Postreplicative recruitment of cohesin to double-strand breaks is required for DNA repair

Chromosome stability depends on accurate chromosome segregation and efficient DNA double-strand break (DSB) repair. Sister chromatid cohesion, established during S phase by the protein complex cohesin, is central to both processes. In the absence of cohesion, chromosomes missegregate and G2-phase DSB repair fails. Here, we demonstrate that G2-phase repair also requires the presence of cohesin at the damage site. Cohesin components are shown to be recruited to extended chromosome regions surrounding DNA breaks induced during G2. We find that in the absence of functional cohesin-loading proteins (Scc2/Scc4), the accumulation of cohesin at DSBs is abolished and repair is defective, even though sister chromatids are connected by S phase generated cohesion. Evidence is also provided that DSB induction elicits establishment of sister chromatid cohesion in G2, implicating that damage-recruited cohesin facilitates DNA repair by tethering chromatids.     

Apparent X-linked primary ciliary dyskinesia associated with retinitis pigmentosa and a hearing loss

Three brothers, one 10-year-old and a pair of 14-year-old dizygotic twins--expressed the classical, early-onset retinitis pigmentosa (RP) with typical ophthalmoscopic findings, night blindness, visual field constricted to 10 degrees and flat ERG response. All three brothers were also diagnosed with primary ciliary dyskinesia (PCD) and had recurrent respiratory infections, chronic sinusitis and bronchiectasis. In all of them, resection of the middle lobe of the right lung was performed. A similar clinical picture of coexisting RP and PCD was noted in the brother of the probands' mother. All probands displayed situs solitus. Consistent with the X-linked mode of RP inheritance, there were also three obligatory female carriers of the disorder in this family: the mother of the affected boys, her mother and a daughter of her brother. In all of them, retinitis pigmentosa "sine pigmento" was found with milder but clinically significant symptoms (mild night blindness, visual field constricted to 30 degrees, and scotopic and photopic ERG responses reduced to 30-60%). No extraocular symptoms were detected in any of the heterozygous female carriers. This family presents an example of two rare phenomena: X-linked dominant retinitis pigmentosa (with milder expression in females) and a rare combination of RP with recurrent respiratory infections due to PCD.     

The role of ephrins and Eph receptors in cancer

Eph receptors are the largest receptor tyrosine kinase family of transmembrane proteins with an extracellular domain capable of recognizing signals from the cells’ environment and influencing cell–cell interaction and cell migration. Ephrins are the ligands to Eph receptors and stimulate bi-directional signaling of the Eph/ephrin axis. Eph receptor and ephrin overexpression can result in tumorigenesis as related to tumor growth and survival and is associated with angiogenesis and metastasis in many types of human cancer. Recent data suggest that Eph/ephrin signaling could play an important role in the development of novel inhibition strategies and cancer treatments to potentially target this receptor tyrosine kinase and/or its ligand. A deeper understanding of the molecular basis for normal versus defective cell–cell interaction through the Eph/ephrin axis will enable the potential development of novel cancer treatments. This review emphasizes the biology of Eph/ephrin as well as the potential for novel targeted therapy through this pathway.     

The PM2 virion has a novel organization with an internal membrane and pentameric receptor binding spikes

Biological membranes are notoriously resistant to structural analysis. Excellent candidates to tackle this problem in situ are membrane-containing viruses where the membrane is constrained by an icosahedral capsid. Cryo-EM and image reconstruction of bacteriophage PM2 revealed a membrane bilayer following the internal surface of the capsid. The viral genome closely interacts with the inner leaflet. The capsid, at a resolution of 8.4 A, reveals 200 trimeric capsomers with a pseudo T = 21 dextro organization. Pentameric receptor-binding spikes protrude from the surface. It is evident from the structure that the PM2 membrane has at least two important roles in the life cycle. First, it acts as a scaffold to nucleate capsid assembly. Second, after host recognition, it fuses with the host outer membrane to promote genome entry. The structure also sheds light on how the viral supercoiled circular double-stranded DNA genome might be packaged and released.     

Determination of new derivatives of genistein in culture media by liquid chromatography

Methods for determination of genistein and its four new analogues in culture media have been developed to support studies on their potential anticancer activities. The investigated compounds were extracted from the media using liquid-liquid extraction with appropriate solvent. After evaporation of organic solvents each of the dry extracts was reconstituted in appropriate mobile phase. Reversed-phase HPLC was applied to quantitative determining of tested compounds. The methods are specific, sensitive and technically simple. They were used to evaluate concentration level of investigated compounds in experiments with human promyelocytic leukemia cells (HL-60 cell line).     

Insights into the genome of the enteric bacterium Escherichia blattae: cobalamin (B12) biosynthesis, B12-dependent reactions, and inactivation of the gene region encoding B12-dependent glycerol dehydratase by a new mu-like prophage

The enteric bacterium Escherichia blattae has been analyzed for the presence of cobalamin (B12) biosynthesis and B12-dependent pathways. Biochemical studies revealed that E. blattae synthesizes B12 de novo aerobically and anaerobically. Genes exhibiting high similarity to all genes of Salmonella enterica serovar Typhimurium, which are involved in the oxygen-independent route of B12 biosynthesis, were present in the genome of E. blattae DSM 4481. The dha regulon encodes the key enzymes for the anaerobic conversion of glycerol to 1,3-propanediol, including coenzyme B12-dependent glycerol dehydratase. E. blattae DSM 4481 lacked glycerol dehydratase activity and showed no anaerobic growth with glycerol, but the genome of E. blattae DSM 4481 contained a dha regulon. The E. blattaedha regulon is unusual, since it harbors genes for two types of dihydroxyacetone kinases. The major difference to dha regulons of other enteric bacteria is the inactivation of the dehydratase-encoding gene region by insertion of a 33,339-bp prophage (MuEb). Sequence analysis revealed that MuEb belongs to the Mu family of bacteriophages. The E. blattae strains ATCC 33429 and ATCC 33430 did not contain MuEb. Accordingly, both strains harbored an intact dehydratase-encoding gene region and fermented glycerol. The properties of the glycerol dehydratases and the correlating genes (dhaBCE) of both strains were similar to other B12-dependent glycerol and diol dehydratases, but both dehydratases exhibited the highest affinity for glycerol of all B12-dependent dehydratases characterized so far. In addition to the non-functional genes encoding B12-dependent glycerol dehydratase, the genome of E. blattae DSM 4481 contained the genes for only one other B12-dependent enzyme, the methylcobalamin-dependent methionine synthase.     

Acridine orange induces translocation of phosphatidylserine to red blood cell surface

Clustering of band-3 on red blood cell (RBC) surface has been assumed to catalyze RBC phagocytosis. In studying this subject, acridine orange (AO) has commonly been employed on the assumption that it specifically induces band-3 clustering. In the present study, we show that AO strongly induces translocation of phosphatidylserine (PS) to RBC surface. Because surface PS is well known to induce RBC intercellular interaction, these findings suggest that the use of AO as a specific inducer of band-3 clustering is questionable. It is possible that band-3 clustering and PS translocation are interdependent, and this interrelationship has yet to be explored. erythrocytes; adherence; acridine orange; band-3; phosphatidylserine     

T cells infiltrate the brain in murine and human transmissible spongiform encephalopathies

CD4 and CD8 T lymphocytes infiltrate the parenchyma of mouse brains several weeks after intracerebral, intraperitoneal, or oral inoculation with the Chandler strain of mouse scrapie, a pattern not seen with inoculation of prion protein knockout (PrP(-/-)) mice. Associated with this cellular infiltration are expression of MHC class I and II molecules and elevation in levels of the T-cell chemokines, especially macrophage inflammatory protein 1beta, IFN-gamma-inducible protein 10, and RANTES. T cells were also found in the central nervous system (CNS) in five of six patients with Creutzfeldt-Jakob disease. T cells harvested from brains and spleens of scrapie-infected mice were analyzed using a newly identified mouse PrP (mPrP) peptide bearing the canonical binding motifs to major histocompatibility complex (MHC) class I H-2(b) or H-2(d) molecules, appropriate MHC class I tetramers made to include these peptides, and CD4 and CD8 T cells stimulated with 15-mer overlapping peptides covering the whole mPrP. Minimal to modest K(b) tetramer binding of mPrP amino acids (aa) 2 to 9, aa 152 to 160, and aa 232 to 241 was observed, but such tetramer-binding lymphocytes as well as CD4 and CD8 lymphocytes incubated with the full repertoire of mPrP peptides failed to synthesize intracellular gamma interferon (IFN-gamma) or tumor necrosis factor alpha (TNF-alpha) cytokines and were unable to lyse PrP(-/-) embryo fibroblasts or macrophages coated with (51)Cr-labeled mPrP peptide. These results suggest that the expression of PrP(sc) in the CNS is associated with release of chemokines and, as shown previously, cytokines that attract and retain PrP-activated T cells and, quite likely, bystander activated T cells that have migrated from the periphery into the CNS. However, these CD4 and CD8 T cells are defective in such an effector function(s) as IFN-gamma and TNF-alpha expression or release or lytic activity.     

Function of herpes simplex virus type 1 gD mutants with different receptor-binding affinities in virus entry and fusion

We have studied the receptor-specific function of four linker-insertion mutants of herpes simplex virus type 1 glycoprotein D (gD) representing each of the functional regions of gD. We used biosensor analysis to measure binding of the gD mutants to the receptors HVEM (HveA) and nectin-1 (HveC). One of the mutants, gD(inverted Delta 34t), failed to bind HVEMt but showed essentially wild-type (WT) affinity for nectin-1t. The receptor-binding kinetics and affinities of the other three gD mutants varied over a 1,000-fold range, but each mutant had the same affinity for both receptors. All of the mutants were functionally impaired in virus entry and cell fusion, and the levels of activity were strikingly similar in these two assays. gD(inverted Delta 34)-containing virus was defective on HVEM-expressing cells but did enter nectin-1-expressing cells to about 60% of WT levels. This showed that the defect of this form of gD on HVEM-expressing cells was primarily one of binding and that this was separable from its later function in virus entry. gD(inverted Delta 243t) showed WT binding affinity for both receptors, but virus containing this form of gD had a markedly reduced rate of entry, suggesting that gD(inverted Delta 243) is impaired in a postbinding step in the entry process. There was no correlation between gD mutant activity in fusion or virus entry and receptor-binding affinity. We conclude that gD functions in virus entry and cell fusion regardless of its receptor-binding kinetics and that as long as binding to a functional receptor occurs, entry will progress.