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PubMed1996

Intron-encoded, antisense small nucleolar RNAs: the characterization of nine novel species points to their direct role as guides for the 2'-O-ribose methylation of rRNAs.

A growing number of small nucleolar RNAs (snoRNAs) are intron-encoded, contain the characteristic box C (UGAUGA) and box D (CUGA) motifs and exhibit long complementarities to conserved sequences in mature rRNAs. We have identified nine additional members of this family, U32 to U40. All but one are encoded in introns of ribosomal protein genes in vertebrates: U32 to U35 in rpL13a, U36 in rpL7a and U38 to U40 in rpS8. By contrast, U37 is encoded in elongation factor 2 gene. Interestingly, U32 and U36 each contain two complementarities (one to 18 S and the other to 28 S rRNA). U32 to U40 are fibrillarin-associated, devoid of a 5'-trimethyl-cap and display an exclusively nucleolar localization. They are all metabolically stable and roughly as abundant as previously reported members of this family. Characterization of their homologs in distant species shows that their 10 to 14 nt long rRNA complementarities are conserved. A clue on the function of this snoRNA family is provided by the comparative analysis of the largely expanded collection of their conserved duplexes with rRNA. Not only does each duplex span at least one site of 2'-O-ribose methylation in the rRNA but the modification site is always at the same position in the duplex, paired to the fifth nucleotide upstream from a box D motif in the snoRNA. Consistent with the notion that each snoRNA of this family guides one particular methylation along the rRNA sequence, we have detected several pairs of snoRNAs with overlapping complementarities to rRNA tracts with vicinal sites of ribose methylations. In each case, the two overlapping complementarities are shifted from each other by a distance equal to the spacing between the methylated sites which are thus found at the same position within each of the mutually exclusive duplexes. Finally, we have also identified, within three previously known snoRNAs, novel antisense elements able to form a canonical duplex around ribose-methylated sites in rRNA, which further supports the conclusion that the duplex structure provides the 2'-O-methyltransferase with the appropriate site-specificity on the substrate.

Animals↗
PubMedDate not supplied

Novel intron-encoded small nucleolar RNAs with long sequence complementarities to mature rRNAs involved in ribosome biogenesis.

Recently, several new snoRNAs encoded in introns of genes coding for ribosomal, ribosome-associated, or nucleolar proteins have been discovered. We are presently studying four of these intronic snoRNAs. Three of them, U20, U21, and U24, are closely related to each other on a structural basis. They are included in genes encoding nucleolin and ribosomal proteins L5 and L7a, respectively, in warm-blooded vertebrates. These three metabolically stable snoRNAs interact with nucleolar protein fibrillarin. In addition, they display common features that make them strikingly related to snoRNA U14. U14 contains two tracts of complementarity to 18S rRNA, which are required for the production of 18S rRNA. U20 displays a 21 nucleotide (nt) long complementarity to 18S rRNA. U21 contains a 13 nt complementarity to an invariant sequence in eukaryotic 28S rRNA. U24 has two separate 12 nt long complementarities to a highly conserved tract of 28S rRNA. Phylogenetic evidences support the fundamental importance of the pairings of these three snoRNAs to pre-rRNA, which could be involved in a control of pre-rRNA folding during preribosome assembly. By transfection of mouse cells, we have also analyzed the processing of U20 and found that the -cis acting signals for its processing from intronic RNA are restricted to the mature snoRNA sequence. Finally, we have documented changes of host genes for these three intronic snoRNAs during the evolution of eukaryotes.

Animals↗
PubMed1999

Antisense RNA strategies for metabolic engineering of Clostridium acetobutylicum.

We examined the effectiveness of antisense RNA (as RNA) strategies for metabolic engineering of Clostridium acetobutylicum. Strain ATCC 824(pRD4) was developed to produce a 102-nucleotide asRNA with 87% complementarity to the butyrate kinase (BK) gene. Strain ATCC 824(pRD4) exhibited 85 to 90% lower BK and acetate kinase specific activities than the control strain. Strain ATCC 824(pRD4) also exhibited 45 to 50% lower phosphotransbutyrylase (PTB) and phosphotransacetylase specific activities than the control strain. This strain exhibited earlier induction of solventogenesis, which resulted in 50 and 35% higher final concentrations of acetone and butanol, respectively, than the concentrations in the control. Strain ATCC 824(pRD1) was developed to putatively produce a 698-nucleotide asRNA with 96% complementarity to the PTB gene. Strain ATCC 824(pRD1) exhibited 70 and 80% lower PTB and BK activities, respectively, than the control exhibited. It also exhibited 300% higher levels of a lactate dehydrogenase activity than the control exhibited. The growth yields of ATCC 824(pRD1) were 28% less than the growth yields of the control. While the levels of acids were not affected in ATCC 824(pRD1) fermentations, the acetone and butanol concentrations were 96 and 75% lower, respectively, than the concentrations in the control fermentations. The lower level of solvent production by ATCC 824(pRD1) was compensated for by approximately 100-fold higher levels of lactate production. The lack of any significant impact on butyrate formation fluxes by the lower PTB and BK levels suggests that butyrate formation fluxes are not controlled by the levels of the butyrate formation enzymes.

Acetone↗
PubMed2026

Comprehensive quality profiling and comparative metabolic characterization of seven dominant fresh-eating Chinese olive (Canarium album Lour.) cultivars in Southern China.

Fresh-eating Chinese olive (Canarium album Lour.) is a subtropical fruit endemic to southern China with considerable commercial value, yet systematic quality characterization of dominant cultivars remains scarce. This study established a multi-dimensional quality dataset for seven dominant cultivars from Fujian and Guangdong provinces, integrating nutritional components, soluble sugars, organic acids, mineral elements, volatile profiles, and non-targeted metabolomics. Significant cultivar-specific differences were observed across all evaluated dimensions: "Lingfeng" exhibited a sugar-dominant low-acid profile, whereas "Sanleng" showed elevated phenolic constituents accumulation. Volatile profiling identified terpenoid-based candidate discriminatory biomarkers, and metabolomic analysis revealed phenylpropanoid biosynthesis, tryptophan metabolism, and starch and sucrose metabolism as the most variable pathways. Correlations between untargeted profiling and targeted absolute quantification validated untargeted result reliability and revealed their complementarity in nutritional evaluation. These findings provide baseline data for FECO germplasm evaluation and targeted industrial utilization.

China↗
PubMed1998

Inefficient assembly and intracellular accumulation of antibodies with mutations in V(H) CDR2.

We previously described secretion defects in four mutants of the murine anti-phosphocholine Ab, T15. The mutant heavy (H) chains had amino acid replacements in the V(H) complementarity-determining region 2 (HCDR2) and were expressed at normal intracellular levels. Here, the intracellular fate of the secretion-defective mutant heavy chains was investigated. Metabolic labeling demonstrated that the T15 wild-type Ab was secreted within a 4-h chase. In contrast, the mutant H chains accumulated with intracellular t(1/2) values ranging from 10 to 24 h. The mutant H chains were associated with increased levels of the molecular chaperones BiP and GRP94, and remained endoglycosidase H sensitive, suggesting retention in the endoplasmic reticulum. Assembly of the mutant H chains with T15 light (L) chain was arrested at the H2 and H2L intermediate stages of the T15 wild-type pathway (H2 --> H2L --> H2L2). Even though some assembly with L chain occurred, it was not as a secretion-competent H2L2 Ig moiety. The T15 L chains coexpressed with mutant H chains were degraded efficiently except for a minor L chain population with a long t(1/2) that was apparently protected at the H2L stage. To our knowledge, this is the first study demonstrating that intracellular half-lives of Ig H and L chains can be influenced by somatic mutations in HCDR2.

Animals↗
PubMed2006

Early morphologic and spectroscopic magnetic resonance in severe traumatic brain injuries can detect "invisible brain stem damage" and predict "vegetative states".

A precise evaluation of the brain damage in the first days of severe traumatic brain injured (TBI) patients is still uncertain despite numerous available cerebral evaluation methods and imaging. In 5-10% of severe TBI patients, clinicians remain concerned with prolonged coma and long-term marked cognitive impairment unexplained by normal morphological T2 star, flair, and diffusion magnetic resonance imaging (MRI). For this reason, we prospectively assessed the potential value of magnetic resonance spectroscopy (MRS) of the brain stem to evaluate the functionality of the consciousness areas. Forty consecutive patients with severe TBI were included. Single voxel proton MRS of the brain stem and morphological MRI of the whole brain were performed at day 17.5 +/- 6.4. Disability Rating Scale and Glasgow Outcome Scale (GOS) were evaluated at 18 months posttrauma. MRS appeared to be a reliable tool in the exploration of brainstem metabolism in TBI. Three different spectra were observed (normal, cholinergic reaction, or neuronal damage) allowing an evaluation of functional damage. MRS disturbances were not correlated with anatomical MRI lesions suggesting that the two techniques are strongly complementarity. In two GOS 2 vegetative patients with normal morphological MRI, MRS detected severe functional damage of the brainstem (NAA/Cr < 1.50) that was described as "invisible brain stem damage." MRI and MRS taken separately could not distinguish patients GOS 3 (n = 7) from GOS 1-2 (n = 11) and GOS 4-5 (n = 20). However, a principal component analysis of combined MRI and MRS data enabled a clear-cut separation between GOS 1-2, GOS 3, and GOS 4-5 patients with no overlap between groups. This study showed that combined MRI and MRS provide a reliable evaluation of patients presenting in deep coma, specially when there are insufficient MRI lesions of the consciousness pathways to explain their status. In the first few days post-trauma metabolic (brainstem spectroscopy) and morphological (T2 star and Flair) MRI studies can predict the long-term neurological outcome, especially the persistent vegetative states and minimally conscious state.

Adult↗
PubMed2005

Cyanide trapping of iminium ion reactive intermediates followed by detection and structure identification using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Secondary and tertiary alicyclic amines are widely found in pharmaceuticals and environmental compounds. The formation of iminium ions as reactive intermediates in the metabolic activation of alicyclic amines has previously been investigated in radiometric assays where radiolabeled cyanide is typically employed. In this paper, we report a relatively high throughput LC-MS/MS method for the detection of the nonradiolabeled cyanide adduct formed in rat or human liver microsomal incubations via constant neutral loss scan followed by structural characterization using product ion scan on a triple quadrupole mass spectrometer. A total of 14 alicyclic amine compounds were investigated with the cyanide trapping LC-MS/MS screen and also with the glutathione (GSH) trapping screen, a well-established and commonly employed technique for reactive metabolite screening. Our results are found to be in general agreement with the previous metabolism reports for these compounds, demonstrating the effectiveness, speed, and simplicity of the cyanide trapping LC-MS/MS method to study the iminium ion intermediates from alicyclic amines and its complementarities to GSH trapping method for reactive metabolite screenings.

Amines↗
PubMed1994

U21, a novel small nucleolar RNA with a 13 nt. complementarity to 28S rRNA, is encoded in an intron of ribosomal protein L5 gene in chicken and mammals.

Following a search of sequence data bases for intronic sequences exhibiting structural features typical of snoRNAs, we have positively identified by Northern assays and sequence analysis another intron-encoded snoRNA, termed U21. U21 RNA is a 93 nt. long, metabolically stable RNA, present at about 10(4) molecules per HeLa cell. It is encoded in intron 5 of the ribosomal protein L5 gene, both in chicken and in the two mammals studied so far, human and mouse. U21 RNA is devoid of a 5'-trimethyl-cap and is likely to result from processing of intronic RNA. The nucleolar localization of U21 has been established by fluorescence microscopy after in situ hybridization with digoxigenin-labeled oligonucleotide probes. Like most other snoRNAs U21 contains the box C and box D motifs and is precipitated by anti-fibrillarin antibodies. By the presence of a typical 5'-3' terminal stem, U21 appears more particularly related to U14, U15, U16 and U20 intron-encoded snoRNAs. Remarkably, U21 contains a long stretch (13 nt.) of complementarity to a highly conserved sequence in 28S rRNA. Sequence comparisons between chicken and mammals, together with Northern hybridizations with antisense oligonucleotides on cellular RNAs from more distant vertebrates, point to the preferential preservation of this segment of U21 sequence during evolution. Accordingly, this complementarity, which overlaps the complementarity of 28S rRNA to another snoRNA, U18, could reflect an important role of U21 snoRNA in the biogenesis of large ribosomal subunit.

Animals↗
PubMed2006

Membrane binding domains.

Eukaryotic signaling and trafficking proteins are rich in modular domains that bind cell membranes. These binding events are tightly regulated in space and time. The structural, biochemical, and biophysical mechanisms for targeting have been worked out for many families of membrane binding domains. This review takes a comparative view of seven major classes of membrane binding domains, the C1, C2, PH, FYVE, PX, ENTH, and BAR domains. These domains use a combination of specific headgroup interactions, hydrophobic membrane penetration, electrostatic surface interactions, and shape complementarity to bind to specific subcellular membranes.

Animals↗
PubMed1999

Homology modelling of human cytochromes P450 involved in xenobiotic metabolism and rationalization of substrate selectivity.

Molecular modelling of human cytochrome P450 (CYP) isoforms is described, based on amino acid sequence homology with a unique bacterial P450 (CYP102) of known crystal structure. It is found that for the human hepatic P450s involved in the metabolism of xenobiotics, ie. CYPIA2, CYP 1A6, CYP2B6, CYP2C9, CYP2C 19, CYP2D6, CYP2E1 and CYP3A4, there is a satisfactory agreement between specific substrate characteristics and topographical features of the putative active sites, including complementarity with key amino acid residues in the P450 haem environments. A combination of homology model interactions with substrates and certain molecular properties of the compounds themselves provides a methodology for the evaluation of potential P450 selectivity in new chemical entities (NCEs).

Amino Acid Sequence↗
PubMed2002

Characterization by liquid chromatography-nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry of two coupled oxidative-conjugative metabolic pathways for 7-ethoxycoumarin in human liver microsomes treated with alamethicin.

The microsomal metabolism of 7-ethoxycoumarin (7-EC) was investigated using liquid chromatography (LC)-NMR and liquid chromatography-mass spectrometry (LC-MS) to characterize the coupling of oxidative-conjugative metabolism events. Within microsomes, cytochromes P450 (P450s) and UDP-glucuronosyltransferases (UGTs) are spatially disparate, each having surface and luminal localization, respectively. To optimize cofactor and substrate transit to UGT without compromising P450 activity, the pore-forming peptide alamethicin was used for microsomal perforation. Aqueous extracts of microsomal incubations containing NADPH and UDP-glucuronic acid were injected for LC-NMR and LC-MS analysis. The analytical complementarity of LC-NMR and LC-MS permitted the identification of four metabolites (M1 to M4). The metabolites M1 and M2 are novel microsomal metabolites for 7-EC, consistent with 3-hydroxylation and subsequent glucuronidation, respectively. Metabolites M3 and M4 were 7-hydroxycoumarin (7-HC) and 7-HC glucuronide, respectively. Viewed collectively, these results illustrate the utility of alamethicin in the examination of coupled oxidative-conjugative metabolism and the synergy of LC-NMR and LC-MS in metabolite identification.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1995

U24, a novel intron-encoded small nucleolar RNA with two 12 nt long, phylogenetically conserved complementarities to 28S rRNA.

Following computer searches of sequence banks, we have positively identified a novel intronic snoRNA, U24, encoded in the ribosomal protein L7a gene in humans and chicken. Like previously reported intronic snoRNAs, U24 is devoid of a 5'-trimethyl-cap. U24 is immunoprecipitated by an antifibrillarin antibody and displays an exclusively nucleolar localization by fluorescence microscopy after in situ hybridization with antisense oligonucleotides. In vertebrates, U24 is a 76 nt long conserved RNA which is metabolically stable, present at approximately 14,000 molecules per human HeLa cell. U24 exhibits a 5'-3' terminal stem-box C-box D structure, typical for several snoRNAs, and contains two 12 nt long conserved sequences complementary to 28S rRNA. It is, therefore, strikingly related to U14, U20 and U21 snoRNAs which also possess long sequences complementary to conserved sequences of mature 18S or 28S rRNAs. In 28S rRNA the two tracts complementary to U24 are adjacent to each other, they involve several methylated nucleotides and are surprisingly close, within the rRNA secondary structure, to complementarities to snoRNAs U18 and U21. Identification of the yeast Saccharomyces cerevisiae U24 gene directly confirms the outstanding conservation of the complementarity to 28S rRNA during evolution, suggesting a key role of U24 pairing to pre-rRNA during ribosome biogenesis, possible in the control of pre-rRNA folding. Yeast S.cerevisiae U24 is also intron-encoded but not in the same host-gene as in humans or chicken.

Animals↗
PubMed2005

MicroRNA biogenesis and function in plants.

A microRNA (miRNA) is a 21-24 nucleotide RNA product of a non-protein-coding gene. Plants, like animals, have a large number of miRNA-encoding genes in their genomes. The biogenesis of miRNAs in Arabidopsis is similar to that in animals in that miRNAs are processed from primary precursors by at least two steps mediated by RNAse III-like enzymes and that the miRNAs are incorporated into a protein complex named RISC. However, the biogenesis of plant miRNAs consists of an additional step, i.e., the miRNAs are methylated on the ribose of the last nucleotide by the miRNA methyltransferase HEN1. The high degree of sequence complementarity between plant miRNAs and their target mRNAs has facilitated the bioinformatic prediction of miRNA targets, many of which have been subsequently validated. Plant miRNAs have been predicted or confirmed to regulate a variety of processes, such as development, metabolism, and stress responses. A large category of miRNA targets consists of genes encoding transcription factors that play important roles in patterning the plant form.

Animals↗
PubMed2005

Identification of coexpressed gene clusters in a comparative analysis of transcriptome and proteome in mouse tissues.

A major advantage of the mouse model lies in the increasing information on its genome, transcriptome, and proteome, as well as in the availability of a fast growing number of targeted and induced mutant alleles. However, data from comparative transcriptome and proteome analyses in this model organism are very limited. We use DNA chip-based RNA expression profiling and 2D gel electrophoresis, combined with peptide mass fingerprinting of liver and kidney, to explore the feasibility of such comprehensive gene expression analyses. Although protein analyses mostly identify known metabolic enzymes and structural proteins, transcriptome analyses reveal the differential expression of functionally diverse and not yet described genes. The comparative analysis suggests correlation between transcriptional and translational expression for the majority of genes. Significant exceptions from this correlation confirm the complementarities of both approaches. Based on RNA expression data from the 200 most differentially expressed genes, we identify chromosomal colocalization of known, as well as not yet described, gene clusters. The determination of 29 such clusters may suggest that coexpression of colocalizing genes is probably rather common.

Animals↗
PubMed2001

Recovering antibody secretion using a hapten ligand as a chemical chaperone.

Engineered antibodies have come to the forefront as research reagents and clinical therapeutics. However, reduced stability or expression levels pose a major problem with many engineered antibodies. As a model for understanding functional consequences of variable region mutation, we have studied the assembly and trafficking of anti-phenylphosphocholine antibodies. Previously, we identified severe secretion defects because of mutations in the heavy chain second complementarity determining region, which is involved in antigen binding. Here we demonstrate that immunoglobulin secretion is increased up to 27-fold by incubating stably transfected PCG1-1 cells with cognate hapten p-nitrophenylphosphocholine. Secretion was unaffected by nonbinding analogs. Radiotracer and metabolic labeling experiments demonstrated specific cellular uptake of p-nitrophenylphosphocholine and increased intracellular heavy and light chain assembly. Brefeldin A inhibited hapten-mediated immunoglobulin secretion but not assembly, indicating that assembly occurs early within the biosynthetic pathway. Recovery of secretion correlated with antigen binding capacity, suggesting that the rescue mechanism involves stabilization of heavy and light chain variable domains. This model system provides the first demonstration that cognate ligands can increase intracellular assembly of functional anti-hapten antibody within mammalian cells and suggests that small molecules of appropriate specificity and affinity acting as chemical chaperones may find application for increasing or regulating immunoglobulin expression.

Arginine↗
PubMed1992

The metabolic pathways for hormonal steroids appear to be reflected in the stereochemistry of DNA.

Computer graphics and energy calculations were employed to examine the relative fit of progesterone and its major biosynthetic precursors and inactive metabolites into partially unwound double stranded DNA. Progesterone was found to be the best fitting molecule; moreover, it was the only compound which exhibited full stereochemical complementarity by inserting completely between base pairs and forming optimal hydrogen bonds with both deoxyribose-phosphate backbones. Intermediates in each step of the biosynthetic and degradation pathways were progressively increasing and decreasing fits into DNA, respectively. When the fits of various possible stereoisomers were examined, the positions of functional groups manifest in the known biosynthetic precursors were found to provide the best possible fitting structures. Conversely, the positions of functional groups of known inactive metabolites provided the worst possible fitting structures. These findings coupled with previous reports showing that the specific biological function assigned to each class of steroid hormone correlates with the formation of a unique pattern of donor/acceptor linkages confirms that hormonal structures are indeed rare in their capacity to form "lock and key" complexes with DNA. Given that all possible linkages to DNA are not yet accounted for, the existence of other naturally occurring compounds with salient biological function is predicted.

Computer Simulation↗
PubMed2000

Molecular-functional studies of adaptive genetic variation in prokaryotes and eukaryotes.

Knowledge of both prokaryotic and eukaryotic organisms is essential to the study of molecular evolution. Their common ancestry mandates that their molecular functions share many aspects of adaptation and constraint, yet their differences in size, ploidy, and structural complexity also give rise to divergent evolutionary options. We explore the interplay of adaptation, constraint, and neutrality in their evolution by the use of genetic variants to probe molecular function in context of molecular structure, metabolic organization, and phenotype-environment interactions. Case studies ranging from bacteria to butterflies, flies, and vertebrates emphasize, among other points: the importance of moving from initial recording of evolutionary pattern variation to studying the processes underlying the patterns, by experiment, reconstructive inference, or both; the complementarity, not conflict, of finding different performance and fitness impacts of natural variants in prokaryotes or eukaryotes, depending on the nature and magnitude of the variants, their locations and roles in pathways, the nature of molecular function affected, and the resulting organismal phenotype-environment interactions leading to selection or its absence; the importance of adaptive functional interaction of different kinds of variants, as in gene expression variants versus variants altering polypeptide properties, or interaction of changes in enzymes' active sites with complementary changes elsewhere that adjust catalytic function in different ways, or coadaptation of different steps' properties in pathways; the power afforded by combining structural and functional analyses of variants with study of the variants' phenotype-environment interactions to understand how molecular changes affect (or fail to affect) adaptive mechanisms "in the wild." Comparative study of prokaryotes and eukaryotes in this multifaceted way promises to deliver both new insights into evolution and a host of new and productive questions about it.

Adaptation, Physiological↗
PubMed1995

Bacterial genetic exchange in nature.

Most bacteria are haploid organisms containing only one copy of each gene per cell for most of the growth cycle. This means that the chance for correcting random mutations in bacterial genes would depend entirely on the complementarity inherent in DNA structures, unless homologous DNA sequences can be imported from outside the cell. Bacteria, like all living organisms have evolved at least one autonomous mechanism, conjugation, for exchanging portions of genetic materials between two related cells. The ecological benefits of conjugation include the expansion of metabolic versatility and resistance to hazardous environmental conditions. Natural bacterial genetic exchange also occurs through virus infections (transduction) and through the uptake of extracellular DNA (transformation). The origin and ecological benefits of transduction and transformation are difficult to assess because they are driven by factors external to the affected cell. Bacterial genetic exchange has implications for the evolution of phenotypes that are either beneficial to humans, such as biodegradation of toxic xenobiotic chemicals, or that are detrimental, such as the evolution of pathogenesis and the spread of antibiotic resistance. Understanding natural bacterial genetic exchange mechanisms is also relevant to the assessment of dispersal risks associated with genetically engineered bacteria and recombinant genes in the environment.

Animals↗