Unusual composition of peptidoglycan in Bordetella pertussis

The composition of the peptidoglycan of Bordetella pertussis and the nature of its turnover products was determined by a new combination of analytical techniques: high performance liquid chromatography of an enzymatic peptidoglycan hydrolysate and fast atom bombardment mass spectrometry and fast atom bombardment collision-activated dissociation tandem mass spectrometry. Sixteen major components of the peptidoglycan were purified, and assignment of complete or partial chemical structures was achieved for nine and seven species, respectively. At this level of resolution, a previously unrecognized heterogeneity of monomeric (five new species; nine total) and dimeric species (five new species; five total) was detected. No species containing diaminopimelyl-diaminopimelic acid cross-links or lysyl-arginine substitutions were found. Previous estimates of total cross-linkage and average chain length were revised downward to 32% and 21 disaccharide residues, respectively. Detection of a chemically novel species, a disaccharide octapeptide monomer, in both the peptidoglycan hydrolysate and culture supernatant fluid, suggests that an N-acetyl-muramyl-L-alanine amidase acts on the intact peptidoglycan of Bordetella and participates in cell wall turnover. Five peptidoglycan turnover products were identified in the supernatant fluid of late logarithmic phase cultures, including the 1,6-anhydro monomeric species known as tracheal cytotoxin. Peptidoglycan turnover was detected at a low rate of approximately 10%/generation, a value sufficient to account for the generation of all tracheal cytotoxin found in culture supernatant fluids.     

Effects of thyrotropin,prostaglandin E1 and iodide on cyclic 3′,5′-AMP concentration in dog thyroid slices

The kinetics and concentration effect on the relationship of thyrotropin (TSH) action on cyclic 3′,5′-AMP concentration has been studied in dog thyroid slices in vitro. TSH markedly increased cyclic 3′,5′-AMP level after 5 min, the effect reached a plateau after 10–60 min and slowly declined afterwards. TSH enhanced in parallel the cyclic 3′,5′-AMP level and the binding of iodide to proteins. For this latter effect of TSH, the four criteria of the validity of the Sutherland model for a hormonal action are therefore fulfilled. The effect of TSH on cyclic 3′,5′-AMP concentration in thyroid did not require the presence of a methylxanthine inhibitor of cyclic 3′,5′-AMP phosphodiesterase in the medium. Prostaglandin E1 increased cyclic 3′,5′-AMP levels in control and stimulated slices. The omission of Ca²⁺ in the incubation medium decreased the action of TSH but partial replacement of Na⁺ by K⁺ had little effect. Iodide, 1 μM to 100 μM, inhibited the action of TSH. This inhibitory effect was relieved by NaClO₄, methimazole and propylthiouracil (1 mM). The possible role of this inhibitory effect in an intracellular regulatory mechanism is discussed.     

Polymerization of oligodeoxythymidylates and oligoriboadenylates catalyzed by T4 polynucleotide ligase and their use as analytical markers in polyacrylamide-gel electrophoresis

A series of polymers of oligodeoxythymidylates has been prepared by the T4 polynucleotide ligase-catalyzed joining of oligodeoxythymidylates in the presence of poly(dA) or poly(rA). A similar series of polymers of oligoriboadenylates was prepared by the enzymatic joining reaction of oligoriboadenylates in the presence of poly(dT). Analysis of the polymer series by polyacrylamide-gel electrophoresis showed that polynucleotides of up to 250 nucleotides in length were present. Chain length of individual oligomers could be determined by internal to external phosphate ratios. The oligodeoxythymidylate and oligoriboadenylate polymers provide a series of specific molecular weight markers for size estimation of single-stranded RNA and DNA in the size range 10–200 nucleotides on denaturing gels containing 7 m urea.     

The Effect of Divalent Cations on Aggregation of Dictyostelium discoideum

Following the initiation of development, amoebae of Dictyostelium discoideum aggregate chemotactically toward cyclic AMP (cAMP). Adenyl cyclase, cAMP phosphodiesterase, and cAMP binding sites all increase 20–40 fold during the first few hours of development. It has been shown that addition of 1 mM EDTA and 5 mM MgCl₂ accelerates the aggregation process. Likewise, the calcium ionophore, A23187, leads to precocious aggregation while 4 × 10⁻⁵ M progesterone considerably delays it These treatments have now been shown to result in increased accumulation of adenyl cyclase in the case of EDTA and Mg²⁺ or the ionophore and greatly decreased accumulation in the case of the steroid.
Treatment with EDTA and Mg²⁺ or the ionophore has been shown not only to accelerate aggregation in wild-type amoebae but to overcome complete blocks to aggregation in certain mutant strains. We have found that addition of Mn²⁺ will also permit aggregation of mutant cells otherwise unable to aggregate. This divalent ion, unlike EDTA and Mg²⁺ or the ionophore, was shown to directly stimulate adenyl cyclase. Calcium ions were also found to affect the enzyme such that at Ca²⁺ concentrations found within the cells the great majority of the activity is inhibited. Manganese ions can overcome the inhibition by Ca²⁺.
These findings show that conditions which stimulate aggregation result in increased activity of adenyl cyclase either by increased accumulation of the enzyme or by increased activity of the available enzyme, and support the proposed central role of adenyl cyclase in aggregation.     

End group labelling of RNA and double stranded DNA by phosphate exchange catalyzed by bacteriophage T4 induced polynucleotide kinase.

End group labelling of sheared double-stranded DNA, and tRNA has been effected without prior dephosphorylation, utilizing the reversal of T₄ polynucleotide kinase activity. Incubation of DNA with polynucleotide kinase in the presence and absence of a phosphate acceptor (ADP) allowed the determination of the relative ratio of 5′ hydroxyl and 5′ phosphoryl terminii in the polynucleotide. This method of analysis has demonstrated a high preference in the formation of 5′ vs 3′ phosphomonoesters during high pressure shearing of double stranded DNA.     

Nestling development and the timing of tick attachments

Parasites exposed to fast-developing hosts experience a variety of conditions over a short time period. Only few studies in vertebrate-ectoparasite systems have integrated the timing of ectoparasite infestations in the host's development into the search for factors explaining ectoparasite burden. In this study we examined the temporal pattern of attachment in a nidicolous tick (Ixodes arboricola) throughout the development of a songbird (Parus major). In the first experiment, we exposed bird clutches at hatching to a mix of the 3 tick instars (larvae, nymphs and adults), and monitored the ticks that attached in relation to the average broods' age. In a complementary experiment we focused on the attachment in adult female ticks--the largest and most significant instar for the species' reproduction--after releasing them at different moments in the nestlings' development. Our observations revealed a positive association between the size of the attached instar and the broods' age. Particularly, adult females were less likely to be found attached to recently hatched nestlings, which contrasts with the smaller-sized larvae and nymphs. These differences suggest either an infestation strategy that is adapted to host physiology and development, or a result of selection by the hosts' anti-tick resistance mechanisms. We discuss the implications of our results in terms of tick life-history strategies.     

Intercalation of daunomycin into d(CG)4 oligomer duplex containing G x T mismatches by vibrational circular dichroism and infrared absorption spectroscopy

The vibrational circular dichroism (VCD) and infrared absorption (IR) spectra of the mismatched octamer oligonucleotides d(CGTGCGCG)(2) (CGT) and d(CGCGTGCG)(2) (CGC) and their complexes with the antitumor drug daunomycin were measured in D(2)O, interpreted, and compared to the octamer d(CGCGCGCG)(2) (CG). The IR spectra of the mismatched octamers in the carbonyl-stretching region are similar to those of the parent CG, whereas the VCD spectra differ in several respects between each other. The main VCD feature due to carbonyl stretching is informative for the mismatches and CG. Vibrational modes in the sugar-phosphate region remain essentially unchanged especially for PO(2) (-) symmetric stretching. Differences between the free and complexed mismatch octamers occurred mainly in the carbonyl-stretching region (1,700-1,600 cm(-1)). The absorption intensity of the C==O peak of G is more prominent for CGC than CGT and resembles CG in this respect. The detailed composition of this doublet is clearly visible, indicating the geometric rearrangement of the base pairs in the presence of the mismatch and upon forming the daunomycin complex.