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PubMed1982

Interferon-induced alterations in membrane functions and the growth of Daudi lymphoma and Friend leukemia cells.

The Friend murine erythroleukemia cell system and the Daudi Burkitt's lymphoma cell system were used to study the effect of growth-inhibitory concentrations of interferon on membrane functions. Experiments with Friend-cell clones sensitive and resistant to interferon indicated that a number of changes in membrane transport occur rapidly after the addition of interferon to sensitive cells. While no change was observed in the activity of the (Na+/K+) ATPase in Friend cells sensitive or resistant to interferon, a piretanide-inhibitable Na+,K+, 2Cl- co-transport system was specifically inhibited after interferon treatment of sensitive cells. In contrast, treatment of Daudi cells with purified molecularly cloned or standard preparations of human leukocyte interferon gave rise to no early changes in the transport of amino acids, 32Pi, sugars, or 86Rb+. The major change observed in Daudi cells was a marked reduction in the uptake and incorporation of thymidine, which begins to decrease after 8-10 h of exposure to interferon.

Animals↗
PubMed1999

Z-350, a new chimera compound possessing alpha1-adrenoceptor antagonistic and steroid 5alpha-reductase inhibitory actions.

The effects of Z-350, (S)-4-[3-(4-{1-(4-methyl-phenyl)-3-[4-(2-methoxyphenyl)piperazine-1-yl]propoxy} benzoyl)indole-1-yl]butyric acid hydrochloride, a newly synthesized compound possessing alpha-adrenoceptor antagonistic and steroid 5alpha-reductase inhibitory actions, were studied in vitro. In functional experiments, Z-350 shifted the concentration/response curve for the phenylephrine-induced contraction of rabbit prostate, urethra and aorta to the right with pA2 values of 8.04, 7.57 and 7.13, respectively. The binding affinity of Z-350 for alpha1-adrenoceptors in rabbit prostate, urethra and aorta were estimated by the displacement of [3H]prazosin. The pKi values for this action of Z-350 were 7.53, 7.95 and 7.62 for the prostate, urethra and aorta, respectively. alpha1-Adrenoceptor subtype selectivities were studied in the submaxillary gland (r(1A) and liver (alpha1B) of rat. Z-350 inhibited the specific binding of [3H]prazosin to alpha1A and (alpha1B-adrenoceptors with pKi values of 7.82 and 7.29, respectively. Z-350 inhibited rat prostatic steroid 5alpha-reductase non-competitively with a pIC50 of 8.42. These results indicate that Z-350 is a alpha1-adrenoceptor antagonist and is a steroid 5alpha-reductase inhibitor. It is expected that Z-350 will be a candidate drug for the treatment of benign prostatic hyperplasia.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed2002

Sensing life: regulation of sensory neuron survival by neurotrophins.

Neurotrophins are a family of structurally and functionally related neurotrophic factors which, in mammals, include: nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3 (NT-3), and NT-4/5. In addition to their canonical role in promoting neuronal survival, these molecules appear to regulate multiple aspects of the development of the nervous system in vertebrates, including neuronal differentiation, axon elongation and target innervation, among others. Actions of neurotrophins and of their receptors in vivo are being analyzed by loss-of-function or gain-of-function experiments in mice. Here, we review the phenotypes of the primary sensory system in these mutant mouse strains and the different strategies specifically involved in the regulation of neuronal survival by neurotrophins in this portion of the nervous system.

Animals↗
PubMed1999

Functional and morphological abnormalities of mitochondria harbouring the tRNA(Leu)(UUR) mutation in mitochondrial DNA derived from patients with maternally inherited diabetes and deafness (MIDD) and progressive kidney disease.

AIMS/HYPOTHESIS: An A to G transition at nucleotide position 3243 in the mitochondrial tRNA Leu(UUR) gene has been identified in patients with maternally inherited diabetes and deafness, as well as in patients with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes, chronic progressive external ophpthalmoplegia, cardiomyopathy and progressive kidney disease. Variations in the mitochondrial DNA haplotype as well as differences in the degree and distribution of heteroplasmy in a certain tissue are factors that may contribute to the variety in phenotypical expression of the 3243 tRNA(Leu)(UUR) mutation. We have done morphological and functional experiments on mitochondria carrying the 3243 mutation derived from patients with either maternally inherited diabetes and deafness or progressive kidney disease to prove the pathogenicity of the 3243 mutation and to examine whether the mtDNA haplotype modulates the pathobiochemistry of this mutation. METHODS: We constructed clonal cell lines that contain predominantly mutated or exclusively wild-type mtDNA with a distinct mtDNA haplotype by the methodology of mitochondria-mediated transformation. Cells lacking mitochondrial DNA (rho(o)) were used as recipients and donor mitochondria were derived from fibroblasts of a patient with either maternally inherited diabetes and deafness or progressive kidney disease. The fibroblasts from these clinically distinct patients carry different mitochondrial DNA haplotypes with the 3243 mutation in heteroplasmic form. RESULTS: Heteroplasmy in the clonal cybrid cells ranged from 0 to 100%, reflecting the heterogeneity of the mitochondrial donor cell. Cybrid cells containing predominantly mutant mitochondrial DNA showed lactic acidosis, poor respiration and marked defects in mitochondrial morphology and respiratory chain complex I and IV activities. No differences were observed in the extent of the mitochondrial dysfunction between the mutant cells derived from the two donors. CONCLUSION/INTERPRETATION: These results provide evidence for a pathogenic effect of the tRNA(Leu)(UUR) mutation in maternally inherited diabetes and deafness and progressive kidney disease, and show no evidence of a contribution of the mitochondrial DNA haplotype as a modulating the biochemical expression of the mutation.

Cell Line↗
PubMed2000

Characterization of opioid receptor types modulating acetylcholine release in septal regions of the rat brain.

Presynaptic opioid receptors of the delta- and mu-types have been shown to inhibit the release of acetylcholine (ACh) in the rat striatum and hippocampus, respectively, but it is unknown whether opioid receptors modulate the release of ACh also in the region of origin of the hippocampal cholinergic innervation, the septum. To answer this question, slices (350 microm) of the medial septal area and of the diagonal band of Broca, as well as (for comparison) of the hippocampus, were prepared from adult male Wistar rats. The slices were incubated with [3H]choline, superfused in the presence of hemicholinium-3 (10 microM) and stimulated twice (S1, S2) by electrical fields (360 pulses, 3 Hz, 2 ms, 60 mA); opioid receptor agonists were present during S2. The preferential mu-agonist [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO) inhibited the evoked ACh release by maximally about 40% in hippocampal slices and acted even more strongly in the medial septal area, or the diagonal band of Broca (about 60% or 75% maximal inhibition, respectively). These effects were reduced or abolished by the preferential mu-antagonist naloxone, which showed no effects when given alone. Using naloxone in the presence of a cocktail of peptidase inhibitors, no evidence for an endogenous tone of opioid peptides was found in the medial septal area, diagonal band of Broca or the hippocampus. Using the preferential delta-agonist [D-Pen2, D-Pen5]enkephalin (DPDPE) and the delta-antagonist naltrindole, a delta-opioid receptor inhibiting evoked ACh release was clearly detectable both in the medial septal area and the diagonal band of Broca, but not in the hippocampus, whereas the preferential kappa-agonist trans-3,4-dichloro-N-methyl-N-[2(1-pyrrolidinyl)cyclo-hexyl] benzeneacetamide (U50,488H) had only weak or no effects. In addition to the functional experiments, double in-situ hybridization studies were performed, in which cells containing mRNA for choline acetyltransferase (ChAT) were labeled by an antibody-linked enzymatic staining procedure, whereas mRNAs for mu- or delta-opioid receptors were detected with radioactive probes. These experiments revealed that in the septal region mainly mu-opioid receptors were expressed by neurons positive for ChAT mRNA, whereas in the rat striatum the expression of delta-opioid receptors prevailed in those neurons. We conclude that in the septal area of the rat brain, in contrast to the rat striatum and hippocampus, both presynaptic mu- and delta-opioid receptors modulate the evoked release of ACh. Whether presynaptic mu- and delta-opioid receptors occur on the same or on different septal cells or axon terminals remains to be clarified.

Acetylcholine↗
PubMed1999

Pharmacological differences and similarities between the native mouse 5-HT3 receptor in N1E-115 cells and a cloned short splice variant of the mouse 5-HT3 receptor expressed in HEK 293 cells.

Human embryonic kidney (HEK) 293 cells were stably transfected with the cDNA encoding the short splice variant of the mouse 5-HT3 receptor (m5-HT3A(b); isolated by RT-PCR from NG108-15 cells) and its pharmacological properties were compared with those of the native 5-HT3 receptor of the mouse neuroblastoma cell line N1E-115. The m5-HT3A(b) receptor of N1E-115 cells differs from that isolated from NG108-15 cells by one amino acid (Val instead of Ile) at position 52 of the amino acid sequence. Both radioligand binding studies with the selective 5-HT3 receptor antagonist [3H]GR65630 (3-(5-methyl-1H-imidazol-4-yl)-1-(1-methyl-1H-indol-3-yl)-1-propanone) and functional experiments by measurement of [14C]guanidinium influx evoked by 5-HT in the absence and presence of 10 microM substance P were carried out. Binding of [3H]GR65630 to the recombinant receptor in HEK 293 cells and the native receptor in N1E-115 cells was specific and of high affinity (Kd 4.4 and 3.0 nM, respectively) and characterized by Bmax values of 875 and 1414 fmol/mg protein, respectively. At 10 nM [3H]GR65630, specific binding was inhibited by the selective 5-HT3 receptor antagonist ondansetron (Ki 11 and 42 nM, respectively) and by 5-HT (Ki 294 and 563 nM, respectively). In the transfected HEK 293 cells, 5-HT induced an influx of [14C]guanidinium both in the absence (pEC50 5.7) and presence of substance P (pEC50 6.6,) which was counteracted by 0.3 microM ondansetron; in the N1E-115 cells, 5-HT also evoked [14C]guanidinium influx in the absence (pEC50 6.0) and presence of substance P (pEC50 6.0). Both in transfected HEK 293 cells and in N1E-115 cells, the 5-HT receptor ligand RS-056812-198 ((R)-N-(quinuclidin-3-yl)-2-(1-methyl-1 H-indol-3-yl)-2-oxo-acetamide; in the presence of substance P) induced an influx of [14C]guanidinium (pEC50 9.8 and 8.7, respectively) with a maximum of about 70 and 30% of the maximum response to 5-HT, respectively. 5-HT (in the presence of substance P)-induced [14C]guanidinium influx was inhibited by the imidazoline BDF 6143 (4-chloro-2(2-imidazolin-2-ylamino)-isoindoline; pIC50 4.9 and 5.3, respectively) and by the sigma-site ligand (+/-)-ifenprodil (pIC50 5.0 and 5.2, respectively). In conclusion, most of the drugs exhibited practically identical properties at both the recombinant m5-HT3A(b) receptor in HEK 293 cells and the native m5-HT3 receptor of N1E-115 cells. However, the recombinant receptor had a higher affinity for ondansetron, and the potency of 5-HT in inducing cation influx through the recombinant, but not through the native receptor, was increased by substance P. RS-056812-198 was a 10-fold more potent partial agonist at the recombinant than at the native receptor. These differences may be due to cell-specific post-translational modifications of the 5-HT3 receptor protein in the two cell lines, to the expression of other subunits in addition to the m5-HT3A(b) receptor in N1E-115 cells and/or to the difference in the amino acid sequence at position 52 of the short splice variants of the m5-HT3 receptors expressed in the two cell lines.

Alternative Splicing↗
PubMed2026

Machine learning identifies ac4C-related prognostic signature and TUBA1C as therapeutic target in COAD.

To explore the role of N4-acetylcytidine (ac4C)-related genes (acRGs) in colon adenocarcinoma (COAD) and identify reliable prognostic biomarkers and potential therapeutic targets. Multi-source transcriptomic datasets (TCGA-COAD, GSE39582, GSE17536) and single-cell RNA-seq data were analyzed. Ten machine learning algorithms were integrated to construct an acRG-based prognostic signature (acRGBS). Immune microenvironment (TME) and genomic profiling were performed, with in vitro functional experiments validating TUBA1C's role. acRGBS, comprising four hub genes (SARAF, CDC42SE2, TSPYL2, TUBA1C), effectively stratified COAD patients into high- and low-risk groups with distinct survival outcomes and was an independent prognostic factor. High-risk patients exhibited increased genomic instability and immunosuppressive TME, while low-risk patients had favorable immunotherapy response. TUBA1C was overexpressed in COAD cells, and its knockdown inhibited proliferation/migration and induced apoptosis. The acRGBS is a robust prognostic tool for COAD, and TUBA1C serves as a candidate therapeutic target, providing new insights for personalized COAD management.

Humans↗
PubMed2000

Sensitivity to chemically diverse phagostimulants in a single gustatory neuron of a polyphagous caterpillar.

Caterpillars of the arctiid moth, Grammia geneura, are polyphagous, but species of Plantago are amongst their preferred food plants. A neuron in the medial styloconic sensillum on the galea has been shown to have a general phagostimulatory function. Experiments with binary mixtures and cross-adaptation have demonstrated that it responds to some sugars, to several amino acids, and also to catalpol. Catalpol is a plant secondary compound in Plantago and a phagostimulant for the caterpillars. The possible significance of combining sensitivity to nutrient compounds with sensitivity to a secondary compound is discussed.

Action Potentials↗
PubMed2001

Identification of a novel low-temperature-response element in the promoter of the barley (Hordeum vulgare L) gene blt101.1.

Two winter barley (Hordeum vulgare L. cv. Igri) genomic clones, lambda gblt101.1 and lambda gblt101.2, encoding the blt101 gene family, were isolated from a genomic library. Deletion analysis of the blt101.1 promoter, using transient beta-glucuronidase (GUS) reporter expression assays, indicated that it contains at least three regulatory regions. A 107-bp region between nucleotides -168 and -275 with respect to the translation initiation codon, confers high-level GUS reporter expression at low temperature and contains a sequence (designated CR1) that is highly conserved in equivalent positions within the promoters of both members of the blt101 gene family. A 10-bp motif contained within CR1 binds proteins present in nuclear extracts from both control and low-temperature-treated barley tissue. Loss-of-function experiments, using transient-expression analysis, confirmed that this motif acts as a previously unreported low-temperature-responsive element. Nuclease sensitivity analysis of intact chromatin indicated that the blt101.1 promoter becomes more susceptible to DNase and micrococcal nuclease at low temperature, consistent with chromatin reorganisation upon transcriptional induction. It is proposed that both the 10-bp motif and chromatin reorganisation are involved in the regulation of blt101.1 at low temperature. This is the first detailed analysis of a low-temperature-specific plant promoter and identifies a novel low-temperature-response element.

Adaptation, Physiological↗
PubMed2004

Hypoblast controls mesoderm generation and axial patterning in the gastrulating rabbit embryo.

Gastrulation in higher vertebrate species classically commences with the generation of mesoderm cells in the primitive streak by epithelio-mesenchymal transformation of epiblast cells. However, the primitive streak also marks, with its longitudinal orientation in the posterior part of the conceptus, the anterior-posterior (or head-tail) axis of the embryo. Results obtained in chick and mouse suggest that signals secreted by the hypoblast (or visceral endoderm), the extraembryonic tissue covering the epiblast ventrally, antagonise the mesoderm induction cascade in the anterior part of the epiblast and thereby restrict streak development to the posterior pole (and possibly initiate head development anteriorly). In this paper we took advantage of the disc-shape morphology of the rabbit gastrula for defining the expression compartments of the signalling molecules Cerberus and Dickkopf at pre-gastrulation and early gastrulation stages in a mammal other than the mouse. The two molecules are expressed in novel expression compartments in a complementary fashion both in the hypoblast and in the emerging primitive streak. In loss-of-function experiments, carried out in a New-type culturing system, hypoblast was removed prior to culture at defined stages before and at the beginning of gastrulation. The epiblast shows a stage-dependent and topographically restricted susceptibility to express Brachyury, a T-box gene pivotal for mesoderm formation, and to transform into (histologically proven) mesoderm. These results confirm for the mammalian embryo that the anterior-posterior axis of the conceptus is formed first as a molecular prepattern in the hypoblast and then irrevocably fixed, under the control of signals secreted from the hypoblast, by epithelio-mesenchymal transformation (primitive streak formation) in the epiblast.

Amino Acid Sequence↗
PubMed2006

Xenopus Xotx2 and Drosophila otd share similar activities in anterior patterning of the frog embryo.

Despite the obvious anatomical differences between the fly and the vertebrate body plans, several genes involved in their development are largely conserved. In this work we provide evidence that overexpression of the Drosophila orthodenticle (otd) gene in Xenopus laevis has a similar effect to that of its homolog Xotx2. Injections of otd mRNA in whole embryos lead to posterior truncations and to induction of ectopic cement glands, similar to Xotx2 injections. In animal cap assays, otd, like Xotx2, is able to activate the cement gland marker XAG and to suppress the expression of the epidermal marker XK81. Finally, as assayed by Einsteck transplantation assays, otd, like Xotx2, is able to respecify a tail/trunk organizer to a head organizer. In this work we also show that Xotx2 and otd share molecular functions that regulate early regional specification of the Xenopus anterior neural plate. Gain-of-function experiment targeting low doses of either otd or Xotx2 mRNAs in the neural plate promote reduction of Xrx1 and Xbf1 expression domain; no changes are observed for the anterior mesodermal marker Xgsc, the dorsal diencephalic marker Xbh1, and the midbrain/hindbrain marker Xen2. otd/Xotx2 inhibition activity of Xrx1 and Xbf1 expression is consistent with the strong inhibition of Xfgf8 expression in the anterior neural ridge observed upon otd/Xotx2 mRNA injection.

Animals↗
PubMed2003

Intraganglionic laminar endings in the rat esophagus contain purinergic P2X2 and P2X3 receptor immunoreactivity.

Intraganglionic laminar endings (IGLEs) represent the most prominent vagal afferent terminal structures throughout the gastrointestinal tract. They are most prominent in the esophagus and stomach, but can be found down to the distal colon. Their role as mechanosensors as proposed on anatomical grounds was recently substantiated in elegant functional experiments. There is evidence that vagal mechanosensors in the esophagus and stomach respond to ATP. Thus, the present study aimed at detecting purinergic receptors on IGLEs. IGLEs in the rat esophagus were identified by immunohistochemistry for calretinin and sections were co-incubated with antibodies directed against P2X2 or P2X3 receptors. Also, double label immunocytochemistry for purinergic receptors and calcitonin gene-related peptide as a marker for spinal afferents was performed. Terminal nerve fibers immunoreactive for P2X2 and P2X3, respectively, were observed between outer and inner layers of the tunica muscularis, covering myenteric ganglia totally or partly. Both P2X2 and P2X3 receptor immunoreactivities were highly co-localized with calretinin positive IGLEs as shown by confocal laser scanning microscopy. Numerous calcitonin gene-related peptide immunostained fibers were found to closely approach and intermingle with P2X immunopositive IGLEs. However, there was never co-staining for either of the purinergic receptors and calcitonin gene-related peptide within the same fibers. P2X3 but not P2X2 immunoreactivity was also observed within nerve fiber arborizations in the mucosa of the pharynx. In the nodose ganglion, 8.9 +/- 1.1% of P2X2 and 7.2 +/- 1.3% of P2X3 immunopositive neurons, respectively, co-stained for calretinin. On the other hand, 63.4 +/- 4.6% and 60.1 +/- 5.3% of calretinin positive cell bodies contained P2X2 and P2X3 receptor immunoreactivity, respectively. These results indicate that IGLEs are equipped with both P2X2 and P2X3 receptors. Thus, they may act as chemosensors or their mechanosensory properties may be modulated by ATP. It is also suggested that spinal afferents innervating the esophagus are equipped with neither P2X2 nor P2X3 purinergic receptors.

Animals↗
PubMed2003

Regulatory regions and nuclear factors involved in nodule-enhanced expression of a soybean phosphoenolpyruvate carboxylase gene: implications for molecular evolution.

We have determined the genomic organization of two closely related phosphoenolpyruvate carboxylase genes in soybean, GmPEPC7, which is expressed at high levels in root nodules, and the housekeeping gene GmPEPC15. Their nucleotide sequences, including most introns and 5;-flanking regions within 600 bp upstream from the transcription start sites, are well conserved, suggesting that they were duplicated quite recently. To gain insights into the process of evolution of the tissue-specifically expressed GmPEPC7gene, we produced chimeric constructs carrying either the GmPEPC7or GmPEPC15promoter fused to the beta-glucuronidase gene. The expression patterns of the reporter observed in nodules that developed on transgenic hairy roots reflected the levels of mRNA levels produced by the genes in wild-type soybean plants, indicating that the GmPEPC7promoter directs nodule-specific expression. Loss-of-function experiments showed that the segment of GmPEPC7between -466 and -400, designated as the "switch region" (SR), was necessary for expression in nodules, although proteins that bind to SR were not detectable in a gel-retardation assay. Another gel-retardation assay indicated that putative nodule nuclear proteins bind specifically to the region of GmPEPC7between -400 and -318, designated as the "amplifier region" (AR). Both SR and AR have characteristic sequences that are not found in the GmPEPC15promoter. Furthermore, experiments using hybrid promoters derived from GmPEPC15demonstrated that AR confers high-level expression in nodules only in combination with SR. When wild-type soybean plants were subjected to prolonged darkness and subsequently illuminated, the level of GmPEPC7mRNA in nodules decreased and then recovered. This study suggests that the acquisition of two interdependent cis-acting elements resulted in molecular evolution of the nodule-enhanced GmPEPC7gene.

Base Sequence↗
PubMed2004

Bulgy tadpoles: inducible defense morph.

Predator induced morphological defenses are marked morphological shifts induced directly by cues associated with a predator. Generally, remote cues, i.e., chemical substances emitted from predators or injured conspecifics, are considered to be ideal signals to induce morphological change in aquatic environments rather than close cues, i.e., close chemical or tactile cues, since chemical substances that can propagate over relatively long distances and persist for a long period may allow organisms to keep safe and to deliberately change their morph. In fact, most organisms adopting an inducible morphological defense utilize remote chemical cues to detect predation risk and to produce morphological defenses. In this paper, we report a unique and functionally well designed inducible morphological defense strategy where the induction process requires close cues from a predator. The tadpoles of Rana pirica exhibited a bulgy bodied morphology when threatened with predation by larval salamanders, Hynobius retardatus, in close proximity. Predation trials and a function experiment showed that the induced bulgy morph is an adaptive defense phenotype against the gape-limited predator larval H. retardatus. Furthermore, R. pirica tadpoles use two adaptive strategies in terms of cost saving, i.e., adjustment of the extent of bulginess according to predation risk and reversibility by actual shrink of bulgy body after removing the predation threat. In general, R. pirica hatch earlier than H. retardatus. In natural ponds, during the early developmental stage R. pirica tadpoles live in close proximity to young H. retardatus larvae. As they grow, the salamanders gradually become serious predators and the predator-prey interaction becomes intimate. After a while, predation, cannibalism and metamorphosis decrease the number of salamanders in the ponds, and the predator-prey interaction weakens. Such a phenology in the predator-prey interaction allows the evolution of a close-cue detection system and adaptive cost-saving strategies. Our results highlight that the characteristics of the inducible defense depend on the intensity and specificity of the predator-prey system.

Adaptation, Physiological↗
PubMed2026

Pan-cancer analysis identifies KANSL2 as a cell-cycle-associated regulator of tumor progression and immunity in liver hepatocellular carcinoma.

KANSL2, a core component of the NSL histone acetyltransferase complex, has been implicated in tumorigenesis. However, its pan-cancer relevance and functional role in liver hepatocellular carcinoma (LIHC) remain unclear. Multi-omics data from TCGA, GEO, and HPA were integrated to systematically evaluate KANSL2 expression, clinical significance, genomic alterations, and immune associations across cancers. Functional enrichment, immune infiltration analyses, and single-cell transcriptomics were performed. In vitro assays were conducted to validate the biological effects of KANSL2 in LIHC cells. KANSL2 is broadly upregulated across cancers and exhibits strong diagnostic performance. Elevated KANSL2 expression correlates with unfavorable prognosis, particularly in LIHC. Mechanistically, KANSL2 and its co-expressed genes are enriched in cell-cycle progression. KANSL2 expression is also closely associated with immune infiltration and immunoregulatory signaling within the tumor microenvironment, with single-cell data indicating preferential expression in proliferative T-cell subsets. Functional experiments demonstrate that KANSL2 silencing suppresses proliferation, migration, and invasion, and induces G2/M phase arrest in LIHC cells. Notably, its effects on apoptosis are limited, suggesting that KANSL2 primarily drives tumor progression through cell-cycle-dependent mechanisms. This study identifies KANSL2 as a key regulator of tumor progression and immune remodeling in LIHC. By promoting malignancy predominantly via cell-cycle control, KANSL2 represents a promising biomarker for diagnosis and prognosis, and a potential therapeutic target.

Carcinoma, Hepatocellular↗
PubMed2005

The brain slice chamber, a novel variation of the Boyden Chamber Assay, allows time-dependent quantification of glioma invasion into mammalian brain in vitro.

Glioma cell invasion occurs in a complex micromilieu consisting of neural and glial cells, myelinated fiber tracts, blood vessels and extracellular matrix proteins. The present work describes the brain slice chamber (BSC) as a novel experimental model for assessing invasion of glioma cells into adult mammalian white and gray matter on the basis of the well known Boyden chamber system. As a matrix for invasive tumor cells we used freshly prepared brain tissue from adult pigs. The tissue was sectioned into 40 mum slices that were mechanically fixed to a millipore filter. The neural structures and the three-dimensional architecture of the slice was preserved as verified by immunohistochemistry, light- and electron microscopy. Human U-373 and U87 astrocytoma cells stably transfected with green fluorescent protein (GFP) were assessed for their invasiveness into the brain-slices during a 24 h period. Invasion of U-87 GFP cells was quantified at different time intervals by confocal laser scanning microscopy showing more intense invasion into white compared to gray matter. Two cytostatics (vincristin and paclitaxel) which both are known to affect the cytoskeleton, inhibited glioma cell invasion in a dose dependent manner, which makes the presented model system suitable for functional experiments. In conclusion, the BSC represents a valid and rapid experimental model that may be used to describe the invasive behavior of glioma cells within the preserved three-dimensional structure of mammalian brain tissue in vitro.

Animals↗
PubMed2006

P2Y receptor mediated modulation of insulin release by a novel generation of 2-substituted-5'-O-(1-boranotriphosphate)-adenosine analogues.

PURPOSE: A series of C2-substituted ATP analogues was previously shown to have potent insulin-secreting properties, yet with poor tissue-selectivity for the pancreatic beta-cell. The present study was designed to evaluate the binding profile on beta-cell membranes and the effects on insulin release and pancreatic vascular resistance of a second generation of P2Y(1) receptor agonists, based on C2-substitution of the adenosine 5'-O-(1-boranotriphosphate) scaffold. MATERIALS AND METHODS: Functional experiments were performed in the rat isolated pancreas model; binding studies with ATP-alpha-[(35)S] were performed in membrane homogenates from the rat insulinoma INS-1 cell line. The diastereoisomers of the compounds are designated by A and B. RESULTS: Under 8.3 mmol l(-1) glucose, 2-methylthio-ATP-alpha-B, A isomer, induced a biphasic and concentration dependent insulin response; its maximal efficacy reaches ninefold the baseline secretion and its EC(50) is 28.1 nmol l(-1). No significant effect of this isomer was observed on vascular resistance, whereas the B isomer, which was a less potent insulin secretagogue, consistently induced a transient vasoconstriction. Interestingly, the insulin response induced by 2-methylthio-ATP-alpha-B, A isomer, was clearly glucose-dependent. This drug competes with ATP-alpha-[(35)S] binding in a complex two sites interaction model, with a K(0.5) value of 17.7 nmol l(-1). 2-Chloro-ATP-alpha-B had a similar insulin-secreting profile as 2-methylthio-ATP-alpha-B, with a lower tissue-selectivity. The non-substituted ATP-alpha-B analog, A isomer, was less potent than the C2-substituted derivatives (A isomers) and had a vasorelaxant effect. CONCLUSIONS: We conclude that 2-methylthio-ATP-alpha-B, A isomer, is a potent and tissue-selective P2Y receptor agonist with high efficacy. Its insulin-releasing action is glucose-dependent, which gives interest to this compound as a drug candidate for treating type 2 diabetes.

Adenosine Triphosphate↗
PubMed1993

Binding of the radioligand [35S]adenosine 5'-O-(2-thiodiphosphate) and intracellular calcium response in rat liver parenchymal cells.

The use of the radioligand [35S]adenosine 5'-O-(2-thiodiphosphate) (ADP beta 35S) for the determination of P2y-purinoceptors on turkey erythrocyte membranes has recently been described. In the present study, we were able to demonstrate specific binding of this radioligand in intact rat liver parenchymal cells. Within 10 min a thermodynamic equilibrium was obtained which lasted for 25 min with a subsequent decline. Displacement studies with several nucleotides were performed yielding Ki values of 1.5 +/- 0.47 microM for UTP, 1.8 +/- 0.35 microM for adenosine 5'-O-(2-thiodiphosphate) (ADP beta S), 31 +/- 6.2 microM for ATP and 35 +/- 6.1 microM for GTP. In addition, we showed that ADP beta 35S is highly resistant to degradation by ecto-nucleotidases, with only 14.5 +/- 1.4% of total ADP beta 35S present being degraded after 1 hr, and that the binding of ADP beta 35S to its binding sites was modulated by EDTA. The Ki value of ATP shifted to 8.1 +/- 1.2 microM upon the addition of 1 mM EDTA to the incubation medium. In these rat liver parenchymal cells all nucleotides promoted calcium entry in a dose-dependent manner with EC50 values of 3.5 +/- 0.22 microM for UTP, 20.7 +/- 3.1 microM for ATP, 38.3 +/- 6.4 microM for ADP beta S and 73.6 +/- 13.7 microM for GTP, with GTP being a partial agonist. Based on the data derived from the present study we discuss the possible correlation between binding and functional experiments and conclude that the described receptor resembles most closely the P2u-purinoceptor and/or "nucleotide receptor", in that UTP is at least as active as ATP.

Adenosine Diphosphate↗