Naturally occurring peptidoglycan variants of Streptococcus pneumoniae.

Analysis by high-performance liquid chromatography of the stem peptide composition of cell walls purified from a large number of pneumococcal strains indicates that these bacteria produce a highly conserved species-specific peptidoglycan independent of serotype, isolation date, and geographic origin. Characteristic features of this highly reproducible peptide pattern are the dominance of linear stem peptides with a monomeric tripeptide, a tri-tetra linear dimer, and two indirectly cross-linked tri-tetra dimers being the most abundant components. Screening of strains with the high-performance liquid chromatography technique has identified two naturally occurring peptidoglycan variants in which the species-specific stem peptide composition was replaced by two drastically different and distinct stem peptide patterns, each unique to the particular clone of pneumococci producing it. Both isolates were multidrug resistant, including resistance to penicillin. In one of these clones--defined by multilocus enzyme analysis and pulsed-field gel electrophoresis of the chromosomal DNAs--the linear stem peptides were replaced by branched peptides that most frequently carried an alanyl-alanine substituent on the epsilon amino group of the diamino acid residue. In the second clone, the predominant stem peptide species replacing the linear stem peptides carried a seryl-alanine substituent. The abnormal peptidoglycans may be related to the altered substrate preference of transpeptidases (penicillin-binding proteins) in the pneumococcal variants.     

Separation of abnormal cell wall composition from penicillin resistance through genetic transformation of Streptococcus pneumoniae.

Compared with most penicillin-susceptible isolates of Streptococcus pneumoniae, penicillin-resistant clinical isolate Hun 663 contains mosaic penicillin-binding protein (PBP) genes encoding PBPs with reduced penicillin affinities, anomalous molecular sizes, and also cell walls of unusual chemical composition. Chromosomal DNA prepared from Hun 663 was used to transform susceptible recipient cells to donor level penicillin resistance, and a resistant transformant was used next as the source of DNA in the construction of a second round of penicillin-resistant transformants. The greatly reduced penicillin affinity of the high-molecular-weight PBPs was retained in all transformants through both genetic crosses. On the other hand, PBP pattern and abnormal cell wall composition, both of which are stable, clone-specific properties of strain Hun 663, were changed: individual transformants showed a variety of new, abnormal PBP patterns. Furthermore, while the composition of cell walls resembled that of the DNA donor in the first-round transformants, it became virtually identical to that of susceptible pneumococci in the second-round transformants. The findings indicate that genetic elements encoding the low affinity of PBPs and the penicillin resistance of the bacteria are separable from determinants that are responsible for the abnormal cell wall composition that often accompanies penicillin resistance in clinical strains of pneumococci.     

Small-spored Alternaria spp. (section Alternaria) are common pathogens on wild tomato species

The wild relatives of modern tomato crops are native to South America. These plants occur in habitats as different as the Andes and the Atacama Desert and are, to some degree, all susceptible to fungal pathogens of the genus Alternaria. Alternaria is a large genus. On tomatoes, several species cause early blight, leaf spots and other diseases. We collected Alternaria-like infection lesions from the leaves of eight wild tomato species from Chile and Peru. Using molecular barcoding markers, we characterized the pathogens. The infection lesions were caused predominantly by small-spored species of Alternaria of the section Alternaria, like A. alternata, but also by Stemphylium spp., Alternaria spp. from the section Ulocladioides and other related species. Morphological observations and an infection assay confirmed this. Comparative genetic diversity analyses show a larger diversity in this wild system than in studies of cultivated Solanum species. As A. alternata has been reported to be an increasing problem in cultivated tomatoes, investigating the evolutionary potential of this pathogen is not only interesting to scientists studying wild plant pathosystems. It could also inform crop protection and breeding programs to be aware of potential epidemics caused by species still confined to South America.     

Electron microprobe analysis of juvenile walleye pollock,Theragra chalcogramma,otoliths from Alaska: a pilot stock separation study

Synopsis The incorporation of dissolved oceanic constituents in the otoliths of fish has potential as a chemical tracer for reconstructing the early life history of marine fish. Wavelength dispersive spectrometers on an electron microprobe were used to measure Na, Mg, P, S, Cl, K, Ca, and Sr concentrations on the outer margins of 57 juvenile walleye pollock, Theragra chalcogramma, otoliths from five locations in the Gulf of Alaska and Bering Sea. Discriminant analyses that used various combinations of Na, P, K, Sr, and fish standard length and/or age showed that 60–80% of the samples could be assigned to the correct capture locality. While the concentrations of some of the measured elements correlated with standard length or age of the fish, there are measurable differences among localities when concentrations are length or age corrected, mainly due to differences in Na and K concentrations. Elemental composition of otoliths potentially could be used to assign fish from a mixed stock fishery to original stocks, information that is greatly needed for the effective management of fish stocks.     

Cyclic AMP-dependent regulation of activities of synthetase and phosphodiesterase of 2′,5′-oligoadenylate in NIH 3T3 cells

Summary Dihydrofolate synthetase (EC 6.3.2.12) from N. gonorrhoeae was isolated and enzyme characteristics were determined. The purified enzyme was found quite stable when stored at –60 °C. About 50% of the enzyme activity wag destroyed within 6 weeks when kept at 4 °C. Maximum velocity was observed at pH 9.3. The enzyme required a monovalent cation, K⁺ or NH₄ ⁺ , and divalent cation, Mg²⁺ or Mn²⁺ for its function. ATP at 5 mM concentration gave maximum activity. K_m values for dihydropteroate and L-glutamate at pH 9.3 were 3.5 × 10^–5 M and 6.5 × 10^–4 M, respectively. Patterns of product inhibition by dihydrofolate were found to be non-competitive with respect to dihydropteroate, having a K_i value of 5.1 ± 0.8 × 10^–4 M, and competitive with respect to L-glutamate, having a K_i value of 6.2 × 10^–4 M.     

Histone H1--DNA interaction. On the mechanism of DNA strands crosslinking by histone H1.

Crosslinking of DNA fibers by histone H1 or phosphorylated on Ser-37 histone H1, and by the individual fragments of the H1 polypeptide chain was studied by the method of turbidimetry. The dependence of the turbidity of DNA-protein complexes on the ionic strength in solution suggests that the condensation of H1.DNA complexes in vitro is apparently due to both specific histone-DNA interactions with the contribution of hydrogen and/or hydrophobic bonds and the formation of polycationic "bridges" fastening the DNA fibers. The effectiveness of the condensation is postulated to be a function of a proportion between the two mechanisms which in turn can be controlled by slight changes in ionic surroundings. The sharp dependence of shrinkage of H1.DNA complexes on ionic strength at "physiological" salt concentrations could provide a mechanism to regulate density and consequently the total activity of chromatin in the cell nuclei. The phosphorylation of histone H1 on Ser-37 by a specific histone kinase does not noticeably affect the pattern of DNA crosslinking by the H1.     

Mechanism of action of cyclic AMP-dependent histone kinase. Substrate specificity of the catalytic enzyme unit]

Interaction of several nucleotide derivates with homogenous catalytic subunit of cyclo-AMP-dependent histone kinase from pig brain is studied. Inhibition constants of these compounds are calculated, and the affinity of inhibitors to the enzyme active site is evaluated. The nature of heterocyclic base is found to be the main contribution into binding with substrate. The enzyme specificity with respect to a number of bivalent metal ions is studied, and Mg2+ is demonstrated to be the only efficient enzyme activator. It is shown by means of stationary kinetics that histone kinase-catalysed phosphotransferase reaction has a "ping-pong"-like mechanism.     

Isolation and some properties of troponin T kinase from rabbit skeletal muscle.

A method for isolation of troponin T kinase (ATP-protein phosphotransferase, EC 2.7.1.37) from rabbit skeletal muscles in proposed. The method gives a 7000-10 000-fold purification and results in an enzyme with specific activity of 400-800-nmol x min-1 x mg-1 of protein. The molecular weight of tropin T kinase as determined by gel filtration exceeds 500 000. Electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulphate revealed that isolated preparations of the enzyme consisted of at least three distinct proteins with apparent mol.wt. of 50 000, 46 000 and 31 000. The enzyme phosphorylates isolated troponin T at a rate which exceeds the phosphorylation rates of casein, phosvitin, histones, phosphorylase b and protamine 5-30-fold. Within the whole troponin complex, only troponin T is phosphorylated by the enzyme. The enzyme phosphorylates only the N-terminal serine residue of troponin T, i.e. the site that is normally phosphorylated in the whole troponin complex isolated from rabbit skeletal muscles.