Three-dimensional ultrastructure of a unicellular cyanobacterium.

The first complete three-dimensional ultrastructural reconstruction of a cyanobacterium was accomplished with high-voltage electron microscopy and computer-aided assembly of serial sections. The precise arrangement of subcellular features within the cell body was very consistent from one cell to another. Specialized inclusion bodies always occupied specific intracellular locations. The photosynthetic thylakoid membranes entirely surrounded the central portion of the cytoplasm, thereby compartmentalizing it from the rest of the cell. The thylakoid membranes formed an interconnecting network of concentric shells, merging only at the inner surface of the cytoplasmic membrane. The thylakoids were in contact with the cytoplasmic membrane at several locations, apparently to maintain the overall configuration of the thylakoid system. These results clarified several unresolved issues regarding structure-function relationships in cyanobacteria.

Expression of the Escherichia coli lacZ gene on a plasmid vector in a cyanobacterium.

A biphasic plasmid vector was used to introduce the Escherichia coli K-12 lac operon into the unicellular cyanobacterium Agmenellum quadruplicatum PR-6. The PR-6 transformants expressed beta-galactosidase at nearly as high a level as did Escherichia coli transformants. In order to accomplish this, it was necessary to obtain PR-6 mutants that could be transformed by plasmids with unmodified recognition sites for the endogenous PR-6 restriction endonuclease Aqu I. These mutants were generated by a variation of the ectopic mutagenesis techniques that have been used in other naturally transforming bacteria. The ability to assay the expression of lacZ in PR-6 paves the way for the construction of gene fusions with various PR-6 promoters and quantitation of their expression under specific in vivo conditions.

Purification and characterization of the C-phycocyanin from Agmenellum quadruplicatum.

The C-phycocyanin from the marine blue-green alga, Agmenellum quadruplicatum, has been isolated and purified to electrophoretic homogeneity. This is the first C-phycocyanin for which a low resolution three dimensional structure has been published (Hackert, M.L., Abad-Zapatero, C., Stevens, S.E. Jr. and Fox, J.L. (1977), J. Mol. Biol. 111, 365-369 and Abad-Zapatero, C., Fox, J.L. and Hackert, M.L. (1977) Biochem. Biophys. Res Commun. 78, 266-272). The native C-phycocyanin complex shows an absorption maximum at 622 nm and another peak at 355 nm. In urea solutions, the 622 nm maximum of whole C-phycocyanin is shifted to 662 nm. Am662 = 94400 was determined. The fluorescence emission maxima at 650 nm for haloprotein is shifted and largely quenched in acid urea. The monomeric protein consists of two polypeptide chains with molecular weights of 16,000 for the alpha chain and 18,500 for the beta chain. Spectra in 8 M urea indicate that the alpha chain possesses one and the beta chain two phycocyanobilin chromophores. The isolated chains show absorption maxima at 622 nm for the alpha chain and 608 nm for the beta chain. Amino acid compositions of the holoprotein and the separated chains are given and N-terminal amino acid sequences are presented.

Genetic analysis of a 9 kDa phycocyanin-associated linker polypeptide.

The gene encoding LR9, a 9 kDa phycocyanin-associated linker polypeptide, was cloned from the cyanobacterium Synechococcus sp. PCC 7002 (Agmenellum quadruplicatum PR-6). This gene, termed cpcD, was located immediately 3' to cpcC, a gene which encodes another phycocyanin-associated linker, LR33. Mutation of cpcD by insertion led to the loss of LR9 as the only detectable change in phycobilisome composition. Cells and isolated phycobilisomes from the cpcD- strain did not detectably differ from the wild-type in absorption or steady-state fluorescence emission. Purified phycobilisomes from the wild-type and cpcD- strains were compared by electron microscopy. The number of phycocyanin discs in the rod substructures of the mutant was more variable than in the wild-type. Hence, one function of LR9 may be to minimize the heterogeneity of rod length, possibly by binding to the core-distal face of phycocyanin-LR33 complexes to prevent the tandem joining of such units. A mutant in which cpcD and cpcC-cpcD intergenic sequences are deleted shows a partial loss of LR33. Inverted repeats in this intergenic region may be required for optimal stability of the cpcC transcript.

Genes from the cyanobacterium Agmenellum quadruplicatum isolated by complementation: characterization and production of merodiploids.

The isolation of several biosynthetic genes from a cyanobacterium, Agmenellum quadruplicatum, by complementation of auxotrophic mutations in Escherichia coli, and their partial characterization, is described. Although our search for such genes has not been exhaustive, it appears that complementation of E. coli mutations may be of limited utility for the identification and/or isolation of cyanobacterial genes. Despite some overlap in the complementation abilities of these isolated cyanobacterial DNA fragments, the genes that we have studied in some detail are not located in operons. We have used mutagenized versions of these cyanobacterial DNA fragments to produce mutant phenotypes in the cyanobacterium, but clean auxotrophs were not obtained. Complementation of these mutant phenotypes can be obtained when the appropriate wild-type DNA fragment is introduced into the cyanobacterium on a shuttle vector. Recombination between two copies of a cyanobacterial gene occurs at high frequency in the cyanobacterium.

Isolation of full-length RNA from a thermophilic cyanobacterium.

Isolation of full-length mRNA without degradation is critical in the study of in vivo gene regulation and transcription, cDNA synthesis and reverse transcription (RT)-PCR. It is particularly difficult to isolate full-length mRNA from thermophiles, which have higher turnover rates of mRNA degradation. Mastigocladus laminosus is a thermophilic heterocystous cyanobacterium. The assay of M. laminosus cell lysates showed that RNase activity was high and was resistant to the conventional guanidine thiocyanate and 2-mercaptoethanol denaturation methods. The mRNA isolated by several conventional methods was completely degraded. A method was developed to purify full-length mRNA by a combination of fast cooling, vanadyl-ribonucleoside-complex inhibition, phenol-chloroform-isoamyl alcohol extraction, lithium chloride precipitation and the lysing of cells with the French Press. This method produced high-quality, full-length mRNA in high yield. Purified mRNA was suitable for Northern blotting, cDNA synthesis and RT-PCR. This method could be applicable to other thermophiles in which the RNase activity is high and/or is resistant to guanidine thiocyanate.

Effects of the nitro-group on the mutagenicity and toxicity of some benzamines.

The Ames Salmonella/microsomal assay was employed to test the mutagenicity of some benzamines (aniline, and o- and p-phenylenediamine) and their nitro-derivatives (p-nitroaniline, 2-nitro-p-phenylenediamine, 3- and 4-nitro-o-phenylenediamine), using strains TA98 and TA100 and their nitroreductase-deficient mutants, TA98NR and TA100NR, in the presence and absence of rat S9 mix. The addition of the nitro-group to benzamine molecules converted them into direct mutagens. Furthermore, the position of the nitro-group affected their mutagenic activities. Cytotoxicity testing with Chinese hamster ovary cells (CHO-K1) showed that the presence of the nitro-group in these compounds had no specific effect on toxicity. The test compounds all showed a dose-related increase in inducing chromosomal aberrations in CHO cells. However, the presence of the nitro-group did not affect potency in inducing chromosomal aberrations. Compounds containing the nitro-group had higher initial oxidation potentials and dipole moments (mu) than their nonnitro-containing counterparts. The mutagenicity and toxicity of these compounds were not related to physico-chemical properties, including oxidation potential, energy difference (deltaE) between the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO), ionization potential (I.P.), and mu.

Mutagenicity and toxicity studies of p-phenylenediamine and its derivatives.

The mutagenicity of p-phenylenediamine and its derivatives was tested using Ames Salmonella strains TA98 and TA100. p-Phenylenediamine was weakly mutagenic to TA98 with metabolic activation. 2-Nitro-p-phenylenediamine was directly mutagenic to both strains, while 2-methyl-p-phenylenediamine required S9 mix. All the test compounds induced a dose-related increase in chromosomal aberrations in Chinese hamster ovary (CHO) cells in the absence of the S9 mix. The mutagenicity and toxicity of these compounds did not correlate with their oxidation potentials, or any other tested physicochemical properties including the energy difference between the lowest unoccupied and the highest occupied molecular orbital, ionization potential, and dipole moment.

The effects of interpersonal and personal agency on perceived control and psychological well-being in adulthood.

A theoretical model that links social support with global beliefs in primary control and provides a developmental perspective on how normative age-related changes alter control beliefs was examined with data from 482 adults aged 18 to 93. Generalized belief in primary control was hypothesized to have a direct positive effect on psychological well-being and to arise from two distinct sources: (a) interpersonal agency (obtaining positive ends through interactions with others) and (b) personal agency (achieving desired outcomes on one's own behalf). Age was believed to affect both types of agency indirectly as a result of age-related changes in physical health and emotional support. Although physical health was presumed to have a direct positive effect on psychological well-being, the effect of emotional support on well-being was mediated by interpersonal agency and perceived primary control. Structural equation modeling analyses with the EQS 5.4 program revealed good model fit (goodness-of-fit index = .98, comparative fit index = .94, root mean square residual [RMR] = .02, standardized RMR = .05, root mean square error of approximation = .06) after a negative direct path from age to generalized beliefs in primary control was added to the a priori model.

Adolescents with physical disabilities: some psychosocial aspects of health.

PURPOSE: To examine the psychosocial issues related to growing up with a physical disability. METHODS: Adolescents with physical disabilities aged 11-16 years were compared with a Canadian national sample of adolescents using the Health Behaviours in School-Aged Children (HBSC), a World Health Organization Cross-National Study survey. RESULTS: Adolescents with physical disabilities reported good self-esteem, strong family relationships, and as many close friends as adolescents in the national sample. However, adolescents with physical disabilities participated in fewer social activities and had less intimate relationships with their friends. They had more positive attitudes toward school, teachers, and their fellow classmates than the national sample, but fewer had plans for postsecondary education. The majority of adolescents with physical disabilities reported that they had not received information on parenthood, birth control, and sexually transmitted diseases. CONCLUSIONS: There are a number of critical areas of risk for adolescents with physical disabilities to which health promotion efforts should be directed. These include lower levels of peer integration, heightened adult orientation, low educational aspirations, and poor knowledge of sexuality.

Telomere length is shorter in healthy offspring of subjects with coronary artery disease: support for the telomere hypothesis.

BACKGROUND: Telomeres are shorter in subjects with coronary artery disease (CAD) and may indicate premature biological ageing. However, whether shorter telomeres are a primary abnormality or secondary to the disease is unclear. OBJECTIVE: To investigate whether shorter telomeres are a primary abnormality or secondary to CAD, telomere lengths in healthy young adults with contrasting familial risk of CAD were compared. DESIGN: Case-control study. METHODS: Mean telomere restriction fragment (TRF) length in DNA from circulating leucocytes was determined by Southern blotting in 45 healthy offspring of subjects with premature CAD (case offspring) and 59 offspring from families without such a history (control offspring). Correlation in mean TRF length was also assessed in 67 offspring-parent pairs. RESULTS: On average, a decrease of 27.5 (10.7) bp in mean TRF per year of age was found. The unadjusted mean TRF length was 6.34 kb (95% CI 6.13 to 6.55) for case offspring and 6.75 kb (95% CI 6.57 to 6.94) for offspring of controls (p = 0.004). The adjusted difference in mean TRF between case and control offspring was 472 bp (95% CI 253 to 691, p<0.001), equivalent to about 17 years of age-related attrition in telomere length. Furthermore, there was a significant positive correlation in mean TRF length between offspring and their parents (r = 0.37, p = 0.002). CONCLUSION: These findings suggest that inheritance of shorter telomeres is associated with increased familial risk of CAD. They support the hypothesis that telomere length is a primary abnormality involved in the pathogenesis of CAD.

Significance of braided trichomes in the cyanobacterium Mastigocladus laminosus.

Loops and braids in filaments of the cyanobacterium Mastigocladus laminosus were observed. Braided filaments were generally in the form of a right-handed helix (87%) but were occasionally observed as left-handed helices (13%). It was demonstrated by time-lapse photomicrography that braids continued to grow as braids and that loops coiled into braids as growth proceeded. Measurements of the distance between grooves in 74 braids yielded an average distance of 13 +/- 3 micron, a result which suggests that braid formation is not random. We propose that the braids arise as a consequence of the helical growth of cells that make up the filaments of M. laminosus.

Multiple parameters for the comprehensive evaluation of the susceptibility of Escherichia coli to the silver ion.

The susceptibility of Escherichia coli B to the antibacterial activity of silver ions was measured in terms of the initial inhibitory concentration, complete inhibitory concentration, postagent effect for bacteriostatic susceptibility, minimum bactericidal concentration, maximum tolerant concentration, and log killing time for bactericidal activity. At a concentration of 9.45 microM and an inoculum size of 10(4-5) CFU ml-1, silver caused growth delay of E. coli; at a concentration of 18.90 microM, silver completely inhibited bacterial growth. Prolonged postagent effects ranged between 1.5 and 12 h at 0.75 x the initial inhibitory concentration, 1.0 x the initial inhibitory concentration, and 1.5 x the initial inhibitory concentration of the silver ion. One log-unit of viable bacterial population size was lost every 30 min at the minimum bactericidal concentration of the silver ion. Silver tolerance was determined as 20 times the initial inhibitory concentration with 48 h of exposure. This study presents an evaluative model as a reference for the quantitative analysis of the susceptibility of bacteria to silver ions.

Transformation in Agmenellum quadruplicatum.

A DNA-mediated transformation system for the blue-green alga Agmenellum quadruplicatum, strain PR-6, is described and characterized for DNA concentration dependence, dependence on time of exposure to DNA, phenotypic expression, sensitivity to various enzymes, and competence. The stability of the transformants has been investigated, and genetic backcross and selfing experiments have been performed. This system fulfills all of the criteria established for the well-characterized transformation systems in heterotrophic bacteria and demonstrates significant similarities to at least one of these systems for all characteristics examined. The efficiency of transformation is high. This system fills a need for a well-characterized genetic system in an oxygen-evolving photoautotroph. We have used it to transform a strain with a mutational lesion in assimilatory nitrogen metabolism to a wild-type genotype.

Cloning and expression of the cryIVD gene of Bacillus thuringiensis subsp. israelensis in the cyanobacterium Agmenellum quadruplicatum PR-6 and its resulting larvicidal activity.

A mosquitocidal cyanobacterium has been developed by introducing the mosquito-toxic cryIVD gene from Bacillus thuringiensis subsp. israelensis into the unicellular cyanobacterium Agmenellum quadruplicatum PR-6 (Synechococcus sp. strain PCC 7002). The cryIVD gene was introduced into the cyanobacterium on a derivative of the PR-6 expression vector pAQE19 delta Sal in which the cryIVD gene was translationally fused to the initial coding sequence of the highly expressed PR-6 cpcB gene. Coomassie blue staining and immunoblot analysis of gel-fractionated cell extract polypeptides indicate that the cpcB-cryIVD gene fusion is expressed at high levels in the cyanobacterial cells, with little or no apparent degradation of the cryIVD gene product. Larvicidal assays revealed that freshly hatched Culex pipiens mosquito larvae readily ingested the transformed cyanobacteria and that the cells proved to be toxic to the larvae.