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F Eckstein

Publications and source records attributed to F Eckstein.

At least 217 records · Page 12Linked to original sources
PubMed1993

The importance of being ribose at the cleavage site in the Tetrahymena ribozyme reaction.

The ribozyme derived from the intron of Tetrahymena thermophila pre-rRNA catalyzes a site-specific endonuclease reaction with both RNA and DNA oligonucleotides. The total transition-state stabilization by the ribozyme, encompassing the binding and chemical steps, is 4.8 kcal/mol greater with a single ribose at the cleavage site relative to the all-deoxyribose substrate. Here we show that this effect is specific to the chemical transition state, with a contribution of only approximately 0.7 kcal/mol toward binding. Substrates with a series of 2'-substituents, -OH(ribo), -F2 (2',2'-difluoro-2'-deoxyribo), F(2'-fluoro-2'-deoxyribo), and -H(deoxyribo), follow a linear free energy relationship between the rate of the chemical step of the ribozyme-catalyzed reaction and the pK(a) of the leaving group, with slope beta leaving group approximately -0.8. Because proton donation to the 3'-oxygen atom from a general acid of the ribozyme would be expected to render the rate insensitive to the pK(a) of the leaving group, it is suggested that this ribozyme does not employ general acid catalysis. The 2'-OCH3 (2'-methoxy-2'-deoxyribo) substituent does not follow this correlation, apparently due to steric hindrance within the active site. The rate of cleavage of the 2'-substituted substrates by the ribozyme follows the order 2'-F2 > -F > -H, suggestive of an inductive effect, i.e., acceleration of the reaction by electron-withdrawing groups. The 2'-OH group provides the largest transition-state stabilization. Because of uncertainty in the relative effect of the 2'-OH and 2'-H substituents on the pK(a) of the neighboring 3'-oxygen leaving group, we do not discount the possibility of interactions between the 2'-hydroxyl group and the ribozyme that further enhance reactivity. Nevertheless, the 2'-OH effect can be explained at least partially by an intramolecular hydrogen bond to an incipient oxyanion at the neighboring 3'-position. This oxyanion is forming as the phosphodiester bond is breaking, explaining why the stabilization is specific to the transition state. Analogous differential hydrogen bonding might be widely used by enzymes to achieve selective transition-state stabilization.

Animals↗
PubMed1993

Hammerhead ribozymes: importance of stem-loop II for activity.

The activity of several hammerhead ribozyme constructs with constant lengths of stems I and III of 5 nt each but with variously shortened stems II is reported. Stems with 2 bp rather than the conventional 4 bp show essentially unaltered catalytic activity, independent of the composition of the tetraloop. Further reduction in size to 1 bp or 0 bp decreases activity drastically. Inversion of the G10.1.C11.1 bp next to the invariant core leads to a loss in activity, even when the stem consists of 4 bp. Thus, the minimal structural requirement for stem-loop II is a 2-bp stem with a conserved G.C bp. The reduction in catalytic activity is predominantly a result of a decrease of catalytic constant kcat, whereas Km is only slightly affected. Thus, the structural requirement for optimal activity in these constructs where the chemical-cleavage step is rate limiting is determined by the stabilization of the transition state.

Base Sequence↗
PubMed1993

Physiological incongruity of the humero-ulnar joint: a functional principle of optimized stress distribution acting upon articulating surfaces?

Investigations into the distribution of subchondral bone density in the human elbow have suggested that the geometry of the trochlear notch deviates from a perfect fit with the trochlea, and that the load is transmitted ventrally and dorsally rather than through the centre of the humero-ulnar joint. We therefore decided to make a quantitative assessment of the degree of incongruity between the two components in 15 human specimens (age distribution 60 to 93 years) with different types of joint surface. Polyether casts of the joint cavity were prepared under loads of 10, 40, 160 and 640 N. The thickness of the casts was then measured at 50 predetermined points, and an area distribution of the width of the joint space represented in a two-dimensional template of the trochlear notch. The reproducibility of this procedure was tested by image analysis. At a load of 10 N, only a narrow space was present ventrally and dorsally in the joint, but in the depths of the trochlear notch a width of 0.5 to 1 mm was recorded in the centre, and up to 3 mm as its medial and lateral edges. Specimens with continuous articular cartilage showed a lower degree of incongruity than those with a divided articular surface. As the load was increased to 640 N, however, the original incongruity between the articular surfaces disappeared almost completely.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1993

Cartilage degeneration in the human patellae and its relationship to the mineralisation of the underlying bone: a key to the understanding of chondromalacia patellae and femoropatellar arthrosis?

According to the literature subchondral bone plays a significant role in the transmission of load through joints and in the pathogenesis of osteoarthrosis. Therefore the degeneration of the articular cartilage was investigated in the patellae from 30 dissecting-room specimens and of 20 patients, previously submitted to arthroscopy, and subchondral mineralisation of their underlying bone was at the same time assessed by means of CT osteoabsorptiometry. Lateral cartilage lesions were localised over highly mineralised subchondral bone; these appear to be due to long-term stress. They were mainly found in the older specimens and showed a high rate of progression with increasing age. Medially localised cartilage lesions, on the other hand, were situated in a transitional region between moderate and slight subchondral mineralisation; they may be caused by infrequent stress peaks and by shear stress in the articular cartilage, the very medial part of the joint being deprived of mechanical stimulation for much of the time. These lesions were to be found predominantly in the younger specimens and showed little progress with advancing age. Patients with lateral cartilage degeneration exhibited higher, patients with medial chondromalacia patellae lower mineralisation than normals. Their density patterns therefore indicate a different mechanical pathogenesis of the cartilage lesions in the lateral and medial facet. It could be shown that CT osteoabsorptiometry allows an assessment of the mechanical situation, present in individual femoro-patellar joints, and that this situation is highly relevant for the pathogenesis of patellar cartilage degeneration.

Absorptiometry, Photon↗
PubMed1993

Chemically modified RNA: approaches and applications.

The modification of phosphate into phosphorothioate internucleotidic linkages in various RNAs and their usefulness in identifying phosphate positions essential for function are described. Several modifications of the 2'-hydroxyl group of the ribose, particularly the replacement by fluorine atoms and amino groups, is discussed. These studies have been concentrated on hammerhead ribozymes in order to determine hydroxyl groups important for the catalytic activity. In addition these derivatives have been instrumental in rendering ribozymes more stable toward nucleases.

Base Sequence↗
PubMed1993

[Areas of contact in human humero-ulnar joints as a function of pressure, their connection through subchondral mineralization and joint surface morphology of the incisura trochlearis].

Evaluation of the stress distribution in joints can be obtained directly from contact areas and pressure forces, and also indirectly from the functional adaptation of the connective tissues. Therefore 8 human humero-ulnar joints, fixed in formalin, were examined for size and position of contact areas (polyether casting/Vidas image analyser) and their dependence upon the joint forces (Zwick material testing machine). The distribution of subchondral mineralisation was assessed, using CT osteoabsorptiometry. Depending on the joint force, the contact areas increase from about 10% of the total surface (20 N) to approximately 60% (1280 N). With weak forces they are localised ventrally and dorsally in the joint, with more powerful forces they run together centrally. With a divided articular surface they join at about 160-640 N, with a continuous surface, at about 40-80 N. Divided joint surfaces show a bicentric mineralisation pattern of the subchondral bone with ventral and dorsal maxima. Continuous surfaces, on the other hand, usually show central maxima. Both the mineralisation pattern and the position of the contact areas suggest a physiological incongruity of the humero-ulnar joint surfaces, which vanishes with increasing pressure due to viscoelastic deformation of articular cartilage and subchondral bone. More marked incongruity is postulated for the divided surfaces than for the others. The consequent peripheral transmission of pressure seems to involve a functional principle, which, present in several human joints, leads to both optimal distribution of the stress and better nutrition of the articular cartilage.

Aged↗
PubMed1993

[Mechanical stress and subchondral mineralization of the human elbow joint. A CT-osteoabsorptiometric study].

We examined 21 elbow joints (age 60-97 years; mean 79 years) that had been fixed in 3.7% formalin. The articular surface of the trochlear notch was completely divided in 10 cases, undivided in 5, and only partially divided in the medial aspect of the joint surface in 6. Subchondral mineralisation was investigated by means of CT osteoabsorptiometry (Müller-Gerbl et al. 1989) on sections 2 mm apart with a Siemens Somatom SF CT scanner. Two-dimensional reconstruction of the surface distribution was achieved by dividing it into 8 Hounsfield ranges (< 300 HU > 900 HU) and using a joint template. Higher values for the subchondral mineralisation are usually found in the distal and medial parts of the joint, indicating greater stress there. Whereas the radial head and the capitulum show a distribution pattern with mineralisation falling off more or less concentrically from the centre outwards, the trochlea and trochlear notch are often poorly mineralised in the centre. There is one maximum on the proximal and one on the distal aspect of the joint surface. Such "two-peak" distribution patterns are more often found when there is complete central division of the articular cartilage of the trochlear notch than when it is continuous. In the latter case, a more central localisation of higher subchondral density is common. In the humero-radial part of the joint, the mineralisation pattern suggests central pressure transmission, probably a result of the fovea capitis radii being slightly less curved than the opposing capitulum.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorptiometry, Photon↗
PubMed1993

Effects of 3'-azido-3'-deoxythymidine metabolites on simian virus 40 origin-dependent replication and heteroduplex repair in HeLa cell extracts.

Although the capacity of 3'-azido-3'-deoxythymidine (AZT) triphosphate, an active metabolite of the antiviral agent zidovudine (AZT), to inhibit polymerization by a variety of purified DNA polymerases has been described, it is important to understand its effect on replication by the more complex protein assemblies responsible for DNA replication in human cells. In the present study, we have determined the effects of AZT metabolites on the efficiency of simian virus 40 origin-dependent bidirectional replication of double-stranded DNA in extracts of human HeLa cells. Replication was inhibited by AZT diphosphate and AZT triphosphate, but only at concentrations exceeding those thought to be present in vivo. However, replication was inhibited by AZT monophosphate at concentrations previously reported to accumulate in human cells cultured in the presence of AZT, suggesting that AZT monophosphate may contribute to cytotoxicity by inhibiting chromosomal replication. In an attempt to determine whether AZT treatment could have longer term mutagenic effects on cells, we also determined the effects of these AZT derivatives on replication fidelity and on the efficiency of repair of DNA substrates containing single-base mismatches. Despite the ability of a normal deoxynucleoside monophosphate to reduce the fidelity of DNA replication, presumably by reducing exonucleolytic proofreading of errors, neither the mono-, di-, nor triphosphate form of AZT reduced base substitution fidelity when present in replication reactions. Similarly, the efficiency of repair of DNA substrates containing single-base mismatches was unaffected by these compounds. However, replication fidelity was affected by perturbations in relative and absolute concentrations of deoxynucleoside triphosphate substrates similar to those reported to occur in AZT-treated cells. Thus, AZT treatment could potentially be mutagenic in vivo via reduced replication fidelity resulting from alterations in deoxynucleoside triphosphate pools.

Antiviral Agents↗
PubMed1992

2'-Fluoro- and 2'-amino-2'-deoxynucleoside 5'-triphosphates as substrates for T7 RNA polymerase.

2'-Fluoro- and 2'-amino-2'-deoxynucleoside 5'-triphosphates have been investigated as substrates for T7 RNA polymerase. Michaelis-Menten kinetic parameters are reported for the incorporation of 2'-fluoro-2'-deoxyuridine, 2'-fluoro-2'-deoxycytidine, and 2'-amino-2'-deoxyuridine into runoff transcripts. The 2'-amino derivative of uridine is a better substrate than the 2'-fluoro derivative. Gel electrophoretic analysis shows that full-length transcripts with a length of 2500 nucleotides can be obtained with the analogues, although a considerable amount of shorter fragments accompanies the full-length product. In keeping with the kinetic analysis, the 2'-aminouridine triphosphate gives a cleaner product than the 2'-fluoro analogue. Transcription of two tRNA genes shows that such shorter templates can be transcribed to full-length products essentially without premature termination with any of the analogues.

Autoradiography↗
PubMed1992

Determinant nucleotides of yeast tRNA(Asp) interact directly with aspartyl-tRNA synthetase.

The interaction of wild-type and mutant yeast tRNA(Asp) transcripts with yeast aspartyl-tRNA synthetase (AspRS; EC 6.1.1.12) has been probed by using iodine cleavage of phosphorothioate-substituted transcripts. AspRS protects phosphates in the anticodon (G34, U35), D-stem (U25), and acceptor end (G73) that correspond to determinant nucleotides for aspartylation. This protection, as well as that in anticodon stem (C29, U40, G41) and D-stem (U11 to U13), is consistent with direct interaction of AspRS at these phosphates. Other protection, in the variable loop (G45), D-loop (G18, G19), and T-stem and loop (G53, U54, U55), as well as enhanced reactivity at G37, may result from conformational changes of the transcript upon binding to AspRS. Transcripts mutated at determinant positions showed a loss of phosphate protection in the region of the mutation while maintaining the global protection pattern. The ensemble of results suggests that aspartylation specificity arises from both protein-base and protein-phosphate contacts and that different regions of tRNA(Asp) interact independently with AspRS. A mutant transcript of yeast tRNA(Phe) that contains the set of identity nucleotides for specific aspartylation gave a phosphate protection pattern strikingly similar to that of wild-type tRNA(Asp). This confirms that a small number of nucleotides within a different tRNA sequence context can direct specific interaction with synthetase.

Aspartate-tRNA Ligase↗
PubMed1992

Activation of G proteins by (Rp) and (Sp) diastereomers of guanosine 5'-[beta-thio]triphosphate in hamster fibroblasts. Differential stereospecificity of Gi, Gs and Gp.

The effects of guanosine 5'-[beta-thio]triphosphate (GTP beta[S]) on G proteins have been examined in Chinese hamster lung fibroblasts (CCL39 line) permeabilized with alpha-toxin from Staphylococcus aureus. Although much less effective than guanosine 5'-[gamma-thio]triphosphate (GTP gamma[S]), both (Rp) and (Sp) diastereomers of GTP beta[S] were found to activate three G protein-mediated pathways: inhibition of forskolin-stimulated adenylate cyclase (mediated by Gi), potentiation of receptor-mediated activation of adenylate cyclase (mediated by Gs), and activation of phosphoinositide breakdown (mediated by Gp). Activation of Gi and Gs occurred above 3 microM-GTP beta[S], but activation of Gp only occurred above 100 microM-GTP beta[S]. Moreover, the order of effectiveness of the two diastereomers was not the same for the three G protein-mediated processes. Whereas both Gi and Gs were more effectively activated (about 5-fold) by (Sp)-GTP beta[S] than by (Rp)-GTP beta[S], Gp showed a marked preference for the (Rp) isomer. Indeed, (Rp)-GTP beta[S] induced the formation of inositol phosphates with a shorter latency and was a better competitor of GDP for binding to Gp than the (Sp) isomer. These results point to different guanine nucleotide-binding properties for Gi and Gs on the one hand and Gp on the other. At least two distinct Gp proteins, differing by their sensitivity to pertussis toxin, are present in CCL39 cells. Since pretreatment of cells with pertussis toxin completely suppressed the effects of (Rp)-GTP beta[S] on Gi, while only slightly attenuating its effects on Gp, we believe that it is the pertussis toxin-insensitive Gp which prefers the (Rp) isomer. Therefore (Rp)-GTP beta[S] may be a valuable tool for the selective activation and the biochemical characterization of this pertussis toxin-insensitive Gp.

Adenylate Cyclase Toxin↗
PubMed1992

Function of specific 2'-hydroxyl groups of guanosines in a hammerhead ribozyme probed by 2' modifications.

The importance of the 2'-hydroxyl group of several guanosine residues for the catalytic efficiency of a hammerhead ribozyme has been investigated. Five ribozymes in which single guanosine residues were substituted with 2'-amino-, 2'-fluoro-, or 2'-deoxyguanosine were chemically synthesized. The comparison of the catalytic activity of the three 2' modifications at a specific position allows conclusions about the functional role of the parent 2'-hydroxyl group. Substitutions of nonconserved nucleotides within the ribozyme caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, when either of the guanosines within the single-stranded loop between stem I and stem II of the ribozyme was replaced by 2'-deoxyguanosine or 2'-fluoro-2'-deoxyguanosine, the catalytic activities of the resulting ribozymes were reduced by factors of at least 150. The catalytic activities of the corresponding ribozymes containing 2'-amino-2'-deoxyguanosine substitutions at these positions, however, were both reduced by factors of 15. These effects resulted from decreases in the respective kcat values, whereas variations in the Km values were comparatively small. A different pattern of reactivity of the three 2' modifications was observed at the guanosine immediately 3' to stem II of the ribozyme. Whereas both 2'-deoxyguanosine and 2'-amino-2'-deoxyguanosine at this position showed catalytic activity similar to that of the unmodified ribozyme, the activity of the corresponding 2'-fluoro-2'-deoxyguanosine-containing ribozyme was reduced by a factor of 15. The implications of these substitution-specific reactivities on the functional role of the native 2'-hydroxyl groups are discussed.

Base Sequence↗
PubMed1992

Hammerhead ribozyme-mediated cleavage of the long terminal repeat RNA of human immunodeficiency virus type 1.

Three ribozymes targeted against different sites of the long terminal repeat RNA (LTR RNA) of human immunodeficiency virus type 1 cleaved a LTR RNA transcript 1,000 x less efficiently than corresponding short synthetic oligoribonucleotide substrates. Varying the stem lengths of the ribozyme resulted in changes of the catalytic efficiency. Almost no cleavage was observed for a ribozyme forming only 10 base pairs instead of 14 with the LTR RNA. Increasing the base pairs to 16 or elongation of the stem formed within the ribozyme revealed only small changes in kcat/Km. The influence of chemical modifications within the ribozyme on the cleavage of the LTR RNA was also examined. 2'-Fluorocytidine substitutions as well as four terminal phosphorothioate internucleotidic linkages influenced the catalytic efficiency of ribozymes only negligibly. However, substitution of uridine by 2'-fluorouridine resulted in a 5-fold decrease of kcat/Km. A ribozyme containing all these modifications revealed only a 7-fold lower catalytic efficiency but a markedly increased stability in cell culture supernatant. These results demonstrate that it is possible to increase the stability of ribozymes toward nucleases without a serious loss in catalytic efficiency.

Base Sequence↗
PubMed1992

Cloning and antisense oligodeoxynucleotide inhibition of a human homolog of cdc2 required in hematopoiesis.

Mechanisms triggering the commitment of pluripotent bone marrow stem cells to differentiated lineages such as mononuclear macrophages or multinucleated megakaryocytes are still unknown, although several lines of evidence suggested correlation between cholinergic signaling and hematopoietic differentiation. We now present cloning of a cDNA coding for CHED (cholinesterase-related cell division controller), a human homolog of the Schizosaccharomyces pombe cell division cycle 2 (cdc2)-like kinases, universal controllers of the mitotic cell cycle. Library screening, RNA blot hybridization, and direct PCR amplification of cDNA reverse-transcribed from cellular mRNA revealed that CHED mRNA is expressed in multiple tissues, including bone marrow. The CHED protein includes the consensus ATP binding and phosphorylation domains characteristic of kinases, displays 34-42% identically aligned amino acid residues with other cdc2-related kinases, and is considerably longer at its amino and carboxyl termini. An antisense oligodeoxynucleotide designed to interrupt CHED's expression (AS-CHED) significantly reduced the ratio between CHED mRNA and actin mRNA within 1 hr of its addition to cultures, a reduction that persisted for 4 days. AS-CHED treatment selectively inhibited megakaryocyte development in murine bone marrow cultures but did not prevent other hematopoietic pathways, as evidenced by increasing numbers of mononuclear cells. An oligodeoxynucleotide blocking production of the acetylcholine-hydrolyzing enzyme, butyrylcholinesterase, displayed a similar inhibition of megakaryocytopoiesis. In contrast, an oligodeoxynucleotide blocking production of the human 2Hs cdc2 homolog interfered with production of the human 2Hs cdc2 homolog interfered with cellular proliferation without altering the cell-type composition of these cultures. Therefore, these findings strengthen the link between cholinergic signaling and cell division control in hematopoiesis and implicate both CHED and cholinesterases in this differentiation process.

Amino Acid Sequence↗