Feasibility of repair of distal penile hypospadias as a day-case surgery

Objective The aim of this study was to present the outcomes of children with distal hypospadias who were operated on outpatient basis.Methods Atotal of 47 consecutive children underwent surgical repair of distal hypospadias in our department. Urethroplasties were performed by the following techniques: stentless meatal advancement-glanuloplasty (MAGPI) or glans approximation procedure (GAP) for glanular hypospadias (14) and tubularized incised plate (TIP) urethroplasty for coronal, subcoronal and midpenil hypospadias with an indwelling urethral catheter or short stent (33). The indwelling urinary catheters were managed by the double diaper technique. Patients were discharged within 6 h after operation. Dressings and catheters were removed on the postoperative day 2 and 6, respectively.Results Voiding difficulty and urinary retention on early postoperative period were observed in 8 patients. Except meatal stenosis in 4 cases and meatal retractions in 2 cases, there were no major complications in any of our patients during the follow-up period, no postoperative fistula or urethral stricture.Conclusion In children, repair of distal penile hypospadias on outpatient basis is feasible. Catheters, urethral stents, drug therapy and dressing are not justifications for hospitalization in such cases.Keywords: hospital stay, hypospadias, outpatient, urethroplasty, wounddressin

Identification of chromophore binding domains of yeast DNA photolyase

Photolyases contain two chromophores, flavin plus either methenyltetrahydrofolate (MTHF) or 8-OH-5-deazaflavin (HDF). Amino acid sequence comparison reveals that all photolyases sequenced to date have extensive sequence homology in the carboxyl-terminal half; in the amino-terminal region the folate and deazaflavin class enzymes are more homologous to other members of the same class. This modular arrangement of sequence homologies suggests that the amino-terminal half of photolyase is involved in MTHF or HDF binding whereas the carboxyl-terminal half carries the flavin binding site. In this study we attempted to identify such structural domains of yeast photolyase by partial proteolysis and gene fusion techniques. Partial digestion with chymotrypsin yielded an amino-terminal 34-kDa fragment containing tightly bound MTHF and a carboxyl-terminal 20-kDa polypeptide which lacked chromophore or DNA binding activity. However, a fusion protein carrying the carboxyl-terminal 275 amino acids of yeast photolyase bound specifically to FAD but not to MTHF or DNA. We conclude that the amino-terminal half of yeast photolyase constitutes the folate binding domain and that the carboxyl-terminal half carries the flavin binding site

Action mechanism of Escherichia coli DNA photolyase. I. Formation of the enzyme-substrate complex.

Escherichia coli DNA photolyase (photoreactivating enzyme) is a flavoprotein. The enzyme binds to DNA containing pyrimidine dimers in a light-independent step and, upon illumination with 300-600 nm radiation, catalyzes the photosensitized cleavage of the cyclobutane ring thus restoring the integrity of the DNA. We have studied the binding reaction using the techniques of nitrocellulose filter binding and flash photolysis. The enzyme binds to dimer-containing DNA with an association rate constant k1 estimated by two different methods to be 1.4 X 10(6) to 4.2 X 10(6) M-1 S-1. The dissociation of the enzyme from dimer-containing DNA displays biphasic kinetics; for the rapidly dissociating class of complexes k2 = 2-3 X 10(-2) S-1, while for the more slowly dissociating class k2 = 1.3 X 10(-3) to 6 X 10(-4) S-1. The equilibrium association constant KA, as determined by the nitrocellulose filter binding assay and the flash photolysis assay, was 4.7 X 10(7) to 6 X 10(7) M-1, in reasonable agreement with the values predicted from k1 and k2. From the dependence of the association constant on ionic strength we conclude that the enzyme contacts no more than two phosphodiester bonds upon binding; this strongly suggests that the pyrimidine dimer is the main structural determinant of specific photolyase-DNA interaction and that nonspecific ionic interactions do not contribute significantly to substrate binding

Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes

Nucleotide excision DNA repair is mechanistically conserved across all kingdoms of life. In prokaryotes, this multi-enzyme process requires six proteins: UvrA?D, DNA polymerase I and DNA ligase. To examine how UvrC locates the UvrB? DNA pre-incision complex at a site of damage, we have labeled UvrB and UvrC with different colored quantum dots and quantitatively observed their interactions with DNA tightropes under a variety of solution conditions using oblique angle fluorescence imaging. Alone, UvrC predominantly interacts statically with DNA at low salt. Surprisingly, however, UvrC and UvrB together in solution bind to form the previously unseen UvrBC complex on duplex DNA. This UvrBC complex is highly motile and engages in unbiased one-dimensional diffusion. To test whether UvrB makes direct contact with the DNA in the UvrBC?DNA complex, we investigated three UvrB mutants: Y96A, a b-hairpin deletion and D338N. These mutants affected the motile properties of the UvrBC complex, indicating that UvrB is in intimate contact with the DNA when bound to UvrC. Given the in vivo excess of UvrB and the abundance of UvrBC in our experiments, this newly identified complex is likely to be the predominant form of UvrC in the cell. © 2013 The Author(s)

Blue-light-receptive cryptochrome is expressed in a sponge eye lacking neurons and opsin

Many larval sponges possess pigment ring eyes that apparently mediate phototactic swimming. Yet sponges are not known to possess nervous systems or opsin genes, so the unknown molecular components of sponge phototaxis must differ fundamentally from those in other animals, inspiring questions about how this sensory system functions. Here we present molecular and biochemical data on cryptochrome, a candidate gene for functional involvement in sponge pigment ring eyes. We report that Amphimedon queenslandica, a demosponge, possesses two cryptochrome/photolyase genes, Aq-Cry1 and Aq-Cry2. The mRNA of one gene (Aq-Cry2) is expressed in situ at the pigment ring eye. Additionally, we report that Aq-Cry2 lacks photolyase activity and contains a flavin-based co-factor that is responsive to wavelengths of light that also mediate larval photic behavior. These results suggest that Aq-Cry2 may act in the aneural, opsin-less phototaxic behavior of a sponge

Transcription Factors in Light and Circadian Clock Signaling Networks Revealed by Genomewide Mapping of Direct Targets for Neurospora White Collar Complex

Light signaling pathways and circadian clocks are inextricably linked and have profound effects on behavior in most organisms. Here, we used chromatin immunoprecipitation (ChIP) sequencing to uncover direct targets of the Neurospora crassa circadian regulator White Collar Complex (WCC). The WCC is a blue-light receptor and the key transcription factor of the circadian oscillator. It controls a transcriptional network that regulates ∼20% of all genes, generating daily rhythms and responses to light. We found that in response to light, WCC binds to hundreds of genomic regions, including the promoters of previously identified clock- and light-regulated genes. We show that WCC directly controls the expression of 24 transcription factor genes, including the clock-controlled adv-1 gene, which controls a circadian output pathway required for daily rhythms in development. Our findings provide links between the key circadian activator and effectors in downstream regulatory pathways

Preferential binding of ATR protein to UV-damaged DNA

The ATR protein is a member of the phosphoinositide 3-kinase-related kinase family and plays an important role in UV-induced DNA damage checkpoint response. Its role as a signal transducer in cell cycle checkpoint is well established, but it is currently unclear whether ATR functions as a damage sensor as well. Here we have purified the ATR protein and investigated its interaction with DNA by using biochemical analysis and electron microscopy. We find that ATR is a DNA-binding protein with higher affinity to UV-damaged than undamaged DNA. In addition, damaged DNA stimulates the kinase activity of ATR to a significantly higher level than undamaged DNA. Our data suggest that ATR may function as an initial sensor in the DNA damage checkpoint response

ERCC1 expression and RAD51B activity correlate with cell cycle response to platinum drug treatment not DNA repair

Background: The H69CIS200 and H69OX400 cell lines are novel models of low-level platinum-drug resistance. Resistance was not associated with increased cellular glutathione or decreased accumulation of platinum, rather the resistant cell lines have a cell cycle alteration allowing them to rapidly proliferate post drug treatment. Results: A decrease in ERCC1 protein expression and an increase in RAD51B foci activity was observed in association with the platinum induced cell cycle arrest but these changes did not correlate with resistance or altered DNA repair capacity. The H69 cells and resistant cell lines have a p53 mutation and consequently decrease expression of p21 in response to platinum drug treatment, promoting progression of the cell cycle instead of increasing p21 to maintain the arrest. Conclusion: Decreased ERCC1 protein and increased RAD51B foci may in part be mediating the maintenance of the cell cycle arrest in the sensitive cells. Resistance in the H69CIS200 and H69OX400 cells may therefore involve the regulation of ERCC1 and RAD51B independent of their roles in DNA repair. The novel mechanism of platinum resistance in the H69CIS200 and H69OX400 cells demonstrates the multifactorial nature of platinum resistance which can occur independently of alterations in DNA repair capacity and changes in ERCC1