Cellular location and activity of Escherichia coli RecG proteins shed light on the function of its structurally unresolved C-terminus

RecG is a DNA translocase encoded by most species of bacteria. The Escherichia coli protein targets branched DNA substrates and drives the unwinding and rewinding of DNA strands. Its ability to remodel replication forks and to genetically interact with PriA protein have led to the idea that it plays an important role in securing faithful genome duplication. Here we report that RecG co-localises with sites of DNA replication and identify conserved arginine and tryptophan residues near its C-terminus that are needed for this localisation. We establish that the extreme C-terminus, which is not resolved in the crystal structure, is vital for DNA unwinding but not for DNA binding. Substituting an alanine for a highly conserved tyrosine near the very end results in a substantial reduction in the ability to unwind replication fork and Holliday junction structures but has no effect on substrate affinity. Deleting or substituting the terminal alanine causes an even greater reduction in unwinding activity, which is somewhat surprising as this residue is not uniformly present in closely related RecG proteins. More significantly, the extreme C-terminal mutations have little effect on localisation. Mutations that do prevent localisation result in only a slight reduction in the capacity for DNA repair. © 2014 The Author(s)

Systemic Delivery of Oncolytic Adenoviruses Targeting Transforming Growth Factor-β Inhibits Established Bone Metastasis in a Prostate Cancer Mouse Model

Abstract We have examined whether Ad.sT?RFc and TAd.sT?RFc, two oncolytic viruses expressing soluble transforming growth factor-? receptor II fused with human Fc (sTGF?RIIFc), can be developed to treat bone metastasis of prostate cancer. Incubation of PC-3 and DU-145 prostate tumor cells with Ad.sT?RFc and TAd.sT?RFc produced sTGF?RIIFc and viral replication; sTGF?RIIFc caused inhibition of TGF-?-mediated SMAD2 and SMAD3 phosphorylation. Ad(E1-).sT?RFc, an E1? adenovirus, produced sTGF?RIIFc but failed to replicate in tumor cells. To examine the antitumor response of adenoviral vectors, PC-3-luc cells were injected into the left heart ventricle of nude mice. On day 9, mice were subjected to whole-body bioluminescence imaging (BLI). Mice bearing hind-limb tumors were administered viral vectors via the tail vein on days 10, 13, and 17 (2.5?1010 viral particles per injection per mouse, each injection in a 0.1-ml volume), and subjected to BLI and X-ray radiography weekly until day 53. Ad.sT?RFc, TAd.sT?RFc, and Ad(E1-).sT?RFc caused significant inhibition of tumor growth; however, Ad.sT?RFc was the most effective among all the vectors. Only Ad.sT?RFc and TAd.sT?RFc inhibited tumor-induced hypercalcemia. Histomorphometric and synchrotron micro-computed tomographic analysis of isolated bones indicated that Ad.sT?RFc induced significant reduction in tumor burden, osteoclast number, and trabecular and cortical bone destruction. These studies suggest that Ad.sT?RFc and TAd.sT?RFc can be developed as potential new therapies for prostate cancer bone metastasis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98454/1/hum%2E2012%2E040.pd

The Stringent Response and Cell Cycle Arrest in Escherichia coli

The bacterial stringent response, triggered by nutritional deprivation, causes an accumulation of the signaling nucleotides pppGpp and ppGpp. We characterize the replication arrest that occurs during the stringent response in Escherichia coli. Wild type cells undergo a RelA-dependent arrest after treatment with serine hydroxamate to contain an integer number of chromosomes and a replication origin-to-terminus ratio of 1. The growth rate prior to starvation determines the number of chromosomes upon arrest. Nucleoids of these cells are decondensed; in the absence of the ability to synthesize ppGpp, nucleoids become highly condensed, similar to that seen after treatment with the translational inhibitor chloramphenicol. After induction of the stringent response, while regions corresponding to the origins of replication segregate, the termini remain colocalized in wild-type cells. In contrast, cells arrested by rifampicin and cephalexin do not show colocalized termini, suggesting that the stringent response arrests chromosome segregation at a specific point. Release from starvation causes rapid nucleoid reorganization, chromosome segregation, and resumption of replication. Arrest of replication and inhibition of colony formation by ppGpp accumulation is relieved in seqA and dam mutants, although other aspects of the stringent response appear to be intact. We propose that DNA methylation and SeqA binding to non-origin loci is necessary to enforce a full stringent arrest, affecting both initiation of replication and chromosome segregation. This is the first indication that bacterial chromosome segregation, whose mechanism is not understood, is a step that may be regulated in response to environmental conditions

Intravesical oxybutinin chloride in children with intermittent catheterization: Sonographic findings

The sonographic findings in the bladder are presented in four children with myelomeningocele and neurogenic dysfunction of the bladder, who were treated with intermittent self-catheterization and intravesical oxybutinin chloride. All were referred for routine sonography of the urinary tract. Each had infused a crushed tablet of oxybutinin chloride intravesically 30–120 min before the examination. In two children, brightly echogenic, non-shadowing particles were suspended in the bladder urine. In one of these, the particles swirled giving the impression of a “snowstorm”; in the other, most of the particles gradually settled forming an irregular clump on the bladder base. In the remaining two children, the urine appeared diffusely hazy with innumerable tiny particles giving the impression of a fine mist filling the bladder. The sonographic appearance of the urine in the bladder after intravesical instillation of crushed tablets can be dramatic and can simulate pus, blood, fungus, or other debris in the bladder lumen. In the absence of clinical symptoms or hematuria, a history of recent infusion of medication into the bladder should be sought.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46706/1/247_2005_Article_BF02012126.pd

Three independently deleted regions at chromosome arm 16q in human prostate cancer: allelic loss at 16q24.1–q24.2 is associated with aggressive behaviour of the disease, recurrent growth, poor differentiation of the tumour and poor prognosis for the patient

Loss of heterozygosity at chromosome arm 16q is a frequent event in human prostate cancer. In this study, loss of heterozygosity at 16q was studied in 44 prostate cancer patients exhibiting various clinical features. Fifteen polymorphic polymerase chain reaction (PCR) markers were used to identify the separately deleted areas and the findings were compared with clinicopathological variables and 5-year survival of the patients. The results indicated that there are at least three independently deleted regions at 16q. Allelic losses at the central and distal areas were associated significantly with aggressive behaviour of the disease (16q24.1–q24.2, P< 0.01, and 16q24.3–qter, P< 0.05), and the central area of deletion was further significantly associated with poorly differentiated tumour cells (P< 0.05) and with recurrent (P< 0.01) growth of the tumour. During the follow-up period, 28% of the patients initially with M0 disease developed distant metastases. Of the patients showing allelic loss at 16q24.1–q24.2, distant metastasis were found in 45% during the 5-year follow-up period, and 31% of the patients showing loss at 16q21.1 also developed distant metastases. After the 5-year follow-up period, 14 (32%) of the patients remained alive, whereas 19 (43%) had died because of their prostate cancer. The overall survival rate of the patients showing allelic loss at 16q21.1 or 16q24.1–q24.2 was significantly lower than that of the patients with retained heterozygosity. © 1999 Cancer Research Campaig

Synthesis of macrocyclic receptors with intrinsic fluorescence featuring quinizarin moieties

An unprecedented class of macrocycles with intrinsic fluorescence consisting of phenolic trimers and quinizarin is developed. Though they are lacking strong hydrogen bonds as observed in calixarenes, the two examples introduced here each adopt a vase-like conformation with all four aromatic units pointing in one direction (syn orientation). This “cone” conformation has been confirmed by NMR spectroscopy, molecular modeling, and X-ray crystallography. The laminar, electron-rich fluorophore as part of the macrocycle allows additional contacts to enclosed guest molecules