A unique anther-mucilage in the pollination biology of Tylosema esculentum

Summary Tylosema esculentum, a perennial geophyte bearing yellow distylous flowers in racemes, maintains a high degree of outbreeding through reciprocal herkogamy. In addition, a viscous liquid, the anther-mucilage, is produced by the anther connective tissue and released concurrently with the pollen. The polysaccharide- and lipid-rich mucilage, which is functional in the shedding and transfer of pollen, is available for more than 1 day due to the gradual solidification of the mucilage. The assimilation of the pollen with the liquid substance significantly affects the pollination biology of T. esculentum. This is the first report on the unique phenomenon of wet pollen in the Caesalpiniaceae.     

New function of vitamin B12: cobamide-dependent reduction of epoxyqueuosine to queuosine in tRNAs of Escherichia coli and Salmonella typhimurium.

Queuosine (Q), 7-[(4,5-cis-dihydroxy-2-cyclopentene-1-yl)-amino)methyl)-7- deazaguanosine, and Q derivatives usually replace guanosine in the anticodon of tRNAs(GUN) of eubacteria and of cytoplasmic and mitochondrial tRNAs of lower and higher eucaryotes except yeasts. Q appears to be synthesized de novo exclusively in eubacteria, and the free-base queuine serves as a nutrient factor for eucaryotes. Recently, a Q derivative, oQ, containing a 2,3-epoxy-4,5-dihydroxycyclopentane ring, has been identified in Escherichia coli tRNA(Tyr). Here we show that oQ is formed when E. coli or Salmonella typhimurium is grown in glucose-salt medium. The formation of oQ was independent of molecular oxygen, and oQ-tRNAs were converted to Q-tRNAs by adding cobalamin to the growth medium. Under strictly anaerobic conditions, considerable amounts of Q were present in E. coli and S. typhimurium tRNAs when the bacteria were grown in the presence of cobalt ions with glycerol as the carbon source and fumarate as the electron acceptor. Under these conditions, the biosynthesis of cobalamin was induced. The results suggest that oQ is derived from ribose and that oQ is finally reduced to Q by a cobamide-dependent enzyme.     

Dynamics of a hydrophobic peptide in membrane bilayers by solid-state nuclear magnetic resonance

Solid-state NMR studies of the dynamics of a synthetic hydrophobic peptide, tert-butyloxycarbonylleucylphenylalanine methyl ester (Boc-Leu-Phe-OMe), in phospholipid bilayers are described. Motionally averaged powder pattern line shapes from 2H- and 15N-labeled backbone and side-chain sites of the peptide in phospholipid bilayers demonstrate the presence of both overall and internal reorientations of substantial amplitude. The peptide motions are shown to be strongly influenced by the motions of the lipids.     

alpha-Ketoglutarate dehydrogenase complex of Escherichia coli. A hybrid complex containing pyruvate dehydrogenase subunits from pyruvate dehydrogenase complex

The alpha-ketoglutarate dehydrogenase complex of Escherichia coli utilizes pyruvate as a poor substrate, with an activity of 0.082 units/mg of protein compared with 22 units/mg of protein for alpha-ketoglutarate. Pyruvate fully reduces the FAD in the complex and both alpha-keto[5-14C]glutarate and [2-14C]pyruvate fully [14C] acylate the lipoyl groups with approximately 10 nmol of 14C/mg of protein, corresponding to 24 lipoyl groups. NADH-dependent succinylation by [4-14C]succinyl-CoA also labels the enzyme with approximately 10 nmol of 14C/mg of protein. Therefore, pyruvate is a true substrate. However, the pyruvate and alpha-ketoglutarate activities exhibit different thiamin pyrophosphate dependencies. Moreover, 3-fluoropyruvate inhibits the pyruvate activity of the complex without affecting the alpha-ketoglutarate activity, and 2-oxo-3-fluoroglutarate inhibits the alpha-ketoglutarate activity without affecting the pyruvate activity. 3-Fluoro[1,2-14C]pyruvate labels about 10% of the E1 components (alpha-ketoacid dehydrogenases). The dihydrolipoyl transsuccinylase-dihydrolipoyl dehydrogenase subcomplex (E2E3) is activated as a pyruvate dehydrogenase complex by addition of E. coli pyruvate dehydrogenase, the E1 component of the pyruvate dehydrogenase complex. All evidence indicates that the alpha-ketoglutarate dehydrogenase complex purified from E. coli is a hybrid complex containing pyruvate dehydrogenase (approximately 10%) and alpha-ketoglutarate dehydrogenase (approximately 90%) as its E1 components.     

Distances of tyrosine residues from a spin-label hapten in the combining site of a specific monoclonal antibody

The nuclear magnetic resonance spectra of an Fab fragment of a monoclonal antibody specifically directed against a nitroxide spin-label hapten have been recorded at different concentrations of the hapten. The hybridoma producing this antibody was grown on deuterated phenylalanine, tryptophan, and 3,5-dideuteriotyrosine or 2,6-dideuteriotyrosine. Difference spectra--without hapten minus with hapten--were calculated for each concentration of hapten. The difference spectra reveal five well-resolved singlet proton resonance signals from tyrosine deuterated in the 3,5-positions (H 2,6 Tyr) and nine from tyrosine deuterated in the 2,6-positions (H 3,5 Tyr). The measured intensities of these signals as a function of combining site occupation have been interpreted in terms of a theory involving intrinsic line widths (T2), the hapten off-rate (k), and distances to the paramagnetic center. Good agreement with theory is found for all of the isolated proton signals. The best estimate of k is 350 s-1; distances in the range 13 to less than 9 A are calculated. Extension of this analysis to other amino acids is discussed.     

Overinitiation of chromosome and plasmid replication in a dna Acos mutant of Escherichia coli K12. Evidence for dnaA-dnaB interactions

The dnaAcos mutations are phenotypic suppressors of dnaAts46 that are co-transduced with dnaA, render the cell cold sensitive, and cause an excess of chromosome replication relative to cell mass when the cells are shifted from 42 degrees C to 32 degrees C. We have used pulse labelling and DNA-DNA hybridization to follow the effect of a temperature shift on the replication of the chromosome and of the plasmids pSC101, RTF-Tc, and lambda dv in such strains. After a shift of a dnaAcos strain from 42 degrees C to 32 degrees C (non-permissive temperature), initiation of the chromosome and replication of the plasmid pSC101 are stimulated, while the dnaA-independent plasmid RTF-Tc is not affected. The presence of pSC101 does not affect the level of overinitiation of the chromosome. The presence of lambda dv suppresses the cold sensitivity of dnaAcos mutants and allows the cells to grow at both 32 degrees C and 42 degrees C. The presence of lambda dv suppresses the overinitiation of chromosome and of pSC101 replication at 32 degrees C. Previous reports had shown that these suppressions involve an interaction between the dnaA product and the lambda P protein, which is also known to interact with dnaB. We show here that the mutant prophage P1 bac-crr, which produces high levels of a dnaB analogue, suppresses the dnaAcos phenotype, while wild type P1 does not. These results suggest that initiation involves interactions between the dnaA and dnaB products.     

Insertion mutations in the dam gene of Escherichia coli K-12

The dam gene of E. coli can be inactivated by insertion of Tn9 or Mud phage. Strains bearing these mutations are viable indicating that the dam gene product is dispensable.     

Isolation and renaturation of alpha 2-macroglobulin receptor from diploid human fibroblasts.

alpha 2-Macroglobulin receptor was extracted from human diploid fibroblasts and purified by affinity chromatography in a single step. The receptor had mol.wt. 125 000 after sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis. The isolated receptor was separated by SDS/polyacrylamide-gel electrophoresis, transferred on to nitrocellulose sheets and subsequently renatured, as shown by a specific binding test, by incubation with Nonidet P40.