Genome ploidy in different stages of the Giardia lamblia life cycle

The early diverging eukaryotic parasite Giardia lamblia is unusual in that it contains two apparently identical nuclei in the vegetative trophozoite stage. We have determined the nuclear and cellular genome ploidy of G. lamblia cells during all stages of the life cycle. During vegetative growth, the nuclei cycle between a diploid (2N) and tetraploid (4N) genome content and the cell, consequently, cycles between 4N and 8N. Stationary phase trophozoites arrest in the G₂ phase with a ploidy of 8N (two nuclei, each with a 4N ploidy). On its way to cyst formation, a G₁ trophozoite goes through two successive rounds of chromosome replication without an intervening cell division event. Fully differentiated cysts contain four nuclei, each with a ploidy of 4N, resulting in a cyst ploidy of 16N. The newly excysted cell, for which we suggest the term 'excyzoite', contains four nuclei (cellular ploidy 16N). In a reversal of the events occurring during encystation, the excyzoite divides twice to form four trophozoites containing two diploid nuclei each. The formation of multiple cells from a single cyst is likely to be one of the main reasons for the low infectious doses of G. lamblia .     

Differences in osmoacclimation between sporophytes and gametophytes of the brown alga Ectocarpus siliculosus

The osmoacclimation of Ectocarpus siliculosus isolates known to have different salt tolerances was investigated. Included were isolates originating from 5 different locations in the northern hemisphere, and sporophyte and gametophyte phases of different ploidy from two of the locations were compared. The effect of salinity treatment (8–64%₀) on inorganic ions (K⁺, Na⁺, Mg²⁺, Cl⁻, SO²⁻₄, phosphate) and the low molecular weight carbohydrate mannitol was measured, together with complimentary measurements of cell viability. Very different responses between isolates were obtained, both between isolates of different geographic origin and between sporophytes and gametophytes from the same parent material. A similarity in response between haploid and diploid gametophytes, and diploid and triploid sporophytes indicates that physiological differences between gametophyte and sporophyte generations are not necessarily based on ploidy changes alone. There were no identifiable differences in the responses of male and female gametophytes. K⁺ is the major osmolyte within the species, and differences in the regulation of K⁺ largely account for the observed variation in osmoacclimation, both between life history phases and between isolates from different localities. Isolates with broader salt tolerances had the higher concentrations of mannitol. There were differences between isolates in the amounts and regulation of Cl⁻ and phosphate, the latter being present in unusually high concentrations. There were also isolate differences in the concentrations of Mg²⁺ and SO²⁻₄, although these divalent ions were present only in low concentrations.     

Quantum yields for CO2 uptake in some diploid and tetraploid plant species

Abstract. Quantum (photon) yields for CO₂ uptake on an absorbed photon fluence rate (PFR) basis (φ_a) were determined for a number of diploid and tetraploid species under a range of experimental conditions. Little variation in φ_a was found either between species or across ploidy levels, which suggests that there are no intrinsic differences in the kinetics of CO₂ fixation. Variations in photosynthetic rate or quantum yield expressed on an incident PFR basis (φ_j) were found to be largely due to differences in leaf absorptance. On this basis these results do not support the biochemical evidence showing ploidy-related differences in the K_m (CO₂) of ribulose -1,5-bisphosphate carboxylase/oxygenase. However, there are obvious difficulties in relating in vitro enzyme kinetic data to in vivo studies and these problems are discussed.     

Inhibitory effect of sodium bicarbonate on the motility of sperm of Japanese eel

Sperm motility of Japanese eel Anguilla japonica was depressed from 59.1 to 1.1% when NaHCO₃ concentration increased from 0 to 150 mM. In 25 mM NaHCO₃, when pH of the medium was 6.2, 7.2, 8.2, 9.2 and 10.2, relative sperm motility was 0, 0, 55.8, 93.7 and 136.6%, respectively to that of the control (0 mM NaHCO₃). The remarkable effect in acid or neutral condition indicates that free-CO₂ (liberated CO₂ and H₂CO₃) is a key factor for the motility inhibiting effect of NaHCO₃.     

Genetically stable cell lines of cucumber for the large-scale production of diploid somatic embryos

For the initiation of embryogenic cucumber ( Cucumis sativus L.) cell lines, from excised radicles, directly in liquid medium, the culture regime, explant density and type and concentration of hormones were adjusted so that pro-embryogenic masses (PEMs) were formed within about 8 weeks. The established cucumber cell lines were maintained tor several years without loss of embryogenic and genetic stability. The ploidy level of somatic embryos from different cucumber eell lines was either diploid or tetraploid and depended on the ploidy level of Ihe cell line. Cucumber cell lines that produced only diploid embryos were obtained by selecting completely diploid explant material and growing it in the dark during the initiation phase. Mixoploid explains could lead to tetraploid or mixoploid ceil lines. Isolation and additional selection and subculturing of single PEMs resulted in either completely diploid or tetraploid cell lines, indicating that all cells of individual PEMs are either diploid or tetraploid. The ernbryogenic cucumber cell Imes, differing only in ploidy level, were indistinguishable in growth rate and embryogetiic potential and were genetically stable over several years.     

Use of impedance measurements to estimate numbers of antibiotic resistant Salmonella strains

Impedance detection times were compared with traditional plating methods for enumerating antibiotic-resistant strains of Salmonella stanley, Salm. thompson and Salm. infantis grown in laboratory medium and pork slurry. The correleation of log₁₀ counts of salmonellas with detection times was highly significant ( r = -0·96 for broth and r = -0·94 for slurries. The confidence limits (± og₁₀ 1·0 for broth and ± log₁₀ 1·65 for slurry) indicated that detection times could reliably be used as a rapid means of enumerating salmonellas when large numbers of counts of known strains are required for growth studies. Use of antibiotic-resistant strains also permitted their selective detection by impedance from the natural spoilage flora of pork slurry when the same antibiotics were incorporated in the detection medium.     

Mitigation of adverse effects of rising CO2 on a planktonic herbivore by mixed algal diets

Putative future increase in atmospheric CO₂ is expected to adversely affect herbivore growth due to decrease in contents of key nutrients such as nitrogen and phosphorus (P) relative to carbon in primary producers including plant and algal species. However, as many herbivores are polyphagous and as the response of primary producers to elevated CO₂ is highly species-specific, effects of elevated CO₂ on herbivore growth may differ between feeding conditions with monospecific and multiproducer diets. To examine this possibility, we performed CO₂ manipulation experiments under a P-limited condition with a planktonic herbivore, Daphnia , and three algal species, Scenedesmus obliquus (green algae), Cyclotella sp. (diatoms) and Synechococcus sp. (cyanobacteria). Semibatch cultures with single algal species (monocultures) and multiple algal species (mixed cultures) were grown at ambient (360 ppm) and high CO₂ levels (2000 ppm) that were within the natural range in lakes. Both in the mono- and mixed cultures, algal steady state abundance increased but algal P : C and N : C ratios decreased when they were grown at high CO₂. As expected, Daphnia fed monospecific algae cultured at high CO₂ had decreased growth rates despite increased algal abundance. However, when fed mixed algae cultured at high CO₂, especially consisting of diatoms and cyanobacteria or the three algal species, Daphnia maintained high growth rates despite lowered P and N contents relative to C in the algal diets. These results imply that algal diets composed of multiple species can mitigate the adverse effects of elevated CO₂ on herbivore performance, although the magnitude of this mitigation depends on the composition of algal species involved in the diets.     

Response of Campylobacter jejuni to combinations of ferrous sulphate and cadmium chloride

On Mueller Hinton (MH) agar, Campylobacter jejuni showed 20.0 and 30.9mm zones of inhibition surrounding discs impregnated with 2.5 and 20 μg CdCl₂ respectively. The minimal inhibitory concentration (MIC) ranged from 0.64 to 3.2 μg CdCl₂/ml of MH agar for four C. jejuni strains. In the presence of 23 μg FeSO₄/ml of MH the MIC increased to a range of 1.5–5.4 μg CdCl₂/ml of MH. Moreover, the numbers of colonies present on MH supplemented with FeSO₄ were greater than on MH without iron. The growth response of C. jejuni in the presence of 0.025% (w/v) FeSO₄ in MH broth was increased about 10000 fold in three of four strains when compared with the growth in unsupplemented MH broth. Zones of inhibition surrounding 20 μg discs of CdCl₂ were 50.6 and 24.4 mm on MH and Campy-BAP media respectively, with cells grown on MH. These results suggest that the blood-containing medium 'neutralized' the biocidal influence of the CdCl₂. In comparison, C. jejuni inoculum from fluid thioglycollate (FT) medium showed smaller zones of inhibition. These decreased from 34.9 mm on MH agar to 19.6 mm on Campy-BAP agar, suggesting that components in the FT growth medium ameliorated the toxic influence of CdCl₂. Atomic absorption spectroscopy analysis indicated mean values (mg/100 g dry weight) of selected metals bound by C. jejuni as: Cu, 10.4; Mg, 146; Na, 2385; Fe, 45.1; Zn, 13.0; and K, 172.     

The content of prostaglandins and their precursors in Mortierella and Cunninghamella species

Five strains of filamentous fungi belonging to the genera Mortierella and Cunninghamella were examined for the content of dihomo-γ-linolenic, arachidonic, eicosapentaenoic acids and prostaglandins (type E₂ and F _2α). Prostaglandins were detected using an ELISA method in mycelia of all tested strains (range 50–4800 ng g⁻¹ of PGE₂ and 6–30 ng g⁻¹ of PG F _2α). Several micro-organisms also produced prostaglandins in the culture medium (2·2–137·6 μg l⁻¹ for PGE₂ and 0·4–7·8 μg l⁻¹ for PG F _2α).     

Maximal biomass production can occur in corn (Zea mays) in the absence of NO3 accumulation in either leaves or roots

In order to investigate effects of limited NO₃ availability in corn ( Zea mays L. cv. Brulouis) 17-day-old plants were grown for a further 25 days on sand in a growth chamber. The plants received frequent irrigation with a complete nutrient solution containing 0.2, 0.6, 1.5 or 3.0 mM NO₃. With 0.2 mM NO; nitrate levels in both roots and leaves diminished rapidly and were almost zero after 10 days treatment. Concurrently, as signs of nitrogen deficiency appeared, shoot growth was restricted, whereas root growth was enhanced. In addition, the concentration of reduced nitrogen and malate in the leaves declined, and in vitro nitrate reductase activity (NRA. EC 1.6.6.1), soluble protein and chlorophyll levels of leaf tissue were depressed and starch concentration was enhanced. With 0.6 mM NO₃ in the nutrient solution, the decrease in NO₃ levels in the tissues and the increase in root development were similar to those observed with 0.2 mM NO₃. However, shoot growth, reduced nitrogen concentration in leaves, and the above-mentioned biochemical characteristics were almost identical to those obtained at 1.5 and 3.0 mM NO₃. This indicates that when supplied with 0.6 mM NO₃, corn plants were able to absorb sufficient NO3 to support maximal biomass production without appreciable NO₃ accumulation in roots or shoot. It is, thus, suggested that the plants responded to low NO₃, availability in medium by enhancing root growth and by maximizing NO₃ reduction relative to NO₃ accumulation.     

Accumulation of a 31-kDa glycoprotein in association with the expression of embryogenic potential by spinach callus in culture

Calli grown from segments of spinach ( Spinacia oleracea L.) root in the presence of gibberellic acid (GA₃) plus auxin, differentiated to yield somatic embryos after transfer to a medium without growth regulators, while calli formed in the absence of GA₃ failed to generate any embryos. We extracted proteins from the two types of callus and analysed them by polyacrylamide gel electrophoresis. Compared with the proteins from calli formed on medium that contained only naphthaleneacetic acid (NAA) as a growth regulator, the proteins from calli grown in the presence of GA₃ included appreciably higher levels of a 31-kDa basic protein (pI = 8.8). The protein resembled type I ribosome-inactivating proteins (EC 3.2.2.22) in terms of molecular mass, isoelectric point, sequence of amino-terminal amino acids and extent of glycosylation. The 31-kDa protein was barely detectable in extracts of various tissues from seedlings. Thus, it is possible that an increase in the relative level of this protein might be associated with the expression of embryogenic potential expressed by spinach callus.     

Effect of nitrate on nitrogen fixation and nodule carbohydrate and organic acid concentrations in pea mutants deficient in nitrate reductase

Nitrate inhibits symbiotic N₂ fixation and a number of hypotheses concerned with NO₃⁻ assimilation have been suggested to explain this inhibition. These hypotheses were tested using a pea ( Pisum sativum L. cv. Juneau) with normal nitrate reductase NR; (EC 1,6,6,4) activity and two mutants of cv. Juneau, A317 and A334, with impaired NR activity. The plants were inoculated with three strains of Rhizobium leguminosarum and grown for 3 weeks in N-free medium, followed by 1 week in medium supplemented with 0, 5 or 10 m M KNO₃ before harvesting. NO₃ was taken up at comparable rates by the parent and the mutants and accumulated in leaf and stem tissue of the latter. Acetylene reduction rates were inhibited similarly in both the parent and mutants in the presence of KNO₃ but there were differences among rhizobial strains. Starch concentration of the nodules decreased by 46% in the presence of KNO₃ and there were differences among rhizobial strains but not among pea genotypes. Malate and succinate accumulated in nodules in the presence of KNO₃. These data are not consistent with the photosynthate deprivation hypothesis as a primary mechanism for NO₃ inhibition of N₂ fixation since NO₃ affected the nodule carbohydrate composition of all three pea genotypes in a similar manner. The lack of correlation between NR activity and NO₃ inhibition of N₂ fixation suggests that NO₃ assimilation may be only indirectly involved in the inhibition phenomenon.     

Effects of 5-azacytidine on the development of parthenogenetic mouse embryos

This study describes the effects of 5-azacytidine (5-azaC) on the development of diploid parthenogenetic embryos (PE) of CBA, C57BL/6 and (CBA × C57BL/6)F₁ mice in vitro at the 1-cell or the blastocyst stage or in vivo after implantation. Our findings indicate that genomic imprinting is modulated by genetic background. Non-fertilized C57BL/6 eggs form diploid parthenogenetic blastocysts at a much higher frequency than CBA eggs. Eggs from F₁ hybrid females form parthenogenetic blastocysts at an approximately intermediate level between these inbred strains of mice. C57BL/6 PE do not develop to the somite stages. In contrast, CBA PE and F₁ PE develop to various somite stages. Following administration of 5–azaC at 1.0 μmol/L in vitro at the 1- -cell stage, the number of implantations of C57BL/6 PE transferred to pseudopregnant females increased. In contrast, the number of implantations and somite F₁ PE did not significantly change following exposure to 5–azaC. However, administration of 5-azaC at the 1-cell stage stimulates development of somite F₁ PE. Administration of 5-azaC at 0.2 and 1.0 μmol/L in vitro at the blastocyst stage did not change the number of implantations of C57BL/6 PE. However, the number of implantations and somite CBA PE decreased. After injection of 5azaC at 0.24mg/kg in vivo at day 8 of gestation, some F₁ PE developed to 26–35 somites compared with a maximum of 25 somites in controls. The different effects of 5-azaC on the development of PE depend upon the mouse strain used and the stage of development.     

INHIBITION OF THYROXINE-INDUCED RESORPTION OF TADPOLE TAIL BY ADENOSINE 3', 5'-CYCLIC MONOPHOSPHATE

Small segments of tail of Bufo bufo japonicus tadpoles were cultured in medium containing thyroxine (T₄) and dibutyryl cyclic AMP (dbcAMP). Like prolactin, the cyclic nucleotide blocked T₄-induced shrinkage or tail pieces. Histological study of the segments after 4-days culture revealed that dbcAMP suppressed degenerative changes induced by T₄. The inhibitory effect of prolactin on T₄-induced tail regression was promoted by caffeine, an inhibitor of adenosine 3', 5'-cyclic monophosphate (cyclic AMP)-phosphodiesterase.
The effect of prolactin on the level of cyclic AMP in the tail was also studied in vivo . Sixty min after prolactin injection, the cyclic AMP level was 2–3 times the control value. Possible involvement of cyclic AMP in the action of prolactin, which blocks tail resorption induced by T₄, was discussed.     

Development of a method based on alkaline gel electrophoresis for estimation of oxidative damage to DNA in Escherichia coli

A method for estimating DNA strand breakage and subsequent repair based on alkaline gel electrophoresis was developed and tested with isogenic strains of Escherichia coli deficient in DNA repair enzymes. Samples from a cell suspension were removed at 2 min intervals following a 15 min exposure to 20 mmol l^-1 H₂O₂. Catalase was added and the cells were embedded in blocks of low-melting point agarose and lysed. After alkaline gel electrophoresis, photographs of the gels were taken and the relative lengths of the distributions of DNA fragments were measured with a scanner and computer. The lengths were correlated with survival of the cells exposed to H₂O₂ and with the importance of particular DNA repair enzymes. Alkaline gel electrophoresis appears to be a relatively simple method for analysing the level of H₂O₂-caused DNA damage and repair in E. coli.     

Inactivation of Strains of Salmonella senftenberg by Gamma Irradiation

S ummary : Strains of Salmonella senftenberg isolated from Norwegian herring meal and strain 775W were exposed to gamma radiation from a ⁶⁰Co source. When they were irradiated in phosphate buffered saline solution, the average D₁₀ values for all the strains was 19·3 krad. On irradiating strains 56 and 775W in herring meal the D₁₀ values were 192·1 and 188·5 krad, respectively, thus indicating that the suspending medium had a great effect on the radiation resistance of the organisms. Radiation doses of the order of 0·8–1·3 Mrad are recommended for the decontamination of herring meal.     

Effects of hypochlorite disinfection on the response of barley aleurone layers to gibberellic acid

Although the use of hypochlorite to disinfect seeds is widespread, the effects on tissues isolated from them have been largely ignored. Disinfection of barley ( Hordeum vulgare L. cv. Himalaya) half-seeds with hypochlorite solutions of ≥1.0% (w/v) available chlorine caused the pericarp to separate from the underlying tissues. Aleurone layers isolated from these grains had lower rates of oxygen consumption and released significantly less protein, PO₄³⁻ Mg²⁺, K⁺ and amylase (EC 3.2.1.1) into the medium in response to gibberellic acid (GA₃) than layers isolated from grains disinfected with a 0.1% hypochlorite solution. Disinfection with 1.0% hypochlorite also quantitatively altered the spectrum of proteins released into the incubation medium by the layers in response to GA₃.     

Effect of different nitrogen nutrients on the viability, protein synthesis and tannin production of Scots pine callus

The effect of two different media on the growth, metabolism and viability of Scots pine ( Pinus sylvestris ) callus cultures was studied. Inorganic nitrogen in the culture media (modified MS) was in the form of either KNO₃ or NH₄NO₃. The cultures were started from buds of mature Scots pine. Growth was poor on the medium with KNO₃, but this compound had a noticeable effect on the metabolism of the callus, which was reflected in alterations in protein and polyphenol synthesis and the pH of the culture medium. Although the fresh mass, water content and viability of the callus decreased when KNO₃ was the exclusive inorganic nitrogen nutrient, protein synthesis was more abundant. Electrophoretic analyses indicated alterations in the patterns of soluble proteins and purified glycoproteins. Phenylalanine ammonia-lyase (EC 4.3.1.5) activities were high in all the calluses, and concentrations of condensed tannins and their precursors, catechins, were higher than in intact buds. The role of inorganic nitrogen nutrition in the deterioration of tissues is discussed on the basis of the effect of ammonium on the metabolism of pine callus.     

The possible role of fructose 2,6-bisphosphate in the cyanide-mediated removal of embryonic dormancy in apple

The effects of pretreatment with HCN on the level of fructose 2.6-bisphosphate (F-2,6-P₂). on activity of fructose 6-phosphate 2-kinase (EC 2.7.1.150; F-6-P. 2K. enzyme synthesizing F-2,6-P₂), as well as on activities of PP₁-dependent and ATP-dependent phosphofructokinases (EC 2.7.1.90. PP₁-PFK and EC 2.7.1.11, ATP-PFK) were studied in cultured, dormant embryos of apple ( Malus domestica Borb. cv. Antonówka). HCN increased the F-2.6-P₂ level and F-6-P, 2K activity in embryonal axes (3-fold), but had no effect in cotyledons. HCN pretreatment of embryos or the addition of F-2.6-P₂ to enzyme extract stimulated PP₁, -PFK activity, whereas the activity of ATP-PFK was slightly inhibited by HCN in axes and in cotyledons. Glycolysis is one of the first processes in the germination of apple embryos, and the stimulation of glycolysis by HCN may be the result of F-6-P, 2K activation in the axes. This will lead to accumulation of F-2,6-P₂, which, in turn, enhances glycolysis by activation of PP₁PKF.     

Influence of nitrate and ammonium on the growth and 2,4-diaminobutyric acid composition of flatpea (Lathyrus sylvestris L.)

Abstract. Presence of 2.4-diaminobutyric acid (A₂bu), a neurotoxin, in tissues of flatpea ( Lathyrus sylvestris L.) necessitates a thorough understanding of the regulation of this nonprotein amino acid before the species can be recommended to livestock producers for forage applications. To determine how different concentrations and ratios of NO₃ and NH⁺₄ in growth media influence the levels of A₂bu and other free amino acids in the 'Lathco'flatpea cultivar, plants were grown hydroponically in controlled environments. The concentration of A₂bu was highest in tissues when the NO₃ to NH⁺₄ ratio in the nutrient solution was low. Responses of amides and other nonprotein amino acids, especially in the roots, followed a similar trend. Free protein amino acids in leaves and stems were generally unaffected by changes in NO₃ to NH⁺₄ ratios. In roots, protein amino acids increased as the NO₃ to NH⁺₄ ratio in the growth medium increased. Ammonium inhibited shoot and root growth; NO₃ alleviated the toxic effects of NH⁺₄. Soluble protein concentrations were higher in the shoots of NO₃-fed plants and in the roots of plants supplied with NH⁺₄. These results suggest that accumulation of A₂bu and other nonprotein amino acids, as well as asparagine and glutamine, plays a role in detoxification of NH⁺₄ and storage of N.