiomycetes, which unspecifically oxidize via one-electron abstraction certain lignin constituents, as well many xenobiotic compounds. Thereby, organic radicals are generated as the primary oxidation products (3). Among the several groups of fungi found in aquatic environments, aquatic hyphomycetes (AQH) are a phylogenetically diverse group of mitosporic fungi specifically adapted to their exclusively aquatic lifestyle. AQH have been shown to metabolize several organic environmental pollutants, including t-NP (23), polycyclic musk fragrances (31), pesticide metabolites (2), and synthetic dyes (22). Therefore, with respect to the fungal attack on organic pollutants found in aquatic ecosystems, AQH are of special importance. Laccase production by strictly aquatic fungi such as AQH has already been demonstrated and discussed in the context of lignocellulose decay in aquatic ecosystems (1). A role of this enzyme in the AQH-catalyzed breakdown of aquatic environmental pollutants has been recently suggested. Here, laccase isolated from the AQH Clavariopsis aquatica was shown to act on nonylphenol (23) and polycyclic musk fragrances (31). Laccase has also been implicated in nonylphenol degradation by white-rot fungi (44, 45). Isolated extracellular laccases from several aquatic and terrestrial fungi were shown to catalyze the formation of oligomeric coupling products from nonylphenols via organic radical intermediates (6, 11). However, the effects of laccase reactions on the isomeric patterns of t-NP have not been assessed to date.The aim of the present study was to quantify the influence of extracellular laccase catalysis and intracellular biotransformation on nonylphenol removal rates and on the isomeric composition of t-NP. For this, C. aquatica was used as a model organism. The derived data were compared to effects of bacteria on nonylphenol isomers reported by other authors (15), and environmental and biotechnological implications of fungal t-NP biotransformation were deduced. At the same time we addressed metabolite formation from t-NP and the two major t-NP isomers 4-(1-ethyl-1,3-dimethylpentyl)phenol (NP111) and 4-(1-ethyl-1,4-dimethylpentyl)phenol (NP112) (Fig. ​(Fig.1).1). This was done to substantiate the apparent differences between fungi and bacteria in the intracellular oxidation of t-NP (11, 15).Open in a separate windowFIG. 1.Chemical structures of the nonylphenol isomers NP111 and NP112.