Variations in the unstructured C-terminal tail of interferons contribute to differential receptor binding and biological activity

Type I interferons (IFNs) elicit antiviral, antiproliferative and immunomodulatory properties in cells. All of them bind to the same receptor proteins, IFNAR1 and IFNAR2, with different affinities. While the 13 known IFNalphas are highly conserved, the C-terminal unstructured tail was found to have large variation in its net charge, from neutral to +4. This led us to speculate that the tail may have a role in modulation of the IFN biological activity, through fine-tuning the binding to IFNAR2. To evaluate this hypothesis, we replaced the tail of IFNalpha2 with that of IFNalpha8 and IFNbeta tails, or deleted the last five residues of this segment. Mutations to the more positively charged tail of IFNalpha8 resulted in a 20-fold higher affinity to IFNAR2, which results in a higher antiviral and antiproliferative activity. Double and multiple mutant cycle analysis placed the tail near a negatively charged loop on IFNAR2, comprising of residues Glu 132-134. Deleting the tail resulted in only twofold reduction in binding compared to the wild-type. Next, we modeled the location of the tail using a two-step procedure: first we generated 200 models of the tail docked on IFNAR2 using HADDOCK, second the models were scored according to the fit between experimentally determined rates of association of nine mutant complexes, and their calculated rates using the PARE software. From the results we suggest that the unstructured tail of IFNalpha is gaining a specific structure in the bound state, binding to a groove below the 132-134 loop in IFNAR2.     

JS-K,a Nitric Oxide Prodrug,Has Enhanced Cytotoxicity in Colon Cancer Cells with Knockdown of Thioredoxin Reductase 1

Background

The selenoenzyme thioredoxin reductase 1 has a complex role relating to cell growth. It is induced as a component of the cellular response to potentially mutagenic oxidants, but also appears to provide growth advantages to transformed cells by inhibiting apoptosis. In addition, selenocysteine-deficient or alkylated forms of thioredoxin reductase 1 have also demonstrated oxidative, pro-apoptotic activity. Therefore, a greater understanding of the role of thioredoxin reductase in redox initiated apoptotic processes is warranted.

Methodology

The role of thioredoxin reductase 1 in RKO cells was evaluated by attenuating endogenous thioredoxin reductase 1 expression with siRNA and then either inducing a selenium-deficient thioredoxin reductase or treatment with distinct redox challenges including, hydrogen peroxide, an oxidized lipid, 4-hydroxy-2-nonenol, and a nitric oxide donating prodrug. Thioredoxin redox status, cellular viability, and effector caspase activity were measured.

Conclusions/Significance

In cells with attenuated endogenous thioredoxin reductase 1, a stably integrated selenocysteine-deficient form of the enzyme was induced but did not alter either the thioredoxin redox status or the cellular growth kinetics. The oxidized lipid and the nitric oxide donor demonstrated enhanced cytotoxicity when thioredoxin reductase 1 was knocked-down; however, the effect was more pronounced with the nitric oxide prodrug. These results are consistent with the hypothesis that attenuation of the thioredoxin-system can promote apoptosis in a nitric oxide-dependent manner.     

Preparation and chiro-optical characterization of polyaniline doped with (+) or (-)-2-pyrrolidone-5-carboxylic acid (PCA)

Optically active polyaniline (PANI) salts were readily generated in solution via the enantioselective acid doping of neutral emeraldine base (EB) form of PANI with either (+) or (-)-2-pyrrolidone-5-carboxylic acid (PCA) in dimethylsulfoxide (DMSO) and dimethylformamide (DMF) solvents. Strong mirror imaged circular dichroism (CD) spectra were obtained for the deep green polymer solutions obtained with (+) or (-) PCA, suggesting that the acid doping is enantioselective, with one helical screw of the polymer chain being preferentially produced depending on the nature of enantiomer. It was observed that molar concentration of PCA as well as nature of solvent plays a very important role in the generation of optically active PANI. The generated optically active PANI did not show any loss of optical activity up to 200 h.     

Ancient Origins of RGK Protein Function: Modulation of Voltage-Gated Calcium Channels Preceded the Protostome and Deuterostome Split

RGK proteins, Gem, Rad, Rem1, and Rem2, are members of the Ras superfamily of small GTP-binding proteins that interact with Ca²⁺ channel β subunits to modify voltage-gated Ca²⁺ channel function. In addition, RGK proteins affect several cellular processes such as cytoskeletal rearrangement, neuronal dendritic complexity, and synapse formation. To probe the phylogenetic origins of RGK protein–Ca²⁺ channel interactions, we identified potential RGK-like protein homologs in genomes for genetically diverse organisms from both the deuterostome and protostome animal superphyla. RGK-like protein homologs cloned from Danio rerio (zebrafish) and Drosophila melanogaster (fruit flies) expressed in mammalian sympathetic neurons decreased Ca²⁺ current density as reported for expression of mammalian RGK proteins. Sequence alignments from evolutionarily diverse organisms spanning the protostome/deuterostome divide revealed conservation of residues within the RGK G-domain involved in RGK protein – Ca_vβ subunit interaction. In addition, the C-terminal eleven residues were highly conserved and constituted a signature sequence unique to RGK proteins but of unknown function. Taken together, these data suggest that RGK proteins, and the ability to modify Ca²⁺ channel function, arose from an ancestor predating the protostomes split from deuterostomes approximately 550 million years ago.     

Synthesis and evaluation of piperazine and homopiperazine analogues of JS-K,an anti-cancer lead compound

Here we report a number of novel JS-K structural analogues with sub-micromolar anti-proliferative activities against human leukemia cell lines HL-60 and U937; JS-K is the anti-cancer lead compound O²-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate. The ability of these compounds to generate intracellular nitric oxide correlated well with their observed anti-proliferative effects: analogues that had potent inhibitory activity against leukemia cells formed elevated levels of intracellular nitric oxide.     

Inception and Five-year Run of a Semen Cryobank. Clinical and Behavioral Aspects

Recent developments in infertility treatment, as well as medical and ethical concern to preserve the fertilizing ability of male subjects potentially at risk, led to the inception of a sperm cryobank in our medical center in 1996. Up to the end of the year 2000, 64 young men displaying higher semen values than generally required were accepted as donors, while 305 married (mean age 32.5, range 22–54) and 381 single women (mean age 41.2, range 27–50) were treated by artificial insemination donor (AID), resulting in 251 conceptions. Besides, 437 male subjects aged 15–61 requested sperm cryopreservation. Methodological considerations about sperm cryopreservation, and behavioral implications therefrom, are evaluated.     

Paracrine intercellular communication by a Ca2+- and SNARE-independent release of GABA and glutamate prior to synapse formation

GABA and glutamate receptors are expressed in immature "silent" CA1 pyramidal neurons prior to synapse formation, but their function is unknown. We now report the presence of tonic, spontaneous, and evoked currents in embryonic and neonatal CA1 neurons mediated primarily by the activation of GABA(A) receptors. These currents are mediated by a nonconventional release of transmitters, as they persist in the presence of calcium channel blockers or botulinium toxin and are observed in Munc18-1-deficient mice in which vesicular release is abolished. This paracrine communication is modulated by glutamate but not GABA transporters, which do not operate during this period of life. Thus, a Ca(2+)- and SNARE-independent release of transmitters underlies a paracrine mode of communication before synapse formation.     

Neuroendocrine regulation of prolactin secretion in adult female rhesus monkeys during different phases of the menstrual cycle: role of neuroexcitatory amino acid (NMA)

The present study attempts to examine the role of N-methyl-D, L-aspartate (NMDA) receptors in the central regulation of prolactin (PRL) secretion, which may be involved in ovarian function and its alteration by glutamate in various phases of the menstrual cycle of female rhesus monkeys (Macaca mulatta). The results suggest that the glutaminergic component of the control system, which governs PRL secretion by utilizing NMDA receptors, may have an important role in regulating changes in PRL secretion. The response of PRL during the luteal phase of the cycle was different from that observed in follicular and menstrual phases. Steroids may influence the NMDA-dependent drive to release PRL. N-methyl-D-aspartic acid (NMA) involvement in the regulation of PRL secretion may occur through activation of the PRL-stimulating system depending on the physiological state or steroidal milieu. It is possible, therefore, that the NMA-induced release of PRL-releasing factors (PRF) and PRL are enhanced in the presence of ovarian feedback.