Role of TRAF3 and -6 in the activation of the NF-kappa B and JNK pathways by X-linked ectodermal dysplasia receptor

X-linked ectodermal dysplasia receptor (XEDAR) is a recently isolated member of the tumor necrosis factor receptor family that has been shown to be highly expressed in ectodermal derivatives during embryonic development and binds to ectodysplasin-A2 (EDA-A2). By using a subclone of 293F cells with stable expression of XEDAR, we report that XEDAR activates the NF-kappaB and JNK pathways in an EDA-A2-dependent fashion. Treatment with EDA-A2 leads to the recruitment of TRAF3 and -6 to the aggregated XEDAR complex, suggesting a central role of these adaptors in the proximal aspect of XEDAR signaling. Whereas TRAF3 and -6, IKK1/IKKalpha, IKK2/IKKbeta, and NEMO/IKKgamma are involved in XEDAR-induced NF-kappaB activation, XEDAR-induced JNK activation seems to be mediated via a pathway dependent on TRAF3, TRAF6, and ASK1. Deletion and point mutagenesis studies delineate two distinct regions in the cytoplasmic domain of XEDAR, which are involved in binding to TRAF3 and -6, respectively, and play a major role in the activation of the NF-kappaB and JNK pathways. Taken together, our results establish a major role of TRAF3 and -6 in XEDAR signaling and in the process of ectodermal differentiation.     

Influence of steroid hormones on plasma proteins in freshwater tilapia Oreochromis mossambicus

The effect of administration of cortisol, corticosterone, testosterone, progesterone and a synthetic estrogen, diethylstilbestrol on plasma proteins of tilapia (Oreochromis mossambicus) was investigated. SDS-PAGE clearly revealed the appearance of several new bands of protein, which were not present in the control plasma and were comparable to the known bands of the molecular markers. Of the different bands appeared in the steroids treated plasma, the most important ones were the presumed vitellogenin and corticotrophin binding globulin with a molecular weight of 180 and 17 kDa, respectively. Increase in protein bands in the steroid treated plasma of O. mossambicus confirmed the anabolic role of steroids in teleost.     

Allosterism and cooperativity in Pseudomonas aeruginosa GDP-mannose dehydrogenase

GDP-Mannose dehydrogenase catalyzes the formation of GDP-mannuronic acid, which is the monomeric unit from which the polysaccharide alginate is formed. Alginate is secreted by the pathogenic bacterium Pseudomonas aeruginosa and is believed to play an important role in the bacteria's resistance to antibiotics and the host immune response. We have characterized the kinetic behavior of GDP-mannose dehydrogenase in detail. The enzyme displays cooperative behavior with respect to NAD(+) binding, and phosphate and GMP act as allosteric effectors. Binding of the allosteric effectors causes the Hill coefficient for NAD(+) binding to decrease from 6 to 1, decreases K(1/2) for NAD(+) by a factor of 10, and decreases V(max) by a factor of 2. The cooperative binding of NAD(+) is also sensitive to pH; deprotonation of two residues with identical pK's of 8.0 is required for maximally cooperative behavior. The kinetic behavior of GDP-mannose dehydrogenase suggests that it must be at least hexameric under turnover conditions; however, dynamic light-scattering measurements do not provide a clear determination of the size of the active enzyme complex.     

Gene Transfer by Transduction in the Marine Environment

To determine the potential for bacteriophage-mediated gene transfer in the marine environment, we established transduction systems by using marine phage host isolates. Plasmid pQSR50, which contains transposon Tn5 and encodes kanamycin and streptomycin resistance, was used in plasmid transduction assays. Both marine bacterial isolates and concentrated natural bacterial communities were used as recipients in transduction studies. Transductants were detected by a gene probe complementary to the neomycin phosphotransferase (nptII) gene in Tn5. The transduction frequencies ranged from 1.33 × 10⁻⁷ to 5.13 × 10⁻⁹ transductants/PFU in studies performed with the bacterial isolates. With the mixed bacterial communities, putative transductants were detected in two of the six experiments performed. These putative transductants were confirmed and separated from indigenous antibiotic-resistant bacteria by colony hybridization probed with the nptII probe and by PCR amplification performed with two sets of primers specific for pQSR50. The frequencies of plasmid transduction in the mixed bacterial communities ranged from 1.58 × 10⁻⁸ to 3.7 × 10⁻⁸ transductants/PFU. Estimates of the transduction rate obtained by using a numerical model suggested that up to 1.3 × 10¹⁴ transduction events per year could occur in the Tampa Bay Estuary. The results of this study suggest that transduction could be an important mechanism for horizontal gene transfer in the marine environment.     

Characterization of Marine Temperate Phage-Host Systems Isolated from Mamala Bay, Oahu, Hawaii

To understand the ecological and genetic role of viruses in the marine environment, it is critical to know the infectivity of viruses and the types of interactions that occur between marine viruses and their hosts. We isolated four marine phages from turbid plaques by using four indigenous bacterial hosts obtained from concentrated water samples from Mamala Bay, Oahu, Hawaii. Two of the rod-shaped bacterial hosts were identified as Sphingomonas paucimobilis and Flavobacterium sp. All of the phage isolates were tailed phages and contained double-stranded DNA. Two of the phage isolates had morphologies typical of the family Siphoviridae, while the other two belonged to the families Myoviridae and Podoviridae. The head diameters of these viruses ranged from 47 to 70.7 nm, and the tail lengths ranged from 12 to 146 nm. The burst sizes ranged from 7.8 to 240 phage/bacterial cell, and the genome sizes, as determined by restriction digestion, ranged from 36 to 112 kb. The members of the Siphoviridae, T-HSIC, and T-D0, and the member of the Myoviridae, T-D1B, were found to form lysogenic associations with their bacterial hosts, which were isolated from the same water samples. Hybridization of phage T-HSIC probe with lysogenic host genomic DNA was observed in dot blot hybridization experiments, indicating that prophage T-HSIC was integrated within the host genome. These phage-host systems are available for use in studies of marine lysogeny and transduction.     

Transcriptional repression of human cad gene by hypoxia inducible factor-1alpha

De novo biosynthesis of pyrimidine nucleotides provides essential precursors for DNA synthesis and cell proliferation. The first three steps of de novo pyrimidine biosynthesis are catalyzed by a multifunctional enzyme known as CAD (carbamoyl phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase). In this work, a decrease in CAD expression is detected in numerous cell lines and primary culture human stromal cells incubated under hypoxia or desferrioxamine (DFO)-induced HIF-1alpha accumulation. A putative hypoxia response element (HRE) binding matrix is identified by analyzing human cad-gene promoter using a bioinformatic approach. Promoter activity assays, using constructs harboring the cad promoter (-710/+122) and the -67/HRE fragment (25-bases), respectively, demonstrate the suppression of reporter-gene expression under hypoxia. Suppression of cad-promoter activity is substantiated by forced expression of wild-type HIF-1alpha but abolished by overexpression of dominant-negative HIF-1alpha. A chromatin immunoprecipitation assay provides further evidence that HIF-1alpha binds to the cad promoter in vivo. These data demonstrate that the cad-gene expression is repressed by HIF-1alpha, which represents a functional link between hypoxia and cell-cycle arrest.     

Pharmacological consequences of oxidative stress in ocular tissues

The eye is a unique organ because of its constant exposure to radiation, atmospheric oxygen, environmental chemicals and physical abrasion. That oxidative stress mechanisms in ocular tissues have been hypothesized to play a role in diseases such as glaucoma, cataract, uveitis, retrolental fibroplasias, age-related macular degeneration and various forms of retinopathy provides an opportunity for new approaches to their prevention and treatment, In the anterior uvea, both H₂O₂ and synthetic peroxides exert pharmacological/toxicological actions tissues of the anterior uvea especially on the sympathetic nerves and smooth muscles of the iris–ciliary bodies of several mammalian species. Effects produced by peroxides require the presence of trace amounts of extracellular calcium and the functional integrity of mitochondrial calcium stores. Arachidonic acid metabolites appear to be involved in both the excitatory action of peroxides on sympathetic neurotransmission and their inhibitory effect on contractility of the iris smooth muscle to muscarinic receptor activation. In addition to the peroxides, isoprostanes (products of free radical catalyzed peroxidation of arachidonic acid independent of the cyclo-oxygenase enzyme) can also alter sympathetic neurotransmission in anterior uveal tissues. In the retina, both H₂O₂ and synthetic peroxides produced an inhibitory action on potassium depolarization induced release of [³H] d-aspartate, in vitro and on the endogenous glutamate and glycine concentrations in vivo. Effects caused by peroxides in the retina are mediated, at least in part, by second messengers such as nitric oxide, prostaglandins and isoprostanes. The ability of H₂O₂ to alter the integrity of neurotransmitter pools from sympathetic nerves in the anterior uvea and glutaminergic nerves in the retina could underlie its role in the etiology of glaucoma.