Showing papers by "University of Würzburg published in 1994"

Associative odor learning in Drosophila abolished by chemical ablation of mushroom bodies

Abstract: The corpora pedunculata, or mushroom bodies (MBs), in the brain of Drosophila melanogaster adults consist of approximately 2500 parallel Kenyon cell fibers derived from four MB neuroblasts. Hydroxyurea fed to newly hatched larvae selectively deletes these cells, resulting in complete, precise MB albation. Adult flies developing without MBs behave normally in most respects, but are unable to perform in a classical conditioning paradigm that tests associative learning of odor cues and electric shock. This deficit cannot be attributed to reductions in olfactory sensitivity, shock reactivity, or locomotor behavior. The results demonstrate that MBs mediate associative odor learning in flies.

Effects of short chain fatty acids on gut morphology and function

Abstract: Short chain fatty acids (SCFAs) are the products of colonic bacterial degradation of unabsorbed starch and non-starch polysaccharide (fibre). They are important anions in the colonic lumen, affecting both colonocyte morphology and function. The three main acids (acetate, propionate, and butyrate) stimulate colonic sodium and fluid absorption and exert proliferative effects on the colonocyte. Experimental animal studies have shown that they promote adaptive responses to small intestinal resection and colonic anastomosis. Acetate increases colonic blood flow and enhances ileal motility. Butyrate has been shown to be the preferred energy substrate for the colonocyte and to be a potent differentiating agent in cell culture. Butyrate may also have a role in preventing certain types of colitis. A diet low in resistant starch and fibre, which will result in a low production of SCFAs in the colon, may explain the high occurrence of colonic disorders seen in the Western civilization.

Drug excretion mediated by a new prototype of polyspecific transporter

Abstract: Cationic drugs of different types and structures (antihistaminics, antiarrhythmics, sedatives, opiates, cytostatics and antibiotics, for example) are excreted in mammals by epithelial cells of the renal proximal tubules and by hepatocytes in the liver In the proximal tubules, two functionally disparate transport systems are involved which are localized in the basolateral and luminal plasma membrane and are different from the previously identified neuronal monoamine transporters and ATP-dependent multidrug exporting proteins Here we report the isolation of a complementary DNA from rat kidney that encodes a 556-amino-acid membrane protein, OCT1, which has the functional characteristics of organic cation uptake over the basolateral membrane of renal proximal tubules and of organic cation uptake into hepatocytes OCT1 is not homologous to any other known protein and is found in kidney, liver and intestine As OCT1 translocates hydrophobic and hydrophilic organic cations of different structures, it is considered to be a new prototype of polyspecific transporters that are important for drug elimination

Organization of the human serotonin transporter gene.

Abstract: The gene encoding the human serotonin transporter (5-HTT) has been isolated and characterized. The human 5-HTT gene is composed of 14 exons spanning approximately 31 kb. The sequence of all exons including adjacent intronic sequences and a tandem repeat DNA polymorphism (VNTR) has been determined and deposited in the EMBL/GenBank data base with the accession numbers X76753 to X76762. The characterization of 5-HTT gene will aid to advance molecular pharmacologic studies of 5-HT uptake regulation and facilitate investigations of its role in psychiatric disorders.

An improved method, using saturating light pulses, for the determination of photosystem I quantum yield via P700 + -absorbance changes at 830 nm

Abstract: An improved method is introduced for the determination of the quantum yield of photosystem I. The new method employs saturating light pulses with steep rise characteristics to distinguish, in a given physiological state, centers with an open acceptor side from centers with a reduced acceptor side. The latter do not contribute to PSI quantum yield (ΦI). Oxidation of P700 is measured by a rapid modulation technique using the absorbance change around 830 nm. The quantum yield ΦI is calculated from the amplitude of the rapid phase of absorbance change (ΔA; 830 nm) upon application of a saturation pulse in a given state, divided by the maximal ΔA (830 nm) which is induced by a saturation pulse with far-red background illumination. Using this technique, ΦI can be determined even under conditions of acceptor-side limitation, as for example in the course of a dark-light induction period or after elimination of CO2 from the gas stream. Thus determined ΦI values display a close-to-linear relationship with those for the quantum yield of PSII (ΦII) calculated from chlorophyll fluorescence parameters. It is concluded that the proposed method may provide new information on the activity of the PSI acceptor side and thus help to separate the effects of acceptorside limitation from those of cyclic PSI, whenever a non-linear relationship between ΦII and the P700-reduction level is observed.

Nociceptor modulated central sensitization causes mechanical hyperalgesia in acute chemogenic and chronic neuropathic pain

Abstract: Brush-evoked pain (mechanical allodynia, dynamic mechanical hyperalgesia) is a hallmark of neuropathic and inflammatory pain states. Here we have examined the neural mechanisms that induce and maintain this component of mechanical hyperalgesia. The principle finding of these experiments is that the severity of brush-evoked pain correlates with the intensity of background pain in patients suffering from chronic painful neuropathies and in normal subjects with acute experimental chemogenic pain. In experiments on nine normal subjects topical application of mustard oil for 5 min evoked strong burning pain and hyperalgesia to light mechanical stimuli. Differential nerve blocks (by compression of the superficial radial nerve) revealed that the brush-evoked pain was transmitted by A beta-fibres, which normally encode non-painful tactile sensations, while the burning pain was signalled by C-fibres. Psychophysical measurements showed that mustard oil treatment resulted in a pronounced sensitization of nociceptors to heat so that subsequent innocuous changes of skin temperature from 35 to 40 degrees C resulted in a proportional increase of burning background pain. Changes in the magnitude of ongoing burning pain were closely correlated (r = 0.81) to the intensity of brush-evoked pain. While conduction block of A-fibres eliminated only touch-evoked pain, blockade of C-fibre excitation instantaneously abolished both ongoing and touch-evoked pain. In nine patients with chronic neuralgia (15 years mean duration) ongoing and brush-evoked pain were examined. In six patients, differential block of A beta-fibres eliminated touch-evoked pain, but ongoing pain persisted when only C-fibres were conducting. Complete relief of both ongoing and stimulus-induced pain was obtained in two patients with intravenous regional guanethedine block and in two other individuals by local anaesthetic blocks of nerves supplying the symptomatic skin, indicating that input from primary afferents was necessary for the maintenance of the pains and that ongoing pain was not self-perpetuated by central mechanisms alone. Quantitative sensory tests revealed heat hyperalgesia in four patients. In those individuals, an increase of skin temperature produced a graded increase of their ongoing pain which was closely correlated (r = 0.94) with the level of brush-evoked pain. In the remaining five patients there was no heat hyperalgesia and consequently no aggravation of pain by increases of skin temperature. Nevertheless when the intensity of the background pain fluctuated spontaneously there were also parallel changes (r = 0.88) of the severity of brush-evoked pain.(ABSTRACT TRUNCATED AT 400 WORDS)

Neurotrophin-6 is a new member of the nerve growth factor family.

Abstract: During vertebrate development, many neurons depend for survival and differentiation on their target cells The best documented mediator of such a retrograde trophic action is the neurotrophin nerve growth factor (NGF) NGF and the other known members of the neurotrophin family, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5) are conserved as distinct genes over large evolutionary distances Here we report the cloning of neurotrophin-6 (NT-6), a new member of this family from the teleost fish Xiphophorus NT-6 distinguishes itself from the other known neurotrophins in that it is not found as a soluble protein in the medium of producing cells The addition of heparin (but not chondroitin) effects the release of NT-6 from cell surface and extracellular matrix molecules Recombinant purified NT-6 has a spectrum of actions similar to NGF on chick sympathetic and sensory neurons, albeit with a lower potency NT-6 is expressed in the embryonic valvulla cerebelli; expression persists in some adult tissues The interaction of NT-6 with heparin-binding molecules may modulate its action in the nervous system

Excision of large DNA regions termed pathogenicity islands from tRNA-specific loci in the chromosome of an Escherichia coli wild-type pathogen.

Abstract: Uropathogenic Escherichia coli 536 (O6:K15:H31) carries two unstable DNA regions, which were shown to be responsible for virulence. These regions, on which the genes for hemolysin production (hly) and P-related fimbriae (prf) are located, are termed pathogenicity islands (PAI) I and II, and were mapped to positions 82 and 97, respectively, on the E. coli K-12 linkage map. Sequence analysis of the PAI region junction sites revealed sequences of the leuX and selC loci specific for leucine and selenocysteine tRNAs. The tRNA loci function as the targets for excision events. Northern (RNA) blot analysis revealed that the sites of excision are transcriptionally active in the wild-type strain and that no tRNA-specific transcripts were found in the deletion mutant. The analysis of deletion mutants revealed that the excision of these regions is specific and involves direct repeats of 16 and 18 nucleotides, respectively, on both sides of the deletions. By using DNA long-range mapping techniques, the size of PAI I, located at position 82, was calculated to be 70 kb, while PAI II, mapped at position 97, comprises 190 kb. The excision events described here reflect the dynamics of the E. coli chromosome.

Reduced replication of Toxoplasma gondii is necessary for induction of bradyzoite-specific antigens : a possible role for nitric oxide in triggering stage conversion

Abstract: Stage conversion between tachyzoites and bradyzoites of Toxoplasma gondii was investigated in vitro by using murine bone marrow-derived macrophages (BMMs) as host cells. Following infection of untreated BMMs with tachyzoites, spontaneous expression of bradyzoite-specific antigens (Bsa) occurred at low frequency with Toxoplasma strain-dependent ratios from 0.03 to 2%. As previously described for peritoneal macrophages, activation of tachyzoite-infected BMMs with gamma interferon (IFN-gamma) or lipopolysaccharide resulted in the induction of Bsa. When bradyzoites were used for infection, prolonged expression of Bsa could be observed in IFN-gamma-activated BMMs. The induction of Bsa expression seemed to be closely linked to parasite multiplication and increased to maximal values of 50 to 70% in intermediately activated macrophages with nitric oxide (NO) levels that allowed reduced parasite replication. Identical results in stage conversion were obtained when sodium nitroprusside was used as a source of exogenous NO, indicating that NO might be a molecular trigger of stage conversion. NO is reactive with iron-sulfur centers in proteins, thereby inhibiting proteins involved in the mitochondrial respiratory chain. Using oligomycin and antimycin A as inhibitors of mitochondrial function, growth inhibition of parasites and induction of Bsa were obtained. Since microglia are the functional correlates of macrophages in the central nervous system and inhibit T. gondii upon activation with IFN-gamma, a similar mechanism might be involved during cyst development in the brain.

Regulation and possible function of the violaxanthin cycle.

Abstract: This paper discusses biochemical and regulatory aspects of the violaxanthin cycle as well as its possible role in photoprotection. The violaxanthin cycle responds to environmental conditions in the short-term and long-term by adjusting rates of pigment conversions and pool sizes of cycle pigments, respectively. Experimental evidence indicating a relationship between zeaxanthin formation and non-photochemical energy dissipation is reviewed. Zeaxanthin-associated energy dissipation appears to be dependent on transthylakoid ΔpH. The involvement of light-harvesting complex II in this quenching process is indicated by several studies. The current hypotheses on the underlying mechanism of zeaxanthin-dependent quenching are alterations of membrane properties, including conformational changes of the light-harvesting complex II, and singlet-singlet energy transfer from chlorophyll to zeaxanthin

Amantadine and memantine are NMDA receptor antagonists with neuroprotective properties.

Abstract: The pharmacological inhibition of excitatory amino acid neurotransmission has evolved to be a major topic in neuropharmacology since enhanced synaptic action of glutamate and possibly other related neurotransmitters has been suggested to play a role both in acute neurological conditions such as ischemia and epilepsy and in chronic degenerative neurological diseases including Parkinson's disease, Huntington's disease and Alzheimer's disease. While antagonists at N-methyl-D-aspartate (NMDA) type glutamate receptors include psychotomimetic and neurotoxic agents such as phencyclidine and MK-801, the aminoadamantanes represent a class of drugs which may be largely free of such actions and which have already been used clinically as antiviral and antiparkinsonian agents. Multiple in vitro studies have recently delineated the neuroprotective properties of amantadine, and of its more potent congener, memantine, which appear to mediate neuroprotection via inhibition of NMDA receptor-dependent glutamate activity. Thus, neuroprotection targeting glutamate receptors does apparently not have to be associated with prominent psychotogenicity, and the development and evaluation of new neuroprotective drugs will have to performed in consideration both of the relative safety and of the good clinical effect of the already known and established aminoadamantanes.

Recurrent origin of a sexually selected trait in Xiphophorus fishes inferred from a molecular phylogeny

Abstract: DARWIN1 believed that sexual selection accounts for the evolution of exaggerated male ornaments, such as the sword-like caudal fin extensions of male fishes of the genus Xiphophorus, that appear detrimental to survival. Swordtails continue to feature prominently in empirical work and theories of sexual selection; the pre-existing bias hypothesis has been offered as an explanation for the evolution of swords in these fishes2,3. Based upon a largely morphological phylogeny, this hypothesis suggests that female preference to mate with sworded males arose in ancestrally swordless species, thus pre-dating the origin of the sword itself and directly driving its evolution. Here we present a molecular phylogeny (based on mitochondria! and nuclear DNA sequences) of Xiphophorus which differs from the traditional one: it indicates that the sword originated and was lost repeatedly. Our phylogeny suggests that the ancestor of the genus is more likely to have possessed a sword than not, thus questioning the applicability of the pre-existing bias hypothesis as an explanation for the evolution of this sexually selected trait.

Phosphorylation of focal adhesion vasodilator-stimulated phosphoprotein at Ser157 in intact human platelets correlates with fibrinogen receptor inhibition.

Abstract: Integrins and other adhesion receptors are essential components for outside-in and inside-out signaling through the cell membrane. The platelet glycoprotein IIb-IIIa (also known as fibrinogen receptor or integrin αIIbβ3) is activated by platelet agonists, inhibited by cyclic-nucleotide-elevating agents, and is involved in the activation of protein tyrosine kinases including the 125-kDa focal adhesion kinase (pp125FAK). However, the molecular details of glycoprotein IIb-IIIa regulation are not well understood. Here we report that in ADP-activated human platelets cAMP- and cGMP-dependent protein-kinase-mediated phosphorylation of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) at Ser157 correlates well with glycoprotein IIb-IIIa inhibition. Human platelets contain similar concentrations of glycoprotein IIb-IIIa complexes (fibrinogen binding sites) and VASP. Using gel-filtered platelets, cAMP-elevating agents [e.g. prostaglandin E1 and the forskolin analog 6-(3-dimethylaminopropionyl)forskolin (NKH 477)] caused VASP Ser157 phosphorylation and inhibited glycoprotein IIb-IIIa activation up to 70–100%. NO-generating, cGMP-elevating agents [e.g. 3-morpholinosydnonimine hydrochloride (SIN1) and sodium nitroprusside] stimulated VASP Ser157 phosphorylation and inhibited glycoprotein IIb-IIIa activation up to a maximal extent of 30–50%. The effects of cAMP- and cGMP-elevating agents on VASP phosphorylation and fibrinogen binding were reversible and could be mimicked by membrane-permeant selective activators of platelet cAMP- or cGMP-dependent protein kinase, respectively. Using threshold concentrations, the nitrovasodilator SIN 1 potentiated the effects of the forskolin analog NKH 477 with respect to inhibition of platelet aggregation, VASP phosphorylation and glycoprotein IIb-IIIa inhibition. It is proposed that the inhibition of glycoprotein IIb-IIIa induced by cyclic nucleotide involves cAMP-and cGMP-dependent protein-kinase-mediated VASP phosphorylation at Ser157.

Proximal myotonic myopathy: a new dominant disorder with myotonia, muscle weakness, and cataracts.

Abstract: We describe three families with a dominantly inherited disorder. Affected individuals have myotonia, proximal muscle weakness, and cataracts. There was no abnormal CTG repeat expansion of the myotonic dystrophy (DM) gene in DNA from blood and muscle. The structure of the three families permitted linkage analysis, and there is no linkage to the gene loci for DM or to the loci for the muscle chloride channel disorders or muscle sodium channel disorders. The collection of symptoms in these three families seems to represent a new disorder.

Immune responses to Yersinia enterocolitica in susceptible BALB/c and resistant C57BL/6 mice: an essential role for gamma interferon.

Abstract: Susceptibility of mice to infection with Yersinia enterocolitica has been shown to be related to neither the Ity locus encoding for resistance to Salmonella typhimurium and other pathogens nor the H-2 locus. Recent studies in our laboratory have demonstrated that T-cell-mediated immune responses are required for overcoming primary Yersinia infection. In the present study, we investigated the course of infection with Y. enterocolitica and the resulting immune responses in Yersinia-susceptible BALB/c and Yersinia-resistant C57BL/6 mice. In the early phase of infection, the clearance of the pathogen was comparable in both strains of mice, suggesting similar mechanisms of innate resistance. Splenic T cells from Yersinia-infected C57BL/6 mice exhibited marked proliferative responses and produced gamma interferon (IFN-gamma) upon exposure to heat-killed yersiniae. By contrast, the Yersinia-specific T-cell response in BALB/c mice was weak, and IFN-gamma production could not be detected before day 21 postinfection. T cells isolated from C57BL/6 mice 7 days after infection mediated immunity to Y. enterocolitica but those from BALB/c mice did not, while at 21 days postinfection T cells from both strains mediated protection. Neutralization of IFN-gamma abrogated resistance to yersiniae in C57BL/6 mice but to a far smaller extent in BALB/c mice. Administration of recombinant IFN-gamma or anti-interleukin-4 antibodies rendered BALB/c mice resistant to yersiniae, whereas this treatment did not significantly affect the course of the infection in C57BL/6 mice. These results indicate that the cellular immune response, in particular the production of IFN-gamma by Yersinia-specific T cells, is associated with resistance of mice to Y. enterocolitica.

Characterization of two MDCK-cell subtypes as a model system to study principal cell and intercalated cell properties.

Abstract: Madin-Darby canine kidney (MDCK) cells originate from the renal collecting duct and consist of different cell subtypes. We cloned two MDCK cell subtypes denominated as C7 and C11 with different morphology and different function. The two clones maintained their functional differences after cloning. C7 monolayers exhibit a high transepithelial resistance (Rte = 5648 +/- 206 omega.cm2, n = 20) and secrete K+ (delta K+ = 1.31 +/- 0.08 mmol/l, n = 10) into the apical medium. C11 monolayers display a low Rte (330 +/- 52 omega.cm2, n = 20) and secrete Cl- (delta Cl- = 16.9 +/- 1.8 mmol/l, n = 10) into the apical medium. Aldosterone (1 mumol/l) stimulates K+ secretion (delta K+ of 3.58 +/- 0.11 mmol/l, n = 7) in C7 cells and H+ secretion in C11 cells (delta pH = 0.060 +/- 0.007, n = 10). Aldosterone-induced stimulation of K+ secretion is inhibited by apical application of amiloride (1 mumol/l). cAMP stimulates H+ secretion in C11 cells (delta pH = -0.068 +/- 0.004, n = 10). Furthermore, C7 cells are peanut-lectin(PNA)-negative and exhibit an intracellular pH of 7.39 +/- 0.05 (n = 7), whereas C11 cells maintain intracellular pH at 7.16 +/- 0.05 (n = 8) and a major fraction of cells is PNA positive. We conclude that we have cloned two subtypes of MDCK cells which stably express different functional characteristics. The C7 subtype resembles principal cells (PC) of the renal collecting duct, whereas the C11 subtype resembles intercalated cells (ICC) of the renal collecting duct.(ABSTRACT TRUNCATED AT 250 WORDS)

Posttranslational Regulation of Nitrate Reductase in Higher Plants.

Abstract: In many plants, nitrogen represents 2 to 6% of dry matter, most of it being present in the form of amino acids, proteins, or nucleic acids. In these organic compounds, nitrogen exists in its most reduced state (oxidation state -3). It is taken up from the soil primarily in the form of nitrate (oxidation state +5). Thus, nitrate has to be reduced by plants at the expense of reductants such as NADH or NADPH, requiring 8 mol of electrons [or 4 mol of NAD(P)H] per mol of nitrate. Reduction is a two-step mechanism. The first step, reduction of nitrate to nitrite (+3 oxidation state), is catalyzed by assimilatory NR, which is an NAD(P)H-dependent cytosolic enzyme. Nitrite is further reduced to ammonia (-3 oxidation state) in the plastids of leaves or roots by NiR. There are at least two important reasons why plants must exert control on the velocity of nitrate reduction. First, it is an energy-consuming process. As shown above, 8 electrons are required to reduce one nitrate to ammonium, but only 4 electrons are needed to reduce CO2 to the carbohydrate level. Accordingly, a C/N ratio of 10 in the plant biomass (a value found in many herbaceous plants) indicates that about 20% of the photosynthetically produced electrons are consumed for nitrate reduction. Plants have to avoid 'luxury' consumption of nitrate and energy. Second, and perhaps more important, the primary product of nitrate reduction, nitrite (NO2-), is cytotoxic and regarded to be mutagenic as a result of the ability to diazotize amino groups. HNO2 is also a weak acid that, in its undissociated form, can easily penetrate biomembranes, thereby leading to acidification of cells or subcellular compartments. Thus, it makes sense if nitrate reduction is controlled in such a way that it does not exceed nitrite and ammonium consumption. In fact, even under rapidly fluctuating environmental conditions, nitrite levels in plant tissues remain low (below 0.1 mM). Apparently, the overall rates of nitrate and nitrite reduction always match the availability of energy and of carbohydrate, perhaps with the exception of some extreme conditions to be discussed below. Synchronization of nitrate reduction and carbon metabolism may occur at the level of transcription or translation of participating enzymes. The expression of NR genes at the transcription level is highly affected by nitrate, but also by light, plant hormones, and other factors (for review, see Solomonson and Barber, 1990; Lillo, 1994). The enzyme protein is rather short-lived, being degraded with a half-time of a few hours (Li and Oaks, 1993). This high turnover rate permits control of nitrate reduction, e.g. in response to nitrate availability (Li and Oaks, 1993). However, over the years, a number of situations have been described in which the level of extractable NRA did not match NR protein or the rate of nitrate reduction in vivo, indicating that yet other regulatory mechanisms might exist that modulate the catalytic activity of the protein (Lillo, 1994, and refs. cited therein). Such a newly discovered type of NR modulation, a reversible protein phosphorylation, will be described below.

Requirement of metabotropic glutamate receptors for the generation of inflammation-evoked hyperexcitability in rat spinal cord neurons.

Abstract: In the central nervous system the transmitter L-glutamate activates both ionotropic receptors coupled to cation channels and metabotropic receptors coupled to G-proteins. The role of metabotropic receptors in the processing of mechanosensory and nociceptive information was studied in a subset of spinal cord neurons with afferent input from the knee joint in anaesthetized rats using electrophysiological methods. The ionophoretic administration of L-2-amino-3-phosphonopropionic acid (L-AP3), an antagonist at the metabotropic receptor, had no effect on the responses to innocuous and noxious pressure applied to the normal knee joint, although the antagonist prevented the potentiation of these responses evoked by the ionophoretic administration of a specific agonist at the metabotropic receptor, trans-(+/-)-1-amino-(1S,3R)-cyclopentane-dicarboxylic acid (t-ACPD). By contrast, in neurons that were rendered hyperexcitable by acute inflammation in the knee joint L-AP3 reduced the responses to pressure applied to the knee. When L-Ap3 was applied during induction of inflammation and throughout the subsequent 1.5 h the spinal neurons did not develop hyperexcitability over this time period. L-AP3 did not impair the activation of ionotropic N-methyl-D-aspartate (NMDA) and non-NMDA receptors by the specific agonists. We conclude that spinal metabotropic glutamate receptors are not involved in the mediation of responses to innocuous and noxious mechanical stimuli applied under normal conditions. They are required, however, for the generation of inflammation-evoked hyperexcitability of spinal cord neurons, a form of functional plasticity underlying the painfulness in pathophysiological conditions such as inflammation.

Production of purified alginates suitable for use in immunoisolated transplantation

Abstract: Alginate is used as a matrix for immunoisolation of cells and tissues in vivo. We have demonstrated previously that commercial alginates contain various fractions of mitogenic impurities and that they can be removed by free flow electrophoresis. The use of purified material is a necessity in order to reveal the parameters that control biocompatibility of the implanted material (such as stability, size, surface charge and curvature, etc.). In this study, we present a protocol for the chemical purification of alginates on a large-scale. Beads made from alginates purified by this multi-step chemical extraction procedure did not induce a significant foreign body reaction when implanted for 3 weeks either intraperitoneally or beneath the kidney capsule of Lewis or non-diabetic BB/Gi rats.