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At least 1,063 records · Page 59Linked to original sources
PubMed1993

Human snRNP polypeptide D1 promotes pre-mRNA splicing in yeast and defines nonessential yeast Smd1p sequences.

Parallel investigations of yeast and metazoan pre-mRNA splicing have documented enormous complexity in the nucleic acid and protein components of the cellular splicing apparatus, the spliceosome. The degree to which yeast and metazoan spliceosomal proteins differ in composition and structure is currently unknown. In this report we demonstrate that the human small nuclear ribonucleoprotein (snRNP) polypeptide D1 complements the cell lethality, splicing deficiency, and snRNA instability phenotypes associated with a yeast smd1 null allele. Mutational analysis of yeast SMD1, guided by a comparison of the predicted yeast and human proteins, reveals that a large, nonconserved portion of Smd1p is dispensable for biological activity. These observations firmly establish D1 as an essential component of the cellular splicing apparatus and suggest that yeast and metazoa are remarkably similar in the polypeptides guiding early snRNP assembly.

Alleles↗
PubMed2003

Tyrosinase RT-PCR as a supplement to histology for detecting melanoma and nevus cells in paraffin sections of sentinel lymph nodes.

The detection of tyrosinase mRNA in sentinel lymph nodes (SLNs) by reverse transcription polymerase chain reaction (RT-PCR) is a sensitive indicator for the presence of melanoma or nevus cells, but it does not enable a distinction between both. We have established an efficient method for extraction and reverse transcription of tyrosinase mRNA from paraffin sections that permits the close correlation of the RT-PCR results with (immuno)histologic findings in adjacent sections. One hundred fifty-three SLNs and 6 non-SLN specimens originating from 92 melanoma and 4 nonmelanoma patients were studied to test the reliability of this approach. The predictive value of positive RT-PCR results was 0.98 for the presence of melanoma or nevus cells; the corresponding negative predictive value was 0.83. Furthermore, the detection rate of tyrosinase mRNA significantly correlated with tumor burden. Among the 33 melanoma-positive SLNs without nevus cells, positive RT-PCR results were obtained in all specimens with extended peripheral (S2) or deeply invasive (S3) micrometastases but in only 46% of the cases with few localized melanoma cells in the subcapsular zone (S1). Routine (immuno)histologic evaluation alone had missed microclusters of melanoma cells in one SLN and small nevus cell aggregates in six other SLNs. They were detected only during microscopic reexamination caused by a positive RT-PCR result. We conclude that histology and immunohistochemistry remain the indispensable gold standard for the identification of melanoma and nevus cells in SLNs. Additional molecular analyses using adjacent paraffin sections may further improve the diagnostic accuracy by sensitizing and guiding the microscopist's attention.

Humans↗
PubMed2026

Oral bacteriome in pediatric patients with malignancies prior to chemotherapy: a pilot study using full-length 16S rRNA sequencing.

OBJECTIVE: To characterize the composition, diversity, and ecological features of the oral bacteriome in pediatric patients with malignancies prior to chemotherapy initiation. METHODS: In this prospective pilot study,supragingival plaque samples were collected from 10 pediatric cancer patients prior to the initiation of chemotherapy. Bacterial genomic DNA was extracted from each sample, and the full-length 16S rRNA gene was amplified and sequenced on the PacBio Sequel II platform using circular consensus sequencing (CCS). Raw CCS reads were quality-filtered and denoised into amplicon sequence variants (ASVs) using DADA2, and taxonomic assignment was performed against the SILVA 138 reference database. Alpha diversity was assessed using the Chao1, Shannon, Simpson, and Faith's phylogenetic diversity (PD whole tree) indices, while beta diversity was evaluated through principal coordinate analysis (PCoA), and non-metric multidimensional scaling (NMDS). Microbial co-occurrence networks were constructed to characterize bacterial interactions, and functional potential was predicted using PICRUSt2, and BugBase. RESULTS: A total of 614,473 high-quality CCS reads were generated, yielding 1,697 ASVs. Alpha diversity analysis revealed substantial inter-individual variation in microbial richness and diversity among the pediatric cancer patients. The bacterial community was dominated by the phyla Firmicutes, Proteobacteria, Bacteroidota, Actinobacteriota. At the genus level, Streptococcus, Prevotella, Neisseria, and Haemophilus were the most abundant taxa. Beta diversity analysis revealed distinct clustering patterns, indicating highly individualized microbial profiles. Co-occurrence network analysis identified several keystone taxa and potential pathogenic associations within the supragingival plaque community. Functional prediction indicated that the dominant metabolic pathways were related to amino acid metabolism, carbohydrate metabolism, and membrane transport. CONCLUSION: These preliminary findings reveal a taxonomically diverse, highly individualized pre-chemotherapy oral bacteriome, providing foundational baseline profiles to guide future longitudinal investigations of chemotherapy-induced dysbiosis and personalized interventions.

Humans↗
PubMed2003

Structure-based incorporation of 6-methyl-8-(2-deoxy-beta-ribofuranosyl)isoxanthopteridine into the human telomeric repeat DNA as a probe for UP1 binding and destabilization of G-tetrad structures.

Heterogeneous ribonucleoprotein A1 (hnRNP A1) is an abundant nuclear protein that participates in RNA processing, alternative splicing, and chromosome maintenance. hnRNP A1 can be proteolyzed to unwinding protein (UP1), a 22.1-kDa protein that retains a high affinity for purine-rich single-stranded nucleic acids, including the human telomeric repeat (hTR) d(TTAGGG)n. Using the structure of UP1 bound to hTR as a guide, we have incorporated the fluorescent guanine analog 6-MI at one of two positions within the DNA to facilitate binding studies. One is where 6-MI remains stacked with an adjacent purine, and another is where it becomes fully unstacked upon UP1 binding. The structures of both modified oligonucleotides complexed to UP1 were determined by x-ray crystallography to validate the efficacy of our design, and 6-MI has proven to be an excellent reporter molecule for single-stranded nucleic acid interactions in positions where there is a change in stacking environment upon complex formation. We have shown that UP1 affinity for d(TTAGGG)2 is approximately 5 nm at 100 mm NaCl, pH 6.0, and our binding studies with d(TTAGG(6-MI)TTAGGG) show that binding is only modestly sensitive to salt and pH. UP1 also has a potent G-tetrad destabilizing activity that reduces the Tm of the hTR sequence d(TAGGGT)4 from 67.0 degrees C to 36.1 degrees C at physiological conditions (150 mm KCl, pH 7.0). Consistent with the structures determined by x-ray crystallography, UP1 is able to bind the hTR sequence in solution as a dimer and supports a model for hnRNP A1 binding to nucleic acids in arrays that may make a contiguous set of anti-parallel single-stranded nucleic acid binding clefts. These data suggest that seemingly disparate roles for hnRNP A1 in alternative splice site selection, RNA processing, RNA transport, and chromosome maintenance reflect its ability to bind a purine-rich consensus sequence (nYAGGn) and destabilize potentially deleterious G-tetrad structures.

Base Pairing↗
PubMed1993

Extensive editing of CR2 maxicircle transcripts of Trypanosoma brucei predicts a protein with homology to a subunit of NADH dehydrogenase.

Several genes of the Trypanosoma brucei mitochondrial genome (the maxicircle) encode mRNAs that are so extensively altered by RNA editing that the gene cannot be identified by analysis of the DNA sequence. The 322-nucleotide preedited RNA of one of these genes, CR2, is converted into a 647-nucleotide transcript by the addition of 345 uridines and the deletion of 20 genomically encoded uridines. The fully edited transcript has an open reading frame that predicts a 194-amino-acid protein. This protein, which we name ND9 (NADH dehydrogenase subunit 9), has homology to a subunit of NADH dehydrogenase (respiratory complex I). Seven guide RNAs that can specify edited CR2 sequence have been identified. Steady-state levels of unedited ND9 transcripts are greater in bloodstream than in procyclic forms, but edited ND9 mRNA is present in similar abundance in both life cycle stages.

Amino Acid Sequence↗
PubMed2002

Comparison of different labeling methods for two-channel high-density microarray experiments.

In this report we evaluate three methods for labeling nucleic acids to be hybridized to a cDNA microarray: direct labeling, indirect amino-allyl labeling, and the dendrimer labeling method (Genisphere). The dendrimer method requires the smallest quantity of sample, 2.5 microg of total RNA compared with 20 microg with the direct or indirect methods. Therefore, we wanted to know whether the performance of the dendrimer method is comparable to the other methods, or whether significant information is lost. Performance can be considered in terms of sensitivity, dynamic range, and reproducibility of the quantitative signals for gene intensity. We compared the three labeling methods by generating three sets of eight self-to-self hybridizations using the same total RNA sample in all cases ("replicate study"). In our analysis, we controlled for the effects of print-tip and background subtraction biases. We also performed a smaller study, namely, a dilution series study with five dilution points per labeling method, to evaluate one aspect of predictive ability. From the replicate study, the dendrimer method appeared to perform as well, and often better, with respect to reproducibility and ability to detect expression. However, in the dilution series study, this method was outperformed by the other two in terms of predictive ability and did not perform very well. These findings are helping to guide our decisions on what labeling method to use for subsequent studies, based on the purpose of a specific study and its limitations in terms of available material.

Fluorescent Dyes↗
PubMed2004

Crystal structure of Argonaute and its implications for RISC slicer activity.

Argonaute proteins and small interfering RNAs (siRNAs) are the known signature components of the RNA interference effector complex RNA-induced silencing complex (RISC). However, the identity of "Slicer," the enzyme that cleaves the messenger RNA (mRNA) as directed by the siRNA, has not been resolved. Here, we report the crystal structure of the Argonaute protein from Pyrococcus furiosus at 2.25 angstrom resolution. The structure reveals a crescent-shaped base made up of the amino-terminal, middle, and PIWI domains. The Piwi Argonaute Zwille (PAZ) domain is held above the base by a "stalk"-like region. The PIWI domain (named for the protein piwi) is similar to ribonuclease H, with a conserved active site aspartate-aspartate-glutamate motif, strongly implicating Argonaute as "Slicer." The architecture of the molecule and the placement of the PAZ and PIWI domains define a groove for substrate binding and suggest a mechanism for siRNA-guided mRNA cleavage.

Amino Acid Sequence↗
PubMed2000

Simian immunodeficiency virus infection of monkeys as a model system for the study of AIDS pathogenesis, treatment, and prevention.

As presented in this review, there are a number of different models of both natural and experimental infection of monkeys with primate lentiviruses. There are numerous different viruses and multiple different monkey species, making for a potentially large number of different combinations. The fact that each different combination of virus isolate and host macaque species may show different behavior underscores the need to understand the different models and their key features. On the one hand, this diversity of systems underscores the need to provide some standardization of the systems used for certain kinds of studies, such as vaccine evaluations, in order to facilitate the comparison of results obtained in different experiments, but in essentially the same experimental system. On the other hand, the rich diversity of different systems, with different features and behaviors, represents a tremendous resource, among other things allowing the investigator to select the system that best recapitulates particular aspects of human HIV infection for study in a relevant nonhuman primate model. Such studies have provided, and may be expected to continue to provide, important insights to guide HIV treatment and vaccine development in the future.

Acquired Immunodeficiency Syndrome↗
PubMed2000

Human immunodeficiency virus glycoprotein 160 induces cytokine mRNA expression in the rat central nervous system.

1. Elevated proinflammatory cytokines within the central nervous system (CNS) of individuals infected with human immunodeficiency virus (HIV) may contribute to altered CNS processes prior to the onset of AIDS. Most studies of HIV-induced alterations in cytokine expression within the CNS have focused on interleukin (IL)-1 and tumor necrosis factor (TNF). 2. We used a ribonuclease protection assay (RPA) to elucidate further the pattern of cytokine mRNA expression in the rat CNS in response to HIV envelope glycoprotein 160 (gp160). Male Sprague-Dawley rats were surgically implanted with a guide cannula directed into a lateral cerebral ventricle. HIV gp160 was injected intracerebroventricularly and rats were sacrificed immediately (time = 0) or at 1, 2, or 4 hr postinjection. Discrete brain regions were dissected, and peripheral glands removed. All tissues were frozen in liquid nitrogen until RNA extraction and assay. 3. IL-1beta IL-1alpha, TNF-alpha, and TNFbeta mRNAs were constitutively expressed in brain tissues. Central administration of gp160 dramatically increased mRNA expression for IL-1beta and TNFalpha in the hypothalamus, hippocampus, brainstem, and cerebellum. Furthermore, although mRNA expression for IL-5, IL-6, and IL-10 was never detected under basal conditions, these mRNAs were increased in brain tissue after administration of gp160. Peak expression in each brain region was detected 2 hr after administration. Multiple cytokine mRNAs were detected in peripheral tissues, but their expression was not altered by central administration of gp160. 4. Our results indicate that gp160 induces mRNA expression in brain for cytokines other than IL-1 and TNF. Screening for multiple cytokine mRNA in this manner provides extensive information concerning the particular cytokines that may be involved in HIV-induced pathologies and alterations in CNS processes.

AIDS Dementia Complex↗
PubMed1992

Targeted cleavage of mRNA by human RNase P.

Ribonuclease P from Escherichia coli can cleave RNAs in simple, hydrogen-bonded complexes of two oligoribonucleotides that resemble the aminoacyl stem and 5' leader sequence of tRNA precursors. RNase P from human (HeLa) cells cannot catalyze the cleavage in vitro of the 5'-proximal oligoribonucleotide that contains the leader sequence in such simple complexes but can do so when the 3'-proximal oligoribonucleotide (external guide sequence) is altered to resemble three-quarters of a tRNA molecule. In such a complex, the efficiency of cleavage of the mRNA for chloramphenicol acetyltransferase, as the 5'-proximal oligoribonucleotide, depends on the structural details of the external guide sequence and on the choice of target site within the mRNA. The presence of the appropriately designed external guide sequence in cells in tissue culture reduces chloramphenicol acetyltransferase activity and the level of the corresponding intact mRNA in the cells. Thus, it appears that the use of such external guide sequences may provide a general technique for gene inactivation.

Base Sequence↗
PubMed2001

Functional groups required for the stability of yeast RNA triphosphatase in vitro and in vivo.

Cet1, the RNA triphosphatase component of the yeast mRNA capping apparatus, catalyzes metal-dependent gamma-phosphate hydrolysis within the hydrophilic interior of an eight-strand beta barrel (the "triphosphate tunnel"), which rests upon a globular protein core (the "pedestal"). We performed a structure-guided alanine scan of 17 residues located in the tunnel (Ser(373), Thr(375), Gln(405), His(411), Ser(429), Glu(488), Thr(490)), on the tunnel's outer surface (Ser(378), Ser(487), Thr(489), His(491)), at the tunnel-pedestal interface (Ile(304), Met(308)) and in the pedestal (Asp(315), Lys(317), Arg(321), Asp(425)). Alanine mutations at 14 positions had no significant effect on Cet1 phosphohydrolase activity in vitro and had no effect on Cet1 function in vivo. Two of the mutations (R321A and D425A) elicited a thermosensitive (ts) yeast growth phenotype. The R321A and D425A proteins had full phosphohydrolase activity in vitro, but were profoundly thermolabile. Arg(321) and Asp(425) interact to form a salt bridge within the pedestal that tethers two of the strands of the tunnel. Mutations R321Q and D411N resulted in ts defects in vivo and in vitro, as did the double-mutant R321A-D435A, whereas the R321K protein was fully stable in vivo and in vitro. These results highlight the critical role of the buried salt bridge in Cet1 stability. Replacement of Ser(429) by alanine or valine elicited a cold-sensitive (cs) yeast growth phenotype. The S429A and S429V proteins were fully active when produced in bacteria at 37 degrees C, but were inactive when produced at 17 degrees C. Replacement of Ser(429) by threonine partially suppressed the cold sensitivity of the Cet1 phosphohydrolase, but did not suppress the cs growth defect in yeast.

Acid Anhydride Hydrolases↗
PubMed2006

Laser microdissection of plant tissue: what you see is what you get.

Laser microdissection (LM) utilizes a cutting or harvesting laser to isolate specific cells from histological sections; the process is guided by microscopy. This provides a means of removing selected cells from complex tissues, based only on their identification by microscopic appearance, location, or staining properties (e.g., immunohistochemistry, reporter gene expression, etc.). Cells isolated by LM can be a source of cell-specific DNA, RNA, protein or metabolites for subsequent evaluation of DNA modifications, transcript/protein/metabolite profiling, or other cell-specific properties that would be averaged with those of neighboring cell types during analysis of undissected complex tissues. Plants are particularly amenable to the application of LM; the highly regular tissue organization and stable cell walls of plants facilitate the visual identification of most cell types even in unstained tissue sections. Plant cells isolated by LM have been the starting point for a variety of genomic and metabolite studies of specific cell types.

Chromosomes, Plant↗
PubMed2000

A randomized, controlled 24-week study of intermittent subcutaneous interleukin-2 in HIV-1 infected patients in Thailand.

OBJECTIVES: To assess the immunological and virological effects, safety profile and feasibility of subcutaneous interleukin-2 (scIL-2) therapy in an HIV-infected Thai population. DESIGN: Seventy-two patients with baseline CD4 cell count of > or = 350 x 10(6)/l and no history of opportunistic infection were randomized to receive antiretroviral therapy plus scIL-2 (scIL-2 group) or antiretroviral therapy alone (control group). scIL-2 was administered at one of three doses for at least 24 weeks. The main measure of treatment efficacy was change in CD4 cell count. RESULTS: The time-weighted mean change in CD4 cell count from baseline to week 24 was + 252 x 10(6)/l for the scIL-2 group compared with + 42 x 10(6)/l for the control group (P< 0.0001). Changes in plasma HIV RNA were not significantly different between the groups over the same time period: there was a 0.83 log10 copies/ml decrease for the scIL-2 group and a 0.70 log copies/ml decrease for the control group (P= 0.362). CONCLUSIONS: This study provides the most extensive experience of scIL-2 therapy in HIV-1 infected women and Asians, and demonstrates the immunological efficacy, tolerability and feasability of scIL-2 therapy in this population. Data from this study were instrumental in guiding the selection of the scIL-2 dosing regimen for ongoing phase III trials.

Adult↗
PubMed1995

Automatic RNA secondary structure determination with stochastic context-free grammars.

We have developed a method for predicting the common secondary structure of large RNA multiple alignments using only the information in the alignment. It uses a series of progressively more sensitive searches of the data in an iterative manner to discover regions of base pairing; the first pass examines the entire multiple alignment. The searching uses two methods to find base pairings. Mutual information is used to measure covariation between pairs of columns in the multiple alignment and a minimum length encoding method is used to detect column pairs with high potential to base pair. Dynamic programming is used to recover the optimal tree made up of the best potential base pairs and to create a stochastic context-free grammar. The information in the tree guides the next iteration of searching. The method is similar to the traditional comparative sequence analysis technique. The method correctly identifies most of the common secondary structure in 16S and 23S rRNA.

Algorithms↗
PubMed2005

Potent inhibitors of subgenomic hepatitis C virus RNA replication through optimization of indole-N-acetamide allosteric inhibitors of the viral NS5B polymerase.

Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. Compounds that block replication of subgenomic HCV RNA in liver cells are of interest because of their demonstrated antiviral effect in the clinic. In followup to our recent report that indole-N-acetamides (e.g., 1) are potent allosteric inhibitors of the HCV NS5B polymerase enzyme, we describe here their optimization as cell-based inhibitors. The crystal structure of 1 bound to NS5B was a guide in the design of a two-dimensional compound array that highlighted that formally zwitterionic inhibitors have strong intracellular potency and that pregnane X receptor (PXR) activation (an undesired off-target activity) is linked to a structural feature of the inhibitor. Optimized analogues devoid of PXR activation (e.g., 55, EC(50) = 127 nM) retain strong cell-based efficacy under high serum conditions and show acceptable pharmacokinetics parameters in rat and dog.

Acetamides↗
PubMed2004

Quantitation of human immunodeficiency virus type 1 RNA loads in cervicovaginal secretions in pregnant women and relationship between viral loads in the genital tract and blood.

The purpose of this study was to analyze the quantitation of the human immunodeficiency virus type 1 RNA (HIV-1 RNA) in the genital tract of HIV-1-infected pregnant women and to evaluate a possible correlation with the viral load in blood plasma (Spearman's rank correlation coefficient). A total of 38 each of cervical, vaginal, and blood samples from 38 women were obtained during the third trimester of pregnancy for quantitation of the HIV-1 RNA load. Viral loads were determined by reverse transcription-polymerase chain reaction. The HIV-1 RNA viral load was detectable in 29 of the 38 (76.3%) blood samples, in 6 of the 38 (15.7%) cervical secretion samples, and in 8 of the 38 (21%) vaginal secretion samples. Overall, the correlation between the HIV-1 RNA viral load in the blood plasma and in cervical secretion samples was 0.51 ( P<0.001). However, the correlation disappeared ( r=0.27) when three patients with high blood plasma viral loads were eliminated from the statistical study. The viral load in the vaginal secretions did not correlate with that in the blood samples ( r=0.26). There were two cases in which HIV-1 RNA was undetectable in the blood and cervix but was detectable in vaginal secretions: one woman had 220 copies/ml and the other 68 copies/ml. These results suggest that pregnant women with undetectable viral loads in blood plasma are still at risk of transmitting the virus vertically during vaginal delivery. Because of this, antiretroviral prophylaxis during vaginal delivery must be administered to HIV-1-infected women and their newborns, regardless of the mother's viral load in plasma. In conclusion, quantification of cervicovaginal levels of HIV-1 may represent a useful tool for assessing the individual risk associated with a vaginal delivery and for guiding decisions about whether a scheduled caesarean should be recommended.

Adult↗
PubMed2003

mRNA expression of ephrins and Eph receptor tyrosine kinases in the neonatal and adult mouse central nervous system.

Ephrins and Eph receptors are a family of molecules that have been implicated in axonal pathfinding. A unique feature of B-class ephrins and Eph receptors is their ability to transmit bidirectional signals in both ephrin- and Eph receptor-expressing cells upon cell-cell contact. These signals can lead to cytoskeletal alterations that have been attributed to regulating neuronal growth responses. Examination of gene-target knockout mice has supported this hypothesis, revealing numerous developmental defects in the nervous systems of mice mutant for both B-class ephrins and Eph receptors. To examine the potential scope of action for these genes in the nervous system, we have used in situ hybridization to study the mRNA expression of ephrins (B1, B2, and B3) and Eph receptors (B1, B2, B3, A4) in neonatal and adult mice. We found ephrins and Eph receptors to be expressed throughout the CNS. Expression was observed in the epithelium and migratory regions of the neonate and adult tissues as well as in discrete regions of high plasticity, including the adult olfactory bulb, hippocampus, and cerebellum. These studies suggest additional potential roles for these molecules in the postnatal and adult CNS and will serve as a guide in the detailed evaluation of mutant mice.

Aging↗