An endonuclease fromCaenorhabditis elegans: Partial purification and characterization

A deoxyribonuclease was partially purified from the free-living nematodeCaenorhabditis elegans. The DNase functioned as an endonuclease and introduced both single-strand nicks and double-strand breaks into DNA. The enzyme hydrolyzed double-stranded DNA seven times more rapidly than single-stranded DNA. DNase activity was not affected by the addition of divalent cations below 1mm but was inhibited at higher ionic concentrations. In addition, the enzyme was not inhibited in the presence of 10mm EDTA. The enzyme was inhibited by salt concentrations greater than 20mm. Three independent mutations in thenuc-1 gene were shown to reduce nuclease activity to less than 1% of that seen in wild-type organisms. This work was supported by National Institutes of Health Grant AG03161 and a TCU Research Foundation Grant. Some stocks used in these experiments were obtained from theCaenorhabditis Genetics Center, which is supported by Contract NOI-AG-9-2113 between the NIH and the curators of the University of Missouri.     

Herbal medicine among the miskito of Eastern Nicaragua

Medicinal plants identified by Miskito informants in Awastara, Nicaragua, were collected in the field. They are listed and botanically identified in this paper. Particularly interesting among the collection of 23 plant species are those used to cure snakebite and athlete’s foot, as observed in the field.     

Effect of habitable space on substrate selection by Paragnetina media (Walker) (Plecoptera: Perlidae)

A biological measure of space available within substrates was used as an index to examine substrate selection by the stonefly nymph Paragnetina media (Walker). Physical measures, such as total surface area of substrate, have not worked well in the past in explaining distribution of aquatic invertebrates. Although analysis of habitable space within substrate did not explain selection completely, the technique provided a precise measure and might be a more rigorous means by which substrate selection could be examined.     

Spontaneous cell shedding by tumor cells in monolayer culture

Summary Sarcoma 180 monolayers spontaneously shed single cells and small multicellular aggregates into the surrounding medium to produce a dual population of floating and substratum-attached cells. Shedding was a motility-associated event that occurred when cells attempted to migrate over one another. It resulted from a combination of cell shape change and active motility, which increased sensitivity to fluid shear dislodgement by reducing a cell's surface area of adhesive contact and increasing strain tension at its adhesive contact points. Shedding occurred at all phases of the cell cycle. Extracellular matrix but not conditioned medium enhanced the floating subpopulation by slowing the kinetics of rattachment to plastic and cellular substrata. Although sarcoma 180 cells are anchorage independent in the sense that they grow readily in single cell suspension, they nevertheless exhibited anchorage modulation of their cell cycle. Short periods in suspension produced a mild G₁ accumulation, whereas longer periods of anchorage deprivation led to a mild G₂ accumulation which appeared to result from an interference with cytokinesis. This work was supported by grants from the Medical Research Council of Canada, The National Cancer Institute of Canada, the Alberta Heritage Savings and Trust Fund for Applied Cancer Research, and the Alberta Heritage Fund for Medical Research.     

Drug Potencies on Partially Purified Brain D2 Dopamine Receptors

To examine the sensitivities of partially purified dopamine receptors to various dopaminergic agonists and antagonists, canine brain striatum dopamine receptors were enriched by isoelectric focusing. The digitonin-solubilized receptors were prelabelled with [3H]spiperone and focused for two time periods. After 5 h (incomplete focusing), radioactive peaks were detected at pH 6 and 9-11. Only the pH 6 peak revealed drug sensitivities expected of D2 receptors. Receptor recovery of the pH 6 peak was 79% with purification being sevenfold. After focusing overnight to equilibrium, the pH 6 peak further separated into peaks at pH 4.6 and 6.8. The receptor was identified only in the pH 4.6 fraction. The recovery of receptors in the pH 4.6 peak was low (10%), indicating little enrichment of the receptor. The rank order of binding of neuroleptics and dopamine agonists to the purified material was similar to that of the original preparation of soluble receptors. Dopamine did not bind to the purified pH 4.6 fraction unless the phosphate buffer (used during focusing) was replaced with Tris buffer. The absence of receptors in the pH 6.8 and pH 10 fractions, although both contained prelabeled [3H]spiperone, indicates the importance of testing agonists and antagonists on each fraction at each step in purification.     

A rapid naphthol yellows method for measuring the cellular protein content of anchorage cultures

Summary A rapid method has been developed for measuring the cellular protein content of mono- and multilayered anchorage cultures. Fixed or air dried cultures are stained for 30 min with 0.2% Naphthol Yellow S (NYS) dissolved in 1% acetic acid. Unbound dye is removed by a series of four 2.5 min washes in 1% acetic acid, and protein-bound dye extracted with 10 mM unbuffered Tris base for spectrophotometric optical density determination at 433 nm. The NYS method exhibited a least-squares correlation coefficient of 0.99997 with the Oyama-Eagle Lowry method.     

A simple and rapid method for screening bacteria for type II restriction endonucleases: enzymes in Aphanothece halophytica

A method is described which allows a large number of bacterial strains to be rapidly and easily screened for the presence of site-specific endonucleases. The method involves selective permeabilization of the bacterial cell and analysis of the exuded material. Type II restriction endonucleases from cyanobacteria and Gram-negative eubacteria have been detected and new enzymes have been found. The method should be widely applicable and easy to modify for use in genera other than those tested. Three-site-specific endonuclease activities, detected by this method in Aphanothece halophytica PCC 7412, were purified and their recognition and cleavage specificities were determined AhaI and AhaII recognise and cleave the same DNA sequences as CauII and AcyI respectively; the specificity of AhaIII (TTTAAA) has been reported previously (Whitehead and Brown, 1982, FEBS Letters 143:296–300).Abbreviations Brij-58 20 cetyl ether - Pu purine nucleoside - Py pyrimidine nucleoside     

DNA probe localization at 18p113 band by in situ hybridization and identification of a small supernumerary chromosome

Summary Recombinant plasmid clone B74 (also named D18S3) containing a human single-copy DNA segment of 6 kilobases (kb) was localized by in situ hybridization on band p113 of chromosome 18. This probe was then used in cytogenetic diagnosis to identify precisely a small supernumerary chromosome as an isochromosome i(18p).     

Differential hormonal action of the bag cell neurons on the arterial system ofAplysia

Summary The peptide-secreting bag cell neurons ofAplysia californica activate a long-lasting, complex behavior called egg laying. During egg laying some organ systems (reproductive) are more active than others (digestive) suggesting that blood flow to these tissues may change in accordance with their activities during egg laying. To examine this possibility we used a semi-intact preparation of the three major arteries innervated by the abdominal ganglion. We found that electrically stimulated bursts of bag cell activity triggered a long-lasting (>1 h) increase in contractile activity in two arteries, the anterior and gastroesophageal, but did not affect contractions of the third (abdominal) artery. The arterial responses were not affected either in form or duration by denervation of the arteries, suggesting that the increase in contractile activity was mediated by hormonal actions of bag cell transmitters on vasoconstrictor muscles. In intact animals this differential action on the arterial system may cause a long-term decrease in blood flow to relatively inactive tissues (digestive and locomotory organs) while increasing circulation to tissues involved in egg production (ovotestis and oviduct).Abbreviations ASW artificial sea water - BCA bag cell activation - ELH egg laying hormone