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Biomedical subjects

E Glaser

Publications and source records attributed to E Glaser.

At least 19 recordsLinked to original sources
PubMed2001

L and D presequence peptides derived from the precursor of F1beta subunit of the ATP synthase inhibit mitochondrial protein import by interaction with import machinery.

We investigated the effect of L and D enantiomers of a 25-residue peptide derived from the N-terminal region of the presequence of Nicotiana plumbaginifolia F1beta subunit of the ATP synthase, pF1beta(1, 25), on import into spinach leaf mitochondria. Three in vitro synthesized precursor proteins using different import pathways were used. Import of the precursor proteins of F1beta subunit of the ATP synthase, pre-F1beta, and the alternative oxidase, pre-AOX, required addition of external ATP. whereas the chimeric precursor containing the N-terminal 84 amino acids of the cytochrome b2 precursor protein linked to dihydrofolate reductase, pre-b2(1, 84)-DHFR was not dependent on ATP. Import of pre-F1beta, and pre-AOX was inhibited already at 1 microM and 3 microM concentration of the L and D enantiomers, whereas inhibition of import of pre-b2(1, 84)-DHFR, occurred at concentrations >10 microM of both enantiomers. Binding efficiency of the precursor proteins was not affected by addition of the L and D enantiomers. There was no correlation between inhibition of import of pre-F1beta and pre-AOX and dissipation of membrane potential measured as a decrease of Rhodamine 123 fluorescence quenching. The inhibitory effect of the L and D presequence enantiomers on import of pre-F1beta and pre-AOX was concluded to occur within the outer membrane translocase machinery beyond the initial precursor receptor interaction. Furthermore, the fact that the D enantiomer had the same effect as the natural peptide showed that interaction of the presequence with the import machinery was not dependent on chiral properties of the presequence.

Amino Acids↗
PubMed2001

Mutagenesis and computer modelling approach to study determinants for recognition of signal peptides by the mitochondrial processing peptidase.

Determinants for the recognition of a mitochondrial presequence by the mitochondrial processing peptidase (MPP) have been investigated using mutagenesis and bioinformatics approaches. All plant mitochondrial presequences with a cleavage site that was confirmed by experimental studies can be grouped into three classes. Two major classes contain an arginine residue at position -2 or -3, and the third class does not have any conserved arginines. Sequence logos revealed loosely conserved cleavage motifs for the first two classes but no significant amino acid conservation for the third class. Investigation of processing determinants for a class III precursor, Nicotiana plumbaginifolia F1beta precursor of ATP synthase (pF1beta), was performed using a series of pF1beta presequence mutants and mutant presequence peptides derived from the C-terminal portion of the presequence. Replacement of -2 Gln by Arg inhibited processing, whereas replacement of either the most proximally located -5 Arg or -15 Arg by Leu had only a low inhibitory effect. The C-terminal portion of the pF1beta presequence forms a helix-turn-helix structure. Mutations disturbing or prolonging the helical element upstream of the cleavage site inhibited processing significantly. Structural models of potato MPP and the C-terminal pF1beta presequence peptide were built by homology modelling and empirical conformational energy search methods, respectively. Molecular docking of the pF1beta presequence peptide to the MPP model suggested binding of the peptide to the negatively charged binding cleft formed by the alpha-MPP and beta-MPP subunits in close proximity to the H111XXE114H115X(116-190)E191 proteolytic active site on beta-MPP. Our results show for the first time that the amino acid at the -2 position, even if not an arginine, as well as structural properties of the C-terminal portion of the presequence are important determinants for the processing of a class III precursor by MPP.

Amino Acid Sequence↗
PubMed2000

Rapid degradation of the presequence of the f1beta precursor of the ATP synthase inside mitochondria.

We have investigated the fate of the presequence of an overexpressed protein derived from the precursor of the F(1)beta subunit of ATP synthase after import and processing in mitochondria. Our studies revealed a rapid degradation of the presequence inside mitochondria catalysed by matrix-located protease(s). In contrast, the mature portion of the precursor was not degraded. This is the first experimental evidence of the rapid degradation of a mitochondrial presequence in organello after in vitro import and processing.

Blotting, Western↗
PubMed2000

Studies on the topology of the protein import channel in relation to the plant mitochondrial processing peptidase integrated into the cytochrome bc1 complex.

The mitochondrial processing peptidase (MPP) specifically cleaves N-terminal targeting signals from hundreds of nuclear-encoded, matrix-targeted precursor proteins. In contrast to yeast and mammals, the plant MPP is an integral component of the respiratory cytochrome bc1 complex. The topology of the protein import channel in relation to MPP/bc1 in plants was studied using chimeric precursors containing truncated cytochrome b2 (cyt b2) proteins of 55-167 residues in length, fused to dihydrofolate reductase (DHFR). The DHFR domain could be tightly folded by methotrexate (MTX), generating translocation intermediates trapped in the import channel with only the cyt b2 pre-sequence/mature domain protruding into the matrix. Spinach and soybean mitochondria imported and processed unfolded precursors. MTX-folded intermediates were not processed in spinach but the longest (1-167) MTX-folded cyt b2-DHFR construct was processed in soybean, while yeast mitochondria successfully processed even shorter MTX-folded constructs. The MTX-folded precursors were cleaved with high efficiency by purified spinach MPP/bc1 complex. We interpret these results as indicating that the protein import channel is located distantly from the MPP/bc1 complex in plants, and that there is no link between protein translocation and protein processing.

Biological Transport↗
PubMed2000

[Malignant non-Hodgkin's lymphoma presenting with arrhythmia and conduction defects. Report of 2 cases].

Primary cardiac lymphoma is very rare. Secondary localisations are more common, observed in 15 to 30% of autopsy series. Clinical symptoms of cardiac involvement are rare, explaining the usual post-mortem diagnosis. The presentation of cardiac involvement by arrhythmias and conduction defects is very uncommon. The authors report two cases, the first of a 35 year old man in whom primary cardiac lymphoma presented with ventricular tachycardia complicated secondarily by complete atrioventricular block (AVB) with pseudo-inferior wall infarction. The second case was a 37 year old man with a cutaneous T cell lymphoma in whom complete AVB was the first sign of a secondary cardiac localisation of his disease. The finding of cardiac lymphoma should lead to aggressive chemotherapy as soon as possible.

Adult↗
PubMed1999

Chemical cleavage of the overexpressed mitochondrial F1beta precursor with CNBr: a new strategy to construct an import-competent preprotein.

We have isolated a soluble import-competent 15 kDa N-terminal fragment of the overexpressed Nicotiana plumbaginifolia F1beta precursor of the ATP synthase (N15pF1beta). The isolation was achieved after chemical cleavage, with CNBr, of the insoluble precursor collected in inclusion bodies, followed by purification of the fragment using ion-exchange chromatography. The purity of the final product was estimated to be more than 99%. N15pF1beta contained a presequence of 54 amino acid residues (except for the N-terminal methionine residue) and 82 N-terminal residues of the mature protein. N15pF1beta was shown to be imported into isolated potato tuber mitochondria and to be processed by the isolated mitochondrial processing peptidase (MPP) integrated into the cytochrome bc1 complex of the respiratory chain. Addition of N15pF1beta at micromolar concentrations resulted in the inhibition of import of F1beta precursor and alternative oxidase precursor, synthesized in vitro, into isolated mitochondria as well as the processing of these precursors catalysed by the isolated MPP-bc1 complex. N15pF1beta conjugated via a biotin link to avidin blocked import sites even after the reisolation of mitochondria and inhibited the import of the mitochondrial precursors, indicating that it can be used as a substrate for the generation of a stable translocation intermediate. Our results present a novel procedure for the production of an N-terminal fragment of the F1beta precursor that contains all information necessary for mitochondrial targeting and processing and that can be used for structural and functional studies of the mitochondrial protein import system. This procedure has a general value because it can be used for the production of chemical quantities of any mitochondrial import substrate and presequence peptide.

Avidin↗
PubMed1999

Interaction of mitochondrial presequences with DnaK and mitochondrial hsp70.

Mitochondrial heat shock protein 70 (mt-hsp70) functions as a molecular chaperone in mitochondrial biogenesis. The chaperone in co-operation with its co-proteins acts as a translocation motor pulling the mitochondrial precursor into the matrix. Mt-hsp70s are highly conserved when compared to the bacterial hsp70 homologue, DnaK. Here we have used DnaK as a model to study the interaction of mitochondrial presequences with mt-hsp70 applying a DnaK-binding algorithm, computer modeling and biochemical investigations. DnaK-binding motifs have been analysed on all available, statistically relevant mitochondrial presequences found in the OWL database by running the algorithm. A total of 87 % of mammalian, 97 % of plant, 71 % of yeast and 100 % of Neurospora crassa presequences had at least one DnaK binding site. Based on the prediction, five 13-mer presequence peptides have been synthesized and their inhibitory effect on the molecular chaperone (DnaK/DnaJ/GrpE) assisted refolding of luciferase has been analysed. The peptide with the highest predicted binding likelihood showed the strongest inhibitory effect, whereas the peptide with no predicted binding capacity showed no inhibitory effect. A 3D structure of the pea mt-hsp70 has been constructed using homology modeling. The binding affinities of the 13-mer presequence peptides and additional control peptides to DnaK and pea mt-hsp70 have been theoretically estimated by calculating the buried hydrophobic surface area of the peptides docked to DnaK and to the mt-hsp70 structural model. These results suggest that mitochondrial presequences interact with the mt-hsp70 during or after mitochondrial protein import.

Amino Acid Sequence↗
PubMed1999

Gonadal steroids promote glial differentiation and alter neuronal morphology in the developing hypothalamus in a regionally specific manner.

One of the more striking sexual dimorphisms in the adult brain is the synaptic patterning in some hypothalamic nuclei. In the arcuate nucleus (ARC) males have twice the number of axosomatic and one-half the number of axodendritic spine synapses as females. The opposite pattern is observed in the immediately adjacent ventromedial nucleus (VMN). In both cases, early exposure to testosterone dictates adult dimorphism, but the exact timing, mechanism, and site of steroid action remain unknown. Astrocytes also exhibit sexual dimorphisms, and their role in mediating neuronal morphology is becoming increasingly evident. Using Golgi-Cox impregnation to examine neuronal morphology and glial fibrillary acidic protein immunoreactivity (GFAP-IR) to characterize astrocytic morphology, we compared structural differences in dendrites and astrocytes from the ARC and VMN in postnatal day 2 rat pups from four hormonally different groups. Consistent with previous observations, testosterone exposure induced a rapid and dramatic stellation response in ARC astrocytes. Coincident with this change in astrocytic morphology was a 37% reduction in the density of dendritic spines on ARC neurons. In contrast, astrocytes in the VMN were poorly differentiated and did not respond to testosterone exposure, nor were there any changes in neuronal dendrite spine density. However, VMN neurons exposed to testosterone had almost double the number of branches compared with that in controls. These data suggest that the degree of maturation and the differentiation of hypothalamic astrocytes in vivo are correlated with the ability of neurons to sprout branches or spines in response to steroid hormones and may underlie regionally specific differences in synaptic patterning.

Animals↗
PubMed1999

Signals required for the import and processing of the alternative oxidase into mitochondria.

The critical residues involved in targeting and processing of the soybean alternative oxidase to plant and animal mitochondria was investigated. Import of various site-directed mutants into soybean mitochondria indicated that positive residues throughout the length of the presequence were important for import, not just those in the predicted region of amphiphilicity. The position of the positive residues in the C-terminal end of the presequence was also important for import. Processing assays of the various constructs with purified spinach mitochondrial processing peptidase showed that all the -2-position mutants had a drastic effect on processing. In contrast to the import assay, the position of the positive residue could be changed for processing. Deletion mutants confirmed the site-directed mutagenesis data in that an amphiphilic alpha-helix was not the only determinant of mitochondrial import in this homologous plant system. Import of these constructs into rat liver mitochondria indicated that the degree of inhibition differed and that the predicted region of amphiphilic alpha-helix was more important with rat liver mitochondria. Processing with a rat liver matrix fraction showed little inhibition. These results are discussed with respect to targeting specificity in plant cells and highlight the need to carry out homologous studies and define the targeting requirements to plant mitochondria.

Amino Acid Sequence↗
PubMed1999

Molecular genetics of gastrointestinal malignancies.

OBJECTIVES: To review the genetic model of colon cancer formation and the primary advances and clinical relevance of molecular genetics of pancreatic cancer and other gastrointestinal malignancies. DATA SOURCES: Research studies, review articles, and textbook chapters. CONCLUSIONS: Genetic discoveries are influencing the screening and treatment of colorectal cancer and other gastrointestinal malignancies by better targeting chemoprevention and treatment in high-risk and molecular-distinct patient populations. IMPLICATIONS FOR NURSING PRACTICE: A basic knowledge of cancer genetics and the latest genetic discoveries will assist oncology nurses in patient teaching, counseling, and care.

Adenomatous Polyposis Coli↗
PubMed1999

Integration of the mitochondrial-processing peptidase into the cytochrome bc1 complex in plants.

The plant mitochondrial cytochrome bc1 complex, like nonplant mitochondrial complexes, consists of cytochromes b and c1, the Rieske iron-sulfur protein, two Core proteins, and five low-molecular mass subunits. However, in contrast to nonplant sources, the two Core proteins are identical to subunits of the general mitochondrial processing peptidase (MPP). The MPP is a fascinating enzyme that catalyzes the specific cleavage of the diverse presequence peptides from hundreds of the nuclear-encoded mitochondrial precursor proteins that are synthesized in the cytosol and imported into the mitochondrion. Integration of the MPP into the bc1 complex renders the bc1 complex in plants bifunctional, being involved both in electron transport and in protein processing. Despite the integration of MPP into the bc1 complex, electron transfer as well as translocation of the precursor through the import channel are independent of the protein-processing activity. Recognition of the processing site by MPP occurs via the recognition of higher-order structural elements in combination with charge and cleavage-site properties. Elucidation of the three-dimensional (3-D) structure of the mammalian cytochrome bc1 complex is highly useful for understanding of the mechanism of action of MPP.

Animals↗
PubMed1999

The precursor of the F1beta subunit of the ATP synthase is covalently modified upon binding to plant mitochondrial.

We present evidence for a unique covalent modification of a nuclear-encoded precursor protein targeted to plant mitochondria. We investigated the early events of in vitro import for the mitochondrial precursor of the ATP synthase F1beta subunit from Nicotiana plumbaginifolia (pF1beta) into plant mitochondria. When pF1beta of 59 kDa was incubated with mitochondria isolated from different higher-plant species, a band of 61 kDa was generated. The 61 kDa protein was a covalently modified form of the 59 kDa pF1beta. The modification was dependent on the 25 amino acid long N-terminal region of the presequence of pF1beta. The modification was catalysed by an enzyme located in the outer mitochondrial membrane which was specific for higher plants and could not be washed off from the membrane by urea, KCl or EDTA. The modification was ATP- and Ca(2+)-dependent, but it was not affected by inhibitors of protein kinases. No inhibition of the modification was observed with phosphatase, methylation or acylation inhibitors. The modification occurs prior to translocation through the mitochondrial outer membrane. Inhibition of the modification process does not affect the import of the precursor protein, hence precursor modification was not a prerequisite for import. Both the modified and the unmodified pF1beta proteins were strongly associated with the mitochondrial outer membrane.

Adenosine Triphosphate↗
PubMed1998

Inhibition of protein import into mitochondria by amphiphilic cations: potential targets and mechanism of action.

In this paper we describe for the first time the inhibitory effect of three amphiphilic cations, trifluoperazine, propranolol and dibucaine on mitochondrial protein import. The amphiphilic cations did not affect binding of mitochondrial precursor proteins to mitochondria. Import into mitoplasts was affected in a similar manner to intact mitochondria, indicating that the protein import machinery of the inner membrane of mitochondria was responsible for the observed effect. At concentrations which completely inhibited protein import, the amphiphilic cations did not affect the membrane potential (DeltaPsi) across the inner membrane. The inhibitory potency of amphiphilic cations reflects their lipid/water partition coefficient and relatively high concentrations of the drugs were required for complete inhibition, hence we propose that the mechanism of protein import inhibition by amphiphilic cations is due to membrane perturbing effects. We discuss the implications of our findings in view of the possible connection between various inner mitochondrial membrane channels and the protein import pore.

Cations↗
PubMed1998

A matrix-located processing peptidase of plant mitochondria.

Nuclear-encoded mitochondrial precursor proteins are proteolytically processed inside the mitochondrion after import. The general mitochondrial processing activity in plant mitochondria has been shown to be integrated into the cytochrome bc1 complex of the respiratory chain. Here we investigate the occurrence of an additional, matrix-located processing activity by incubation of the precursors of the soybean mitochondrial proteins, alternative oxidase, the FAd subunit of the ATP synthetase and the tobacco F1 beta subunit of the ATP synthase, with the membrane and soluble components of mitochondria isolated from soybean cotyledons and spinach leaves. A matrix-located peptidase specifically processed the precursors to the predicted mature form in a reaction which was sensitive to orthophenanthroline, a characteristic inhibitor of mitochondrial processing peptidase (MPP). The specificity of the matrix peptidase was illustrated by the inhibition of processing of the alternative oxidase precursor in both soybean and spinach matrix extracts upon altering a single amino acid residue in the targeting presequence (-2 Arg to Gly). Additionally, there was no evidence for general proteolysis of precursor proteins incubated with the matrix. The purity of the matrix fractions was ascertained by spectrophotometric and immunological analyses. The results demonstrate that there is a specific processing activity in the matrix of soybean and spinach in addition to the previously well characterized membrane-bound MPP integrated into the cytochrome bcl complex of the respiratory chain.

Cell Nucleus↗
PubMed1998

Mitochondrial protein import in plants. Signals, sorting, targeting, processing and regulation.

Mitochondrial biogenesis requires a coordinated expression of both the nuclear and the organellar genomes and specific intracellular protein trafficking, processing and assembly machinery. Most mitochondrial proteins are synthesised as precursor proteins containing an N-terminal extension which functions as a targeting signal, which is proteolytically cleaved off after import into mitochondria. We review our present knowledge on components and mechanisms involved in the mitochondrial protein import process in plants. This encompasses properties of targeting peptides, sorting of precursor proteins between mitochondria and chloroplasts, signal recognition, mechanism of translocation across the mitochondrial membranes and the role of cytosolic and organellar molecular chaperones in this process. The mitochondrial protein processing in plants is catalysed by the mitochondrial processing peptidase (MPP), which in contrast to other sources, is integrated into the bc1 complex of the respiratory chain. This is the most studied component of the plant import machinery characterised to date. What are the biochemical consequences of the integration of the MPP into an oligomeric protein complex and how are several hundred presequences of precursor proteins with no sequence similarities and no consensus for cleavage, specifically cleaved off by MPP? Finally we will address the emerging area of the control of protein import into mitochondria.

Amino Acid Sequence↗
PubMed1998

Feature-extraction from endopeptidase cleavage sites in mitochondrial targeting peptides.

Cleavage sites in nuclear-encoded mitochondrial protein targeting peptides (mTPs) from mammals, yeast, and plants have been analysed for characteristic physicochemical features using statistical methods, perceptrons, multilayer neural networks, and self-organizing feature maps. Three different sequence motifs were found, revealing loosely defined arginine motifs with Arg in positions -10, -3, and -2. A self-organizing feature map was able to cluster these three types of endopeptidase target sites but did not identify any species-specific characteristics in mTPs. Neural networks were used to define local sequence features around precursor cleavage sites.

Amino Acid Sequence↗
PubMed1998

Histologic effect of diode laser sclerostomy in human cadaver eyes.

BACKGROUND AND OBJECTIVES: To study tissue effects and thresholds of efficacy in producing a full-thickness scleral fistula in human eyes obtained from cadavers. The effect of laser sclerostomies created with indocyanine green (ICG) was also evaluated. MATERIALS AND METHODS: Ab externo laser sclerostomies were produced in 12 fresh human eyes obtained from cadavers using a 200-micron diameter fiber optic connected to a diode laser system. Power settings were 500, 750, 1000, 1250, 1500, and 2000 mW with a constant duration of 100 and 200 ms. The same diode laser settings were repeated in the tissues injected with ICG. RESULTS: The laser sclerostomies were associated with heat coagulation damage adjacent to the burn margins, with disruption of stromal collagen. Tissue damage was greater at higher power and longer duration. Scleral injection of ICG prior to laser sclerostomy did not enhance laser penetration. CONCLUSION: The diode laser can create a sclerostomy in human sclera with an optimum level of 1500 mW and 100 ms. ICG did not significantly enhance the ease of penetration or reduce the association thermal damage to the sclera.

Coloring Agents↗