The kinetic significance of cell size : I. Variation of cell cycle parameters with size measured at mitosis

Using a cell line of human lymphoid cells, the kinetic significance of cell size measured at mitosis has been explored using fraction of labelled mitoses data. It was found that smaller cells tend to have progressively longer generation times. The principal mechanism for this generation time dilation is a progressively protracted G 1 duration as cell size decreases. There is a concomitant, but much slighter increase in S phase duration. G 2 duration remains essentially constant irrespective of cell size.     

Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa

Addition of the divalent cation ionophore, A23187, to washed populations of human spermatozoa resulted in a sudden burst of production of reactive oxygen species which peaked within 3-5 min. This activity was dependent upon the presence of calcium in the external medium and was unaffected by the mitochondrial inhibitors, oligomycin, antimycin and rotenone. Studies with scavengers of reactive oxygen species revealed that, while reagents directed against singlet oxygen and the hydroxyl radical were without effect, cytochrome C reduced the response to A23187 by about 50%, suggesting that the superoxide anion radical is a major product of the activated human spermatozoon. The clinical implications of these studies stem from the considerable variation observed between individuals in the levels of reactive oxygen species produced by the spermatozoa. This variability was shown to be inversely related to the ability of the spermatozoa to exhibit sperm-oocyte fusion on exposure to A23187; defective samples exhibited a basal level of reactive oxygen species production which was 40 times that observed with normal functional cells.     

Mechanism-based fragmentation of coenzyme A transferase. Comparison of alpha 2-macroglobulin and coenzyme A transferase thiol ester reactions

The plasma proteins, alpha 2-macroglobulin and complement components 3 and 4, contain an internal thiol ester involving a glutamyl and cysteinyl residue. The thiol ester is susceptible to cyclization at greater than 37 degrees C and forms an unstable 5-oxyproline intermediate. The latter can be hydrolyzed to produce two peptide fragments. We propose that enzymes having activated glutamyl residues as part of their catalytic mechanisms may undergo an analogous cyclization and peptidyl cleavage. As a model, we have investigated pig heart succinyl-CoA:3-keto acid transferase. When the CoA-enzyme thiolester intermediate is heated at pH 7.4 and 70 degrees C for 1 h, approximately 60% of the Mr = 60,000 subunits are cleaved to give Mr = 40,000 and 20,000 fragments. We have shown that formation of the enzyme thiolester is an obligate precursor for the protein fragmentation. However, the reaction was incomplete with a maximum of approximately 65% cleavage at times greater than 60 min. These results suggest that there is a competing, deactivation reaction; namely, the thiol ester and oxyproline intermediates are hydrolyzed to regenerate the active site glutamic acid. Although the maximum rate of cleavage is at 70 degrees C, approximately 15% autolysis also occurs at 37 degrees C. The Mr = 40,000 fragment had the same amino terminal sequence as the Mr = 60,000 subunit, (Trp-Lys-Phe-Tyr-Thr-Asp-Ala-Val-Glu-Ala-). No amino terminal could be detected for the Mr = 20,000 fragment, even after digesting the fragment with pyroglutaminase. Peptide maps of the fragments and the uncleaved subunit indicate that the fragments are generated in parallel. The size of the fragments puts the active site about two-thirds of the way from the amino terminal of the protein.     

5''-flanking sequences that inhibit in vitro transcription of a xenopus laevis tRNA gene

We determined the nucleotide sequences of all coding regions and a significant part of the flanking regions of the chicken c-src gene, which is a cellular homolog of the v-src gene of Rous sarcoma virus. The c-src gene consists of 12 exons; the boundaries of the exons were determined by assuming that the amino acid sequence of its product, pp60c-src, is basically the same as that of pp60v-src. The deduced amino acid sequence of pp60c-src was very similar to that of pp60v-src, but the last 19 carboxy-terminal amino acids of pp60c-src were replaced by a new set of 12 amino acids of pp60v-src. The sequence encoding the carboxy-terminal sequence of pp60v-src was found 900 bp downstream from the termination codon of the c-src gene. We suggest that the c-src sequence was captured by a virus through recombination at both sides of the c-src gene, and that the recombinations occurred at the level of proviral DNA.     

The effect of differential root and shoot temperature on the nitrate reductase activity,assayed in vivo and in vitro in roots ofHordeum vulgare (barley)

There was a large increase in nitrate reductase activity (NAR) assayed both in vivo and in vitro in roots of barley plants (cv. Midas_ grown with roots at 10°C and shoots at 20°C, compared with whole plants grown at 20°C. There were diurnal fluctuations in NRA in roots from both treatments, but they were much greater in roots grown at 20°C, where NRA fell to a very low value in the dark period. The diurnal fluctuations in the malate content of the roots were also related to the root growth temperature. Plants with roots grown at the lower temperature had a higher malate content, especially in the dark period where it was 20 times greater than in plants with roots at 20°C. At all times there was a three-fold increase in soluble carbohydrate in cooled roots and diurnal fluctuations were much less pronounced than those of malate. Growth at low temperatures increased the total flux of amino N into the xylem sap and increased the proportion of reduced N in the total N flux. At certain times of day both 10°C- and 20°C-grown roots responded to exogeneous malate by increasing the flux of amino acid into the xylem sap, although this effect was always more pronounced in 20°C-grown roots.     

Menstrual Blood Loss and Fibrin Degradation Products

As part of a large-scale study of menstrual blood loss in the community serum fibrin degradation products were measured soon after menstruation in 331 women. No significant correlation was found between the amount of blood lost and the serum level of fibrin degradation products. These findings conflict with reports suggesting that excessive intrauterine fibrinolysis, which may play a part in menorrhagia, is associated with raised serum F.D.P. concentrations.     

Relationships between structural development and the absorption of ions by the root system of Cucurbita pepo

Summary In both the seminal axis and lateral roots of Cucurbita pepo L. the formation of large central xylem elements and the commencement of secondary cambial activity occur 10–20 cm from the root tip. Concomitant with or slightly preceding these developments there are changes in the structure of the walls of endodermal cells where the lignified casparian band spreads along the radial wall and substances staining with Sudan IV are deposited in both radial and tangential walls. At distances more than 30 cm from the tip of primary roots the radius of the stele increases considerably causing splits in the cortex. The endodermis is stretched and the suberin becomes organized in a lamellar form.Against this background of anatomical change certain of the transport capabilities of the root are retained while others are lost. Using an apparatus for measuring the uptake of tracers by segments of intact roots it was found that neither the uptake nor translocation of potassium seem to be affected by the suberization of the endodermis or by secondary thickening, while the translocation of calcium is virtually eliminated when these processes begin. As the root ages its ability to absorb phosphate declines although the translocation of the phosphate absorbed is much less affected by structural development than that of calcium.The observed rates of potassium uptake by complete root systems could be predicted quite accurately from the average of segment uptake data suggesting that the method used gives reliable results.     

Stable concentrations of phytochrome in pisum under continuous illumination with red light

In vivo spectrophotometry showed that the phytochrome concentration in pea epicotyl hooks decreased at a constant rate for 4 hours when the tissue was exposed to continuous red light. Thereafter the rate slowed progressively so that a steady concentration of phytochrome was approached at hour 7. Returning the plants to darkness resulted in an increase in phytochrome due to the apparent synthesis of P_R. A closely similar pattern of changes was found in the amount of phytochrome extracted from the tissue. The establishment of the stable concentration was inhibited by 2,4-dichlorophenoxyacetic acid and did not occur in segments which had been incubated for longer than 24 hours, but was observed when segment growth was inhibited by mannitol. The results may be explained by an equilibrium between P_FR destruction and apparent P_R synthesis.     

Interactions between aluminium and phosphorus on root surfaces and cell wall material

Summary The incorporation of aluminium into barley roots followed similar kinetics over a 24-hour incubation at either 23°C or 3°C. At either temperature most of the aluminium was recovered in the cell wall fraction of the roots. Aluminium is adsorbed by preparations of cell wall material, and once bound is not appreciably exchangeable with calcium or sodium ions. Cell wall material pretreated with aluminium is able to adsorb appreciable amounts of phosphorus. In experiments with root segments the phosphorus associated with aluminium was found to be almost completely exchangeable. A mechanism for the interaction of aluminium and phosphorus on the root surface is proposed.Supported by S.R.C. postdoctoral fellowship.     

Sulphate deprivation depresses the transport of nitrogen to the xylem and the hydraulic conductivity of barley (Hordeum vulgare L.) roots

During the first 4 d after the removal of SO ₄ ^2- from cultures of young barley plants, the net uptake of ¹⁵N-nitrate and the transport of labelled N to the shoot both decline. This occurred during a period in which there was no measurable change in plant growth rate and where the incorporation of [³H]leucine into membrane and soluble proteins was unaffected. Reduced N translocation was associated with six- to eightfold increases in the level of asparagine and two- to fourfold increases in glutamine in root tissue; during the first 4 d of SO ₄ ^2- deprivation there were no corresponding increases in amides in leaf tissue. The provision of 1 mol · m^–3 methionine halted, and to some extent reversed the decline in NO ₃ ^- uptake and N translocation which occurred during continued SO ₄ ^2- deprivation. This treatment had relatively little effect in lowering amide levels in roots. Experiments with excised root systems indicated that SO ₄ ^2- deprivation progressively lowered the hydraulic conductivity, Lp, of roots; after 4 d the Lp of SO ₄ ^2- -deprived excised roots was only 20% of that of +S controls. In the expanding leaves of intact plants, SO ₄ ^2- deprivation for 5 d was found to lower stomatal conductance, transpiration and photosynthesis, in the order given, to 33%, 37% and 18% of control values. The accumulation of amides in roots is probably explained by a failure to export either the products of root nitrate assimilation or phloem-delivered amino-N. This may be correlated with the lowered hydraulic conductivity. Enhanced glutamine and-or asparagine levels probably repressed net uptake of NO ₃ ^- and ¹³NO ₃ ^- influx reported earlier (Clarkson et al. 1989, J. Exp. Bot. 40, 953–963). Attention is drawn to the similar hydraulic signals occurring in the early stages of several different types of mineral-nutrient stresses.Abbreviations Asn asparagine - Gln glutamine - Lp hydraulic conductivity J.L.K. is extremely grateful to the British Council for supporting his working visit to Long Ashton. We thank John Radin for helpful discussion and encouragement.