Characterization of the minimum domain required for targeting budding yeast myosin II to the site of cell division

Background  

All eukaryotes with the exception of plants use an actomyosin ring to generate a constriction force at the site of cell division (cleavage furrow) during mitosis and meiosis. The structure and filament forming abilities located in the C-terminal or tail region of one of the main components, myosin II, are important for localising the molecule to the contractile ring (CR) during cytokinesis. However, it remains poorly understood how myosin II is recruited to the site of cell division and how this recruitment relates to myosin filament assembly. Significant conservation between species of the components involved in cytokinesis, including those of the CR, allows the use of easily genetically manipulated organisms, such as budding yeast (Saccharomyces cerevisiae), in the study of cytokinesis. Budding yeast has a single myosin II protein, named Myo1. Unlike most other class II myosins, the tail of Myo1 has an irregular coiled coil. In this report we use molecular genetics, biochemistry and live cell imaging to characterize the minimum localisation domain (MLD) of budding yeast Myo1.     

Comparison of PCR and clinical laboratory tests for diagnosing <Emphasis Type="Italic">H. pylori</Emphasis> infection in pediatric patients

Background

Histology and/or culture are generally considered the gold standard for the detection of H. pylori infection. Especially in children, these tests may result in a false negative outcome because of patchy distribution of the organism in the stomach mucosa. We have developed a PCR assay utilizing nested primer pairs directed against a subunit of the H. pylori urease gene (ureA). As part of a prospective evaluation of diagnostic tests to aid in detecting H. pylori infection in children, the aim of this study was to compare our PCR and Western blot assays with results obtained from histologic examination of biopsy specimens, rapid urease tests, and an FDA approved serologic assay and published PCR results to determine if we could validate the assays for diagnostic use on our patient population.

Results

Gastric biopsy specimens obtained from 101 pediatric patients were evaluated for the presence of H. pylori using histologic techniques, rapid urease (CLOtest) test and the PCR assay. Serum samples from each patient were assayed using both ELISA and Western Blot for antibodies to H. pylori. A total of 32 patients tested were positive by at least one of the methods evaluated. Thirteen patients had positive histology, 13 had a positive CLOtest, and 17 patients had positive H. pylori PCR. Out of the 13 CLO positive patients, 12 were positive by histologic analysis and all 13 were positive by PCR. Results of serologic tests on the same population did not correlate well with other assays. Twenty-eight patients showed serologic evidence of H. pylori infection, of which 9 were both CLO and histology positive and 12 were positive by PCR. Of the seropositive patients, 26 were ELISA positive, 13 were positive by Western blot, and 11 by both serologic methods.

Conclusions

The results obtained suggest that our nested PCR assay has the specificity and sensitivity necessary for clinical application when compared to standard histologic examination and rapid urease test. In addition, we found the current commercially available approved ELISA method appears unable to accurately detect H. pylori in this population. The Western blot assay yielded better concordance with CLOtest and histology, but not as good as the nested PCR assay.

     

Determination of free space, growth, solute concentrations and parameters of water relations of suspension-cultured tobacco cells

Abstract Methods were developed for measuring water content of the free space of suspension-cultured tobacco cells using ³H- or ¹⁴C-sorbitol. Sorbitol was not taken up by cells in significant quantities over the 3 min taken to label free space. Free space accounted for 50–60% of the water content of cell pellets irrespective of whether ³H- or ¹⁴-C-sorbitol was used. ¹⁴C-inulin labelled 13.5% less of the water in cell pellets than ³H-sorbitol, probably due to inadequate penetration by inulin into the free space in the cell wall matrix and within clumps of cells. Measurement of free space is necessary for measuring growth on a fresh or dry weight basis, solute concentrations and parameters of water relations of cells. Techniques for making these measurements on tobacco cells were also developed in this study. Solutes were measured after extraction from cells by expressing sap or by boiling cells in ethanol. Similar solute concentrations were found using both methods of extraction. By expressing sap from cells grown in culture medium with an osmotic pressure of 0.24 MPa, the cells were found to have an internal osmotic pressure of 0.70 MPa. Glucose, fructose, sucrose, amino acids and K⁺ accounted for 60% of this osmotic pressure. Elastic moduli were estimated using the Boyle-Van't Hoff relationship after suspending cells in solutions with different osmotic pressures and assessing their water content or internal osmotic pressure. For two different lines of tobacco cells, elastic modulus varied between 1 MPa and 5.4 MPa at turgor pressures of 0.15–0.52 MPa (line 1) and between 0.2 MPa and 4.2 MPa at turgor pressures of 0.04–0.26 MPa (line 2).     

THE COMPOSITION OF PHOSPHATE GRANULES IN THE DIGESTIVE GLANDS OF MARINE PROSOBRANCH GASTROPODS: VARIATION IN RELATION TO TAXONOMY

The composition of some 1150 phosphate granules in the digestiveglands of over 40 species of marine prosobranch gastropods hasbeen surveyed using a simple preparation technique and semi-quantitativeSEM x-ray microanalysis. Spectral peaks for Mg, K, Ca, Mn, Feand Zn were compared to that of P. Four major types of phosphategranule can be recognised, each generally characteristic ofa taxonomic grouping: high Mg in archaeogastropods and littorinids,multiple metal in higher mesogastropods, and, in neogastropods,Mg-Ca in muricoideans and high Zn in buccinoideans. At leastone Conus species (C. ventricosus) has high-Mg granules. Somecauses of variation in granule composition are discussed: speculatively,it is suggested a palaeoenvironmental influence seems possible. (Received 5 November 1997; accepted 6 January 1998)     

Inheritance of pathogenicity and cultural characters in Ceratocystis ulmi: hybridization of aggressive and non-aggressive strains

When British isolates of Ceratocystis ulmi were surveyed for compatibility type, both A- and B-types were found in the non-aggressive strain, but only the B-type in the aggressive strain. Single ascospore progeny from crosses between compatible aggressive and non-aggressive isolates showed a near-normal growth rate distribution, with a mean lying between the parents. Many grew either faster than the aggressive or slower than the non-aggressive parent. The progeny were highly variable in culture morphology and could not be classified in terms of the parental types. When inoculated into English elm they showed a marked skewness towards low pathogenicity. None approached the aggressive strain in pathogenicity. It is concluded that the above characters are under polygenic control, and that the aggressive strain could not arise from the non-aggressive by a simple mutation. The results suggest that the two strains may be reproductively isolated.     

Eggplant mosaic virus, and its relationship to Andean potato latent virus

Eggplant mosaic virus (EMV), obtained from Solanum melongena L. from Trinidad, is readily transmitted by inoculation of sap to several solanaceous and a few non-solanaceous plant species. Purified preparations of EMV contain isometric particles 30 nm in diameter, and with sedimentation co efficients of either 111 or 53 S. The particles have thirty-two major morphological subunits. EMV is closely serologically related to Andean potato latent virus and has a similar host range, but is more virulent. Also, whereas EMV accumulates fastest in Nicotiana clevelandii leaves at 20–24 °C, Andean potato latent virus accumulates fastest at 15 °C, and fails to attain a serologically detectable concentration at 24 °C. A few symptomatologically or serologically distinguishable strains of EMV were obtained. EMV has properties typical of viruses of the Andean potato latent subgroup of the turnip yellow mosaic group of viruses, and its present cryptogram is */*:*/*:S/S:S/Cl.     

Inoculum sources for coffee berry disease*

Isolates of Colletotrichum spp. from coffee berries and bark in Kenya fell into four categories, distinguishable by their cultural characteristics on agar. Three of these proved non-pathogenic; the fourth invariably infected both wounded and unwounded berries and caused coffee berry disease. The sporulating capacity on bark of the various Colletotrichum strains showed peaks in the periods before both the long and the short rains. The pathogenic strain made up only a very small proportion of the whole Colletotrichum complex on the bark and its sporulating capacity never exceeded twenty spores/cm²/h. By contrast, a green berry with an active coffee berry disease lesion produced 5×10⁴conidia/h and an infected ripe berry twice as many. It is concluded that in any year, regardless of cropping pattern, diseased berries are likely to play an important role in the development of coffee berry disease.     

Interactive Growth of Staphylococcus aureus Strains with a Poultry Skin Microflora in a Diffusion Apparatus

The growth of different biotypes of Staphylococcus aureus strains isolated from poultry was studied using the NBS Ecologen in which the interacting mixed culture was derived from the microfiora of hen skin and separated from the Staph. aureus culture by a membrane of 0.4 μm pore size. Inhibition of growth of the Staph. aureus cultures occurred with strains from each biotype. Marked inhibition of growth was always accompanied by the production of large numbers (>10⁹/ml) of plaque forming units (phage). In the mixed culture chamber poultry phage group C strains became the predominant Staph. aureus type. The phages produced by the mixed culture showed a wide spectrum of lytic activity for the propagating strains of the human, bovine and poultry phage sets.     

Broad-bean stain and true broad-bean mosaic viruses

Autumn-sown crops of broad beans (Vicia faba L.) in England often contain plants with some leaves characteristically distorted and with a chlorotic mosaic. From some of these plants true broad-bean mosaic virus was isolated in 1959 and 1960 but not in 1965 and 1966. From other plants a similar but distinct virus, which caused staining of the seeds and we call broad-bean stain virus, was isolated in 1960, 1965 and 1966. The two viruses were readily distinguished in serological tests, and in some test plants. Both were seed-borne, and spread in crops, but were not transmitted by several animal species tested as vectors. Both viruses have isometric particles about 25 mμ in diameter. Some of these particles contain about 35% ribonucleic acid, some about 26% and some of those of broad-bean stain virus contain none; these three types of particles had sedimentation coefficients of about 120–130 S, 100 S and 60 S respectively. The ribonucleic acid of each virus had molar base content of G 23%, A 26%, C 18% and U 32%. These two viruses are members of the cowpea mosaic group of plant viruses; broad-bean strain virus was serologically related to cowpea mosaic, F I, red-clover mottle, and squash mosaic viruses. The particles of all these viruses and of true broad-bean mosaic virus were similar in appearance, sedimentation behaviour, and nucleic acid content and composition. The nucleic acid of red-clover mottle virus had a molar base content of G 20%, A 29%, C 20%, U 30%.