Biomedical subjects

R Menzel

Publications and source records attributed to R Menzel.

At least 73 records · Page 4Linked to original sources

PubMed1997

A semi-in-vivo preparation for optical recording of the insect brain.

We describe a method for optically recording neuronal activity from an intact insect brain, upon natural sensory stimulation. In this preparation, the head capsule of the honeybee, Apis mellifera, is isolated from the body while leaving the entire brain undamaged. In short, a hole is first cut into the cuticule to allow optical access to the brain and to allow the removal of tracheae and glands. Then the head is cut free and placed into a dye-loaded and cooled ringer solution in a staining chamber for 1 h. Subsequently, the head is fixed in a recording chamber, covered with a cover-slip, and imaged under the microscope with a cooled CCD camera. The whole preparation leaves the antennae dry, free to move, and functional throughout the experiment, allowing for natural odour stimulation of the olfactory system. Using calcium sensitive or potential sensitive dyes (calcium-green or RH795), we could record the processing of olfactory information at the glomerular level in the antennal lobe of the bee.

Animals↗

PubMed1997

Regulation of Escherichia coli topA gene transcription: involvement of a sigmaS-dependent promoter.

To investigate the regulation of Escherichia coli topA gene transcription, primer extension was employed to determine the transcription initiation sites from the chromosomal topA gene. When cells were grown in LB medium to log phase, four transcription initiation sites could be identified. Three of these sites corresponded to promoters P1, P2 and P4 previously characterized using topA-galK fusion plasmids. The P3 promoter that is active on the plasmid was not utilized at the chromosomal topA gene under the conditions employed. There was a new transcription initiation site corresponding to a new promoter Px1. When cells started to enter stationary phase, promoter Px1 gradually became the major transcription initiation site for topA, while transcription from promoters P2 and P4 decreased. In an E. coli mutant lacking sigmaS (the rpoS gene product), the stationary phase specific sigma factor, the induction of transcription from promoter Px1 was abolished. In another mutant lacking H-NS activity, resulting in increased sigmaS level in log-phase, the transcription from promoter Px1 during log phase was increased. Thus Px1 appeared to be regulated by sigmaS. The activity of promoter P1 on the chromosome increased during heat shock, consistent with the previous result obtained using the topA-galK fusion plasmid showing that P1 is a sigma32-dependent heat shock promoter. Promoters P2 and P4 were most likely to be recognized by sigma70. The total level of topoisomerase I protein in the rpoS mutant was not reduced significantly in stationary phase due to increased transcription initiation from the other topA promoters. The utilization of multiple sigma factors for transcription initiation of topA could be important for adaptation of E. coli to change in growth conditions.

Bacterial Proteins↗

PubMed1997

Mutation of amino acids 39-44 of human CD14 abrogates binding of lipopolysaccharide and Escherichia coli.

As a key receptor for lipopolysaccharide (LPS) on the surface of monocytes and macrophages, the CD14 molecule is primarily involved in non-specific host defense mechanisms against gram-negative bacteria. To delineate the structural basis of LPS binding, 23 mutants in the N-terminal 152 amino acids of human CD14 were generated and stably transfected into CHO cells. In each mutant, a block of five amino acids was substituted by alanine. Reactivity of the mutants with anti-CD14 mAbs, and their ability to interact with LPS and Escherichia coli were tested. 4 of 21 expressed CD14 mutants, ([Ala9-Ala13]CD14, [Ala39-Ala41, Ala43, Ala44]CD14, [Ala51-Ala55]CD14 and [Ala57, Ala59, Ala61-Ala63]CD14), are not recognized by anti-CD14 mAbs that interfere with the binding of LPS to human monocytes. However, only [Ala39-Ala41, Ala43, Ala44]CD14 is unable to react with fluorescein-isothiocyanate-labeled LPS or with FITC-labeled E. coli (055:B5). In addition, [Ala39-Ala4l, Ala43, Ala44]CD14 does not mediate LPS (E. coli 055:B5; 10 ng/ml)-induced translocation of nuclear factor kappaB in CHO-cell transfectants. The results indicate that the region between amino acids 39 and 44 forms an essential part of the LPS-binding site of human CD14.

Amino Acid Sequence↗

PubMed1997

Inducible membranes in yeast: relation to the unfolded-protein-response pathway.

Overproduction of an endoplasmic reticulum (ER)-resident membrane protein (cytochrome P450 52A3) and of a secretory protein (invertase) was used to study the regulation of the luminal ER protein Kar2p under conditions that lead to ER proliferation and secretory overload, respectively. In both cases we found (i) a significant increase of Kar2 protein and mRNA levels, (ii) a transcriptional regulation based on the the function of the 22 bp unfolded-protein-response element of the KAR2 promoter and (iii) an essential role of the transmembrane kinase Ire1p for upregulation of KAR2 gene expression. These results show that the same mechanism operates when KAR2 induction is triggered by overproduction of cytochrome P450 or invertase and that this mechanism shares the known features of the unfolded-protein-response pathway. Disruption of the IRE1 gene resulted in a marked decrease of the invertase protein levels produced. In contrast, a functional IRE1 gene was not required to reach high-level production of the integral membrane protein cytochrome P450 52A3, Moreover, IRE1 gene disruption did not prevent P450-induced ER proliferation. We suggest that Ire1p-mediated KAR2 induction is, in the case of cytochrome P450 52A3 overproduction, a process which follows on ER proliferation, thereby monitoring the increase of ER size and adjusting the level of Kar2p accordingly.

Cytochrome P-450 Enzyme System↗

PubMed1997

Insect visual perception: complex abilities of simple nervous systems.

Despite their relatively simple nervous systems, insects display a rich behavioural repertoire, in which vision plays a major role. In the past two years, much knowledge has been gained about how insects are capable of a variety of flexible, visually guided tasks that involve a high level of complexity. From long-range navigation to median-range orientation and close-up recognition, insects apply different strategies that complement each other, that are used sequentially during their approach flight towards their goals, and that may replace each other, depending on the salience of, and the attention towards, particular visual cues.

Animals↗

PubMed1997

DNA supercoiling and bacterial adaptation: thermotolerance and thermoresistance.

When bacterial cells are shifted to higher temperatures their degree of DNA supercoiling changes. Topoisomerases are involved in bacterial adaptation to environmental changes requiring rapid shifts in gene expression. This role in heat shock has been elucidated by genetic studies on the Escherichia coli topA gene and its sigma 32-dependent promoter, P1. Other studies have shown that certain gyrA mutants have increased thermoresistance.

Adaptation, Physiological↗

PubMed1997

Odorant intensity as a determinant for olfactory conditioning in honeybees: roles in discrimination, overshadowing and memory consolidation.

Stimulus intensity is an important determinant for perception, learning and behaviour. We studied the effects of odorant concentration on classical conditioning involving odorants and odorant-mechanosensory compounds using the proboscis-extension reflex in the honeybee. Our results show that high concentrations of odorant (a) support better discrimination in a feature-positive task using rewarded odorant-mechanosensory compounds versus unrewarded mechanosensory stimuli, (b) have a stronger capacity to overshadow learning of a simultaneously trained mechanosensory stimulus, and (c) induce better memory consolidation. Furthermore, honeybees were trained discriminatively to two different concentrations of one odorant. Honeybees are not able to solve this task when presented with rewarded low versus unrewarded high concentrations. Taken together, our results suggest that high concentrations of odorant support stronger associations (are more 'salient') than low concentrations. Our results, however, do not indicate that honeybees can treat two different concentrations of one odorant as qualitatively different stimuli. These findings fill a gap in what is known about honeybee olfactory learning and are a first step in relating behaviour to recent advances in the physiological analysis of coding for odorant concentration in honeybees.

Animals↗

PubMed1997

Mutations in the hepatocyte nuclear factor-1alpha gene in MODY and early-onset NIDDM: evidence for a mutational hotspot in exon 4.

We have recently shown that mutations in the gene encoding the transcription factor hepatocyte nuclear factor (HNF)-1alpha are the cause of one form of maturity-onset diabetes of the young (MODY3). Here, we report the exon-intron organization and partial sequence of the human HNF-1alpha gene. In addition, we have screened the ten exons and flanking introns of this gene for mutations in a group of 25 unrelated white subjects from Germany who presented with NIDDM before 35 years of age and had a first-degree relative with NIDDM. Mutations were identified in nine of these individuals, suggesting that mutations in the HNF-1alpha gene are a common cause of diabetes in German subjects with early-onset NIDDM and a family history of diabetes. Thus, screening for mutations in this gene may be indicated in subjects with early-onset NIDDM. Interestingly, three of the nine mutations occurred at the same site in exon 4 with insertion of a C in a polyC tract, centered around codon 290 (designated Pro291fsinsC), thereby resulting in a frameshift during translation and premature termination. Analyses of linked DNA polymorphisms in the HNF-1alpha gene indicated that the Pro291fsinsC mutation was present on a different haplotype in each subject, implying that the polyC tract represents a mutational hot spot. We have also identified the mutation in the HNF-1alpha gene in the Jutland pedigree, one of the original MODY pedigrees reported in the literature, as being a T-->G substitution in codon 241, resulting in the replacement of a conserved Cys by Gly (C241G). The information on the sequence of the HNF-1alpha gene and its promoter region will facilitate the search for mutations in other subjects and studies of the role of the gene in determining normal beta-cell functions.

Adolescent↗

PubMed1996

Topogenesis of a microsomal cytochrome P450 and induction of endoplasmic reticulum membrane proliferation in Saccharomyces cerevisiae.

Cytochrome P450 52A3 (P450Cm1) is one of the membrane proteins known to trigger by its high-level expression a marked proliferation of the endoplasmic reticulum, (ER). To gain insight into the relationship between the expression of a membrane protein and the induction of ER proliferation we have characterized the membrane topology of P450Cm1 and identified the structural determinants required for ER targeting, formation of correct membrane orientation, and ER retention. We show that all these features are interrelated and determined by sequence elements within the NH2-terminal region of P450Cm1. Using several approaches--a protease protection assay followed by probing with peptide-specific antibodies, immunolabeling of the intact membrane-bound P450 protein, and expression of fusion proteins in Saccharomyces cerevisiae--membrane topology was defined as follows: residues 2-16 are located in the ER lumen, only the first hydrophobic segment (residues 17-34) spans the membrane, a second hydrophobic segment (48-66) is exposed at the cytoplasmic side, and the remaining part (67-523) forms a large cytosolic domain. Fused to a cytosolic reporter protein, the first 44-amino-acid sequence of P450Cm1 was sufficient to mediate ER targeting, wild-type membrane orientation, and retention in the ER. Similar to wild-type P450Cm1, various fusion proteins were able to induce distinctly organized structures of proliferated ER provided that they were either permanently retained in the ER or accumulated in this compartment due to a delay in further transportation. Thus, we conclude that membrane insertion of the first hydrophobic segment is sufficient to deliver a signal for increased membrane formation.

Amino Acid Sequence↗

PubMed1996

Candida maltosa NADPH-cytochrome P450 reductase: cloning of a full-length cDNA, heterologous expression in Saccharomyces cerevisiae and function of the N-terminal region for membrane anchoring and proliferation of the endoplasmic reticulum.

A full-length cDNA for NADPH-cytochrome P450 reductase from Candida maltosa was cloned and sequenced. The derived amino acid sequence showed a high similarity to the reductases from other eukaryotes. Expression in Saccharomyces cerevisiae under control of the GAL10 promoter resulted in an approximately 70-fold increase in NADPH-cytochrome c reductase activity in the microsomal fraction. The functional integrity of the heterologously expressed reductase as an electron transfer component for alkane hydroxylating cytochrome P450 from C. maltosa was shown in a reconstituted system containing both enzymes in a highly purified state. The signal-anchor sequence of the reductase was identified within the N-terminal region of the protein by means of constructing and expressing fusion proteins with the cytosolic form of yeast invertase. The first 33 amino acids turned out to be sufficient for stable membrane insertion, wild-type membrane orientation and retention in the endoplasmic reticulum. As shown by immunoelectron microscopy, the heterologously expressed reductase was integrated into the endoplasmic reticulum of the host organism. It triggered a strong proliferation of the membrane system. This membrane-inducing property of the reductase was transferable to the cytosolic reporter protein with the same N-terminal sequences that confer membrane insertion.

Amino Acid Sequence↗

PubMed1996

Behavioral, neural and cellular components underlying olfactory learning in the honeybee.

A top-down approach as applied to learning and memory in honeybees provides the opportunity of relating different levels of complexity to each other, and of analyzing the rules and mechanisms from the viewpoint of the respective next higher level. Olfactory conditioning of harnessed bees exemplifies essential elements of associative learning and, in general, forms a bridge between the systems and the cellular levels of analysis. Intracellular recordings of identified neurons during olfactory conditioning play a key role in this effort. They allow testing of the assumptions made by modern behavioral theories of associative learning and provide access to cellular and molecular studies, owing to the identification of their transmitters and the peculiarities of the connectivities. Analysis at this intermediate level of complexity is particularly profitable in the bee, because essential neural elements of the associative network are known and can be tested during ongoing learning behavior. In this respect, the honeybee offers unique properties for the building of bridges between the molecular, cellular, neuronal, network and behavioral levels of associative learning.

Animals↗

PubMed1996

Effect of the deletion of the sigma 32-dependent promoter (P1) of the Escherichia coli topoisomerase I gene on thermotolerance.

Topoisomerase I and DNA gyrase are the major topoisomerase activities responsible for the regulation of DNA supercoiling in the bacterium Escherichia coli. The P1 promoter of topA has previously been shown to be a delta 32-dependent heat-shock promoter. A mutant strain with a deletion of P1 was constructed. This mutant is > 10-fold more sensitive to heat treatment (52 degrees C) than the wild type. After brief treatment at 42 degrees C, wild-type Escherichia coli acquires an enhanced resistance to the effects of a subsequent 52 degrees C treatment. This is not the case for the P1 deletion mutant, which, and under these conditions, is about 100-fold less thermotolerant than the wild type. The presence of a plasmid expressing topoisomerase I restored the heat-survival level of the mutant to that of the wild type. During heat shock, the superhelical density of a plasmid with the heat-inducible rpoD promoter is increased in the P1 deletion mutant. We also note that the pulse-labelling pattern of proteins at 42 C (displayed on SDS-polyacrylamide gels) is different in the mutant, and, most notably, the amounts of DnaK and of GroEL protein are reduced. A model is proposed in order to unify these observations.

Bacterial Proteins↗

PubMed1996

Learning and memory in honeybees: from behavior to neural substrates.

Learning and memory in honeybees is analyzed on five levels, using a top-down approach. (a) Observatory learning is applied during navigation and dance communication. (b) Local cues at the feeding site are learned associatively. (c) Classical conditioning of the proboscis extension response to olfactory stimuli provides insight into behavioral, neural, and neuropharmacological mechanisms of associative learning. (d) At the neural level, the pathways coding the conditioned and the unconditioned stimulus are identified. The reinforcing function of the unconditioned stimulus is traced to a particular neuron. (e) At the cellular level, the cAMP pathway is found to be critically involved. Nitric oxide is an essential mediator for the transfer from short- to long-term memory.

Animal Communication↗

PubMed1996

The knowledge base of bee navigation

Navigation in honeybees is discussed against the background of the types of memories employed in the navigational task. Two questions are addressed. Do bees have goal-specific expectations, and when are novel routes travelled? Expectations are deduced from (1) context stimuli as determinants for local cue memories, (2) landmark-dependent path integration, (3) sequential learning of landmarks, and (4) motivation- and context-dependent memory retrieval. Novel routes are travelled under two conditions: (1) goal-cue-based piloting and (2) integration of simultaneously activated vector memories. Our data do not support the conclusion that memory integration in bees is organised by a cognitive map. The assumption of purely separate memories that are only retrieved according to the chain of events during navigational performance also appears to be inadequate. We favour the view that multiple memories are integrated using external and internal sources of information. Such configural memories lead to both specific expectations and novel routes.

Journal Article↗

PubMed1995

The creation of a camptothecin-sensitive Escherichia coli based on the expression of the human topoisomerase I.

The synthesis of the human topoisomerase I (Topo I) in Escherichia coli (Ec) results in phenotypes consistent with the appearance of a DNA-relaxing activity. High-level expression was problematic and recloning the gene in a reduced-copy-number plasmid under more stringently regulated control produced a stable plasmid. An Ec strain with an inducible sensitivity to the eukaryotic Topo I poison, camptothecin (CPT), was constructed by introducing a mutation (imp4312) known to enhance the permeability of Ec to a wide variety of compounds. We are able to infer that CPT sensitivity in Ec involves DNA damage by noting elevated sensitivity in a repair-deficient recA strain and by observing the induction of a sulA::lac fusion following exposure to the drug.

Camptothecin↗

PubMed1995

Proliferation of intracellular membrane structures upon homologous overproduction of cytochrome P-450 in Candida maltosa.

In an alkane-assimilating yeast, Candida maltosa, a cultivation on alkane causes both induction of endoplasmic reticulum (ER)-resident membrane proteins, such as cytochrome P-450, and proliferation of ER. In this study, individual genes for alkane-inducible forms of cytochrome P-450 (P-450alk) were homologously overexpressed in C. maltosa using a galactose-inducible expression system developed in this yeast. Immunoelectron microscopy revealed that, upon the overexpression, a dramatic proliferation of ER occurred, in which overproduced P-450alk protein accumulated. The proliferated membranes were mainly tubular forms and stacks of paired membranes were also observed after prolonged expression. The tubular forms were morphologically very similar to the proliferated ER in alkane-induced C. maltosa cells. The observed proliferation of ER membranes by homologous overproduction of P-450alk, here depicted, will provide a unique opportunity for investigating the mechanisms by which cells regulate ER biogenesis, in comparison with the intrinsic form of ER proliferation.

Candida↗

PubMed1995

Overexpression of RNase H partially complements the growth defect of an Escherichia coli delta topA mutant: R-loop formation is a major problem in the absence of DNA topoisomerase I.

Previous biochemical studies have suggested a role for bacterial DNA topoisomerase (TOPO) I in the suppression of R-loop formation during transcription. In this report, we present several pieces of genetic evidence to support a model in which R-loop formation is dynamically regulated during transcription by activities of multiple DNA TOPOs and RNase H. In addition, our results suggest that events leading to the serious growth problems in the absence of DNA TOPO I are linked to R-loop formation. We show that the overexpression of RNase H, an enzyme that degrades the RNA moiety of an R loop, can partially compensate for the absence of DNA TOPO I. We also note that a defect in DNA gyrase can correct several phenotypes associated with a mutation in the rnhA gene, which encodes the major RNase H activity. In addition, we found that a combination of topA and rnhA mutations is lethal.

Cold Temperature↗

PubMed1995

Localization of MODY3 to a 5-cM region of human chromosome 12.

Maturity-onset diabetes of the young (MODY) is a heterogeneous disorder that appears to be characterized by a primary defect in insulin secretion. Mutations in an unknown locus (MODY1) on chromosome 20 and the glucokinase gene (MODY2) on chromosome 7 can cause this form of non-insulin-dependent diabetes. Recent genetic studies have identified a third locus on chromosome 12 (MODY3) that is linked to MODY in a group of French families. We have identified three families from Denmark, Germany, and the U.S. (Michigan) showing evidence of linkage with MODY3 and a family from Japan showing suggestive evidence. Analysis of key recombinants in these families localized MODY3 to a 5-cM interval between the markers D12S86 and D12S807/D12S820.

Adult↗