Biomedical subjects

N Klemm

Publications and source records attributed to N Klemm.

At least 19 recordsLinked to original sources

PubMed1993

Characterization of two cytosolic diacylglycerol kinase forms.

Two forms of rat brain cytosolic diacylglycerol kinase (EC2.7.1.107) were separated by heparin-agarose column chromatography. These forms, designated DGK-I and DGK-II, were not interconvertible as determined by rechromatography. DGK-I and DGK-II had respective molecular masses of 88 and 180 kDa, as measured by Sepharose 6B chromatography. Both forms preferred diacylglycerol over monoacylglycerol and were insensitive to R59022. DGK-II, but not DGK-I, was activated by an activator substance prepared from chicken egg yolk. DGK-II was activated by a rat brain cytosolic activator and was exclusively sensitive to 5'-AMP-mediated inactivation. Further studies revealed that these two forms had the following distinct characteristics: (a) substrate specificity, (b) inhibition by heparin, (c) sensitivity to lysine-containing polyamino acids, and (d) responses to different phospholipids. In general, DGK-II was more responsive to various inhibitors and activators, making it a prime candidate for a regulatable enzyme.

Adenosine Monophosphate↗

PubMed1991

Irreversible inactivation of diacylglycerol kinase-II requires a mediator.

Cytosolic diacylglycerol kinase was irreversibly inactivated by 5'-AMP since the enzyme remained less active after the removal of 5'-AMP by P-10 gel chromatography. The inactivation was time-dependent, suggesting the involvement of a covalent bond modification. A reconstitution experiment detected a rat brain cytosolic mediator for the effect of 5'-AMP. A protein kinase rich fraction prepared from rat liver was also capable of restoring the sensitivity of diacylglycerol kinase-II to 5'-AMP. We propose that 5'-AMP-activated protein kinase is the mediator which inactivates diacylglycerol kinase-II, possibly by phosphorylation.

Adenosine Monophosphate↗

PubMed1990

Indirect inactivation of rat brain cytosolic diacylglycerol kinase by nucleoside-5'-monophosphate.

Cytosolic diacylglycerol kinase was inhibited drastically by nucleoside monophosphate. The inhibition was relatively specific for adenosine-5'-monophosphate (5'-AMP), although uridine-5'-monophosphate was also effective. The effect of 5'-AMP on diacylglycerol kinase appeared to be indirect since the degree of inhibition lessened with the dilution of the cytosol and the more purified enzyme failed to respond to 5'-AMP. A 5'-AMP-dependent mediator is proposed to be involved in the inactivation of diacylglycerol kinase.

Adenosine Monophosphate↗

PubMed1989

Quantitative benedict test using bicinchoninic acid.

Cupric ion (Cu2+), in complex form, functions as a selective oxidizing agent for a variety of compounds in the qualitative Benedict test. Cupric ion is reduced to cuprous ion (Cu+) in the reaction. We found that the cuprous ion formed in this type of redox reaction could be detected and quantified using 2,2'-bicinchoninic acid. This reagent produced an intense purple complex with cuprous ion. The color development in this modified Benedict test was dependent on pH, temperature, and time. The reaction is insensitive to ethanol and sodium dodecyl sulfate. This improved method of the Benedict test has enhanced sensitivity and makes the quantitation of compounds possible. This method should be useful for studies of Benedict-positive compounds which are available only in small amounts.

Biochemistry↗

PubMed1988

An endogenous regulator of diacylglycerol kinase.

During the initial steps of the subcellular fractionation of rat brain homogenate, we recovered more than 100% of diacylglycerol kinase activity. The unusually high yields prompted us to examine the possibility that we had removed an endogenous inhibitor from diacylglycerol kinase during those steps. Our study revealed the existence of a potent inhibitor of diacylglycerol kinase in the crude synaptosomal-mitochondrial fraction (P2 pellet). The inhibitory substance was water soluble upon organic solvent extraction. The inhibitory activity of the substance was retained after extensive dialysis, suggesting the macromolecular nature of this compound. This substance may represent an important physiological regulator of diacylglycerol kinase.

Animals↗

PubMed1988

Inhibition of rat diacylglycerol kinase activity by synthetic L-lysine containing polypeptides.

We investigated whether or not synthetic polymers of amino acids affected the activity of rat diacylglycerol kinase. Among all the polypeptides of amino acids tested, only the polymers containing significant amounts of basic amino acid residues inhibited diacylglycerol kinase activity. Poly-L-lysine was a better inhibitor than poly-L-arginine. Both the density of positive charge and the nature of spacing amino acids of the inhibitory peptides were determining factors in interacting with the enzyme. Various polyamines were ineffective. Our observation indicated that the activity of diacylglycerol kinase might be controlled by a site which binds to a polycation.

Animals↗

PubMed1986

Neurons reactive to antibodies against serotonin in the stomatogastric nervous system and in the alimentary canal of locust and crickets (Orthoptera, Insecta).

Immunoreactive neurons in the stomatogastric nervous system and in the alimentary tract of the locust Schistocerca gregaria and the crickets Gryllus bimaculatus and Acheta domesticus have been examined using antibodies against serotonin (5-hydroxytryptamine; 5-HT). For comparative anatomical analysis cobalt chloride infusion was applied. The innervation of the visceral muscles was studied electron microscopically. In all three species the majority of the 5-HT-immunoreactive cell bodies of the stomatogastric nervous system occur in the frontal ganglion in which 30-40% of the total number of cell bodies react with anti-5-HT. In the occipital ganglion only two to four cell bodies (1-2%) are 5-HT-immunoreactive. Single immunoreactive cell bodies were observed in the ventricular ganglia in only a few preparations. The 5-HT-immunoreactive neurons in the frontal ganglion are pseudounipolar or multipolar. The main process of the 5-HT-immunoreactive neurons of the frontal ganglion descend along the entire stomatogastric nervous system. Some arborizations of these processes ascend into the brain and others supply the neuropil of all stomatogastric ganglia. Side branches leave the stomatogastric nervous system and form a plexus along the surface of the entire intestinal tract from where 5-HT-immunoreactive fibers supply: all muscle layers of the muscularis; the external dilator muscles of the foregut and probably some somatic muscles, neurohaemal organs and Malpighian tubules (excretory system). Serotonin-immunoreactive fibers further proceed into salivary gland and the retrocerebral complex (corpora cardiaca and corpora allata). The retrocerebral glandular complex and the hindgut receive additional immunoreactive neurons from the central nervous system. Electron microscopic analysis of nerves innervating the muscle layers of the alimentary tract revealed one type of 5-HT-immunoreactive and one or two types of non-5-HT-immunoreactive fibers. All fiber types contact the sarcolemma of muscle fibers without any obvious synaptic membrane specializations. The 5-HT-immunoreactive fibers are in some regions in close contact with the haemolymph. These regions can be regarded as sites of neurohormonal release. The distribution of serotonin-immunoreactive neurons suggests that 5-HT acts as a neurotransmitter and/or modulator on intestinal muscles and some somatic muscles and glandular cells, and as a neurohormone released from neurohaemal sites into the body fluid.

Animals↗

PubMed1985

Effect of a hypocholesterolemic agent on cholesteryl ester metabolism in glioblastoma cells.

The hypocholesterolemic agent 3 beta-(2-diethylaminoethoxy)androst-5-en-17-one . HCl (U18666A) is known to induce experimental epilepsy. The possibility that this drug interferes with cholesteryl ester formation in glioblastoma cells was examined. The incorporation of radioactive oleic acid into cellular cholesteryl ester was drastically and specifically inhibited by U18666A. The inhibitory effect of U18666A persisted in different oleic acid concentrations. Kinetic studies revealed the rapidity of U18666A action. U18666A was found to be ineffective in inhibiting acyl-CoA:cholesterol acyltransferase activity when it was added directly to the cell homogenates. In contrast, the acyltransferase activity was greatly diminished in homogenates derived from U18666A-treated cells. Thus, U18666A appeared to block cellular cholesteryl ester biosynthesis by indirectly inactivating acyl-CoA:cholesterol acyltransferase activity in a cell-dependent manner. The potent inhibition of cholesteryl ester formation by U18666A represents one unique aspect of the drug which might contribute to its ability to induce chronic epileptiform activity.

Androstenes↗

PubMed1985

Mapping and ultrastructure of serotonin-immunoreactive neurons in the optic lobes of three insect species.

With antibodies to serotonin (5-HT) we have mapped immunoreactive neurons in the optic lobes of three species, the blowfly Calliphora, the desert ant Cataglyphis, and the worker bee Apis. The main emphasis in this investigation is on a system of 5-HT-positive neurons connecting the most peripheral neuropil of the optic lobes, the lamina, to more central neuropil regions. To aid in electron microscopical identification of these neurons we used immunocytochemistry at the EM-level and Golgi-EM for Calliphora and horseradish peroxidase (HRP) labelling for the other two insects. The immunoreactive terminals in Calliphora and the HRP-labelled ones in the other insects contain large (c. 100 nm) granular vesicles and smaller (c.60 nm) clear vesicles. In Cataglyphis and Apis the profiles with granular vesicles are presynaptic to second order neurons of the lamina, whereas in Calliphora no synaptic contacts were found. In this animal the 5-HT-positive terminals are situated distal to the synaptic layer of the lamina, in a region of retinal photoreceptor axons and perikarya of the lamina monopolar neurons. In Catagylphis and Apis the interactions of the 5-HT-neurons with the laminar neurons might occur through chemical synapses, whereas in Calliphora neuroactive substance could be released non-synaptically from varicosities distal to the synaptic layer. The possible involvement of 5-HT in control of neuronal activity in the optic lobes is discussed.

Animals↗

PubMed1985

Dopamine-beta-hydroxylase-like immunoreactive neurons in two insect species, Calliphora erythrocephala and Periplaneta americana.

The localization of dopamine-beta-hydroxylase in the cephalic central nervous system of the blowfly (Calliphora erythrocephala) and the cockroach (Periplaneta americana) was investigated. Immunoreactive neurons were demonstrated in both species. The results were compared with the known distribution of catecholamines in the brain of both species. In certain cell groups and neuropilar regions of both species D beta H-immunoreactivity coincides with the presence of catecholamines. Additionally D beta H immunoreactivity was found in several cell bodies and neuropilar regions in which no catecholamines could be detected. A correlation between the presence of octopamine and anti-D beta H labelling was not found. Thus it seems that the D beta H-immunoreactivity neither indicates the presence of octopamine nor is it limited to noradrenaline-containing neurons. Parallel findings in vertebrates are discussed.

Animals↗

PubMed1985

Induction of intracellular acyl-CoA:cholesterol acyltransferase activity in glioblastoma cells by lidocaine.

The perturbation of cellular cholesteryl ester biosynthesis in glioblastoma C-6 cells by lidocaine was investigated. Lidocaine specifically inhibited the incorporation of radioactive oleic acid into cellular cholesteryl ester but had no significant effect on the incorporation of oleic acid into phosphatidylcholine. Oxygenated cholesterol-enhanced cholesteryl ester formation was less sensitive to lidocaine inhibition. Several other local anesthetics were compared. Lidocaine altered cholesteryl ester formation in time- and dose-dependent manners. Lidocaine was a powerful inhibitor initially and its potency declined with time. Lidocaine was capable of directly inhibiting acyl-CoA:cholesterol acyltransferase (ACAT) activity in broken cell homogenates. The lidocaine-mediated inhibition of cellular cholesteryl ester formation triggered an enhanced intracellular ACAT activity that was not fully expressed in the presence of lidocaine. The activation of ACAT activity by lidocaine might represent a compensatory mechanism by which the inhibitory effect of lidocaine was partially overcome with time.

Anesthetics, Local↗

PubMed1984

Serotonin-immunoreactive neurons in the brain of the honeybee.

The distribution of serotonin-immunoreactive neurons in the brain of the worker honey bee Apis mellifera was studied by means of immunocytochemical staining by using a well-characterized antibody to serotonin (5-HT). About 75 immunoreactive perikarya are grouped into clusters in the optic lobe and in the median and dorsal protocerebrum. Immunoreactive fibers were resolved in all areas of the brain. The optic lobe shows restricted layers of 5-HT-immunoreactive fibers in the lamina and medulla organized perpendicular to the retinotopic elements. Immunoreactive fibers in the lobula represent invasions of protocerebral giant wide-field neurons. The nonglomerular neuropil of the brain exhibits a meshwork of immunoreactive fibres invading glomerular neuropil of the mushroom bodies, central body complex, and antennal lobes. Mushroom body stalks and lobes contain immunoreactive fibers arranged perpendicular to the Kenyon cell fibers and matching subcompartments of these corpora pedunculata areas. The calyces are devoid of immunofluorescence. Serotonin-positive fibres in the central body complex are arranged in its subcompartments. No 5-HT immunoreactivity was found in the pons. Antennal glomeruli contain immunoreactive fibers restricted around the margin of the glomeruli. The selective mapping of 5-HT-immunoreactive neurons complements studies on the distribution of monoamine-containing neurons in the bee brain. Serotonin- and catecholamine-containing neurons often occur together in the same brain areas and subcompartments. The immunohistochemical approach in chemoneuroanatomy gives new evidence for a more complicated architecture of the brain than could be deduced from the classical neuroanatomical studies.

Animals↗

PubMed1984

Distribution of serotonin-containing neurons and their pathways in the supraoesophageal ganglion of the cockroach Periplaneta americana (L.) as revealed by immunocytochemistry.

The distribution of serotonin (5-HT)-containing neurons in the supraoesophageal (cerebral) ganglion of the cockroach Periplaneta americana was studied using immunocytochemistry and the formaldehyde histofluorescence method ( Klemm , '83). In this material immunocytochemistry was more sensitive than the formaldehyde histofluorescence procedure. A relatively small number of 5-HT-immunoreactive cell bodies (220-280) were found. For the first time, their processes could be followed. They highly arborize and innervate many brain regions. Three patterns of monoamine innervation have been demonstrated: (1) 5-HT and catecholamine fibres ( Klemm , '83) occurring in the same region (e.g., outer lateral protocerebral neuropil, stratum caudale , mushroom body, fan-shaped body, olfactory lobe), but having certain differences with respect to the organization of their projection fields; (2) 5-HT fibres innervating a region lacking catecholamine-containing fibres (pons); and (3) catecholamine neurons innervating a region lacking 5-HT fibres (ellipsoid body). In the mushroom body only the extrinsic neurons contain 5-HT immunoreactivity. They form a commissural fibre system linking the left- and right-hand mushroom bodies and other brain regions. The pons is part of a 5-HT-neuron fibre system innervating many areas including the mushroom bodies. The present study demonstrates novel, complex, and widely distributed connections within the insect brain.

Animals↗

PubMed1984

Acyl-CoA cholesterol acyltransferase in cultured glioblastoma cells.

We investigated the incorporation of radioactive precursors into cholesteryl ester in cultured glioblastoma cells. It was found that polar cholesterol derivatives and exogenous cholesterol contained in lipoprotein complexes greatly enhanced intracellular cholesteryl ester formation. The direct transfer of the acyl moiety from acyl-CoA to free cholesterol was demonstrated in broken cell preparations. Further evidence of the existence of the acyl-CoA:cholesterol acyltransferase (ACAT) in glioblastoma cells came from the conversion of radioactive cholesterol to cholesteryl ester by glial cell homogenates. The characteristics of the enzymic assay were studied in detail. This enzymic activity was greatly enhanced in homogenates prepared from 7-ketocholesterol-treated cells. Thus, cells more active in cholesterol esterification in cell-free preparations. The marked inhibition of intracellular cholesteryl ester formation in intact cells by progesterone is a strong argument for the exclusive role of ACAT in glioblastoma cells. Similar properties of cholesteryl ester biosynthesis have been observed in neuroblastoma cells and primary brain cell cultures. In conclusion, the same enzyme is involved in cholesteryl ester biosynthesis in all neural cells. Neural and nonneural cells share many fundamental characteristics of cholesteryl ester formation.

Animals↗

PubMed1984

Insensitivity of ubiquinone biosynthesis in glioblastoma cells to an epileptogenic drug, U18666A.

To investigate the perturbation of ubiquinone biosynthesis by a hypocholesterolemic drug, 3 beta-(2-diethylaminoethoxy)androst-5-en-17-one hydrochloride (U18666A), we measured the incorporation of radioactive mevalonate, methionine, tyrosine, and 4-hydroxybenzoic acid into ubiquinone in glioblastoma cells. These four precursors unanimously showed that ubiquinone biosynthesis was not significantly altered by U18666A, which blocked cholesterol biosynthesis at steps beyond mevalonate formation. The fluctuation of the endogenous mevalonate level had little effect on ubiquinone biosynthesis, implying the relative stability of cellular ubiquinone biosynthesis. Furthermore, exogenously added mevalonate did not have an appreciable effect on ubiquinone biosynthesis. The major ubiquinone produced in rat glioblastoma cells was identified as ubiquinone-9. The mevalonate-derived products accumulated in the U18666A-treated cells differed significantly from those reported in a broken cell study, suggesting the existence of delicate mechanisms regulating the formation of cholesterol intermediates.

Androstenes↗

PubMed1984

Stimulation of fatty acid release in glioblastoma cells by caffeine.

Caffeine, a well-known behavior stimulant, was found to activate the fatty acid release mechanism in glioblastoma cells. The enhancement of the liberation of fatty acids from esterified complex lipids by caffeine was time dependent. Cell density had a strong influence on the responsiveness to caffeine. The mobilization of unsaturated fatty acids were preferentially stimulated by caffeine. Therefore, caffeine may alter membrane structure and enhance eicosanoid biosynthesis.

Arachidonic Acid↗

PubMed1983

Organization of catecholamine and serotonin-immunoreactive neurons in the corpora pedunculata of the desert locust, Schistocerca gregaria Forsk.

Catecholamine (CA)-containing and serotonin (5-HT)-immunoreactive fibers were shown in the mushroom body of Schistocerca gregaria. Both fiber systems occupy distinct areas in the mushroom body. None of these amines could be detected in the Kenyon cell bodies. The 5-HT fibers and many of the CA fibers originate from few extrinsic neurons with large and highly arborized processes concentrated in the commissural pathway linking mushroom bodies to each other and to different brain regions (e.g. optic lobe, lateral neuropil).

Animals↗

PubMed1983

Monoamine-containing neurons and their projections in the brain (supraoesophageal ganglion) of cockroaches. AN aldehyde fluorescence study.

Fluorogenic monoamines were studied in the brain of three cockroach species by use of aldehyde-fluorescence techniques. All three optic ganglia contain fluorogenic monoamines. The lamina contains fibres with an indolylalkylamine-fluorophore. The medulla is innervated by local CA neurons which contribute to four fluorescent strata. The lobula receives both CA- and 5-HT-fibres, predominantly of central origin. CA occur in almost all areas of the brain. The areas are interconnected by a CA-fibre system. All parts of the mushroom body are innervated by CA-fibres from the surrounding neuropil. The CA innervation in the mushroom body divides it into a fronto-ventral part (alpha-lobe, beta-lobe, anterio-ventral peduncle) and a dorso-caudal part (caudo-dorsal peduncle, calices) leaving a fluorescence-free central part of the peduncle in between. CA-fibres run between the mushroom bodies of both hemispheres and also between the mushroom body and the lobula. The central body complex contains CA. The pons aggregates indolylalkylamine-containing fibres. The olfactory glomeruli are surrounded by CA-fibres originating from deutocerebral cell bodies. CA-fibres are further linked to the protocerebral neuropil. CA-fibre tracts pass from the brain to the suboesophageal ganglion and the stomatogastric nervous system. The cell bodies of the frontal ganglion are of indolylalkylamine type. Non-fluorescent neuropils (n. ocellaris, tractus olfactorio-globularis, lobus glomerulatus) are innervated by the CA-fibre system.

Aldehydes↗