Biomedical subjects

M E Harris

Publications and source records attributed to M E Harris.

At least 37 records · Page 2Linked to original sources

PubMed1995

Enhancement of beta-amyloid peptide A beta(1-40)-mediated neurotoxicity by glutamine synthetase.

The beta-amyloid peptide (A beta), a main constituent in both senile and diffuse plaques in Alzheimer's disease brains, was previously shown to be neurotoxic and to be able to interact with several macromolecular components of brain tissue. Previous investigations carried out in our laboratory demonstrated free radical species formation in aqueous solutions of A beta(1-40) and its C-end fragment, A beta(25-35). Toxic forms of A beta rapidly inactivate the oxidation-sensitive cytosolic enzyme glutamine synthetase (GS). In this regard, we suggested and subsequently demonstrated that A beta radicals can cause an oxidative damage of cell proteins and lipids resulting in disruption of membrane functions, enzyme inactivation, and cell death. Because GS can be a substrate for A beta-derived oxidizing species, the present study was conducted to determine if GS could protect against A beta neurotoxicity. In contrast to this initial hypothesis, we here report that GS significantly enhances the neurotoxic effects of A beta(1-40). The A beta-mediated inactivation of GS was found to be accompanied by the loss of immunoreactive GS and the significant increase of A beta(1-40) neurotoxicity.

Amyloid beta-Peptides↗

PubMed1995

Editing domains of Trypanosoma brucei mitochondrial RNAs identified by secondary structure.

The posttranscriptional insertion and deletion of U residues in trypanosome mitochondrial transcripts called RNA editing initiates at the 3' end of precisely defined editing domains that can be identified independently of the cognate guide RNA. The regions where editing initiates in Trypanosoma brucei cytochrome b and cytochrome oxidase subunit II preedited mRNAs are specifically cleaved by a trypanosome mitochondrial endonuclease that acts like mung bean nuclease and therefore is single strand specific. The regions where editing initiates in virtually all examined preedited mRNAs are predicted to form loop structures, suggesting that editing domains could generally be recognized as prominent single-stranded loops. In contrast to preedited mRNA, edited mRNA can be either resistant or sensitive to cleavage by trypanosome mitochondrial endonuclease, depending on the reaction conditions. This selectivity appears dependent on the availability of extract RNAs, and in model reactions, edited mRNA becomes resistant to cleavage upon base pairing with its guide RNA. Natural partially edited mRNAs are also specifically cleaved with a sensitivity like preedited and unlike edited mRNAs, consistent with their being intermediates in editing. These results suggest that in vivo, the structure of editing domains could initially be recognized by the mitochondrial endonuclease, which could target its associated RNA ligase and terminal U transferase to begin cycles of enzymatic editing modifications.

Animals↗

PubMed1995

Identification of phosphates involved in catalysis by the ribozyme RNase P RNA.

The RNA subunit of ribonuclease P (RNase P RNA) is a catalytic RNA that cleaves precursor tRNAs to generate mature tRNA 5' ends. Little is known concerning the identity and arrangement of functional groups that constitute the active site of this ribozyme. We have used an RNase P RNA-substrate conjugate that undergoes rapid, accurate, and efficient self-cleavage in vitro to probe, by phosphorothioate modification-interference, functional groups required for catalysis. We identify four phosphate oxygens where substitution by sulfur significantly reduces the catalytic rate (50-200-fold). Interference at one site was partially rescued in the presence of manganese, suggesting a direct involvement in binding divalent metal ion cofactors required for catalysis. All sites are located in conserved sequence and secondary structure, and positioned adjacent to the substrate phosphate in a tertiary structure model of the ribozyme-substrate complex. The spatial arrangement of phosphorothioate-sensitive sites in RNase P RNA was found to resemble the distribution of analogous positions in the secondary and potential tertiary structures of other large catalytic RNAs.

Base Sequence↗

PubMed1994

Rational design of self-cleaving pre-tRNA-ribonuclease P RNA conjugates.

Ribonuclease P (RNaseP) generates the mature 5' end of tRNAs by removing 5'leader sequences from pre-tRNAs. In vitro, the RNA subunit is sufficient to catalyze this reaction and is therefore a ribozyme. The kinetic analysis of RNase P-mediated catalysis is complicated because product release is normally rate-limiting. Furthermore, the intermolecular nature of the cleavage reaction precludes many applications of in vitro selection schemes to the analysis of RNaseP. To examine and manipulate the RNase P function more effectively, we designed a pair of ribozymes in which the RNase P RNA is covalently linked to a pre-tRNA substrate. To facilitate intramolecular cleavage, pre-tRNA molecules were tethered to circulatory permuted RNaseP RNA molecules at nucleotides implicated in substrate binding. These "active-site-tethered" pre-tRNA-RNaseP RNA conjugates undergo accurate and efficient self-cleavage in vitro, with first-order reaction rates equivalent to the rate of the chemical step of the native RNase P reaction. Unlike most ribozymes, RNase P recognizes its substrate through tertiary RNA-RNA interactions, rather than through extensive Watson-Crick base-pairing. However, the development of the active-site-tethered conjugates has led us to create a sequence-specific endonuclease, termed Endo.P. In the Endo.P configuration, the 3'half of the pre-tRNA acceptor stem binds exogenous RNA substrates via Watson-Crick base-pairing; the bound substrate is subsequently cleaved at the predicted site. The demonstration of sequence-specific cleavage by Endo.P expands the potential of RNase P and its derivatives as reagents in gene therapy.

Base Sequence↗

PubMed1994

Use of photoaffinity crosslinking and molecular modeling to analyze the global architecture of ribonuclease P RNA.

Bacterial ribonuclease P (RNase P), an endonuclease involved in tRNA maturation, is a ribonucleoprotein containing a catalytic RNA. The secondary structure of this ribozyme is well established, but comparatively little is understood about its 3-D structure. In this analysis, orientation and distance constraints between elements within the Escherichia coli RNase P RNA-pre-tRNA complex were determined by intra- and intermolecular crosslinking experiments. A molecular mechanics-based RNA structure refinement protocol was used to incorporate the distance constraints indicated by crosslinking, along with the known secondary structure of RNase P RNA and the tertiary structure of tRNA, into molecular models. Seven different structures that satisfy the constraints equally well were generated and compared by superposition to estimate helix positions and orientations. Manual refinement within the range of conformations indicated by the molecular mechanics analysis was used to derive a model of RNase P RNA with bound substrate pre-tRNA that is consistent with the crosslinking results and the available phylogenetic comparisons.

Affinity Labels↗

PubMed1994

Detection of oxidation products in individual neurons by fluorescence microscopy.

In the study of the central nervous system, it is necessary to address mechanisms by which cells are injured. In vitro investigations using cells in culture allow sharply focused mechanistic questions to be addressed; however, these studies have often been limited by the sensitivity constraints of assays. Many assays for oxidative products require large amounts of cells that must be disrupted. The extent of oxidation in individual cells is therefore unknown and the results yield an average among different cell types. This is inconvenient in cultures of the nervous system which often have multiple cell types. Using a newly developed method for visualizing oxidation products in individual cells, we have examined oxidation in neurons in culture. The method uses a hydrazide, biotin-4-amidobenzoic hydrazide, to bind carbonyls generated from oxidation. Biotin is detected by streptavidin conjugated with a fluorescent dye. Neurons in culture were exposed to 0.1 to 100 microM ferrous sulfate and fluorescence was visualized and quantitated using confocal laser microscopy. Low levels of oxidation (0.1 microM ferrous sulfate) were easily detected with this method. Iron concentration and fluorescence intensity correlated highly (r = 0.991). As an indicator of the sensitivity of this new method, carbonyl content in the cultures was also quantitated using the 2,4-dinitrophenylhydrazine assay (DNPH). The DNPH assay failed to detect the low levels of oxidation which were detected by the biotin-4-amidobenzoic hydrazide method. Fluorescence intensity partially paralleled loss of neuronal viability. Low concentrations of iron (0.1 and 1.0 microM) did not produce significant neuronal death; however, higher concentrations (10 and 100 microM) produced 19 and 53% neuronal loss, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

RNA editing in kinetoplastid mitochondria.

RNA editing in the mitochondrion of kinetoplastid protozoa results in the posttranscriptional addition and deletion of uridine residues in mRNAs. Editing of mRNAs can lead to the formation of initiation codons for mitochondrial translation, the correction of frame-shifted genes at the RNA level, and in extensively edited mRNAs, the formation of complete reading frames. Kinetoplastid RNA editing requires that genetic information from two or more separately transcribed genes be brought together to form the mature, edited mRNA. The information necessary for the proper insertion or deletion of uridines in the mRNA is present in small mitochondrial transcripts termed guide RNAs (gRNAs). Editing of mRNAs appears to be associated with a high molecular weight complex, called the editosome, containing specific gRNAs, unedited mRNAs, and proteins. Editing is likely a two-step process involving first the breakage of a phosphodiester bond at the editing site and formation of a chimeric molecule with a gRNA covalently joined to the 5' end of the 3' portion of an mRNA. The chimera is resolved by the rejoining of the 5' end of the mRNA to the 3' portion of the mRNA with the addition or deletion of a uridine at the junction point. Two models are proposed for the biochemical mechanism of RNA editing. The first is an enzymatic cascade of cleavage and ligation while the other supports successive rounds of transesterification. The obvious functional necessity for editing in kinetoplastid mitochondria is the formation of translatable mRNAs. Far less clear is the evolutionary origin of editing and the role editing plays in regulating mitochondrial gene expression.

Animals↗

PubMed1993

Long-term results in the treatment of hydrocephalus.

The majority of infants presenting with progressive hydrocephalus are now treated surgically with ventricular shunting. It is difficult to predict the eventual developmental outcome of most treated patients. This article reviews the results of surgical therapy for infantile hydrocephalus with particular attention to neurologic and intellectual outcome.

Cause of Death↗

PubMed1992

Kinetoplastid RNA editing: in vitro formation of cytochrome b gRNA-mRNA chimeras from synthetic substrate RNAs.

RNA editing in the kinetoplastid Trypanosoma brucei results in the addition and deletion of uridine residues within several mitochondrial mRNAs. The site and number of uridines added appears to be directed by small (approximately 70 nt) guide RNAs (gRNAs), which can base pair to the edited sequences. We examined reactions involving synthetic cytochrome b (CYb) gRNA and pre-edited mRNA in vitro. A major product of the in vitro reaction is a chimeric RNA molecule containing both gRNA and mRNA sequences. Formation of the CYb gRNA-mRNA chimera was specific, since such molecules did not accumulate when either the gRNA or mRNA was substituted with control RNAs. The reaction required a free 3' hydroxyl on the gRNA and was unaffected by capping of the gRNA's 5' end. Direct RNA sequencing indicated that the CYb gRNA is covalently linked via its 3' poly(U) tail to one of the editing sites on the CYb mRNA. These results suggest that the U's added during editing are donated by the poly(U) tail of a gRNA via a chimeric gRNA-mRNA intermediate.

Animals↗

PubMed1992

Native mRNA editing complexes from Trypanosoma brucei mitochondria.

The aim of this study was to identify multicomponent complexes involved in kinetoplastid mitochondrial mRNA editing. Mitochondrial extracts from Trypanosoma brucei were fractionated on 10-30% glycerol gradients and assayed for RNAs and activities potentially involved in editing, including pre-edited mRNA, guide RNA (gRNA), endonuclease, terminal uridylyltransferase (TUTase), RNA ligase and gRNA-mRNA chimera-forming activities. These experiments suggest that two distinct editing complexes exist. Complex I (19S) consists of gRNA, TUTase, RNA ligase and chimera-forming activity. Complex II (35-40S) is composed of gRNA, preedited mRNA, RNA ligase and chimera-forming activity. These studies provide the first evidence that editing occurs in a multicomponent complex. The possible roles of complex I, complex II and RNA ligase in editing are discussed.

Animals↗

PubMed1992

Trypanosoma brucei mitochondrial ribosomal RNA synthesis, processing and developmentally regulated expression.

The steady-state levels of the mitochondrial ribosomal RNAs of Trypanosoma brucei are repressed in the early bloodstream developmental stage of the parasite and accumulate approximately 30-fold during differentiation to the stage found in the midgut of the insect vector. In order to determine the mechanism regulating this developmental process, we have examined the transcription and processing of the 9S and 12S mitochondrial rRNAs of T. brucei. A short-lived RNA was detected in pulse labeling experiments which contains the mature 12S and 9S rRNAs and at least 1200 nucleotides of RNA transcribed from upstream of the 12S rRNA gene. This putative processing precursor RNA was identified in both intact cells and in run-on experiments using isolated mitochondria. The transcripts containing the upstream sequences are unstable and reach isotopic equilibrium within 15 min. Mature rRNAs in the insect developmental stage are stable and show no detectable turnover during a 36-h chase. Comparison of rRNA synthesis in bloodstream and insect life-stages indicates that mitochondrial rRNA levels are controlled not at the transcriptional level, but rather by a mechanism which likely modulates the stability of the mature rRNAs. These results suggest that a short-lived rRNA precursor is synthesized and processed at comparable rates in both bloodstream and insect stages of the parasite. Thus, it appears that differential stability of the mature 9S and 12S rRNAs plays a major role in modulating mitochondrial gene expression during the developmental cycle of T. brucei.

Animals↗

PubMed1992

Molecular modeling to predict the structural and biological effects of mutations in a highly conserved histone mRNA loop sequence.

The 3'-end of histone mRNAs contains a highly conserved sequence motif which is believed to form a 6 base pair stem and a 4 base loop. These sequences are involved in both the efficiency of 3'-end formation and stability of the mature histone mRNA. We have modeled four stem basepairs and the loop portion of this structure using the wildtype sequences and several mutant sequences. A structure for the wildtype stem-loop is proposed that is based on energy minimization using a representative wildtype sequence and comparison with structures obtained using naturally occurring mutations which do not alter loop function. A wildtype structure is proposed in which the top basepair of the stem is broken, forming a six base loop. Mutant sequences with altered bases in the loop and in the stem were also modeled. The effect of these mutations on the proposed wildtype structure is discussed and possible biological consequences considered.

Base Sequence↗

PubMed1991

Modification of Trypanosoma brucei mitochondrial rRNA by posttranscriptional 3' polyuridine tail formation.

Trypanosoma brucei mitochondrial transcripts can be posttranscriptionally processed by uridine addition or deletion. With editing of mRNAs, uridine addition and deletion create precisely altered reading frames. The addition of nonencoded uridines to mitochondrial guide RNAs results in a less precise modification. Although uridines are specifically added to the 3' termini, their number varies, which results in heterogeneous oligo(U) tails on guide RNAs. In this paper, we show that the mitochondrial 9S and 12S rRNAs are also modified by uridine addition. These modifications appear to have aspects in common with both RNA editing and oligo(U) tail formation. Metabolic labeling studies with intact mitochondria and [alpha-32P]UTP, in the absence of transcription, demonstrated the posttranscriptional timing of the event. T1 RNase comparison analyses of cytidine 3',5'-[5'-32P]biphosphate 3'-end-labeled and [alpha-32P]UTP metabolically labeled rRNAs, along with direct RNA sequencing of the 3' termini, identified the site of uridine addition and revealed the creation of an oligo(U) tail for both rRNAs. 12S and 9S rRNAs hybrid selected from total cell RNA exhibited the same modification, demonstrating the presence of this processing in vivo. Moreover, only 3'-poly(U)-tailed 9S and 12S rRNAs were detected in total cellular and mitochondrial RNAs, which suggests that they are the most abundant and probable mature forms. The 12S and 9S rRNA oligo(U) tails differed significantly from each other, with the 12S having a heterogeneous tail of 2 to 17 uridines and the 9S having a tail of precisely 11 uridines. The mechanism of formation and the function of the rRNA poly(U) tails remain to be determined.

Animals↗

PubMed1991

Regulation of histone mRNA in the unperturbed cell cycle: evidence suggesting control at two posttranscriptional steps.

The levels of histone mRNA increase 35-fold as selectively detached mitotic CHO cells progress from mitosis through G1 and into S phase. Using an exogenous gene with a histone 3' end which is not sensitive to transcriptional or half-life regulation, we show that 3' processing is regulated as cells progress from G1 to S phase. The half-life of histone mRNA is similar in G1- and S-phase cells, as measured after inhibition of transcription by actinomycin D (dactinomycin) or indirectly after stabilization by the protein synthesis inhibitor cycloheximide. Taken together, these results suggest that the change in histone mRNA levels between G1- and S-phase cells must be due to an increase in the rate of biosynthesis, a combination of changes in transcription rate and processing efficiency. In G2 phase, there is a rapid 35-fold decrease in the histone mRNA concentration which our results suggest is due primarily to an altered stability of histone mRNA. These results are consistent with a model for cell cycle regulation of histone mRNA levels in which the effects on both RNA 3' processing and transcription, rather than alterations in mRNA stability, are the major mechanisms by which low histone mRNA levels are maintained during G1.

Animals↗

PubMed1991

Techniques of peripheral nerve repair.

At present, the principles of microsurgical reconstruction of the peripheral nerve incorporate a clear understanding of the pathophysiology of the peripheral nerve, accurate preoperative assessment of the lesion, aggressive early treatment to avoid irreversible atrophy of the end organ, use of nontraumatic microtechniques for optimal alignment of individual fascicular bundles, introduction of a minimum amount of foreign material at the suture line, resection of the scar-producing epineurium, total avoidance of tension at the suture line, and placement of the nerve repair in a well-vascularized soft tissue bed. If tension is eliminated, a minimal amount of suture material is required to repair the nerve ends, because the bundles are maintained in anatomical alignment by a fibrin clot. We have reviewed the various nerve repair methods, stressing that with strict attention to microsurgical technique, the surgeon can hope to maximize reinnervation. Although the importance of all aspects of careful surgical technique cannot be overemphasized, we believe that it is unlikely that improved clinical results will come from further refinements in microsurgical techniques. We are not limited by a working knowledge and understanding of the details of the neurobiology and the neurochemistry of nerve regeneration.

Humans↗

PubMed1990

Addition of uridines to edited RNAs in trypanosome mitochondria occurs independently of transcription.

RNA editing is a novel RNA processing event of unknown mechanism that results in the introduction of nucleotides not encoded in the DNA into specific RNA molecules. We have examined the post-transcriptional addition of nucleotides into the mitochondrial RNA of Trypanosoma brucei. Utilizing an isolated organelle system we have determined that addition of uridines to edited RNAs does not require ongoing transcription. Trypanosome mitochondria incorporate CTP, ATP, and UTP into RNA in the absence of transcription. GTP is incorporated into RNA only as a result of the transcription process. Post-transcriptional CTP and ATP incorporation can be ascribed to known enzymatic activities. CTP is incorporated into tRNAs as a result of synthesis or turnover of their 3' CCA sequences. ATP is incorporated into the 3' CCA of tRNAs and into mitochondrial messenger RNAs due to polyadenylation. In the absence of transcription, UTP is incorporated into transcripts known to undergo editing, and the degree of UTP incorporation is consistent with the degree of editing occurring in these transcripts. Cytochrome b mRNAs, which contain a single editing site near their 5' ends, are initially transcribed unedited at that site. Post-transcriptional labeling of cytochrome b mRNAs in the organelle with [alpha-32P]UTP results in the addition of uridines near the 5' end of the RNA but not in a 3' region which lacks an editing site. These results indicate that RNA editing is a post-transcriptional process in the mitochondria of trypanosomes.

Adenosine Triphosphate↗

PubMed1988

B-naphthoflavone induction and its effect on hepatic phospholipid metabolism in rainbow trout (Salmo gairdneri).

1. B-naphthoflavone (BNF) induction of microsomal cytochrome P-488 and its effect on liver phospholipid metabolism were examined in rainbow trout (Salmo gairdneri) 24 and 96 hr after intraperitoneal injection. 2. Cytochrome P-448 content increased at 96 hr to approximately double the cytochrome content of control fish. 3. Computer-analyzed laser densitometry scans of LDS-polyacrylamide gels of 96 hr BNF-treated liver microsomes showed a 90% increase in cytochrome P-448 levels. 4. A 34% increase in microsomal phospholipid (mumol/mg protein) was observed 24 hr after BNF injection, with a marked increase in choline, ethanolamine and inositol phospholipids. 5. Following 96 hr of exposure to BNF some differences in enzyme activity were noted; choline kinase and cytidylyltransferase activities were reduced, while a marked increase was observed in choline phosphotransferase activity. In light of current information on induction of liver microsomal phospholipid metabolism in mammals, the results of the this study suggest that trout do not respond like mammals to inducers of monooxygenase activity.

Animals↗

PubMed1987

Two-stage tuberculin testing with control antigens in patients residing in two chronic disease hospitals.

We studied the prevalence of tuberculin reactivity and anergy in 360 elderly patients residing in two municipal chronic disease hospitals. Eighty-five (26%) of the 323 patients tested had a positive reaction to a stage 1 tuberculin test and 12 (6%) of the 207 stage 1 tuberculin-negative patients exhibited a booster response to a stage 2 tuberculin test. Thirty percent of the same 207 patients had no response to an anergy panel of skin test antigens that included candida, mumps, and trichophyton. Nonresponders to tuberculin and the anergy panel had significantly higher one-year mortality rates compared to responders (44 v 20%, P = 0.001). Tuberculin-positivity among the 770 employees working in these facilities was 43%; 12 (4%) had a booster response. A survey of 29 randomly selected long-term care facilities in the Boston area indicated that all had a policy for pre-employment screening of employees, but less than 50% had a policy for patients and only one institution used two-stage testing. Routine tuberculin testing is recommended for long-term care facilities and the two-stage method is preferable in institutions with adequate resources.

Adult↗