Lead poisoning in whooper and tundra swans

Six weak whooper swans (Cygnus cygnus) and two weak tundra swans (Cygnus columbianus) were found at Swamp Miyajima (Hokkaido, Japan) in May 1998. Anorexia, depression, green watery feces, pale conjunctiva, and anemia were observed. Radiographs showed from six to 38 suspected lead pellets in the gizzard. Blood lead concentrations were 2.5-6.7 microg/g (mean+/-SD=4.6+/-1.14 microg/g) on day 1. After blood collection, the birds were treated with calcium disodium ethylenediaminetetraacetate (CaEDTA) given intravenously and force fed. Despite treatment, seven birds died the next day. Green, bile-stained livers and pale or green kidneys were observed on necropsy. Microscopically, bile pigment was widespread in the liver and acid-fast intranuclear inclusion bodies were observed in renal tubular epithelium. Lead concentrations in livers and kidneys were 14.0-30.4 microg/g and 30.2-122 microg/g wet weight, respectively. Only one bird survived and this whooper swan continued to be treated with CaEDTA and activated charcoal. No lead shot was observed in the proventriculus and gizzard by radiography on day 64 and the blood lead concentration decreased from 2.9 microg/g to 0.09 microg/g during that same period. After 4 mo of rehabilitation, the whooper swan was returned to the wild. Lead intoxication continues to be a problem at Swamp Miyajima.     

The P5 activator of a group IC ribozyme can replace the P7.1/7.2 activator of a group IA ribozyme

The P5 or P7 extensions in the group I intron ribozyme serve as "modular activator units" by stabilizing the conserved core of the ribozyme. The P5 extension of a group IC1 intron was introduced to a barely active group IA2 intron lacking its original P7 extension. The inserted P5 extension significantly activated the chimeric construct. Because the CYT-18 protein factor is also known to activate mutant group IA2 and IC1 introns lacking their P7 and P5 extensions, respectively, the RNA and protein activator units function in an analogous manner.     

Translocation of analogues of the antimicrobial peptides magainin and buforin across human cell membranes

Cationic antimicrobial peptides play important roles in innate immunity. Compared with extensive studies on peptide-bacteria interactions, little is known about peptide-human cell interactions. Using human cervical carcinoma HeLa and fibroblastic TM12 cells, we investigated the cellular uptake of fluorescent analogues of the two representative antimicrobial peptides magainin 2 and buforin 2 in comparison with the representative Arg-rich cell-penetrating Tat-(47-57) peptide (YGRKKRRQRRR). The dose, time, temperature, and energy dependence of translocation suggested that the three peptides cross cell membranes through different mechanisms. The magainin peptide was internalized within a time scale of tens of minutes. The cooperative concentration dependence of uptake suggested that the peptide forms a pore as an intermediate similar to the observations in model membranes. Furthermore, the translocation was coupled with cytotoxicity, which was larger for tumor HeLa cells. In contrast, the buforin peptide translocated within 10 min by a temperature-independent, less concentration-dependent passive mechanism without showing any significant cytotoxicity at the highest concentration investigated (100 microm). The uptake of the Tat peptide was proportional to the peptide concentration, and the concentration dependence was lost upon ATP depletion. The peptide exhibited a moderate cytotoxicity at higher concentrations. The time course did not show saturation even after 120 min. The buforin peptide, covalently attached to the 28-kDa green fluorescent protein, also entered cells, suggesting a potency of the peptide as a vector for macromolecular delivery into cells. However, the mechanism appeared to be different from that of the parent peptide.     

Archaeal-type rhodopsins in Chlamydomonas: model structure and intracellular localization

Phototaxis in the unicellular green alga Chlamydomonas reinhardtii is mediated by rhodopsin-type photoreceptor(s). Recent expressed sequence tag database from the Kazusa DNA Research Institute has provided the basis for unequivocal identification of two archaeal-type rhodopsins in it. Here we demonstrate that one is located near the eyespot, wherein the photoreceptor(s) has long been thought to be enriched, along with the results of bioinformatic analyses. Secondary structure prediction showed that the second putative transmembrane helices (helix B) of these rhodopsins are rich in glutamate residues, and homology modeling suggested that some additional intra- or intermolecular interactions are necessary for opsin-like folding of the N-terminal ca. 300-aa membrane spanning domains of 712 and 737-aa polypeptides. These results complement physiological and electrophysiological experiments combined with the manipulation of their expression [O.A. Sineshchekov, K.H. Jung, J.H. Spudich, Proc. Natl. Sci. USA 99 (2002) 8689; G. Nagel, D. Olig, M. Fuhrmann, S. Kateriya, A.M. Musti, E. Bamberg, P. Hegemann, Science 296 (2002) 2395].     

Multiple factors contribute to outcrossing in a tropical emergent Dipterocarpus tempehes, including a new pollen-tube guidance mechanism for self-incompatibility

The self-rejection system of Dipterocarpus tempehes (Dipterocarpaceae), an emergent tree of the lowland tropical forests of Borneo, were studied by means of pollination experiments, fluorescence microscopy of pollen tubes, and monitoring of ovary maturation patterns. Fruit set was higher in cross-pollinated flowers than in control and self-pollinated flowers, indicating the existence of pollen limitation and a self-rejection system. Although the adhesion and the germination of self-pollen and the growth of self-pollen tubes were not inhibited, the proportion of cross-pollen tubes that entered the style was 1.7-2.3 times higher than that of self-pollen tubes, indicating a partial self-incompatibility that inhibits self-pollen tubes from entering the style hollow. These results suggest, for the first time, that self-incompatibility is caused by a defect of pollen-tube guidance. We also suggest a threshold effect in number of pollen tubes or late-acting self-incompatibility to fully explain the drastic and uniform selection against self-pollinated flowers before ovary swelling. After that, maternal selection and/or inbreeding depression caused the abortion and delayed maturation of self-pollinated flowers. The advantages of the self-rejection process composed of partial early-acting self-incompatibility and relatively strong delayed abortion is discussed with respect to the general-flowering phenomenon in lowland dipterocarp forests.     

Adaptive evolution of the IgA hinge region in primates

IgA is a major component that prevents the penetration of pathogenic bacteria into mucosal surfaces. The IgA antibody is cleaved at the IgA hinge region with high specificity by IgA-specific proteases produced by several pathogenic bacteria. We conducted a genomic sequence analysis of the IgA genes of a wide spectrum of primates, including the first intron and second exon, which consist of the hinge region and the CH2 domain, to find evidence of positive selection. Because the hinge region is quite small, we combined the largest collection of sequences that could be clearly aligned and evaluated the total numbers of synonymous and nonsynonymous substitutions on the phylogenetic tree. The nonsynonymous to synonymous substitution ratio (d(N)/d(S) test) showed that hominoids, Old World monkeys, and New World monkeys have d(N)/d(S) ratios of 5.4, 6.3, and 4.2, respectively. Fisher's exact probability tests showed statistical significance for the Old World monkey. Because the substitution rates of the flanking sequences are more or less similar to the synonymous rates of the hinge region, these high values of d(N)/d(S) should be the result of positive selection at the hinge region. Combining the high sequence variability in each population and the highly accelerated nonsynonymous substitution rates in the hinge region, we conclude that this unusual IgA evolution is a molecular evidence of adaptive evolution possibly caused by the host-parasite relationship.     

On the interaction of ribosomal protein L5 with 5S rRNA

Ribosomal protein L5, a 5S rRNA binding protein in the large subunit, is composed of a five-stranded antiparallel beta-sheet and four alpha-helices, and folds in a way that is topologically similar to the ribonucleprotein (RNP) domain [Nakashima et al., RNA 7, 692-701, 20011. The crystal structure of ribosomal protein L5 (BstL5) from Bacillus stearothermophilus suggests that a concave surface formed by an anti-parallel beta-sheet and long loop structures are strongly involved in 5S rRNA binding. To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurred at beta-strands and loop structures in BstL5. The mutation of Lys33 at the beta 1-strand caused a significant reduction in 5S rRNA binding. In addition, the Arg92, Phe122, and Glu134 mutations on the beta2-strand, the alpha3-beta4 loop, and the beta4-beta5 loop, respectively, resulted in a moderate decrease in the 5S rRNA binding affinity. In contrast, mutation of the conserved residue Pro65 at the beta2-strand had little effect on the 5S rRNA binding activity. These results, taken together with previous results, identified Lys33, Asn37, Gln63, and Thr90 on the beta-sheet structure, and Phe77 at the beta2-beta3 loop as critical residues for the 5S rRNA binding. The contribution of these amino acids to 5S rRNA binding was further quantitatively evaluated by surface plasmon resonance (SPR) analysis by the use of BIAcore. The results showed that the amino acids on the beta-sheet structure are required to decrease the dissociation rate constant for the BstL5-5S rRNA complex, while those on the loops are to increase the association rate constant for the BstL5-5S rRNA interaction.     

Electroporation-mediated delivery of catalytic oligodeoxynucleotides for manipulation of vascular gene expression

The development of inexpensive and effective approaches to transiently decrease gene expression in vivo would be useful for the study of physiological processes in living animals. DNAzymes are a novel class of DNA oligonucleotides that can catalytically cleave target mRNAs and thereby reduce protein production. However, current methods for their delivery in vivo are limited and inefficient. In this study, we show that electroporation can be used to deliver DNAzymes to the intact mesenteric vasculature of rats. With the use of PKC-epsilon as a target, a set of wild-type and mutant control DNAzymes was designed and shown to reduce both PKC-epsilon mRNA and protein levels in cultured smooth muscle cells in a specific manner. The wild-type DNAzyme reduced PKC-epsilon protein levels by 70% at 24 h in two different cell lines without decreasing the levels of the five other PKC isoforms tested. When delivered to the intact vasculature using electroporation, the DNAzyme reduced PKC-epsilon protein levels by >60% without affecting these other PKC isoforms. Electroporation was required for oligonucleotide transfer and was able to deliver the DNAzymes to multiple cell layers in the vessel wall. Protein levels were reduced maximally by 24 h postelectroporation and returned to normal by 48 h. These results suggest that electroporation can be used to deliver DNAzymes and other DNA oligonucleotides to the vasculature in vivo and can decrease gene expression for a window of time that can be used for experimental studies.     

Lipoprotein lipase mRNA in white adipose tissue but not in skeletal muscle is increased by pioglitazone through PPAR-gamma

Lipoprotein lipase (LPL), a key enzyme for triglyceride hydrolysis, is an insulin-dependent enzyme and mainly synthesized in white adipose tissue (WAT) and skeletal muscles (SM). To explore how pioglitazone, an enhancer of insulin action, affects LPL synthesis, we examined the effect of pioglitazone on LPL mRNA levels in WAT or SM of brown adipose tissue (BAT)-deficient mice, which develop insulin resistance and hypertriglyceridemia. Both LPL mRNA of WAT and SM were halved in BAT-deficient mice. Pioglitazone increased LPL mRNA in WAT by 8-fold, which was substantially associated with a 4-fold increase of peroxisome proliferator activated receptor (PPAR)-gamma mRNA (r=0.97, p<0.0001), whereas pioglitazone did not affect LPL mRNA in SM. These results suggest that pioglitazone exclusively increases LPL production in WAT via stimulation of PPAR-gamma synthesis. Since pioglitazone does not affect LPL production in SM, this would contribute to prevent the development of insulin resistance due to lipotoxicity.