Genetic and developmental characterization of Dmca1D,a calcium channel alpha1 subunit gene in Drosophila melanogaster.

To begin unraveling the functional significance of calcium channel diversity, we identified mutations in Dmca1D, a Drosophila calcium channel alpha1 subunit cDNA that we recently cloned. These mutations constitute the l(2)35Fa lethal locus, which we rename Dmca1D. A severe allele, Dmca1D(X10), truncates the channel after the IV-S4 transmembrane domain. These mutants die as late embryos because they lack vigorous hatching movements. In the weaker allele, Dmca1D(AR66), a cysteine in transmembrane domain I-S1 is changed to tyrosine. Dmca1D(AR66) embryos hatch but pharate adults have difficulty eclosing. Those that do eclose have difficulty in fluid-filling of the wings. These studies show that this member of the calcium channel alpha1 subunit gene family plays a nonredundant, vital role in larvae and adults.     

Dstac is required for normal circadian activity rhythms in Drosophila

The genetic, molecular and neuronal mechanism underlying circadian activity rhythms is well characterized in the brain of Drosophila. The small ventrolateral neurons (s-LN_Vs) and pigment dispersing factor (PDF) expressed by them are especially important for regulating circadian locomotion. Here we describe a novel gene, Dstac, which is similar to the stac genes found in vertebrates that encode adaptor proteins, which bind and regulate L-type voltage-gated Ca²⁺ channels (CaChs). We show that Dstac is coexpressed with PDF by the s-LN_Vs and regulates circadian activity. Furthermore, the L-type CaCh, Dmca1D, appears to be expressed by the s-LN_Vs. Since vertebrate Stac3 regulates an L-type CaCh we hypothesize that Dstac regulates Dmca1D in s-LN_Vs and circadian activity.     

Model organisms: new insights into ion channel and transporter function. L-type calcium channels regulate epithelial fluid transport in Drosophila melanogaster

The neuropeptide CAP2b stimulates fluid transport obligatorily via calcium entry, nitric oxide, and cGMP in Drosophila melanogaster Malpighian (renal) tubules. We have shown by RT-PCR that the Drosophila L-type calcium channel alpha1-subunit genes Dmca1D and Dmca1A (nbA) are both expressed in tubules. CAP2b-stimulated fluid transport and cytosolic calcium concentration ([Ca2+]i) increases are inhibited by the L-type calcium channel blockers verapamil and nifedipine. cGMP-stimulated fluid transport is verapamil and nifedipine sensitive. Furthermore, cGMP induces a slow [Ca2+]i increase in tubule principal cells via verapamil- and nifedipine-sensitive calcium entry; RT-PCR shows that tubules express Drosophila cyclic nucleotide-gated channel (cng). Additionally, thapsigargin-induced [Ca2+]i increase is verapamil sensitive. Phenylalkylamines bind with differing affinities to the basolateral and apical surfaces of principal cells in the main segment; however, dihydropyridine binds apically in the tubule initial segment. Immunocytochemical evidence suggests localization of alpha1-subunits to both basolateral and apical surfaces of principal cells in the tubule main segment. We suggest roles for L-type calcium channels and cGMP-mediated calcium influx in both calcium signaling and fluid transport mechanisms in Drosophila.     

Distinct roles of Drosophila cacophony and Dmca1D Ca2+ channels in synaptic homeostasis: Genetic interactions with slowpoke Ca2+‐activated BK channels in presynaptic excitability and postsynaptic response

Ca²⁺ influx through voltage‐activated Ca²⁺ channels and its feedback regulation by Ca²⁺‐activated K⁺ (BK) channels is critical in Ca²⁺‐dependent cellular processes, including synaptic transmission, growth and homeostasis. Here we report differential roles of cacophony (Ca_V2) and Dmca1D (Ca_V1) Ca²⁺ channels in synaptic transmission and in synaptic homeostatic regulations induced by slowpoke (slo) BK channel mutations. At Drosophila larval neuromuscular junctions (NMJs), a well‐established homeostatic mechanism of transmitter release enhancement is triggered by experimentally suppressing postsynaptic receptor response. In contrast, a distinct homeostatic adjustment is induced by slo mutations. To compensate for the loss of BK channel control presynaptic Sh K⁺ current is upregulated to suppress transmitter release, coupled with a reduction in quantal size. We demonstrate contrasting effects of cac and Dmca1D channels in decreasing transmitter release and muscle excitability, respectively, consistent with their predominant pre‐ vs. postsynaptic localization. Antibody staining indicated reduced postsynaptic GluRII receptor subunit density and altered ratio of GluRII A and B subunits in slo NMJs, leading to quantal size reduction. Such slo‐triggered modifications were suppressed in cac;;slo larvae, correlated with a quantal size reversion to normal in double mutants, indicating a role of cac Ca²⁺ channels in slo‐triggered homeostatic processes. In Dmca1D;slo double mutants, the quantal size and quantal content were not drastically different from those of slo, although Dmca1D suppressed the slo‐induced satellite bouton overgrowth. Taken together, cac and Dmca1D Ca²⁺ channels differentially contribute to functional and structural aspects of slo‐induced synaptic modifications. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 1–15, 2014     

Presynaptic N-type calcium channels regulate synaptic growth

Voltage-gated calcium channels couple changes in membrane potential to neuronal functions regulated by calcium, including neurotransmitter release. Here we report that presynaptic N-type calcium channels not only control neurotransmitter release but also regulate synaptic growth at Drosophila neuromuscular junctions. In a screen for behavioral mutants that disrupt synaptic transmission, an allele of the N-type calcium channel locus (Dmca1A) was identified that caused synaptic undergrowth. The underlying molecular defect was identified as a neutralization of a charged residue in the third S4 voltage sensor. RNA interference reduction of N-type calcium channel expression also reduced synaptic growth. Hypomorphic mutations in syntaxin-1A or n-synaptobrevin, which also disrupt neurotransmitter release, did not affect synapse proliferation at the neuromuscular junction, suggesting calcium entry through presynaptic N-type calcium channels, not neurotransmitter release per se, is important for synaptic growth. The reduced synapse proliferation in Dmca1A mutants is not due to increased synapse retraction but instead reflects a role for calcium influx in synaptic growth mechanisms. These results suggest N-type channels participate in synaptic growth through signaling pathways that are distinct from those that mediate neurotransmitter release. Linking presynaptic voltage-gated calcium entry to downstream calcium-sensitive synaptic growth regulators provides an efficient activity-dependent mechanism for modifying synaptic strength.     

Genomic Organization and Evolution of Alternative Exons in a Drosophila Calcium Channel Gene

The genomic organization of a gene coding for an α1 subunit of a voltage-gated calcium channel of Drosophila melanogaster (Dmca1A) was determined. Thirty-four exons, distributed over 45 kb of genomic sequence, have been identified and mapped, including exons in three regions involved in alternative splicing and new sites potentially involved in RNA editing. The comparison of the intron/exon boundaries of this channel with a mammalian counterpart shows that the genomic structure of these two genes has remained fairly similar during evolution, with more than half of the Drosophila intron positions being perfectly conserved compared to the human channel. Phylogenetic analysis of the mutually exclusive alternative exons revealed that they have diverged considerably. It is suggested that this divergence, rather than reflecting evolutionary age, is the likely result of accelerated rates of evolution following duplication.     

A model genetic system for testing the in vivo function of peptide toxins

We have developed a model genetic system for analyzing the function of peptide toxins from animal venoms. We engineered and propagated strains of Drosophila melanogaster expressing heat-inducible transgenes encoding either kappa-ACTX-Hv1c or omega-ACTX-Hv1a, two insect-specific neurotoxic peptides found in the venom of the Australian funnel-web spider Hadronyche versuta. Heat induction of transgene expression for 20 min was sufficient to kill all transgenic flies, indicating that the ion channels targeted by these toxins are viable insecticide targets. The unusual phenotype of flies induced to express omega-ACTX-Hv1a recapitulates that of a hypomorphic allele of the high-voltage-activated calcium channel Dmca1D, suggesting that this is likely to be the target of omega-ACTX-Hv1a.     

Anthobothrium galeorhini n. sp. (Eucestoda: Tetraphyllidea) a parasite of Galeorhinus galeus (Triakidae) from the Argentine coast

Anthobothrium galeorhini n. sp. (Eucestoda: Phyllobothriidae) collected from the spiral intestine of Galeorhinus galeus (Linnaeus, 1758) at the Puerto Madryn (Atlantic coastal region) is described. The new species differs from A. cornucopia as described by Euzet (1959) by the size of body, bothridia, gravid proglottids and eggs; by the neck length and by the different number of testes and proglottids. The size of bothridia and the gravid proglottids, the cirrus length and the number of testes allow differentiating the species here proposed from A. laciniatum Linton, 1890 as described by Euzel (1959). The body and cirrus sac size, the bothridia shape and the number of testes separate the species here described from A. parvum Stossich, 1895 as described by Yamaguti (1952). The anatomic differences between the cestode described by Arandas Rego (1977) and located laxonomycally under the name Anthobothrium laciniatum are listed and the systematic position of the cestode is discussed. Species parasitic of skates listed in scientific literature must be taxonomically reaccommodated, since their characteristics are not consistent with the diagnosis of Anthobothrium.     

A taxonomic revision of the pipunculid fly genus Chalarus Walker, with particular reference to the European fauna

Twenty-one species of Chalarus Walker are described from Europe, 11 of them new. The puparial remains of 15 species are described. Type material of all previously described Chalarus species except C. velutinus (Macquart, 1835) was examined. The status of C. holosericeus (Meigen, 1824) is revised, and exiguus (Haliday, 1833) is confirmed as a valid species. Lectotypes are designated for C. holosericeus and C. spurius (Fallén, 1816). Five species-groups are tentatively proposed. Separate keys to females and males are given. The status of Chalarus species described from outside Europe is discussed, and two of these, C. kamijoi Morakote, in Morakote & Hirashima (1990) and C. konishii Morakote, in Morakote & Hirashima (1990), are synonymized with fimbriatus Coe and exiguus (Haliday) respectively. The functional significance of some taxonomic characters, particularly of the eyes and ovipositor, is discussed.     

An off-line system for in-time analysis of cardiac catheterization data and for establishment of a cardiological database for retrospective studies.

The system described may be divided in two major parts: (i) automatic analysis of cardiac catheterization data running off-line on a Siemens 305 computer; (ii) storage and retrieval of the results from these analyses and additional data from related departments (thoracic surgery, internal medicine and clinical physiology) in a cardiological database on an IBM 370/155 computer. The first part is described with special regard to (i) operation and control during the phases of data collection, pre-processing, processing and storage of results; (ii) the presentation of the results in a complete and readable form and (iii) the modular design of the software. The results of an evaluation of the computer methods versus manual methods are also presented. The second part has been described with regard to the type of data entered in the cardiological database. The structure of the database and the programs used for storage and retrieval have not been described in detail in the present publication.     

Metabolic performance and insulin binding of mammary cells isolated from Wistar and Zucker rats

A method for the isolation of cells from lactating mammary gland tissue of rats is described. Metabolic properties of mammary cells isolated by the described method either from Wistar of Zucker rats as well as insulin binding were studied. Whereas synthesis of protein and lipid showed no significant differences, the rate of glucose oxidation is slightly increased in cells isolated from Zucker rats. Incorporation of (14)C-uridine in the RNA-fraction is stimulated by insulin; the half-maximal concentration is given by 5 x 10(-10) mol/1). Specific binding of insulin to both types of cells can be described by a nonlinear Scatchard-plot. The apparent affinity constants for the high affinity receptors (1 x 10(-10) mol/1) correlate well with the described metabolic effect of insulin on RNA-synthesis. The total capacity for insulin binding is reduced in cells isolated from Zucker rats when compared to those from Wistar rats.     

A new mermithid species (Nematoda) from chironomid imagos eclosing from Lake Ozawindib in northern Minnesota

Hydromermis biesboeri n. sp. (Nematoda: Mermithidae) is described from the hemocoel of imagos of chironomid host species eclosing from Lake Ozawindib in northern Minnesota in July and August 2008 and 2009. The new species is distinguished from the other 28 described species in the genus by terminal mouth, wide oval (male) and long oval (female) amphids with a dorsal commissure, tapered and rounded posterior ends, split base of the spicule, short protractor muscles in males, and by females having an S-shaped vagina with equal-length terminal limbs. The new species represents the 10th Hydromermis mermithid species described, or reported, from the region and the second from Lake Ozawindib.     

The cDNA sequence of a neutral horseradish peroxidase

A cDNA clone encoding a horseradish (Armoracia rusticana) peroxidase has been isolated and characterized. The cDNA contains 1378 nucleotides excluding the poly(A) tail and the deduced protein contains 327 amino acids which includes a 28 amino acid leader sequence. The predicted amino acid sequence is nine amino acids shorter than the major isoenzyme belonging to the horseradish peroxidase C group (HRP-C) and the sequence shows 53.7% identity with this isoenzyme. The described clone encodes nine cysteines of which eight correspond well with the cysteines found in HRP-C. Five potential N-glycosylation sites with the general sequence Asn-X-Thr/Ser are present in the deduced sequence. Compared to the earlier described HRP-C this is three glycosylation sites less. The shorter sequence and fewer N-glycosylation sites give the native isoenzyme a molecular weight of several thousands less than the horseradish peroxidase C isoenzymes. Comparison with the net charge value of HRP-C indicates that the described cDNA clone encodes a peroxidase which has either the same or a slightly less basic pI value, depending on whether the encoded protein is N-terminally blocked or not. This excludes the possibility that HRP-n could belong to either the HRP-A, -D or -E groups. The low sequence identity (53.7%) with HRP-C indicates that the described clone does not belong to the HRP-C isoenzyme group and comparison of the total amino acid composition with the HRP-B group does not place the described clone within this isoenzyme group. Our conclusion is that the described cDNA clone encodes a neutral horseradish peroxidase which belongs to a new, not earlier described, horseradish peroxidase group.     

Linkage analysis of five new genetic markers of the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae).

Five new autosomal recessive genes are described in the oriental fruit fly, Bactrocera dorsalis (Hendel). These genetic markers are associated into three linkage groups. The matte (mt) gene is linked to the previously described mandarin red (ma) gene, and the white puparium (wp) gene is linked to the white eye (we) and amethyst (am) loci. The third designated linkage group has the yellow eye (ye) marker. The we/we homozygote is epistatic to ye/ye, and each is epistatic to am/am and ma/ma.     

Synthesis of new steroid haptens for radioimmunoassay—VII. 19-O-carboxymethyl ether derivative of androstenedione. Specific antiserum for measurement of androstenedione in plasma

The synthesis of 19-O-carboxymethyl ether derivative of androstenedione and the preparation of its bovine serum albumin (BSA) conjugate is described. The BSA conjugate was employed to raise specific antibodies in rabbits suitable for radioimmunoassay (RIA). A non-chromatographic RIA for androstenedione is described.     

A fluorogenic active-site titrant for serine proteases

The synthesis of 3'-(4-guanidinobenzoyloxy)-spiro(isobenzofuran-1(3H),9H-[9H]xan then]-3-one or 3HFGB, an active-site titrant for serine proteases, is described. 3HFGB has two advantages over previously described titrants. It is very sensitive (it can measure serine proteases at the 10(-11)-10(-12) M level), and it can be used at stoichiometric ratios of titrant to protease of unity or less. Moreover, the fluorogenic leaving group, 3'-hydroxyspiro[isobenzofuran-1(3H),9'-[9H]xanthen]-3- one, is suitable for use in a range of esteropeptidase substrates. The use of 3HFGB in the active-site titration of trypsin is described.     

A Redescription of Adult Phyllodistomum umblae (Fabricius) (Digenea, Gorgoderidae) from Salvelinus alpinus (L.) in Norway

Phyllodistomum umblae (Fabricius, 1780), originally described on the basis of specimens recovered from the ureter of Arctic char Salvelinus alpinus in Drangedalen, Norway, is here redescribed with both light (LM) and scanning electron microscope (SEM) from material obtained from the type host and type locality. This species has been overlooked in general text-books of this century, e.g. those of Dawes and Yamaguti, but was discussed by Nybelin in a paper in 1926 in which he suggested its conspecificity with P. conostomum Olsson. The dimensions, shapes, position and arrangement of the internal structures and selected size ratios are described and illustrated with LM-pictures. The SEM-investigation reveals the tegumental microstructure, and special emphasis is given to the arrangement of papillae. The body papillae comprise: (a) constant numbers in a constant bilateral arrangement; (b) a variable number in an orientated bilateral concentration called rows; (c) regional concentrations; (d) randomly distributed ones. This system and its variability are described and figured. Four types of papillae, and their distribution as seen by SEM, are described. These results are compared with the results from other SEM–investigations on gorgoderids.     

Management of disease control and epidemics in AI in Denmark

The author's presentation constituted a survey of the control of contagious diseases at Danish AI stations. The control is performed in accordance with EU regulations and requires documented freedom from Aujeszky's disease, Classical Swine Fever and Brucellosis (based on blood testing) before transfer to quarantine sections and to AI stations. The Danish regulations regarding Porcine Reproductive and Respiratory Syndrome (PRRS) are described for both sero-positive and sero-negative stations. At some stations, all boars are vaccinated against both American PRRS (Ingelvac PRRS vet.) and European PRRS (Progressis vet.), and are subsequently sero-positive for PRRS. The period from vaccination until the time that semen can be used is described, and the reason for its length is explained. Furthermore, the process applied when distinguishing Yersinia from Brucellosis reactions is described. The purpose of this control is to prevent Yersinia-positive herds that supply boars from compromising supplies from Yersinia-negative herds through cross-reactions with Brucellosis. The vaccinations required before transfer to isolation and AI stations (Glasser's disease, pleuropneumonia caused by Actinobacillus pleuropneumoniae, erysipelas and PPV) as well as vaccinations done regularly at the AI stations (erysipelas and PPV) are described. Finally, a survey is presented of other diseases found at Danish AI stations (primarily pneumonia and leg problems) and their medical treatment.