Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo

Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides within specific sequence contexts (CpG motifs) are detected, like bacterial or viral DNA, as a danger signal by the vertebrate immune system. CpG ODN synthesized with a nuclease-resistant phosphorothioate backbone have been shown to be potent Th1-directed adjuvants in mice, but these motifs have been relatively inactive on primate leukocytes in vitro. Moreover, in vitro assays that predict in vivo adjuvant activity for primates have not been reported. In the present study we tested a panel of CpG ODN for their in vitro and in vivo immune effects in mice and identified in vitro activation of B and NK cells as excellent predictors of in vivo adjuvant activity. Therefore, we tested >250 phosphorothioate ODN for their capacity to stimulate proliferation and CD86 expression of human B cells and to induce lytic activity and CD69 expression of human NK cells. These studies revealed that the sequence, number, and spacing of individual CpG motifs contribute to the immunostimulatory activity of a CpG phosphorothioate ODN. An ODN with a TpC dinucleotide at the 5' end followed by three 6 mer CpG motifs (5'-GTCGTT-3') separated by TpT dinucleotides consistently showed the highest activity for human, chimpanzee, and rhesus monkey leukocytes. Chimpanzees or monkeys vaccinated once against hepatitis B with this CpG ODN adjuvant developed 15 times higher anti-hepatitis B Ab titers than those receiving vaccine alone. In conclusion, we report an optimal human CpG motif for phosphorothioate ODN that is a candidate human vaccine adjuvant.     

Maurotoxin versus Pi1/HsTx1 scorpion toxins. Toward new insights in the understanding of their distinct disulfide bridge patterns

Maurotoxin (MTX) is a scorpion toxin acting on several K(+) channel subtypes. It is a 34-residue peptide cross-linked by four disulfide bridges that are in an "uncommon" arrangement of the type C1-C5, C2-C6, C3-C4, and C7-C8 (versus C1-C5, C2-C6, C3-C7, and C4-C8 for Pi1 or HsTx1, two MTX-related scorpion toxins). We report here that a single mutation in MTX, in either position 15 or 33, resulted in a shift from the MTX toward the Pi1/HsTx1 disulfide bridge pattern. This shift is accompanied by structural and pharmacological changes of the peptide without altering the general alpha/beta scaffold of scorpion toxins.     

Fluorescence properties and functional roles of tryptophan residues 60d, 96, 148, 207, and 215 of thrombin

Conservative Trp-to-Phe mutations were individually created in human thrombin at positions 60d, 96, 148, 207, and 215. Fluorescence intensities for these residues varied by a factor of 6. Residues 60d, 96, 148, and 215 transferred energy to the thrombin inhibitor 5-dimethylaminonaphthalene-1-sulfonylarginine-N-(3-ethyl-1,5- pentanediyl)amide efficiently, but residue 207 did not. Intensities correlated inversely with exposure to solvent, and measured and theoretical energy transfer efficiencies agreed well. Function was measured with respect to fibrinogen clotting, platelet and factor V activation, inhibition by antithrombin, and the thrombomodulin-dependent activation of protein C and thrombin-activable fibrinolysis inhibitor (TAFI). All activities of W96F and W207F ranged from 74 to 154% of the wild-type activity. This was also true for W148F, except for inhibition by antithrombin, where it showed 60% activity. W60dF was deficient by 30, 57, and 43% with fibrinogen clotting, platelet activation, and factor V cleavage (Arg(1006)), respectively. W215F was deficient by 90, 55, and 56% with fibrinogen clotting, platelet activation, and factor V cleavage (Arg(1536)). With protein C and TAFI, W96F, W148F, and W207F were normal. W60dF, however, was 76 and 23% of normal levels with protein C and TAFI, respectively. In contrast, W215F was 25 and 124% of normal levels in these reactions. Thus, many activities of thrombin are retained upon substitution of Trp with Phe at positions 96, 148, and 207. Trp(60d), however, appears to be very important for TAFI activation, and Trp(215) appears to very important for clotting and protein C activation.     

Molecular genetic study of the factor VIII gene in families from Bashkir with hemophilia A

The distribution of hemophilia A was studied in Bashkortostan. The factor VIII gene of the blood coagulation system was analyzed in 34 patients with hemophilia A and 48 of their close relatives. Inversion of intron 22 of the factor VIII gene was revealed in nine cases, which comprised 30% of the total sample analyzed. The type II and type III of this mutation occurred at a relatively high frequency, which may be explained by the founder effect and genetic drift. The allelic frequencies of the polymorphic locus HindIII at intron 19 were similar; a substantial allelic heterogeneity of both microsatellite (CA)-repeats at intron 13 and the DXS52 locus were found on normal and mutant X chromosomes. The molecular genetic analysis of (CA)-repeats and the loci HindIII and DXS52 in families with hemophilia A makes it possible to reveal up to 89% of the informative families.     

Protein kinase A-dependent phosphorylation of aquaporin-1

The molecular mechanisms for regulating water balance in many tissues are unknown. Like the kidney, the eye contains multiple water channel proteins (aquaporins) that transport water through membranes, including two (AQP1 and AQP4) in the ciliary body, the site of aqueous humor production. Previous results from our laboratory demonstrated that water channel activity of AQP1 was significantly increased by protein kinase A (PKA) activators such as cyclic-AMP (cAMP) and forskolin. The purpose of this study is to determine whether PKA-dependent protein phosphorylation is involved in the regulation of water channel activity of AQP1. Results presented here suggest that catalytic subunit of protein kinase A significantly increased the amount of phosphorylated AQP1 protein. In addition, these results indicated that cAMP-responsive redistribution of AQP1 may be regulated by phosphorylation of AQP1. Moreover, they provide new insights on the molecular mechanisms for regulating water balance in several tissues involving rapid water transport such as ciliary epithelium. In addition, they suggest important potential roles for AQP1 in several clinical disorders involving rapid water transport such as glaucoma.     

Antioxidative effects of green tea polyphenols on free radical initiated and photosensitized peroxidation of human low density lipoprotein

Antioxidative effects of the main polyphenolic components extracted from green tea leaves, i.e. (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin gallate (EGCG) and gallic acid (GA), against free radical initiated peroxidation of human low density lipoprotein (LDL) were studied. The peroxidation was initiated either thermally by a water-soluble initiator 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH), or photochemically by a triplet sensitizer benzophenone (BP). The reaction kinetics was monitored by the uptake of oxygen and the depletion of alpha-tocopherol (TOH) presented in the native LDL. Kinetic analysis of the antioxidation process demonstrates that these green tea polyphenols are effective antioxidants against both AAPH-initiated and BP-photosensitized LDL peroxidation. The antioxidative action of the green tea polyphenols includes trapping the initiating and/or propagating peroxyl radicals with the activity sequence EC>EGCG>ECG>EGC>GA for the AAPH initiated peroxidation, and reducing the alpha-tocopheroxyl radical to regenerate alpha-tocopherol with the activity sequence of ECG>EC>EGCG>EGC>GA and ECG>EGCG>GA>EC>EGC for the AAPH-initiated and BP-photosensitized peroxidations respectively.     

Design of potent beta-lactamase inhibitors by phage display of beta-lactamase inhibitory protein

Beta-lactamase inhibitory protein (BLIP) binds tightly to several beta-lactamases including TEM-1 beta-lactamase (K(i) 0.1 nm). The TEM-1 beta-lactamase/BLIP co-crystal structure indicates that two turn regions in BLIP insert into the active site of beta-lactamase to block the binding of beta-lactam antibiotics. Residues from each turn, Asp(49) and Phe(142), mimic interactions made by penicillin G when bound in the beta-lactamase active site. Phage display was used to determine which residues within the turn regions of BLIP are critical for binding TEM-1 beta-lactamase. The sequences of a set of functional mutants from each library indicated that a few sequence types were predominant. These BLIP mutants exhibited K(i) values for beta-lactamase inhibition ranging from 0.01 to 0.2 nm. The results indicate that even though BLIP is a potent inhibitor of TEM-1 beta-lactamase, the wild-type sequence of the active site binding region is not optimal and that derivatives of BLIP that bind beta-lactamase extremely tightly can be obtained. Importantly, all of the tight binding BLIP mutants have sequences that would be predicted theoretically to form turn structures.     

Growth-enhanced salmon modify stream ecosystem functioning

Use of fast-growing domesticated and/or genetically modified strains of fish is becoming increasingly common in aquaculture, increasing the likelihood of deliberate or accidental introductions into the wild. To date, their ecological impacts on ecosystems remain to be quantified. Here, using a controlled phenotype manipulation by implanting growth hormone in juvenile Atlantic salmon (Salmo salar), we found that growth-enhanced fish display changes in several phenotypic traits known to be important for ecosystem functioning, such as habitat use, morphology and excretion rate. Furthermore, these phenotypic changes were associated with significant impacts on the invertebrate community and key stream ecosystem functions such as primary production and leaf-litter decomposition. These findings provide novel evidence that introductions of growth-enhanced fish into the wild can affect the functioning of natural ecosystems and represent a form of intraspecific invasion. Consequently, environmental impact assessments of growth-enhanced organisms need to explicitly consider ecosystem-level effects.     

c-mos proto-oncogene product is partly degraded after release from meiotic arrest and persists during interphase in mouse zygotes.

Recently, it has been shown that the product of the c-mos proto-oncogene is a component of cytostatic factor, an activity present in unfertilized eggs from vertebrates that arrests the cell cycle in metaphase of the second meiotic division (metaphase II) possibly by stabilizing maturation-promoting factor (MPF). We have studied the behavior of the c-mos product in metaphase II mouse oocytes and soon after activation. The amount of c-mos in the oocyte was still very high after second polar body extrusion, when cyclin B has been degraded and MPF activity had decreased dramatically. Degradation of c-mos takes place later, during the G1 phase of the first cell cycle and a residual amount of c-mos is detectable during the first zygotic interphase. Our data show that the degradation of c-mos is not involved in the release from the metaphase arrest.     

The NSR1 gene encodes a protein that specifically binds nuclear localization sequences and has two RNA recognition motifs

We previously identified a protein (p67) in the yeast, Saccharomyces cerevisiae, that specifically recognizes nuclear localization sequences. We report here the partial purification of p67, and the isolation, sequencing, and disruption of the gene (NSR1) encoding this protein. p67 was purified using an affinity column conjugated with a peptide containing the histone H2B nuclear localization sequence from yeast. Using antibodies against p67 we have cloned the gene for this protein. The protein encoded by the NSR1 gene recognizes the wild-type H2B nuclear localization sequence, but does not recognize a mutant H2B sequence that is incompetent for nuclear localization in vivo. Interestingly, the NSR1 protein has two RNA recognition motifs, as well as an acidic NH2 terminus containing a series of serine clusters, and a basic COOH terminus containing arg-gly repeats. We have confirmed the nuclear localization of p67 by immunofluorescence and found that a restricted portion of the nucleus is highlighted. We have also shown that NSR1 (p67) is required for normal cell growth.