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PubMed1987

Defective superoxide-dismutase molecules accumulate with age in human lenses.

The specific activity of superoxide dismutase (SOD) in human transparent lenses declines as a function of age. Immunotitration using monospecific antibodies showed that, with increasing age, lenses exhibit an accumulation of catalytically inactive, but antigenically reactive, enzyme molecules. Antiserum produced against denatured enzyme removed the inactive molecules from the lens homogenates without affecting the enzyme activity. These aberrant molecules are at least partially denatured and are totally devoid of catalytic activity.

Adolescent↗
PubMed1994

Obidoxime Antagonizes the Neuromuscular Failure Induced by Neostigmine and Diisopropyl Fluorophosphate via Different Mechanisms.

The efficacies and mechanisms of obidoxime in antagonizing the neuromuscular failure induced by neostigmine and diisopropyl fluorophosphate (DFP) were studied in mouse phrenic nerve/diaphragm preparations. Obidoxime antagonized neostigmine-induced tetanic fade (EC(50): 300 &mgr;M) by inhibiting the regenerative and sustained depolarization during repetitive stimulation. The antagonism was associated with a depression and shortening of single endplate potentials (EPPs) and miniature EPPs (MEPPs). In contrast, the neuromuscular failure induced irreversibly after treatment with DFP and followed by washout was restored by obidoxime at concentrations (EC(50): 0.6 &mgr;M) 500-fold lower than that against neostigmine. The regenerative depolarization was abolished with no depression of single EPPs and MEPPs, and the antagonistic action persisted after washout of obidoxime. The EC(50) of obidoxime was proportionately increased in the presence of increasing concentrations of DFP. Nevertheless, the EC(50) against DFP, at a concentration (30 &mgr;M) 15-fold in excess of that which caused tetanic fade, was still 10-fold lower than that which antagonized neostigmine. In both cases, the amplitudes of train EPPs were increased. It is concluded that obidoxime antagonizes neostigmine-induced neuromuscular failure by a curare-like action but antagonizes DFP by an enzyme reactivation. Copyright 1994 S. Karger AG, Basel

Journal Article↗
PubMed1990

Enhanced removal of detergent and recovery of enzymatic activity following sodium dodecyl sulfate-polyacrylamide gel electrophoresis: use of casein in gel wash buffer.

The inclusion of 1% casein or bovine serum albumin in buffer used to reactivate enzymes subjected to sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis resulted in accelerated removal of SDS and restoration of nuclease and beta-galactosidase enzyme activities. Nuclease and beta-galactosidase activities which are absent from gels after longer wash procedures are detectable with this technique. Enzyme activity in gels prepared with SDS which contained inhibitory contaminants was partially restored by the casein wash procedure. The threshold of detection of two-dimensionally separated deoxyribonuclease I using the casein wash procedure was 1 picogram.

Autoradiography↗
PubMed1975

Determination of the subunit molecular weight of hypoxanthine-guanine phosphoribosyltransferase from human erythrocytes by recovery of enzyme activity from sodium dodecyl sulphate gels.

The molecular weights of the subunits of the enzyme hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) from human erythrocytes were determined with a simple novel method, including electrophoresis in sodium dodecyl sulphate gels, gel slicing, elution of protein from the gel slices and enzyme reactivation in the presence of the substrate 5-phosphorylribose-1-pyrophosphate. As molecular weight standards glutaraldehyde-polymerized polypeptides of human haemoglobin were used. The experiments clearly showed the existence of molecular weight differences in human erythrocyte hypoxanthine-quanine phosphoribosyltransferase.

Chromatography, Gel↗
PubMed1996

Structural modifications of the omega loop in human acetylcholinesterase.

Conformational mobility of the surface omega loop (Cys-69-Cys-96) in human acetylcholinesterase (HuAChE) was recently implicated in substrate accessibility to the active center and in the mechanism of allosteric modulation of enzymatic activity. We therefore generated and kinetically evaluated the following modifications or replacements in HuAChE: (a) residues at the loop ends, (b) residues involved in putative hydrogen-bond interactions within the loop and between the loop and the protein core, (c) ChEs conserved proline residues within the loop and (d) a deletion of a conserved segment of 5 residues. All the residue replacements, including those of the prolines, had either limited or no effect on enzyme reactivity. These results suggest that unlike the case of lipase, the omega loop in the HuAChE is not involved in large lid-like displacements. In cases where modifications of the loop sequence had some effect on reactivity, the effects could be attributed to an altered position of residue Trp-86 supporting the proposed coupling between the structure of the omega loop and the positioning of the Trp-86 indole moiety, in catalytic activity and in allosterism.

Acetylcholinesterase↗
PubMed1987

Phospholipase A2 activity in ram spermatozoa plasma membranes.

A highly active phospholipase A2 in ram spermatozoa plasma membranes has been established. Phospholipase A2 was optimally active at pH 7.4 and was Ca2+ dependent. Low concentrations of K+ (25 mM) were found to inhibit phospholipase A2 activity, whereas higher concentrations induced enzyme reactivation. An inhibitory effect of Zn2+ has also been observed.

Animals↗
PubMed1987

Enzyme histochemistry of monoamine oxidase in the heart of aged rats.

The distribution of monoamine oxidase types A and B in the heart of young (3-month-old) and aged (26-month) Wistar rats was studied by histochemical methods. In young rats, MAO activity was higher in the left ventricle than in the right. Enzyme reactivity was present within both muscle cells and the blood vessel wall. Histochemical staining was abolished by clorgyline administration. In the heart of aged rats, we observed a very remarkable increase of clorgyline sensitive MAO activity primarily at the level of myocardial cells. The right ventricle showed the highest increase of MAO reactivity.

Aging↗
PubMed1989

Enzyme histochemistry of glutamate dehydrogenase in ageing rat cerebellar cortex.

The influence of ageing on glutamate dehydrogenase activity was studied in the cerebellar cortex of 3-month-old (young), 12-month-old (adult) and 26-month-old (aged) male Sprague-Dawley rats by using an enzyme histochemical technique. In young rats the enzyme reactivity was observed in the neuropil of the molecular layer as well as in the perikarya of basket cells and of stellate cells; within the cytoplasm of Purkinje neurons and in synaptic glomeruli of the granular layer. Glutamate dehydrogenase activity was significantly increased in the cerebellar cortex of adult rats and decreased in old animals. The synaptic glomeruli of the granular layer were the structures of the cerebellar cortex more remarkably affected by age-related changes. The possibility that decreased glutamate catabolism occurring in the ageing cerebellar cortex may result in an excess of the amino acid and may contribute to the nerve cell loss occurring in the cerebellum of old rats is discussed.

Aging↗
PubMed1991

Regeneration experiments of the platinated enzyme fumarase, using sodium diethyldithiocarbamate, thiourea, and sodium thiosulfate.

The enzyme fumarase is inhibited by [cis-Pt(NH3)2(H2O)2] (NO3)2. The Pt compound most likely binds at a S-methionine site. Sodium diethyldithiocarbamate (Naddtc) appears to be a powerful regenerator of enzymatic activity. Thiourea is less active, while sodium thiosulfate (STS) is almost inactive in restoring the activity of the enzyme. The regeneration phenomena are based on the dissociation of the Pt-S bonds of the methionine type, and formation of species like [Pt(ddtc)2]. In the model adduct [Pt(dien)GS-Me]2+ Naddtc, thiourea and STS easily break the Pt-S bond of the methionine type. It is concluded that the model system for Naddtc and thiourea does resemble fumarase quite well. S-donor ligands, which may be used as rescue agents in Pt antitumor therapy, are known to suppress nephrotoxicity caused by [cis-PtCl2(NH3)2]. A parallel is drawn between the enzyme reactivation, modeled by fumarase, and the [cis-PtCl2(NH3)2] nephrotoxicity suppression by rescue agents. It is proposed that a Pt-methionine type binding is broken by the rescue agents Naddtc and thiourea, but that the rescue agent STS only inhibits the nephrotoxicity by inactivating unbound Pt species in the cell.

Animals↗
PubMed1993

Organophosphate poisoning.

The present review discusses the structure of the anticholinesterase organophosphates (OPs), which are used predominantly as insecticides. OP poisoning can occur in a variety of situations and can be accidental or suicidal. It is common in developing countries. The cholinergic syndrome is caused by acetylcholinesterase inhibition, and diagnosis is based on the clinical signs and symptoms as well as the measurement of inhibition of erythrocyte acetylcholinesterase and/or plasma cholinesterase activity. Antidotal treatment is with atropine, an enzyme reactivator such as pralidoxime and diazepam. Anticholinesterase OPs may produce effects other than the acute cholinergic syndrome, including the intermediate syndrome. Later effects may include organophosphorus-induced delayed neuropathy. Certain OPs are exploited for their anticholinesterase effects, including defoliants such as 'DEF', herbicides such as glyphosate, fire retardants and industrial intermediates. The toxicology of this group is heterogeneous and they may or may not possess anticholinesterase activity.

Acetylcholinesterase↗
PubMed1988

Temporal disparity in pineal and retinal ontogeny.

The development of photoreceptors and two putative neurotransmitter systems in the pineal organ and retina was studied during embryogenesis in the three-spined stickleback Gasterosteus aculeatus L. The investigation was performed by aid of immunocytochemistry using well characterized antisera to the retinal proteins alpha-transducin (TD alpha) and S-antigen (SA) (photoreceptor-markers), antisera against L-glutamic acid decarboxylase (GAD), gamma-aminobutyric acid (GABA), choline-O-acetyltransferase (ChAT) and with acetylcholinesterase (AChE) histochemistry (neurotransmitter-markers). It was possible to set up the following developmental time-table concerning the first appearance of positive immuno- and enzyme-reactive cells in the pineal organ and retina: I AChE-activity and TD alpha- and SA-immunoreactive cells in the pineal organ; II GAD- and GABA-immunoreactive cells in the pineal organ and retina; ChAT immunoreactivity and AChE activity in the retina; III hatching; IV SA-immunoreactive cells in the retina. The obtained results provide good evidence that while photoreceptor cells develop much earlier in the pineal organ than in the retina, neurons develop simultaneously in the pineal organ and retina.

Acetylcholinesterase↗
PubMed1984

Effects of HI-6 on brain neuronal RNA and acetylcholinesterase: metabolic responses during acute soman intoxication.

Quantitative cytochemical techniques were used to monitor effects of the bis-pyridinium oxime HI-6 with and without atropine sulfate (AS) on soman-induced cerebrocortical (layer V) and striatal neuron RNA and acetyl-cholinesterase (AChE) impairments. In addition, plasma cholinesterase (ChE) activity was measured to determine the extent of peripheral enzyme reactivation. Antidotal pretreatment effected complete (HI-6) or partial (AS) amelioration of neuronal RNA depletion evidenced following 1.5 LD50 (195 micrograms/kg) soman, whereas combined HI-6 + AS treatment only partially restored (cortical) or did not change (striatal) neuron RNA contents. HI-6 produced appreciable plasma ChE reactivation but brain AChE activity was not significantly altered. In rats treated only with antidotes, HI-6 or AS alone significantly reduced neuronal RNA in both brain regions. These data indicate that HI-6 influences the metabolic status of central cholinergic compartments and can completely protect against soman-induced neuronal RNA depletion. However, there are no precise relationships among RNA restitution, AChE reactivation or the protective potency of antidotal treatments. Effects of HI-6 on neuronal RNA may signify central cholinolytic activity in vivo, but indirect effects mediated by peripheral mechanisms can not be excluded at present.

Acetylcholinesterase↗
PubMed1995

beta-Eudesmol as an antidote for intoxication from organophosphorus anticholinesterase agents.

beta-Eudesmol, a sesquiterpenol present in Chinese herbs, improved the tetanic contraction impaired by diisopropylfluorophosphate in isolated mouse diaphragm preparations by an inhibition of the regenerative acetylcholine release. The antagonism was enhanced when a small concentration of obidoxime was present. Neither enzyme reactivation nor curare-like action was evident. beta-Eudesmol (300 mg/kg, i.p.) elevated the LD50 of diisopropylfluorophosphate (s.c.) in control mice from 4.2 to 6.4 mg/kg and in mice pretreated with atropine from 7.8 to 10.6 mg/kg. In mice pretreated with atropine and obidoxime, beta-eudesmol showed a greater synergistic effect, increasing the LD50 from 281 to more than 800 mg/kg. beta-Eudesmol also markedly alleviated diisopropylfluorophosphate-induced muscle fasciculation, tremor and convulsion and prolonged the time to death. It is proposed that beta-eudesmol may be added to the standard antidotal regimen (atropine plus obidoxime) for treating organophosphate intoxication.

Animals↗
PubMed2003

Human glycine N-methyltransferase is unfolded by urea through a compact monomer state.

Human recombinant glycine N-methyltransferase (GNMT) unfolding by urea was studied by enzyme activity, size-exclusion chromatography, fluorescence spectroscopy, and circular dichroism. Urea unfolding of GNMT is a two-step process. The first transition is a reversible dissociation of the GNMT tetramer to compact monomers in 1.0-3.5M urea with enzyme inactivation. The compact monomers were characterized by Stokes radius (R(s)) of 40.7A equal to that of globular proteins with the same molecular mass as GNMT monomers, absence of exposure of tryptophan residues into solvent, and presence of about 50% of secondary structure of native protein. The second step of GNMT unfolding is a reversible transition of monomers from compact to a fully unfolded state with R(s) of 50A, exposed tryptophan residues, and disrupted secondary structure in 8M urea.

Dimerization↗
PubMed2006

Inflammation, proteases and cancer.

Tumours are complex tissues composed of ever-evolving neoplastic cells, matrix proteins that provide structural support and sequester biologically active molecules, and a cellular stromal component. Reciprocal interactions between neoplastic cells, activated host cells and the dynamic micro-environment in which they live enables tumour growth and dissemination. It has become evident that early and persistent inflammatory responses observed in or around developing neoplasms regulates many aspects of tumour development (matrix remodelling, angiogenesis, malignant potential) by providing diverse mediators implicated in maintaining tissue homeostasis, e.g., soluble growth and survival factors, matrix remodelling enzymes, reactive oxygen species and other bioactive molecules. This review highlights recent insights into the role of chronic inflammation associated with cancer development and examines proteolytic pathways activated by infiltrating leukocytes during neoplastic programming of tissues.

Cytokines↗
PubMed2004

History of forensic serology and molecular genetics in the sphere of activity of the German Society for Forensic Medicine.

In the field of forensic serology, essential developmental impulses have come from the sphere of activity of the German Society for Forensic Medicine. Among these are the orientating enzyme-reactive and specific tests for blood using crystallization tests, the determination of the species-specificity of the donor of the stain and the beginning of the individualization of a stain to its donor. Pioneering work has also been done in the development of blood group serology of the conventional markers. DNA analysis originated in the Anglo-Saxon region. The German Society for Forensic Medicine also contributed to its further progress and essentially influenced it e.g. by the discovery of microsatellite markers, including STRs, by the validation of numerous test methods, by optimization of these methods and by preliminary work for the use of mass spectrometry in DNA analysis.

Europe↗
PubMed2005

Cyto- and chemoarchitecture of the sensory trigeminal nuclei of the echidna, platypus and rat.

We have examined the cyto- and chemoarchitecture of the trigeminal nuclei of two monotremes using Nissl staining, enzyme reactivity for cytochrome oxidase, immunoreactivity for calcium binding proteins and non-phosphorylated neurofilament (SMI-32 antibody) and lectin histochemistry (Griffonia simplicifolia isolectin B4). The principal trigeminal nucleus and the oralis and interpolaris spinal trigeminal nuclei were substantially larger in the platypus than in either the echidna or rat, but the caudalis subnucleus was similar in size in both monotremes and the rat. The numerical density of Nissl stained neurons was higher in the principal, oralis and interpolaris nuclei of the platypus relative to the echidna, but similar to that in the rat. Neuropil immunoreactivity for parvalbumin was particularly intense in the principal trigeminal, oralis and interpolaris subnuclei of the platypus, but the numerical density of parvalbumin immunoreactive neurons was not particularly high in these nuclei of the platypus. Neuropil immunoreactivity for calbindin and calretinin was relatively weak in both monotremes, although calretinin immunoreactive somata made up a large proportion of neurons in the principal, oralis and interpolaris subnuclei of the echidna. Distribution of calretinin immunoreactivity and Griffonia simplicifolia B4 isolectin reactivity suggested that the caudalis subnucleus of the echidna does not have a clearly defined gelatinosus region. Our findings indicate that the trigeminal nuclei of the echidna do not appear to be highly specialized, but that the principal, oralis and interpolaris subnuclei of the platypus trigeminal complex are highly differentiated, presumably for processing of tactile and electrosensory information from the bill.

Animals↗