The role of Mg2+ in the hydrolytic activity of the isolated chloroplast ATPase: Study by high-performance liquid chromatography

The influences of total magnesium ion concentration at different total ATP concentrations, and of total ATP concentration, for different total magnesium ion concentrations, on the enzymatic rate of the isolated chloroplast F₁ ATPase, have been followed by a chromatographic method consisting in the separation and determination of ADP. From the various series of curves, it is concluded that the experimental results (position of the maxima,K _m values) are better fitted by a mechanism involving the activation of the enzyme by magnesium ion and hydrolysis of free ATP, rather than by the classical mechanism, for which the enzyme hydrolyzes the MgATP complex and is inhibited by Mg²⁺. Although the equations giving the reaction rate are similar in the two cases, the calculated values ofK _m are widely different. The value obtained from the classical mechanism does not agree withK _D , the dissociation constant of the enzyme-substrate complex, measured by the Hummel and Dreyer method. Moreover, when the total ATP concentration tends toward the total magnesium ion concentration, the nucleotide binding to the enzyme tends toward zero, although it should be maximum if MgATP were the true substrate. Finally, the inhibitory effect of Na⁺ is more easily explained as a competition between this ion and the activating Mg²⁺, than by the classical mechanism.     

Structure and function of the photosynthetic reaction center fromRhodobacter sphaeroides

The three-dimensional structure of the photosynthetic reaction center fromRhodobacter sphaeroides is described. The reaction center is a transmembrane protein that converts light into chemical energy. The protein has three subunits: L, M, and H. The mostly helical L and M subunits provide the scaffolding and the finely tuned environment in which the chromophores carry out electron transfer. The details of the protein-chromophore interactions are from studies of a trigonal crystal form that diffracted to 2.65-Å resolution. Functional studies of the multi-subunit complex by site-specific replacement of key amino acid residues are summarized in the context of the molecular structure.This work was supported in part by the U.S. Department of Energy, Office of Health and Environmental Research, under Contract No. W-31-109-ENG-38 and by Public Health Service Grant GM36598.     

Phylogenetic relationships of the garter snakes based on DNA sequence and allozyme variation

We estimated phylogenetic relationships among 26 species of garter snakes (genus Thamnophis ) using allozyme and mitochondrial cytochrome b gene nucleotide sequence variation. Parsimony analyses of the two data sets give substantially different estimates of phylogeny. Several lines of evidence indicate that much of this conflict is due to error associated with the restricted number of characters in each data set. Such sampling error may be reduced by combining all the characters; we therefore present an estimate of phylogeny based on parsimony analysis of all the data combined. All our analyses support several conclusions in conflict with previous views: a very distant relationship between T.errans and T. elegans , non-monophyly of the elegans group (even excluding T: errans ), and nesting of the form validus (previously considered a member of the genus Nerodia ) within Thamnophis.
The combined analysis gives an almost fully resolved tree. However, bootstrapping indicates only weak support for many clades in this tree. Furthermore, paraphyly of the assemblages of cytochrome b gene lineages within T. elegans and T. radix indicate the potential for discordance between the mitochondrial DNA (mtDNA) and species phylogenies through the sorting of ancestral mtDNA polymorphisms. These problems suggest the need for assaying additional characters, especially ones likely to be independent of those used in the present study.     

Monitoring the persistence of Laccaria bicolor as an ectomycorrhizal symbiont of nursery-grown Douglas fir by PCR of the rDNA intergenic spacer

The large-scale inoculation of selected beneficial ectomycorrhizal fungi in forest nurseries has generated renewed interest in the ecology of these symbiotic fungi. However, information on the dissemination and persistence of introduced symbionts is scarce due to the limitation of the current identification methods. To identify ectomycorrhizal fungi on single root tips, we investigated the polymorphism of the PCR-amplified ribosomal DNA intergenic spacer (IGS) from a wide range of ectomycorrhizal fungi. To investigate the reliability of this molecular approach in large-scale surveys, the dissemination and persistence on Douglas fir seedlings of the introduced Laccaria bicolor S238N were assessed in a forest nursery in the Massif Central (France). Several hundred ectomycorrhizas and fruiting bodies were sampled from plots where control and L. bicolor inoculated-Douglas fir seedlings were grown for 1.5 years. PCR typing of mycorrhizas indicated that trees inoculated with L. bicolor S238N remained exclusively colonized by that isolate (or sexually derived isolates) for the entire test period. In contrast, control seedlings were infected by indigenous isolates of Laccaria laccata and Thelephora terrestris. The molecular evidence for the persistence of the introduced mycobiont despite the competition from indigenous isolates of the same species provides further illustration of the potential of exotic species for large-scale microbial application.     

Recruitment of helper T cells for induction of tumour rejection by cytolytic T lymphocytes

Immunotherapy of cancer could be possible in cases in which competent effector T cells can be induced. Such an approach depends on expression of tumour-specific antigens by the tumour cells and on the availability of sufficient costimulatory support for activation of cytotoxic T lymphocytes. Here, a strategy for helper T cell recruitment for induction of tumour-specific cytotoxic immune responses is presented. Allogenic MHC class II molecules were introduced into tumour cells by cell fusion. These hybrid cells, when injected into mice, induced rejection of an established tumour. The contribution of CD4-expressing helper T cells in the induction phase and of CD8-expressing T cells in the effector phase of the immune response was demonstrated. The approach described could be applicable to cases in which a suitable tumour antigen is present but not identified; it employs regulatory interactions that govern physiological immune responses and is designed to be minimally invasive.     

Analysis of a primer-independent GTF-I from Streptococcus salivarius

Abstract A glucosyltransferase (GTF) gene, designated gtfL , from Streptococcus salivarius was cloned and expressed in Escherichia coli and its nucleotide sequence determined. The GTF-L enzyme catalysed the synthesis of water-insoluble glucan in a primer-independent manner. The nucleotide sequence and derived amino acid sequence of GTF-L were similar in size and domain structure to previously sequenced glucosyltransferases. However, a 464-bp region of high variability was identified which could be selectively amplified from strains of S. salivarius by the polymerase chain reaction and could therefore form the basis for species identification. No sequence-specific motifs related to the solubility and linkage of the glucan product or its need for a dextran primer could be ascertained.     

Evaluation of the Sensitivity and Specificity of Polymerase Chain Reaction Test Kit,AMPLICOR Chlamydia trachomatis

The sensitivity and specificity of the polymerase chain reaction (PCR) test kit, AMPLICOR Chlamydia trachomatis, were examined by the use of purified elementary bodies (EBs), cells having inclusions containing reticulate bodies alone and 20 clinical isolates. The numbers of EB and inclusion of C. trachomatis at the detection limit were determined to be approximately 2 to 4 EBs and one inclusion per assay, respectively. No reaction occurred for C. psittaci and C. pneumoniae. All clinical isolates were positively reacted in the PCR assay.