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Biomedical subjects

J Engel

Publications and source records attributed to J Engel.

At least 109 records · Page 6Linked to original sources
PubMed1999

A phase I single-dose trial of gadolinium texaphyrin (Gd-Tex), a tumor selective radiation sensitizer detectable by magnetic resonance imaging.

Gadolinium Texaphyrin (Gd-Tex) is a radiation sensitizer with a novel mechanism of action that sensitizes both oxic and hypoxic cells, localizes selectively in tumors, and is detectable by magnetic resonance imaging (MRI). This Phase I single-dose trial of Gd-Tex administered concurrently with radiation therapy was carried out to determine the maximally tolerated dose (MTD), dose-limiting toxicities, pharmacokinetics, and biolocalization of Gd-Tex as determined by MRI. Adults with incurable cancers of any histology requiring radiation therapy were eligible. A single i.v. dose of Gd-Tex was followed at least 2 h later by radiation therapy. The Gd-Tex dose was escalated in cohorts of 3 to 5 patients. Thirty-eight patients (median age, 58 years; range, 35-77 years) with incurable cancers of the lung (26), cervix (3), or other solid tumors (9) received a total of 41 single administrations of Gd-Tex. The Gd-Tex dose was escalated from 0.6 to 29.6 mg/kg. Irradiated sites included the thorax, brain, pelvis, bone, soft tissue, and sites of nodal metastases. The MTD was 22.3 mg/kg, determined by reversible acute tubular necrosis as the dose-limiting toxicities. Gd-Tex selectively accumulated in primary and metastatic tumors as demonstrated by MRI. No increase in radiation toxicity to normal tissues was seen. The median half-life of Gd-Tex after single-dose administration is 7.4 h. This study demonstrates that Gd-Tex is well tolerated in doses below the MTD, and that there is selective biolocalization in tumors. The maximum recommended dose for single administrations is 16.7 mg/kg.

Adult↗
PubMed1999

Retigabine N-glucuronidation and its potential role in enterohepatic circulation.

The metabolism of retigabine in humans and dogs is dominated by N-glucuronidation (), whereas in rats, a multitude of metabolites of this new anticonvulsant is observed (). The comparison of the in vivo and in vitro kinetics of retigabine N-glucuronidation in these species identified a constant ratio between retigabine and retigabine N-glucuronide in vivo in humans and dog. An enterohepatic circulation of retigabine in these species is likely to be the result of reversible glucuronidation-deglucuronidation reactions. Rats did not show such a phenomenon, indicating that enterohepatic circulation of retigabine via retigabine N-glucuronide does not occur in this species. In the rat, 90% of retigabine N-glucuronidation is catalyzed by UDP-glucuronosyltransferase (UGT)1A1 and UGT1A2, whereas family 2 UGT enzymes contribute also. Of ten recombinant human UGTs, only UGTs 1A1, 1A3, 1A4, and 1A9 catalyzed the N-glucuronidation of retigabine. From the known substrate specificities of UGT1A4 toward lamotrigine and bilirubin and our activity and inhibition data, we conclude that UGT1A4 is a major retigabine N-glucuronosyl transferase in vivo and significantly contributes to the enterohepatic cycling of the drug.

Animals↗
PubMed1999

Metabolism of retigabine (D-23129), a novel anticonvulsant.

Retigabine (D-23129, N-(2-amino-4-(4-fluorobenzylamino)-phenyl) carbamic acid ethyl ester) is a potent anticonvulsant in a variety of animal models. Rats metabolized [14C]retigabine mainly through glucuronidation and acetylation reactions. Glucuronides were detected in incubates with liver microsomes or slices, in plasma, and in bile and feces but were absent in urine (0-24 h) that contained about 2% of the dose as retigabine and approximately 29% of the dose in > 20 metabolites, which are derived mainly from acetylation reactions. About 67% of the radioactivity was excreted into feces, approximately 10% of the dose as glucuronide. The metabolite pattern in the urine (0-24 h) of dogs was comparatively simple in that retigabine (13%), retigabine-N-glucuronide (5%), and retigabine-N-glucoside (1%) were present. In the same 24-h interval, about 39% of unchanged retigabine was excreted into feces. Plasma profiling and spectroscopic analysis (liquid chromatography with tandem mass spectrometry NMR) of two isolated urinary metabolites obtained after single oral dosing of 600 mg retigabine in healthy volunteers indicated that both acetylation and glucuronidation are major metabolic pathways of retigabine in humans. We found that in vitro assays with liver slices from rat and humans reveal the major circulating metabolites in vivo.

Animals↗
PubMed1999

When is imaging enough?

Despite tremendous recent advances in both structural and functional neuroimaging, these tests alone are not enough to recommend surgery for medically refractory epilepsy. A detailed history, careful description of the ictal events, and the neurological examination will always be necessary. Interictal EEG and neuropsychological testing also add important information. In many situations, however, video-EEG monitoring of habitual seizures now plays a confirmatory, rather than a primary, role in the presurgical evaluation, and can occasionally contribute confounding data that delay or prevent surgical intervention. In the present climate of cost-containment, it is reasonable to ask if this expensive diagnostic tool is always needed when it is clear by other means that seizures are not psychogenic, particularly in certain pediatric surgically remediably syndromes such as those catastrophic epilepsys of infants and young children amenable to hemispherectomy, and drop attack that can be treated with corpus callosotomy, as well as mesial temporal lobe epilepsy and seizures due to discrete resectable lesions that often become medically intractable in adolescence. Retrospective studies should be carried out to determine when long-term, in-patient, video-EEG monitoring in these conditions add information that appropriately changes the therapeutic approach.

Adolescent↗
PubMed1998

An autonomous folding unit mediates the assembly of two-stranded coiled coils.

Subunit oligomerization of many proteins is mediated by coiled-coil domains. Although the basic features contributing to the thermodynamic stability of coiled coils are well understood, the mechanistic details of their assembly have not yet been dissected. Here we report a 13-residue sequence pattern that occurs with limited sequence variations in many two-stranded coiled coils and that is absolutely required for the assembly of the Dictyostelium discoideum actin-bundling protein cortexillin I and the yeast transcriptional activator GCN4. The functional relationship between coiled-coil "trigger" sequences was manifested by replacing the intrinsic trigger motif of GCN4 with the related sequence from cortexillin I. We demonstrate that these trigger sequences represent autonomous helical folding units that, in contrast to arbitrarily chosen heptad repeats, can mediate coiled-coil formation. Aside from being of general interest for protein folding, trigger motifs should be of particular importance in the protein de novo design.

Amino Acid Sequence↗
PubMed1998

All-trans retinol, vitamin D and other hydrophobic compounds bind in the axial pore of the five-stranded coiled-coil domain of cartilage oligomeric matrix protein.

The potential storage and delivery function of cartilage oligomeric matrix protein (COMP) for cell signaling molecules was explored by binding hydrophobic compounds to the recombinant five-stranded coiled-coil domain of COMP. Complex formation with benzene, cyclohexane, vitamin D3 and elaidic acid was demonstrated through increases in denaturation temperatures of 2-10 degreesC. For all-trans retinol and all-trans retinoic acid, an equilibrium dissociation constant KD = 0.6 microM was evaluated by fluorescence titration. Binding of benzene and all-trans retinol into the hydrophobic axial pore of the COMP coiled-coil domain was proven by the X-ray crystal structures of the corresponding complexes at 0.25 and 0.27 nm resolution, respectively. Benzene binds with its plane perpendicular to the pore axis. The binding site is between the two internal rings formed by Leu37 and Thr40 pointing into the pore of the COMP coiled-coil domain. The retinol beta-ionone ring is positioned in a hydrophobic environment near Thr40, and the 1.1 nm long isoprene tail follows a completely hydrophobic region of the pore. Its terminal hydroxyl group complexes with a ring of the five side chains of Gln54. A mutant in which Gln54 is replaced by Ile binds all-trans retinol with affinity similar to the wild-type, demonstrating that hydrophobic interactions are predominant.

Amino Acid Sequence↗
PubMed1998

Tenascin-C hexabrachion assembly is a sequential two-step process initiated by coiled-coil alpha-helices.

We have investigated the oligomerization process of tenascin-C using a variety of recombinant wild-type and mutant polypeptide chain fragments produced by heterologous gene expression in Escherichia coli. Biochemical and biophysical analyses of the structures and assemblies of these fragments indicated a sequential two-step oligomerization mechanism of tenascin-C involving the concerted interaction of two distinct domains and cysteines 64, 111, and 113. First, the sequence between alanine 114 and glutamine 139 initiates hexabrachion formation via a parallel three-stranded coiled coil. Subsequently, the tenascin assembly domain, which is unique to the tenascins, is responsible for the connection of two triplets to a hexamer. The oligomerization of the tenascin assembly domains by the three-stranded coiled coil increases their homophilic binding affinity and is an important prerequisite for tenascin-C hexamerization. Although formation of the characteristic hexabrachion structure involves the covalent linkage of the six subunits by cysteine residues, mutational analysis indicates that hexamer formation is not dependent on intermolecular disulfide bonds. Most interestingly, substitution of glutamate 130 within the coiled-coil domain by leucine or alanine resulted in the formation of parallel four-stranded helix structures, which further associated to dodecamers. Aside from supporting a sequential process of tenascin-C assembly, this finding provides experimental evidence that non-core residues can have profound effects on the oligomerization states of coiled coils.

Amino Acid Sequence↗
PubMed1998

Mac-2 binding protein is a cell-adhesive protein of the extracellular matrix which self-assembles into ring-like structures and binds beta1 integrins, collagens and fibronectin.

Human Mac-2 binding protein (M2BP) was prepared in recombinant form from the culture medium of 293 kidney cells and consisted of a 92 kDa subunit. The protein was obtained in a native state as indicated by CD spectroscopy, demonstrating alpha-helical and beta-type structure, and by protease resistance and immunological analysis. It was highly modified by N- and O-glycosylation but not by glycosaminoglycans. Ultracentrifugation showed non-covalent association into oligomers with molar masses of 1000-1500 kDa. Electron microscopy showed ring-like shapes with diameters of 30-40 nm. M2BP bound in solid-phase assays to collagens IV, V and VI, fibronectin and nidogen, but not to fibrillar collagens I and III or other basement membrane proteins. The protein also mediated adhesion of cell lines at comparable strength with laminin. Adhesion to M2BP was inhibited by antibodies to integrin beta1 subunits but not to alpha2 and alpha6 subunits, RGD peptide or lactose. This distinguishes cell adhesion of M2BP from that of laminin and excludes involvement of lactose-binding galectin-3. Immunological assays demonstrated variable secretion by cultured human cells of M2BP, which was detected in the extracellular matrix of several mouse tissues.

Animals↗
PubMed1998

Electron microscopic structure of agrin and mapping of its binding site in laminin-1.

Agrin is a large, multidomain heparan sulfate proteoglycan that is associated with basement membranes of several tissues. Particular splice variants of agrin are essential for the formation of synaptic structures at the neuromuscular junction. The binding of agrin to laminin appears to be required for its localization to synaptic basal lamina and other basement membranes. Here, electron microscopy was used to determine the structure of agrin and to localize its binding site in laminin-1. Agrin appears as an approximately 95 nm long particle that consists of a globular, N-terminal laminin-binding domain, a central rod predominantly formed by the follistatin-like domains and three globular, C-terminal laminin G-like domains. In a few cases, heparan sulfate glycosaminoglycan chains were seen emerging from the central portion of the core protein. Moreover, we show that agrin binds to the central region of the three-stranded, coiled-coil oligomerization domain in the long arm of laminin-1, which mediates subunit assembly of the native laminin molecule. In summary, our data show for the first time a protein-protein interaction of the extracellular matrix that involves a coiled-coil domain, and they assign a novel role to this domain of laminin-1. Based on this, we propose that agrin associates with basal lamina in a polarized way.

Agrin↗
PubMed1998

Synthesis, anticonvulsant activity, and structure-activity relationships of sodium channel blocking 3-aminopyrroles.

Starting from the corresponding acetophenone and glycine derivatives, a series of new 3-aminopyrroles was synthesized in few steps. Using this procedure with hydrazine and hydroxylamine instead of the glycinates provides access to 3-aminopyrazoles and 5-amino 1,2-oxazoles. The various derivatives were tested for anticonvulsant activity in a variety of test models. Several compounds exhibit considerable activity with a remarkable lack of neurotoxicity. 4-(4-Bromophenyl)-3-morpholinopyrrole-2-carboxylic acid methyl ester, 3, proved to be the most active compound. It was protective in the maximal electroshock seizure (MES) test in rats with an oral ED50 of 2.5 mg/kg with no neurotoxicity noted at doses up to 500 mg/kg. Compound 3 blocks sodium channels in a frequency-dependent manner. The essential structural features which could be responsible for an interaction with an active site of the voltage-dependent sodium channel are established within a suggested pharmacophore model.

Animals↗
PubMed1998

1,5-Disubstituted indazol-3-ols with anti-inflammatory activity.

A series of new indazol-3-ol derivatives was synthesized. Some of these compounds exhibit interesting anti-inflammatory activities in various models of inflammation. 5-Methoxy-1-[quinoline-2-yl-methoxy)-benzyl]-1H-indazol-3-ol (27) strongly inhibits the oxidation of arachidonic acid to 5-hydroperoxyeicosatetraenoic acid catalyzed by 5-lipoxygenase (IC50 = 44 nM). 27 also inhibits the contraction of sensitized guinea pig tracheal segments (IC50 = 2.9 microM). In guinea pigs treated with 27 (1 mg/kg i.p.) 2 h before antigen provocation, there was a marked inhibition (47%) of the antigen-induced airway eosinophilia. After topical application of 1 microgram/ear 27 inhibits the arachidonic acid induced mouse ear edema (41%).

Animals↗
PubMed1998

Research on the human brain in an epilepsy surgery setting.

Recent advances in our understanding of the fundamental mechanisms of epilepsy have derived, to a large extent, from improvements in designing parallel human and animal studies. This is the result not only of better animal models of human epileptic phenomena, but of an increasing ability to carry out detailed invasive studies on patients in the course of surgical treatment for medically refractory epilepsy. In addition to interictal and ictal video-EEG recordings with chronic depth and subdural electrodes, it is also possible to sample single-unit activity with chronically implanted microelectrodes, and measure constituents of extracellular fluid with chronically implanted microdialysis probes, using protocols that in the past were possible only in the experimental animal laboratory. Subsequent surgical resection provides tissue that can be used for electrophysiological, morphological, biochemical, and molecular biological investigations. Patients in epilepsy surgery facilities represent a precious resource for research that should be utilized to the fullest extent possible by basic scientists interested in mechanisms of epilepsy. It is particularly important that invasive research be pursued now, because improved diagnostic technology is greatly reducing the need for chronic intracranial electrode recordings, and surgical approaches that do not yield tissue could be used more commonly in the future. Therefore, the capacity to carry out invasive research in the context of epilepsy surgery may diminish greatly over time. To take full advantage of these opportunities, carefully designed iterative experimental protocols are necessary to characterize abnormalities in the human epileptic brain, to create appropriate experimental animal models to study these phenomena in greater detail, and to return to the human brain to validate the clinical relevance of observations made on animals. It is also important, however, to recognize certain unavoidable limitations of human research, including ethical considerations, variability inherent in the clinical setting, imprecision in defining target areas, lack of control data, and small subject numbers, which continue to make animal investigations essential to the achievement of our goals of defining fundamental mechanisms of human epileptic phenomena.

Animals↗
PubMed1998

Paired pulse suppression and facilitation in human epileptogenic hippocampal formation.

Paired pulse stimulation has commonly been employed to investigate changes in excitability in epileptic hippocampal tissue employing the in vitro slice preparation. We used paired pulse stimulation in the intact temporal lobe of patients with temporal lobe seizures to compare the excitability of pathways in the epileptogenic hippocampus (located in the temporal lobe in which seizures arise) with those in the non-epileptogenic hippocampus of the contralateral temporal lobe (in the hemisphere to which seizures spread). A total of 20 patients with temporal lobe seizure onsets were studied during chronic depth electrode monitoring for seizure localization. Intracranial in vivo stimulation and recording sites included the hippocampus, entorhinal cortex, subicular cortex and parahippocampal gyrus. A comparison of all hippocampal pathways located in the temporal lobe where seizures typically started (n = 37) with those in temporal lobes contralateral to seizure onset (n = 53) showed significantly greater paired pulse suppression of population post-synaptic potentials on the epileptogenic side (F(1,87) = 6.1, P < 0.01). Similarly, mean paired pulse suppression was significantly greater for epileptogenic perforant path responses than for contralateral perforant path responses (F(1,13) = 7.5, P < 0.01). In contrast, local stimulation activating intrinsic associational pathways of the epileptogenic hippocampus showed decreased paired pulse suppression in comparison to the epileptogenic perforant path. These results may be a functional consequence of the formation of abnormal recurrent inhibitory and recurrent excitatory pathways in the sclerotic hippocampus. Enhanced inhibition may be adaptive in suppressing seizures during interictal periods, while abnormal recurrent excitatory circuits in the presence of enhanced inhibition may drive the hypersynchronization of principal neurons necessary for seizure genesis.

Action Potentials↗
PubMed1998

Sequence analysis suggests the presence of an IG-like domain in the N-terminal region of alpha-dystroglycan which was crystallized after mutation of a protease susceptible site (Arg168-->His).

Recently, we demonstrated that the N-terminal region of mouse alpha-dystroglycan represents an autonomously folding globular domain, organized into at least two subdomains (Brancaccio et al., Eur. J. Biochem. 246, 166-172, 1997). We have now found a similarity between a part of the alpha-dystroglycan N-terminal sequence (approximately from position 80 to 180) and several protein sequences belonging to the immunoglobulin kappa family. Moreover, we have recombinantly expressed and purified a 31 kDa protein fragment which matches the entire alpha-dystroglycan N-terminal globular domain. To prevent the action of bacterial endogenous proteases and/or thrombin, which cleaves the protein into two fragments at an Arg-Ala trypsin-sensitive site in positions 168-169, we have introduced a single mutation (Arg168-->His), thus making the whole domain more stable and suitable for crystallization. Crystals of this mutant protein were obtained by vapor diffusion using the hanging drop technique, and they diffract to 0.28 nm Bragg spacing.

Amino Acid Sequence↗
PubMed1998

NMR structure of a parallel homotrimeric coiled coil.

The solution structure of the oligomerization domain of cartilage matrix protein (also known as matrilin-1) has been determined by heteronuclear NMR spectroscopy. The domain folds into a parallel, disulfide-linked, three-stranded, alpha-helical coiled coil, spanning five heptad repeats in the amino acid sequence. The sequence of the first two heptad repeats shows some deviations from the consensus of hydrophobic and hydrophilic residue preferences. While the corresponding region of the coiled coil has a higher intrinsic flexibility, backbone alpha-helix and superhelix parameters are consistent with a regular coiled coil structure.

Amino Acid Sequence↗