Dimerization and inter-chromophore distance of Cph1 phytochrome from Synechocystis,as monitored by fluorescence homo and hetero energy transfer

We investigated the dimerization of phytochrome Cph1 from the cyanobacterium Synechocystis by fluorescence resonance energy transfer (FRET). As donor we used the chromophore analogue phycoerythrobilin (PEB) and as acceptor either the natural chromophore phycocyanobilin (PCB; hetero transfer) or PEB (homo transfer). Both chromophores bind in a 1:1 stoichiometry to apo-monomers expressed in Escherichia coli. Energy transfer was characterized by time-resolved fluorescence intensity and anisotropy decay after excitation of PEB by picosecond pulses from a tunable Ti-sapphire laser system. ApoCph1 was first assembled with PEB at a low stoichiometry of 0.1. The remaining sites were then sequentially titrated with PCB. In the course of this titration, the mean lifetime of PEB decreased from 3.33 to 1.25 ns in the P(r) form of Cph1, whereas the anisotropy decay was unaffected. In the P(fr)/P(r) photoequilibrium (about 65% P(fr)), the mean lifetime decreased significantly less, to 1.67 ns. These observations provide strong support for inter-chromophore hetero energy transfer in mixed PEB/PCB dimers. The reduced energy transfer in P(fr) may be due to a structural difference but is at least in part due to the difference in spectral overlap, which was 4.1 x 10(-13) and 1.6 x 10(-13) cm(3) M(-1) in P(r) and P(fr), respectively. From the changes in the mean lifetime, rates of hetero energy transfer of 0.68 and 0.37 ns(-1) were calculated for the P(r) form and the P(fr)/P(r) photoequilibrium, respectively. Sequential titration of apo Cph1 with PEB alone to full occupancy did not affect the intensity decay but led to a substantial increase in depolarization. This is the experimental signature of homo energy transfer. Values for the rate of energy transfer k(HT) (0.47 ns(-1)) and the angle 2theta between the transition dipole moment directions (2theta = 45 +/- 5 degrees) were determined from an analysis of the concentration dependence of the anisotropy at five different PEB/Cph1 stoichiometries. The independently determined rates of hetero and homo energy transfer are thus of comparable magnitude and consistent with the energy transfer interpretation. Using these results and exploiting the 2-fold symmetry of the dimer, the chromophore-chromophore distance R(DA) was calculated and found to be in the range 49 A < R(DA) < 63 A. Further evidence for energy transfer in Cph1 dimers was obtained from dilution experiments with PEB/PEB dimers: the lifetime was unchanged, but the anisotropy increased as the dimers dissociated with increasing dilution. These experiments allowed a rough estimate of 5 +/- 3 microM for the dimer dissociation constant. With the deletion mutant Cph1Delta2 that lacks the carboxy terminal histidine kinase domain less energy transfer was observed suggesting that in this mutant dimerization is much weaker. The carboxy terminal domain of Cph1 that is involved in intersubunit trans-phosphorylation and signal transduction thus plays a dominant role in the dimerization. The FRET method provides a sensitive assay to monitor the association of Cph1 monomers.     

Impacts of avidity and specificity on the antiviral efficiency of HIV-1-specific CTL

Although CD8(+) CTLs are presumed to be an important mediator of protective immunity in HIV-1 infection, the factors that determine CTL antiviral efficiency are poorly understood. Two factors that have been proposed to influence CTL antiviral function are antigenic avidity and epitope specificity. In this study we evaluate these by examining the activity of HIV-1-specific CTL against acutely infected cells. The ability of CTL to kill infected cells is variable and depends more on epitope specificity than functional avidity within the range for the tested clones (50% of maximal killing, 50 pg/ml to 100 ng/ml); killing efficiency is similar for different clones recognizing the same epitope, despite their variation in avidity. When CTL clones are tested for their ability to suppress viral replication, similar results are observed. Inhibition is more dependent on epitope specificity than functional avidity among the tested clones (50% of maximal killing, 20 pg/ml to 20 ng/ml). Thus, CTL specificity can be an overriding factor in the ability of CTL to interact with HIV-1-infected cells, indicating that factors determining the process of epitope presentation on infected cells have a key influence on CTL efficiency. These results suggest that CTL specificity may have a pivotal role in the immunopathogenesis of infection, and that simple quantitative measures of CTL may be insufficient indicators of the CTL response to HIV-1.     

GM-CSF restores innate,but not adaptive,immune responses in glucocorticoid-immunosuppressed human blood in vitro

Infection remains the major complication of immunosuppressive therapy in organ transplantation. Therefore, reconstitution of the innate immunity against infections, without activation of the adaptive immune responses, to prevent graft rejection is a clinically desirable status in transplant recipients. We found that GM-CSF restored TNF mRNA and protein expression without inducing IL-2 production and T cell proliferation in glucocorticoid-immunosuppressed blood from either healthy donors or liver transplant patients. Gene array experiments indicated that GM-CSF selectively restored a variety of dexamethasone-suppressed, LPS-inducible genes relevant for innate immunity. A possible explanation for the lack of GM-CSF to restore T cell proliferation is its enhancement of the release of IL-1betaR antagonist, rather than of IL-1beta itself, since exogenously added IL-1beta induced an IL-2-independent Con A-stimulated proliferation of glucocorticoid-immunosuppressed lymphocytes. Finally, to test the in vivo relevance of our findings, we showed that GM-CSF restored the survival of dexamethasone- or cyclosporine A-immunosuppressed mice from an otherwise lethal infection with Salmonella typhimurium. In addition to this increased resistance to infection, GM-CSF did not induce graft rejection of a skin allotransplant in cyclosporine A-immunosuppressed mice. The selective restoration potential of GM-CSF suggests its therapeutic use in improving the resistance against infections upon organ transplantation.     

Diversity of microbial communities correlated to physiochemical parameters in a digestion basin of a zero-discharge mariculture system

Bacterial community structure and physiochemical parameters were examined in a sedimentation basin of a zero-discharge mariculture system. The system consisted of an intensively stocked fish basin from which water was recirculated through two separate treatment loops. Surface water from the basin was pumped over a trickling filter in one loop while bottom-water was recirculated through a sedimentation basin followed by a fluidized bed reactor in the other. Ammonia oxidation to nitrate in the trickling filter and organic matter digestion together with nitrate reduction in the sedimentation basin and fluidized bed reactor, allowed zero-discharge operation of the system. Relatively high concentrations of oxygen, nitrate, sulphate and organic matter detected simultaneously in the digestion basin suggested the potential for a wide range of microbially-mediated transformation processes. In this study, physiochemical parameters were correlated to bacterial diversity and distribution in horizontal and vertical profiles within this basin in an effort to obtain a basic understanding of the chemical and microbial processes in this system. Chemical activity and microbial diversity, the latter measured by denaturing gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction (PCR) amplified 16S rDNA fragments, were higher in the sludge layer than in the overlying aqueous layer of the basin. Chemical parameters in sludge samples close to the basin inlet suggested enhanced microbial activity relative to other sampling areas with evidence of both nitrate and sulphate reduction. Four of the nine DGGE bands identified in this zone were affiliated with the Bacteroidetes phylum. Detected sequences closely related to sequences of organisms involved in the sulphur cycle included Desulfovibrio, Dethiosulfovibrio and apparent sulphur oxidizers from the gamma-proteobacteria. In addition, a number of sequences from the beta and alpha-proteobacteria were identified.     

Duplication is more common among laterally transferred genes than among indigenous genes

Background

Recent developments in the understanding of paralogous evolution have prompted a focus not only on obviously advantageous genes, but also on genes that can be considered to have a weak or sporadic impact on the survival of the organism. Here we examine the duplicative behavior of a category of genes that can be considered to be mostly transient in the genome, namely laterally transferred genes. Using both a compositional method and a gene-tree approach, we identify a number of proposed laterally transferred genes and study their nucleotide composition and frequency of duplication.

Results

It is found that duplications are significantly overrepresented among potential laterally transferred genes compared to the indigenous ones. Furthermore, the GC₃ distribution of potential laterally transferred genes was found to be largely uniform in some genomes, suggesting an import from a broad range of donors.

Conclusions

The results are discussed not in a context of strongly optimized established genes, but rather of genes with weak or ancillary functions. The importance of duplication may therefore depend on the variability and availability of weak genes for which novel functions may be discovered. Therefore, lateral transfer may accelerate the evolutionary process of duplication by bringing foreign genes that have mainly weak or no function into the genome.

     

Synthesis of an asymmetrically substituted AZA crown ether as metal and amino acid binding site in DNA conjugates

Crown ether 4 as a receptor core for protonated primary amines such as amino acids has been synthesized and incorporated into oligodeoxynucleotides as dangling ends.     

Modulation of the pharmacokinetic properties of PNA: preparation of galactosyl, mannosyl, fucosyl, N-acetylgalactosaminyl, and N-acetylglucosaminyl derivatives of aminoethylglycine peptide nucleic acid monomers and their incorporation into PNA oligomers

A series of N-(2-aminoethyl)-alpha-amino acid thymine peptide nucleic acid (PNA) monomers bearing glycosylated side chains in the alpha-amino acid position have been synthesized. These include PNA monomers where glycine has been replaced by serine and threonine (O-glycosylated), derivatives of lysine and nor-alanine (C-glycosylated), and amide derivatives of aspartic acid (N-glycosylated). The Boc and Fmoc derivatives of these monomers were used for incorporation in PNA oligomers. Twelve PNA decamers containing the glycosylated units in one, two, or three positions were prepared, and the thermal stability (T(m)) of their complexes with a complementary RNA was determined. Incorporation of the glycosyl monomers reduced the duplex stability by 0-6 degrees C per substitution. A cysteine was attached to the amino terminus of eight of the PNA decamers (Cys-CTCATACTCT-NH(2)) for easy conjugation to a [(18)F]radiolabeled N-(4-fluorobenzyl)-2-bromoacetamide. The in vivo biodistribution of these PNA oligomers was determined in rat 2 h after intravenous administration. Most of the radioactivity was recovered in the kidneys and in the urine. However, N-acetylgalactosamine (and to a lesser extent galactose and mannose)-modified PNAs were effectively targeting the liver (40-fold over unmodified PNA). Thus, the pharmacodistribution in rats of PNA oligomers can be profoundly changed by glycosylation. These results could be of great significance for PNA drug development, as they should allow modulation and fine-tuning of the pharmacokinetic profile of a drug lead.     

Structure of HCV IRES domain II determined by NMR

Complex RNA structures regulate many biological processes, but are often too large for structure determination by NMR methods. The 5' untranslated region (5' UTR) of the hepatitis C viral (HCV) RNA genome contains an internal ribosome entry site (IRES) that binds to 40S ribosomal subunits with high affinity and specificity to control translation. Domain II of the HCV IRES forms a 25-kDa folded subdomain that may alter ribosome conformation. We report here the structure of domain II as determined using an NMR approach that combines short- and long-range structural data. Domain II adopts a distorted L-shape structure, and its overall shape in the free form is markedly similar to its 40S subunit-bound form; this suggests how domain II may modulate 40S subunit conformation. The results show how NMR can be used for structural analysis of large biological RNAs.