Asparaginase synthesis by semi-continuous fermentor cultures of Vibrio succinogenes

Summary Vibrio succinogenes produces an asparaginase that does not hydrolyze glutamine, is not immunosuppressive, and has antitumor activity. Fermentor cultures initiated by small inocula exhibit a pattern of increasing enzyme activity consistent with induction during exponential phase. Semi-continuous cultures permit the harvesting of fully induced cells.     

Characterization of the mouse ClC-K1/Barttin chloride channel

Several Cl⁻ channels have been described in the native renal tubule, but their correspondence with ClC-K1 and ClC-K2 channels (orthologs of human ClC-Ka and ClC-Kb), which play a major role in transcellular Cl⁻ absorption in the kidney, has yet to be established. This is partly because investigation of heterologous expression has involved rat or human ClC-K models, whereas characterization of the native renal tubule has been done in mice. Here, we investigate the electrophysiological properties of mouse ClC-K1 channels heterologously expressed in Xenopus laevis oocytes and in HEK293 cells with or without their accessory Barttin subunit. Current amplitudes and plasma membrane insertion of mouse ClC-K1 were enhanced by Barttin. External basic pH or elevated calcium stimulated currents followed the anion permeability sequence Cl⁻ > Br⁻ > NO₃⁻ > I⁻. Single-channel recordings revealed a unit conductance of ~ 40 pS. Channel activity in cell-attached patches increased with membrane depolarization (voltage for half-maximal activation: ~ − 65 mV). Insertion of the V166E mutation, which introduces a glutamate in mouse ClC-K1, which is crucial for channel gating, reduced the unit conductance to ~ 20 pS. This mutation shifted the depolarizing voltage for half-maximal channel activation to ~ + 25 mV. The unit conductance and voltage dependence of wild-type and V166E ClC-K1 were not affected by Barttin. Owing to their strikingly similar properties, we propose that the ClC-K1/Barttin complex is the molecular substrate of a chloride channel previously detected in the mouse thick ascending limb (Paulais et al., J Membr. Biol, 1990, 113:253–260).     

The Wound Healing and Antibacterial Activity of Five Ethnomedical Calophyllum inophyllum Oils: An Alternative Therapeutic Strategy to Treat Infected Wounds

Background

Calophyllum inophyllum L. (Calophyllaceae) is an evergreen tree ethno-medically used along the seashores and islands of the Indian and Pacific Oceans, especially in Polynesia. Oil extracted from the seeds is traditionally used topically to treat a wide range of skin injuries from burn, scar and infected wounds to skin diseases such as dermatosis, urticaria and eczema. However, very few scientific studies reported and quantified the therapeutic properties of Calophyllum inophyllum oil (CIO). In this work, five CIO from Indonesia (CIO1), Tahiti (CIO2, 3), Fiji islands (CIO4) and New Caledonia (CIO5) were studied and their cytotoxic, wound healing, and antibacterial properties were presented in order to provide a scientific support to their traditional use and verify their safety.

Methods

The safety of the five CIO was ascertained using the Alamar blue assay on human keratinocyte cells. CIO wound healing properties were determined using the scratch test assay on human keratinocyte cells. CIO-stimulated antibacterial innate immune response was evaluated using ELISA by measuring β defensin-2 release in human derivative macrophage cells. CIO antibacterial activity was tested using oilogramme against twenty aerobic Gram- bacteria species, twenty aerobic Gram+ bacteria species, including a multi-drug resistant Staphylococcus aureus strain and two anaerobic Gram+ bacteria species e.g. Propionibacterium acnes and Propionibacterium granulosum. To detect polarity profile of the components responsible of the antibacterial activity, we performed bioautography against a Staphylococcus aureus strain.

Results

Based on Alamar Blue assay, we showed that CIO can be safely used on keratinocyte cells between 2.7% and 11.2% depending on CIO origin. Concerning the healing activity, all the CIO tested accelerated in vitro wound closure, the healing factor being 1.3 to 2.1 higher compared to control when keratinocytes were incubated after scratch with CIO at 0.1%. Furthermore, our results showed that CIO exhibit two distinct antibacterial effects: one against Gram+ bacteria by direct inhibition of mitotic growth and another potent effect against Gram- bacteria due to increased release of β-defensin 2 peptide by macrophages. Interestingly, the needed concentrations of CIO to inhibit bacteria growth and to promote wound healing are lower than concentrations exhibiting cytotoxic effects on keratinocyte cells. Finally, we performed bioautography assay against Staphylococcus aureus to determine polarity profile of the components responsible for CIO antibacterial activity. Our results showed for the five tested CIO that components responsible of the bacterial growth inhibition are the more polar one on the TLC chromatographic profile and are contained in the resinous fraction of the oil.

Conclusions

This study was conducted to evaluate cytotoxicity, wound healing and antibacterial properties of five CIO traditionally used to treat infected wounds. Using cell and bacteria cultures, we confirmed the pharmacological effects of CIO as wound healing and antimicrobial agent. Moreover, we showed that concentration of CIO needed to exhibit therapeutic effects are lower than concentrations exhibiting cytotoxic effects in vitro. For the first time, this study provides support for traditional uses of CIO. These wound healing and antibiotic properties make CIO a valuable candidate to treat infected wounds especially in tropical areas.     

Differential regulation of the bumetanide-sensitive cotransporter (NKCC2) by ovarian hormones

The Na-K-2Cl cotransporter (NKCC2) regulates sodium transport along the thick ascending limb of Henle's loop and is important in control of sodium balance, renal concentrating ability and renin release. To determine if there are sex differences in NKCC2 abundance and/or distribution, and to evaluate the contribution of ovarian hormones to any such differences, we performed semiquantitative immunoblotting and immunoperoxidase immunohistochemistry for NKCC2 in the kidney of Sprague Dawley male, female and ovariectomized (OVX) rats with and without 17-β estradiol or progesterone supplementation. Intact females demonstrated greater NKCC2 protein in homogenates of whole kidney (334 ± 29%), cortex (219 ± 20%) and outer medulla (133 ± 9%) compared to males. Ovarian hormone supplementation to OVX rats regulated NKCC2 in the outer medulla only, with NKCC2 protein abundance decreasing slightly in response to progesterone but increasing in response to 17-β estradiol. Immunohistochemistry demonstrated prominent NKCC2 labeling in the apical membrane of thick ascending limb cells. Kidney section NKCC2 labeling confirmed regionalized regulation of NKCC2 by ovarian hormones. Localized regulation of NKCC2 by ovarian hormones may have importance in controlling sodium and water balance over the lifetime of women as the milieu of sex hormones varies.     

Molecular epidemiology of malaria prevalence and parasitaemia in a wild bird population

Avian malaria (Plasmodium spp.) and other blood parasitic infections of birds constitute increasingly popular model systems in ecological and evolutionary host-parasite studies. Field studies of these parasites commonly use two traits in hypothesis testing: infection status (or prevalence at the population level) and parasitaemia, yet the causes of variation in these traits remain poorly understood. Here, we use quantitative PCR to investigate fine-scale environmental and host predictors of malaria infection status and parasitaemia in a large 4-year data set from a well-characterized population of blue tits (Cyanistes caeruleus). We also examine the temporal dynamics of both traits within individuals. Both infection status and parasitaemia showed marked temporal and spatial variation within this population. However, spatiotemporal patterns of prevalence and parasitaemia were non-parallel, suggesting that different biological processes underpin variation in these two traits at this scale. Infection probability and parasitaemia both increased with host age, and parasitaemia was higher in individuals investing more in reproduction (those with larger clutch sizes). Several local environmental characteristics predicted parasitaemia, including food availability, altitude, and distance from the woodland edge. Although infection status and parasitaemia were somewhat repeatable within individuals, infections were clearly dynamic: patent infections frequently disappeared from the bloodstream, with up to 26% being lost between years, and parasitaemia also fluctuated within individuals across years in a pattern that mirrored annual population-level changes. Overall, these findings highlight the ecological complexity of avian malaria infections in natural populations, while providing valuable insight into the fundamental biology of this system that will increase its utility as a model host-parasite system.     

TLRs mediate IFN-gamma production by human uterine NK cells in endometrium

The human endometrium (EM) contains macrophages, NK cells, T cells, B cells, and neutrophils in contact with a variety of stromal and epithelial cells. The interplay between these different cell types and their roles in defense against pathogen invasion in this specialized tissue are important for controlling infection and reproduction. TLRs are a family of receptors able to recognize conserved pathogen-associated molecular patterns. In this study, we determined the expression of TLRs on uterine NK (uNK) cells from the human EM and the extent to which uNK cells responded to TLR agonist stimulation. uNK cells expressed TLRs 2, 3, and 4, and produced IFN-gamma when total human endometrial cells were stimulated with agonists to TLR2 or TLR3 (peptidoglycan or poly(I:C), respectively). Activated uNK cell clones produced IFN-gamma upon stimulation with peptidoglycan or poly(I:C). However, purified uNK cells did not respond directly to TLR agonists, but IFN-gamma was produced by uNK cells in response to TLR stimulation when cocultured with APCs. These data indicate that uNK cells express TLRs and that they can respond to TLR agonists within EM by producing IFN-gamma. These data also indicate that the uNK cells do not respond directly to TLR stimulation, but rather their production of IFN-gamma is dependent upon interactions with other cells within EM.     

A Polyketide Synthase Gene Required for Biosynthesis of the Aflatoxin-like Toxin, Dothistromin

Dothistromin is a polyketide toxin, produced by a fungal forest pathogen, with structural similarity to the aflatoxin precursor versicolorin B. Biochemical and genetic studies suggested that there are common steps in the biosynthetic pathways for these metabolites and showed similarities between some of the genes. A polyketide synthase gene (pksA) was isolated from dothistromin-producing Dothistroma septosporum by hybridization with an aflatoxin ortholog from Aspergillus parasiticus. Inactivation of this gene in D. septosporum resulted in mutants that could not produce dothistromin but that could convert exogenous aflatoxin precursors, including norsolorinic acid, into dothistromin. The mutants also had reduced asexual sporulation compared to the wild type. So far four other genes are known to be clustered immediately alongside pksA. Three of these (cypA, moxA, avfA) are predicted to be orthologs of aflatoxin biosynthetic genes. The other gene (epoA), located between avfA and moxA, is predicted to encode an epoxide hydrolase, for which there is no homolog in either the aflatoxin or sterigmatocystin gene clusters. The pksA gene is located on a small chromosome of ~1.3 Mb in size, along with the dothistromin ketoreductase (dotA) gene.     

A bimodal influence of thyroid hormone on cerebellum oligodendrocyte differentiation

Thyroid hormone (T(3)) can trigger a massive differentiation of cultured oligodendrocytes precursor cells (OPC) by binding the nuclear T(3) receptor α1 (TRα1). Whether this reflects a physiological function of TRα1 remains uncertain. Using a recently generated mouse model, in which CRE/loxP recombination is used to block its function, we show that TRα1 acts at two levels for the in vivo differentiation of OPC in mouse cerebellum. At the early postnatal stage, it promotes the secretion of several neurotrophic factors by acting in Purkinje neurons and astrocytes, defining an environment suitable for OPC differentiation. At later stages, TRα1 acts in a cell-autonomous manner to ensure the complete arrest of OPC proliferation. These data explain contradictory observations made on various models and outline the importance of T(3) signaling both for synchronizing postnatal neurodevelopment and restraining OPC proliferation in adult brain.