Developmental changes in amniotic and allantoic fluid insulin-like growth factor (IGF)-I and -II concentrations of avian embryos

In the literature, IGFs in the developing embryo are usually determined by blood serum concentrations. For this study, IGF-I/-II was quantified in the amniotic and allantoic fluids of fertile commercial broiler chicken (Gallus domesticus) (n=222), Pekin duck (Anas platyrhyncha) (n=250), and turkey (Meleagridis gallopavo) eggs (n= 200) during incubation. Amniotic and allantoic fluids were collected from embryos starting at 6 days of incubation for chickens and 8 days of incubation for ducks and turkeys. IGF concentrations within the fluids were determined by radioimmunoassay. Chicken amniotic IGF-I concentration at stage 29 of development was significantly higher (P< or =0.05) than the duck or turkey. At stage 36 of development the concentration of IGF-II in the amniotic fluid was 2.8 times greater in the chicken versus the duck (P< or =0.05) and 2 times greater than in the turkey (P< or =0.05). Within species, chicken IGF-I concentration in the amniotic fluid had a cubic trend (P< or =0.001), duck IGF-I increased linearly (P< or =0.001), and turkey concentrations declined quadratically (P< or =0.001) throughout development. In all species, the IGF-II concentration was higher than the IGF-I concentration in the amniotic and allantoic fluids.     

Molecular characterization of autoinduction of bioluminescence in the Microtox indicator strain Vibrio fischeri ATCC 49387

Repeated attempts to clone the luxI from Vibrio fischeri ATCC 49387 failed to produce a clone carrying a functional LuxI. Sequence data from the clones revealed the presence of a polymorphism when compared with previously published luxI sequences, prompting further characterization of bioluminescence regulation in V. fischeri ATCC 49387. Further investigation of V. fischeri ATCC 49387 revealed that its LuxI protein lacks detectable LuxI activity due to the presence of a glutamine residue at position 125 in the deduced amino acid sequence. Specific bioluminescence in V. fischeri ATCC 49387 increases with increasing cell density, indicative of a typical autoinduction response. However, conditioned medium from this strain does not induce bioluminescence in an ATCC 49387 luxR-plux-based acyl homoserine lactone reporter strain, but does induce bioluminescence in ATCC 49387. It has been previously shown that a V. fischeri MJ-1 luxI mutant exhibits autoinduction of bioluminescence through N-octanoyl-L-homoserine lactone, the product of the AinS autoinducer synthase. However, a bioreporter based on luxR-plux from V. fischeri ATCC 49387 responded poorly to conditioned medium from V. fischeri ATCC 49387 and also responded poorly to authentic N-octanoyl-DL-homoserine lactone. A similar MJ-1-based bioreporter showed significant induction under the same conditions. A putative ainS gene cloned from ATCC 49387, unlike luxI from ATCC 49387, expresses V. fischeri autoinducer synthase activity in Escherichia coli. This study suggests that a regulatory mechanism independent of LuxR and LuxI but possibly involving AinS is responsible for the control of autoinduction of bioluminescence in V. fischeri ATCC 49387.     

Common Cutaneous Bacteria Isolated from Snakes Inhibit Growth of <Emphasis Type="Italic">Ophidiomyces ophiodiicola</Emphasis>

There is increasing concern regarding potential impacts of snake fungal disease (SFD), caused by Ophidiomyces ophiodiicola (Oo), on free-ranging snake populations in the eastern USA. The snake cutaneous microbiome likely serves as the first line of defense against Oo and other pathogens; however, little is known about microbial associations in snakes. The objective of this study was to better define the composition and immune function of the snake cutaneous microbiome. Eight timber rattlesnakes (Crotalus horridus) and four black racers (Coluber constrictor) were captured in Arkansas and Tennessee, with some snakes exhibiting signs of SFD. Oo was detected through real-time qPCR in five snakes. Additional histopathological techniques confirmed a diagnosis of SFD in one racer, the species’ first confirmed case of SFD in Tennessee. Fifty-eight bacterial and five fungal strains were isolated from skin swabs and identified with Sanger sequencing. Non-metric multidimensional scaling and PERMANOVA analyses indicated that the culturable microbiome does not differ between snake species. Fifteen bacterial strains isolated from rattlesnakes and a single strain isolated from a racer inhibited growth of Oo in vitro. Results shed light on the culturable cutaneous microbiome of snakes and probiotic members that may play a role in fighting an emergent disease.     

Mechanism of spring relapse in avian malaria: effect of gonadotropin and corticosterone

Previous work demonstrated that corticosterone induces relapse of avian malaria, and that this effect varies markedly from winter to spring. In the present study, English sparrows with latent Plasmodium relictum infections were treated in winter with corticosterone, gonadotropin, corticosterone + gonadotropin, or a control regimen consisting of the oil vehicle. Gonadotropin neither induced relapse nor potentiated the induction of relapse by corticosterone. These data cast doubt on the hypothesis that spring relapse in malarial infections is mediated by seasonal changes in reproductive hormone levels.     

Presence of a [3Fe–4S] cluster in a PsaC variant as a functional component of the photosystem I electron transfer chain in <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7002

A site-directed C14G mutation was introduced into the stromal PsaC subunit of Synechococcus sp. strain PCC 7002 in vivo in order to introduce an exchangeable coordination site into the terminal F_B [4Fe–4S] cluster of Photosystem I (PSI). Using an engineered PSI-less strain (psaAB deletion), psaC was deleted and replaced with recombinant versions controlled by a strong promoter, and the psaAB deletion was complemented. Modified PSI accumulated at lower levels in this strain and supported slower photoautotrophic growth than wild type. As-isolated PSI complexes containing PsaC_C14G showed resonances with g values of 2.038 and 2.007 characteristic of a [3Fe–4S]¹⁺ cluster. When the PSI complexes were illuminated at 15 K, these resonances partially disappeared and two new sets of resonances appeared. The majority set had g values of 2.05, 1.95, and 1.85, characteristic of F_A ^?, and the minority set had g values of 2.11, 1.90, and 1.88 from F_B′ in the modified site. The S?=?1/2 spin state of the latter implied the presence of a thiolate as the terminal ligand. The [3Fe–4S] clusters could be partially reconstituted with iron, producing a larger population of [4Fe–4S] clusters. Rates of flavodoxin reduction were identical in PSI complexes isolated from wild type and the PsaC_C14G variant strain; this implied equivalent capacity for forward electron transfer in PSI complexes that contained [3Fe–4S] and [4Fe–4S] clusters. The development of this cyanobacterial strain is a first step toward translation of in vitro PSI-based biosolar molecular wire systems in vivo and provides new insights into the formation of Fe/S clusters.     

Ultraviolet radiation-induced lethality and repair of pyrimidine dimers in fish embryos

Pimephales promelas (fathead minnow) embryos were used to show a correlation between induction of pyrimidine dimers in DNA and embryo death. Embryo killing was measured by a lack of heart-beat and blood circulation at 48 h post-ultraviolet radiation (UVR). When the embryos were exposed to various doses of UVR from a FS-40 sunlamp followed by exposure to photoreactivating light (PRL) (320-400 nm), the number of pyrimidine dimers decreased significantly. The photorepair of dimers was accompanied by a substantial increase in embryo survival. When embryo killing was examined as a function of the number of dimers present, dimers were identified as a major lesion involved in UVR-induced killing in these fish embryos. This in vivo study on photoreactivation treatment of fish embryos shows a direct association between UVR-induced pyrimidine dimers and embryo killing. In addition, when embryos were held in the dark for 9 h after UVR, 50% of the dimers were removed by excision repair.     

A scaffoldless technique for self-generation of three-dimensional keratinospheroids on liquid crystal surfaces

We describe a new scaffold-free three-dimensional (3D) cell culture model using cholesteryl ester based lyotropic liquid crystal (LC) substrates. Keratinocytes were deposited randomly on the LC surface where they self-assembled into 3D microtissues or keratinospheroids. The cell density required to form spheroids was optimized. We investigated cell viability using dead/live cell assays. The adhesion characteristics of cells within the microtissues were determined using histological sectioning and immunofluorescence staining. Fourier transform infrared spectroscopy (FTIR) was used to characterize the biochemistry of the keratinospheroids. We found that both cells and microtissues could migrate on the LC surface. The viability study indicated approximately 80% viability of cells in the microtissues up to 20 days of culture. Strong intercellular adhesion was observed in the stratification of the multi-layered microspheroids using field emission-scanning electron microscopy (FE-SEM) and histochemical staining. The cytoskeleton and vinculins of the cells in the microtissues were expressed diffusely, but the microtissues were enriched with lipids and nucleic acids, which indicates close resemblance to the conditions in vivo. The basic 3D culture model based on LC may be used for cell and microtissue migration studies in response to cytochemical treatment.     

Enolase isozymes in Coho salmon.

1. Analysis of the enolase isozymic distribution has been performed in tissues of the Coho salmon, using electrophoretic separation on cellulose acetate strips followed by localization of enzymatic activity. 2. A total of six electrophoretically distinct forms are seen in Coho salmon in patterns that differ both qualitatively and quantitatively from one tissue to another. 3. The isozymes in skeletal muscle and liver are sufficiently similar to one another that a purification procedure previously developed for trout muscle enolase by Cory & Wold (1966) can be used to partially purify enolase from either of the above-mentioned Coho tissues. The main form of enolase in Coho muscle has an isoelectric point of 7.57. 4. Both liver and skeletal muscle enolases can be reversibly denatured in guanidine HCl and subsequently renatured. Liver enolase appeared to renature somewhat faster than muscle enolase under the same conditions. 5. While polyploidy among salmonids may contribute to the complexity of enolase patterns in fish, the differences in isozymic patterns seen from one tissue to another indicate the presence of distinct, nonallelic genes, probably arising through gene duplication.     

Eicosapentaenoic acid and docosahexaenoic acid reduce interleukin-1β-mediated cartilage degradation

Introduction  

In inflammatory joint disease, such as osteoarthritis (OA), there is an increased level of proinflammatory cytokines, such as interleukin (IL)-1β. These cytokines stimulate the production of matrix metalloproteinases (MMPs), which leads to the degradation of the cartilage extracellular matrix and the loss of key structural components such as sulphated glycosaminoglycan (sGAG) and collagen II. The aim of this study was to examine the therapeutic potential of n-3 polyunsaturated fatty acids (PUFAs) in an in vitro model of cartilage inflammation.