Comparison of the compact dry TC method with the standard method ISO 21149:2006 for determining aerobic colony counts in cosmetic emulsion.

OBJECTIVE: Compact Dry TC, a rapid method kit for determining aerobic colony counts, has been developed by Nissui Pharmaceutical Co. for food application. These plates are pre-sterilized and contain culture medium, a cold-soluble gelling agent and a colour redox indicator for rapid enumeration. In this study, the alternative method is compared with the standard method ISO 21149:2006 - Cosmetic - Microbiology - Enumeration and detection of aerobic mesophilic bacteria, for cosmetic emulsions application. METHODS: An oil-in-water (o/w) cosmetic emulsion was contaminated with a pool of bacterial strains (Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 9027). One millilitre of samples was spread on agar as described in ISO 21149. The colonies were enumerated after 3 days of incubation. At the same time, 1.2 mL samples were spread on Compact Dry TC kits. The kit was incubated at 35°C ± 1°C for 48 h, and the colonies were enumerated. Accuracy determination was carried out using six replicates at four levels of concentrations (10, 50, 100 and 250 CFU mL-1 ). The repeatability study was carried out using 12 replicates at four levels of concentrations (10, 50, 100 and 250 CFU mL-1 ). Variations relative to the analyst and to the batch of emulsion have been investigated. RESULTS: The linear correlation coefficients of Compact Dry TC Kit enumeration with standard method ISO 21149:2006 was 0.9999. In comparison study, no apparent differences were noted between the Compact Dry TC kit and the reference method ISO 21149, for the detection level of aerobic microorganisms. Relative accuracy, repeatability and intermediate precision studies were acceptable. In the repeatability study, the Shapiro-Wilk test has confirmed the normally distribution of the twelve assays. No significant variations in Compact Dry TC count results were observed with different analysts and different batches of emulsion. CONCLUSION: The results showed that the two compared methods 'Compact Dry TC' vs. 'conventional pour plate' performed equally well. Demonstration was achieved that the Compact Dry TC method may constitute a useful alternative tool for rapid enumeration of aerobic mesophilic bacteria in cosmetic emulsions.

Sustained xanthophyll pigments-related photoprotective NPQ is involved in photoinhibition in the haptophyte Tisochrysis lutea.

Dynamic xanthophyll cycle (XC) related non-photochemical quenching (NPQd, also called qE) is present in most phototrophs. It allows dissipating excess light energy under adverse growing conditions. Generally, NPQd rapidly reverses for photosynthesis to resume when light intensity decreases back toward optimal intensity. Under certain environmental conditions and/or in some species, NPQ can be strongly sustained (NPQs showing hours-to-days relaxation kinetics). Tisochrysis lutea is a South Pacific haptophyte phytoplankton with a strong potential for aquaculture and biotechnology applications. It was previously reported to show a surprisingly low NPQd capacity while synthesizing large amounts of diatoxanthin (Dt), a pigment involved in the XC. In order to better understand this paradox, we investigated the characteristics of NPQ in T. lutea under various growth conditions of light and nutrient availability (different photoperiods, low and high light, nutrient starvations). We found a strong NPQs, unmeasurable with usual fluorometry protocols. Along with confirming the involvement of Dt in both NPQd and NPQs (by using the dithiothreitol inhibitor), we highlighted a strong relationship between Dt and the maximum quantum yield of photochemistry (Fv/Fm) across growing conditions and during relaxation experiments in darkness. It suggests that changes in Fv/Fm, usually attributed to the 'photoinhibitory' quenching (qI), are simultaneously largely impacted by photoprotective NPQ. The overlap of xanthophyll pigments-related photoprotective NPQ with several other mechanisms involved in the cell response (Photosystem II photoinactivation, changes in pigments composition, and detoxification by antioxidants) to energy unbalance is further discussed. Our findings question both how widespread NPQs is in the global ocean, particularly in nutrient starved environments (oligotrophic waters) and situations (post-bloom), and the use of adapted active fluorescence protocols (i.e. with extended NPQ relaxation period prior to measurement).

Incidence and outcomes of infective endocarditis after transcatheter aortic valve implantation versus surgical aortic valve replacement.

OBJECTIVES: Transcatheter aortic valve implantation (TAVI) is an alternative to surgical aortic valve replacement (AVR) in aortic stenosis (AS). Infective endocarditis (IE) in patients with prosthetic heart valves is associated with significant morbidity and mortality. Data on the incidence, risk factors, and outcomes of IE after TAVI are conflicting. We evaluated these issues in patients with percutaneous TAVI vs. isolated surgical AVR (SAVR) at a nationwide level. METHODS: Based on the administrative hospital discharge database, the study collected information for all patients with aortic stenosis treated with AVR in France between 2010 and 2018. RESULTS: A total of 47 553 patients undergoing TAVI and 60 253 patients undergoing isolated SAVR were identified. During a mean follow-up of 2.0 years (median (25th to 75th percentile) 1.2 (0.1-3.4) years), the incidence rates of IE were 1.89 (95% confidence interval (CI) 1.78-2.00) and 1.40 (95% CI 1.34-1.46) events per 100 person-years in unmatched TAVI and SAVR patients, respectively. In 32 582 propensity-matched patients (16 291 with TAVI and 16 291 with SAVR), risk of IE was not different in patients treated with TAVI vs. SAVR (incidence rates of IE 1.86 (95% CI 1.70-2.04) %/year vs 1.71 (95% CI 1.58-1.85) %/year respectively, relative risk (RR) 1.09, 95% CI 0.96-1.23). In these matched patients, total mortality was higher in TAVI patients with IE (43.0% 95% CI 37.3-49.3) than in SAVR patients with IE (32.8% 95% CI 28.6-37.3; RR 1.32, 95% CI 1.08-1.60). DISCUSSION: In a nationwide cohort of patients with AS, treatment with TAVI was associated with a risk of IE similar to that following SAVR. Mortality was higher for patients with IE following TAVI than for those with IE following SAVR.

Engineering and biochemical characterization of the rat microsomal cytochrome P4501A1 fused to ferredoxin and ferredoxin-NADP(+) reductase from plant chloroplasts.

Fusion proteins of rat cytochrome P4501A1 with maize ferredoxin I (Fd) and pea ferredoxin NADP(+) reductase (FNR), the last electron transfer proteins of the photosynthetic channel in plant chloroplasts, were obtained by gene fusion in the yeast expression vector pAAH5N. The encoded fusion proteins P4501A1-Fd, P4501A1-FNR, P4501A1-Fd-FNR and P4501A1-FNR-Fd were produced in microsomes of the yeast Saccharomyces cerevisiae AH22. Enzymatic assays were carried out in vitro with the isolated microsomes. P4501A1-Fd-FNR and P4501A1-FNR-Fd were found to catalyze P450-monooxygenase activities towards 7-ethoxycoumarin and the herbicide chlortoluron. P4501A1-Fd-FNR was the most efficient enzyme as measured in vitro in ferricyanide and cytochrome c reductions, as well as P450-monooxygenase assays. Apparent K(m) and k(cat) of P4501A1-Fd-FNR were 70 microM and 7800 min(-1) for NADPH, 13.2 microM and 51.1 min(-1) for 7-ethoxycoumarin, and 21.3 microM and 23. 8 min(-1) for the herbicide chlortoluron, respectively. Fd in P4501A1-Fd-FNR fusion enzyme was found to be a limiting factor compared to P4501A1 fused to the yeast NADPH-cytochrome P450 reductase, an artificial enzyme described previously. The efficiency of electron transfer in the P4501A1 fusion proteins and a possible in vivo molecular coupling of Fd and FNR with microsomal cytochrome P4501A1 produced in plant chloroplasts are discussed.

A gene encoding a yeast equivalent of mammalian NADPH-adrenodoxin oxidoreductases.

Adrenodoxin oxidoreductase (ADR) and adrenodoxin (ADX) are the two proteins involved in electron transport to mammalian mitochondrial P-450s capable of steroid modifications. The cloning and sequencing of a S. cervisiae ADR homologue (YADR) is presented here. The YADR protein sequence shares 36 and 37% of identical amino acids with human and bovine ADR respectively. The physiological role of this ADR homologue in yeast is unknown. We intend to study the interaction of this YADR with bovine ADX in vitro and in vivo.

Hydroxylated human homotrimeric collagen I in Agrobacterium tumefaciens-mediated transient expression and in transgenic tobacco plant.

Potential contamination of animal-derived collagen with pathogens has led to the demand for safe recombinant sources of this complex molecule. In continuation of our previous work [Ruggiero et al. (2000) FEBS Lett. 469, 132-136], here we show that it is possible to produce recombinant hydroxylated homotrimeric collagen in tobacco plants that are co-transformed with a human type I collagen and a chimeric proline-4-hydroxylase (P4H). This is to our knowledge the first time that transient expression in tobacco was used to improve the quality of a recombinant protein produced in plants through co-expression with an animal cell-derived modifying enzyme. We demonstrated the functionality of the new chimeric P4H and thus improved the thermal stability of recombinant collagen I from plants to 37 degrees C.

Consequences of acid catalysis in concurrent ring opening and halogenation of spiroketals.

[formula: see text] Lewis and/or Bronsted acid additives permit ring opening and halogenation of spiroketals at substantially reduced temperatures to produce omega-iodo enol ethers in improved yield and purity, which can undergo further reaction in the presence of distal electrophilic centers to give new steroid skeletons.

Outer-ring stereochemical modulation of cytotoxicity in cephalostatins.

[structure: see text] 20- and 25'-epimers of cephalostatin 7, prepared by directed unsymmetrical pyrazine synthesis, address outer-ring topographical and stability questions and intimate an oxacarbenium ion rationale for the role in bioactivity of the spiroketal (E/F, E'/F') rings of this class of antitumor agents.

Characterization of recombinant adrenodoxin reductase homologue (Arh1p) from yeast. Implication in in vitro cytochrome p45011beta monooxygenase system.

The mammalian electron transfer chain of mitochondrial cytochrome P450 forms involved in steroidogenesis includes very specific proteins, namely adrenodoxin reductase and adrenodoxin. Adrenodoxin reductase transfers electrons from NADPH to adrenodoxin, which subsequently donates them to the cytochrome P450 forms. The Saccharomyces cerevisiae ARH1 gene product (Arh1p) presents homology to mammalian adrenodoxin reductase. We demonstrate the capacity of recombinant Arh1p, made in Escherichia coli, to substitute for its mammalian homologue in ferricyanide, cytochrome c reduction, and, more importantly, in vitro 11beta-hydroxylase assays. Electrons could be transferred from NADPH and NADH as measured in the cytochrome c reduction assay. Apparent Km values were determined to be 0.5, 0.6, and 0.1 microM for NADPH, NADH, and bovine adrenodoxin, respectively. These values differ slightly from those of mammalian adrenodoxin reductase, except for NADH, which is a very poor electron donor to the mammalian protein. Subcellular fractionation studies have localized Arh1p to the inner membrane of yeast mitochondria. The biological function of Arh1p remains unknown, and to date, no mitochondrial cytochrome P450 has been identified. ARH1 is, however, essential for yeast viability because an ARH1 gene disruption is lethal not only in aerobic growth conditions but also, surprisingly enough, during fermentation.

On the relationship of OSW-1 to the cephalostatins.

Antineoplastic bis-steroidal (cephalostatin-type) analogues of the saponin OSW-1 were produced from a dihydroaglycone of OSW-1. The key aglycone 6H was obtained from 5alpha-androstan-3beta-ol-17-one in 8 steps (38% yield). The SAR of the aglycones, intermediates, and hybrid analogues provide insights regarding the proposed common role of C22-oxocarbenium ions in the bioactivity of both OSW-1 and cephalostatins.

The role of microvascular environment in the metastasizing ability of an experimental tumor.

A rapidly growing, locally very invasive and easily transplantable fibrosarcoma that was developed through chemical carcinogenesis in Balb/c mice in this laboratory several years ago did not metastasize into the viscera of its hosts when implanted into the subcutaneous connective tissue or skeletal muscle of syngeneic mice. When, however the same tumour was implanted into the liver or the kidneys of Balb/c mice it metastasized extensively into many different organs within 2 weeks of its transplantation. Evidence is presented that because of some unknown deficiency the cells of the fibrosarcoma under study are unable to penetrate through the endothelial wall into the lumen of the particular type of vessels which surround and vascularize the tumours in the subcutaneous connective tissue and muscle, and that, in contrast, they can easily cross into the lumen of the vessels that surround and vascularize them in the liver and kidney. Thus, this in vivo study indicates that the type of microvascular environment in which certain experimental tumours are transplanted can control their ability to accomplish vascular invasion, the first step of the metastatic process.

11 beta-hydroxylase activity in recombinant yeast mitochondria. In vivo conversion of 11-deoxycortisol to hydrocortisone.

In mammals, the final 11 beta-hydroxylation step of the hydrocortisone biosynthesis pathway is performed by a mitochondrial enzyme, namely cytochrome P-450(11 beta), together with the electron carriers adrenodoxin and NADPH adrenodoxin oxidoreductase. Successful production of a functional steroid 11 beta-hydroxylase activity was obtained in recombinant yeast in vivo. This conversion was achieved by coexpression of a mitochondrially targeted adrenodoxin and a modified bovine P-450(11 beta) whose natural presequence was replaced by a yeast presequence, together with an unexpected yeast endogenous NADPH-adrenodoxin-reductase-like activity. Adrenodoxin and P-450(11 beta) behave as a mitochondrial matrix and membrane protein, respectively. Saccharomyces cerevisiae apparently produces a mitochondrial protein which is capable of transferring electrons to bovine adrenodoxin, which in turn transfers the electrons to P-450(11 beta). The endogenous adrenodoxin oxidoreductase gains electrons specifically from NADPH. The notion that a yeast microsomal NADPH P-450 oxidoreductase can transfer electrons to mammalian microsomal P-450s can be extended to mitochondria, where an NADPH adrenodoxin oxidoreductase protein transfers electrons to adrenodoxin and renders a mitochondrial mammalian P-450 functional in vivo. The physiological function of this yeast NADPH adrenodoxin oxidoreductase activity is not known.

On topography and functionality in the B-D rings of cephalostatin cytotoxins.

Analogues 12'beta-hydroxycephalostatin 1 (9), 7'-deoxyritterazine G (10), and 14-epi-7'-deoxyritterazine B (11) were prepared via our protocol for unsymmetrical pyrazine synthesis. Cytotoxicity against human tumors was also determined for the first time for ritterazines, with femtomolar potency and a high correlation to cephalostatins observed. The SAR of these and related compounds provide insight into the importance of topography and certain chemical functionality in the B-D and B'-D' rings of cephalostatin type antineoplastics.