Showing papers by "University of Würzburg published in 1991"

Development and function of T cells in mice rendered interleukin-2 deficient by gene targeting.

Abstract: Interleukin-2 (IL-2) is a lymphocytotropic hormone which is thought to have a key role in the immune response of mammalian cells. It is produced by a subpopulation of activated T-lymphocytes and acts in vitro as the principal auto- and paracrine T-cell growth factor (for reviews see refs 1-3). IL-2 is, however, not the sole T-cell growth factor, nor does it act exclusively on T cells, also promoting growth of NK cells and differentiation of B cells. A role for IL-2 in T-cell development has been postulated but remains controversial. Here we test the requirement for IL-2 in vivo using IL-2-deficient mice generated by targeted recombination. We find that mice homozygous for the IL-2 gene mutation are normal with regard to thymocyte and peripheral T-cell subset composition, but that a dysregulation of the immune system is manifested by reduced polyclonal in vitro T-cell responses and by dramatic changes in the isotype levels of serum immunoglobulins.

Isolation and direct characterization of resident microglial cells from the normal and inflamed central nervous system.

Abstract: In addition to the major population of infiltrating leukocytes recovered from inflamed rat central nervous system (CNS), all of which expressed high levels of leukocyte common antigen CD45, many cells were coisolated that were MRC OX42+ (complement receptor 3/CD11b) but expressed low-to-moderate levels of CD45 and major histocompatibility complex (MHC) class I molecules. Most cells from normal CNS, in contrast, lay within this latter, CD45low population. From previous in situ immunohistochemical studies, the fortuitously isolated CD45low cells were probably resident (ramified) microglia. Using irradiation chimeras, we show that resident microglia respond to inflammation by upregulating CD45, CD4, and MHC class I molecules with a minority of these cells increasing their expression of MHC class II molecules. A 3- to 4-fold increase in the number of microglia isolated from inflamed CNS provided indirect evidence that the cells had proliferated. In normal CNS, a very small population of blood-derived CD45high-expressing cells are present; most MHC class II expression is associated with these few cells and not with the resident microglia.

Selective increase of iron in substantia nigra zona compacta of parkinsonian brains.

Abstract: Histochemical and biochemical determinations of total iron, iron (II), and iron (III) contents in brain regions from Parkinson's and Alzheimer's diseases have demonstrated a selective increase of total iron content in parkinsonian substantia nigra zona compacta but not in the zona reticulata. The increase of iron content is mainly in iron (III). The ratio of iron (II):iron (III) in zona compacta changes from almost 2:1 to 1:2. This change is thought to be relevant and may contribute to the selective elevation of basal lipid peroxidation in substantia nigra reported previously. Iron may be available in a free state and thus can participate in autooxidation of dopamine with the resultant generation of H2O2 and oxygen free radicals.

Kurzmitteilung / Short Communication A Convenient Preparation of Acetone Solutions of Dimethyldioxirane

Abstract: A novel simplified procedure affords consistently 0.09-0.10 M solutions of distilled dimethyldioxirane in acetone; other than control of the reaction temperature below 15° and vigorous mechanical stirring, no other precautions are maniatory.

Iron‐Melanin Interaction and Lipid Peroxidation: Implications for Parkinson's Disease

Abstract: The vulnerability of substantia nigral (SN) melaninized dopamine neurons to neurodegeneration in Parkinson's disease and the selective increases of iron and basal lipid peroxidation in SN indicate that iron-melanin interaction could be crucial to the pathogenesis of this disease. The present study describes, for the first time, the identification and characterization of a high-affinity (KD = 13 nM) and a lower affinity (KD = 200 nM) binding site for iron on dopamine melanin. The binding of iron to melanin is dependent on pH and the concentration of melanin. Iron chelators, U74500A, desferrioxamine, and to less extent 1,10-phenanthroline and chlorpromazine, but not the Parkinson-inducing neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, can inhibit the binding of iron to melanin and iron-induced lipid peroxidation. Although melanin alone diminishes basal lipid peroxidation in rat cortical homogenates, it can also potentiate that initiated by iron, a reaction inhibited by desferrioxamine. In the absence of an identifiable exogenous or endogenous neurotoxin in idiopathic Parkinson's disease, iron-melanin interaction in pars compacta of SN may be a strong candidate for the cytotoxic component of oxygen radical-induced neurodegeneration of melaninized dopamine neurons.

Shedding of ICAM-1 from human melanoma cell lines induced by IFN-gamma and tumor necrosis factor-alpha. Functional consequences on cell-mediated cytotoxicity.

Abstract: ICAM-1-mediated cell-cell adhesion is essential for various immunologic functions, including non-MHC-restricted cytotoxicity. The present study was designed to establish whether shedding of ICAM-1 from melanoma cells occurred and to characterize the effects of soluble ICAM-1 on some cell adhesion-dependent functions. The shed soluble ICAM-1 molecule was detected and quantified by a specific ELISA. Shedding of ICAM-1 could be induced by IFN-gamma and TNF-alpha alone, or more effectively, by a combination of the two cytokines together. The use of purified soluble ICAM-1 enabled us to test for the functional significance of the ICAM-1 shedding from tumor cells. Conjugate formation between the cloned NK cell line CNK6 and the erythromyeloid cell line K562, as well as between lymphokine-activated killer cells and the melanoma cell line M26, could be inhibited by purified soluble ICAM-1 and cell-free supernatants from melanoma cell cultures containing shed ICAM-1. Furthermore, the non-MHC-restricted cytotoxicity mediated by NK and lymphokine-activated killer cells could be abrogated either by purified soluble ICAM-1 or by melanoma cell culture supernatants containing shed ICAM-1. Thus, shedding of ICAM-1 may be one of the mechanisms by which neoplastic cells escape immunosurveillance.

The extraction of soil water by the suction-cup method: a review

Abstract: SUMMARY This article deals with the extraction of soil water using the suction-cup method and its accompanying problems. This method has become well-developed over the past 20 years. It allows continuous sampling during any period and, if necessary, at several different depths of a soil profile. The installation of the suction probe is easy and the profile is only negligibly disturbed. Some problems may occur when this technique is used. The spatial variability of the properties investigated is often underestimated and must be clarified by sufficient replication. An important point of discussion is the by passing of water flowing through macropores. The problems of alteration of the sample by the sampling system also deserve attention. These can be the ‘filter effect’ as regards macromolecules and colloids, the sorption of trace substances, and the gas exchange between the sample and the atmosphere of the sampling system.

Use of the polymerase chain reaction for direct detection of Listeria monocytogenes in soft cheese

Abstract: The polymerase chain reaction (PCR) amplification technique was investigated as a tool for direct detection of Listeria monocytogenes in soft cheeses. Different sets of oligonucleotide primers were used, and parts of the L. monocytogenes Dth 18-gene could be amplified specifically when either a plasmid vector carrying the cloned gene or chromosomal DNA was used a template. The detection limit for L. monocytogenes in dilutions of pure cultures was between 1 and 10 colony-forming units. In extracts from soft cheeses containing L. monocytogenes DNA, the amplification was strongly inhibited. This inhibition could be reduced by an additional purification step. Despite this the detection limit showed a large variation, depending on the brand of cheese used. In some cheeses 10(3) cfu/0.5g could be visualized whereas in others the presence of 10(8) cfu/0.5 g did not yield a detectable quantity of amplified product.

Basic organization of operant behavior as revealed in Drosophila flight orientation.

Abstract: Operant behavior is studied in tethered Drosophila flies using visual motion, heat or odour as operandum and yaw torque, thrust or direction of flight as operans in various combinations (Fig. 1). On the basis of these results a conceptual framework of operant behavior is proposed: (1) It requires a goal (desired state) of which the actual state deviates. (2) To attain the goal a range of motor programs is activated (initiating activity, see Fig. 7). (3) Efference copies of the motor programs are compared to the sensory input referring to the deviation from the desired state (e.g. by cross-correlation). (4) In case of a significant coincidence the respective motor program is used to modify the sensory input in the direction towards the goal. (5) Consistent control of a sensory stimulus by a behavior may lead to a more permanent behavioral change (conditioning). In this scheme operant activity (1-4) and operant conditioning (1-5) are distinguished.

Human T lymphocytes recognize a peptide of single point-mutated, oncogenic ras proteins.

Abstract: P21ras proteins are thought to play an important role in cell proliferation and differentiation. Single nucleotide mutations in the encoding cellular proto-oncogenes often result in p21ras proteins with transforming activity. Such activated ras oncogenes have been demonstrated in a variety of human malignancies and also in preneoplastic changes. Using a synthetic peptide corresponding to amino acids 5-16 of mutated p21ras proteins with an exchange of the normal glycine at position 12 by valine, it is shown here that human CD4+ T cells specifically recognize the mutated protein sequence and can be generated as antigen-specific T lymphocyte lines. The fact that these T lines did not crossreact to the sequence of normal p21ras proteins offers new perspectives for specific immunotherapy of human malignancies and even precancerous lesions.

Estimates of nitrogen fixation by trees on an aridity gradient in Namibia.

Abstract: Nitrogen (N2) fixation was estimated along an aridity gradient in Namibia from the natural abundance of 15N (δ15N value) in 11 woody species of the Mimosacease which were compared with the δ15N values in 11 woody non-Mimosaceae Averaging all species and habitats the calculated contribution of N2 fixation (N f ) to leaf nitrogen (N) concentration of Mimosaceae averaged about 30%, with large variation between and within species While in Acacia albida N f was only 2%, it was 49% in Acacia hereroensis and Dichrostachys cinerea, and reached 71% in Acacia melifera In the majority of species N f was 10–30% There was a marked variation in background δ15N values along the aridity gradient, with the highest δ15N values in the lowland savanna The difference between δ15N values of Mimosaceae and non-Mimosaceae, which is assumed to result mainly from N2 fixation, was also largest in the lowland savanna Variations in δ15N of Mimosaceae did not affect N concentrations, but higher δ15N-values of Mimosaeae are associated with lower carbon isotope ratios (δ13C value) N2 fixation was associated with reduced intrinsic water use efficiency The opposite trends were found in non-Mimosaceae, in which N-concentration increased with δ15N, but δ13C was unaffected The large variation among species and sites is discussed

Signal transduction by cGMP in heart.

Abstract: Early studies in whole heart indicated that cGMP antagonized the positive inotropic effects of catecholamines and cAMP. However, the regulation of cGMP levels by a variety of agents was not always consistent with their effects on contractility. It is now clear that at least two major cell types in whole heart, cardiac myocytes and vascular smooth muscle cells, differ markedly in their mechanisms of cGMP regulation and response to cGMP. Furthermore, experiments on isolated cardiac myocytes indicate that the mechanism of cGMP action even in this single cell type can be multifaceted. Cyclic GMP inhibits the L-type calcium channel current (ICa), which is the major source of Ca++ entry into heart cells, and which plays a predominant role in the initiation and regulation of cardiac electrical and contractile activities. Patch-clamp measurements of ICa indicate that in isolated frog myocytes cGMP inhibits ICa by stimulation of cAMP phosphodiesterase (cGS-PDE), whereas in purified rat ventricular myocytes, cGMP predominantly inhibits ICa via a mechanism involving cGMP-dependent protein kinase (cGMP-PK). Under certain conditions, cGMP can also inhibit a cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) and thereby produce a stimulatory effect on ICa. Biochemical characterization of the endogenous PDEs and cGMP-PK in purified cardiac myocytes provided further evidence in support of these mechanisms of cGMP action on ICa.

Hippocampal neuron number in schizophrenia. A stereological study.

Abstract: • Neuropathologic and neuroradiologic studies have reported hippocampal abnormalities in schizophrenics. We estimated the total number of neurons in the hippocampus of schizophrenics and controls to elucidate the neuronal basis of such changes. Thirteen brains of schizophrenics and 13 control brains closely matched for sex and age were studied. A new stereological method was applied to serial coronal sections through the whole hippocampus. Total hippocampal volume was reduced in the schizophrenic sample, more pronounced on the left side, but mean differences were not significant. The volumes of the pyramidal cell layer in the four subdivisions subiculum and cornu Ammonis sectors CA 1, CA 2/3, and CA 4 were almost identical in both groups. Schizophrenics did not differ from controls with regard to nerve cell density in any of the four subdivisions. The estimates of the total number of neurons in the hippocampal subdivisions were not different between schizophrenics and controls. The data do not support the hypothesis that hippocampal abnormalities are caused by neuronal cell loss. However, they are consistent with the suggestion that white matter changes in the hippocampus may play a role in the pathogenesis of schizophrenia.

Ion Relations of Symplastic and Apoplastic Space in Leaves from Spinacia oleracea L. and Pisum sativum L. under Salinity

Abstract: Salt tolerant spinach (Spinacia oleracea) and salt sensitive pea (Pisum sativum) plants were exposed to mild salinity under identical growth conditions. In order to compare the ability of the two species for extra- and intracellular solute compartmentation in leaves, various solutes were determined in intercellular washing fluids and in aqueously isolated intact chloroplasts. In pea plants exposed to 100 millimolar NaCl for 14 days, apoplastic salt concentrations in leaflets increased continuously with time up to 204 (Cl(-)) and 87 millimolar (Na(+)), whereas the two ions reached a steady concentration of only 13 and 7 millimolar, respectively, in spinach leaves. In isolated intact chloroplasts from both species, sodium concentrations were not much different, but chloride concentrations were significantly higher in pea than in spinach. Together with data from whole leaf extracts, these measurements permitted an estimation of apoplastic, cytoplasmic, and vacuolar solute concentrations. Sodium and chloride concentration gradients across the tonoplast were rather similar in both species, but spinach was able to maintain much steeper sodium gradients across the plasmamembrane compared with peas. Between day 12 and day 17, concentrations of other inorganic ions in the pea leaf apoplast increased abruptly, indicating the onset of cell disintegration. It is concluded that the differential salt sensitivity of pea and spinach cannot be traced back to a single plant performance. Major differences appear to be the inability of pea to control salt accumulation in the shoot, to maintain steep ion gradients across the leaf cell plasmalemma, and to synthesize compatible solutes. Perhaps less important is a lower selectivity of pea for K(+)/Na(+) and NO(3) (-)/Cl(-) uptake by roots.

Detection of a gene encoding a phosphatidylinositol‐specific phospholipase C that is co‐ordinately expressed with listeriolysin in Listeria monocytogenes

Abstract: A phosphatidylinositol-specific phospholipase C (PI-PLC) that is unique to the pathogenic Listeria species L. monocytogenes and L. ivanovii has been detected. Deletion analysis performed with Escherichia coli recombinants expressing PI-PLC activity together with maxicell analysis showed that a 34 kDa polypeptide was responsible for this activity. Nucleotide sequencing revealed that the gene encoding this polypeptide comprises 317 amino acid residues with a 22-amino-acid signal peptide. This gene, designated pic for phosphatidylinositol-specific phospholipase C, is located back to back with the listeriolysin gene on the chromosome of L. monocytogenes where these genes are transcribed by divergent non-overlapping promoters. Expression of the pic gene is dependent on the product of the prfA gene, which also regulates expression of the listeriolysin gene in L. monocytogenes.

Changes in tonic descending inhibition of spinal neurons with articular input during the development of acute arthritis in the cat

Abstract: 1. In 15 alpha-chloralose-anesthetized cats we studied the presence of tonic descending inhibition (TDI) of spinal neurons with input from the knee and its modulation during an acute inflammation of this joint. TDI of spinal neurons with articular input was assessed by applying reversible cold blocks to the lower thoracic cord. The amount of descending inhibition was estimated from the induction and/or increase of resting discharges and of the responses to mechanical stimuli to the knee and other structures during the transitory and reversible blocks. In each experiment one or a few neurons were investigated while the joint was in normal condition [altogether 15 nociceptive-specific (NS) and 6 wide-dynamic-range (WDR) neurons]. One of the neurons was then selected for long-term recordings during which an acute inflammation in the knee was induced by the intra-articular injection of kaolin and carrageenan. Before and during developing arthritis, cold blocks were applied to examine whether the amount of TDI would change during the inflammatory process. 2. The neurons with input from the normal knee were under TDI because application of the cold block induced or increased resting discharges and the responses to noxious compression of the knee and the adjacent thigh and lower leg. In 10 of 15 NS neurons, the response threshold was lowered into the innocuous range. In 9 of 17 cells tested, the excitatory receptive field expanded to the ipsilateral paw, and 4 neurons became inhibited by paw compression. Seven of 18 neurons tested revealed inhibitory receptive fields on the contralateral leg during cold block. The neurons were located in laminae IV-VII. 3. Fourteen neurons were continuously monitored during development of inflammation, and changes in the effectiveness of TDI were assessed by blocking the cord before and during the development of arthritis. In most neurons baseline resting activity in the intact state of the cord increased while the arthritis developed. This inflammation-evoked enhancement of resting discharges was more pronounced during periods of spinalization. Consequently, the differences between the resting discharges in the cold-blocked and the intact state were progressively enhanced in arthritis. 4. After induction of arthritis, the responses to compression of the knee joint increased in the intact state as well as during cold blocks. In 11 of 14 neurons, the differences between the responses in the spinal and intact state were progressively enlarged during the development of inflammation. A similar result was obtained for flexion of the injected knee.(ABSTRACT TRUNCATED AT 400 WORDS)

Serum interleukin-2 concentrations in Guillain-Barré syndrome and chronic idiopathic demyelinating polyradiculoneuropathy: Comparison with other neurological diseases of presumed immunopathogenesis

Abstract: Serum concentrations of the cytokine interleukin-2 (IL-2) were quantitated by enzyme-linked immunosorbent assay in 42 patients with Guillain-Barre syndrome, 15 patients with chronic idiopathic demyelinating polyradiculoneuropathy, 37 patients with other neuropathies, 54 patients with other noninflammatory, nondemyelinating neurological disorders, and 26 healthy control subjects. We found markedly increased serum levels of IL-2 in patients with Guillain-Barre syndrome and to a much lesser extent, in patients with chronic idiopathic demyelinating polyradiculoneuropathy. Increased serum concentrations of IL-2 in patients with Guillain-Barre syndrome returned to normal in parallel with recovery from the disease. These findings suggest ongoing T-cell proliferation in patients with Guillain-Barre syndrome and some patients with chronic idiopathic demyelinating polyradiculoneuropathy. IL-2 levels were also raised in patients with active multiple sclerosis, myasthenia gravis, and herpes simplex encephalitis, and some patients with polymyositis, invoking T cells in the pathogenesis of these diseases.

A subcutaneous delta-positive T-cell lymphoma that produces interferon gamma.

Abstract: WE report an unusual form of cutaneous T-cell lymphoma characterized by subcutaneous tumors that regress spontaneously, fever, progressive leukopenia, high levels of interferon gamma in the serum, and an increased natural-killer-cell activity of peripheral-blood mononuclear cells. Histologically, the infiltrate consists of nonepidermotropic, pleomorphic lymphoid cells located in the subcutaneous fatty tissue. These cells express the phenotype of immature (CD1+, CD3+, CD4-, CD5-, CD8-), activated (CD30+, CD25+, HLA-DR+) T cells positive for delta T-cell receptors. They express the natural-killer-cell phenotype (CD56+) and produce interferon gamma in cell culture. Case Report A 70-year-old woman presented in March 1985 with recurrent, spontaneously regressing, . . .

Molecular cloning, sequencing, and identification of a metalloprotease gene from Listeria monocytogenes that is species specific and physically linked to the listeriolysin gene.

Abstract: The entire nucleotide sequence of an open reading frame located immediately downstream of the listeriolysin gene from a virulent Listeria monocytogenes serotype 1/2a strain was determined. The product of the open reading frame was 510 amino acids with a predicted molecular weight of 57,400. The deduced amino acid sequence of this open reading frame is highly similar to that of a family of secreted metalloproteases produced by various members of the genus Bacillus, of which thermolysin is the prototype. Immunoblots performed with specific antisera raised against thermolysin from Bacillus stearothermophilus allowed the detection of a 60-kDa polypeptide, corresponding to the pro-form of the protease, in culture supernatants of L. monocytogenes strains. In maxicell experiments, Escherichia coli recombinants harboring this open reading frame also specifically directed production of a 60-kDa protein. Protease activity was low to undetectable in both Listeria strains and E. coli recombinants. This is due to lack of processing of the inactive pro-form of the protease to its mature active form in both species. We have designated this gene mpl for metalloprotease of L. monocytogenes. The gene was present only in pathogenic L. monocytogenes strains, in which it was physically linked to the listeriolysin gene.

Characterization of Sp-5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole- 3',5'-monophosphorothioate (Sp-5,6-DCl-cBiMPS) as a potent and specific activator of cyclic-AMP-dependent protein kinase in cell extracts and intact cells.

Abstract: A newly designed cyclic AMP (cAMP) analogue, Sp-5,6-dichloro-1-beta-D- ribofuranosylbenzimidazole-3',5'-monophosphorothioate (Sp-5,6-DCl-cBiMPS), and 8-(p-chlorophenylthio)-cAMP (8-pCPT-cAMP) were compared with respect to their chemical and biological properties in order to assess their potential as activators of the cAMP-dependent protein kinases (cAMP-PK) in intact cells. Sp-5,6-DCl-cBiMPS was shown to be both a potent and specific activator of purified cAMP-PK and of cAMP-PK in platelet membranes, whereas 8-pCPT-cAMP proved to be a potent activator of cAMP-PK and cyclic-GMP-dependent protein kinase (cGMP-PK) both as purified enzymes and in platelet membranes. Sp-5,6-DCl-cBiMPS was not significantly hydrolysed by three types of cyclic nucleotide phosphodiesterases, whereas 8-pCPT-cAMP (and 8-bromo-cAMP) was hydrolysed to a significant extent by the Ca2+/calmodulin-dependent phosphodiesterase and by the cGMP-inhibited phosphodiesterase. The apparent lipophilicity, a measure of potential cell-membrane permeability, of Sp-5,6-DCl-cBiMPS was higher than that of 8-pCPT-cAMP. Extracellular application of Sp-5,6-DCl-cBiMPS to intact human platelets reproduced the pattern of protein phosphorylation induced by prostaglandin E1, a cAMP-increasing inhibitor of platelet activation. In intact platelets, Sp-5,6- DCl-cBiMPS was also more effective than 8-pCPT-cAMP in inducing quantitative phosphorylation of the 46/50 kDa vasodilator-stimulated phosphoprotein (VASP), a major substrate of cAMP-PK in platelets. As observed with prostaglandin E1, pretreatment of human platelets with Sp-5,6-DCl-cBiMPS prevented the aggregation induced by thrombin. The results suggest that Sp-5,6-DCl-cBiMPS is a very potent and specific activator of cAMP-PK in cell extracts and intact cells and, in this respect, is superior to any other cAMP analogue used for intact-cell studies. In contrast with 8-pCPT-cAMP, Sp-5,6-DCl-cBiMPS can be used to distinguish the signal-transduction pathways mediated by cAMP-PK and cGMP-PK.