USE OF A PETHIDINE AND MIDAZOLAM COMBINATION FOR THE REVERSIBLE SEDATION OF CRABEATER SEALS (LOBODON CARCINOPHAGUS)

Between October and December of 1996–1999, off eastern Antarctica (60°-150°E), we darted 31 crabeater seals with midazolam and pethidine at estimated dose rates of 0.15–0.4 mg/kg and 1–3 mg/kg, respectively. Maximum sedation was reached at 23 ± 9 min (n = 18) and first signs of recovery were noted at 54 ± 24 min (n = 4). Seals greater than 250 kg body-mass were sedated by administration of approximately 90–100 mg midazolam and 600 mg pethidine, but the degree of sedation was unpredictable and did not permit invasive procedures in some cases. Behavior of the seal and adjacent conspecifics affected the success of procedures and our ability to monitor vital signs. Naloxone and flumazenil reversed sedation, making this combination attractive for use in animals adjacent to water. Additional ketamine was administered to two seals, resulting in improved restraint.     

Manipulating photosynthesis

The levels of individual photosynthetic proteins can be independently decreased by theAgrobacterium-mediated transformation of plants with antisens RNA constructs. Protocols for the introduction of such constructs intoAgrobacterium, theAgrobacterium-mediated transformation of tobacco leaf disks, and the screening and analysis of the transgenic plants produced are described.     

Chemistry in environmentally benign media Part 1. Synthesis and characterization of 1,2-bis[bis(hydroxymethyl)phosphino]ethane (‘HMPE’). X-ray structure of [Pt{(HOH2C)2PCH2CH2P(CH2OH)2}2](Cl)2

The water-soluble bisphosphine, 1,2-bis(bis(hydroxymethyl)phosphino)ethane (1), was synthesized in near quantitative yield by the reaction of bisphosphine, H₂PCH₂CH₂PH₂, with an aqueous formaldehyde in the presence of K₂PtCl₄. The reaction of this water-soluble bisphosphine 1 with cis-Pt(COD)Cl₂ affords the mononuclear bischelate complex, [Pt{(HOH₂C)₂PCH₂CH₂P(CH₂OH)₂}₂](Cl)₂ (2), in near quantitative yield. The new ligand and complex have been characterized spectroscopically and the structure of the metal complex, 2, was determined by X-ray crystallography. The Pt(II) complex 2 crystallizes in the orthorhombic space group Pbca(a=14.623(1), B=16.216(2), C=9.319(4) Å) with Z=4. The final R value is 0.024.     

Experimental Oesophagostomum dentatum infection in the pig: Worm populations resulting from single infections with three doses of larvae

This report describes the effect of different dose levels of infection upon worm burdens and development and fecundity of the parasites. Three groups each of 40, 9-week-old, helminth naïve pigs were inoculated once with either 2000 (group A), 20,000 (group B), or 200,000 (group C) infective third stage larvae of Oesophagostomum dentatum. Subgroups of 5 pigs from each major group were killed 3, 6, 11, 14, 18, 25, 34 and 47 days post inoculation (p.i.) and the large intestinal worm burdens were determined. Faecal egg counts were determined at frequent intervals after day 13 p.i. There were no overt clinical signs of gastrointestinal helminthosis during the experiment. Faecal egg counts became positive in groups A and B at around day 19 p.i., whereas most pigs in the high dose group C did not have positive egg counts until day 27–33 p.i. and some pigs remained with zero egg counts until the end of the study. Throughout the experiment the worm populations in group C consisted mainly of immature larval stages, while those in groups A and B were predominantly adult stages after days 14–18. Adult worms from the low dose group A were significantly longer than those from group C. At high population densities, stunted development of worms and reduced fecundity among female worms were found. Furthermore, there was a tendency for the distribution of the worms within the intestine to be altered with increasing population size.     

Magnesium-Dependent stimulation of protein synthesis by the insulin mimic,pervanadate

The insulin mimic, peroxide of vanadate (pervanadate), stimulated ³⁵S-methionine incorporation into Xenopus oocyte protein in a Mg²⁺-dependent manner. Reducing the extracellular Mg²⁺ concentration from 1.0 to 0.1 mM decreased the pervanadate-stimulated component of incorporation by 35%; with 0.01 mM Mg²⁺ or lower, the pervanadate-stimulated component was abolished. In addition, reducing extracellular Mg²⁺ to 0.01 mM inhibited about 50% of the insulinstimulated component of methionine incorporation. Mg²⁺ depletion had no effects on incorporation in controls or when protein synthesis was stimulated by Zn²⁺ or bovine growth hormone. Thus, not all substances that stimulated protein synthesis showed a dependence on extracellular Mg²⁺. Reducing extracellular Ca²⁺ had no effects on methionine incorporation in control cells or in cells stimulated by pervanadate or insulin. When oocytes maintained in a paraffin oil medium were brought into contact with a 0.5 m?I droplet of buffer containing the Mg²⁺ indicator dye, mag-fura-2, and pervanadate, apparent droplet Mg²⁺ decreased rapidly, indicating net uptake by the cells. Insulin also caused a net uptake of Mg²⁺. In contrast, apparent extracellular Mg²⁺ was constant when cells were in contact with droplets containing no effectors. Together, these data indicate that extracellular Mg²⁺, but not Ca²⁺, is involved in the stimulation of protein synthesis by pervanadate, and to a lesser extent by insulin. Pervanadate appears to induce a net uptake of Mg²⁺, and this change in membrane transport may be an early event in signalling the increase in translation. © 1995 Wiley-Liss, Inc.     

PEST sequences in calmodulin-binding proteins

Many short-lived proteins which are devoid of proteolytic activity contain PEST sequences which are segments along the polypeptide chain that are rich in proline (P), glutamate (E), serine (S) and threonine (T). These designated PEST sequences are believed to be putative intramolecular signals for rapid proteolytic degradation. Calmodulin is a ubiquitous, 17kDa, acidic Ca²⁺-binding protein which plays an important role in the regulation of many physiological processes through its interaction with a wide range of calmodulin-binding proteins. Several calmodulin-binding proteins are known to contain PEST sequences and are susceptible to proteolysis by endogenous neutral proteases such as calpain I and calpain II. In this report, we discuss the functions of PEST sequences in calmodulin-binding proteins and assess the correlation between calmodulin-binding proteins and PEST sequences.     

The Caenorhabditis elegans Avermectin Resistance and Anesthetic Response Gene unc-9 Encodes a Member of a Protein Family Implicated in Electrical Coupling of Excitable Cells

Abstract: Mutations in the unc-9 gene of the nematode Caenorhabditis elegans cause abnormal forward locomotion and an egg-retention phenotype. unc-9 mutations also reduce the worms' sensitivity to avermectin and block a form of hypersensitivity to volatile anesthetics. We report here the cloning and molecular characterization of unc-9 and show that it encodes a member of the OPUS family of proteins that is 56% identical to another OPUS protein, UNC-7. It is significant that unc-9 mutants share all phenotypes with unc-7 mutants. Mutants in another gene, unc-124 , also share all tested phenotypes with unc-9 mutants, including identical locomotory and egg-laying defects, suggesting that multiple genes are required for the same biochemical function. OPUS proteins are implicated in the function of invertebrate gap junctions, and, based on a new alignment including 24 members from C. elegans , we present a refined model for the structure of OPUS proteins suggesting that oligomers could form a hydrophilic pore. We also show that alteration of highly conserved proline residues in UNC-9 leads to a cold sensitivity that likely affects a step in protein expression rather than function. Finally, we speculate on the basis of the avermectin resistance and anesthetic response phenotypes.     

Seasonality of feeding activity in Antarctic suspension feeders

The feeding activity of four benthic suspension-feeding groups (bryozoans, hydroids, polychaetes and holothurians) was monitored in situ every month for a 2-year period at Signy Island in the maritime Antarctic. The bryozoans were monitored at species level, whereas the other taxa could be differentiated only to genus. A marked seasonal variation in feeding activity was observed in most taxa. Although environmental parameters such as sea water temperature, fastice duration and water column chlorophyll concentrations suggested that winter in the maritime Antarctic lasts for about 6 months, many animals ceased feeding only for a short period of 2 or 3 months around the middle of the austral winter (June/July). These suspension feeders must therefore be efficient at utilising the low concentration of the microplankton existing in the water column for much of the year. Comparison with environmental variables suggested several possible cues for changes in feeding activity, but these cues may differ between taxa. Photoperiod and changes in disturbance by water movement (both mediated by ice), and food concentration are likely to be important environmental cues for polar suspension feeders.     

Electrophoresis of proteins and nucleic acids on acrylamide-agarose gels lacking covalent crosslinking

The preparation of acrylamide-agarose gels lacking covalent crosslinking with methylenebisacrylamide is described. These hybrid gels melt at 85 degrees C and, consequently, allow quantitative analysis of tritium-labeled protein after electrophoresis. Recovery of tritium-labeled ribonucleic acids extracted from hybrid gels is 20 to 25% greater than from standard acrylamide-methylenebisacrylamide gels. Standard curves of electrophoretic mobilities as a function of molecular weights of dissociated proteins and ribonucleic acids are compared for acrylamide-agarose gels and acrylamide-methylenebisacrylamide gels.