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.     

A biologically active thrombin cleavage product of human serum spreading factor

Purified human serum spreading factor preparations consisting of two immunologically-related, biologically-active proteins of molecular weights approximately 65,000 and 75,000 were incubated with purified hydrolytic enzymes: papain, neuraminidase and thrombin. Biologically active products of the enzymatic digestions were obtained in each case. Digestion of serum spreading factor preparations with thrombin produced a single active form of molecular weight approximately 57,000. Generation of a single molecular weight form of serum spreading factor by thrombin cleavage of the two higher molecular weight forms should simplify studies of the biochemistry and biology of this protein, and may represent a reaction of physiological significance.     

Asparagine-linked glycosylation in Saccharomyces cerevisiae: genetic analysis of an early step.

Asparagine-linked glycosylation is a form of covalent modification that distinguishes proteins that are either membrane bound or are in cellular compartments topologically outside of the cell from those proteins that remain soluble in the cytoplasm. This type of glycosylation occurs stepwise, with core oligosaccharide added in the endoplasmic reticulum and subsequent modifications occurring in the golgi. We used tunicamycin, an inhibitor of one of the earliest steps in the synthesis of N-linked oligosaccharide, to select for mutants that are resistant to this antibiotic. Genetic, biochemical, and physiological experiments led to the following conclusions. The synthesis of N-linked oligosaccharide is an essential function in cells. In contrast to mammalian cells, yeast cells do not transport tunicamycin by a glucosamine transport function. We identified a gene, ALG7, that is probably the structural gene for UDP-N-acetylglucosamine-1-P transferase, the enzyme inhibited by tunicamycin. Dominant mutations in this gene result in increased activity of the transferase and loss of the ability of the cell to sporulate. In addition, we identified another gene, TUN1, in which recessive mutations result in resistance to tunicamycin. The ALG7 and TUN1 genes both map on chromosome VII.     

Inhibition of ceruloplasmin and other copper oxidases by thiomolybdate

The dietary antagonism between copper and molybdate salts prompted a study of the inhibition of copper enzymes by thiomolybdate (TM). TM strongly inhibited the oxidase activity of five copper oxidase with I50% values in the 1-5 microM range. The mechanism of the TM effect on the copper oxidase, ceruloplasmin (Cp) (E.C. 1.16.3.1), was studied in detail. In Vmax vs. E plots, TM gave parallel data suggesting irreversibility but a large number of TM molecules per Cp were required. The inhibition of Cp by TM could not be reversed by dialysis. Isolation of TM-inhibited Cp on Sephadex G-10 did not yield any active Cp molecules. Cu(II) did not restore any inhibited oxidase activity. Gel electrophoresis supported the covalent binding of Cp by TM without any extensive change in protein structure. EPR results confirmed that Cu(II) is reduced to Cu(I) after reaction with TM. However, the Mo(VI) in MoS4(2-) did not change in oxidation number. Analysis of the TM-Cp compound accounted for all six Cu atoms as found in native Cp. The data suggest the covalent binding of sulfide to Cp copper. TM also inhibited the activity of ascorbate oxidase, cytochrome oxidase, superoxide dismutase, and tyrosinase. However, no inhibition of carbonic anhydrase, a zinc enzyme, was observed at 1 mM TM.