Electron microscopy of extracellular matrix components.
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Aggrecans↗
Biomedical subjects
J Engel
Publications and source records attributed to J Engel.
The cartilage proteoglycan aggregate: assembly through combined protein-carbohydrate and protein-protein interactions.
In vitro reassembled aggregates of cartilage proteoglycan (aggrecan) were studied by glycerol spraying/rotary shadowing electron microscopy and compared to the corresponding native (i.e. never dissociated) structures. In both cases a tightly packed central filament structure was observed consisting of the hyaluronate binding region (HABR) of the proteoglycan, link protein (LP) and hyaluronate (HA). This differs from earlier results where a discontinuous central filament structure was seen after spreading proteoglycan aggregates at a water/air interphase. Binding of isolated HABR to HA is random but upon addition of link protein a clustering of the HA-binding proteins is observed, indicating a cooperativity. In a fully saturated aggregate the HA is covered by a continuous protein shell consisting of HABR and LP. When added in amounts below saturation HABR and LP bind to the HA in clusters which are interrupted by free strands of HA. The proteoglycan aggregate is thus an example for a structure where a polysaccharide forms a template for a supramolecular assembly largely stabilized by protein-protein interactions.
Aggrecans↗
Chronic pretreatment with naloxone modifies benzodiazepine receptor binding in amygdaloid kindled rats.
Male Sprague-Dawley rats received either naloxone (75 micrograms/h) or saline (0.5 microliter/h) s.c. for 14 days delivered with osmotic minipumps. Two days after termination of either treatment, daily amygdala kindling stimulation was applied until the animals experienced stage V kindled seizures. Benzodiazepine (BDZ) binding sites were labeled with [3H]flunitrazepam (2 nM), and changes in specific brain areas were determined by in vitro quantitative autoradiography. Twenty-four hours after the last electrical stimulation, the saline pretreated fully kindled rats showed enhanced BDZ receptor binding in dentate gyrus, and decreased binding in cingulate cortex ipsilateral to the stimulation compared to saline controls. Twenty-eight days after the last stage V kindled seizure, the significant alterations were no longer evident. In agreement with a previous study, we found that naloxone pretreated amygdala kindled rats showed stage V kindled seizures followed by intervals of 3-5 days in which the same electrical stimulation failed to induce any behavioral and EEG alterations. In comparison with the saline pretreated kindled and saline control groups, the naloxone pretreated kindled rats had significantly higher BDZ binding in different cortical areas, amygdala complex, hippocampus, substantia nigra and periaqueductal gray, 24 h after the last electrical stimulation. The present study indicates that previous chronic exposure to naloxone increases BDZ receptor binding in kindled rats, and suggests that this effect may be associated with the enhanced seizure suppression observed in these animals.
Amygdala↗
A new nomenclature for the laminins.
The authors have adopted a new nomenclature for the laminins. They are numbered with arabic numerals in the order discovered. The previous A, B1 and B2 chains, and their isoforms, are alpha, beta and gamma, respectively, followed by an arabic numeral to identify the isoform. For example, the first laminin identified from the Engelbreth-Holm-Swarm tumor is laminin-1 with the chain composition alpha 1 beta 1 gamma 1. The genes for these chains are LAMA1, LAMB1 and LAMC1, respectively.
Animals↗
Anisotropic propagation of Ca2+ waves in isolated cardiomyocytes.
Digital imaging microscopy of fluor-3 fluorescence was used to study the propagation of intracellular Ca2+ waves in isolated adult rat cardiomyocytes from 17 to 37 degrees C. Ca2+ waves spread in both transverse and longitudinal direction of a myocyte. Transverse propagation was pronounced in waves starting from a focus at the edge of a myocyte and in waves following an irregular, curved path (spiral waves). For the former type of waves, propagation velocities were determined. Both transverse and longitudinal wave components propagated at constant velocity ranging from 30 to 125 micron/s. Myocytes were anisotropic with respect to wave propagation: waves propagated faster in the longitudinal than in the transverse direction. The ratio between longitudinal and transverse velocity increased from 1.30 at 17 degrees C to 1.55 at 37 degrees C. Apparent activation energies for transverse and longitudinal wave propagation were estimated to be -20 kJ/mol, suggesting that these processes are limited by diffusion of Ca2+. Direction-dependent propagation velocities are interpreted to result from the highly ordered structure of the myocytes, especially from the anisotropic arrangement of diffusion obstacles such as myofilaments and mitochondria.
Aniline Compounds↗
Epilepsy surgery.
The publication of many review papers and five books on the surgical treatment of epilepsy during the past year attests to the tremendous increase in interest in this alternative mode of therapy. The number of therapeutic surgical procedures in the United States alone has more than tripled since 1986; however, there may be 10- to 100-fold more patients who could benefit from surgical intervention. Advances in diagnostic testing and surgical technique have greatly improved the safety and efficacy of surgical treatment, particularly for clearly defined, surgically remediable syndromes such as mesial temporal lobe epilepsy and certain pediatric disorders. Greater efforts must now be made to 1) communicate these advances to primary care physicians in order to identify appropriate surgical candidates early in the course of their disorder, and 2) document the cost-effectiveness of surgical treatment in order to justify compensation by third-party payers.
Brain↗
Capture of solar and higher-energy neutrinos by 127I.
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Journal Article↗
Structural investigation of Cetrorelix, a new potent and long-acting LH-RH antagonist.
The new decapeptide SB-75 (INN: Cetrorelix) has been characterized as a potent antagonist of luteinizing-hormone releasing hormone (LH-RH). Such derivatives are of great medicinal interest owing to their potential application in areas such as hormone-dependent tumors, uterine fibroids, and in diseases and conditions which result from inappropriate hormone levels or which can be treated by suppression of estrogens. SB-75 is the subject of intensive ongoing clinical evaluation and is an accepted standard for the design of new LH-RH antagonists. We characterized SB-75 by means of modern MS and NMR techniques to demonstrate the significance of both sequencing methods on a complicated unnatural decapeptide. Our structural elucidations with nuclear Overhauser (NOE) experiments revealed clear evidence for a highly flexible molecule with no single predominant conformation even in sodium dodecyl sulfate (SDS) mimicking a cellular membrane.
Amino Acid Sequence↗
Neuroendocrine evidence for reduced serotonergic neurotransmission during heavy drinking.
A fenfluramine (60-mg oral dose) challenge test was performed in 19 male heavy drinkers (mean daily consumption 88 g of pure alcohol). Twelve healthy males served as controls. The prolactin and temperature responses to fenfluramine were significantly reduced in the group of heavy drinkers. The results suggest impaired central serotonergic neurotransmission in alcoholism, possibly involving subsensitivity in various serotonin receptor subtypes.
Adult↗
Effect of citalopram on alcohol intake in heavy drinkers.
The effect of the selective serotonin reuptake inhibitor citalopram (40 mg daily dose) on alcohol intake was investigated in a double-blind, placebo-controlled cross-over study. Thirty men with heavy alcohol consumption (mean daily alcohol intake 111 +/- 51 g pure alcohol) completed the study. After a 2-week baseline period, subjects were randomly allocated to treatment with either citalopram or placebo for 5 weeks. In the total sample of heavy drinkers, no difference was found between citalopram and placebo treatment in alcohol consumption or days of abstinence. However, the response to citalopram was negatively correlated (rs = -0.67, p < 0.01) with baseline levels of mean daily alcohol intake. Therefore, we divided the total sample into two subgroups with baseline mean daily alcohol intake above and below median (107 g pure alcohol), respectively. In the group with the higher baseline values (138 +/- 25 g pure alcohol), citalopram was not different from placebo in reducing the daily alcohol intake, but in subjects with the lower baseline values (85 +/- 15 g pure alcohol), citalopram was significantly (p < 0.01) superior to placebo. Consequently, citalopram at the present dose appears capable of reducing alcohol intake only in a subgroup of heavy drinkers with a mean daily consumption of between 60 and 100 g pure alcohol.
Adult↗
The gene amyE(TV1) codes for a nonglucogenic alpha-amylase from Thermoactinomyces vulgaris 94-2A in Bacillus subtilis.
We isolated the gene amyE(TV1) from Thermoactinomyces vulgaris 94-2A encoding a nonglucogenic alpha-amylase (AmyTV1). A chromosomal DNA fragment of 2,247 bp contained an open reading frame of 483 codons, which was expressed in Escherichia coli and Bacillus subtilis. The deduced amino acid sequence of the AmyTV1 protein was confirmed by sequencing of several peptides derived from the enzyme isolated from a T. vulgaris 94-2A culture. The amino acid sequence was aligned with several known alpha-amylase sequences. We found 83% homology with the 48-kDa alpha-amylase part of the Bacillus polymyxa beta-alpha-amylase polyprotein and 50% homology with Taka amylase A of Aspergillus oryzae but only 45% homology with another T. vulgaris amylase (neopullulanase, TVA II) recently cloned from strain R-47. The putative promoter region was characterized with primer extension and deletion experiments and by expression studies with B. subtilis. Multiple promoter sites (P3, P2, and P1) were found; P1 alone drives about 1/10 of the AmyTV1 expression directed by the native tandem configuration P3P2P1. The expression levels in B. subtilis could be enhanced by fusion of the amyE(TV1) coding region to the promoter of the Bacillus amyloliquefaciens alpha-amylase gene.
Amino Acid Sequence↗
Efficacy study of THINKable in the attention and memory retraining of traumatically head-injured patients.
The efficacy of computer-assisted attention and memory retraining was evaluated with 15 severely head-injured patients. Training with selected exercises tailored to the individual's needs were provided from the THINKable program for up to 20 hours in both the attention and memory remediation modules. The experimental design evaluated outcome by juxtaposing multiple baseline procedures with a pre- and post-group comparison. Significant results were documented on the computerized tasks, psychometric measures and on patient and observer ratings of everyday behaviours of attention and memory.
Adolescent↗
Overview of seizures.
Epilepsy is a chronic disorder characterized by recurrent unprovoked epileptic seizures. Not all epileptic seizures indicate the existence of an epileptic disorder; many represent a natural response of the normal brain to transient noxious insults that are not likely to be repeated (reactive seizures). Not only is it important to distinguish conditions associated with these relatively benign epileptic events from epilepsy, but also it is important to recognize the many types of epileptic seizures and many types of epilepsy, reflecting different underlying anatomic and pathophysiologic substrates, which determine therapy and prognosis. Acute care for an epileptic seizure, therefore, includes search for an underlying treatable cause and protection against recurrence, if necessary. Neurologic consultation in the emergency department can help avoid unnecessary diagnostic procedures and treatments, particularly in patients with reactive seizures and established epileptic disorders. Communication with the patient's continuing care physician is an essential part of the emergency department management plan.
Diagnosis, Differential↗
Direct detection of the intracellular formation of carboxyphosphamides using nuclear magnetic resonance spectroscopy.
31P nuclear magnetic resonance (NMR) spectroscopy was used in conjunction with cell perfusion techniques to monitor the intracellular chemistry of the cyclophosphamide (CP, CAS 6055-19-2) metabolites 4-hydroxy-cyclophosphamide (4-HO-CP) and aldophosphamide (AP) in U937 human histiocytic (CP-sensitive) and K562 human erythroleukemia (CP-resistant) cells. Similar experiments were carried out using the ifosfamide (IF, CAS3778-73-2) metabolites 4-hydroxyifosfamide (4-HO-IF) and aldoifosfamide (AIF). The hydroxy and aldehydic metabolites were generated by the triphenylphosphine reduction of 4-hydroperoxycyclophosphamide (4-HO2-CP) or 4-hydroperoxyifosfamide (4-HO2-IF) or by a spontaneous elimination/addition reaction involving water and 4-thiocyclophosphamide analogs 4-(2-hydroxyethyl) thiocyclophosphamide (4-ESCP) or mafosfamide. Cell death resulting from 4-HO-CP/AP perfusions was mimicked by perfusion with acrolein or an acrolein producing but non-alkylating, dechloro-CP analog. Acrolein toxicity was minimized by the presence of 2-mercaptoethanol or mesna (sodium 2-mercaptoethanesulfonate) in perfusion solutions as well as by fractional dose drug perfusions (sequential 2.5-3.0 h perfusions separated by cell washes with drug-free medium). The intracellular half-life for phosphoramide mustard (PM) at an intracellular pH value of 7.1 +/- 0.1 and an ambient probe temperature of 23 +/- 1 degree C in U937 cells was 2.1 h [k = (5.4 +/- 0.3) x 10(-3) min-1] and in K562 cells was 3.1 h [k = (3.7 +/- 0.4) x 10(-3) min-1]. Similar half-lives (2-4 h) were determined for intracellular isophosphoramide mustard (IPM). Fractional dose perfusion of U937 or K562 cells with 1.5 mmol/l 4-HO-CP/AP (generated from 4-HO2-CP) and 0.3 mmol/l mesna allowed for the observation of intracellular carboxyphosphamide (CBP); CBP was formed in higher concentrations in the CP-resistant K562 cells. Similar results were obtained using 4-ESCP and mafosfamide as sources of 4-HO-CP/AP. Identification of CBP was based on chemical shift, chemical stability, and membrane permeability studies of synthetic CBP. Concentrations of carboxyifosfamide (CBIF) formed in K562 cells were also greater than that in U937 cells.
Acrolein↗
Quasielastic neutrino scattering from oxygen and the atmospheric neutrino problem.
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Journal Article↗
Benzodiazepine receptor binding following chronic treatment with naloxone, morphine and met-enkephalin in normal rats.
The effects of chronic administration of naloxone, morphine and met-enkephalin on benzodiazepine (BDZ) receptor binding in rat brain were determined 2 and 50 days after treatments were accomplished. Two days after naloxone treatment (75 micrograms/h s.c. for 14 days), enhanced BDZ receptor binding was observed in cingulate, frontal, piriform, entorhinal and sensorimotor cortices; amygdala complex, hippocampus, substantia nigra and central gray. Two days after morphine treatment (20 mg/kg i.p. daily for 6 days), increased BDZ receptor binding was detected in cingulate, frontal, piriform, entorhinal and sensorimotor cortices; amygdala complex, hippocampus and substantia nigra. Two days after met-enkephalin treatment (10 micrograms/h i.c.v. for 6 days) enhanced BDZ receptor binding was shown only in sensorimotor cortex. No significant changes were observed 50 days after the treatments were completed. These data indicate an important interaction between GABAergic and opioid peptide systems.
Animals↗
Ionic interactions in the coiled-coil domain of laminin determine the specificity of chain assembly.
Laminins are a family of large (800 to 900 kDa) multidomain glycoproteins specifically found in basement membranes. They consist of one heavy A chain and two light chains B1 and B2, and several tissue-specific laminin isoforms exist. Chain assembly is mediated through the formation of a rod-like triple-stranded alpha-helical coiled-coil domain about 75 nm long. The interacting edges of the chains are mostly formed by hydrophobic residues in positions a and d of an (abcdefg)n heptad sequence repeat and by a distinct pattern of charged residues in positions e and g. Here, we have analyzed the sequences of known laminin chains in an effort to relate them to interaction potential. Initially, those sequences localized in the long arm were arranged in an optimum heptad-repeating scheme. The interacting edges between chains were then analyzed for interchain hydrophobic and ionic interactions. The short heptad blocks were allowed to shift axially with respect to each other to maximize the number of interactions. The number of hydrophobic interactions was very high and similar for all chain combinations, but especially so for homodimers. As these were not observed experimentally, it seems that hydrophobic interactions probably represent only a prerequisite for coiled-coil formation. The number of ionic interactions, however, directly resembles the interaction potential observed in in vitro experiments. In particular, the number of interchain ionic interactions is high for parallel heterodimer configurations of A and B chains, but low for homodimer arrangements. When the laminin isoform chains, rat s-laminin (B1s) and human merosin (Am), are included in the analysis, they show rather low numbers of mutual interactions but high ionic interaction potentials between them and distinct mouse laminin chains are predicted. For mouse laminin the analysis was extended to a full three-stranded coiled-coil structure. The highest number of interchain ionic interactions occurs for an anti-clockwise chain arrangement of A-->B1-->B2 when viewed from the N terminus. None of the laminin chains appears to be designed for the formation of homodimers, although such conformations are frequently found in other alpha-fibrous proteins.
Amino Acid Sequence↗
Two-step binding mechanism of fibrinogen to alpha IIb beta 3 integrin reconstituted into planar lipid bilayers.
The platelet integrin alpha IIb beta 3 binds to fibrinogen and thus mediates platelet aggregation after stimulation. This integrin was isolated from human platelets and reconstituted into lipid vesicles. As judged by electron microscopy the integrin incorporated adequately only into 1,2-dimyristoylglycero-3-phosphocholine/1,2-dimyristoylphosphatidy lglycerol vesicles after removal of the detergent by adsorption to Bio-Beads. These vesicles were then used to generate planar lipid bilayers. The binding of fluorochrome labeled fibrinogen or the peptide ligand Gly-Arg-Gly-Asp-Ser-Pro-Cys (GRGDSPC) was monitored by total internal reflection fluorescence microscopy and a solid phase binding assay. Analysis of the kinetics revealed fast reversible formation of a fibrinogen/integrin precomplex (KD = 50 nM) followed by formation of a stable irreversible complex. This transition was monitored by measuring the fraction of precomplex which could be dissociated by addition of excess Gly-Arg-Gly-Asp-Ser (GRGDS). For the peptide, the KD was 1200 nM, and the rates of association and dissociation were faster than the time resolution of the method. Similar KD values were found by inhibition of fibrinogen binding to alpha IIb beta 3 in the immobilized receptor assay. Since the binding of fibrinogen was irreversible, KD values were dependent on the time period between fibrinogen incubation and peptide addition. These and results by other authors point to the biological importance of the biphasic binding process of fibrinogen to its receptor on platelets.
Amino Acid Sequence↗