Structure and organization of rhodophyte and chromophyte plastid genomes: implications for the ancestry of plastids

Summary Recombinational repair is the means by which DNA double-strand breaks (DSBs) are repaired in yeast. DNA divergence between chromosomes was shown previously to inhibit repair in diploid G1 cells, resulting in chromosome loss at low nonlethal doses of ionizing radiation. Furthermore, 15–20% divergence prevents meiotic recombination between individual pairs of Saccharomyces cerevisiae and S. carlsbergensis chromosomes in an otherwise S. cerevisiae background. Based on analysis of the efficiency of DSB-induced chromosome loss and direct genetic detection of intragenic recombination, we conclude that limited DSB recombinational repair can occur between homoeologous chromosomes. There is no difference in loss between a repair-proficient Pms⁺ strain and a mismatch repair mutant, pms1. Since DSB recombinational repair is tolerant of diverged DNAs, this type of repair could lead to novel genes and altered chromosomes. The sensitivity to DSB-induced loss of 11 individual yeast artificial chromosomes (YACs) containing mouse or human (chromosome 21 or HeLa) DNA was determined. Recombinational repair between a pair of homologous HeLa YACs appears as efficient as that between homologous yeast chromosomes in that there is no loss at low radiation doses. Single YACs exhibited considerable variation in response, although the response for individual YACs was highly reproducible. Based on the results with the yeast homoeologous chromosomes, we propose that the potential exists for intra- YAC recombinational repair between diverged repeat DNA and that the extent of repair is dependent upon the amount of repeat DNA and the degree of divergence. The sensitivity of YACs containing mammalian DNA to ionizing radiation-induced loss may thus be an indicator of the extent of repeat DNA.     

Proliferating cell nuclear antigen is required for DNA excision repair.

Fractionation of extracts from human cell lines allows nucleotide excision repair of damaged DNA to be resolved into discrete incision and polymerization stages. Generation of incised intermediates depends on the XP-A protein, a polypeptide that recognizes sites of damaged DNA, and on the human single-stranded DNA-binding protein HSSB. The proliferating cell nuclear antigen (PCNA) is required for the DNA synthesis that converts the nicked intermediates to completed repair events. This need for PCNA implies that repair synthesis is carried out by DNA polymerase delta or epsilon. The ability to visualize repair intermediates in the absence of PCNA facilitates dissection of the multiprotein reaction that leads to incision of damaged DNA in a major pathway of cellular defense against mutagens.     

A novel combinatorial biocatalytic approach for producing antibacterial compounds effective against Mycobacterium tuberculosis (TB)

Two bacterial hosts expressing cloned aromatic oxygenases were used to catalyze the oxidation and polymerization of indole and related substrates, creating mixtures of indigoid compounds comprised of novel dimers and trimers. Crude extracts and purified compounds were tested for their ability to inhibit the growth of Gram-positive organisms, in general, and Mycobacterium tuberculosis (TB), in particular. Of the 74 compounds tested against M. tuberculosis, ~66 % had minimum inhibitory concentrations (MIC) of 5 μg/ml or less. The most effective antibiotic found was designated SAB-P1, a heterodimer of indole and anthranil, which had a MIC of 0.16 μg/ml, and did not inhibit kidney cells (IC⁵⁰) at concentrations of >8 μg/ml. Combinatorial biocatalysis was used to create a series of halogenated derivatives of SAB-P1 with a wider therapeutic window. None of the derivatives had MIC values that were superior to SAB-P1, but some had a wider therapeutic window because of decreased kidney cell toxicity. Generally, the indigoid dimers that were effective against TB appeared to be specific for TB. Some of the trimers generated, however, had a broader spectrum of activity inhibiting not only TB (MIC?=?1.1 μg/ml) but also the growth of Mycobacterium smegmatis MC2 155, Bacillus cereus, Enterococcus faecalis, Staphylococcus epidermidis, Bacillus subtilis 168, and Clostridium acetobutylicum. The structure of two of the novel dimers (SAB-C4 and SAB-P1) and a trimer (SAB-R1) were solved using X-ray crystallography.     

alpha beta Spectrin coiled coil association at the tetramerization site

On the basis of sequence homology studies, it has been suggested that the association of human erythrocytes alpha and beta spectrin at the tetramerization site involves interactions between helices. However, no empirical details are available, presumably due to the experimental difficulties in studying spectrin molecules because of its size and/or its structural flexibility. It has been speculated that erythrocyte tetramerization involves helical bundling rather than coiled coil association. We have used recombinant spectrin peptides to model alpha and beta spectrin to study their association at the tetramerization site. Two alpha peptides, Sp alpha 1-156 and Sp alpha 1-368, and one beta peptide, Sp beta 1898-2083, were used as model peptides to demonstrate the formation of the alpha beta complex. We also found that the replacement of R28 in Sp alpha 1-368 to give Sp alpha 1-368R28C abolished complex formation with the beta peptide. Circular dichroism techniques were used to monitor the secondary structures of the individual peptides and of the complex, and the results showed that both Sp alpha 1-156 and Sp beta 1898-2083 peptides in solution, separately, included helices that were not paired with other helices in the absence of their binding partners. However, in a mixture of Sp alpha 1-156 and Sp beta 1898-2083 and formation of the alpha beta complex, the unpaired helices associated to form coiled coils. Since the sequences of these two peptides that are involved in the coiled coil association are derived from a native protein, the information obtained from this study also provides insight toward a better understanding of naturally occurring coiled coil subunit-subunit association.     

Definition of a short region of XPG necessary for TFIIH interaction and stable recruitment to sites of UV damage

XPG is the human endonuclease that cuts 3' to DNA lesions during nucleotide excision repair. Missense mutations in XPG can lead to xeroderma pigmentosum (XP), whereas truncated or unstable XPG proteins cause Cockayne syndrome (CS), normally yielding life spans of <7 years. One XP-G individual who had advanced XP/CS symptoms at 28 years has been identified. The genetic, biochemical, and cellular defects in this remarkable case provide insight into the onset of XP and CS, and they reveal a previously unrecognized property of XPG. Both of this individual's XPG alleles produce a severely truncated protein, but an infrequent alternative splice generates an XPG protein lacking seven internal amino acids, which can account for his very slight cellular UV resistance. Deletion of XPG amino acids 225 to 231 does not abolish structure-specific endonuclease activity. Instead, this region is essential for interaction with TFIIH and for the stable recruitment of XPG to sites of local UV damage after the prior recruitment of TFIIH. These results define a new functional domain of XPG, and they demonstrate that recruitment of DNA repair proteins to sites of damage does not necessarily lead to productive repair reactions. This observation has potential implications that extend beyond nucleotide excision repair.     

Immune modulation in pemphigus vulgaris: role of CD28 and IL-10

Pemphigus vulgaris (PV) is an autoimmune bullous skin disease characterized by Abs to the desmosomal cadherin desmoglein-3. Although the autoantibodies have been shown to be pathogenic, the role of the cellular immune system in the pathology of pemphigus-induced acantholysis is unclear. To further delineate the potential role of T cell-signaling pathways in the pathogenesis of PV, we performed passive transfer experiments with PV IgG in gene-targeted mutant mice. Our results demonstrated that CD28-deficient mice (lacking a costimulatory signal for T cell activation) are 5-fold more sensitive to the development of PV than wild-type mice. To evaluate whether the higher incidence of disease was due to an impairment in intercellular adhesion of keratinocytes, we performed an in vitro acantholysis, using CD28-/- mice keratinocytes. No alteration in in vitro adhesion was detected in CD28-/--type keratinocytes. Because the CD28 molecule plays a pivotal role in the induction of Th2 cytokines, we examined the levels of a prototypic Th2 cytokine (IL-10) in CD28-/- mice. Lower levels of IL-10 mRNA were found in lesions from CD28-/- mice. To determine whether pemphigus susceptibility in CD28-/- was related to IL-10 deficiency, we performed passive transfer experiments in IL-10-/- mice that demonstrated increased blisters compared with controls. To confirm that IL-10 is involved in the pathogenesis, rIL-10 was given with PV IgG. IL-10 significantly suppressed the disease activity. These data suggest a potential role of IL-10 in PV.     

CD4+ Th1 and CD8+ type 1 cytotoxic T cells both play a crucial role in the full development of contact hypersensitivity

The role of CD4(+) vs CD8(+) T cells in contact hypersensitivity (CHS) remains controversial. In this study, we used gene knockout (KO) mice deficient in CD4(+) or CD8(+) T cells to directly address this issue. Mice lacking either CD4(+) or CD8(+) T cells demonstrated depressed CHS responses to dinitrofluorobenzene and oxazolone compared with wild-type C57BL/6 mice. The depression of CHS was more significant in CD8 KO mice than in CD4 KO mice. Furthermore, in vivo depletion of either CD8(+) T cells from CD4 KO mice or CD4(+) T cells from CD8 KO mice virtually abolished CHS responses. Lymph node cells (LNCs) from hapten-sensitized CD4 and CD8 KO mice showed a decreased capacity for transferring CHS. In vitro depletion of either CD4(+) T cells from CD8 KO LNCs or CD8(+) T cells from CD4 KO LNCs resulted in a complete loss of CHS transfer. LNCs from CD4 and CD8 KO mice produced significant amounts of IFN-gamma, indicating that both CD4(+) and CD8(+) T cells are able to secrete IFN-gamma. LNCs from CD8, but not CD4, KO mice were able to produce IL-4 and IL-10, suggesting that IL-4 and IL-10 are mainly derived from CD4(+) T cells. Intracellular cytokine staining of LNCs confirmed that IFN-gamma-positive cells consisted of CD4(+) (Th1) and CD8(+) (type 1 cytotoxic T) T cells, whereas IL-10-positive cells were exclusively CD4(+) (Th2) T cells. Collectively, these results suggest that both CD4(+) Th1 and CD8(+) type 1 cytotoxic T cells are crucial effector cells in CHS responses to dinitrofluorobenzene and oxazolone in C57BL/6 mice.     

Association of non-heterocystous cyanobacteria with crop plants

Cyanobacteria have the ability to form associations with organisms from all domains of life, notably with plants, which they provide with fixed nitrogen, among other substances. This study was aimed at developing artificial associations between non-heterocystous cyanobacteria and selected crop plants. We isolated several non-heterocystous cyanobacteria from various rice fields. The cultures were tested for their capacity to produce the plant hormone indole-3-acetic acid (IAA), and the possible role of IAA in the association of cyanobacteria with seedling roots was evaluated. Axenic cultures were co-inoculated with 10-day-old plant seedlings of Triticum aestivum, Vigna radiata and Pisum sativum and incubated for 1 week. Cyanobacterial association with the roots of these seedlings was quantified by measuring chlorophyll-a. Cyanobacterial association with the roots was observed by light microscopy as well as by confocal laser scanning microscopy (CLSM). Based on sequence analysis of the 16S rRNA gene, the isolates were identified as Synechocystis sp., Chroococcidiopsis sp., Leptolyngbya sp., and Phormidium sp. CLSM observations revealed the intimate association of cyanobacteria with the seedling roots as well as invasion of the roots and root cells. Strains producing IAA were more efficient in the colonization of the roots than those that lacked this ability. IAA-producing cyanobacteria possess a tryptophan-dependent pathway, and these cyanobacteria showed IAA synthesis activity in the presence of roots in media lacking tryptophan. Based on the results of this study, we conclude that non-heterocystous cyanobacteria also have the potential for use in agriculture to improve the growth and yield of crop plants that do not naturally form associations with cyanobacteria.     

In vitro antioxidant activity of Juglans regia L. bark extract and its protective effect on cyclophosphamide-induced urotoxicity in mice

Walnut (Juglans regia L.) bark has been claimed to possess anti-inflammatory, blood purifying, anticancer, depurative, diuretic and laxative activities. It contains several therapeutically active constituents, especially polyphenols. We studied the antioxidant potential of aqueous extract of walnut bark and its modulatory effect on cyclophosphamide (CP)-induced urotoxicity in Swiss albino male mice. Free radical-scavenging activity of extract was assessed in four in vitro assays. The phenolic and flavonolic contents of the extract were also measured. Walnut bark extract treatment (150 mg/kg p.o. x 10 days) resulted in protective restoration of decreased antioxidants in CP-treated (18 mg/kg i.p. x 10 days) animals. CP treatment caused decreases in the activities of catalase (CAT), glutathione peroxidase (GP), glutathione reductase (GR) and glutathione S-transferase (GST) and in the glutathione (GSH) content in urinary bladder and a significant concomitant increase in lipid peroxidation (LPO). Administration of extract restored all the antioxidants significantly and lowered the elevated LPO in the bladder. A correlation between radical scavenging capacities of the extract with phenolic content was observed thus justifying its antioxidant potential against oxidative stress-mediated urotoxicity in mice. Walnut is reported to possess antiproliferative activity. Its protective effect on CP-induced toxicity in bladder is a promising activity, which warrants possible clinical investigations on this medicinal plant.