A wat1 mutant of fission yeast is defective in cell morphology

The organization of the actin cytoskeleton plays an integral role in cell morphogenesis of all eukaryotes. We have isolated a temperature-sensitive mutant in Schizosaccharomyces pombe, wat1-1, in which acting patches are delocalized, resulting in an elliptically shaped cell phenotype. Molecular cloning and DNA sequencing of wat1 ⁺ showed that the gene encodes a 314 residue protein containing WD-40 repeats. Cells lacking wat1 ⁺ are slow growing but viable at 25° C and temperature-sensitive for growth above 33° C. At restrictive temperature, wat1-d strains are phenotypically indistinguishable from wat1-1. When combined with a deletion for the wat1 ⁺ gene, cdc mutants failed to elongate at restrictive temperature and exhibited alterations in actin patch localization. This analysis suggests that wat1 ⁺ is required directly or indirectly for polarized cell growth in S. pombe. Wat1p and a functional, epitope-tagged, version of Wat1p can be overproduced without inducing alterations in cell morphology. Received: 18 September 1996 / Accepted: 22 October 1996     

Environmental factors affecting the occurrence of different morphological forms of cyanoprokaryotes in the northern Baltic Sea

Cyanoprokaryotes (blue-green algae, cyanobacteria) and environmentalfactors were studied in the Baltic Sea, off the SW coast ofFinland, from April to December 1993. The aim was to outlinethe seasonal development of cyanoprokaryote populations andthe occurrence of species and species groups in relation toprevailing hydrographic conditions and general features of theplanktonic community. Altogether, 27 taxa of cyanoprokaryoteswere encountered, representing the orders Chroococcales, Nostocalesand Oscillatoriales. Different morphological forms of cyanoprokaryotespredominated during certain periods of the year the spherical,gomphosphaerioid colonies of the genera Coelomoron, Coelosphaerium,Snowella and Woronichinia were rather abundant in early springand the most abundant group in late autumn; minute-celled colonieswith mucilaginous structures, e.g. the genera Aphanothece, Cyanonephron,Lemmermanniella and Merismopedia, were abundant during earlysummer; and filamentous, nitrogen-fixing species in late summerand early autumn. Each of the periods represents a distinctsuccessional phase of the planktonic community with inherentenvironmental conditions, and it was obvious that the cyanoprokaryoticassemblage was modified by the general development of the planktoniccommunity. According to canonical correspondence analysis (CCA),temperature and the weight ratio of inorganic nitrogen to inorganicphosphorus were the most important factors explaining variationin the cyanoprokaryote assemblage.     

Fertile plant regeneration from cell suspension and protoplast cultures of barley (t Hordeum vulgare cv. Schooner)

Fertile plants were regenerated from both cell suspension and protoplast-derived cultures of the two-row barley, Hordeum vulgare L. cv. Schooner. Embryogenic calluses, derived from immature embryos, were used to establish suspension cultures. More than 100 plants, with variable seed set, have been regenerated from six embryogenic cell suspension cultures. Protoplasts isolated from three suspension cultures divided and when the resultant embryogenic proto-calluses were transferred to regeneration medium both green and albino shoots were produced. The green shoots were transferred to growth regulator-free medium and plantlets that developed strong root systems were potted in soil and grown to maturity in the glasshouse. Root tip analysis of plants regenerated from cell suspension cultures revealed the expected 2N = 14 complement of chromosomes. However, chromosomal analysis of protoplast-derived plants showed numerical variation among a proportion of the regenerants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Sensitive chiral high-performance liquid chromatographic assay for labetalol in biological fluids

The four stereoisomers of the combined α- and β-adrenoceptor antagonist labetalol were separated and quantified at therapeutic concentrations by normal-phase high-pressure liquid chromatography using a chiral stationary phase and fluorescence detection. Drug in plasma or urine was recovered by solid-phase extraction with 83±5% efficiency. Limits of detection from biological samples (3 ml) were between 1.5–1.8 ng ml⁻¹. Intra-day and inter-day variation at 25 ng ml⁻¹ were ≤2.7% and ≤5.80% respectively for all stereoisomers. The assay was applied to an examination of the disposition of labetalol stereoisomers after a single oral dose of racemate to a human volunteer. Labetalol appears to undergo enantioselective metabolism leading to relatively low plasma concentrations of the pharmacologically active enantiomers.     

Analysis of Streptococcus mutans Proteins Modulated by Culture under Acidic Conditions

Streptococcus mutans, a major etiological agent of dental caries, causes demineralization of the tooth tissue due to the formation of acids from dietary carbohydrates. Dominant among the virulence determinants of this organism are aciduricity and acidogenicity, the abilities to grow at low pH and to produce acid, respectively. The mechanisms underlying the ability of S. mutans to survive and proliferate at low pH are currently under investigation. In this study we cultured S. mutans at pH 5.2 or 7.0 and extracted soluble cellular proteins. These were analyzed using high-resolution two-dimensional gel electrophoresis, and replicate maps of proteins expressed under each of the two conditions were generated. Proteins with modulated expression at low pH, as judged by a change in the relative integrated optical density, were excised and digested with trypsin by using an in-gel protocol. Tryptic digests were analyzed using matrix-assisted laser desorption ionization mass spectrometry to generate peptide mass fingerprints, and these were used to assign putative functions according to their homology with the translated sequences in the S. mutans genomic database. Thirty individual proteins exhibited altered expression as a result of culture of S. mutans at low pH. Up-regulated proteins (n = 18) included neutral endopeptidase, phosphoglucomutase, 60-kDa chaperonin, cell division proteins, enolase, lactate dehydrogenase, fructose bisphosphate aldolase, acetoin reductase, superoxide dismutase, and lactoylglutathione lyase. Proteins down-regulated at pH 5.2 (n = 12) included protein translation elongation factors G, Tu, and Ts, DnaK, small-subunit ribosomal protein S1P, large-subunit ribosomal protein L12P, and components of both phosphoenolpyruvate:protein phosphotransferase and multiple sugar binding transport systems. The identification of proteins differentially expressed following growth at low pH provides new information regarding the mechanisms of survival and has identified new target genes for mutagenesis studies to further assess their physiological significance.     

Maximum body size among insular Komodo dragon populations covaries with large prey density

This study documents variation in maximum body size of Komodo dragons ( Varanus komodoensis ) among the four extant island populations in Komodo National Park and compares an indirect measure of deer density, the major prey item for large dragons, to differences in maximum body size among islands. The largest 15% of dragons from the large islands of Komodo and Rinca were significantly longer and heavier than the largest 15% of dragons on the small islands of Gili Motang and Nusa Kode. There was a 33% difference in snout vent length (SVL) between dragons found on Komodo and those found on Gili Motang, with mass varying by more than four-fold. Density of deer pellet groups between islands ranged from 5.86±0.75 groups per transect on Gili Motang to 20.73±1.02 groups per transect on Komodo Island. Maximal dragon SVL and mass was highly positively correlated with this index of deer density. Low prey density on the two small islands could constrain body size via energetic constraints. At present we can not deduce if insular body size variation has arisen through genotypic or phenotypic mechanisms.     

Development and application of a positive–negative selectable marker system for use in reverse genetics in Plasmodium

A limitation of transfection of malaria parasites is the availability of only a low number of positive selectable markers for selection of transformed mutants. This is exacerbated for the rodent parasite Plasmodium berghei as selection of mutants is performed in vivo in laboratory rodents. We here report the development and application of a negative selection system based upon transgenic expression of a bifunctional protein (yFCU) combining yeast cytosine deaminase and uridyl phosphoribosyl transferase (UPRT) activity in P.berghei followed by in vivo selection with the prodrug 5-fluorocytosine (5-FC). The combination of yfcu and a positive selectable marker was used to first achieve positive selection of mutant parasites with a disrupted gene in a conventional manner. Thereafter through negative selection using 5-FC, mutants were selected where the disrupted gene had been restored to its original configuration as a result of the excision of the selectable markers from the genome through homologous recombination. This procedure was carried out for a Plasmodium gene (p48/45) encoding a protein involved in fertilization, the function of which had been previously implied through gene disruption alone. Such reversible recombination can therefore be employed for both the rapid analysis of the phenotype by targeted disruption of a gene and further associate phenotype and function by genotype restoration through the use of a single plasmid and a single positive selectable marker. Furthermore the negative selection system may also be adapted to facilitate other procedures such as ‘Hit and Run’ and ‘vector recycling’ which in principle will allow unlimited manipulation of a single parasite clone. This is the first demonstration of the general use of yFCU in combination with a positive selectable marker in reverse genetics approaches and it should be possible to adapt its use to many other biological systems.     

Detection of the change point in oxygen uptake during an incremental exercise test using recursive residuals: relationship to the plasma lactate accumulation and blood acid base balance

The purpose of this study was to develop a method to determine the power output at which oxygen uptake (V˙O₂) during an incremental exercise test begins to rise non-linearly. A group of 26 healthy non-smoking men [mean age 22.1 (SD 1.4) years, body mass 73.6 (SD 7.4) kg, height 179.4 (SD 7.5) cm, maximal oxygen uptake (V˙O_2max) 3.726 (SD 0.363) l · min⁻¹], experienced in laboratory tests, were the subjects in this study. They performed an incremental exercise test on a cycle ergometer at a pedalling rate of 70 rev · min⁻¹. The test started at a power output of 30 W, followed by increases amounting to 30 W every 3 min. At 5 min prior to the first exercise intensity, at the end of each stage of exercise protocol, blood samples (1 ml each) were taken from an antecubital vein. The samples were analysed for plasma lactate concentration [La]_pl, partial pressure of O₂ and CO₂ and hydrogen ion concentration [H⁺]_b. The lactate threshold (LT) in this study was defined as the highest power output above which [La⁻]_pl showed a sustained increase of more than 0.5 mmol · l⁻¹ · step⁻¹. The V˙O₂ was measured breath-by-breath. In the analysis of the change point (CP) of V˙O₂ during the incremental exercise test, a two-phase model was assumed for the 3rd-min-data of each step of the test: X _i =at _i +b+ɛ _i for i=1,2,…,T, and E(X _i )>at _i +b for i =T+1,…,n, where X ₁, … , X _n are independent and ɛ _i ∼N(0,σ²). In the first phase, a linear relationship between V˙O₂ and power output was assumed, whereas in the second phase an additional increase in V˙O₂ above the values expected from the linear model was allowed. The power output at which the first phase ended was called the change point in oxygen uptake (CP-V˙O₂). The identification of the model consisted of two steps: testing for the existence of CP and estimating its location. Both procedures were based on suitably normalised recursive residuals. We showed that in 25 out of 26 subjects it was possible to determine the CP-O₂ as described in our model. The power output at CP-V˙O₂ amounted to 136.8 (SD 31.3) W. It was only 11 W – non significantly – higher than the power output corresponding to LT. The V˙O₂ at CP-V˙O₂ amounted to 1.828 (SD 0.356) l · min⁻¹ was [48.9 (SD 7.9)% V˙O_{2 max} ]. The [La⁻]_pl at CP-V˙O₂, amounting to 2.57 (SD 0.69) mmol · l⁻¹ was significantly elevated (P<0.01) above the resting level [1.85 (SD 0.46) mmol · l⁻¹], however the [H⁺]_b at CP-V˙O₂ amounting to 45.1 (SD 3.0) nmol · l⁻¹, was not significantly different from the values at rest which amounted to 44.14 (SD 2.79) nmol · l⁻¹. An increase of power output of 30 W above CP-V˙O₂ was accompanied by a significant increase in [H⁺]_b above the resting level (P=0.03). Accepted: 25 March 1998