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PubMed2005

Ligaria cuneifolia flavonoid fractions modulate cell growth of normal lymphocytes and tumor cells as well as multidrug resistant cells.

Flavonoids are ubiquitous compounds present in plant extracts. They represent a major active component of the plant extract and are often known for their anti-inflammatory and anti-tumor effects. Previously, we demonstrated that Ligaria cuneifolia (R et P) Tiegh. (Loranthaceae) extracts inhibit proliferation of murine mitogen-activated lymphocytes as well as murine T leukaemia (LB) and breast tumor cells (MMT). The aim of this study was to assess the anti-proliferative and pro-apoptotic activities of three separate flavonoid fractions derived from L. cuneifolia whole extract (aqueous, methanolic and ethyl acetate) on normal and tumor cells. This was performed as a bio-guided approach leading to the isolation and identification of the active compounds responsible for the effects observed with the whole extract. Results showed that the three fractions differed in the amount and type of compounds found. Only the ethyl acetate flavonoid fraction (100 microg/ml) was able to inhibit significantly the proliferation of Con A stimulated splenocytes or LB and MMT cells. Inhibition of proliferation was mediated by apoptosis as determined by morphology and DNA hypodiploidy. The ethyl acetate fraction modified mRNA expression of IL-2, IL-10 and TGF-beta, while the methanol fraction only modified IL-10 mRNA on LB cells. Our results show that the ethyl acetate flavonoid fraction contains the most active compound/s and is the potential candidate to isolate the active compound/s responsible for the effects observed with L. cuneifolia whole extract.

Animals↗
PubMed2000

Review article: transcription factors and growth factors in ulcer healing.

This review is focused on recent investigations demonstrating a pharmacological and pathophysiologic role in gastroduodenal ulceration for growth factors such as basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF), as well as for transcription factors. Our experiments revealed accelerated healing, without decreased gastric acid secretion, of chronic cysteamine-induced duodenal ulcers in rats treated daily for 3 weeks with intragastric administration of bFGF, PDGF or VEGF. Our recent studies also indicate a pathophysiological role of endogenous growth factors in the natural history of experimental duodenal ulcer development and healing. More recently, we investigated the genetic regulation of these growth factors in experimental duodenal ulceration. Since gene expression is most effectively controlled by transcription factors, proteins that bind to cis-acting elements of DNA and guide the binding of polymerase II to start the transcription of specific mRNA, we tested the hypothesis that the expression of IEGs and their transcription factor products, such as Egr-1 and Sp1, might precede the increased synthesis of bFGF, PDGF and VEGF in duodenal ulcer healing. Indeed, the duodenal ulcerogen cysteamine, but not its nonulcerogen and toxic analogue ethanolamine, rapidly increased duodenal (but not gastric) mucosal levels of ET-1, which was followed by enhanced expression of Egr-1 and a decrease in Sp1 in the preulcerogenic stage of duodenal ulceration. These changes in levels of ET-1 and expression of transcription factors were also accompanied by increased expression of the CDK inhibitor p21. Thus, not only growth factors such as bFGF, PDGF and VEGF, but also transcription factors such as Egr-1 and Sp1 and the cell cycle regulator p21, may play a role in the natural history of experimental duodenal ulceration.

Animals↗
PubMed1992

Expression of s-laminin and laminin in the developing rat central nervous system.

The extracellular matrix component, s-laminin, is a homologue of the B1 subunit of laminin. S-laminin is concentrated in the synaptic cleft at the neuromuscular junction and contains a site that is adhesive for motor neurons, suggesting that it may influence neuromuscular development. To ascertain whether s-laminin may also play roles in the genesis of the central nervous system, we have examined its expression in the brain and spinal cord of embryonic and postnatal rats. S-laminin was not detectable in synapse-rich areas of adults. However, s-laminin was present in discrete subsets of three laminin-containing structures: (1) In the developing cerebral cortex, laminin and s-laminin were expressed in the subplate, a transient layer through which neuroblasts migrate and cortical afferents grow. Both laminin and s-laminin disappeared as embryogenesis proceeded; however, laminin was more widely distributed and present longer than s-laminin. (2) In the developing spinal cord, laminin was present throughout the pia. In contrast, s-laminin was concentrated in the pia that overlies the floor plate, a region in which extracellular cues have been postulated to guide growing axons. (3) In central capillaries, s-laminin appeared perinatally, an interval during which the blood-brain barrier matures. In contrast, laminin was present in capillary walls of both embryos and adults. To extend our immunohistochemical results, we used biochemical methods to characterize s-laminin in brain. We found that authentic s-laminin mRNA is present in the embryonic brain, but that brain-derived s-laminin differs (perhaps by a posttranslational modification) from that derived from nonneural tissues. We also used tissue culture methods to show that glia are capable of synthesizing "brain-like" s-laminin, and of assembling it into an extracellular matrix. Thus, glia may be one cellular source of s-laminin in brain. Together, these results demonstrate that s-laminin is present in the developing central nervous system, and raise the possibility that this molecule may influence developmental processes.

Animals↗
PubMed2003

A common sequence motif determines the Cajal body-specific localization of box H/ACA scaRNAs.

Post-transcriptional synthesis of 2'-O-methylated nucleotides and pseudouridines in Sm spliceosomal small nuclear RNAs takes place in the nucleoplasmic Cajal bodies and it is directed by guide RNAs (scaRNAs) that are structurally and functionally indistinguishable from small nucleolar RNAs (snoRNAs) directing rRNA modification in the nucleolus. The scaRNAs are synthesized in the nucleoplasm and specifically targeted to Cajal bodies. Here, mutational analysis of the human U85 box C/D-H/ACA scaRNA, followed by in situ localization, demonstrates that box H/ACA scaRNAs share a common Cajal body-specific localization signal, the CAB box. Two copies of the evolutionarily conserved CAB consensus (UGAG) are located in the terminal loops of the 5' and 3' hairpins of the box H/ACA domains of mammalian, Drosophila and plant scaRNAs. Upon alteration of the CAB boxes, mutant scaRNAs accumulate in the nucleolus. In turn, authentic snoRNAs can be targeted into Cajal bodies by addition of exogenous CAB box motifs. Our results indicate that scaRNAs represent an ancient group of small nuclear RNAs which are localized to Cajal bodies by an evolutionarily conserved mechanism.

Base Sequence↗
PubMed2003

Scribble is essential for olfactory behavior in Drosophila melanogaster.

The ability to discriminate and respond to chemical signals from the environment is an almost universal prerequisite for survival. Here, we report that the scaffold protein Scribble is essential for odor-guided behavior in Drosophila. Previously, we identified a P-element insert line with generalized sexually dimorphic smell impairment, smi97B. We found that the transposon in this line is located between the predicted promoter region and the transcription initiation site of scrib. A deficiency in this region, Df(3R)Tl-X, and two scrib null alleles fail to complement the smell-impaired phenotype of smi97B. Wild-type behavior is restored by precise excision of the P element, scrib mRNA levels correspond with mutant and wild-type phenotypes, and introduction of a full-length scrib transgene in the smi97B mutant rescues the olfactory deficit. Expression of Scrib is widespread in olfactory organs and the central nervous system. Finally, alternative splicing of scrib generates transcripts that differ in the number of leucine-rich repeats and PDZ domains.

Alleles↗
PubMed2002

Upregulation of EphA receptor expression in the injured adult rat spinal cord.

After spinal cord injury (SCI), the inability of supraspinal neurons to regenerate or reform functional connections is likely due to proteins in the surrounding microenvironment restricting regeneration. EphAs are a family of receptor tyrosine kinases that are involved in axonal guidance during development. These receptors and their ligands, the Ephrins, act via repulsive mechanisms to guide growing axons towards their appropriate targets and allow for the correct developmental connections to be made. In the present study, we investigated whether EphA receptor expression changed after a thoracic contusion SCI. Our results indicate that several EphA molecules are upregulated after SCI. Using semiquantitative RT-PCR to investigate mRNA expression after SCI, we found that EphA3, A4, and A7 mRNAs were upregulated. EphA3, A4, A6, and A8 receptor immunoreactivity increased in the ventrolateral white matter (VWM) at the injury epicenter. EphA7 had the highest level of immunoreactivity in both control and injured rat spinal cord. EphA receptor expression in the white matter originated from glial cells as coexpression in both astrocytes and oligodendrocytes was observed. In contrast, gray matter expression was localized to neurons of the ventral gray matter (motor neurons) and dorsal horn. After SCI, specific EphA receptor subtypes are upregulated and these increases may create an environment that is unfavorable for neurite outgrowth and functional regeneration.

Animals↗
PubMed2001

Expression of bone matrix protein mRNAs by primary and cloned cultures of the regenerative phenotype of human periodontal fibroblasts.

The successful regeneration of periodontal tissues is dependent, in part, on the ability of cells to reconstitute the mineralized tissues of cementum and bone. The aim of the present study was to characterize regeneration-associated cells in terms of their ability to express mineralized tissue macromolecules. Following guided tissue regeneration, cell cultures were established from regenerating tissue, periodontal ligament, and gingiva. Additionally, these cells were transfected, and single-cell-derived clones were established. Following treatment with platelet-derived growth factor-BB and insulin-derived growth factor-1, the presence of mRNA for alkaline phosphatase, osteocalcin, bone sialoprotein, osteopontin, and bone morphogenetic proteins-2 and -4 was assessed. The three cell types expressed similar mRNA levels for alkaline phosphatase, bone morphogenetic protein-2, and bone morphogenetic protein-4, whereas the expression of osteopontin, osteocalcin, and bone sialoprotein was greater in the periodontal ligament and regenerating tissue fibroblasts compared with the gingival fibroblasts. The two growth factors did not affect the expression of any of the genes. This study has identified markers that correlate with the known ability of periodontal ligament and regenerating tissue-derived fibroblasts to facilitate regeneration of the mineralized tissues of the periodontium.

Alkaline Phosphatase↗
PubMed2003

Fold recognition, homology modeling, docking simulations, kinetics analysis and mutagenesis of ATP/CTP:tRNA nucleotidyltransferase from Methanococcus jannaschii.

ATP/CTP:tRNA nucleotidyltransferases (NTases) and poly(A) polymerases (PAPs) belong to the same superfamily and their catalytic domains are remotely related. Based on the results of fold-recognition analysis and comparison of secondary structure patterns, we predicted that these two NTase families share three domains, corresponding to "palm," "fingers," and "fingernails" in the PAP crystal structure. A homology model of tRNA NTase from Methanococcus jannaschii was constructed. Energy minimization calculations of enzyme-nucleotide complexes and computer-aided docking of nucleotides onto the enzyme's surface were carried out to explore possible ATP and CTP binding sites. Theoretical models were used to guide experimental analysis. Recombinant His-tagged enzyme was expressed in Escherichia coli, and kinetic properties were characterized. The apparent K(M) for CTP was determined to be 38 microM, and the apparent K(M) for ATP was 21 microM. Three mutations of basic amino acids to alanine were created in a highly conserved region predicted to be in the vicinity of the nucleotide binding site. A deletion was also constructed to remove the C-terminal structural domain defined by the model; it retained about 1% of wild type enzymatic activity using CTP as co-substrate, confirming that detectable catalytic activity is exhibited by the N-terminal domain, as defined by the model. Our results suggest a mechanism of differential ATP and CTP binding, which explains how the tRNA NTase, having only one catalytic site, utilizes different nucleotide triphosphates depending on the nature of the tRNA substrate.

Adenosine Triphosphate↗
PubMed1998

Purification and characterization of a novel 13 S hetero-oligomeric protein complex that stimulates in vitro Golgi transport.

Intracellular protein traffic involves a tightly regulated series of events in which a membrane-bounded vesicles bud from one compartment and are specifically targeted to the next compartment, where they dock and fuse. A cell-free system that reconstitutes vesicle trafficking between the cis and medial Golgi cisternae has been used previously to identify several proteins involved in vesicular transport (N-ethylmaleimide-sensitive fusion protein, soluble N-ethylmaleimide-sensitive fusion protein attachment proteins, p115, and p16); however, these factors are insufficient to drive the transport reaction. We have used a modified version of this in vitro intra-Golgi transport assay to guide purification of a new transport-stimulating activity. The active component is a 13 S hetero-oligomeric complex consisting of at least five polypeptides (approximately 110, 109, 90, 82, and 71 kDa), which we term Golgi transport complex (GTC). Hydrodynamic properties suggest that GTC is approximately 800 kDa and nonglobular. We obtained peptide sequence information from the 90-kDa subunit (GTC-90) that allowed us to identify a number of GTC-90 cDNAs. Comparison of these cDNAs with one another and with the genomic sequence suggests that the GTC-90 mRNA is alternatively spliced. Anti-GTC-90 antibodies inhibit the in vitro Golgi transport assay, confirming the functionality of the purified complex. Subcellular fractionation indicates that GTC-90 exists in both membrane and cytosolic pools, with the cytosolic pool associated exclusively with the GTC complex. The membrane-associated pool of GTC-90 is localized to the Golgi apparatus.

Adaptor Proteins, Vesicular Transport↗
PubMed2001

Cellular distribution of GAP-43 mRNA in hippocampus and cerebellum of adult rat brain by in situ RT-PCR.

The growth-associated protein GAP-43 is a presynaptic membrane phosphoprotein that plays a key role in guiding the growth of axons and in modulating the formation of new synapses. To identify the cells that synthesize GAP-43 mRNA, we applied direct in situ reverse transcription-polymerase chain reaction (in situ RT-PCR) in cerebellum and hippocampus of adult rat brain. In situ RT-PCR revealed GAP-43 mRNA in cerebellar granule cells, in Purkinje cells and in some interneurons of the molecular layer. Previous in situ hybridization studies had demonstrated a dense label throughout the granular layer of the cerebellar cortex but no labeling of other cerebellar neurons. Hippocampal cells showing distinct GAP-43 mRNA signal after in situ RT-PCR were CA1 and CA3 pyramidal neurons, CA4 hilar cells, and dentate gyrus granule cells, whereas in situ hybridization studies had detected GAP-43 mRNA only in CA3 and CA1 pyramidal neurons. Our data indicate that GAP-43 mRNA is widely distributed, suggesting that many cell types are potentially involved in synaptic plasticity events. (J Histochem Cytochem 49:1195-1196, 2001)

Animals↗
PubMed2000

The alternative migratory pathways of the Drosophila tracheal cells are associated with distinct subsets of mesodermal cells.

The Drosophila tracheal system is a model for the study of the mechanisms that guide cell migration. The general conclusion from many studies is that migration of tracheal cells relies on directional cues provided by nearby cells. However, very little is known about which paths are followed by the migrating tracheal cells and what kind of interactions they establish to move in the appropriate direction. Here we analyze how tracheal cells migrate relative to their surroundings and which tissues participate in tracheal cell migration. We find that cells in different branches exploit different strategies for their migration; while some migrate through preexisting grooves, others make their way through homogeneous cell populations. We also find that alternative migratory pathways of tracheal cells are associated with distinct subsets of mesodermal cells and propose a model for the allocation of groups of tracheal cells to different branches. These results show how adjacent tissues influence morphogenesis of the tracheal system and offer a model for understanding how organ formation is determined by its genetic program and by the surrounding topological constraints.

Animals↗
PubMed2004

[Effect of Xiangdan Injection on mRNA expression of endothelial vaso-active factors of patients with coronary heart disease and blood stasis].

OBJECTIVE: To evaluate the effect of Xiangdan Injection on mRNA expression of the endothelial vaso-active factors of patients with coronary heart disease and blood stasis. METHODS: Fifty-six patients were randomly divided into two groups:twenty-eight patients were treated according to the therapeutic guide for coronary heart disease as the control group and 28 were given the same treatment plus Xiangdan Injection as the treated group. The expressions of ET-1 and eNOS mRNA were examined with RT-PCR before experiment and ten days later. RESULTS: The positive rate of eNOS mRNA of the treated group increased, while the positive rate of ET-1 mRNA of the treated group decreased after ten day's treatment, with significant differences as compared with that before the experiment. Xiangdan Injection up-regulated the eNOS mRNA expression and suppressed the ET-1 mRNA expression. Changes of expression were not observed in the control group. CONCLUSION: Xiangdan Injection improves the endothelial function of patients with coronary heart disease and blood stasis by regulating the expressions of ET-1 and eNOS mRNA.

Aged↗
PubMed2002

A comparative guide to gene prediction tools for the bioinformatics amateur.

Several hundred programs using different algorithms have been designed to predict individual coding features within any genomic sequence, but none of these tools covers all aspects of a gene or is 100% accurate in its prediction. Automated simultaneous processing of the results from a number of these programs minimizes the chance of a false positive prediction and quickly generates integrated data. We report here on the analysis of two known genes in 5 and 25 kb segments of genomic sequence using four genome annotation packages, NIX, RUMMAGE, Genotator and EMBOSS. Gene predictions were confirmed using cDNA sequences and a comparison was made between the packages. This study showed a similarity in the ability of NIX, RUMMAGE and Genotator to predict well-characterised genes and basic structures, but poor exon prediction for a small, 3 exon gene. However, the BLAST subprograms of all three packages correctly identified the 3 exons. In addition, EST BLAST subprograms identified a previously undescribed, possible 5' untranslated exon for the smaller gene and a number of putative alternatively spliced exons in the larger gene. Overall, NIX was found to be the most user-friendly package, in terms of easy access to databases and the interactive graphical display of results.

Algorithms↗
PubMed1993

Requirements of a group I intron for reactions at the 3' splice site.

Structural elements of the Tetrahymena group I intron have been implicated in directing the exon-ligation transesterification reaction (the second step of self-splicing) to the correct 3' splice site. The 3' splice site also serves as the reaction site in a distinct transesterification reaction known as G-exchange and in a specific hydrolysis reaction. The dependence of the reactions on the internal guide sequence (IGS), an exon-binding element, was examined. Hydrolysis did not require the IGS, while G-exchange did, suggesting that one of the guanosine-binding sites employed during G-exchange requires the IGS. For the exon ligation reaction, the specification of the 3' splice site is thought to be influenced by the 3' exon-IGS pairing P10 and three structural elements in the intron: the universally conserved guanosine residue preceding the 3' splice site, the long-range pairing P9.0, and the triple stem-loop region composed of P9, P9.1 and P9.2. A systematic mutational study of the three structural elements was performed in order to clarify the mechanism of the specification of the 3' splice site for the reactions. For both transesterification reactions, the elements are important, with some superfluity in function. The specific hydrolysis reaction, however, shows even more dependence on the individual elements, especially the conserved guanosine. The results are discussed in terms of models for the mechanism of self-splicing.

Animals↗
PubMed2003

Organic anion and cation transporters occur in pairs of similar and similarly expressed genes.

Organic anion and cation transporters (OATs, OCTs, OCTNs, and ORCTLs), transmembrane proteins essential to renal xenobiotic excretion, are encoded by a group of related genes. As yet there have been no studies of the transcriptional regulation of this important gene family. While such studies have traditionally been labor-intensive, comparative genomics approaches are now available that have proven reliable guides to critical regulatory elements. We report here the genomic sequencing of murine OAT1 (the cDNA of which was originally cloned by us as NKT) and OAT3 (Roct), and derivation of phylogenetic footprints (evolutionarily conserved non-coding sequences) by comparison to the human genome. We find binding sites within these footprints for several transcription factors implicated in kidney development, including PAX1, PBX, WT1, and HNF1. Additionally, we note that OATs and OCTs occur in the human and mouse genomes as tightly linked pairs (OAT1 and OAT3, UST3 and OAT5, OAT4 and URAT1/RST, OCT1 and 2, OCTN1 and 2, ORCTL3 and 4) that are also close phylogenetic relations, with Flipt1 and 2, and OAT2 the only unpaired family members. Finally, we find that pair-members have similar tissue distributions, suggesting that the pairing might exist to facilitate the co-regulation of the genes within each pair.

5' Flanking Region↗
PubMed2005

MicroRNA directs mRNA cleavage of the transcription factor NAC1 to downregulate auxin signals for arabidopsis lateral root development.

Although several plant microRNAs (miRNAs) have been shown to play a role in plant development, no phenotype has yet been associated with a reduction or loss of expression of any plant miRNA. Arabidopsis thaliana miR164 was predicted to target five NAM/ATAF/CUC (NAC) domain-encoding mRNAs, including NAC1, which transduces auxin signals for lateral root emergence. Here, we show that miR164 guides the cleavage of endogenous and transgenic NAC1 mRNA, producing 3'-specific fragments. Cleavage was blocked by NAC1 mutations that disrupt base pairing with miR164. Compared with wild-type plants, Arabidopsis mir164a and mir164b mutant plants expressed less miR164 and more NAC1 mRNA and produced more lateral roots. These mutant phenotypes can be complemented by expression of the appropriate MIR164a and MIR164b genomic sequences. By contrast, inducible expression of miR164 in wild-type plants led to decreased NAC1 mRNA levels and reduced lateral root emergence. Auxin induction of miR164 was mirrored by an increase in the NAC1 mRNA 3' fragment, which was not observed in the auxin-insensitive mutants auxin resistant1 (axr1-12), axr2-1, and transport inhibitor response1. Moreover, the cleavage-resistant form of NAC1 mRNA was unaffected by auxin treatment. Our results indicate that auxin induction of miR164 provides a homeostatic mechanism to clear NAC1 mRNA to downregulate auxin signals.

Arabidopsis↗
PubMed2005

Semaphorin signaling guides cranial neural crest cell migration in zebrafish.

Cranial neural crest cells (NCCs) migrate into the pharyngeal arches in three primary streams separated by two cranial neural crest (NC)-free zones. Multiple tissues have been implicated in the guidance of cranial NCC migration; however, the signals provided by these tissues have remained elusive. We investigate the function of semaphorins (semas) and their receptors, neuropilins (nrps), in cranial NCC migration in zebrafish. We find that genes of the sema3F and sema3G class are expressed in the cranial NC-free zones, while nrp2a and nrp2b are expressed in the migrating NCCs. sema3F/3G expression is expanded homogeneously in the head periphery through which the cranial NCCs migrate in lzr/pbx4 mutants, in which the cranial NC streams are fused. Antisense morpholino knockdown of Sema3F/3G or Nrp2 suppresses the abnormal cranial NC phenotype of lzr/pbx4 mutants, demonstrating that aberrant Sema3F/3G-Nrp2 signaling is responsible for this phenotype and suggesting that repulsive Sema3F/3G-Npn2 signaling normally contributes to the guidance of migrating cranial NCCs. Furthermore, global over-expression of sema3Gb phenocopies the aberrant cranial NC phenotype of lzr/pbx4 mutants when endogenous Sema3 ligands are knocked down, consistent with a model in which the patterned expression of Sema3 ligands in the head periphery coordinates the migration of Nrp-expressing cranial NCCs.

Animals↗
PubMed2003

Identification of signature and primers specific to genus Pseudomonas using mismatched patterns of 16S rDNA sequences.

BACKGROUND: Pseudomonas, a soil bacterium, has been observed as a dominant genus that survives in different habitats with wide hostile conditions. We had a basic assumption that the species level variation in 16S rDNA sequences of a bacterial genus is mainly due to substitutions rather than insertion or deletion of bases. Keeping this in view, the aim was to identify a region of 16S rDNA sequence and within that focus on substitution prone stretches indicating species level variation and to derive patterns from these stretches that are specific to the genus. RESULTS: Repeating elements that are highly conserved across different species of Pseudomonas were considered as guiding markers to locate a region within the 16S gene. Four repeating patterns showing more than 80% consistency across fifty different species of Pseudomonas were identified. The sub-sequences between the repeating patterns yielded a continuous region of 495 bases. The sub-sequences after alignment and using Shanon's entropy measure yielded a consensus pattern. A stretch of 24 base positions in this region, showing maximum variations across the sampled sequences was focused for possible genus specific patterns. Nine patterns in this stretch showed nearly 70% specificity to the target genus. These patterns were further used to obtain a signature that is highly specific to Pseudomonas. The signature region was used to design PCR primers, which yielded a PCR product of 150 bp whose specificity was validated through a sample experiment. CONCLUSIONS: The developed approach was successfully applied to genus Pseudomonas. It could be tried in other bacterial genera to obtain respective signature patterns and thereby PCR primers, for their rapid tracking in the environmental samples.

Base Pair Mismatch↗