Significance of chemical affinity on metal subcellular distribution in yellow perch (Perca flavescens) livers from Lake Saint-Pierre (QUEBEC, Canada).

The subcellular partitioning approach provides useful information on the location of metals within cells and is often used on organisms with high levels of bioaccumulation to establish relationships between the internal concentration and the potential toxicity of metals. Relatively little is known about the subcellular partitioning of metals in wild fish with low bioaccumulation levels in comparison with those from higher contaminated areas. This study aims to examine the subcellular partitioning of various metals considering their chemical affinity and essentiality at relatively low contamination levels. Class A (Y, Sr), class B (Cu, Cd, MeHg), and borderline (Fe, Mn) metal concentrations were measured in livers and subcellular fractions of yellow perch (n = 21) collected in Lake Saint-Pierre, QC, Canada. The results showed that all metals, apart from MeHg, were distributed among subcellular fractions according to their chemical affinity. More than 60% of Y, Sr, Fe, and Mn were found in the metal-sensitive fractions. Cd and Cu were largely associated with the metallothionein-like proteins and peptides (60% and 67% respectively) whereas MeHg was found mainly in the metal-sensitive fractions (86%). In addition, the difference between the subcellular distribution of Cu and other essential metals like Fe and Mn denotes that, although the essentiality of some metals is a determinant of their subcellular distribution, the chemical affinity of metals is also a key driver. The similarity of the subcellular partitioning results with previous studies on yellow perch and other fish species from higher contaminated areas supports the idea that metals are distributed in the cellular environment according to their chemical properties regardless of the bioaccumulation gradient.

Synthesis and Diversification of Macrocyclic Alkynediyl Sulfide Peptides.

The synthesis of rare macrocyclic alkynediyl sulfides by a Cu-catalyzed Csp -S cross-coupling is presented. The catalytic protocol (Cu(MeCN)4 PF6 /dtbbpy) promotes macrocyclization of peptides, dipeptides and tripeptides at ambient temperature (14 examples, 23→73 % yields) via thiols and bromoalkynes, and is chemoselective with regards to terminal alkynes. Importantly, the underexplored alkynediyl sulfide functionality incorporates a rigidifying structural element and opens new opportunities for diversification of macrocyclic frameworks through S oxidation, halide addition and azide-alkyne cycloaddition chemistries to integrate sulfones, halides or valuable fluorophores (7 examples, 37→92 % yields).

General Cu-Catalyzed Csp-S Coupling.

Copper-catalyzed cross-coupling of thiols and bromoalkynes affords a mild, rapid, and selective Csp-S coupling with broad scope, enabling the use of aryl-, alkyl-, and silyl-substituted alkynyl coupling partners (38 total examples, 50-99% yields). Importantly, the method enables the preparation of difficult-to-access bis-heteroatom-functionalized (S,S-, S,P-, and S,N-) alkynes.

Role of prey subcellular distribution on the bioaccumulation of yttrium (Y) in the rainbow trout.

Our knowledge of the processes leading to the bioaccumulation of rare earth elements (REE) in aquatic biota is limited. As the contamination of freshwater ecosystems by anthropogenic REE have recently been reported, it becomes increasingly urgent to understand how these metals are transferred to freshwater organisms in order to develop appropriate guidelines. We exposed rainbow trout (Oncorhynchus mykiss) to an REE, yttrium (Y), to either a range of Y-contaminated prey (Daphnia magna) or a range of Y-contaminated water. For the feeding experiment, the relationship between the Y assimilation by O. mykiss and the Y subcellular fractionation in D. magna was evaluated. Assimilation efficiency of Y by O. mykiss was low, ranging from 0.8 to 3%. These values were close to the proportion of Y accumulated in D. magna cytosol, 0.6-2%, a theoretical trophically available fraction. Moreover, under our laboratory conditions, water appeared as a poor source of Y transfer to O. mykiss. Regardless of the source of contamination, a similar pattern of Y bioaccumulation among O. mykiss tissues was revealed: muscles < liver < gills < intestine. We conclude that the trophic transfer potential of Y is low and the evaluation of Y burden in prey cytosol appears to be a relevant predictor of Y assimilation by their consumers.

Toxicity and Subcellular Fractionation of Yttrium in Three Freshwater Organisms: Daphnia magna, Chironomus riparius, and Oncorhynchus mykiss.

The demand for rare earth elements (REEs) has increased since the 1990s leading to the development of many mining projects worldwide. However, less is known about how organisms can handle these metals in natural aquatic systems. Through laboratory experiments, we assessed the chronic toxicity and subcellular fractionation of yttrium (Y), one of the four most abundant REEs, in three freshwater organisms commonly used in aquatic toxicology: Daphnia magna, Chironomus riparius, and Oncorhynchus mykiss. In bioassays using growth as an end point, C. riparius was the only organism showing toxicity at Y exposure concentrations close to environmental ones. The lowest observable effect concentrations (LOECs) of Y assessed for D. magna and O. mykiss were at least 100 times higher than the Y concentration in natural freshwater. A negative correlation between Y toxicity and water hardness was observed for D. magna. When exposed to their respective estimated LOECs, D. magna bioaccumulated 15-45 times more Y than the other two organisms exposed to their own LOECs. This former species sequestered up to 75% of Y in the NaOH-resistant fraction, a putative metal-detoxified subcellular fraction. To a lesser extent, C. riparius bioaccumulated 20-30% of Y in this detoxified fraction. In contrast, the Y subcellular distribution in O. mykiss liver did not highlight any notable detoxification strategy; Y was accumulated primarily in mitochondria (ca. 32%), a putative metal-sensitive fraction. This fraction was also the main sensitive fraction where Y accumulated in C. riparius and D. magna. Hence, the interaction of Y with mitochondria could explain its toxicity. In conclusion, there is a wide range of subcellular handling strategies for Y, with D. magna accumulating high quantities but sequestering most of it in detoxified fractions, whereas O. mykiss tending to accumulate less Y but in highly sensitive fractions.

The fish or the egg: Maternal transfer and subcellular partitioning of mercury and selenium in Yellow Perch (Perca flavescens).

Mercury (Hg) is a trace element of particular concern since it is ubiquitous in the environment and because its methylated form (MeHg) readily bioaccumulates and biomagnifies in food webs. This latter process leads to elevated Hg concentrations in fish and may thus induce toxicity. Maternal transfer of bioaccumulated contaminants to offspring is a suggested mechanism of impaired reproductive success in fish. The purpose of this study was to assess the toxicity potential of Hg during maternal transfer in Yellow Perch from Lake Saint-Pierre (Quebec, Canada) using a subcellular partitioning approach. We also evaluated potential protective effects of selenium, as this element has been shown to alleviate Hg toxicity through sequestration. A customized subcellular partitioning protocol was used to separate liver and gonad of Yellow Perch into various subcellular fractions. Results show that, in the liver, MeHg was primarily (51%) associated to the subcellular fraction containing cytosolic enzymes. Furthermore, 23% and 15% of MeHg was found in hepatic and gonadal mitochondria, respectively, suggesting that Yellow Perch is not effectively detoxifying this metal. There was also a strong relationship (R2 = 0.73) between MeHg bioaccumulation in the liver and MeHg concentrations in gonadal mitochondria, which corroborates the potential risk linked to MeHg maternal transfer. On the other hand, we also found that selenium might have a protective effect on Hg toxicity at a subcellular level. In fact, Se:Hg molar ratios in subcellular fractions were systematically above 1 in all tissues and fractions examined, which corresponds to the suggested protective threshold. This study provides the first assessment of subcellular Se:Hg molar ratios in fish. Since early developmental stages in aquatic biota are particularly sensitive to Hg, this study represents a step forward in understanding the likelihood for toxic effects in wild fish through maternal transfer.

Subcellular partitioning of metals and metalloids (As, Cd, Cu, Se and Zn) in liver and gonads of wild white suckers (Catostomus commersonii) collected downstream from a mining operation.

In the present study, we examined the subcellular distribution of metals and metalloids (As, Cd, Cu, Se and Zn) in the liver and gonads of wild white suckers (Catostomus commersonii) collected downstream from a metal mining operation (exposure area) and in a reference area. Metal partitioning among potentially metal-sensitive fractions (heat-denatured proteins (HDP), mitochondria and microsomes) and potentially biologically detoxified fractions (heat-stable proteins (HSP) and metal-rich granules) within cells was determined after differential centrifugation, NaOH digestion and heat-denaturation steps. Metal-handling strategies between liver and gonads, and between sexes, were examined. Hepatic metal concentrations were significantly higher in exposed compared to reference fish, especially for Se (14x), Cd (5x) and Cu (3x), and did not vary between sexes. In contrast, gonadal Cd, Cu, Se and Zn concentrations were consistently lower in testes than in ovaries; marked differences in Cd and Se concentrations between exposed and reference fish were observed for both sexes. Overall, metal-handling strategies were similar in both liver (male and female pooled) and female gonads, but differed from those in male gonads, likely due to the different functions assigned to ovaries and testes. Subcellular partitioning of As, Cd and Cu showed that the HSP fraction was most responsive to increased metal exposure, presumably reflecting Cu regulation, and possibly Cd and As detoxification. Zinc concentrations were tightly controlled and mainly found in the HDP fraction. Interestingly, changes in Cd-handling strategy in female gonads were particularly evident, with Cd shifting dramatically from the metal-sensitive HDP fraction in reference fish to the metal-detoxified HSP fraction in exposed fish. It seems that Cd detoxification in female gonads was not fully induced in the less contaminated fish, but became more effective above a threshold Cd concentration of 0.05 nmol/g dry weight. Partitioning of Se was different, with the largest contributor to the total liver and gonad Se burdens being the putative metal-sensitive HDP fraction, suggesting that excess Se in this fraction in exposed fish may lead to Se-related stress. The present subcellular partitioning results demonstrate that metal handling strategies vary among metals, between organs and (in some cases) as a function of metal exposure. They also show promise in identifying metals of potential concern in a risk assessment context.

Heteroleptic Copper(I)-Based Complexes for Photocatalysis: Combinatorial Assembly, Discovery, and Optimization.

A library of 50 copper-based complexes derived from bisphosphines and diamines was prepared and evaluated in three mechanistically distinct photocatalytic reactions. In all cases, a copper-based catalyst was identified to afford high yields, where new heteroleptic complexes derived from the bisphosphine BINAP displayed high efficiency across all reaction types. Importantly, the evaluation of the library of copper complexes revealed that even when photophysical data is available, it is not always possible to predict which catalyst structure will be efficient or inefficient in a given process, emphasizing the advantages for catalyst structures with high modularity and structural variability.

Binding of trace elements (Ag, Cd, Co, Cu, Ni, and Tl) to cytosolic biomolecules in livers of juvenile yellow perch (Perca flavescens) collected from lakes representing metal contamination gradients.

Biomolecules involved in handling cytosolic metals in the liver of the yellow perch (Perca flavescens) were characterized in juvenile fish collected from 4 lakes constituting metal contamination gradients. Using size-exclusion liquid chromatography coupled to an inductively coupled mass spectrometer, we determined metal distributions among ligands of different molecular weights in the cytosol, before and after a heat denaturation step designed to isolate metallothionein-like peptides and proteins. Silver, Cd, and Cu found in the heat-stable protein supernatants were indeed largely present as metallothionein-like peptide complexes; but Co, Ni, and Tl, also present in the heat-stable protein supernatants, did not coelute with metallothionein-like peptides and proteins. This difference in metal partitioning is consistent with the known preference of "soft" metals such as Ag, Cd, and Cu(I) for thiolated ligands and the contrasting tendency of Co and Ni to bind to ligands with oxygen and nitrogen as donor atoms. Metal handling in the whole cytosol also reflected these differences in metal-binding behavior. For Cd and Cu, the importance of the molecular weight pool that includes metallothionein-like peptides and proteins increased relative to the other pools as the total cytosolic metal concentration ([M]cytosol ) increased, consistent with a concentration-dependent detoxification response. In contrast, for Ni and Tl the increase in [M]cytosol was accompanied by a marked increase in the high-molecular weight (670-33 kDa) pool, suggesting that hepatic Ni and Tl are not effectively detoxified. Overall, the results suggest that metal detoxification is less effective for Ni, Tl, and Co than for Ag, Cd, and Cu. Environ Toxicol Chem 2018;37:576-586. © 2017 SETAC.

A comparison of metal concentrations in the tissues of yellow American eel (Anguilla rostrata) and European eel (Anguilla anguilla).

Historically abundant and widespread, populations of Atlantic eels have suffered a sharp decline in recent decades, in the ranges 40-80% and 90-99% for American and European eels, respectively. As a result, American eels are now classified as threatened, whereas European eels are considered to be in critical danger of extinction. Several causes have been identified as likely contributors of this decline, including overfishing, obstacles to migration (hydroelectric dams), climate change and habitat contamination. In the context of a larger project investigating the role of organic and inorganic contaminants in this decline, in this study, we measured the liver, kidney and muscle concentrations of essential (Cu, Se and Zn) and non-essential (Ag, As, Cd, Cr, Hg, Ni and Pb) metals in eels sampled at four sites in the South-West of France and four sites in Eastern Canada varying in contamination. Tissue concentrations of Cd, Hg and Se increased with fish size and age. Tissue metal concentrations generally reflected the contamination of their sampling sites. This was the case for Ag, As, Cd, Cu, Hg, Pb and Se. Comparison of tissue concentrations of these metals with the toxicological literature suggests that all of them except As could pose a risk to the health of eels from the most contaminated sites. In particular, European eels may be particularly at risk of Cd and Pb toxicity. Globally, our study suggests that a substantial accumulation of inorganic contaminants in the tissues of both eel species at sites contaminated by historical anthropogenic inputs may play a role in their decline.

Hepatic oxidative stress and metal subcellular partitioning are affected by selenium exposure in wild yellow perch (Perca flavescens).

Yellow perch (Perca flavescens) collected from 11 lakes in the Canadian mining regions of Sudbury (Ontario) and Rouyn-Noranda (Quebec) display wide ranges in the concentrations of cadmium (Cd), nickel (Ni), selenium (Se), and thallium (Tl) in their livers. To determine if these trace elements, as well as copper (Cu) and zinc (Zn), are causing oxidative stress in these fish, we measured three biochemical indicators (glutathione (GSH), glutathione disulfide (GSSG) and thiobarbituric acid-reactive substances (TBARS)) in their livers. We observed that 44% of the yellow perch that we collected were at risk of cellular oxidative stress and lipid peroxidation. Considering all fish from all lakes, higher liver Se concentrations were coincident with both lower proportions of GSSG compared to GSH and lower concentrations of TBARS, suggesting that the essential trace-element Se acts as an antioxidant. Furthermore, fish suffering oxidative stress had higher proportions of Cd, Cu and Zn in potentially sensitive subcellular fractions (organelles and heat-denatured proteins) than did fish not suffering from stress. This result suggests that reactive oxygen species may oxidize metal-binding proteins and thereby reduce the capacity of fish to safely bind trace metals. High Cd concentrations in metal-sensitive subcellular fractions likely further exacerbate the negative effects of lower Se exposure.

Hemagglutinin and neuraminidase containing virus-like particles produced in HEK-293 suspension culture: An effective influenza vaccine candidate.

Virus-like particles (VLPs) constitute a promising alternative as influenza vaccine. They are non-replicative particles that mimic the morphology of native viruses which make them more immunogenic than classical subunit vaccines. In this study, we propose HEK-293 cells in suspension culture in serum-free medium as an efficient platform to produce large quantities of VLPs. For this purpose, a stable cell line expressing the main influenza viral antigens hemagglutinin (HA) and neuraminidase (NA) (subtype H1N1) under the regulation of a cumate inducible promoter was developed (293HA-NA cells). The production of VLPs was evaluated by transient transfection of plasmids encoding human immunodeficiency virus (HIV) Gag or M1 influenza matrix protein. To facilitate the monitoring of VLPs production, Gag was fused to the green fluorescence protein (GFP). The transient transfection of the gag containing plasmid in 293HA-NA cells increased the release of HA and NA seven times more than its counterpart transfected with the M1 encoding plasmid. Consequently, the production of HA-NA containing VLPs using Gag as scaffold was evaluated in a 3-L controlled stirred tank bioreactor. The VLPs secreted in the culture medium were recovered by ultracentrifugation on a sucrose cushion and ultrafiltered by tangential flow filtration. Transmission electron micrographs of final sample revealed the presence of particles with the average typical size (150-200nm) and morphology of HIV-1 immature particles. The concentration of the influenza glycoproteins on the Gag-VLPs was estimated by single radial immunodiffusion and hemagglutination assay for HA and by Dot-Blot for HA and NA. More significantly, intranasal immunization of mice with influenza Gag-VLPs induced strong antigen-specific mucosal and systemic antibody responses and provided full protection against a lethal intranasal challenge with the homologous virus strain. These data suggest that, with further optimization and characterization the process could support mass production of safer and better-controlled VLPs-based influenza vaccine candidate.

Photochemical Synthesis of Complex Carbazoles: Evaluation of Electronic Effects in Both UV- and Visible-Light Methods in Continuous Flow.

An evaluation of both a visible-light- and UV-light-mediated synthesis of carbazoles from various triarylamines with differing electronic properties under continuous-flow conditions has been conducted. In general, triarylamines bearing electron-rich groups tend to produce higher yields than triarylamines possessing electron-withdrawing groups. The incorporation of nitrogen-based heterocycles, as well as halogen-containing arenes in carbazole skeletons, was well tolerated, and often synthetically useful complementarity was observed between the UV-light and visible-light (photoredox) methods.

Gonadal transcriptome analysis of wild contaminated female European eels during artificial gonad maturation.

Since the early 1980s, the population of European eels (Anguilla anguilla) has dramatically declined. Nowadays, the European eel is listed on the red list of threatened species (IUCN Red List) and is considered as critically endangered of extinction. Pollution is one of the putative causes for the collapse of this species. Among their possible effects, contaminants gradually accumulated in eels during their somatic growth phase (yellow eel stage) would be remobilized during their reproductive migration leading to potential toxic events in gonads. The aim of this study was to investigate the effects of organic and inorganic contaminants on the gonad development of wild female silver eels. Female silver eels from two sites with differing contamination levels were artificially matured. Transcriptomic analyses by means of a 1000 candidate gene cDNA microarray were performed on gonads after 11weeks of maturation to get insight into the mechanisms of toxicity of contaminants. The transcription levels of several genes, that were associated to the gonadosomatic index (GSI), were involved in mitotic cell division but also in gametogenesis. Genes associated to contaminants were mainly involved in the mechanisms of protection against oxidative stress, in DNA repair, in the purinergic signaling pathway and in steroidogenesis, suggesting an impairment of gonad development in eels from the polluted site. This was in agreement with the fact that eels from the reference site showed a higher gonad growth in comparison to contaminated fish.

Transcriptome profile analysis reveals specific signatures of pollutants in Atlantic eels.

Identifying specific effects of contaminants in a multi-stress field context remain a challenge in ecotoxicology. In this context, "omics" technologies, by allowing the simultaneous measurement of numerous biological endpoints, could help unravel the in situ toxicity of contaminants. In this study, wild Atlantic eels were sampled in 8 sites presenting a broad contamination gradient in France and Canada. The global hepatic transcriptome of animals was determined by RNA-Seq. In parallel, the contamination level of fish to 8 metals and 25 organic pollutants was determined. Factor analysis for multiple testing was used to identify genes that are most likely to be related to a single factor. Among the variables analyzed, arsenic (As), cadmium (Cd), lindane (γ-HCH) and the hepato-somatic index (HSI) were found to be the main factors affecting eel's transcriptome. Genes associated with As exposure were involved in the mechanisms that have been described during As vasculotoxicity in mammals. Genes correlated with Cd were involved in cell cycle and energy metabolism. For γ-HCH, genes were involved in lipolysis and cell growth. Genes associated with HSI were involved in protein, lipid and iron metabolisms. Our study proposes specific gene signatures of pollutants and their impacts in fish exposed to multi-stress conditions.

Abnormal ovarian DNA methylation programming during gonad maturation in wild contaminated fish.

There is increasing evidence that pollutants may cause diseases via epigenetic modifications. Epigenetic mechanisms such as DNA methylation participate in the regulation of gene transcription. Surprisingly, epigenetics research is still limited in ecotoxicology. In this study, we investigated whether chronic exposure to contaminants experienced by wild female fish (Anguilla anguilla) throughout their juvenile phase can affect the DNA methylation status of their oocytes during gonad maturation. Thus, fish were sampled in two locations presenting a low or a high contamination level. Then, fish were transferred to the laboratory and artificially matured. Before hormonal treatment, the DNA methylation levels of the genes encoding for the aromatase and the receptor of the follicle stimulating hormone were higher in contaminated fish than in fish from the clean site. For the hormone receptor, this hypermethylation was positively correlated with the contamination level of fish and was associated with a decrease in its transcription level. In addition, whereas gonad growth was associated with an increase in DNA methylation in fish from the clean site, no changes were observed in contaminated fish in response to hormonal treatment. Finally, a higher gonad growth was observed in fish from the reference site in comparison to contaminated fish.

A monovalent agonist of TrkA tyrosine kinase receptors can be converted into a bivalent antagonist.

BACKGROUND: Receptor tyrosine kinases (RTK) act through dimerization. Previously it was thought that only bivalent ligands could be agonistic, whereas monovalent ligands should be antagonistic. This notion changed after the demonstration that monovalent ligands can be agonistic, including our report of a small molecule monovalent ligand "D3" that is a partial agonist of the NGF receptor TrkA. A bivalent "D3-linker-D3" was expected to increase agonism. METHODS: Dimeric analogs were synthesized and tested in binding, biochemical, and biological assays. RESULTS: One analog, 1-ss, binds TrkA with higher affinity than D3 and induces or stabilizes receptor dimers. However, 1-ss exhibited antagonistic activity, through two mechanisms. One mechanism is that 1-ss blocks NGF binding, unlike D3 which is non-competitive. Inhibition of NGF binding may be due to the linker of 1-ss filling the inter-receptor space that NGF traverses before docking. In a second mechanism, 1-ss acts as a pure antagonist, inhibiting NGF-independent TrkA activity in cells over-expressing receptors. Inhibition is likely due to 1-ss "freezing" the TrkA dimer in the inactive state. CONCLUSIONS: Dimerization of an RTK can result in antagonism, through two independent mechanisms. GENERAL SIGNIFICANCE: we report a small molecule monovalent agonist being converted to a bivalent antagonist.

Fluorescent labeling in semi-solid medium for selection of mammalian cells secreting high-levels of recombinant proteins.

BACKGROUND: Despite the powerful impact in recent years of gene expression markers like the green fluorescent protein (GFP) to link the expression of recombinant protein for selection of high producers, there is a strong incentive to develop rapid and efficient methods for isolating mammalian cell clones secreting high levels of marker-free recombinant proteins. Recently, a method combining cell colony growth in methylcellulose-based medium with detection by a fluorescently labeled secondary antibody or antigen has shown promise for the selection of Chinese Hamster Ovary (CHO) cell lines secreting recombinant antibodies. Here we report an extension of this method referred to as fluorescent labeling in semi-solid medium (FLSSM) to detect recombinant proteins significantly smaller than antibodies, such as IGF-E5, a 25 kDa insulin-like growth factor derivative. RESULTS: CHO cell clones, expressing 300 microg/ml IGF-E5 in batch culture, were isolated more easily and quickly compared to the classic limiting dilution method. The intensity of the detected fluorescent signal was found to be proportional to the amount of IGF-E5 secreted, thus allowing the highest producers in the population to be identified and picked. CHO clones producing up to 9.5 microg/ml of Tissue-Plasminogen Activator (tPA, 67 kDa) were also generated using FLSSM. In addition, IGF-E5 high-producers were isolated from 293SF transfectants, showing that cell selection in semi-solid medium is not limited to CHO and lymphoid cells. The best positive clones were collected with a micromanipulator as well as with an automated colony picker, thus demonstrating the method's high throughput potential. CONCLUSION: FLSSM allows rapid visualization of the high secretors from transfected pools prior to picking, thus eliminating the tedious task of screening a high number of cell isolates. Because of its rapidity and its simplicity, FLSSM is a versatile method for the screening of high producers for research and industry.

High-level recombinant protein production in CHO cells using an adenoviral vector and the cumate gene-switch.

To facilitate and accelerate the production of eukaryotic proteins with correct post-translational modifications, we have developed a protein production system based on the transduction of Chinese hamster ovary (CHO) cells using adenovirus vectors (AdVs). We have engineered a CHO cell line (CHO-cTA) that stably expresses the transactivator (cTA) of our newly developed cumate gene-switch transcription system. This cell line is adapted to suspension culture and can grow in serum-free and protein-free medium. To increase the transduction level of AdVs, we have also generated a cell line (CHO-cTA-CAR) that expresses additional amounts of the coxackievirus and adenovirus receptor (CAR) on its surface. Recombinant protein production was tested using an AdV carrying the secreted alkaline phosphatase (SEAP) under the control of the CR5 promoter, which is strongly and specifically activated by binding to cTA. The SEAP expression was linked to the expression of the green fluorescent protein (GFP) through an internal ribosome entry site (IRES) to facilitate titration of the AdV. We monitored SEAP expression on a daily basis for 9 days after transduction of CHO-cTA and CHO-cTA-CAR using different quantities of AdVs at 37 and 30 degrees C. Incubation at the latter temperature increased the production of SEAP at least 10-fold, and the presence of CAR increased the transduction level of the AdV. Maximum SEAP production (63 mg/L) was achieved at 6-7 days post-infection at 30 degrees C by transducing CHO-cTA-CAR with 500 infectious particles/cell. Because numerous AdVs can now be generated within a few weeks and large-scale production of AdVs is now a routine procedure, this system could be used to produce rapidly milligram quantities of a battery of recombinant proteins as well as for large-scale protein production.

The ribonucleotide reductase domain of the R1 subunit of herpes simplex virus type 2 ribonucleotide reductase is essential for R1 antiapoptotic function.

The R1 subunit (ICP10) of herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (RR), which in addition to its C-terminal reductase domain possesses a unique N-terminal domain of about 400 aa, protects cells against apoptosis. As the NH(2) domain on its own is not antiapoptotic, it has been postulated that both domains of R1 or part(s) of them could be necessary for this function. Here, N- and C-terminal deletions were introduced in HSV-2 R1 to map the domain(s) involved in its antiapoptotic potential. The results showed that, whereas most of the NH(2) domain including part of the recently described putative alpha-crystallin domain is dispensable for antiapoptotic activity, it is the integrity of the structured RR domain that is required for protection. As the alpha-crystallin domain appears to play an important role in protein folding and oligomerization, the N-terminal boundary of the antiapoptotic domain could not be defined precisely. In addition, this study provided evidence that overexpression of HSV-2 R2 at levels up to 30-fold more than HSV-2 R1 did not decrease protection from tumour necrosis factor alpha, indicating that the R1 surface where R2 binds is not involved in antiapoptotic activity. Importantly, this result suggests that the co-expression of both RR subunits during the lytic cycle should not affect protection from this cytokine.