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Epsilon sequence (UUAACUUUA) has originally been found in the bacteriophage T7 gene 10 leader region. It enhances translation in Escherichia coli via base pairing with nucleotides 458-466 located in the helical domain #17 of 16S rRNA. We have recently reported that when the complementarity to 16S rRNA is extended, the epsilon is converted from an enhancer to an independent initiator of translation. Here we report the effect of two other structural parameters, positioning in mRNA and the degree of complementarity to 16S rRNA on the translation initiation activity of epsilon in E. coli cells. Our results show that epsilon displays maximal activity as a translational initiator at its natural 9-nucleotide-long complementarity to 16S rRNA and at a 16-nucleotide-long distance to the initiation codon. Under these conditions its efficiency is comparable with that of the consensus Shine-Dalgarno sequence.

Abstract Using SDS–PAGE and MALDI-TOF mass spectrometry, we investigated the difference in the molecular structure between human and bovine ceruloplasmin. In both cases, we found that the protein is present in two majors forms of different molecular mass. The difference between human and bovine ceruloplasmin was more obvious when characterized by MALDI-TOF than with the SDS–PAGE analysis. Furthermore, we established that the N-glycoside content of both enzymes is dissimilar and that the N-glycosyl moieties are distributed in a distinctive fashion in two glycoproteins. Finally, it appeared that both proteins exhibited different cleavage patterns after treatment with trypsin. This study indicates that human and bovine ceruloplasmin differ not only in sugar composition but also in primary structure.

Abstract The basic fibroblast growth factor (FGF-2) gene is transcribed bidirectionally to yield multiple sense (coding) transcripts and a unique 1.5 kb antisense transcript which may regulate sense RNA stability. The antisense RNA also contains a long open reading frame that predicts a hypothetical protein with homology to the prokaryotic MutT antimutator proteins. However, translation of this protein has not previously been demonstrated. We employed antibodies against the conserved MutT-domain of the deduced human FGF-2 antisense protein (GFG) to demonstrate expression of an immunoreactive 24 kDa protein in liver extracts fromXenopus laevis,and two proteins of 28 and 35 kDa in rat liver. In rats, GFG protein expression detected by western blot was tissue-specific and correlated with the level of FGF-2 antisense mRNA expression. These findings demonstrate that, in addition to its possible RNA regulatory function, the FGF-2 antisense transcript is translated into a conserved MutT-related protein.

Recent literature demonstrates the accelerated aggregation of α-synuclein, a protein implicated in the pathogenesis of Parkinson's disease (PD), by the presence of preformed fibrillar conformers in vitro. Furthermore, these preformed fibrillar seeds are suggested to accelerate pathological induction in vivo when injected into the brains of mice. Variation in the results of in vivo studies is proposed to be caused by α-synuclein conformational variants. To investigate the impact of amino acid sequence on seeding efficiency, human and mouse α-synuclein seeds, which vary at 7 amino acid residues, were generated and cross-seeding kinetics studied. Using transmission electron microscopy (TEM), we confirmed that mouse α-synuclein aggregated more rapidly than human α-synuclein. Subsequently, we determined that seeding of human and mouse α-synuclein was more rapid in the presence of seeds generated from the same species. In addition, an established amyloid inhibitor, scyllo-inositol, was examined for potential inhibitory effects on α-synuclein aggregation. TEM analysis of protein:inhibitor assays demonstrated that scyllo-inositol inhibits the aggregation of α-synuclein, suggesting the therapeutic potential of the small molecule in PD.

Abstract A variety of signals result in the transcriptional induction of the p21CIP1/WAF1 promoter and both Sp1 and the related Sp3 proteins have been implicated in this induction. We have characterized the role of the C-terminal D-domains of both Sp1 and Sp3 proteins in the activation of this promoter in response to butyrate treatment of Hep G2 cells. We have defined a negative regulatory domain present in the C-terminus of Sp3. This domain decreases Sp3 protein levels, and this property can be transferred to Sp1. Changes in Sp3 protein levels may bring about growth arrest through the induction of inhibitors of the cell cycle such as p21CIP1/WAF1.

Abstract PIWI family proteins are important members of Argonaute family that play an essential role in spermatogenesis and development when loaded with piRNAs. Here we solved the crystal structure of the human PIWIL2 PAZ domain and found its PAZ domain adopts a canonical PAZ fold. We furhter built a homology model of PIWIL2 bound to 2 nt 3′ overhangs. We found that PIWIL2 utilizes a deep hydrophobic concave to accommodate the 2 nt at 3′-end of RNAs. The recognition of 2 nt 3′ overhangs by PIWIL2 is conserved in other human PIWIL proteins, implicating the evolutionarily conserved role of PAZ domain in binding to target RNAs.

To test the hypothesis that oxyhemoglobin causes contraction of vascular smooth muscle by production of inositol 1,4,5-trisphosphate which results in a release of intracellular calcium, smooth muscle cells were exposed to oxyhemoglobin and inositol trisphosphate was measured. Oxyhemoglobin, but not methemoglobin which has much less contractile action, stimulated inositol trisphosphate production. The time course was consistent with an early role for this compound in the contraction produced by hemoglobin. The increase in production of inositol trisphosphate was inhibited by pertussis toxin and also by neomycin, an inhibitor of phospholipase C, although the actions of the latter compound cannot be attributed only to an inhibition of the enzyme responsible for the production of inositol trisphosphate.

Abstract To prevent autoxidation leghemoglobin was extracted from soybean root nodules of different ages in an atmosphere of carbon monoxide. Spectrophotometric examination of the leghemoglobins revealed that, during the functional life of the nodules, leghemoglobin exists only in its reduced form. Therefore, the decline of nitrogenase activity in older root nodules cannot be attributed to the accumulation of oxidized leghemoglobin.

Protein phosphatase one (PP1) is a major eukaryotic serine/threonine protein phosphatase whose activity is controlled by targeting or regulatory subunits. Currently, very few plant protein phosphatase one regulatory subunits are known. Here, Arabidopsis GL2 EXPRESSION MODULATOR (GEM) was identified and confirmed as a protein phosphatase one binding partner. GEM is a phosphoprotein, contains a highly conserved phosphoinositide binding GRAM domain and a classic protein phosphatase one binding RVXF motif. Lipid overlays show GEM has the ability to interact with phosphoinositides through its GRAM domain. GEM is the first plant specific protein phosphatase one interactor to be discovered.

N,N-diethyl-2-[(4 phenylmethyl)-phenoxy]-ethanamine X HCl (DPPE), a compound selective for the antiestrogen binding site, is structurally similar to the aminoethyl ether group of antihistamines. Our studies now reveal that H1-, but not H2-antagonists, also compete for this site in the order: DPPE = hydroxyzine = perchlorperazine greater than phenyltoloxamine greater than pyrilamine greater than diphenhydramine. The affinity of these compounds for the antiestrogen binding site correlates with their in vitro cytotoxicity against MCF-7 and EVSA-T human breast cancer cells. Tamoxifen, DPPE and hydroxyzine also bind to H1 receptors present in digitonin-solubilized rat liver microsomes, but with less affinity than pyrilamine, which is selective for this site; the ratio of H1 to antiestrogen binding sites in this preparation is 4:1. The data suggest that the antiestrogen binding site may be, in whole or in part, a receptor for histamine different from H1 and H2.