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Biomedical subjects

J Engel

Publications and source records attributed to J Engel.

At least 55 records · Page 3Linked to original sources
PubMed1995

Diet restriction in rhesus monkeys lowers fasting and glucose-stimulated glucoregulatory end points.

Male rhesus monkeys (Macaca mulatta) of different age groups representing the species life span were fed ad libitum or a 30% reduced calorie diet over a 7-yr period. During the first 2-3 yr of this longitudinal study, glucose and insulin levels were not altered by diet restriction (DR). However, reductions in fasting blood glucose became apparent in DR animals after 3-4 yr. At the end of the 6th yr of study, glycated hemoglobin was measured, and intravenous glucose tolerance tests (IVGTTs) were conducted. Maximum glucose levels reached during IVGTTs increased with age but were lower in DR animals compared with controls. Several measures of the insulin response (baseline, maximum, and integrated areas under curve) increased with age and were lower in DR monkeys. With the exception of glycated hemoglobin, which was not different in monkeys subjected to DR, these findings confirm previous studies in rodents demonstrating that DR alters glucose metabolism and may be related to the antiaging action of this intervention.

Aging↗
PubMed1995

Synthesis and antitumor activity of two ifosfamide analogs with a five-membered ring.

Two ifosfamide (CAS 3778-73-2) analogs with a five-membered ring, i.e. the oxazaphospholidine derivatives 6 and 7, were synthesized and their cytotoxic activity in vitro, acute toxicity and antitumor activity in vivo determined in comparison with the oxazaphorinane ifosfamide 1. The observed low biological activity gives evidence that both, the six-membered oxazaphosphorinane ring and the two N-2-chloroethyl-side chains are necessary for the generation of the ultimate alkylator, i.e. the ifosfamide mustard 5.

Animals↗
PubMed1995

In vivo studies on the effects of memantine on dopamine metabolism in the striatum and n. accumbens of the rat.

The action of the amantadine derivative memantine on the dopamine metabolism in the striatum and the n. accumbens of the rat was studied in vivo with different voltammetrical techniques. It was shown by differential pulse voltammetry that memantine enhanced extracellular levels of dopamine (DA) in the striatum of the anaesthetized rat, whereas an increase of 3,4 dihydroxyphenylacetic acid (DOPAC) could be observed only under freely moving conditions using square wave voltammetry. Under chloral hydrate anaesthesia the effect of memantine on extracellular DOPAC levels in the striatum and the n. accumbens was compared with MK-801, a well-known non-competitive NMDA-antagonist. Memantine did not affect striatal DOPAC concentrations under these conditions whereas MK-801 reduced the DOPAC signal. In the n. accumbens memantine enhanced the levels of extracellular DOPAC, while after MK-801 the signal was only slightly different from control. These findings suggest that in addition to its NMDA receptor antagonism, memantine affects dopaminergic transmission also by other mechanisms.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1994

Fibritin encoded by bacteriophage T4 gene wac has a parallel triple-stranded alpha-helical coiled-coil structure.

The bacteriophage T4 late gene wac (whisker's antigen control) encodes a fibrous protein which forms a collar/whiskers complex. Whiskers function as a helper protein for the long tail fibres assembly and plays a role in regulating retraction of the long tail fibres in response to environmental conditions. In this work we show that expression of the cloned wac gene in Escherichia coli yields a protein oligomer of 53 nm length which we call fibritin, and which is able to complement gpwac T4 particles in vitro. CD spectroscopy of fibritin indicates a 90% alpha-helical content, and scanning calorimetry shows that the protein has several distinct domains. The analysis of the 486 amino acid sequence of fibritin reveals three structural components: a 408 amino acid region that contains 12 putative coiled-coil segments with a canonical heptad (a-b-c-d-e-f-g)n substructure where the "a" and "d" positions are preferentially occupied by apolar residues, and the N and C-terminal domains (47 and 29 amino acid residues, respectively) have no heptad substructure. The distribution of hydrophobic residues within heptads is more similar to a triple than to a double coiled-coil. The alpha-helical segments are separated by short "linker" regions, variable in length, that have a high proportion of glycine and proline residues. Each coiled-coil segment has, on the borders with linker regions, residues that are common to the N and C-terminal caps of the alpha-helices. Full-length and amino-terminally truncated fibritins can be reassembled in vitro after temperature-induced denaturation. Co-assembly of full-length fibritin and the N-terminal deletion mutant, as well as analytical centrifugation, indicates that the protein is a parallel triple-standard alpha-helical coiled-coil. Deletions of various N-terminal portions of fibritin did not block trimerisation but the mutant trimers are unable to bind to T4 particles. The last 18 C-terminal residues of fibritin are required for correct trimerisation of gpwac monomers in vivo. We propose that fibritin might serve as a convenient model for the investigation of folding and assembly mechanisms of alpha-fibrous proteins.

Amino Acid Sequence↗
PubMed1994

Synthesis and quantitative structure-activity relationships of anticonvulsant 2,3,6-triaminopyridines.

The synthesis of 2,3,6-triaminopyridine derivatives, representing a unique chemical structure for anticonvulsants, is described. The synthetic program was performed (a) to identify more potent analogs, (b) to determine structural properties controlling potency as well as neurotoxicity, and (c) to reduce the requirements for animal testing. As a result, besides other structural properties, the overall molecular lipophilicity (log k', octanol-coated column) explained changes in anticonvulsant potency and neurotoxicity. Mimicking the interaction of the amphiphilic triaminopyridines with biological membranes, NMR experiments in the presence of lecithin vesicles were conducted in order to measure the phospholipid-binding parameter log delta (1/T2). Replacement of log k' with log delta (1/T2) in the correlation analysis afforded a more significant equation describing the anticonvulsant activity of 21 derivatives.

Aminopyridines↗
PubMed1994

Sodium/calcium exchanger in olfactory receptor neurones of Xenopus laevis.

Ca2+ ions enter neurones through various types of calcium and cation channels. The mechanisms by which Ca2+ ions are spatially buffered and expelled from neurones have been studied considerable less. Using calcium imaging in conjunction with the patch clamp technique, we investigated the Na/Ca exchanger in olfactory neurones and found evidence for its localization on the dendrite. It is suggested that this tends to decouple increases in [Ca2+]i occurring in the transduction compartments of the cell from processes in the soma.

Animals↗
PubMed1994

Selective recognition of cyclic RGD peptides of NMR defined conformation by alpha IIb beta 3, alpha V beta 3, and alpha 5 beta 1 integrins.

The binding of purified fibrinogen receptor alpha IIb beta 3, vitronectin receptor alpha V beta 3, and fibronectin receptor alpha 5 beta 1 to their corresponding ligands in solid-phase binding assays was used to examine the inhibitory activity of various linear and cyclic penta- and hexapeptides of different conformation containing RGD or RAD sequences. Cyclic peptides with different defined backbone conformations were designed by introducing a single D-amino acid or a proline at different positions in the ring. The data were calibrated for alpha IIb beta 3 integrin incorporated into a planar lipid bilayer by a physical method (total internal reflection fluorescence microscopy) which yielded KD = 1.7 microM for a linear RGD peptide and KD = 0.03 microM for fibrinogen. With this integrin, three cyclic hexapeptides ([GRGDFL], [ARGDFV], [GRGDFV]) were 2-4-fold more inhibitory than the linear GRGDS peptide in solid-phase assays and showed similar inhibition as the fibrinogen ligand. Six peptides had the same or a 2-fold lower activity as the linear reference peptide, and three peptides were up to 7-fold less active. Replacement of Arg or Asp by their stereoisomers or Gly by Ala resulted in a 100-1000-fold reduction in activity. With the two other integrins, a single cyclic pentapeptide [RGDFV] was 10-fold more active (alpha V beta 3) or equal in activity (alpha 5 beta 1) to linear GRGDS, while all of the other cyclic peptides were moderately or distinctly less active. Changes in the RGD sequence caused a less dramatic reduction in binding strength for alpha V beta 3 and alpha 5 beta 1 than for alpha II beta 3. Inhibitory activity was compared with the distance between the C beta atoms of Arg and Asp residues as determined by NMR and indicated that the optimum distance is in the range of 0.75-0.85 nm for alpha IIb beta 3 and at or below 0.67 nm for alpha V beta 3 and alpha 5 beta 1. This indicates that alpha IIb beta 3 less sensitive to variations in the RGD backbone structure and can accommodate a larger distance than the integrins alpha V beta 3 and alpha 5 beta 1.

Amino Acid Sequence↗
PubMed1994

Conformational changes of the recombinant extracellular domain of E-cadherin upon calcium binding.

The cell-adhesion protein E-cadherin/uvomorulin exhibits a calcium-dependent homoassociation. The effect of Ca2+ on the extracellular fragment of E-cadherin was studied using the recombinant protein expressed in the baculovirus expression system. The recombinant and native fragment of E-cadherin were found to be similar by many biochemical criteria [Herrenknecht, K. & Kemler, R. (1993) J. Cell Sci. 17, 147-154]. A large and reversible conformational transition was observed upon Ca2+ depletion. A change from a rod-like structure, 22 nm in length, to a more globular assembly of the five subdomains became evident by electron-microscopical analysis. In the presence of Ca2+, the circular dichroic spectra indicated predominantly beta-structure but a more negative ellipticity was observed in the absence of Ca2+. The intrinsic tryptophan fluorescence decreased by 12% upon Ca2+ depletion. Both effects were used for calcium titrations which indicated calcium binding to several sites with average K(d) values of 45-150 microM. Cleavage of the protein fragment by trypsin occurred only at low Ca2+ concentrations and from the calcium-dependence of cleavage rates, a K(d) value of 24 microM was derived. The major site of cleavage was identified by partial sequencing to be located between the two putative calcium-binding sites in the third subdomain from the N-terminus. In agreement with earlier results with the native fragment, the recombinant protein did not associate in the presence or absence of Ca2+. We suggest the calcium-dependent homoassociation therefore depends on additional effects connected with the cell surface association of E-cadherin.

Amino Acid Sequence↗
PubMed1994

Changes in calcium and collagen IV binding caused by mutations in the EF hand and other domains of extracellular matrix protein BM-40 (SPARC, osteonectin).

Recombinant expression in a human kidney cell-line was used to prepare mutant human BM-40 with deletions including the N-terminal acidic domain, a central alpha-helical domain and the C-terminal EF hand domain. Two putative EF hand motifs were altered by point mutations. Only elimination of the whole EF hand domain or its single disulfide bond decreased production and secretion indicating that the C-terminal region of BM-40 is essential for correct folding. Deletions in the alpha-helical domain changed a single disulfide bond in this domain and caused oligomerization. Several mutations resulted in significant conformational changes as shown by CD spectroscopy and epitope analysis. Fluorescence titration demonstrated a single high-affinity (Kd = 0.08 microM) calcium-binding site with a low dissociation rate constant. A Glu to Lys mutation in the -Z position of the C-terminal EF hand motif and lack of a stabilizing disulfide bridge caused a 30 to 100-fold reduction in calcium affinity, while an Asp to Lys mutation in the X position had only a small effect. Deletions in the alpha-helical domain caused an even more dramatic reduction in calcium binding and abolished calcium-dependent binding of BM-40 to collagen IV. Both binding properties are critically dependent on a conformational interaction between the EF hand and the alpha-helical domain, which contains the collagen-binding site.

Amino Acid Sequence↗
PubMed1994

Characterization of type I procollagen N-proteinase from fetal bovine tendon and skin. Purification of the 500-kilodalton form of the enzyme from bovine tendon.

Procollagen N-proteinase (EC 3.4.24.14) is the enzyme that specifically cleaves the NH2-terminal propeptides from type I procollagen. Two forms of N-proteinase with apparent molecular sizes of 300 and 500 kDa were found in partially purified preparations from fetal bovine tendon extracts. The 500-kDa form of enzyme was purified 16,000-fold with a recovery of 8% from the extracts of the tendons by six purification steps. The purified enzyme was a neutral, Ca(2+)-dependent proteinase (5-10 mM) that was inhibited by metal chelators. The 500-kDa enzyme contained unreduced polypeptides of 58, 125, 170, and 190 kDa which were separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Electron microscopic study indicated that the enzyme molecules were generally globular and had diameters of 33 +/- 4 nm. Other properties of the 500-kDa enzyme were: 1) the Km for type I procollagen is 35 nM at pH 7.5 and 35 degrees C, and the kcat is 290 h-1; 2) the activation energy for reaction with type I procollagen is 10,050 cal mol-1; 3) the isoelectric point is 3.8; 4) the enzyme cleaves the NH2-terminal propeptides of type II procollagen as well as type I procollagen but not of type III procollagen; and 5) the enzyme specifically cleaves a -Pro-Gln- bond in the pro-alpha 1(I) chain and an -Ala-Gln- bond in the pro-alpha 2(I) chain. The bovine N-proteinase with a mass of 300 kDa was found to be similar to the 500-kDa enzyme and appeared to be a degraded form of the 500-kDa enzyme generated during purification. The N-proteinase from fetal bovine skin extracts also contained 300-kDa and 500-kDa enzyme forms.

Amino Acid Sequence↗
PubMed1994

Self-assembly of collagen I from a proband homozygous for a mutation that substituted serine for glycine at position 661 in the alpha 2(I) chain. Possible relationship between the effects of mutations on critical concentration and the severity of the phenotype.

Procollagen I was isolated from cultured skin fibroblasts from a proband who was homozygous for a mutation in the COL1A2 gene that substituted a serine codon for a glycine codon at position 661 of the alpha 2(I) chain. The procollagen I was cleaved to pCcollagen I by procollagen N-proteinase and the pCcollagen I was used as a substrate for assay of self-assembly of collagen I into fibrils. The mutated pCcollagen I was cleaved to collagen I by procollagen C-proteinase at the same rate as control pCcollagen I. However, self-assembly of the mutated collagen I had a lag period that was 15-fold greater than the lag period observed with normal collagen I under the same conditions. Also, self-assembly of the mutated collagen I had a propagation rate of about one-fourth of the propagation rate of normal collagen I. In addition, the critical concentration for fibril assembly was slightly increased. Rotary shadowing electron microscopy of the mutated procollagen I did not reveal any increased flexibility of the triple helix as was seen previously with two mutated procollagens I in which there were substitutions of cysteine for glycine residues in the alpha 1(I) chain (Vogel, B. E., Doelz, R., Kadler, K. E., Hojima, Y., Engel, J., and Prockop, D. J. (1988) J. Biol. Chem. 263, 19249-19255; Lightfoot, S. J., Holmes, D. F., Brass, A., Grant, M. E., Byers, P. H., and Kadler, K. E. (1992) J. Biol. Chem. 267, 25521-25528). However, morphometric analysis by dark-field light microscopy and electron microscopy showed that the fibrils formed from the mutated collagen I appeared thicker in diameter than the fibrils formed from the normal collagen I. Comparison of the results with similar data on four mutated procollagens previously studied raised the possibility that mutations which markedly increase the critical concentration of fibril assembly produce more severe phenotypes than mutations which change other parameters of fibril assembly.

Amino Acid Sequence↗
PubMed1994

The thrombospondin-like chains of cartilage oligomeric matrix protein are assembled by a five-stranded alpha-helical bundle between residues 20 and 83.

The N-terminal fragment of rat cartilage oligomeric matrix protein (COMP), comprising residues 20-83, was over-expressed in E. coli and purified under non-denaturing conditions. The fragment forms pentamers similar to the assembly domain of the native protein. Its five chains can be covalently linked in vitro by oxidation of cysteines 68 and 71. The fragment adopts a predominantly alpha-helical structure as judged by circular dichroism spectroscopy. On the basis of these findings we propose the model of a five-stranded alpha-helical bundle for the assembly domain of COMP. The studied sequence is conserved in thrombospondins 3 and 4 thus raising the possibility that these proteins are also pentamers.

Amino Acid Sequence↗
PubMed1994

Characterization of native laminin from bovine kidney and comparison with other laminin variants.

A comprehensive characterization of laminin isoforms requires access to native preparations of laminins of a defined subunit composition. For this purpose an optimized isolation procedure was developed and shown to be broadly applicable to normal mammalian tissues. The protocol does in addition yield side fractions highly enriched in collagens XII and XIV. The major laminin purified from bovine kidney is indistinguishable from mouse Engelbreth-Holm-Swarm (EHS) tumor laminin in electron microscopy, but contains an A chain that migrates in a position intermediate to the Ae and the Am chains on SDS/PAGE. Antisera raised against mouse EHS-tumor laminin crossreact with B chains, but not with the A chain, of kidney laminin. Further, this A chain is not recognized by antisera raised against the Am chain. Laminins from heart and kidney both contain a significant subpopulation with a 190-kDa polypeptide identified as the B1s chain. The Am-containing laminins from heart and placenta differ morphologically from the Ae-containing EHS laminin in having one short arm that does not have the characteristic globule-rod-globule appearance. Further, the Am-containing laminins show a significantly higher thermal stability of the coiled-coil alpha-helical region in the long arm than does Ae-containing EHS laminin, indicating that certain combinations of laminin chains interact more strongly than others.

Animals↗
PubMed1994

NGF increases [Ca2+]i in regenerating mature oligodendroglial cells.

The effect of NGF on [Ca2+]i of mature regenerating oligodendroglial cells was investigated by measuring fluo-3 fluorescence. NGF caused transient increases in [Ca2+]i, which could be inhibited by anti-NGF antibody. The rise in [CA2+]i was in part due to influx of extracellular Ca2+ since it was markedly attenuated in Ca2+-free solution. It also depended on release of Ca2+ from intracellular stores as tested by prior depletion with cyclopiazonic acid. These results support a role for Ca2+ in the effects of NGF on oligodendroglial cells.

Animals↗
PubMed1994

Asymmetric interictal glucose hypometabolism and cognitive performance in epileptic patients.

OBJECTIVE: To relate hemispheric metabolic asymmetries to cognitive performance in patients with unilateral mesial temporal lobe epilepsy. DESIGN: Asymmetrical cerebral glucose metabolisms on interictal fludeoxyglucose F 18 positron emission tomograms (FDG-PET) were correlated with cognitive measures. Analyses included partial correlations that controlled for the correlation between metabolic asymmetries of the lateral temporal lobe and other brain regions and the correlation of IQ scores with affected cognitive scores. SETTING: A university epilepsy surgery center. PATIENTS: Subjects included 13 patients who had intractable complex partial seizures originating from a mesial temporal lobe. INTERVENTION: Patients underwent FDG-PET scanning as part of their diagnostic work-up. Asymmetry indexes for cerebral metabolic rates were determined for whole hemisphere, lateral temporal lobe, mesial temporal lobe, frontal lobe, thalamus, and parietal lobe. MAIN OUTCOME MEASURES: The following cognitive domains were assessed: psychometric intelligence, mental control/attention, language, and verbal and nonverbal memory. Neuropsychological measures were obtained within 14 days of FDG-PET scanning. RESULTS: Relative reductions in glucose metabolism of the left hemisphere and left lateral temporal lobe correlated with a lower verbal IQ score and a lower score on a verbal memory task, recall of logical prose. Relative reduction in metabolism of the left side of the thalamus also correlated with lower scores for recall of logical prose. CONCLUSION: Relative hypometabolism of the left hemisphere correlates with lower cognitive performance. Hypometabolism of the left lateral temporal lobe and thalamus independently correlates with the verbal memory difficulties seen in this epileptic patient population.

Adult↗
PubMed1994

Depression in complex partial seizures. Electroencephalography and cerebral metabolic correlates.

OBJECTIVE: We sought to determine whether the frequency of depressive disorders among patients with complex partial seizures is associated with the laterality of ictal onset or the laterality of interictal cerebral hypometabolism. DESIGN: Fifty-three patients with medically intractable complex partial seizures were assessed for history of interictal depressive disorders meeting strict Diagnostic and Statistical Manual of Mental Disorders, Revised Third Edition (DSM-III-R), criteria with use of a modification of the Structured Clinical Interview for DSM-III-R. All subjects underwent video-electroencephalographic telemetry and fludeoxyglucose F18 positron emission tomographic scanning. The main outcome measure was the frequency of a history of major depressive episodes (MDEs) in subjects with left-sided vs right-sided ictal onset and in subjects with left-sided vs right-sided temporal lobe hypometabolism. RESULTS: Thirty-three subjects (62%) had a history of interictal depressive disorders, 16 (30%) of whom met criteria for one or more MDEs. More of those with left-sided ictal onset had a history of depression. Thirty-six subjects had definite unilateral temporal lobe hypometabolism demonstrated by positron emission tomographic scanning. More of those with left temporal lobe hypometabolism had a history of MDEs, and the combination of left temporal lobe hypometabolism and high-degree hypometabolism was strongly associated with a history of MDEs. Furthermore, among those with right temporal lobe hypometabolism, more of those with high-degree hypometabolism had a history of MDEs. CONCLUSION: Laterality of ictal onset and degree of interictal temporal lobe hypometabolism may be interdependent factors that contribute to the risk of depressive disorder in patients with complex partial seizures.

Adult↗