Relationship between osmoprotection and the structure and intracellular accumulation of betaines by Escherichia coli

Abstract Naturally occuring betaines, especially glycine betaine and proline betaine, were accumulated by Escherichia coli from urine. In synthetic hyperosmotic medium, with an homologous series of added betaines, (CH₃)₃N⁺-(CH₂) _n -COO⁻, osmoprotective activity and intracellular accumulation decreased monotonically as n increased from 1 to 5. In contrast, α -substituted glycine betaines were accumulated in a similar manner to glycine betaine, but with different osmoprotective activities. Arsenobetaine, with a quaternary arsonium group, was also accumulated but amino acids which can become negatively charged in a chemically basic environment were not.     

Relationship between chimpanzee Rh-like genes and the R-C-E-F blood group system

The antigenic closeness between the chimpanzee alloantigen R^c of the R-C-E-F system, and the human alloantigen Rh_o(D) suggests a phylogeconnection between their genes. To confirm at the molecular level the common origin of these genes, genomic DNA from 16 unrelated chimpanzees of various R-C-E-F phenotypes were digested by three restriction enzymes and analyzed by Southern blot using a human Rh cDNA probe and three exon-specific probes. Restrictions profiles displayed reach polymorphism. Correlations between some bands and certain R-C-E-F phenotypes demonstrate that the human Rh cDNA probe defines in chimpanzee genomic DNA some genes of the R-C-E-F system.     

Leukaemia inhibitory factor and the regulation of pre-implantation development of the mammalian embryo

This paper reviews the evidence that certain growth factors, particularily leukaemia inhibitory factor (LIF), play a crucial role in regulating the development of the pre-implantation mammalian embryo. LIF was originally implicated in regulating the early development of the mouse embryo because it inhibited the differentiation of embryonic stem (ES) cells, pluripotential cells derived from the inner cell mass of the blastocyst. Subsequent studies on its role in vivo revealed, surprisingly, that it is essential for the growth rather than the differentiation of the blastocyst. In vivo, overtly normal blastocysts can be produced in a LIF-deficient environment that are capable of forming viable fertile adults. However, in the absence of LIF, they fail to implant and enter into a state resembling that exhibited by blastocysts undergoing delayed implantation, which is characterized by a cessation of cell proliferation. This failure to implant occurs because the principle sites of LIF production are the endometrial glands of the uterus. These synthesize and secrete LIF at implantation, with LIF synthesis essential for implantation. Preliminary evidence indicates that LIF synthesis is required both by the uterus for it to undergo decidualization and by the blastocyst for implantation. These data indicate that the maternal environment plays a crucial role in the development and growth of the pre-implantation embryo, by supplying factors that regulate these processes in the embryo. © 1994 Wiley-Liss, Inc.     

Purification and Properties of an S-Adenosylmethionine: 2,4-Disubstituted Phenol O-Methyltransferase from Phanerochaete chrysosporium

An enzyme catalyzing the O-methylation of acetovanillone (3-methoxy-4-hydroxyacetophenone) by S-adeno-sylmethionine was isolated from Phanerochaete chrysosporium and purified 270-fold by ultrafiltration, anion-exchange chromatography, and gel filtration. The enzyme exhibited a pH optimum between 7 and 9 and was rapidly denatured at temperatures above 55°C. The K_m values for acetovanillone and S-adenosylmethionine were 34 and 99 μM, respectively. S-Adenosylhomocysteine acted as a powerful competitive inhibitor of S-adenosylmethionine, with a K_i of 41 μM. The enzyme was also susceptible to inhibition by thiol reagents and low concentrations of heavy metal ions. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the enzyme was monomeric and had a molecular weight of approximately 53,000. Substrate specificity studies showed that 3-methoxy- and 3,5-dimethoxy-substituted 4-hydroxy-benzaldehydes, -benzoic acids, and -acetophenones were the preferred substrates for the enzyme. The corresponding 3,4-dihydroxy compounds were methylated relatively slowly, while the 3-hydroxy-4-methoxy compounds were almost inactive as substrates. Substituents in both the 2 and 4 positions relative to the hydroxyl group appeared to be essential for significant enzyme attack of a substrate. Provided that certain steric criteria were satisfied, the nature of the substituent was not critical. Hence, xenobiotic compounds such as 2,4-dichlorophenol and 2,4-dibromophenol were methylated almost as readily as acetovanillone. However, an extended side chain in the 4 position was not compatible with activity as a substrate, and neither homovanillic, caffeic, nor ferulic acid was methylated. The substrate range of the O-methyltransferase tends to imply a role in the catabolism or detoxification of lignin degradation products such as vanillic and syringic acids.     

Accumulation of the β-subunit of polygalacturonase 1 in normal and mutant tomato fruit

Polyclonal antiserum raised against the native PG1 isoform of tomato fruit (Lycopersicon esculentum Mill.) polygalacturonase [poly(1,4--d-galacturonide) glycanohydrolase, EC 3.2.1.15] bound to each of the subunits of the protein and also to a range of other fruit proteins. Affinity purification was used to remove antibody molecules that bound to the native form of the PG2 isoform. The resulting serum bound to native PG1, denatured PG2 and -subunits of PG1 but not to native PG2 or other fruit proteins. This anti-PG1 serum was used to monitor the occurrence of the PG1 -subunit and PG2 in detergent extracts of tomato tissues. The -subunit polypeptide was detected in pericarp but not locule tissue of fruit, including fruit of the rin and nor mutants. It increased in amount in the pericarp tissues from an early stage to the mature green stage, clearly prior to any appreciable accumulation of the PG2 subunit. The -subunit polypeptide was not detected in stem or leaf tissues. A PG2-specific antiserum was used to study the interaction of PG2 with the isolated -subunit. The PG2 isoform was bound to the -subunit over a wide range of salt concentrations and pH; the interaction was independent of the presence of reducing agents. It is concluded that strong non-covalent forces are involved in the interaction. The results are consistent with a model in which the -subunit is positioned in the cell wall structure and provides a specific binding site for the active PG2 subunit when this is synthesised during ripening.Abbreviations B breaker - MG mature green - M_r relative molecular mass - nor non-ripening mutant - PAGE polyacrylamide gel electrophoresis - PG polygalacturonase - rin ripening inhibitor mutant - SDS sodium dodecyl sulphate     

Age and growth of the Australian sharpnose shark,Rhizoprionodon taylori,from north Queensland,Australia

Synopsis Age and growth were studied inRhizoprionodon taylori using specimens caught in Cleveland Bay, North Queensland, Australia. Von Bertalanffy growth parameters were estimated using three different techniques: vertebral ageing, back calculation and length frequency. Vertebrae from 138 specimens were sectioned and narrow circuli counted to estimate age. Marginal increment analysis verified that circuli were produced annually in late summer, probably as a result of stress during the mating season. The oldest female was 7 and male 6 years old. Von Bertalanffy growth parameters estimated from vertebral ageing data for males were t_O = 0.410 yr, K = 1.337, L = 652.2 mm, and for females t_O = 0.455 yr, K = 1.013 and L = 732.5 mm. Growth parameters determined by length frequency and back calculation techniques concurred with those from vertebral ageing. Growth of the 0+ age class was very rapid, averaging 140% of the size at birth in the first year. Males and females matured after only one year, the lowest age at maturity reported in the family Carcharhinidae. Annual growth increments decreased rapidly after maturity, and little growth occurred after three years.     

Tissue distribution of bupivacaine enantiomers in sheep

rac-Bupivacaine HCl was infused intravenously to constant arterial blood drug concentrations in sheep using a regimen of 4 mg/min for 15 min followed by 1 mg/min to 24 h. At 24 h, arterial blood was sampled, the animal was killed with a bolus of KCl solution, then rapidly dissected and samples were obtained from heart, brain, lung, kidney, liver, muscle, fat, gut, and rumen. Tissue:blood distribution coefficients for (+)-(R)-bupivacaine exceeded those of (?)-(S)-bupivacaine (P < 0.05) for heart, brain, lung, fat, gut, and rumen by an overall mean of 43%. Blood:plasma distribution coefficients of (?)-(S)-bupivacaine exceeded those of (+)-(R)-bupivacaine by a mean of 29% and this offset the tissue:blood distribution coefficients so that the previously significant enantioselective differences disappeared. It is concluded that although enantioselectivity of bupivacame distribution is shown by the measured tissue:blood distribution coefficients, it is not shown when tissue:plasma water distribution coefficients are calculated, suggesting that there is no intrinsic difference between the bupivacaine enantiomers in tissue affinity. Sheep given fatal intravenous bolus doses of rac-bupivacaine had significantly greater concentrations of (+)-(R)-bupivacaine than (?)-(S)-bupivacaine in brain (P = 0.028) and ventricle (P = 0.036); these could augment the greater myocardial toxicity of this enantiomer found in vitro. © 1993 Wiley-Liss, Inc.     

A 4D HCC(CO)NNH experiment for the correlation of aliphatic side-chain and backbone resonances in 13C/15N-labelled proteins

Summary We recently proposed a novel four-dimensional (4D) NMR strategy for the assignment of backbone nuclei in spectra of ¹³C/¹⁵N-labelled proteins (Boucher et al. (1992) J. Am. Chem. Soc., 114, 2262–2264 and J. Biomol. NMR, 2, 631–637). In this paper we extend this approach with a new constant time 4D HCC(CO)NNH experiment that also correlates the chemical shifts of the aliphatic sidechain (¹H and ¹³C) and backbone (¹H, ¹³C and ¹⁵N) nuclei. It separates the sidechain resonances, which may heavily overlap in spectra of proteins with large numbers of similar residues, according to the backbone nitrogen and amide proton chemical shifts. When used in conjunction with a 4D HCANNH or HNCAHA experiment it allows, in principle, complete assignment of aliphatic sidechain and backbone resonances with just two 4D NMR experiments.