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R Menzel

Publications and source records attributed to R Menzel.

At least 55 records · Page 3Linked to original sources
PubMed1999

Associative learning modifies neural representations of odors in the insect brain.

Recording brain activity in vivo during learning is fundamental to understanding how memories are formed. We used functional calcium imaging to track odor representations in the primary chemosensory center of the honeybee, the antennal lobe, while training animals to discriminate a rewarded odor from an unrewarded one. Our results show that associative learning transforms odor representations and decorrelates activity patterns for the rewarded versus the unrewarded odor, making them less similar. Additionally, activity for the rewarded but not for the unrewarded odor is increased. These results indicate that neural representations of the environment may be modified through associative learning.

Animals↗
PubMed1999

The glomerular code for odor representation is species specific in the honeybee Apis mellifera.

Odors are coded by glomerular activity patterns in the insect antennal lobe (AL) and in the mammalian olfactory bulb. We measured glomerular responses to 30 different odors in the AL of honeybees using calcium-sensitive dyes. By subsequently staining glomeruli and identifying individual glomerular outlines, we were able to compare the patterns between animals. Regardless of whether the odors were mixtures or pure substances, environmental odors or pheromones, their representations were highly conserved among individuals. Therefore, it may be possible to create a functional atlas of the AL in which particular molecular receptive ranges are attributed to each glomerulus.

Animals↗
PubMed1999

Olfactory discrimination ability and odor structure-activity relationships in honeybees.

Using the training procedure introduced by von Frisch in 1919, we tested the ability of free-flying honeybees to discriminate a conditioning odor from an array of 44 simultaneously presented substances. The stimuli included homologous series of aliphatic alcohols, aldehydes and ketones, isomeric forms of some of these substances, as well as several terpenes and odor mixtures, and thus comprised stimuli of varying degrees of structural similarity to any conditioning odor. We found (i) that the honeybees significantly distinguished between 97.0% of the 1848 odor pairs tested, thus showing an excellent discrimination performance when tested in a free-flying situation with an array of structurally related substances; (ii) a significant negative correlation between discrimination performance and structural similarity of odorants in terms of differences in carbon chain length with all aliphatic substance classes tested; (iii) that both the position and type of a functional group also affected discriminability of odorants in a substance class-specific manner; and (iv) striking similarities in odor structure-activity relationships between honeybees and human and nonhuman primates tested previously on a subset of substances employed here. Our findings demonstrate that the similarities found in the structural organization of the olfactory systems of insects and vertebrates are paralleled by striking similarities in relative discrimination abilities. This strongly suggests that similar mechanisms of odor coding and discrimination may underlie olfaction in vertebrates and insects.

Animals↗
PubMed1999

Structures in Bacillus subtilis are recognized by CD14 in a lipopolysaccharide binding protein-dependent reaction.

The CD14 molecule expressed on monocytes and macrophages is a high-affinity receptor for bacterial lipopolysaccharide (LPS) and hence an important component of the innate immune system. LPS binding protein (LBP) is required to facilitate the binding of LPS to CD14 in vitro and is necessary for the induction of an inflammatory response to LPS in vivo. Here we show that CD14 and LBP can also bind to lipoteichoic acid from the gram-positive bacterium Bacillus subtilis. Although CD14 does not interact with intact B. subtilis organisms, a brief exposure of the bacteria to serum converts them into a form which can bind to CD14 in an LBP-dependent reaction. When serum-pretreated B. subtilis organisms are incubated with the myelomonocytic cell line U937, which expresses CD14, the bacteria are rapidly phagocytosed. The phagocytosis is strictly dependent both on LBP and on CD14. These in vitro results suggest that LBP plays a role in the innate response not only to gram-negative but also to gram-positive infections.

Acute-Phase Proteins↗
PubMed1999

IA in Kenyon cells of the mushroom body of honeybees resembles shaker currents: kinetics, modulation by K+, and simulation.

Cultured Kenyon cells from the mushroom body of the honeybee, Apis mellifera, show a voltage-gated, fast transient K+ current that is sensitive to 4-aminopyridine, an A current. The kinetic properties of this A current and its modulation by extracellular K+ ions were investigated in vitro with the whole cell patch-clamp technique. The A current was isolated from other voltage-gated currents either pharmacologically or with suitable voltage-clamp protocols. Hodgkin- and Huxley-style mathematical equations were used for the description of this current and for the simulation of action potentials in a Kenyon cell model. Activation and inactivation of the A current are fast and voltage dependent with time constants of 0.4 +/- 0.1 ms (means +/- SE) at +45 mV and 3.0 +/- 1.6 ms at +45 mV, respectively. The pronounced voltage dependence of the inactivation kinetics indicates that at least a part of this current of the honeybee Kenyon cells is a shaker-like current. Deactivation and recovery from inactivation also show voltage dependency. The time constant of deactivation has a value of 0.4 +/- 0.1 ms at -75 mV. Recovery from inactivation needs a double-exponential function to be fitted adequately; the resulting time constants are 18 +/- 3.1 ms for the fast and 745 +/- 107 ms for the slow process at -75 mV. Half-maximal activation of the A current occurs at -0.7 +/- 2.9 mV, and half-maximal inactivation occurs at -54.7 +/- 2.4 mV. An increase in the extracellular K+ concentration increases the conductance and accelerates the recovery from inactivation of the A current, affecting the slow but not the fast time constant. With respect to these modulations the current under investigation resembles some of the shaker-like currents. The data of the A current were incorporated into a reduced computational model of the voltage-gated currents of Kenyon cells. In addition, the model contained a delayed rectifier K+ current, a Na+ current, and a leakage current. The model is able to generate an action potential on current injection. The model predicts that the A current causes repolarization of the action potential but not a delay in the initiation of the action potential. It further predicts that the activation of the delayed rectifier K+ current is too slow to contribute markedly to repolarization during a single action potential. Because of its fast activation, the A current reduces the amplitude of the net depolarizing current and thus reduces the peak amplitude and the duration of the action potential.

Action Potentials↗
PubMed1999

Pharmacological dissociation between the reinforcing, sensitizing, and response-releasing functions of reward in honeybee classical conditioning.

Reserpine depletes biogenic amines from their stores in the honeybee (Apis mellifera carnica) brain and leads to impaired appetitive conditioning using sucrose as a reinforcer. Compensatory injection of octopamine or dopamine directly into the brain restores these behavioral losses. Dopamine rescues the slowing-down effect on motor patterns, but not sensitization or conditioning. Octopamine leaves the motor patterns as well as sensitization unchanged but rescues conditioning. Specifically, octopamine rescues acquisition but not retrieval. Serotonin has no significant effect on sensitization but impairs conditioning. The authors conclude that octopamine is involved in selectively mediating the reinforcing but not the sensitizing or response-releasing function of the sucrose reward, whereas dopamine is selectively involved in the expression of the motor response.

Adrenergic Uptake Inhibitors↗
PubMed1998

A fluorescence polarization competition immunoassay for tyrosine kinases.

We have recently reported a homogeneous, nonradioactive fluorescence polarization method to assay protein tyrosine kinase activity. Our original approach can only be used with a peptide substrate and requires large amounts of anti-phosphotyrosine antibody. To overcome these problems an alternate fluorescence polarization competition immunoassay was designed and evaluated. In this assay, phosphorylated peptide or protein produced by kinase reaction will compete with a fluorescent phosphopeptide used as a tracer for immunocomplex formation with phosphotyrosine antibody. In this format kinase activity will result in the loss of the polarization signal. To validate the fluorescence polarization competition immunoassay, Lck activity was compared with a more commonly used 32PO4-transfer assay using Lck peptide or enolase as the substrate. In both the assays, Lck activity showed a similar dependence on ATP, Lck enzyme, and the peptide/enolase substrate concentrations with the FP signal inversely proportional to the amount of 32PO4 transferred to the substrate. Inhibition by staurosporine and the Lck inhibitor 4-amino-5-(methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine was similar in these two assays. The advantages of this assay over other kinase assays include use of nonisotopic substrates and a more simple procedure in which the kinase assay is done in a single tube (well of a microtiter plate), without separation, precipitation, or washing. This method is easily automated for high-throughput drug discovery screening.

Adenosine Triphosphate↗
PubMed1998

A low renal threshold for glucose in diabetic patients with a mutation in the hepatocyte nuclear factor-1alpha (HNF-1alpha) gene.

One form of maturity-onset diabetes of the young, Type 3 (MODY3), results from mutations in the gene coding for hepatocyte nuclear factor-1alpha (HNF-1alpha), a transcription factor first described in the liver. MODY3 is characterized by a defective glucose-stimulated insulin secretion. Earlier observations of glycosuria with normal blood glucose levels in some MODY families suggest an additional renal manifestation of the respective genetic defect. We measured the renal threshold for glucose in five diabetic carriers of a missense mutation (Arg 272 His) in HNF-1alpha and, for comparison, in eight Type 1 diabetic patients, applying a non-invasive protocol of frequent parallel blood and urine sampling during a slow shift in blood glucose levels. We found that the mean renal threshold for glucose was lowered in the HNF-1alpha diabetic patients compared to those with Type 1 diabetes (6.5 +/- 0.9 mmol l(-1) vs 10.7 +/- 0.5 mmol l(-1); p < 0.01). This lowered glucose threshold might be an indication of an extra-pancreatic effect of HNF-1alpha gene mutations in humans. Defects in HNF-1alpha may lead to an altered tubular glucose reabsorption, possibly due to decreased expression of the renal glucose transporter proteins involved in reabsorption of glucose from the urine.

Absorption↗
PubMed1998

Bees travel novel homeward routes by integrating separately acquired vector memories

The question of whether bees can take novel short cuts between familiar sites has been central to the discussion about the existence of cognitive maps in these insects. The failure of bees to show this capacity in the majority of previous studies may be a result of the training procedure, because extensive training to one feeding site may have eliminated or weakened memories for other sites that were previously trained. Here we present a novel approach to this problem, by rewarding honey bees, Apis mellifera carnica, at two feeding sites, one (Sm, 630 m southeast from the hive) at which they could feed in the morning, and the other (Sa, 790 m northeast) at which they could feed in the afternoon. We then displaced bees to Sa in the morning and to Sm in the afternoon either from the other feeding site or from the hive. Bees were also displaced to two novel sites, one at a completely unfamiliar location (S4) and another that was located halfway between the two feeding sites (S3). Bees displaced from either of the feeding sites never took novel short cuts; instead, they used the homeward directions that would have been correct had they not been displaced. Bees caught at the hive entrance, however, chose the correct homeward direction not only when displaced to both feeding sites, but also when displaced to S3, although not from S4. Control bees that had been trained to only one of the feeding sites were not able to travel directly home from S3 excluding the possibility that bees used landmarks close to the hive. This is the first evidence that bees take a novel short cut by activating two vector memories simultaneously. The potential mechanisms of integrating the two memories are discussed. Since bees took novel short cuts in only one direction (to the hive) and only when displaced from the hive (not the feeders), we conclude that inference of a cognitive map in bees would be premature. Copyright 1998 The Association for the Study of Animal Behaviour.

Journal Article↗
PubMed1998

Temporal determinants of olfactory long-term retention in honeybee classical conditioning: nonmonotonous effects of the training trial interval.

The question of which acquisition parameters govern long-term retention is important to an understanding of memory function. We investigate the effects of the time interval between learning trials on mediate (1 day)- and long-term (4 days) retention. In classical conditioning of the proboscis extension reflex, we train honeybees to associate an odorant with a sucrose reward using intertrial intervals of either 30 s, 1 min, 3 min, or 20 min. Intervals of 20 and 1 min result in stable retention but 3-min and 30-s intervals result in reduced retention after 4 days compared to that seen after 1 day. Thus, stability of long-term retention depends nonmonotonously on the intertrial interval. Reduced retention with 3-min intervals might be caused by a disruption of memory consolidation which is known to be especially sensitive to interference 3 min after a conditioning trial. Habituation and backward inhibitory learning are discussed as explanations for reduced retention with 30-s intervals.

Animals↗
PubMed1998

Enhanced expression of Ki-67, topoisomerase IIalpha, PCNA, p53 and p21WAF1/Cip1 reflecting proliferation and repair activity in UV-irradiated melanocytic nevi.

To investigate the effect of ultraviolet (UV) irradiation on the expression of cell cycle-associated proteins, melanocytic nevi from healthy volunteers were partially covered, irradiated with a defined UV dose, and excised 1 week thereafter. The irradiated and the protected parts were examined separately by conventional microscopy and immunohistochemistry using the antibodies Ki-S11 (Ki-67), Ki-S7 (topoisomerase IIalpha), PC10 (proliferating cell nuclear antigen [PCNA]), DO-7 (p53), 6B6 (p21WAF1/Cip1), and the melanocytic marker HMB-45. DNA nick-end labeling was used as a marker of apoptosis. Irradiation resulted in morphological changes and increased HMB-45 reactivity. Proliferation, as assessed by Ki-67 and topoisomerase IIalpha expression, was also clearly enhanced in the UV-exposed areas. This was confirmed by the appearance of occasional mitotic figures. PCNA expression levels markedly exceeded those of the proliferation markers and did not correlate with the latter in most cases. p21 immunolabeling indices were also consistently augmented after UV exposure; hence it is likely that growth-inhibitory mechanisms partly compensate for the proliferative impulse, and the disproportional rise in PCNA expression probably reflects DNA repair activity. Enhanced p53 immunostaining in four cases suggests that the induction of p21 after irradiation may be p53 mediated, whereas no concomitant apoptotic events were observed. We conclude that UV light can stimulate the proliferative activity of melanocytes in melanocytic nevi, but that simultaneously cell cycle inhibitors are activated to permit DNA repair.

Adult↗
PubMed1998

Gene activation by the AraC protein can be inhibited by DNA looping between AraC and a LexA repressor that interacts with AraC: possible applications as a two-hybrid system.

The Escherichia coli activator and repressor proteins AraC and LexA bind DNA as homodimers. Here we show that their heterodimerization through fused cognate dimerization domains results in repression of AraC-dependent gene activation by LexA. Repression also requires a LexA operator half-site located several helical turns downstream of the AraC operator. This requirement for a specific spatial organization of the operators suggests the formation of a DNA loop between operator-bound Ara/LexA heterodimers, and we propose that heterodimerization with the AraC hybrid provides co-operativity for operator binding and repression by the LexA hybrid. Consistent with a mechanism that involves DNA looping, repression increases when the E. coli DNA looping and transcriptional effector protein IHF binds between the AraC and LexA operators. Thus, we have combined the functions of three distinct transcriptional effector proteins to achieve a new mode of gene regulation by DNA looping, in which the activator protein is an essential part of the repressor complex. The flexibility of the DNA loop may facilitate this novel combinatorial arrangement of those proteins on the DNA. The requirement for protein interactions between the AraC and LexA hybrids for gene regulation suggests that this regulatory circuit may prove useful as an E. coli-based two-hybrid system.

AraC Transcription Factor↗
PubMed1998

Short communication: long- but not medium-term retention of olfactory memories in honeybees is impaired by actinomycin D and anisomycin.

Although work in a wide variety of species and paradigms has demonstrated that long-term memory is sensitive to the blocking of protein synthesis, previous studies have suggested that the honeybee might represent an exception to this rule. Retention tested one day after training was not impaired by the inhibition of translation by cycloheximide. Using blockers of either transcription (actinomycin D) or translation (anisomycin), we present experiments that reconcile this unusual finding by testing over longer retention periods. Honeybees were conditioned to associate an odourant with a sucrose reward. Typically, this leads to stable retention over days. However, injection of either drug led to lower retention after 4 days, whereas retention after 2 or sometimes even 3 days was unaffected. This dissociates two forms of memory: a protein synthesis-independent, medium-term memory (up to 3 days) and a protein synthesis-dependent, long-term memory lasting for at least 4 days.

Animals↗
PubMed1998

Odour coding is bilaterally symmetrical in the antennal lobes of honeybees (Apis mellifera).

The primary olfactory neuropil, the antennal lobe (AL) in insects, is organized in glomeruli. Glomerular activity patterns are believed to represent the across-fibre pattern of the olfactory code. These patterns depend on an organized innervation from the afferent receptor cells, and interconnections of local interneurons. It is unclear how the complex organization of the AL is achieved ontogenetically. In this study, we measured the functional activity patterns elicited by stimulation with odours in the right and the left AL of the same honeybee (Apis mellifera) using optical imaging of the calcium-sensitive dye calcium green. We show here that these patterns are bilaterally symmetrical (n=25 bees). This symmetry holds true for all odours tested, irrespective of their role as pheromones or as environmental odours, or whether they were pure substances or complex blends (n=13 odours). Therefore, we exclude that activity dependent mechanisms local to one AL determine the functional glomerular activity. This identity is genetically predetermined. Alternatively, if activity dependent processes are involved, bilateral connections would have to shape symmetry, or, temporal constraints could lead to identical patterns on both sides due to their common history of odour exposure.

Acyclic Monoterpenes↗
PubMed1998

Low endotoxic potential of Legionella pneumophila lipopolysaccharide due to failure of interaction with the monocyte lipopolysaccharide receptor CD14.

Legionella pneumophila, a gram-negative bacterium causing Legionnaires' disease and Pontiac fever, was shown to be highly reactive in in vitro gelation of Limulus lysate but not able to induce fever and the local Shwartzman reaction in rabbits and mice. We analyzed the capacity of purified L. pneumophila lipopolysaccharide (LPS-Lp) to induce activation of the human monocytic cell line Mono Mac 6, as revealed by secretion of proinflammatory cytokines and desensitization to subsequent LPS stimulation. We showed that despite normal reactivity of LPS-Lp in the Limulus amoebocyte lysate assay, induction of cytokine secretion in Mono Mac 6 cells and desensitization to an endotoxin challenge required LPS-Lp concentrations 1,000 times higher than for LPS of Salmonella enterica serovar Minnesota. Therefore, we examined the interaction of LPS-Lp with the LPS receptor CD14. We demonstrated that LPS-Lp did not bind to membrane-bound CD14 expressed on transfected CHO cells, nor did it react with soluble CD14. Our results suggest that the low endotoxic potential of LPS-Lp is due to a failure of interaction with the LPS receptor CD14.

Animals↗
PubMed1998

The molecular basis for therapeutic concepts utilizing CD14.

The CD14 molecule is a key receptor on myeloid lineage cells involved in the recognition of lipopolysaccharide (LPS) and Gram-negative bacteria. The application of its soluble form, sCD14, has been shown to protect mice from lethality in LPS-induced shock. Therefore the protein or its derivatives may be considered as a possible therapeutic alternative for the treatment of patients suffering from Gram-negative septic shock. In this study we performed an alanine scan of amino acids 1 to 152 of human CD14. Twenty-three substitution mutants were generated and stably transfected into CHO-cells. In each mutant five amino acids were substituted by alanine. We analyzed (a) whether mutant proteins expressed on the surface of transfectants were recognized by a panel of anti-CD14 monoclonal antibodies (mAb's), (b) the ability of mCD14-mutants to bind LPS and E. coli in a serum- or LBP-dependent manner, and (c) the capacity of soluble mutants to mediate the LPS-induced IL 6 release of U 373 astrocytoma cells. Twenty-one CD14-mutants were expressed on the surface of transfectants and 18 were present as soluble forms in the culture supernatants. We demonstrated that only CD14(39-41,43-44)A completely lacked the ability to bind LPS and E. coli. In addition, a combined mutant CD14(9-13/57,59,61-63)A had very limited capacity to interact with LPS indicating that the LPS-binding site of human CD14 is a conformational epitope. Analysis of LPS-induced activation of CD14-negative U 373 cells revealed that the regions 9-13 and 91-101 are most important for sCD14-mediated signalling.

Acute-Phase Proteins↗
PubMed1997

A homogeneous, fluorescence polarization assay for src-family tyrosine kinases.

A nonradioactive, simple, sensitive fluorescence polarization assay was developed to assay protein tyrosine kinase activity. This assay involves incubation of a fluorescenylated peptide substrate with the kinase, ATP, and anti-phosphotyrosine antibody. The phosphorylated peptide product is immunocomplexed with the anti-phosphotyrosine antibody resulting in an increase in the polarization signal as measured in a fluorescence polarization analyzer. Among several anti-phosphotyrosine antibodies examined, monoclonal antibody PY54 was found to give the best polarization signal with the test peptide. For validation of the fluorescence polarization assay, Lck activity was compared with a 32PO4 transfer assay. In both the fluorescence polarization and 32PO4 transfer assays, Lck activity showed a similar dependence on ATP, Lck enzyme, and peptide substrate concentrations. Both assays gave similar inhibition constants with a known tyrosine kinase inhibitor staurosporine and the Lck inhibitor, 4-amino-5-(methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. These results show that the fluorescence polarization assay can detect inhibitors and is comparable to the 32PO4 transfer assay. The fluorescence polarization method is advantageous compared to the 32PO4 transfer assay or ELISA or DELFIA because it is a one-step assay that does not involve several washings, liquid transfer, and sample preparation steps. It has the added advantage of using nonisotopic substrates. The fluorescence polarization assay thus is environmentally safe and minimizes handling problems. The homogeneous nature of the assay makes it readily adaptable to high-throughput screening for small-molecule drug discovery.

Adenosine Triphosphate↗