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At least 631 records · Page 35Linked to original sources
PubMed2007

Is autophagy the key mechanism by which the sphingolipid rheostat controls the cell fate decision?

Sphingolipids are major constituents of biological membrane and some of them behave as second messengers involved in the cell fate decision. Ceramide and sphingosine 1-phosphate (S1P) constitute a rheostat system in which ceramide promotes cell death and S1P increases cell survival. We have shown that both sphingolipids are able to trigger autophagy with opposing outcomes on cell survival. Here we discuss and speculate on the diverging functions of the autophagic pathways induced by ceramide and S1P, respectively.

Autophagy↗
PubMed2005

Triple layer control: phosphorylation, acetylation and ubiquitination of FOXO proteins.

FOXO proteins are transcriptional regulators that control cell cycle progression, DNA repair, defense against oxidative damage and apoptosis. These divergent functions of FOXO proteins are regulated by signal-induced, post-translational modifications. Phosphorylation of cytoplasmic FOXO at specific sites by JNK initiates translocation into the nucleus. Acetylation and deacetylation of nuclear FOXO affects the selection of transcriptional programs that are controlled by FOXO proteins. Activation of Akt by growth factors results in phosphorylation of nuclear FOXO at specific sites followed by additional phosphorylations mediated by other kinases. Akt-dependent phosphorylation reduces the DNA-binding activity of FOXO, interferes with binding to the co-activators p300/CBP, and inactivates the FOXO nuclear translocation signal. The Akt-phosphorylated FOXO is exported from the nucleus in a CRM1- and 14-3-3-dependent process. Cytoplasmic, Akt-phosphorylated FOXO interacts with the ubiquitin ligase Skp2 and is targeted for proteasomal degradation. The nuclear-cytoplasmic "FOXO shuttle" is driven by stress signals that result in nuclear import and FOXO transcriptional activity and growth signals that initiate nuclear export and proteasomal degradation of FOXO.

Acetylation↗
PubMed2003

Liver chip and gene shaving.

A comprehensive profile of genes expressed at the mRNA level in various human tissues is considered to be important for understanding the molecular mechanisms of the tissue-specific function and the pathogenesis of related diseases. Here, the gene expression profiling in three human digestive tissues, liver, stomach, and pancreas, was catalogued by generating a large number of expressed sequence tags, and clarified how quantitatively the gene expressions are different. After assembling the redundant clones among three tissues, the results showed that only 1.7% among the assembled genes was expressed commonly in the investigated tissues. These results suggest that the significant functional divergences in different tissues must be related to the divergence of the gene expression profiles. Recently, microarray technologies are widely used. Considering the results that different genes express in different tissues, however, it is important to spot the cDNAs derived from the same tissues or cells examined to acquire information efficiently. For the study of digestive diseases, we constructed an in-house microarray by using the cDNA sets derived from the digestive tissues (liver and gastric chip). In addition, because the amount of information acquired by the microarray analyses is huge, the power of bioinformatics for unifying the obtained data is indispensable. Some examples of the strategies for handling the microarray data obtained by our in-house microarrays are shown in this article.

Gene Expression Profiling↗
PubMed1992

Immunogold quantification of glutamate in two types of excitatory synapse with different firing patterns.

A quantitative immunocytochemical method was used to study the regional levels of glutamate in two types of lamprey (Ichtyomyzon unicuspis) axon, which both activate excitatory amino acid receptors, but which when active exhibit different firing patterns. Giant reticulospinal axons fire in brief bursts, while dorsal column axons, mainly belonging to cutaneous afferents, show a sustained firing at high frequency. In both types of axon, clusters of synaptic vesicles showed a strong accumulation of glutamate immunogold labeling, and the density of gold particles correlated strictly with the packing density of synaptic vesicles. The most densely packed vesicle areas had a particle density corresponding to a concentration of fixed glutamate of about 30 mM in coprocessed glutamate conjugates, suggesting an intravesicular glutamate concentration of at least 60 mM. The level of labeling in axoplasmic matrix was considerably lower, but differed significantly between the two types of axon. Dorsal column axons showed a particle density in axoplasmic matrix that was approximately four times higher than that in giant reticulospinal axons. The mitochondrial glutamate labeling was also significantly stronger in the dorsal column axons. In addition, the number of mitochondrial profiles surrounding vesicle clusters was about four times higher in dorsal column synapses than in reticulospinal synapses. Antisera to aspartate, GABA, glutamine, and homocysteate failed to produce a specific labeling of synaptic vesicle clusters in reticulospinal or dorsal column axons. In conjunction with previous demonstrations of a stimulus-induced vesicle depletion in giant reticulospinal synapses (Wickelgren et al., 1985), these results imply that glutamate is released from synaptic vesicles. The different extravesicular glutamate levels in reticulospinal axons and dorsal column axons may relate to different requirements for the refilling of synaptic vesicles in these functionally divergent neurons.

Animals↗
PubMed2004

Characterization of human TMEM16G gene in silico.

TMEM16A, TMEM16B, TMEM16C, TMEM16D, TMEM16E, TMEM16F and TP53I5 are TMEM16 family eight-transmembrane proteins with N- and C-terminal tails facing the cytoplasm. TMEM16A gene at human chromosome 11q13.3 is amplified in head and neck tumors, and TMEM16E gene at human chromosome 11p14.3 is mutated in gnathodiaphyseal dysplasia (GDD). Ngep cDNA (NM_207031.1) is derived from mouse Tmem16g gene. Here, we characterized human TMEM16G gene by using bioinformatics. TMEM16G gene, consisting of 25 exons, was located at human chromosome 2q37.3. Intra-species comparative genomics revealed that the PASK-PPP1R7-TMEM16G-HDLBP-NEDD5 locus was the unique region without paralogous region. TMEM16G mRNA was preferentially expressed in normal prostate and prostate cancer. Complete coding sequence of TMEM16G cDNA was determined by assembling 25 exons of TMEM16G gene. Human TMEM16G gene was found to encode 932-amino-acid TMEM16G protein with TM16H1, TM16H2 and TM16H3 domains. Comparative proteomics revealed that T844N amino-acid substitution occurred in human TMEM16G during evolution. TMHMM2 program predicted that mouse Tmem16g and artificial human TMEM16G (844T) were eight-transmembrane proteins, but that wild-type human TMEM16G (844N) was a seven-transmembrane protein. These facts indicate that amino-acid substitution at codon 844 of human TMEM16G resulted in the mis-folding of the eighth transmembrane helix. Human TMEM16G with altered membrane topology might show functional divergence compared with other members of the TMEM16 family.

Amino Acid Sequence↗
PubMed2005

In vivo analyses of UV-irradiation-induced p53 promoter binding using a novel quantitative real-time PCR assay.

The p53 tumor suppressor protein mediates cell cycle arrest and apoptosis through transactivation of downstream target genes. While many target genes have been identified to date, the mechanisms and time course of their induction are still unclear. We investigated the kinetics of p53 binding to the p21CIP1, MDM2, BAX and PIG3 promoters in vivo using a novel quantitative real-time chromatin immunoprecipitation-PCR assay. Our results demonstrate distinct kinetics of p53 promoter binding dependent on the target gene promoters. The timed induction of target genes due to genotoxic stress is likely to play a pivotal role for the divergent functions of p53.

Blotting, Southern↗
PubMed2005

[Implication of alternative splice transcripts of caspase-3 and survivin in chemoresistance].

Recent studies have shown that resistance to apoptosis may contribute to chemoresistance. Alteration of caspases, such as caspase-3, results on decreased apoptosis. Genes of IAP (inhibitor of apoptosis proteins) family, such as survivin, were also implicated in tumor development where they are mutated or have deregulated expression. Initial studies revealed strong survivin expression in several fetal tissues and some proliferating adult tissues, whereas no survivin expression was detected in a range of adult tissues. Although the factors at the origins on survivin re-expression in tumors are still unknown, the anti-apoptotic function of survivin is mediated in part by inhibiting caspase-3 activity. Recently, functionally divergent splice variants resulting from alternative splicing, with apoptotic (for caspase-3) or anti-apoptotic (for survivin) opposite activities have been described. The alternative splice variant, caspase-3s results from exon 6 deletion and shows antagonist of apoptotic property of caspase-3. Three alternative splice variants of survivin (survivin-DeltaEx3, survivin-2B and survivin-3B) differing in their anti-apoptotic properties were reported. While the anti-apoptotic effect of survivin-DeltaEx3 is preserved, survivin-2B has lost its anti-apoptotic potential and may act as a naturally occurring antagonist of survivin and survivin-DeltaEx3. At present, little is known about properties of survivin-3B. Several evidences indicate that in several cancers, the ratio of splice variants is significantly altered, and modifications of splicing pathways have been developed for cancer treatment. Recent investigations have shown that expression of alternative splice variants of caspase-3 and of survivin were also altered in many human cancers, and that variations in their expression were associated with tumor progression and chemoresistance. In this article, we describe recent data concerning alternative splice variants of these two proteins.

Alternative Splicing↗
PubMed1991

Pre-ligation of CR1 enhances IgG-dependent phagocytosis by cultured human monocytes.

Opsonization of the C3b receptor (CR1) on phagocytic cells with C3b enhances both attachment of targets to the cells and subsequent IgG-dependent ingestion of these targets. To explore mechanisms involved in this increased phagocytosis, we adhered cultured human monocytes to surfaces pre-coated with CR1 ligand or control proteins and quantitated ingestion of sheep E opsonized with IgG alone. Three ligands for CR1 resulted in markedly enhanced phagocytosis of targets when compared individually to a panel of non-ligands, as determined by both the proportion of monocytes ingesting targets (percent phagocytosis) and by the number of targets ingested per 100 monocytes (phagocytic index). The ligands included purified C3b, iC3, and Fab fragments of 1B4, a monoclonal anti-CR1, which resulted in a percent phagocytosis of 56.3 (p less than 0.01), 59.0 (p less than 0.01), and 54.4 (p less than 0.02) and a phagocytic index of 281.2 (p less than 0.01), 281.1 (p less than 0.01), and 247.1 (p less than 0.02), respectively. Control proteins including human serum albumin, hemoglobin, Fab fragments of anti-fibronectin, anti-beta 2 microglobulin, and MOPC 21, and Fc fragments of 1B4 and MOPC 21 produced no significant stimulation of phagocytosis, nor did F(ab')2 fragments of monoclonal anti-CR3, M1/70. CR1-specific augmentation of target ingestion was apparent with monocytes cultured in serum-free medium for 1 to 7 days, but was not seen with freshly elutriated cells. Phagocytosis of unopsonized or IgM-coated targets was minimal. These results suggest that the adherent monocytes are primed by CR1 cross-linking for enhanced FcR-mediated phagocytosis even when the CR1 ligand is not present on the targets. This contrasts with the behavior of CR3, and demonstrated functional divergence between these C3 fragment receptors in the phagocytic process.

Antibodies, Monoclonal↗
PubMed1991

Primary structure and cellular distribution of two fatty acid-binding proteins in adult rat kidneys.

Fatty acid-binding proteins (FABPs) were purified from the kidneys of female and male rats and characterized by primary structure and histological distribution in the kidney. Two FABPs (14 and 15.5 kDa) were found in male rat kidney cytosol whereas only 14-kDa FABP could be recognized in female rat kidneys throughout the purification steps. The amino acid sequence of the 14-kDa FABP was identical to that of rat heart FABP deduced from the cDNA sequence (Heuckeroth, R. O., Birkenmeier, E. H., Levin, M. S., and Gordon, J. I. (1987) J. Biol. Chem. 262, 9709-9717). Structural analysis of the male-specific 15.5-kDa FABP identified this second FABP as a proteolytically modified form of alpha 2u-globulin, an 18.7-kDa major urinary protein of adult male rats (Unterman, R. D., Lynch, K. R., Nakhasi, H. L., dolan, K. P., Hamilton, J. W., Cohn, D. V., and Feigelson, P. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 3478-3482) which shares a common ancestry with a number of hydrophobic ligand-binding proteins such as serum retinol-binding proteins. Immunohistochemical investigation disclosed that heart-type FABP (14-kDa FABP) is localized in the cytoplasm of the epithelia of the distal tubules in both male and female rat kidneys whereas 15.5-kDa FABP immunostaining was observed predominantly in the endosomes or lysosomes of proximal tubules in male rat kidneys. These results suggest strongly the functional divergence of two FABPs in the rat kidney.

Amino Acid Sequence↗
PubMed1988

A mammalian viral enhancer confers transcriptional regulation in yeast.

Transcriptional enhancers are DNA sequences that regulate RNA transcription from linked promoters by binding to cellular proteins (trans-activators). In the mammalian virus SV40, initiation of transcription is controlled, in part, by a strong 72-base pair enhancer. We show that yeast cells contain a factor that binds specifically to a key DNA motif in the SV40 enhancer, the P element that is essential for viral transcription in mammalian cells. The P element shows sequence similarity to a yeast DNA transcriptional regulatory element, GCN4, that controls transcription of genes that code for amino acid biosynthetic enzymes. Insertion of the SV40 enhancer or single or multiple copies of the P element itself upstream from the cytochrome Cyc-1 promoter places the hybrid viral-yeast transcription unit under metabolic control in yeast cells. These studies suggest that the SV40 P element and its complementary trans-activator represent a conserved transcriptional control mechanism that operates on widely divergent functions in evolution.

Base Sequence↗
PubMed1979

Evolutionary relationships of the gut hormones.

Peptides identical or related to mammalian gut hormones occur widely, not just in gut endocrine cells but also in central or peripheral nerves, amphibian skin glands, and a variety of invertebrate tissues. The dual distribution in brain and gut was probably already established early in the vertebrate line; representatives of the oldest vertebrate group, the cyclostomes, have cholecystokinin-like factors in gut endocrine cells and in brain. The related sequences of certain gut peptides, notably gastrin and cholecystokinin (CCK), and secretin, glucagon, vasoactive intestinal polypeptide (VIP), and gastric inhibitory peptide (GIP), indicate evolution from common ancestral molecules by gene duplication and divergence. Functionally important residues are conserved. Thus the COOH-terminal pentapeptide common to gastrin and CCK also contains their minimal active fragment. There are also evolutionary changes at the level of the target organ receptor mechanisms: these are also evolutionary changes at the level of the target organ receptor mechanisms; these are illustrated by evidence suggesting that secretin regulates the flow of pancreatic juice in mammals whereas the structurally related peptide VIP has a similar role in birds.

Animals↗
PubMed1984

Urological complications in gynecological surgery and radiotherapy.

Urological complications in connection with gynaecologically or obstetrically indicated operations are rare and usually only of clinical importance when they are not recognized and treated during the operation or in the immediate postoperative period. In such cases, for example, a ureteral lesion can result in loss of the function of the ipsilateral kidney, which is particularly serious when the primary operation was carried out for a benign primary disease. The aim of the present study was therefore to demonstrate to the gynaecologist above all the preventive and diagnostic measures serving to prevent lesions to the efferent urinary pathways or to aid their immediate diagnosis. Amongst the preventive measures is included first a discussion of the pelvic anatomy which, in consideration of the special problems involved in the preparation of the caudal ureter in extended abdominal hysterectomy, is supplemented by experimental and clinical studies on vascularization of the ureters. This section also includes a detailed description of the topographic and sometimes functional divergences of the urinary tract from the norm under physiological and pathological conditions, such as are typically encountered in gynaecologico-obstetric patients, since these deviations predispose patients towards urological complications during surgical interventions. The diagnostic measures include above all the dye tests which can be performed without special technical equipment, and radiological examinations. The radiological tests are illustrated by examples selected from some 300 urograms taken over the past 20 years. The data reported in the literature on the lesions to the efferent urinary pathways are supplemented by data from about 4,000 case histories of patients treated at the Zürich University Gynaecological Clinic by surgery or radiotherapy between 1960 and 1980. The possibilities of a synoptic evaluation of the complication rates of such cases reported in the literature, or a comparison with personal experience, are very limited, since not only is the patient material under consideration very heterogeneous, but in many cases there are sometimes very considerable differences in the preventive measures implemented, in the surgical techniques, in the surgical radicality, and--in the case of combined surgery and radiotherapy--in the treatment plans and types of irradiation. The guidelines relating to the treatment of lesions to the efferent urinary pathways caused by surgery or radiotherapy are presented in detail insofar as they are of importance for the gynaecologist as immediate measures.(ABSTRACT TRUNCATED AT 400 WORDS)

Female↗
PubMed1993

Multiple structural elements determine ligand binding of fibroblast growth factor receptors. Evidence that both Ig domain 2 and 3 define receptor specificity.

The murine fibroblast growth factor receptor 2 (FGFR2) and keratinocyte growth factor receptor (KGFR) are two products of the same gene which display distinct binding specificities. We and others have shown that a major structural element underlying this functional divergence is a variable 50 amino acids long region constituting the C-terminal half of the third immunoglobulin (Ig)-like domain of the receptor. This region of the two receptors is encoded by two distinct exons which are alternatively used in cells of different tissues and origin. To further investigate the role of this confined variable region in determining ligand binding specificity we have generated a chimeric molecule between FGFR1 and KGFR where the variable segment of KGFR replaces the homologous region in FGFR1. Binding studies as well as chemical crosslinking of radiolabeled ligands revealed that the recombinant FGFR1/KGFR chimera has retained the binding affinity to acidic FGF and FGF4 (hst/kfgf) but lost the capacity to bind basic FGF (bFGF). This chimeric receptor bound keratinocyte growth factor (KGF), however, with significantly lower affinity as compared with KGFR. High affinity binding of KGF was acquired only when also domain 2 in this chimera was replaced by its homologous domain from FGFR2. These results demonstrate that ligand binding and specificity involves multiple receptor elements which are located at both Ig-like domain 2 and 3 of FGF receptors.

Animals↗
PubMed1994

Amyloid precursor-like protein 2 (APLP2) is modified by the addition of chondroitin sulfate glycosaminoglycan at a single site.

beta-Amyloid, the principal component of senile plaques in individuals with Alzheimer's disease, is derived from larger integral membrane glycoproteins, termed amyloid precursor proteins (APP). APP is a member of a family of proteins that includes the amyloid precursor-like proteins APLP1 and APLP2. The present study examines the metabolism of mouse APLP2 in cultured mammalian cells. We report that in stably transfected Chinese hamster ovary and transiently transfected African green monkey kidney (COS-1) cells, APLP2 is modified by glycosaminoglycan (GAG) addition. The sensitivity of GAG-modified APLP2 to digestion with chondroitinase AC indicates that chondroitin sulfate (CS) chains are the preponderant GAG on APLP2. CS GAG modification of APLP2 occurs in a region with little homology to APP. Contained within this heterologous region is a predicted CS modification site, ENEGSGMAEQ (APLP2 residues 610-619); APLP2 polypeptides harboring a serine-to-alanine substitution at position 614 fail to undergo CS GAG modification. Our observation that APLP2 is modified by a pathway distinct from APP suggests that the two molecules may be functionally divergent.

Alzheimer Disease↗
PubMed1993

Effects of the residue adjacent to the reactive serine on the substrate interactions of Drosophila esterase 6.

Esterase 6 from Drosophila melanogaster is a carboxylesterase that belongs to the serine esterase multigene family. It has a basic histidine (His) at residue 187, adjacent to the reactive serine (Ser) at residue 188, whereas most other characterized members of the family have an acidic glutamate (Glu) in the equivalent position. We have used site-directed in vitro mutagenesis to replace the His codon of the esterase 6 gene with either Gln or Glu codons. The enzymes encoded by these active-site mutants and a wild-type control have been expressed, purified, and characterized. Substitution of Gln for His at position 187 has little effect on the biochemical properties of esterase 6, but the presence of Glu at this position is associated with three major differences. First, the pH optimum is increased from 7 to 9. Second, the mutant enzyme shows decreased activity for beta-naphthyl esters and p-nitrophenyl acetate but has gained the ability to hydrolyze acetylthiocholine. Finally, the Gibb's free energy of activation for the enzyme is increased. These results suggest that residue 187 interacts directly with the substrate alkyl group and that this interaction is fully realized in the transition state. We further propose that the presence of His rather than Glu at position 187 in esterase 6 contributes significantly to its functional divergence from the cholinesterases and that this divergence is due to different interactions between residue 187 and the substrate alkyl group.

Acetylthiocholine↗
PubMed2006

Evolutionary divergence of LFY function in the mustards Arabidopsis thaliana and Leavenworthia crassa.

LEAFY (LFY), a transcription factor involved in the regulation of flower development in Arabidopsis thaliana, has been identified as a candidate gene in the diversification of plant architecture in Brassicaceae. Previous research with Leavenworthia crassa, which produces solitary flowers in the axils of rosette leaves, has shown that the L. crassa LFY ortholog, LcrLFY, rescues most aspects of flower development in A. thaliana but showed two novel traits: flowers produced additional petals and inflorescences produced terminal flowers. In this paper, we explore the molecular mechanisms responsible for these novel phenotypes. We used microarray hybridizations to identify 32 genes differentially expressed between a transgenic LcrLFY line and a control transgenic LFY line. Of particular interest, TERMINAL FLOWER 1 (TFL1) transcripts were found at elevated levels in LcrLFY lines. To distinguish regulatory versus functional changes within the LcrLFY locus, reciprocal chimeric transgenes between LcrLFY and LFY were constructed. These lines implicate divergence of LcrLFY cis-regulation as the primary cause of both novel transgenic phenotypes but implicate divergence of LcrLFY protein function as the primary cause of elevated TFL1 levels. Taken together these results show that LcrLFY has diverged from A. thaliana in both the cis-regulatory and protein-coding regions and imply that molecular coevolution of LcrLFY and the L. crassa TFL1 ortholog, LcrTFL1, contributed to the evolution of rosette flowering.

Arabidopsis↗
PubMed2005

Conservation and divergence of FCA function between Arabidopsis and rice.

Although several genes have been identified in rice which are functionally equivalent to the flowering time genes in Arabidopsis, primarily genes involved in the photoperiod pathway, little data is available regarding the genes that function in the autonomous pathway in rice. In order to acquire further insight into the control of heading dates in rice, we isolated and conducted an expression analysis on OsFCA, which exhibited 38% sequence homology with Arabidopsis FCA. The N-terminal region of the OsFCA protein appears to be unusually rich in glycine-residues, unlike the N-terminal region found in FCA. However, the genetic structure of OsFCA is, in general, similar to that of FCA. RT-PCR and in silico analyses also showed that alternative splicing and polyadenylation at intron3 were conserved in the genetic expression of OsFCA. We were able to detect alpha, beta, and gamma transcripts, but not the delta transcript, of the OsFCA gene. The beta and gamma transcripts of the OsFCA gene were detected via Northern analysis in the leaves, roots, and flowers of the plant. Flowers in younger stages exhibited higher transcript levels. These data suggest that intron3 may constitute a primary control point in the OsFCA pre-mRNA processing of rice. The overexpression of OsFCA cDNA, driven by the 35S promoter, was shown to partially rescue the late flowering phenotype of the fca mutant, suggesting that the functions of the OsFCA and the FCA are partially overlapped, despite the lack of an apparent FLC homologue in the rice genome. The constitutive expression of OsFCA resulted in no downregulation of FLC, but did result in the weak upregulation of SOC1 in the transgenic Arabidopsis. OsFCA overexpression did not result in a reduction of the gamma transcript levels of FCA in the transgenic Arabidopsis either, thereby suggesting that OsFCA had no effects on the autoregulation of Arabidopsis FCA. All of these results imply conservation and divergence in the functions of FCA between rice and Arabidopsis.

Alternative Splicing↗
PubMed1983

Divergence thresholds as functions of temporal separation, spatial separation, and retinal locus.

Human thresholds were measured for the detection of angular divergence between straight lines using pairs of line segments. The dependence of these thresholds on temporal separation between the two lines, spatial separation, and retinal locus was assessed. Results were comparable to prior divergence thresholds obtained by Harrington and Harrington in their study of "blur patterns." In blur patterns motion parameters may be processed partly or wholly as form information rather than as motion information per se. Harrington and Harrington had used moderate blurring velocities, for which information on both motion and form were present. Observers may have been responding either to motion or to form. The study reported here used briefly presented two-line "blur patterns" with only form information. Analysis suggested that the form components of fast motion-produced blur patterns could be processed by the human visual system. Neither temporal nor spatial separation was a significant determiner of thresholds in accordance with Harrington and Harrington who found no effect of blur-line density in the range studied. Retinal locus was a factor as it was with blur patterns. Some possible mechanisms for the detection of divergence indicated by these results are discussed.

Discrimination Learning↗