The role of temperature in the regulation of the circadian rhythm of CO2 fixation in Bryophyllum fedtschenkoi

Detached leaves of Bryophyllum fedtschenkoi Hamet et Perrier kept in normal air show a single period of net CO₂ fixation on transfer to constant darkness at temperatures in the range 0–25 °C. The duration of this initial fixation period is largely independent of temperature in the range 5–20 °C, but lengthens very markedly at temperatures below 4 °C, and is reduced at temperatures above 25 °C. The onset of net fixation of CO₂ on transfer of leaves to constant darkness is immediate at low temperatures, but is delayed as the temperature is increased. The ambient temperature also determines whether or not a circadian rhythm of CO₂ exchange occurs. The rhythm begins to appear at about 20 °C, is most evident at 30 °C and becomes less distinct at 35 °C. The occurrence of a distinct circadian rhythm in CO₂ output at 30° C in the absence of a detectable rhythm in PEPCase kinase activity shows that the kinase rhythm is not a mandatory requirement for the rhythm of PEPCase activity. However, when it occurs, the kinase rhythm undoubtedly amplifies the PEPCase rhythm.Abbreviation PEPCase phosphoenolpyruvate carboxylase We thank the Agricultural and Food Research Council for financial support for this work.     

Lipid structure and Ca2+-ATPase function

Effects of lipid structure on the function of the Ca²⁺-ATPase of skeletal muscle of sarcoplasmic reticulum are reviewed. Binding of phospholipids to the ATPase shows little specificity. Phosphatidylcholines with short (C14) or long (C24) fatty acyl chains have marked effects on the activity of the ATPase, including a change in the stoichiometry of Ca binding. Low ATPase activity in gel phase lipid follows from low rate of phosphorylation. Phosphatidylinositol 4-phosphate increases ATPase activity by increasing the rate of dephosphorylation of the phosphorylated ATPase. Stimulation is not seen with other anionic phospholipids; phosphatidic acid decreases ATPase activity in a Mg²⁺-dependent manner.Abbreviations di(C141)PC dimyristoleoylphosphatidycholine - di(C160)PC dipalmitoylphosphatidylcholine - di(C181)PC dioleoylphosphatidylcholine - di(Br₂C180)PC dibromostearoylphosphatidylcholine - di(C241)PC dinervonylphosphatidylcholine - di(C181)PA dioleoylphosphatidic acid - di(C181)PE dioleoylphosphatidylethanolamine - Ptdlns phosphatidylinositol - PtdIns-4P phos-phatidylinositol 4-phosphate     

Extracellular polysaccharide of Nostoc commune (Cyanobacteria) inhibits fusion of membrane vesicles during desiccation

Cells of the cyanobacterium Nostoc commune secrete a complex, high molecular weight, extracellular polysaccharide (EPS) which accumulates to more than 60% of the dry weight of colonies. The EPS was purified from the clonal isolate N. commune DRH1. The midpoint of the membrane phase transition (T_m) of desiccated cells of N. commune CHEN was low (T_{m dry} = 8 °C) and was comparable to the T_m of rehydrated cells((T_m)_H20 = 6 °C). The EPS was not responsible for the depression of T_m. However, the EPS, at low concentrations, inhibited specifically the fusion of phosphatidylcholine membrane vesicles when they were dried in vitro at0% relative humidity (−400 MPa). Low concentrations of a trehalose:sucrose mixture, in a molar ratio which corresponded with that present in cells in vivo, together with small amounts of the EPS, were efficient in preventing leakage of carboxyfloroscein (CF) from membrane vesicles. Freeze-fracture electron microscopy resolved complex changes in the structure of the EPS and the outer membrane in response to rehydration of desiccated cells. The capacity of the EPS to prevent membrane fusion, the maintenance of a low T_{m dry} in desiccated cells, and the changes in rheological properties of the EPS in response to water availability, constitute what are likely important mechanisms for desiccation tolerance in this cyanobacterium. This revised version was published online in August 2006 with corrections to the Cover Date.     

Spatial expression dynamics of Men-9 delineate the third floral whorl in male and female flowers of dioecious Silene latifolia

Sex determination in Silene latifolia is controlled by heteromorphic sex chromosomes. Female flowers have five fused carpels and ten arrested stamen primordia. The male-determining Y chromosome overrides female development to suppress carpel formation and promote stamen development. The isolation and characterization of two S. latifoliaM ale en hanced cDNAs, Men-9a and Men-9b, which probably represent different alleles of a novel gene are reported here. Men-9a and Men-9b share 91.8% coding sequence nucleotide identity, yet only 85.4% amino acid identity. The Men-9 cDNAs are related to the previously reported MROS3 cDNA from S. latifolia. However, MROS3 is not present in the S. latifolia population used in these studies and the expression dynamics of Men-9a and Men-9b contrast dramatically with those reported for MROS3. Men-9 cDNAs are expressed primarily in anthers of young male flowers, with highest expression in 1–2 mm buds. Men-9 expression is also observed at a low level in female flowers. In situ hybridization analysis reveals two phases of Men-9 expression. The first phase is during a common stage of early stamen development in male and female flowers prior to stamen arrest in female flowers. The second phase of Men-9 expression is maximal in the epidermis and endothecium of Y chromosome- and Ustilago violacea-induced stamens; expression in male and female flowers extends to the epidermis of the staminal nectaries with strict boundaries at the second and fourth whorls. Men-9 gene expression therefore delineates the boundaries of the third floral whorl in S. latifolia flowers.     

Regulation of expression of a cDNA from barley roots encoding a high affinity sulphate transporter

A cDNA encoding a high-affinity sulphate transporter has been isolated from barley by complementation of a yeast mutant. The cDNA, designated HVST1, encodes a polypeptide of 660 amino acids (M_r = 72 550), which is predicted to have 12 membrane-spanning domains and has extensive sequence homology with other identified eukaryotic sulphate transporters. The K_m for sulphate was 6.9 µM when the HVST1 cDNA was expressed in a yeast mutant deficient in the gene encoding for the yeast SUL1 sulphate transporter. The strong pH-dependency of sulphate uptake when HVST1 was expressed heterologously in yeast suggests that the HVST1 polypeptide is a proton/sulphate co-transporter. The gene encoding HVST1 is expressed specifically in root tissues and the abundance of the mRNA is strongly influenced by sulphur nutrition. During sulphur-starvation of barley, the abundance of mRNA corresponding to HVST1, and the capacity of the roots to take up sulphate, both increase. Upon re-supply of sulphate, the abundance of the mRNA corresponding to HVST1, and the capacity of the roots to take up sulphate, decrease rapidly, concomitant with rises in tissue sulphate, cysteine and glutathione contents. Addition of the cysteine precursor, O-acetylserine, to plants grown with adequate sulphur supply, leads to increases in sulphate transporter mRNA, sulphate uptake rates and tissue contents of glutathione and cysteine. It is suggested, that whilst sulphate, cysteine and glutathione may be candidates for negative metabolic regulators of sulphate transporter gene expression, this regulation may be overridden by O-acetylserine acting as a positive regulator.     

Nonsyndromic cleft lip with or without cleft palate: evidence of linkage to BCL3 in 17 multigenerational families.

Nonsyndromic cleft lip with or without cleft palate (CL/P) is a common craniofacial developmental defect. Recent segregation analyses have suggested that major genes play a role in the etiology of CL/P. Linkage to 22 candidate genes was tested in 11 multigenerational families with CL/P, and 21 of these candidates were excluded. APOC2, 19q13.1, which is linked to the proto-oncogene BCL3, gave suggestive evidence for linkage to CL/P. The study was expanded to include a total of 39 multigenerational CL/P families. Linkage was tested in all families, using an anonymous marker, D19S178, and intragenic markers in BCL3 and APOC2. Linkage was tested under two models, autosomal dominant with reduced penetrance and affecteds only. Homogeneity testing on the two-point data gave evidence of heterogeneity at APOC2 under the affecteds-only model. Both models showed evidence of heterogeneity, with 43% of families linked at zero recombination to BCL3 when marker data from BCL3 and APOC2 were included. A maximum multipoint LOD score of 7.00 at BCL3 was found among the 17 families that had posterior probabilities > = 50% in favor of linkage. The transmission disequilibrium test provided additional evidence for linkage with the 3 allele of BCL3 more often transmitted to affected children. These results suggest that BCL3, or a nearby gene, plays a role in the etiology of CL/P in some families.     

Exercise,agonistic behaviour and food acquisition in Arctic charr,Salvelinus alpinus

Synopsis Although swimming is energetically costly, a number of studies on salmonid species have demonstrated increased growth rates in fishes forced to swim for prolonged periods at moderate speeds (typically 1–2 body lengths per sec). This suggests that additional energetic costs of swimming are more than met by alternative compensatory gains. The mechanisms underlying such effects are not fully understood. In this paper, we describe an experiment designed to examine one possible mechanism, namely a swimming-induced inhibition of aggression, with consequent beneficial effects on growth. The study used Arctic charr,Salvelinus alpinus, a species for which a positive relationship between exercise and growth has been clearly established. Using direct behavioural observations on small groups, we demonstrate that individuals displaying high levels of aggressive behaviour are able to monopolise access to food and that enforced swimming at a moderate speed (1 body length per sec) reduces the incidence of aggression although not the degree of monopolisation of food shown by aggressive individuals. These results suggest that the enhanced growth rates accompanying enforced swimming may reflect lower energetic costs of reduced aggressive activity rather than improved access to food by subordinates.     

Methylation and expression of a metallothionein promoter ovine growth hormone fusion gene (MToGH1) in transgenic mice

We have examined transgene methylation in the DNA from the livers of a pedigree of mice carrying three copies of an integrated MToGH1 transgene. Utilizing the methylation-sensitive isoschizomersMsp I andHpa II, Southern blot analysis revealed that all second generation animals derived from a transgenic female had hypermethylated DNA, whereas first generation animals sired by a transgenic male displayed a range of methylation phenotypes ranging from no methylation to hypermethylation of the transgene sequences. Of the mice that exhibited hypermethylation of the transgene in CpG dinucleotides (CmCGG), a minority of these animals also exhibited apparent CpC methylation (i.e. inhibition ofMsp I cutting, presumably blocked by methylation of the outer C of CCGG). Methylation was also examined in the inner C of CC(A/T)GG sequences in the MToGH1 transgene using the isoschizomer pairBstN I andEcoR II. A minority of MToGH1 animals in the F₁ generation showed clear evidence of methylation in these sites as well as in the inner and outer Cs of CCGG sites. An examination of MToGH1 expression in terms of oGH levels in serum revealed that there was a high degree of variation in the levels of circulating oGH between animals of this pedigree. There was a weak inverse relationship between the serum level of oGH and the extent of methylation of the transgene. In particular, mice exhibiting CpC together with CpG methylation were found to have very low levels of circulating oGH. Our results highlight the nature and complexity of epigenetic factors associated with transgene sequences which may ultimately influence expression of introduced genes in the mammalian genome.     

Cooperativity and effects of ionic strength on the binding of sodium n-dodecyl sulphate to lysozyme

The binding of sodium n-dodecyl sulphate to lysozyme has been measured by equilibrium dialysis at 25°C and pH 3.2 over a range of ionic strengts from 0.0119 to 0.2119. Binding isotherms in the region corresponding to ionic binding between the surfactant anions and cationic amino acid residues on the protein have been interpreted in terms of the Hill equation and exhibit positive cooperativity with Hill coefficients in the region of 7–11. The Gibbs energies of binding have been calculated from the Hill binding constants and from the Wyman binding potentials. The stability of the surfactant-protein complexes is discussed in relation to the stability of surfactant micelles. Ionic binding of the surfactant is weakened and hydrophobic binding strengthened by increasing ionic strength.     

Germacranolides from Melampodium americanum and Melampodium pilosum

Chemical analysis of Melampodium americanum yielded, besides the known melampodinin A, four new melampolides, melampodinin B, melampodinin C, 9-des