Short-term results after STARR versus internal Delorme for obstructed defecation: a non-randomized prospective study.

Obstructed defecation syndrome due to internal intussusception and rectocele is a common disease, and various transanal surgical techniques have been proposed. Aim of the present study was to compare the internal Delorme (ID) and the stapled transanal rectal resection (STARR) results in the treatment of patients with obstructed defecation syndrome. From September 2011 to May 2012, 23 patients were operated with STARR procedure and 12 patients with Delorme's procedure for obstructed defecation syndrome. All patients underwent preoperative assessment: clinical evaluation (Altomare ODS score, Wexner constipation scoring system), proctoscopy, defecography, anorectal manometry and endoanal ultrasonography. Surgery was proposed with: failure of medical therapy, incomplete defecation, and unsuccessful attempts with long periods spent in bathroom, defecation with digital assistance, use of enemas and defecography findings of rectoanal intussusception and rectocele. The average operative time was 28 min (range 15-65) for the STARR group and 56 min (range 28-96) for the ID group with a mean hospital stay of 2 days for both the procedures. The Wexner score significantly fell postoperatively from 17 to 4, 7 in STARR group and from 15.3 to 3.3 in the ID group. The Altomare score postoperatively fell from 18.2 to 5.5 for STARR group and from 16.5 to 5.3 for ID group. No statistically significant differences were observed between the two procedures considering the outcomes parameters and the complications. Both ID and STARR procedure seem to be effective in the treatment of ODS.

An EPR study of the dinuclear iron site in the soluble methane monooxygenase from Methylococcus capsulatus (Bath) reduced by one electron at 77 K: the effects of component interactions and the binding of small molecules to the diiron(III) center.

Reduction of the soluble methane monooxygenase hydroxylase (MMOH) from Methylococcus capsulatus (Bath) in frozen 4:1 buffer/glycerol solutions at 77 K by mobile electrons generated by gamma-irradiation produces an EPR-detectable, mixed-valent Fe(II)Fe(III) center. At this temperature the conformation of the enzyme remains essentially unaltered during reduction, so the mixed-valent EPR spectra serve to probe the active site structure of the EPR-silent, diiron(III) state. The EPR spectra of the cryoreduced samples reveal that the diiron(III) cluster of the resting hydroxylase has at least two chemically distinct forms, the structures of which differ from that of the equilibrium Fe(II)Fe(III) site. Their relative populations depend on pH, the presence of component B, and formation of the MMOH/MMOB complex by reoxidation of the reduced, diiron(II) hydroxylase. The formation of complexes between MMOB, MMOR, and the oxidized hydroxylase does not measurably affect the structure of the diiron(III) site. Cryogenic reduction in combination with EPR spectroscopy has also provided information about interaction of MMOH in the diiron(III) state with small molecules. The diiron(III) center binds methanol and phenols, whereas DMSO and methane have no measurable effect on the EPR properties of cryoreduced hydroxylase. Addition of component B favors the binding of some exogenous ligands, such as DMSO and glycerol, to the active site diiron(III) state and markedly perturbs the structure of the diiron(III) cluster complexed with methanol or phenol. The results reveal different reactivity of the Fe(III)Fe(III) and Fe(II)Fe(III) redox states of MMOH toward exogenous ligands. Moreover, unlike oxidized hydroxylase, the binding of exogenous ligands to the protein in the mixed-valent state is allosterically inhibited by MMOB. The differential reactivity of the hydroxylase in its diiron(III) and mixed-valent states toward small molecules, as well as the structural basis for the regulatory effects of component B, is interpreted in terms of a model involving carboxylate shifts of a flexible glutamate ligand at the Fe(II)Fe(III) center.

Modulated growth of Saccharomyces cerevisiae by altering the driving force of the reactions of cytochrome c: Marcus' theory in vitro and in vivo.

According to Marcus' theory, rates of electron transfer reactions depend parabolically on the free energy of reaction. Amino acid replacements in the electron transport protein cytochrome c produced a series of proteins which changed the free energy of reaction for cytochrome c in oxidative phosphorylation. This study shows that Marcus' theory of electron transfer can be applied to the reactions of redox-altered cytochromes c with cytochrome c1 both in vitro and in vivo. In vitro, isolation of physiologically relevant partners of cytochrome c suggests that a change in free energy of reaction of cytochrome c changes the rate of electron transfer with cytochrome bc1 complex as would be predicted by Marcus' theory of electron transfer. Furthermore, the reactivity pattern observed in vitro is paralleled in in vivo studies. In vivo the rates of growth of Saccharomyces cerevisiae, in which these alternatives have been incorporated, also are consistent with the change in free energy of the reactions of cytochrome c with cytochrome bc1 complex. This study suggests that Marcus' theory of electron transport can predict rates not only in vitro, in isolated protein-protein systems, but also in vivo, where the relative growth rates of yeast may be predicted from the in vitro results.

Structural and functional effects of multiple mutations at distal sites in cytochrome c.

Multiple mutations at distally located sites have been introduced into yeast iso-1 cytochrome c to determine the contributions of three amino acids to the structural and functional properties of this protein. The mutant proteins, for which high-resolution structures were determined, included all possible combinations of the substitutions Arg38Ala, Asn52Ile, and Phe82Ser. Arg38, Asn52, and Phe82 are all conserved in the primary sequences of eukaryotic cytochromes c and have been shown to significantly affect several properties of these proteins including protein stability, heme reduction potential, and oxidation state dependent conformational changes. The present studies show that the structural consequences of each amino acid substitution in combinatorial mutant proteins were similar to those observed in the related single-mutant proteins, and therefore no synergistic effect between mutation sites was observed for this feature. With respect to protein stability, the effect of individual mutations can be understood from the structural changes observed for each. It is found that stability effects of the three mutation sites are independent and cumulative in multiple-mutant proteins. This reflects the independent nature of the structural changes induced at the three distally located mutation sites. In terms of heme reduction potential two effects are observed. For substitution of Phe82 by serine, the mechanism by which reduction potential is lowered is different from that occurring at either the Arg38 or the Asn52 site and is independent of residue replacements at these latter two positions. For Arg38 and Asn52, overlapping interactions lead to a higher reduction potential than expected from a strict additive effect of substitutions at these residues. This appears to arise from interaction of these two amino acids with a common heme element, namely, the heme propionate A group. The present results underscore the difficulty of predicting synergistic effects of multiple mutations within a protein.

Thermodynamics of the equilibrium unfolding of oxidized and reduced Saccharomyces cerevisiae iso-1-cytochromes c.

We report thermodynamic data for the chemical denaturation of iso-1-cytochromes c from Saccharomyces cerevisiae having amino acid substitutions R38A, N52I, and F82S in all possible combinations. The guanidine hydrochloride denaturation of isolated proteins was monitored by fluorescence measurements. The redox potentials, Eo', for both the folded and unfolded conformations have been measured. Free energy changes of chemical unfolding together with direct electrochemical measurement of the free energy changes of reduction for both the native and unfolded proteins yield a complete thermodynamic cycle, which includes four states of cytochrome c: oxidized folded, oxidized unfolded, reduced folded, and reduced unfolded. Completed cycles illustrate that the stability of cytochrome c to denaturing conditions is different for each amino acid substitution by an amount that depends on the heme oxidation state. Thus, the differential protein stability cannot be interpreted simply in terms of a hydrophobic effect, without also considering coupled Coulombic effects.

Electron transfer in cytochrome c depends upon the structure of the intervening medium.

BACKGROUND: Long-distance electron-transfer (ET) reactions through proteins are involved in a great many biochemical processes; however, the way in which the protein structure influences the rates of these reactions is not well understood. We have therefore measured the rates of intramolecular ET from the ferroheme to a bis(2,2'-bipyridine)imidazoleruthenium(III) acceptor at histidine 39 or 54 in derivatives of yeast iso-1-cytochrome c, and studied the effect of an asparagine to isoleucine mutation at position 52, a residue situated between the heme and the electron acceptor. RESULTS: The Fe2+-->Ru3+ rate constants demonstrate that residue 52 affects ET from the heme to His54 (Ile52 > Asn52), but not to His39 (Ile52 = Asn52). The enhanced Fe(2+)-Ru3+(His54) electronic coupling for the N52I/K54H protein is in good agreement with sigma-tunneling calculations, which predict the length of the ET pathways between the heme and His54. CONCLUSION: The structure of the intervening medium between the heme and electron acceptors at the protein surface influences the donor-acceptor couplings in cytochrome c.

Strand-specificity in the transformation of yeast with synthetic oligonucleotides.

Cyc1 mutants of the yeast Saccharomyces cerevisiae were directly transformed with both sense and antisense oligonucleotides to examine the involvement of the two genomic DNA strands in transformation. Sense oligonucleotides yielded approximately 20-fold more transformants than antisense oligonucleotides. This differential effect was observed with oligonucleotides designed to make alterations at six different sites along the gene and was independent of the oligonucleotide sequence and length, number of mismatches and the host strain. Competition studies showed that antisense oligonucleotides did not inhibit transformation. Although the mechanism for this strand specificity is unknown, this difference was maintained even when CYC1 transcription was diminished to approximately 2% of the normal level.