Mapping the excitation energy migration pathways in phycobilisomes from the cyanobacterium Acaryochloris marina

In this study, we use ultrafast time-resolved absorption and fluorescence spectroscopies to examine A. marina phycobilisomes isolated from cells grown under light of different intensities and spectral regimes. Investigations were performed at room temperature and at 77?K. The study demonstrates that if complexes are stabilized by high phosphate (900?mM) buffer, there are no differences between them in temporal and spectral properties of fluorescence. However, when the complexes are allowed to disassemble into trimers in low phosphate (50?mM) buffer, differences are clearly observed. The fluorescence properties of intact or disassembled phycobilisomes from cells grown in low intensity white light are unresponsive to variation in phosphate concentration. This antenna complex was further studied in detail with application of femtosecond time-resolved absorption at room temperature. Combined spectroscopic and kinetic analysis of time-resolved fluorescence and absorption data of this antenna allowed us to identify spectrally different forms of phycocyanobilins and to propose a simplified model of how they could be distributed within the phycobilisome structure.     

Genetic relationships among 527 Gram-negative bacterial plasmids

Plasmids are mosaic in composition with a maintenance "backbone" as well as "accessory" genes obtained via horizontal gene transfer. This horizontal gene transfer complicates the study of their genetic relationships. We describe a method for relating a large number of Gram-negative (GN) bacterial plasmids based on their genetic sequences. Complete coding gene sequences of 527 GN bacterial plasmids were obtained from NCBI. Initial classification of their genetic relationships was accomplished using a computational approach analogous to hybridization of "mixed-genome microarrays." Because of this similarity, the phrase "virtual hybridization" is used to describe this approach. Protein sequences generated from the gene sequences were randomly chosen to serve as "probes" for the virtual arrays, and virtual hybridization for each GN plasmid was achieved using BLASTp. Each resulting intensity matrix was used to generate a distance matrix from which an initial tree was constructed. Relationships were refined for several clusters by identifying conserved proteins within a cluster. Multiple-sequence alignment was applied to the concatenated conserved proteins, and maximum likelihood was used to generate relationships from the results of the alignment. While it is not possible to prove that the genetic relationships among the 527 GN bacterial plasmids obtained in this study are correct, replication of identical results produced in a separate study for a small group of IncA/C plasmids provides evidence that the approach used can correctly predict genetic relationships. In addition, results obtained for clusters of Borrelia plasmids are consistent with the expected exclusivity for plasmids from this genus. Finally, the 527-plasmid tree was used to study the distribution of four common antibiotic resistance genes.     

Osmostress Induces Autophosphorylation of Hog1 via a C-Terminal Regulatory Region That Is Conserved in p38��

Many protein kinases require phosphorylation at their activation loop for induction of catalysis. Mitogen-activated protein kinases (MAPKs) are activated by a unique mode of phosphorylation, on neighboring Tyrosine and Threonine residues. Whereas many kinases obtain their activation via autophosphorylation, MAPKs are usually phosphorylated by specific, dedicated, MAPK kinases (MAP2Ks). Here we show however, that the yeast MAPK Hog1, known to be activated by the MAP2K Pbs2, is activated in pbs2Δ cells via an autophosphorylation activity that is induced by osmotic pressure. We mapped a novel domain at the Hog1 C-terminal region that inhibits this activity. Removal of this domain provides a Hog1 protein that is partially independent of MAP2K, namely, partially rescues osmostress sensitivity of pbs2Δ cells. We further mapped a short domain (7 amino acid residues long) that is critical for induction of autophosphorylation. Its removal abolishes autophosphorylation, but maintains Pbs2-mediated phosphorylation. This 7 amino acids stretch is conserved in the human p38α. Similar to the case of Hog1, it’s removal from p38α abolishes p38α’s autophosphorylation capability, but maintains, although reduces, its activation by MKK6. This study joins a few recent reports to suggest that, like many protein kinases, MAPKs are also regulated via induced autoactivation.     

The PI3K–NF-κB signal transduction pathway is involved in mediating the anti-inflammatory effect of IB-MECA in adjuvant-induced arthritis

The anti-inflammatory effect of adenosine was previously found to be mediated via activation of the A₃ adenosine receptor (A₃AR). The aim of the present study was to decipher the molecular mechanism involved with the inhibitory effect of IB-MECA, an A₃AR agonist, on adjuvant-induced arthritis.     

Validation of mixed-genome microarrays as a method for genetic discrimination

Comparative genomic hybridizations have been used to examine genetic relationships among bacteria. The microarrays used in these experiments may have open reading frames from one or more reference strains (whole-genome microarrays), or they may be composed of random DNA fragments from a large number of strains (mixed-genome microarrays [MGMs]). In this work both experimental and virtual arrays are analyzed to assess the validity of genetic inferences from these experiments with a focus on MGMs. Empirical data are analyzed from an Enterococcus MGM, while a virtual MGM is constructed in silico using sequenced genomes (Streptococcus). On average, a small MGM is capable of correctly deriving phylogenetic relationships between seven species of Enterococcus with accuracies of 100% (n=100 probes) and 95% (n=46 probes); more probes are required for intraspecific differentiation. Compared to multilocus sequence methods and whole-genome microarrays, MGMs provide additional discrimination between closely related strains and offer the possibility of identifying unique strain or lineage markers. Representational bias can have mixed effects. Microarrays composed of probes from a single genome can be used to derive phylogenetic relationships, although branch length can be exaggerated for the reference strain. We describe a case where disproportional representation of different strains used to construct an MGM can result in inaccurate phylogenetic inferences, and we illustrate an algorithm that is capable of correcting this type of bias. The bias correction algorithm automatically provides bootstrap confidence values and can provide multiple bias-corrected trees with high confidence values.     

Validation of Mixed-Genome Microarrays as a Method for Genetic Discrimination

Comparative genomic hybridizations have been used to examine genetic relationships among bacteria. The microarrays used in these experiments may have open reading frames from one or more reference strains (whole-genome microarrays), or they may be composed of random DNA fragments from a large number of strains (mixed-genome microarrays [MGMs]). In this work both experimental and virtual arrays are analyzed to assess the validity of genetic inferences from these experiments with a focus on MGMs. Empirical data are analyzed from an Enterococcus MGM, while a virtual MGM is constructed in silico using sequenced genomes (Streptococcus). On average, a small MGM is capable of correctly deriving phylogenetic relationships between seven species of Enterococcus with accuracies of 100% (n = 100 probes) and 95% (n = 46 probes); more probes are required for intraspecific differentiation. Compared to multilocus sequence methods and whole-genome microarrays, MGMs provide additional discrimination between closely related strains and offer the possibility of identifying unique strain or lineage markers. Representational bias can have mixed effects. Microarrays composed of probes from a single genome can be used to derive phylogenetic relationships, although branch length can be exaggerated for the reference strain. We describe a case where disproportional representation of different strains used to construct an MGM can result in inaccurate phylogenetic inferences, and we illustrate an algorithm that is capable of correcting this type of bias. The bias correction algorithm automatically provides bootstrap confidence values and can provide multiple bias-corrected trees with high confidence values.     

Alterations in the expression of MHC class I glycoproteins by B16BL6 melanoma cells modulate insulin receptor-regulated signal transduction and augment [correction of augments] resistance to apoptosis

In a variety of malignancies, the immune-escape phenotype is associated, in part, with the inability of tumor cells to properly present their Ags to CTLs due to a deranged expression of MHC class I glycoproteins. However, these molecules were found to possess broader nonimmune functions, including participation in signal transduction and regulation of proliferation, differentiation, and sensitivity to apoptosis-inducing factors; processes, which are characteristically impaired during malignant transformation. We investigated whether the deranged expression of MHC class I expression by tumor cells could affect proper receptor-mediated signal transduction and accentuate their malignant phenotype. The malignant and H-2K murine MHC class I-deficient B16BL6 melanoma cells were characterized by an attenuated capacity to bind insulin due to the retention of corresponding receptor in intracellular stores. The restoration of H-2K expression in these cells, which abrogated their capacity to form tumors in mice, enhanced membrane translocation of the receptor, presumably, by modulating its glycosylation. The addition of insulin to H-2K-expressing melanoma cells cultured in serum-free conditions precluded apoptotic death by up-regulating the activity of protein kinase B (PKB)/Akt. In contrast, the deficiency for H-2K characteristic to the malignant clones was associated with a constitutive high activity of PKB/Akt, which rendered them resistant to apoptosis, induced by deprivation of serum-derived growth factors. The possibility to correct the regulation of PKB/Akt activity by restoration of H-2K expression in B16BL6 melanoma cells may be considered as an attractive approach for cancer therapy, since an aberrant activation of this enzyme is characteristic to resistant malignancies.     

The cell-surface isoform of colony stimulating factor 1 (CSF1) restores but does not completely normalize fecundity in CSF1-deficient mice

The complete genetic absence of colony stimulating factor 1 (CSF1) in CSF1-deficient Csf1(op)/Csf1(op) mice leads to reproductive defects in males and females. Although the cell-surface or membrane-bound isoform of CSF1 (mCSF1) is biologically active in bone, little is known about its role in reproduction. Transgenic mice expressing mCSF1 under the control of the 2.4-kb rat collagen type I alpha promoter were developed [Tg(Col1a1-mCSF1)1Gqy] and bred onto a Csf1(op)/Csf1(op) background [Csf1(op)/Csf1(op); Tg(Col1a1-mCSF1)1Gqy] to examine the effects of the mCSF1 isoform in bone in vivo. Surprisingly, when interbred, these mice were fertile. The Csf1(op)/Csf1(op); Tg(Col1a1-mCSF1)1Gqy transgenic male mice have normal libido, sperm number and percent of motile sperm. In Csf1(op)/Csf1(op); Tg(Col1a1-mCSF1)1Gqy females, puberty and estrus cycles are at expected age and duration. Further, females are able to carry pregnancies to term and nurse their offspring. Crosses of Csf1(op)/Csf1(op); Tg(Col1a1-mCSF1)1Gqy males or females with their control littermates showed no significant differences in either number or viability of offspring. However, crossing Csf1(op)/Csf1(op); Tg(Col1a1-mCSF1)1Gqy males with Csf1(op); Tg(Col1a1-mCSF1)1Gqy females resulted in a decline in both the number and viability of offspring, suggesting that a subtle reproductive defect might persist in the transgenic animals that was only manifest when the animals were interbred. Although the gravid murine uterus expresses extremely high levels of CSF1 that are thought to be important for reproduction, uterine tissue levels of CSF1 remained low and unchanged during pregnancy in Csf1(op)/Csf1(op); Tg(Col1a1-mCSF1)1Gqy mice. Low levels of CSF1 protein were detected in serum and in lung and uterine tissue in Csf1(op)/Csf1(op); Tg(Col1a1-mCSF1)1Gqy mouse, which likely result from the known proteolytic shedding of mCSF1 from the cell surface. These data are consistent with the conclusion that mCSF1, when shed from the cell surface, can support reproduction and that high uterine tissue levels of CSF1 may not be required for mouse reproduction.