Karyotype variation in aminoethylcysteine resistant cell and callus cultures and regenerated plants of a dihaploid potato (Solanum tuberosum)

Karyotypic studies on cell suspensions and calli of an S-(2-aminoethyl)cysteine resistant cell line of the interdihaploid potato H²578 (2n=24) revealed a high degree of variation in the number of chromosomes (33–217) and dicentric chromosomes (0–8). The suspensions also exhibited megachromosomes and fused chromosomes. Differential staining showed that in suspensions dicentrics survived mitotic cycles mainly by parallel separation of the chromatids during anaphase and hardly by the breakage-fusion-bridge cycle. Two phenotypes of plantlets regenerated, each with 34 or 35 chromosomes with gross structural mutations and with the triploid amount of DNA. Chromosomal variation was related to the degree of tissue organization.     

Structural and dynamic properties of a bromouracil-adenine base pair in DNA studied by proton NMR

We have synthesized and studied by proton NMR a duplex heptaoligonucleotide containing a 5-bromouracil (brU)-adenine base pair. This represents the first structural characterization of a B-form DNA containing brU. The brU.A base pair is Watson-Crick rather than Hoogsteen as seen for the monomers in the crystalline state. From analysis of the NOESY sepctra at very short mixing times evidence is presented that substitution of brU for T induces significant conformational changes from that of a normal B DNA. The helix twist between brU4.A11 and G3.C12 is ca. 15 degrees and for both brU4 and G3 the glycosyl torsion angles are significantly changed. The imino proton of the bru.A base pair shows a pH insensitive line with which shows that the pK of brU in this base pair is very much higher than that of the monomer.     

Changes in the management of labour: 1. Length and management of the second stage.

An analysis of 18,940 deliveries between 1981 and 1984 in a large British obstetric unit showed marked changes in the management of the second stage of labour. These changes included an increase in the length of the second stage (especially among primiparous women), a rise in the rate of spontaneous delivery and a large decline in the rate of episiotomy. There was no change in neonatal outcome or in the rate of maternal postpartum complications. These findings may have resulted from a more conservative approach to the management of the second stage. They may also reflect a better understanding among obstetric professionals of what constitutes a normal second stage and therefore better decisions about when to act and when to wait.     

Conversion of phosphatidylglycerol to lyso(bis)phosphatidic acid by alveolar macrophages

We report here studies of the synthesis of lyso(bis)phosphatidic acid [L(b)PA] by normal and BCG-elicited rabbit alveolar macrophages. This study was prompted by our earlier observations that 1) alveolar macrophages did not synthesize L(b)PA de novo despite its abundance in these cells, 2) BCG-elicited cells contained only one-quarter the amount of L(b)PA as normal cells, and 3) the turnover of arachidonate in L(b)PA led to hydroxyeicosatetraenoic acid and leukotriene synthesis. We found that exogenous phosphatidylglycerol (PG) was specifically converted to L(b)PA by both types of cells although BCG-elicited cells had only one-quarter the synthetic capacity of normal cells. Other phospholipids were found to become cell associated but were not significantly metabolized. Both glycerol moieties and the phosphate were incorporated into the product L(b)PA. However, substitution of the ester with an alkyl linkage in position 1 blocked the conversion of PG to L(b)PA. Most of the alkylphosphatidylglycerol was converted to phosphatidylcholine and phosphatidylethanolamine. This result implied that catabolism of the acyl group in position 1 was essential for L(b)PA synthesis. Because alveolar macrophages are present in a surfactant-rich milieu, we suggest that surfactant provides a source of PG for macrophage synthesis of L(b)PA in situ. It is interesting that the surfactants from rabbits challenged with BCG have a significant decrease in PG content.     

Use of a biotinylated probe and in situ hybridization for light and electron microscopic localization of P 0 mRNA in myelin-forming Schwann cells

Summary A biotinylated P ₀ glycoprotein cDNA was hybridized in situ to aldehyde-fixed vibratome sections and to aldehyde-fixed thin sections of Lowicryl-embedded trigeminal ganglia of 15 day old rats. Alkaline phosphatase and peroxidase detectors were used for light microscopic (LM) studies and peroxidase or colloidal gold were employed for electron microscopic (EM) detection. In both LM and EM sections, probe was found in cytoplasmic areas of myelinforming Schwann cells that were enriched in granular endoplasmic reticulum, demonstrating that these regions contain P ₀ mRNA. Interestingly, P ₀ mRNA tended to cluster in regions close to the developing myelin sheath. Relatively simple methods are here described for EM detection of mRNA with reasonable tissue preservation and high resolution. These methods may be useful for developmental and disease-related studies of specific mRNAs in mammalian tissues.     

Effects of net blotch on growth and yield of spring barley

The effect of net blotch on the growth and yield of cv. Beatrice spring barley was examined in a greenhouse experiment. Separate inoculations at growth stages 21 and 34 reduced green leaf area, root weight, leaf sheath and stem weight and tiller number. The early inoculated plants, which responded and recovered more rapidly than later treated ones, suffered a loss in grain yield and this was related to the amount of disease, the loss in green leaf area and the reduction in unit leaf rate.     

Slow- and fast-binding inhibitors of thermolysin display different modes of binding: crystallographic analysis of extended phosphonamidate transition-state analogues

The modes of binding to thermolysin of two phosphonamidate peptide inhibitors, carbobenzoxy-GlyP-L-Leu-L-Leu (ZGPLL) and carbobenzoxy-L-PheP-L-Leu-L-Ala (ZFPLA), have been determined by X-ray crystallography and refined at high resolution to crystallographic R-values of 17.7% and 17.0%, respectively. (GlyP is used to indicate that the trigonal carbon of the peptide linkage is replaced by the tetrahedral phosphorus of a phosphonamidate group.). These inhibitors were designed to be structural analogues of the presumed catalytic transition state and are potent inhibitors of thermolysin (ZGPLL, Ki = 9.1 nM; ZFPLA, Ki = 0.068 nM) [Bartlett, P. A., & Marlowe, C. K. (1987) Biochemistry (following paper in this issue)]. ZFPLA binds to thermolysin in the manner expected for the transition state and, for the first time, provides direct support for the presumed mode of binding of extended substrates in the S2 subsite. The mode of binding of ZFPLA displays all the interactions that are presumed to stabilize the transition state and supports the postulated mechanism of catalysis [Hangauer, D. G., Monzingo, A. F., & Matthews, B. W. (1984) Biochemistry 23, 5730-5741]. The two oxygens of the phosphonamidate moiety are liganded to the zinc to give overall pentacoordination of the metal. For the second inhibitor the situation is different. Although both ZFPLA and ZGPLL have similar modes of binding in the S1' and S2' subsites, the configurations of the carbobenzoxy-Phe and carbobenzoxy-Gly moieties are different. For ZFPLA the carbonyl group of the carbobenzoxy group is hydrogen bonded directly to the enzyme, whereas in ZGPLL the carbonyl group is rotated 117 degrees, and there is a water molecule interposed between the inhibitor and the enzyme. For ZGPLL only one of the phosphonamidate oxygens is liganded to the zinc. Correlated with the change in inhibitor-zinc ligation from monodentate in ZGPLL to bidentate in ZFPLA there is an increase in the phosphorus-nitrogen bond length of about 0.25 A, strongly suggesting that the phosphonamide nitrogen in ZFPLA is cationic, analogous to the doubly protonated nitrogen of the transition state. The observation that the nitrogen of ZFPLA appears to donate two hydrogen bonds to the protein also indicates that it is cationic. The different configurations adopted by the respective inhibitors are correlated with large differences in their kinetics of binding [Bartlett, P. A., & Marlowe, C. K. (1987) Biochemistry (following paper in this issue)]. These differences in kinetics are not associated with any significant conformational change on the part of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism and base specificity of DNA Breakage in intact cells by neocarzinostatin

When electrophoresed on an agarose gel, the DNA isolated from neocarzinostatin- (NCS-) treated HeLa cells migrates in a ladder of discrete bands indicative of preferential breakage in the linker region of the nucleosomes. The 5'-termini of the drug-induced DNA strand breaks were characterized by reduction of the nucleoside 5'-aldehyde ends to 5'-hydroxyls followed by incorporation of 32P from [gamma-32P]ATP by polynucleotide kinase and treatment of the DNA with hot alkali and alkaline phosphatase prior to the kinase assay to give the total 5'-termini. In DNA isolated from NCS-treated cells, nucleoside aldehyde accounts for 30-45% of the drug-generated 5' ends; the remainder have PO4 termini. By contrast, 5'-terminal nucleoside aldehyde in DNA cut with the drug in vitro exceeds 80% of the total 5' ends. Of the 32P representing nucleoside aldehyde in DNA from NCS-exposed cells, 77% is in TMP; the rest is in AMP much greater than CMP greater than GMP, a distribution in excellent agreement with that obtained for in vitro drug-treated DNA. DNA sequencing experiments, using the 340 base pair alphoid DNA fragment isolated from drug-treated cells, show that the pattern of breakage produced by NCS within a defined sequence of DNA in intact cells is similar to that in the in vitro reaction, with a preferential attack at thymidylate residues, but a much higher concentration of the drug was required to cause comparable breakage in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epsilon subunit of Escherichia coli F1-ATPase: effects on affinity for aurovertin and inhibition of product release in unisite ATP hydrolysis

The epsilon subunit of Escherichia coli F1-ATPase is a tightly bound but dissociable partial inhibitor of ATPase activity. The effects of epsilon on the enzyme were investigated by comparing the ATPase activity and aurovertin binding properties of the epsilon-depleted F1-ATPase and the epsilon-replete complex. Kinetic data of multisite ATP hydrolysis were analyzed to give the best fit for one, two, or three kinetic components. Each form of F1-ATPase contained a high-affinity component, with a Km near 20 microM and a velocity of approximately 1 unit/mg. Each also exhibited a component with a Km in the range of 0.2 mM. The velocity of this component was 25 units/mg for epsilon-depleted ATPase but only 4 units/mg for epsilon-replete enzyme. The epsilon-depleted enzyme also contained a very low affinity component not present in the epsilon-replete enzyme. In unisite hydrolysis studies, epsilon had no effect on the equilibrium between substrate ATP and product ADP.P1 at the active site but reduced the rate of product release 15-fold. These results suggest that epsilon subunit slows a conformational change that is required to reduce the affinity at the active site, allowing dissociation of product. It is suggested that inhibition of multisite hydrolysis by epsilon is also due to a reduced rate of product release. epsilon-depleted F1-ATPase showed little of no modulation of aurovertin fluorescence by added ADP and ATP. Aurovertin fluorescence titrations in buffer containing ethylenediaminetetraacetic acid (EDTA) revealed that epsilon-depleted enzyme had high affinity for aurovertin (Kd less than 0.1 microM) regardless of the presence of nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)