Protoplast formation and cell wall regeneration in Clostridium perfringens

A protocol was developed for the efficient production and regeneration of Clostridium perfringens protoplasts. Cell wall regeneration frequencies of up to 5% were obtained.     

Monoclonal antibodies to alpha-chain regions of human fibrinogen that participate in polymer formation

Monoclonal antibodies have been generated against a cross-link-containing derivative of alpha polymer (alpha XLCNBr), isolated following CNBr digestion of fibrin [Sobel, J. H., Ehrlich, P. H., Birken, S., Saffran, A. J., & Canfield, R. E. (1983) Biochemistry (preceding paper in this issue)]. One cloned cell line (F-102) was chosen for characterization based on its apparent specificity for the A alpha-chain region A alpha 518-584 (CNBr X). A second line (F-103) was selected because of its anti-A alpha 241-476 (CNBr VIII) properties. These two regions of the A alpha chain have previously been implicated as major contributors to the cross-linking process that leads to alpha-polymer formation. Radioimmunoassays have been developed, employing the immunoglobulins produced by clones F-102 and F-103. These assays have been applied, in conjunction with high-performance liquid chromatography purified tryptic and chymotryptic derivatives of CNBr VIII and CNBr X, to localize the respective determinants involved in antibody binding. In each case, virtually full immunoreactivity was exhibited by both the CNBr fragment and a single tryptic or chymotryptic peptide originating from it. These findings indicate that sequence-specific, rather than conformational, determinants were operative in the generation of antibodies F-102 and F-103. The epitope recognized by F-102 was localized to the region of A alpha 540-554, while the F-103 binding site resided within A alpha 259-276. When these radioimmunoassays were applied to study the relative immunoreactivity exhibited by a variety of fibrinogen derivatives, the results obtained support earlier suggestions that the COOH-terminal portion of the A alpha chain contains regions of random conformation.     

Antiphytovirale Aktivität von Rhamnolipid aus Pseudomonas aeruginosa

A rhamnolipid released by Pseudomonas aeruginosa 196 Aa into the culture medium reduced the number of local lesions induced by tobacco mosaic virus on leaves of the hypersensitive host Nicotiana glutinosa L. by up to 90%. The content of potato virus X in the systemically infected host Nicotiana tabacum L. ‘Samsun’ is decreased in inoculated as well as in secondarily infected leaves by up to 50%. In a smaller degree red clover mottle virus is influenced in the systemic host Pisum sativumconvar.speciosum (Dierb.) Alef ‘Nadja’.     

18O-Labeling of guanosine monophosphate upon hydrolysis of cyclic guanosine 3':5'-monophosphate by phosphodiesterase

The hydrolysis of cGMP by phosphodiesterase was conducted in [18O]water to determine the site of bond cleavage and the stoichiometry of 18O incorporation into 5'-GMP. Three different forms of phosphodiesterase including a calmodulin-calcium-dependent enzyme in its basal and activated states were examined. The hydrolysis of cGMP catalyzed by each of the forms of phosphodiesterase proceeded with incorporation of 1 18O atom recoverable in the phosphate moiety of each molecule of 5'-GMP generated. No molecular species of phosphate deriving from the 5'-GMP generated containing two or three 18O were detectable. These results indicate that the phosphodiesterase-catalyzed hydrolysis of cGMP proceeds by nucleophilic substitution at phosphorus resulting in P-O bond cleavage. The stoichiometry of 18O incorporation indicates that the reaction proceeds without phosphate-water oxygen exchange when the hydrolytic reaction is catalyzed by diverse forms of phosphodiesterase in the basal or activated state. These considerations of the phosphodiesterase reaction help to establish the validity of monitoring the rate of enzyme-catalyzed hydrolysis of cGMP as a function of the rate of 18O-labeling of the phosphate of 5'-GMP when the reaction proceeds in a medium of predetermined 18O enrichment.     

Understanding plant reproductive diversity

Flowering plants display spectacular floral diversity and a bewildering array of reproductive adaptations that promote mating, particularly outbreeding. A striking feature of this diversity is that related species often differ in pollination and mating systems, and intraspecific variation in sexual traits is not unusual, especially among herbaceous plants. This variation provides opportunities for evolutionary biologists to link micro-evolutionary processes to the macro-evolutionary patterns that are evident within lineages. Here, I provide some personal reflections on recent progress in our understanding of the ecology and evolution of plant reproductive diversity. I begin with a brief historical sketch of the major developments in this field and then focus on three of the most significant evolutionary transitions in the reproductive biology of flowering plants: the pathway from outcrossing to predominant self-fertilization, the origin of separate sexes (females and males) from hermaphroditism and the shift from animal pollination to wind pollination. For each evolutionary transition, I consider what we have discovered and some of the problems that still remain unsolved. I conclude by discussing how new approaches might influence future research in plant reproductive biology.     

Increasing saliva (free) oestriol to progesterone ratio in late pregnancy: a role for oestriol in initiating spontaneous labour in man?

Oestriol and progesterone concentrations were measured in samples of saliva obtained daily from six normal women during the final four weeks before the spontaneous onset of labour. Progesterone concentrations were found to plateau whereas oestriol concentrations continued to rise so that the mean ratio of saliva oestriol to progesterone increased from 0.80 to 1.43 between 29 days and one day before labour. Saliva oestriol concentrations were 15 times higher than saliva oestradiol concentrations. As saliva steroid concentrations reflect the unbound unconjugated (free) plasma steroid concentrations these data suggest that a changing ratio of oestriol to progesterone may play a part in initiating spontaneous labour in man.     

Fluorophotometric enzyme immunoassay of thyroid-stimulating hormone.

A method for enzyme immunoassay of thyroid-stimulating hormone (TSH) is described, TSH was conjugated with horseradish peroxidase according to periodate oxidation method. Separation of the bound and free was obtained by double-antibody solid-phase technique using Sepharose 4B-anti-rabbit immunogiobulin G (IgG)-geat IgG. The fluorescence reaction using tyramine and hydrogen peroxide as substrates was used for the determination of enzyme activity in order to increase the sensitivity of enzyme immunoassay. The standard curve for serum TSH was satisfactory to recognize TSH concentrations as 0.06 μU/tube. TSH values obtained by this method correlated well with those obtained by radioimmunoassay (r, 0.96). The coefficients of variation were 1.8 to 5.3% (within assay) and 5.1 to 10.5% (between assay). The method is about equal to radioimmunoassay with respect to sensitivity. Since it requires minimal equipment and is less expensive than radioimmunoassay, it is possible to perform routine assays even in laboratories with limited facilities.     

Biochemical studies of taste sensation. XI. Isolation, characterization and taste ligand binding activity of plasma membranes from catfish taste tissue

Variants of creatine kinase-MM (variant of ATP:creatine N-phosphotransferase, EC 2.7.3.2), present in human heart and skeletal muscle, have been purified to homogeneity using DEAE-Sepharose column chromatography and column chromatofocusing techniques. Creatine kinase-MM I-IV were present in both heart and skeletal muscle, while MM-V was found only in heart. The number, ratio and elution profile of the variants during chromatofocusing remained identical even when they were purified in the presence of proteinase inhibitors. MM-I-V, on chromatofocusing, were eluted at pH 8.3, 7.9, 7.6, 7.2 and 6.8, respectively. Isoelectric focusing revealed the pI of MM-I-V to be 7.2, 6.9, 6.7, 6.4 and 6.2. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis showed a doublet pattern for creatine kinase-MM variants III-V. However, polyacrylamide gel electrophoresis without SDS indicated homogeneity because each variant showed a single band. The doublet pattern observed in the presence of SDS may reflect the presence of two subunits of slightly different mass.