Isolation of adenosine 5'-diphosphate-L-glycero-D-mannoheptose, the assumed substrate of heptose transferase(s), from Salmonella minnesota R595 and Shigella sonnei Re mutants

From heptose transferase-less Re mutants of Salmonella minnesota and Shigella sonnei, a mixture of nucleotide-linked heptoses was isolated. After paper chromatography in different solvent systems, ADP derivatives of D-glycero-D-mannoheptose and L-glycero-D-mannoheptose could be isolated in pure form. The structure of ADP-L-glycero-D-mannoheptose was verified by analytical methods and by transformation of ADP-D-glycero-D-mannoheptose with ADP-D-glycero-D-mannoheptose-6-epimerase.     

The effect of age on the antigen-presenting mechanism in limiting dilution precursor cell frequency analysis

We have utilized limiting dilution analysis (LDA)2 to compare the intrinsic precursor cytotoxic T lymphocyte (pCTL) frequency for influenza-plus-self in young and old C57BL/6 mice. Under conditions of excess interleukin 2 (IL-2) and antigen presenting cells (APC) derived from spleens of mice matched in age to those being tested, we found more than a twofold difference in pCTL frequency between young and old animals. However, there was no difference in pCTL frequency between the two age groups if antigen was presented to the old responder cells on spleen cells derived from young mice. The apparent decrease in pCTL frequency in old mice by standard LDA may in fact be due to a defect in the antigen processing and/or presentation mechanism of old spleen cells. We conclude that the age-associated defective CTL activity previously reported by us and by others may be due at least in part to a defect in the antigen presentation mechanism of aging mice.     

DnaB proteolysis in vivo regulates oligomerization and its localization at oriC in Bacillus subtilis

Initiation of bacterial DNA replication at oriC is mediated by primosomal proteins that act cooperatively to melt an AT-rich region where the replicative helicase is loaded prior to the assembly of the replication fork. In Bacillus subtilis, the dnaD, dnaB and dnaI genes are essential for initiation of DNA replication. We established that their mRNAs are maintained in fast growing asynchronous cultures. DnaB is truncated at its C-terminus in a growth phase-dependent manner. Proteolysis is confined to cytosolic, not to membrane-associated DnaB, and affects oligomerization. Truncated DnaB is depleted at the oriC relative to the native protein. We propose that DNA-induced oligomerization is essential for its action at oriC and proteolysis regulates its localization at oriC. We show that DnaB has two separate ssDNA-binding sites one located within residues 1–300 and another between residues 365–428, and a dsDNA-binding site within residues 365–428. Tetramerization of DnaB is mediated within residues 1–300, and DNA-dependent oligomerization within residues 365–428. Finally, we show that association of DnaB with the oriC is asymmetric and extensive. It encompasses an area from the middle of dnaA to the end of yaaA that includes the AT-rich region melted during the initiation stage of DNA replication.     

Method for isolating single cardiac cells in the guinea pig using pronase. The role of calcium ions

A method for isolation of guinea-pig cardiomyocytes with pronase has been developed. The method has been assessed in hearts perfused with solutions containing pronase (1 U/ml) and 200 microM Ca2+. Eighty per cent of the cells released were rod-shaped and 1.2 mM Ca2+ tolerant. Enriched medium 199 was used for all solutions. Sodium and slow inward currents recorded from cells dispersed with pronase were similar to those recorded from cells isolated after prolonged exposure to collagenase. Two principal factors are to be marked: (a) presence of high enough amounts of Ca2+ in enzyme solution (up to 200 microM); (b) use of the enriched medium in all the stages of the procedure.     

The role of molecular conformation in ion capture by carboxylic ionophores: a circular dichroism study of narasin A in single-phase solvents and liposomes

Conformational and thermodynamic aspects of cation binding by the carboxylic ionophore narasin A were studied by circular dichroism (CD). In single-phase solvents, dramatic increases in the maximum differential absorption (delta epsilon) of the C-11 carbonyl were observed upon the binding of K+, Na+ and protons to the free anionic form. These changes were associated with major shifts in the conformation equilibrium between extended and pseudocyclic conformers of narasin. Similar CD changes observed upon the binding of K+ to narasin A in dimyristoylphosphatidylcholine vesicles provided evidence that in the membrane environment, comparable conformation changes were associated with ion binding. Variation of the polar and protic properties of single-phase solvents was also found to influence the delta epsilon of the cation bound species of narasin A, supporting previous evidence for polarity-mediated modulation of conformation. Comparison of cation binding affinities indicated that in both single-phase solvents and liposomes, narasin had a marked equilibrium selectivity for K+ over Na+.     

Potential of Agrotis ipsilon nucleopolyhedrovirus for suppression of the black cutworm (Lepidoptera: Noctuidae) and effect of an optical brightener on virus efficacy.

Studies were performed in the laboratory, greenhouse and field to assess the potential of Agrotis ipsilon multicapsid nucleopolyhedrovirus (AgipMNPV) and a viral enhancing agent, M2R, for suppression of Agrotis ipsilon (Hufnagel). In laboratory droplet feeding bioassays, AgipMNPV was shown to be highly active against third-instar A. ipsilon. The optical brightener M2R significantly reduced LD50 estimates by approximately 160-fold, but had no direct effect on survival time estimates. In greenhouse trials, spray and bait formulations of AgipMNPV significantly reduced feeding damage to corn seedlings caused by third-instar A. ipsilon. In two sets of replicated field trials, bait formulations of AgipMNPV significantly reduced feeding damage to corn seedlings by third-instar A. ipsilon. However, there were no beneficial effects attributable to the inclusion of M2R in AgipMNPV formulations under greenhouse or field conditions. It seems likely that in an appropriately designed pest management program AgipMNPV could be used to suppress field populations of early and mid-instar A. ipsilon.