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At least 1,297 records · Page 72Linked to original sources
PubMed1994

A full-coordinate model of the polymerase domain of HIV-1 reverse transcriptase and its interaction with a nucleic acid substrate.

We present a full-coordinate model of residues 1-319 of the polymerase domain of HIV-I reverse transcriptase. This model was constructed from the x-ray crystallographic structure of Jacobo-Molina et al. (Jacobo-Molina et al., P.N.A.S. USA 90, 6320-6324 (1993)) which is currently available to the degree of C-coordinates. The backbone and side-chain atoms were constructed using the MAXSPROUT suite of programs (L. Holm and C. Sander, J. Mol. Biol. 218, 183-194 (1991)) and refined through molecular modeling. A seven base pair A-form dsDNA was positioned in the nucleic acid binding cleft to represent the template-primer complex. The orientation of the template-primer complex in the nucleic acid binding cleft was guided by the positions of phosphorus atoms in the crystal structure.

Amino Acid Sequence↗
PubMed2000

Identification of brain-specific and imprinted small nucleolar RNA genes exhibiting an unusual genomic organization.

We have identified three C/D-box small nucleolar RNAs (snoRNAs) and one H/ACA-box snoRNA in mouse and human. In mice, all four snoRNAs (MBII-13, MBII-52, MBII-85, and MBI-36) are exclusively expressed in the brain, unlike all other known snoRNAs. Two of the human RNA orthologues (HBII-52 and HBI-36) share this expression pattern, and the remainder, HBII-13 and HBII-85, are prevalently expressed in that tissue. In mice and humans, the brain-specific H/ACA box snoRNA (MBI-36 and HBI-36, respectively) is intron-encoded in the brain-specific serotonin 2C receptor gene. The three human C/D box snoRNAs map to chromosome 15q11-q13, within a region implicated in the Prader-Willi syndrome (PWS), which is a neurogenetic disease resulting from a deficiency of paternal gene expression. Unlike other C/D box snoRNAs, two snoRNAs, HBII-52 and HBII-85, are encoded in a tandemly repeated array of 47 or 24 units, respectively. In mouse the homologue of HBII-52 is processed from intronic portions of the tandem repeats. Interestingly, these snoRNAs were absent from the cortex of a patient with PWS and from a PWS mouse model, demonstrating their paternal imprinting status and pointing to their potential role in the etiology of PWS. Despite displaying hallmarks of the two families of ubiquitous snoRNAs that guide 2'-O-ribose methylation and pseudouridylation of rRNA, respectively, they lack any telltale rRNA complementarity. Instead, brain-specific C/D box snoRNA HBII-52 has an 18-nt phylogenetically conserved complementarity to a critical segment of serotonin 2C receptor mRNA, pointing to a potential role in the processing of this mRNA.

Animals↗
PubMed2006

Characterization of 43 non-protein-coding mRNA genes in Arabidopsis, including the MIR162a-derived transcripts.

Messenger RNAs that do not contain a long open reading frame (ORF) or non-protein-coding RNAs (npcRNAs) are an emerging novel class of transcripts. Their functions may involve the RNA molecule itself and/or short ORF-encoded peptides. npcRNA genes are difficult to identify using standard gene prediction programs that rely on the presence of relatively long ORFs. Here, we used detailed bioinformatic analyses of expressed sequence tag/cDNA databases to detect a restricted set of npcRNAs in the Arabidopsis (Arabidopsis thaliana) genome and further characterized these transcripts using a combination of bioinformatic and molecular approaches. Compositional analyses revealed strong nucleotide strand asymmetries in the npcRNAs, as well as a biased GC content, suggesting the existence of functional constraints on these RNAs. Thirteen of these transcripts display tissue-specific expression patterns, and three are regulated in conditions affecting root architecture. The npcRNA 78 gene contains the miR162 sequence in an alternative intron and corresponds to the MIR162a locus. Although DICER-LIKE 1 (DCL1) mRNA is known to be regulated by miR162-guided cleavage, its level does not change in a mir162a mutant. Alternative splicing of npcRNA 78 leads to several transcript isoforms, which all accumulate in a dcl1 mutant. This suggests that npcRNA 78 is a genuine substrate of DCL1 and that splicing of this microRNA primary transcript and miR162 processing are competitive nuclear events. Our results provide new insights into Arabidopsis npcRNA biology and the potential roles of these genes.

Alternative Splicing↗
PubMed1996

Cellular activation of mesangial gelatinase A by cytochalasin D is accompanied by enhanced mRNA expression of both gelatinase A and its membrane-associated gelatinase A activator (MT-MMP).

Activation of gelatinase A represents a crucial regulatory step in the control of its enzymic activity. Rat kidney mesangial cells secrete predominantly latent gelatinase A that can be activated following treatment with cytochalasin D. In the present paper we provide new evidence, using reverse transcription-PCR, that treatment of rat mesangial cells with cytochalasin D enhances the steady-state level of mRNA of the membrane-type matrix metalloproteinase (MT-MMP), as well as of gelatinase A, with no change in the level of tissue inhibitor of metalloproteinases-2 (TIMP-2) mRNA. Since the TIMP-2 protein level is reduced in conditioned medium from cytochalasin D-treated cells, the results of the present study are consistent with a model in which the action of cytochalasin D is to cause extracellular gelatinase A and TIMP-2 to be sequestered at the plasma membrane, forming a heterotrimeric complex with MT-MMP. In this manner, TIMP-2 may assume a bifunctional role causing: (i) inhibition of gelatinase A in the extracellular compartment; and (ii) guiding gelatinase A to activation through a membrane association with MT-MMP.

Animals↗
PubMed1997

Identification of specific Rp-phosphate oxygens in the tRNA anticodon loop required for ribosomal P-site binding.

tRNA binding to the ribosomal P site is dependent not only on correct codon-anticodon interaction but also involves identification of structural elements of tRNA by the ribosome. By using a phosphorothioate substitution-interference approach, we identified specific nonbridging Rp-phosphate oxygens in the anticodon loop of tRNA(Phe) from Escherichia coli which are required for P-site binding. Stereospecific involvement of phosphate oxygens at these positions was confirmed by using synthetic anticodon arm analogues at which single Rp- or Sp-phosphorothioates were incorporated. Identical interference results with yeast tRNA(Phe) and E. coli tRNA(fMet) indicate a common backbone conformation or common recognition elements in the anticodon loop of tRNAs. N-ethyl-N-nitrosourea modification-interference experiments with natural tRNAs point to the importance of the same phosphates in the loop. Guided by the crystal structure of tRNA(Phe), we propose that specific Rp-phosphate oxygens are required for anticodon loop ("U-turn") stabilization or are involved in interactions with the ribosome on correct tRNA-mRNA complex formation.

Anticodon↗
PubMed1993

Functional properties of pro region of Escherichia coli heat-stable enterotoxin.

Escherichia coli heat-stable enterotoxin Ip (STp) is synthesized as the 72-amino-acid residue precursor consisting of three regions: pre region (amino acid residues 1 to 19), pro region (amino acid residues 20 to 54), and mature ST (mST) region (amino acid residues 55 to 72). We examined the role of the pro sequence of STp in enterotoxigenicity of a strain by deleting the gene fragment encoding amino acids 22 to 57. This deletion caused a remarkable reduction of its enterotoxic activity of culture supernatant. In order to analyze the sequence responsible for the function of the pro region, two additional deletion mutants were made. The deletion of the sequence covering amino acids 29 to 38, which is conserved in all sequences of ST reported, brought about a significant reduction of enterotoxic activity but the deletion of the non-conserved sequence (amino acids 40 to 53) did not. This result shows that conserved sequence is mainly responsible for the function. Subsequently, to examine the mechanism of action of the pro region, plasmids carrying DNA sequences of hybrid proteins consisting of pre-pro-nuclease, pre-mST-nuclease, pre-pro-mST-nuclease and pre-pro-nuclease-mST were constructed. Amino acid sequence determination and SDS-polyacrylamide gel analysis revealed that these fusion proteins were cleaved between pre sequence and pro sequence during secretion and the cleaved fusion proteins were accumulated in periplasmic space. But the amount of hybrid protein accumulated in the periplasmic space varied among the strains. That is, the amount of the pre-pro-nuclease gene product that accumulated in the periplasmic space was the highest of all fusion gene products. These results indicate that the existence of the mST region strongly interferes with the translocation of the gene product into the periplasmic space and that the pro region functions to guide the mST region into the periplasmic space.

Amino Acid Sequence↗
PubMed2007

MicroRNA: past and present.

MicroRNAs (miRNAs) are approximately 22 nucleotide (nt) non-coding RNAs that participate in gene regulation. MiRNAs confer their regulation at a post-transcriptional level, where they either cleave or repress translation of mRNAs. Over 3000 identified mature miRNAs exist in species ranging from plants to humans, suggesting that they are ancient players in gene regulation. A relatively small number of miRNAs have been experimentally tested for their function. Of those tested, functions including cell differentiation, proliferation, apoptosis, anti-viral defense and cancer have been proposed. Improved software programs are now able to predict the targets of miRNAs in a more efficient manner, thus facilitating the elucidation of miRNA function. Furthermore, methods such as real-time PCR and microarray have been enhanced for studying miRNA expression. Using these tools, scientists are able to discover novel functions for miRNAs. It is possible that miRNAs will be revealed as having a role in virtually every aspect of gene regulation. This review guides readers through the biogenesis of miRNAs, their mechanism of action on their target mRNAs, the functional outcomes of their action on mRNAs and the current techniques to investigate these processes.

Animals↗
PubMed1996

Muscle development and attachment to the epidermis is accompanied by expression of beta 3 and beta 1 tubulin isotypes, respectively.

In Drosophila beta tubulins are encoded by a small gene family whose members are differentially expressed in a highly cell and tissue specific manner. Here we focus on the expression of the beta 3 tubulin isotype during mesoderm differentiation and beta 1 tubulin expression in the apodemes during embryonic development. The beta 3 tubulin isotype is first detectable at the extended germband stage shortly before the separation of somatic and visceral derivatives. Comparing the distribution of the beta 3 mRNA and the beta 3 isotype shows that the transcription of the beta 3 tubulin gene is cell type specifically repressed during differentiation of individual mesodermal derivatives, from which the dorsal vessel remains transcriptionally active until shortly before hatching. In contrast the beta 3 tubulin protein is detectable in all mesodermal derivatives. The beta 3 tubulin is an excellent marker to study mesoderm differentiation on a regulatory and cellular level using both genetics and molecular biology. In the visceral mesoderm, the expression of the beta 3 tubulin gene is regulated by homeotic gene products, while other transactivators regulate expression in the dorsal vessel and the body wall musculature. In the somatic mesoderm, the beta 3 tubulin allows to visualize myotube formation and insertion into the epidermis. This contact to the epidermal attachment sites (apodemes) induces beta 1 tubulin expression, as can be seen in double staining experiments. We determined a 14bp cis-regulatory enhancer element guiding expression of the beta 1 tubulin gene in these attachment sites. Using the beta 1 and beta 3 tubulin isotypes as markers we started to isolate mutants which are disturbed in muscle formation.

Animals↗
PubMed2003

Mutation patterns of the reverse transcriptase genes in HIV-1 infected patients receiving combinations of nucleoside and non nucleoside inhibitors.

A genotyping assay was used to define human immunodeficiency virus type 1 (HIV-1) reverse transcriptase codons in plasma samples from 80 HIV-1 patients extensively treated with two nucleoside reverse transcriptase (zidovudine and lamivudine) and one non nucleoside reverse transcriptase (nevirapine) inhibitor. The frequencies of T215S/Y/F, M41L, D67N, L210W K70R, K219Q mutations, detectable in plasma samples, conferring resistance to zidovudine were 61.2, 56.2, 36.2, 31.5, 27.5 and 17.5%, respectively. Mutations (M184V or M184I) conferring resistance to lamivudine were detected in an extremely high percentage of patients (61%). Among mutations correlated to high (K103N, V106A, Y181C/I, Y188C/H/L, G190A/C/E/Q/S/T) or moderate (V108I, V118I) levels of nevirapine resistance, the predominant amino acid change was a substitution at 103 codon, present in 24 of 80 samples tested. Finally Q151M, the marker mutation able to confer resistance to all nucleoside analogues, was detected in seven patients with a viral load of between 1 x 10(4) and 9 x 10(4) HIV-1 RNA copies/ml. The relationship between the genotype and the viral load showed that the incidence of some specific mutations [M41L, T215Y (correlated to zidovudine resistance) and K103N (correlated to all NNRTIs drugs)] significantly (P=0.001) increased with higher viral load. Our results, albeit limited to a small cohort, showed a high frequency of mutations correlated to drugs in use, suggesting a need for therapeutic change in the near future and demonstrating that the development of genotyping tests helps to guide the therapeutic management of HIV-1 infected people. Our data highlight the dangers of selecting antiretroviral therapy without previous antiretroviral drug testing. Although the cost of these assays is a concern, prescribing inefficacious drugs could create serious problems for HIV-1 patients.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Different strategies of differential display: areas of application.

The main goal of this review is to provide some guide-lines for choosing a proper method when searching for differentially expressed transcripts. The choice of an approach should depend upon particular features of the experiment and project requirements. We outline some general considerations to help decide between differential display-related techniques and subtractive hybridization. While discussing advantages and problems of differential display, special attention is given to several recently developed methods based on alternative principles of fingerprint production, which provide a possibility for complete systematic investigation of RNA samples.

Animals↗
PubMed2005

Receptor tyrosine phosphatases guide vertebrate motor axons during development.

Receptor-type protein tyrosine phosphatases (RPTPs) are required for appropriate growth of axons during nervous system development in Drosophila. In the vertebrate, type IIa RPTPs [protein tyrosine phosphatase (PTP)-delta, PTP-sigma, and LAR (leukocyte common-antigen-related)] and the type III RPTP, PTP receptor type O (PTPRO), have been implicated in the regulation of axon growth, but their roles in developmental axon guidance are unclear. PTPRO, PTP-delta, and PTP-sigma are each expressed in chick motor neurons during the period of axonogenesis. To examine potential roles of RPTPs in axon growth and guidance in vivo, we used double-stranded RNA (dsRNA) interference combined with in ovo electroporation to knock down RPTP expression levels in the embryonic chick lumbar spinal cord. Although most branches of the developing limb nerves appeared grossly normal, a dorsal nerve identified as the anterior iliotibialis was clearly affected by dsRNA knock-down of RPTPs. In experimental embryos treated with dsRNA targeting PTP-delta, PTP-sigma, or PTPRO, this nerve showed abnormal fasciculation, was reduced in size, or was missing entirely; interference with PTPRO produced the most severe phenotypes. Control embryos electroporated with vehicle, or with dsRNA targeting choline acetyltransferase or axonin-1, did not exhibit this phenotype. Surprisingly, embryos electroporated with dsRNA targeting PTP-delta together with PTPRO, or all three RPTPs combined, had less severe phenotypes than embryos treated with PTPRO alone. This result suggests that competition between type IIa and type III RPTPs can regulate motor axon outgrowth, consistent with findings in Drosophila. Our results indicate that RPTPs, and especially PTPRO, are required for axon growth and guidance in the developing vertebrate limb.

Amino Acids↗
PubMed2005

Use of virologic assays in the diagnosis and management of hepatitis C virus infection.

This article discusses the use of virologic assays in the diagnosis and management of hepatitis C virus (HCV) infection. The use of virologic tests has become essential in the management of HCV infection to diagnose HCV infection, guide treatment decisions, and assess the virologic response to antiviral therapy. The continuing development of test systems accompanied by new antiviral drugs and novel therapeutic approaches should lead to an optimization of the treatment of HCV infection.

Genetic Markers↗
PubMed2004

Increased immune transcript levels are correlated with acute graft-versus-host disease and cytomegalovirus response after allogeneic stem cell transplantation.

BACKGROUND: Donor T cells are primarily responsible for graft-versus-host disease (GVHD). Three effector pathways have been described for T-cell cytotoxicity: granzyme B/perforin, Fas/Fas ligand (FasL), and secreted molecules such as tumor necrosis factor (TNF)-alpha. Therefore, this study evaluates the gene expression pattern in the peripheral blood of patients after allogeneic stem cell transplantation and correlates the results to acute GVHD. METHODS: Real-time quantitative reverse transcriptase-polymerase chain reaction was used to quantify the gene expression of granzyme B, perforin, FasL, and TNF-alpha in peripheral blood from 53 patients. RESULTS: Samples were available from 27 of the 38 patients with acute GVHD diagnoses. Increased gene expression (>50%) during acute GVHD was detected in 23 of 27, 26 of 27, and 24 of 27 patients for granzyme B, perforin, and FasL, respectively. TNF-alpha showed a diffuse correlation. The median increases were as follows: granzyme B, 7.2x (1.6-183.2); perforin, 5.8x (1.6-254.9); and FasL, 8.5x (1.5-895.6). We also showed that all of the 10 patients with increasing levels of granzyme B, perforin, and FasL during steroid treatment demonstrated persistent or deteriorating GVHD. Patients with increasing transcription levels during cytomegalovirus (CMV) reactivation responded significantly better to therapy than those with declining levels. A total of 13 of 17 patients with increasing levels versus 0 of 11 patients with decreasing levels responded well to CMV treatment (P<0.01). CONCLUSION: Although not specific for acute GVHD, quantitative assessment of immune transcripts may be of value in diagnosing and monitoring acute GVHD. It may also serve as a guide for the clinician in detecting patients who respond poorly to CMV therapy.

Acute Disease↗
PubMed2003

Culture-independent identification of pathogenic bacteria and polymicrobial infections in the genitourinary tract of renal transplant recipients.

Renal transplant recipients are predisposed to urinary tract infections caused by both common uropathogens and opportunistic bacteria resulting frequently in significant polymicrobial infections. In this study, a culture-independent 16S rRNA-based approach was established to identify unusual, fastidious, or anaerobic bacteria and to investigate bacterial diversity in urinary tract specimens. Similarly sized amplicons encompassing the V6 to V8 region of the 16S rRNA were analyzed with denaturing high-performance liquid chromatography (DHPLC) (WAVE System). Artificial mixtures of single amplicons from commonly encountered uropathogenic bacteria produced distinct peak profiles whose identities were confirmed by sequencing individually collected peak products. We evaluated the application of the method on 109 urinary tract specimens from renal transplant recipients; 100% correlation was found for culture-positive specimens, and DHPLC generated peak profiles. However, for culture-negative specimens, DHPLC facilitated the detection of novel peak profiles. DNA sequencing of these individual peaks was used to identify the bacteria involved. Thus, in PCR-positive but culture-negative samples the method allowed detection of previously known uropathogens such as Corynebacterium urealyticum and Gardnerella vaginalis, but also unusual agents including Anaerococcus lactolyticus, Bacteroides vulgatus, Dialister invisus, Fusobacterium nucleatum, Lactobacillus iners, Leptotrichia amnionii, Prevotella buccalis, Prevotella ruminicola, Rahnella aquatilis, and Streptococcus intermedius were detected as single pathogens or as constituents of polymicrobial infections. The method described is reproducible and rapidly and enables both DHPLC-based profiling and sequence-based investigation of microbial communities and polymicrobial infections. A detailed understanding of infections found in recipients of renal transplants will guide antibiotic therapy regimens and provide new perspectives for decreasing the risk of graft rejection.

Bacteria↗
PubMed2006

Translational control by neuroguidin, a eukaryotic initiation factor 4E and CPEB binding protein.

CPEB-mediated translation is important in early development and neuronal synaptic plasticity. Here, we describe a new eukaryotic initiation factor 4E (eIF4E) binding protein, Neuroguidin (Ngd), and its interaction with CPEB. In the mammalian nervous system, Ngd is detected as puncta in axons and dendrites and in growth cones and filopodia. Ngd contains three motifs that resemble those present in eIF4G, 4EBP, Cup, and Maskin, all of which are eIF4E binding proteins. Ngd binds eIF4E directly, and all three motifs must be deleted to abrogate the interaction between these two proteins. In injected Xenopus oocytes, Ngd binds CPEB and, most importantly, represses translation in a cytoplasmic polyadenylation element (CPE)-dependent manner. In Xenopus embryos, Ngd is found in both neural tube and neural crest cells. The injection of morpholino-containing antisense oligonucleotides directed against ngd mRNA disrupts neural tube closure and neural crest migration; however, the wild-type phenotype is restored by the injection of a rescuing ngd mRNA. These data suggest that Ngd guides neural development by regulating the translation of CPE-containing mRNAs.

Amino Acid Sequence↗
PubMed2001

Cutting edge: editing of recycling class II:peptide complexes by HLA-DM.

HLA-DM catalyzes the exchange and selection of ligands for MHC class II molecules within mature endosomal/lysosomal compartments. Here, evidence is provided that DM edits peptides in early endosomes, thus influencing presentation via recycling class II molecules. Maximal class II-restricted presentation of an albumin-derived peptide, dependent on endocytosis and recycling class II molecules, was observed in cells lacking HLA-DM. DM editing of this epitope was observed in early endocytic compartments as shown using inhibitors of early to late endosomal transport. Editing was tempered by coexpression of HLA-DO, suggesting that DM:DO ratio may be important in guiding epitope editing in early endosomal compartments. Thus, HLA-DM appears to interact with, and edit epitopes displayed by, recycling class II molecules.

Antigen Presentation↗
PubMed2001

Semaphorin 3A elicits stage-dependent collapse, turning, and branching in Xenopus retinal growth cones.

The semaphorin receptor, neuropilin-1 (NP-1), was first identified in Xenopus as the A5 antigen and is expressed abundantly in developing retinal ganglion cells (RGCs). Here we show that growth cones acquire responsiveness to semaphorin 3A (Sema 3A) with age and that the onset of responsiveness correlates with the appearance of NP-1 immunoreactivity. Growth cones from "old" (stage 35/36) retinal explants collapse rapidly (5-10 min) in response to Sema 3A and turn away from a gradient of Sema 3A, whereas "young" growth cones (stage 24) are insensitive to Sema 3A. Moreover, transfection of full-length NP-1 into young neurons confers premature Sema 3A sensitivity. When young neurons are aged in culture they develop Sema 3A sensitivity in parallel with those in vivo, suggesting that an intrinsic mechanism of NP-1 regulation mediates this age-dependent change. Sema 3A-induced collapse is transient, and after recovery approximately 30% of growth cones extend new branches within 1 hr, implicating Sema 3A as a branching factor. Pharmacological inhibitors were used to investigate whether these three Sema 3A-induced behaviors (collapse, turning, and branching) use distinct second messenger signaling pathways. All three behaviors were found to be mediated via cGMP. In situ hybridization shows that Sema 3A is expressed in the tectum and at the anterior boundary of the optic tract where axons bend caudally, suggesting that Sema 3A/NP-1 interactions play a role in guiding axons in the optic tract and in stimulating terminal branching in the tectum.

Aging↗
PubMed2003

Tenascin-R as a repellent guidance molecule for developing optic axons in zebrafish.

To investigate the role of tenascin-R in nervous system development, we studied axon pathfinding in the developing optic system of zebrafish. Zebrafish tenascin-R has the same domain structure as tenascin-R in amniotes. Amino acid sequence identity with human tenascin-R is 60%. In 3-d-old larvae, tenascin-R mRNA is expressed in scattered cells throughout the periventricular cell layer of the diencephalon and tectum. Tenascin-R immunoreactivity is not detectable in the optic nerve, optic tract, or tectal optic neuropil but immediately borders the optic tract caudally. Reducing expression of tenascin-R in 3-d-old larvae in vivo by injecting morpholinos into fertilized eggs led to excessive branching of the optic tract in 86% of all injected larvae compared with 20-37% in controls. Branches were almost exclusively caudal, where tenascin-R immunoreactivity normally borders the optic tract, suggesting a role for tenascin-R in guiding optic axons in the ventral diencephalon by a contact-repellent mechanism.

Animals↗