Insertional mutation of the Drosophila nuclear lamin Dm0 gene results in defective nuclear envelopes, clustering of nuclear pore complexes, and accumulation of annulate lamellae.

Nuclear lamins are thought to play an important role in disassembly and reassembly of the nucleus during mitosis. Here, we describe a Drosophila lamin Dm0 mutant resulting from a P element insertion into the first intron of the Dm0 gene. Homozygous mutant animals showed a severe phenotype including retardation in development, reduced viability, sterility, and impaired locomotion. Immunocytochemical and ultrastructural analysis revealed that reduced lamin Dm0 expression caused an enrichment of nuclear pore complexes in cytoplasmic annulate lamellae and in nuclear envelope clusters. In several cells, particularly the densely packed somata of the central nervous system, defective nuclear envelopes were observed in addition. All aspects of the mutant phenotype were rescued upon P element-mediated germline transformation with a lamin Dm0 transgene. These data constitute the first genetic proof that lamins are essential for the structural organization of the cell nucleus.

2-Deoxy-D-glucose maps movement-specific nervous activity in the second visual ganglion of Drosophila.

Adult Drosophila were fed with tritium labeled deoxyglucose prior to a 5-hour period of visual stimulation. A flickering disk of light and a moving grating were presented to the left and right eyes, respectively. Autoradiography revealed enhanced labeling solely in that part of the second optic ganglion (medulla) whose visual field was stimulated by movement.

Paralysis and early death in cysteine string protein mutants of Drosophila.

Multimeric complexes of synaptic vesicle and terminal membrane proteins are important components of the neurotransmitter release mechanism. The csp gene of Drosophila encodes proteins homologous to synaptic vesicle proteins in Torpedo. Monoclonal antibodies demonstrate different distributions of isoforms at distinct subsets of terminals. Deletion of the csp gene in Drosophila causes a temperature-sensitive block of synaptic transmission, followed by paralysis and premature death.

Presynaptic dysfunction in Drosophila csp mutants.

Cysteine string proteins are synapse-specific proteins. In Drosophila, csp deletion mutants exhibit temperature-sensitive paralysis and early death. Here, we report that neuromuscular transmission is impaired presynaptically in these csp mutant larvae. At 22 degrees C, evoked transmitter release is depressed relative to wild type and rescued controls, and high frequency stimulation of the nerve leads to sporadic failures. At 30 degrees C, stimulus-evoked responses decline gradually before failing completely. When the temperature is returned to 22 degrees C, evoked responses recover. Spontaneous release events persist at both 22 degrees C and 30 degrees C. Since nerve conduction and postsynaptic sensitivity are unaffected, these data indicate that csp mutations disrupt depolarization-secretion coupling. This disruption explains the cellular basis of the temperature-sensitive paralysis of these organisms.

A Dilatometric Study of Tempering Complemented by Mössbauer Spectroscopy and other Characterization Techniques.

A new approach for non-isothermal tempering analysis utilizing dilatometry is proposed and was carried out on a medium carbon steel with high silicon and additions of Mo and V for secondary hardening. The method includes a second non-isothermal step performed with the same heating rate (2 °C/min) used for the first step in order to create a baseline for analysis. The results were correlated with several other characterization techniques. Mössbauer spectroscopy confirmed the formation of transition carbides by auto-tempering as well as the presence of retained austenite decomposition (stage II) and cementite precipitation (stage III), which demonstrated significant overlap. Electrical resistivity measurements were correlated with dislocation densities obtained through X-ray diffraction analysis. Transmission electron microscopy dark field images confirmed the secondary hardening assessment from dilatometry.

The DnaJ-like cysteine string protein and exocytotic neurotransmitter release.

The fast, tightly regulated release of neurotransmitters from presynaptic nerve terminals is effected by a complex molecular apparatus. The precise roles of the various proteins involved remain largely conjectural. Cysteine string proteins (CSPs) are novel synaptic vesicle components that have been conserved in evolution. They are characterized by an N-terminus 'J'-domain and a central, multiply palmitoylated string of cysteine residues. Vertebrate CSPs have been implicated in a functional interaction of synaptic vesicles with presynaptic Ca2+ channels. Genetic 'knockout' of CSPs in Drosophila results in a temperature-sensitive breakdown of elicited transmitter release. Here we try to integrate these observations into speculative functional models on the role of this new protein family in synaptic vesicle exocytosis.

DNA cleavage by type III restriction-modification enzyme EcoP15I is independent of spacer distance between two head to head oriented recognition sites.

The type III restriction-modification enzyme EcoP15I requires the interaction of two unmethylated, inversely oriented recognition sites 5'-CAGCAG in head to head configuration to allow an efficient DNA cleavage. It has been hypothesized that two convergent DNA-translocating enzyme-substrate complexes interact to form the active cleavage complex and that translocation is driven by ATP hydrolysis. Using a half-automated, fluorescence-based detection method, we investigated how the distance between two inversely oriented recognition sites affects DNA cleavage efficiency. We determined that EcoP15I cleaves DNA efficiently even for two adjacent head to head or tail to tail oriented target sites. Hence, DNA translocation appears not to be required for initiating DNA cleavage in these cases. Furthermore, we report here that EcoP15I is able to cleave single-site substrates. When we analyzed the interaction of EcoP15I with DNA substrates containing adjacent target sites in the presence of non-hydrolyzable ATP analogues, we found that cleavage depended on the hydrolysis of ATP. Moreover, we show that cleavage occurs at only one of the two possible cleavage positions of an interacting pair of target sequences. When EcoP15I bound to a DNA substrate containing one recognition site in the absence of ATP, we observed a 36 nucleotide DNaseI-footprint that is asymmetric on both strands. All of our footprinting experiments showed that the enzyme did not cover the region around the cleavage site. Analyzing a DNA fragment with two head to head oriented recognition sites, EcoP15I protected 27-33 nucleotides around the recognition sequence, including an additional region of 26 bp between both cleavage sites. For all DNA substrates examined, the presence of ATP caused altered footprinting patterns. We assume that the altered patterns are most likely due to a conformational change of the enzyme. Overall, our data further refine the tracking-collision model for type III restriction enzymes.

Apoptotic cell death in activated monocytes following incorporation of clodronate-liposomes.

The present study was performed to elucidate whether sterically stabilized liposomes laden with clodronate, which lead to depletion of macrophages (Mphis) and amelioration of experimental autoimmune arthritis in vivo, selectively affect cells of the mphi lineage in vitro. The rates of incorporation of drug-free, fluorescent liposomes and the rates of cell death following exposure to clodronate-liposomes were assessed in human peripheral blood monocytes, as well as in polymorphonuclear leukocytes (PMNs), T cells, endothelial cells, and fibroblasts, both at rest and following activation. Gel electrophoresis of nuclear extracts and ultrastructural analyses were performed to identify the modality of cell death. Monocytes, particularly upon activation, were more efficient in incorporating sterically stabilized liposomes than all other cells except PMNs. Twenty percent of resting monocytes and up to 65% of activated monocytes died within 24 h of exposure to clodronate-liposomes, whereas the other cell types, including PMNs, remained unaffected. Activated monocytes exposed to clodronate-liposomes, but not resting or activated monocytes exposed to drug-free liposomes, showed clear signs of apoptotic cell death. In most of the assays, sterically stabilized liposomes were more efficient than conventional phosphatidylcholine-liposomes. Sterically stabilized clodronate-liposomes preferentially affect cells of the mphi lineage, particularly if activated. Selective elimination of activated Mphis by apoptosis may explain both therapeutic efficacy and safety of clodronate-liposomes in experimental models of autoimmunity.

Inflated wings, tissue autolysis and early death in tissue inhibitor of metalloproteinases mutants of Drosophila.

In vertebrates, tissue inhibitors of metalloproteinases (TIMPs) play key roles in extracellular matrix (ECM) homeostasis and growth control. Deletion of the recently cloned Timp gene of Drosophila results in a subviable phenotype. Adult flies display inflated wings similar to integrin mutants, suffer from a bloated gut and progressive dissolution of internal tissues, and die prematurely. Our results demonstrate that the Timp gene product controls selective aspects of ECM function in Drosophila, and suggest that it is involved in cell adhesion/cell signaling pathways. Hence, Drosophila Timp mutants may prove useful as a model system for a wide variety of pathological conditions related to ECM dysregulation.

The black-pearl gene of Drosophila defines a novel conserved protein family and is required for larval growth and survival.

Using a transposon insertion line of the Drosophila Genome Project we have cloned the black-pearl gene (blp), analyzed cDNA clones, generated various mutants, and characterized their phenotypes. The blp gene codes for a protein of 15.7 kDa calculated molecular weight that has been conserved from yeast to plants and mammals with high homology. A domain of these new proteins shows distant similarity to DnaJ domains indicating a functionally relevant interaction with other proteins. The P element insertion in line P1539 lies within the 5' untranslated leader of the black-pearl gene. Flies homozygous for this insertion are semi-lethal, escapers produce very few offspring and show melanotic inclusions in the hemocoel ('black pearls') similar to various melanotic 'tumor' mutants. Two small deletions confined to the blp gene and two EMS-induced mutations are homozygous lethal. These null mutants appear normal up to a prolonged first instar larval stage but fail to grow and die. Thus in Drosophila the blp gene is specifically required for larval growth. The evolutionary conservation in both unicellular and multicellular organisms suggests for the new protein family described here a fundamental role in cell growth.

Rat antigen-induced arthritis: cartilage alterations assessed with iodine-123-antileukoproteinase.

UNLABELLED: Imaging of cartilage alterations was attempted in joints of rats with chronic antigen-induced arthritis (AIA) using the cationic 123I-labeled serine proteinase inhibitor antileukoproteinase (123I-ALP; pI > 10), which selectively accumulates in normal cartilage, presumably through interaction with negatively charged proteoglycans. METHODS: Iodine-123-ALP or 123I-myoglobin, a control protein of comparable size but with different isoelectric point (pI=7.3) was injected intravenously into normal or AIA rats. Joint accumulation was followed by scintigraphy for 14 hr. Tissue radioactivity was assessed by well-counter measurements after dissection. The content of charged molecules in articular cartilage was determined by toluidine blue staining; the degree of joint destruction was assessed in parallel by x-ray, ex vivo MRI and histopathology. RESULTS: In intact articular cartilage, ALP accumulated to a significantly higher degree than myoglobin. This preferential accumulation was lost in rats with chronic AIA. The target-to-background ratio for 123I-ALP negatively correlated with the loss of toluidine blue staining in cartilage, which documents depletion of charged matrix molecules (r=-0.92, p < 0.01 at 4 hr; r=-0.97, p < 0.01 at 13 hr). ALP scintigraphy was sensitive in detecting cartilage alterations, even though the degree of joint destruction and inflammatory infiltration was mild, as demonstrated by x-ray, MRI and histopathology. CONCLUSION: In rat AIA, loss of ALP accumulation appears to document proteoglycan depletion in mildly altered arthritic cartilage. ALP scintigraphy may represent a functional assay for early, premorphological cartilage alterations in human arthritis as well.

The sap47 gene of Drosophila melanogaster codes for a novel conserved neuronal protein associated with synaptic terminals.

Proteins expressed specifically in neurons and transported to synaptic terminals are likely to constitute important molecular elements of nervous system function. In an effort to characterize synapse-associated proteins (SAPs) of Drosophila, we have isolated from a hybridoma library several monoclonal antibodies (MABs) that selectively stain synaptic terminals in immunohistochemical preparations. MAB nc46 binds to most but not all synaptic terminals of the Drosophila nervous system, it also recognizes a protein with homologous distribution in other dipteran flies and binds to large parts of fish CNS. In Western blots the antibody labels a Drosophila brain protein of 47 kDa and cross-reacts with brain proteins from several species including insects, fish, mouse and man. From these data we conclude that the corresponding gene has been conserved in evolution at least among diptera. Using MAB nc46 and expression cloning we have identified the 'sap47' gene coding for the 'synapse-associated protein of 47 kDa' of Drosophila melanogaster. Sequence analysis of genomic and cDNA clones reveals the intron-exon structure of the gene and characterizes the complete open reading frames of two alternatively spliced transcripts. The sap47 gene is located in 89A8-B3 on chromosome 3R and codes for two almost identical inferred polypeptides of 347 and 351 amino acids with no significant sequence homology to known proteins.

[3H]2-deoxyglucose mapping of odor-induced neuronal activity in the antennal lobes of Drosophila melanogaster.

Olfactory stimulation results in an enhanced uptake of [3H]2-deoxyglucose in specific glomeruli in the antennal lobes of Drosophila melanogaster. Unilateral stimulation induces activity in receptor axons and lobe interneurons on the ipsilateral side. Collaterals from the receptor axons to the contralateral lobe are also active but stimulate either weak or no postsynaptic activity. This difference in signal transfer properties could be relevant to odor detection by flies.

Identification of [3H]deoxyglucose-labelled interneurons in the fly from serial autoradiographs.

Using the [3H]deoxyglucose technique we find in the third visual ganglion of the fly, Musca domestica, a number of neuronal profiles whose labelling strongly depends on the direction of visual movement. By reconstruction from serial autoradiographs of semithin sections the three-dimensional morphology of the labelled profiles, we demonstrate that cell bodies, neurites, axons and arborizations of two interneurons are labelled whose homologues in Calliphora have been identified as movement-sensitive centrifugal horizontal cells ('CH-cells'). A set of three other cells whose homologues in Calliphora show similar electrophysiological responses to horizontal movement ('HS-cells') exhibit very little label on either side. It is suggested that the relation between deoxyglucose mapping and physiological activity can be investigated at the cellular level by using this system of fly interneurons.

Identification of H1 visual interneuron in Drosophila by [3H]2-deoxyglucose uptake during stationary flight.

High-resolution 2-deoxyglucose (2-DG) neuronal activity labeling is used to identify a visual interneuron in Drosophila by its stimulus-specific uptake of [3H]2-DG during stationary flight in a well-characterized behavioral situation. With a single rotating stripe as visual stimulus a neuron is heavily labeled that has not been described in Drosophila before but is homologous to the extensively studied H1 visual interneuron of larger diptera. Labeling of this cell is inconspicuous in Drosophila if the animal is stimulated with a rotating striped drum.

Autoradiographic localization of [3H]choline uptake in the brain of Drosophila melanogaster.

We describe the localization of sites for choline uptake in the brain of Drosophila melanogaster by the technique of autoradiography of diffusible substances. After systemic injection of [3H]choline, heavy labelling is found in certain regions of the ventral mid-brain, the antennal mechanosensory system including certain commissures, the olfactory lobes and the antennal glomerular tract. This accumulation of label is sensitive to hemicholinium-3 and can be suppressed by incubation at 0 degrees C. It is proposed that acetylcholine is a major neurotransmitter of the antennal mechanosensory and chemosensory systems of Drosophila.

Cardiotoxicity of dichloromethane in rats and in cultured rat cardiac myocytes.

The purpose of the present study was to examine whether cardiac actions of dichloromethane (DCM) in vivo correlate with in vitro alterations of Ca(2+) dynamics in cardiac myocytes. Electrically induced fluctuations of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) were investigated in neonatal rat ventricular myocytes using spectrofluorometric analysis of fura-2 binding. [Ca(2+)](i) transients were inhibited in a concentration-dependent and reversible manner with IC(10) and IC(50)values of 3.2 and 18.1 mm. Complete inhibition of [Ca(2+)](i) transients and cessation of beating were observed at 40.96 mm without morphological alterations. Left ventricular pressure in urethane-anaesthetized rats was measured by introducing a tip catheter by way of the carotid artery into the left ventricle and ECG (lead II) was recorded by two needle electrodes. Administration of 3.1, 6.2 or 12.4 mmol DCM/kg orally resulted in DCM blood concentrations between 1.0 and 1.6 mm accompanied by a dose-dependent decrease of contractility parameters. Moreover, DCM administration provided protection against arrhythmia development due to CaCl(2) infusion. These observations are consistent with the view that both the negative inotropic effects of DCM and the protection from CaCl(2)-induced arrhythmia are mediated by an inhibition of Ca(2+) dynamics in cardiomyocytes.

[Health promotion by sports in schools. A model project in 3 Cologne public schools--what is the responsibility of the school physician?]. / Gesundheitsförderung durch Sport an Schulen. Ein Modellprojekt an drei Kölner Realschulen--Was kann der Schularzt hierbei tun?

This project was started under the aspect of judging mental (psychic) and physical items of pupils from three schools, aged between 10 and 16. Therefore, it includes not only the subject of sports but also all the other subjects concerning health, such as biology and hygiene. Support by other institutions besides schools was necessary. The observation period was fixed up to three years, the main points being sports and motorial tests and accompanying medical care and survey by school doctors from the Public Health Centre. The project was started at the beginning of the school period 1987/88 and will last until the end of the school period 1989/90. The surveying period is still too short draw any conclusions or to make any statements.