Biomedical subjects
Publications and source records attributed to P C Lo.
[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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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