Immunological Response of Pigs to Human Cells, Including Issues Such as the Production of Natural Antibodies in Newborns.

Pigs have recently become very popular for use not only in xenotransplantation field, but in regeneration studies as well, sometimes with pigs being used as the scaffold. We have already presented our findings related to the pig immune system against human cells, including the complement systems, natural antibodies (NAs), and NK cells. In this study, we investigated the pig innate immunological reaction against human cells further. Our investigations included issues such as the production of NAs in newborns, day 0 and day 1, and sow colostrum. The alternative pathway for pig complement reacted with human cells, and pig NK cells and macrophages directly injured human aortic endothelial cells. Pig serum clearly contains the natural antibodies IgG and IgM to human peripheral blood mononuclear cells (PBMCs). Pig plasma from day 1 newborns contained almost the same levels of these natural antibodies to human PBMCs as those of sow plasma. On the other hand, pig plasma from day 0 newborns did not contain IgG and IgM to human PBMCs. In addition, sow colostrum clearly contained both IgG and IgM to human PBMCs. As expected, the pig innate immunity system reacted to human cells, including natural antibodies. However, the NAs of pigs, both IgM and IgG, against human cells do not exist in pig serum at day 0, but at day 1 and in mother's milk, indicating that NAs in newborns did not come from the placenta but from sow colostrum.

HLA-G1, but Not HLA-G3, Suppresses Human Monocyte/Macrophage-mediated Swine Endothelial Cell Lysis.

The inhibitory function of HLA-G1, a class Ib molecule, on monocyte/macrophage-mediated cytotoxicity was examined. The expression of inhibitory receptors that interact with HLA-G, immunoglobulin-like transcript 2 (ILT2), ILT4, and KIR2DL4 (CD158d) on in vitro-generated macrophages obtained from peripheral blood mononuclear cells and the phorbol 12-myristate 13-acetate (PMA)-activated THP-1 cells were examined by flow cytometry. cDNAs of HLA-G1, HLA-G3, HLA-E, and human ß2-microglobulin were prepared, transfected into pig endothelial cells (PECs), and macrophage- and the THP-1 cell-mediated PEC cytolysis was then assessed. In vitro-generated macrophages expressed not only ILT2 and ILT4 but CD158d as well. The transgenic HLA-G1 on PEC indicated a significant suppression in macrophage-mediated cytotoxicity, which was equivalent to that of transgenic HLA-E. HLA-G1 was clearly expressed on the cell surface of PEC, whereas the levels of HLA-G3 were much lower and remained in the intracellular space. On the other hand, the PMA-activated THP-1 cell was less expressed these inhibitory molecules than in vitro-generated macrophages. Therefore, the HLA-G1 on PECs showed a significant but relatively smaller suppression to THP-1 cell-mediated cytotoxicity compared to in vitro-generated macrophages. These results indicate that by generating HLA-G1, but not HLA-G3, transgenic pigs can protect porcine grafts from monocyte/macrophage-mediated cytotoxicity.

Supplemental Analysis for N-linked Sugars in Adult Pig Islets.

The pig pancreas is considered to be one of the most suitable sources of islets for clinical xenotransplantation. However, after producing α1-3galactosyltransferase knockout pigs, most of the organs of these pigs showed less antigenicity to the human body. Wild-type adult pig islets (APIs) that originally produced negligible levels of α-Gal, different from neonatal porcine islet-like cell clusters, showed a clear antigenicity to human serum. Concerning the so-called non-Gal epitopes, many studies related to glycoproteins and glycolipids are ongoing in efforts to identify them. However, our knowledge of non-Gal glycoantigens remains incomplete. In our previous study, N-glycans were isolated from APIs, and the structures of 28 of the N-glycans were detected. In this study, to identify additional structures, further analyses were performed by liquid chromatography-mass spectrometry (LC-MS). N-glycans were isolated from APIs by the method described by O'Neil et al with minor modifications and LC-MS-based structural analyses were then performed. The detected N-glycan peaks in the LC-MS spectra were selected using the FLexAnalysis software program and the structures of the glycans were predicted using the GlyocoMod Tool. The API preparation contained 11 peaks and 16 structures were then nominated as containing N-linked sugars. Among them, 5 sulfated glycans were estimated, confirming the existence of sulfate structures in N-glycans in API. In addition, these data may supplement several N-glycan structures that contain two deoxyhexose units, such as fucose, to our previous report. The data herein will be helpful for future studies of antigenicity associated with API.

Human HLA-Ev (147) Expression in Transgenic Animals.

BACKGROUND: In our previous study, we reported on the development of substituting S147C for HLA-E as a useful gene tool for xenotransplantation. In this study we exchanged the codon of HLA-Ev (147), checked its function, and established a line of transgenic mice. METHODS: A new construct, a codon exchanging human HLA-Ev (147) + IRES + human beta 2-microgloblin, was established. The construct was subcloned into pCXN2 (the chick beta-actin promoter and cytomegalovirus enhancer) vector. Natural killer cell- and macrophage-mediated cytotoxicities were performed using the established the pig endothelial cell (PEC) line with the new gene. Transgenic mice with it were next produced using a micro-injection method. RESULTS: The expression of the molecule on PECs was confirmed by the transfection of the plasmid. The established molecules on PECs functioned well in regulating natural killer cell-mediated cytotoxicity and macrophage-mediated cytotoxicity. We have also successfully generated several lines of transgenic mice with this plasmid. The expression of HLA-Ev (147) in each mouse organ was confirmed by assessing the mRNA. The chick beta-actin promoter and cytomegalovirus enhancer resulted in a relatively broad expression of the gene in each organ, and a strong expression in the cases of the heart and lung. CONCLUSION: A synthetic HLA-Ev (147) gene with a codon usage optimized to a mammalian system represents a critical factor in the development of transgenic animals for xenotransplantation.

EEG dynamics of experienced Zen meditation practitioners probed by complexity index and spectral measure.

Changes in the electroencephalogram (EEG) characteristics in experienced Zen meditation practitioners (n = 23) during 40 minutes of meditation were compared with those in the matched controls (n = 23) taking a rest for 40 minutes. Averaged complexity index ([image omitted] ) evaluation and spectral analysis were measured in three intervals: the first, middle and the last 5-min segments of Zen meditation or relaxing rest. Significant increase in frontal alpha-1 (8-10 Hz) and occipital beta power was found during meditation as compared with the EEG under the rest, whereas an average increase of theta power was observed in the controls. In meditation, brain dynamics exhibited high [image omitted] , which correlated with more beta activity. Control subjects showed no significant change in [image omitted] level. This distinction became more significant during the last 5 minutes of meditation over most electrodes. Deeper meditation state has been reported as having implications of increased beta power that can be more prominent by the approach of [image omitted] estimation. Our results substantiate the idea that long-term training with Zen-Buddhist meditation induces changes in the electro-cortical activity of the brain.

Photodynamic activity of BAM-SiPc, an unsymmetrical bisamino silicon(IV) phthalocyanine, in tumour-bearing nude mice.

BACKGROUND AND PURPOSE: Ever since the discovery of photodynamic therapy, there has been a continuous search for more potent photosensitizers. Towards that end, we have synthesized a number of novel phthalocyanine derivatives. The unsymmetrical bisamino silicon(IV) phthalocyanine BAM-SiPc is one of the most potent compounds. In in vitro cell culture, it exhibits high phototoxicity against a number of cancer cell lines. EXPERIMENTAL APPROACH: In the present investigation, the in vivo effect of BAM-SiPc was studied in the tumour-bearing nude mice model. The biodistribution of BAM-SiPc was followed to evaluate its tumour selectivity and rate of clearance. The tumour volume in the hepatocarcinoma HepG2- and the colorectal adenocarcinoma HT29-bearing nude mice was measured after photodynamic therapy. The level of intrinsic toxicity induced was also investigated. Finally, the metabolism of BAM-SiPc in the 'normal' WRL68 liver cells and the hepatocarcinoma HepG2 cells was compared. KEY RESULTS: The results not only showed significant tumour regression of HepG2 and growth inhibition of HT29 in the tumour-bearing nude mice, but also no apparent hepatic or cardiac injury with the protocol used. Histological analyses showed that apoptosis was induced in the solid tumour. BAM-SiPc could be metabolized by WRL68 liver cells but not by the hepatocarcinoma HepG2 cells. Unfortunately, BAM-SiPc did not show any specific targeting towards the tumour tissue. CONCLUSIONS AND IMPLICATIONS: The efficiency of BAM-SiPc in inhibiting tumour growth makes it a good candidate for further evaluation. Enhancement of its uptake in tumour tissue by conjugation with biomolecules is currently under investigation.

Cardiogenesis in the Drosophila model: control mechanisms during early induction and diversification of cardiac progenitors.

The dorsal vessel of Drosophila displays developmental, functional, and morphological similarities to the primitive linear heart tube of early vertebrate embryos. Because these similarities extend to the genetic and molecular level, Drosophila has become a fruitful model to study control mechanisms of early heart development. Herein we summarize recently obtained insights into control mechanisms during early induction and diversification of cardiac progenitors in Drosophila. We also show that induction of tinman, a key cardiogenic gene, in the dorsal mesoderm by Dpp (Drosophila BMP) involves protein/protein interactions between Tinman and the Smad proteins Mad and Medea, in addition to their DNA-binding activities to specific tinman enhancer sequences. Furthermore, we present evidence that binding of a high-mobility-group protein, HMG-D, to the Dpp-responsive enhancer of tinman as well as to the Tinman protein may be involved in the formation of a fully active enhancer complex.

Intramolecular [2 + 2]-photocycloaddition/thermal fragmentation approach toward 5-8-5 ring systems.

[reaction: see text]. An intramolecular [2 + 2]-photocycloaddition is used to provide a photoadduct, which upon fragmentation, lactone cleavage, and subsequent Cope rearrangement provides a dicyclopenta[a,d]cyclooctene ring system with substituents in place (e.g., C3 and C11) to access several 5-8-5 diterpene and sesterterpene natural products.

A role for the COUP-TF-related gene seven-up in the diversification of cardioblast identities in the dorsal vessel of Drosophila.

The Drosophila gene tinman is essential for dorsal vessel (heart) formation and is structurally and functionally conserved in vertebrates. In the mature embryonic dorsal vessel, tinman is expressed in four of the six pairs of cardioblasts in each segment. We provide evidence that seven-up, which is homologous to the vertebrate COUP-TF transcription factor and is expressed in the non-Tinman-expressing cardioblasts, represses tinman in these cells. Loss of function seven-up mutations derepress tinman expression in these cardioblasts while ectopic expression of seven-up represses tinman in the cardioblasts that normally express it. These changes are correlated with alterations in the expression of additional molecular markers for each of these two types of cardioblasts, such as the novel T-box-containing gene Tb66F2 and the potassium channel-encoding gene sur. These observations suggest that seven-up has a role in diversifying cardioblast identities within each segment. We also describe the tinman cis sequences that mediate tinman repression by seven-up and examine whether Seven-up can bind these sequences to directly inhibit tinman.

An efficient method for quantifying the multichannel EEG spatial-temporal complexity.

The complexity index (delta) quantifies the intrinsic dimensionality of the global complexity of a point set, and was shown to be able to characterize electroencephalogram spatial-temporal features. The complexity index is conceptually comprehensible and easily implemented, yet, it is time consuming. In this paper, we present an efficient computational method based on the projection of the high-dimensional state-space points onto a one-dimensional axis. The computational time decreases by at least 50%, without affecting the measure accuracy.

Sequence and expression of myoglianin, a novel Drosophila gene of the TGF-beta superfamily.

Various members of the TGF-beta superfamily of signaling molecules are known to have important roles in mesoderm patterning and differentiation during vertebrate and invertebrate embryogenesis. Here we characterize a new TGF-beta member from Drosophila, Myoglianin, that is most closely related to the vertebrate muscle differentiation factor Myostatin and to vertebrate BMP-11. Northern analysis shows that myoglianin is expressed throughout the Drosophila life cycle. In situ hybridization detects maternally-derived transcripts that are enriched in the pole plasm and later become enclosed in the pole cells. Between stages 11 and 14, myoglianin mRNA is exclusively detected in glial cells and their precursors. Following stage 14, high levels of myoglianin expression are observed in the developing somatic muscles as well as in visceral muscles and cardioblasts. We also show that the zygotic expression of a recently described Drosophila activin, which maps to the same interval 102 on chromosome 4 as myoglianin, is restricted to the developing central and peripheral nervous system.

Three-dimensional filtering approach to brain potential mapping.

The spatial distribution of electroencephalogram (EEG) features on the scalp surface, both in time or frequency, is of great importance in clinical applications and medical research. Traditionally, mathematical methods based on interpolation algorithms have been widely applied to obtain the EEG mappings. This paper presents an innovative approach to reconstructing the brain potential mappings from multichannel EEG's. The three-dimensional (3-D) filtering approach, differing from the numerical interpolating methods, considers the spatial distribution of brain potentials as a 3-D signal, which is processed and interpolated according to its spatial frequency characteristics. The performance of the 3-D filtering method evaluated on simulated brain potentials is shown to be comparable to the four-nearest-neighbors method. Moreover, the 3-D filtering method is superior to the spherical splines method in efficiency. Two main advantages of this method are: the prospect of developing real-time, animated EEG mappings utilizing powerful digital signal processors and its capability of processing and interpolating the brain potentials on the realistic irregular scalp surface.

Confucian ethic of death with dignity and its contemporary relevance.

This paper advances three claims. First, according to contemporary Western advocates of physician-assisted suicide and voluntary euthanasia, "death with dignity" is understood negatively as bringing about death to avoid or prevent indignity, that is, to avoid a degrading existence. Second, there is a similar morally affirmative view on death with dignity in ancient China, in classical Confucianism in particular. Third, there is a consonance as well as dissonance between these two ethics of death with dignity, such as that the Confucian perspective would regard the argument for physician-assisted suicide and voluntary euthanasia as less than compelling because of the latter's impoverished vision of human life.

bagpipe-Dependent expression of vimar, a novel Armadillo-repeats gene, in Drosophila visceral mesoderm.

Two homeobox-containing genes, tinman and bagpipe, play important roles during the specification of the midgut visceral musculature from the mesoderm during Drosophila embryogenesis. Expression of tinman in the dorsal mesoderm activates the expression of the bagpipe gene in segmental subsets of those cells, which then become determined to form the midgut visceral mesoderm. Understanding how the bagpipe gene affects this specification requires the isolation and characterization of its downstream target genes. Using an enhancer trap line that expresses its marker in the midgut visceral mesoderm, we have cloned and characterized a novel gene (vimar) that is expressed embryonically in the mid and hindgut visceral mesoderm, as well as in the CNS and PNS. The expression of this gene in the midgut visceral mesoderm initiates shortly after bagpipe expression and depends on bagpipe function. Maternal and zygotic transcripts are produced from this gene by alternative polyadenylation, and encode the same 634-amino acid protein. The vimar protein contains 15 tandem copies of the Armadillo repeat, a protein interaction domain, and is similar to mammalian Smg guanine dissociation stimulator protein, which stimulates the activity of a number of different p21 small G-proteins. These results, together with the observed lethality of vimar mutations, indicate that vimar is one of the bagpipe target genes that are required for normal development and differentiation of the midgut visceral mesoderm.

Synthesis of a potent wide-spectrum serotonin-, norepinephrine-, dopamine-reuptake inhibitor (SNDRI) and a species-selective dopamine-reuptake inhibitor based on the gamma-amino alcohol functional group.

A series of gamma-amino alcohols were synthesized and screened for reuptake inhibition and noncompetitive NMDA antagonism. Compound (+/-)-3f simultaneously and potently inhibits reuptake of 5-HT, NE, and DA, representing a potential wide-spectrum reuptake inhibitor antidepressant. In addition, comparative rat and human studies uncovered a species-selective DA reuptake inhibitor (+/-)-2e, KD(hDAT)/KD(rDAT) = 97.

A novel KH-domain protein mediates cell adhesion processes in Drosophila.

Adhesion of cells to one another and to extracellular matrices has major roles in morphogenetic processes during development. One important family of cell adhesion receptors are the integrins, which in Drosophila have crucial functions in at least two adhesion-mediated developmental events: embryonic muscle attachment and adhesion of the wing epithelia. We have cloned and characterized a gene (struthio) that is expressed in embryonic mesodermal and muscle cells, including cardioblasts, and epidermal muscle attachment sites in a pattern that is reminiscent of the expression pattern of the PS integrins. Maternal and zygotic transcripts are produced by this gene and encode similar proteins with two alternative carboxy tails. Both proteins contain identical KH domains, a protein sequence motif that is found in numerous proteins that interact with RNA. The struthio protein is highly homologous in a region including the KH domain to the mouse quaking and C. elegans gld-1 proteins, two developmentally important genes. Somatic homozygous clones of an embryonic lethal mutation in this gene (stru1A122) cause wing blisters and flight impairment, phenotypes which are associated with PS integrin subunit mutations. Thus, the struthio gene encodes a putative RNA-binding protein that appears to regulate some aspects of Drosophila integrin functioning.

Suppressor U1 snRNAs in Drosophila.

Although the role of U1 small nuclear RNAs (snRNAs) in 5' splice site recognition is well established, suppressor U1 snRNAs active in intact multicellular animals have been lacking. Here we describe suppression of a 5' splice site mutation in the Drosophila melanogaster white gene (wDR18) by compensatory changes in U1 snRNA. Mutation of positions -1 and +6 of the 5' splice site of the second intron (ACG[GTGAGT to ACC]GTGAGC) results in the accumulation of RNA retaining this 74-nucleotide intron in both transfected cells and transgenic flies. U1-3G, a suppressor U1 snRNA which restores base-pairing at position +6 of the mutant intron, increases the ratio of spliced to unspliced wDR18 RNA up to fivefold in transfected Schneider cells and increases eye pigmentation in wDR18 flies. U1-9G, which targets position -1, suppresses wDR18 in transfected cells less well. U1-3G,9G has the same effect as U1-3G although it accumulates to lower levels. Suppression of wDR18 has revealed that the U1b embryonic variant (G134 to U) is active in Schneider cells and pupal eye discs. However, the combination of 9G with 134U leads to reduced accumulation of both U1b-9G and U1b-3G,9G, possibly because nucleotides 9 and 134 both participate in a potential long-range intramolecular base-pairing interaction. High levels of functional U1-3G suppressor reduce both viability and fertility in transformed flies. These results show that, despite the difficulties inherent in stably altering splice site selection in multicellular organisms, it is possible to obtain suppressor U1 snRNAs in flies.

Species-specific signals for the splicing of a short Drosophila intron in vitro.

The effects of branchpoint sequence, the pyrimidine stretch, and intron size on the splicing efficiency of the Drosophila white gene second intron were examined in nuclear extracts from Drosophila and human cells. This 74-nucleotide intron is typical of many Drosophila introns in that it lacks a significant pyrimidine stretch and is below the minimum size required for splicing in human nuclear extracts. Alteration of sequences of adjacent to the 3' splice site to create a pyrimidine stretch was necessary for splicing in human, but not Drosophila, extracts. Increasing the size of this intron with insertions between the 5' splice site and the branchpoint greatly reduced the efficiency of splicing of introns longer than 79 nucleotides in Drosophila extracts but had an opposite effect in human extracts, in which introns longer than 78 nucleotides were spliced with much greater efficiency. The white-apricot copia insertion is immediately adjacent to the branchpoint normally used in the splicing of this intron, and a copia long terminal repeat insertion prevents splicing in Drosophila, but not human, extracts. However, a consensus branchpoint does not restore the splicing of introns containing the copia long terminal repeat, and alteration of the wild-type branchpoint sequence alone does not eliminate splicing. These results demonstrate species specificity of splicing signals, particularly pyrimidine stretch and size requirements, and raise the possibility that variant mechanisms not found in mammals may operate in the splicing of small introns in Drosophila and possibly other species.

Drosophila melanogaster genes for U1 snRNA variants and their expression during development.

We have cloned and characterized a complete set of seven U1-related sequences from Drosophila melanogaster. These sequences are located at the three cytogenetic loci 21D, 82E, and 95C. Three of these sequences have been previously studied: one U1 gene at 21D which encodes the prototype U1 sequence (U1a), one U1 gene at 82E which encodes a U1 variant with a single nucleotide substitution (U1b), and a pseudogene at 82E. The four previously uncharacterized genes are another U1b gene at 82E, two additional U1a genes at 95C, and a U1 gene at 95C which encodes a new variant (U1c) with a distinct single nucleotide change relative to U1a. Three blocks of 5' flanking sequence similarity are common to all six full length genes. Using specific primer extension assays, we have observed that the U1b RNA is expressed in Drosophila Kc cells and is associated with snRNP proteins, suggesting that the U1b-containing snRNP particles are able to participate in the process of pre-mRNA splicing. We have also examined the expression throughout Drosophila development of the two U1 variants relative to the prototype sequence. The U1c variant is undetectable by our methods, while the U1b variant exhibits a primarily embryonic pattern reminiscent of the expression of certain U1 variants in sea urchin, Xenopus, and mouse.

Structure and expression of the Drosophila melanogaster gene for the U1 small nuclear ribonucleoprotein particle 70K protein.

A genomic clone encoding the Drosophila U1 small nuclear ribonucleoprotein particle 70K protein was isolated by hybridization with a human U1 small nuclear ribonucleoprotein particle 70K protein cDNA. Southern blot and in situ hybridizations showed that this U1 70K gene is unique in the Drosophila genome, residing at cytological position 27D1,2. Polyadenylated transcripts of 1.9 and 3.1 kilobases were observed. While the 1.9-kilobase mRNA is always more abundant, the ratio of these two transcripts is developmentally regulated. Analysis of cDNA and genomic sequences indicated that these two RNAs encode an identical protein with a predicted molecular weight of 52,879. Comparison of the U1 70K proteins predicted from Drosophila, human, and Xenopus cDNAs revealed 68% amino acid identity in the most amino-terminal 214 amino acids, which include a sequence motif common to many proteins which bind RNA. The carboxy-terminal half is less well conserved but is highly charged and contains distinctive arginine-rich regions in all three species. These arginine-rich regions contain stretches of arginine-serine dipeptides like those found in transformer, transformer-2, and suppressor-of-white-apricot proteins, all of which have been identified as regulators of mRNA splicing in Drosophila melanogaster.