[Medical rehabilitation in patients with anal incompetence after surgery for stage IV hemorrhoids]. / Konservativnaya reabilitatsiya patsientov s nedostatochnost'yu anal'nogo sfinktera posle khirurgicheskogo lecheniya gemorroya IV stadii.

The choice of medical rehabilitation in patients with anal incontinence is impossible without diagnostic data revealing the mechanism of fecal incontinence. The most promising are programs of comprehensive physiotherapeutic rehabilitation based on biofeedback training. The rate of anal incompetence (AI) after hemorrhoidectomy is 1.3-12.5%. However, in addition to the organic cause (surgical trauma), functional disorders of the external sphincter and pelvic floor muscles may contribute to the pathogenesis of anal incontinence, aggravating the incontinence symptoms after surgery. Therefore, these functional disorders should be diagnosed before surgery. However, medical rehabilitation programs for anal incontinence after hemorrhoidectomy are not standardized, and functional outcomes have not been studied. OBJECTIVE: To evaluate the outcomes of comprehensive rehabilitation in patients with AI after hemorrhoidectomy to improve quality of life after surgery. MATERIALS AND METHODS: A retrospective study was carried out on 46 patients (mean age 53.8±15.4 years) after hemorrhoidectomy with fecal incontinence, 13 (28.3%) males and 33 (71.7%) females. The main group included 25 patients who received comprehensive rehabilitation, including biofeedback training and tibial neuromodulation (TNM) for 15 days. The control group consisted of 21 patients who received TNM at home also for 15 days. The severity of fecal incontinence was determined using the Wexner score. The functional state of the sphincter before and after surgery was assessed using the anorectal manometry (sphincterometry) (WPM Solar, the Netherlands). RESULTS: Comprehensive rehabilitation resulted in a statistically significant clinical improvement: a decrease in the Wexner score in both males and females. No significant differences in manometry results were observed: the anal sphincter tone increased by 16.0% in females and 10.6% in males, and contractility increased by 17.7% and 15.1%, respectively. Monotherapy with TNM in control group patients improved tone indices by 8.7% in females and 6.8% in males, and contractility by 6.2 and 5.4%, respectively, which was lower than in the main group. CONCLUSION: Contraindications to physiotherapeutic procedures based on electrical stimulation, extracorporeal magnetic stimulation, and magnetic translumbosacral neuromodulation determine the only possible choice of medical rehabilitation, which is the combination of biofeedback training and TNM (as superior to TNM monotherapy). If out-patient medical rehabilitation is not feasible, patients are recommended to complement the home course with a specially designed set of exercises for anal incontinence treatment.

Identification of large deletions in the APC gene in Russian patients with familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) is a hereditary syndrome characterized by the presence of multiple adenomatous polyps in the colon. The main cause of the disease is a germline mutation in the APC gene. Here we report 4 unrelated FAP patients with different large deletions in the APC gene detected by Multiplex Ligation-dependent Probe Amplification (MLPA) method: deletion of exons 7-15, deletion of promoters B, A, and 5'-UTR region and deletion of promoter B (in 2 patients). The deletion of promoters B, A, and 5'-UTR was not described in the literature earlier, so we report it for the first time. In 2 families with promoter B deletion, we could identify the tendency for decreasing the age of disease manifestation in each next generation, in contrast to the previous one. The incidence of large deletions in APC among Russian patients with FAP reached 4.8% and our finding suggests the need to study this gene by MLPA in "no mutation patients" after Sanger's sequencing.

[Pilot clinical and genetic study of Russian patients with Peutz-Jeghers syndrome].

Peutz-Jeghers syndrome is a rare hereditary syndrome characterized by presence of hamartoma polyps in intestinal tract and usually by mucocutaneous pigmentation. Clinical-genetic characteristics of Russian patients with Peutz-Jeghers syndrome were studied for the first time. Four germline mutations in STK11gene were found in probands from six families and three of them had not been described previously. Clinical pattern of disease in Russian patients included: frequent polyposis of colon and stomach (62,5% and 75%, respectively) along with small bowel; frequent presence of malignant tumors (62,5%). These clinical aspects can help physicians to find out Peutz-Jeghers syndrome. Molecular-genetic testing of individuals should be recommended.

Cell transplantation in surgical treatment of familial adenomatous polyposis coli.

We developed a method of surgical treatment of familial adenomatous polyposis coli giving an opportunity to prevent the growth of new polyps in the preserved part of the rectum and consisting in transplantation of fetal cells of the epithelial origin into the rectum wall after mucosectomy. Since the rectum is partially preserved, ileorectal anastomosis can be formed after colectomy, which preserves natural bowel passage. Complex examination 4 weeks after surgery revealed the formation of normal rectal mucosa. No new polyps were detected in the rectum 1-3 years after surgery.

Single-strand interruptions in replicating chromosomes cause double-strand breaks.

Replication-dependent chromosomal breakage suggests that replication forks occasionally run into nicks in template DNA and collapse, generating double-strand ends. To model replication fork collapse in vivo, I constructed phage lambda chromosomes carrying the nicking site of M13 bacteriophage and infected with these substrates Escherichia coli cells, producing M13 nicking enzyme. I detected double-strand breaks at the nicking sites in lambda DNA purified from these cells. The double-strand breakage depends on (i) the presence of the nicking site; (ii) the production of the nicking enzyme; and (iii) replication of the nick-containing chromosome. Replication fork collapse at nicks in template DNA explains diverse phenomena, including eukaryotic cell killing by DNA topoisomerase inhibitors and inviability of recombination-deficient vertebrate cell lines.

DNA replication meets genetic exchange: chromosomal damage and its repair by homologous recombination.

Proceedings of the National Academy of Sciences Colloquium on the roles of homologous recombination in DNA replication are summarized. Current findings in experimental systems ranging from bacteriophages to mammalian cell lines substantiate the idea that homologous recombination is a system supporting DNA replication when either the template DNA is damaged or the replication machinery malfunctions. There are several lines of supporting evidence: (i) DNA replication aggravates preexisting DNA damage, which then blocks subsequent replication; (ii) replication forks abandoned by malfunctioning replisomes become prone to breakage; (iii) mutants with malfunctioning replisomes or with elevated levels of DNA damage depend on homologous recombination; and (iv) homologous recombination primes DNA replication in vivo and can restore replication fork structures in vitro. The mechanisms of recombinational repair in bacteriophage T4, Escherichia coli, and Saccharomyces cerevisiae are compared. In vitro properties of the eukaryotic recombinases suggest a bigger role for single-strand annealing in the eukaryotic recombinational repair.

Recombinational repair of DNA damage in Escherichia coli and bacteriophage lambda.

Although homologous recombination and DNA repair phenomena in bacteria were initially extensively studied without regard to any relationship between the two, it is now appreciated that DNA repair and homologous recombination are related through DNA replication. In Escherichia coli, two-strand DNA damage, generated mostly during replication on a template DNA containing one-strand damage, is repaired by recombination with a homologous intact duplex, usually the sister chromosome. The two major types of two-strand DNA lesions are channeled into two distinct pathways of recombinational repair: daughter-strand gaps are closed by the RecF pathway, while disintegrated replication forks are reestablished by the RecBCD pathway. The phage lambda recombination system is simpler in that its major reaction is to link two double-stranded DNA ends by using overlapping homologous sequences. The remarkable progress in understanding the mechanisms of recombinational repair in E. coli over the last decade is due to the in vitro characterization of the activities of individual recombination proteins. Putting our knowledge about recombinational repair in the broader context of DNA replication will guide future experimentation.

Double-strand end repair via the RecBC pathway in Escherichia coli primes DNA replication.

To study the relationship between homologous recombination and DNA replication in Escherichia coli, we monitored the behavior of phage lambda chromosomes, repressed or not for lambda gene activities. Recombination in our system is stimulated both by DNA replication and by experimentally introduced double-strand ends, supporting the idea that DNA replication generates occasional double-strand ends. We report that the RecBC recombinational pathway of E. coli uses double-strand ends to prime DNA synthesis, implying a circular relationship between DNA replication and recombination and suggesting that the primary role of recombination is in the repair of disintegrated replication forks arising during vegetative reproduction.

Study of plasmid replication in Escherichia coli with a combination of 2D gel electrophoresis and electron microscopy.

We studied theta-mode DNA replication in p15A-based Escherichia coli plasmids by analyzing their replication intermediates using a combination of neutral agarose 2D gel electrophoresis and electron microscopy. Our analysis: (1) confirms the original assignment of various features of the 2D gel pattern; (2) shows that while one replication fork progresses around the plasmid DNA, the other is immobile, as if the replication were unidirectional; and (3) reveals that termination often occurs at a location away from the replication origin, suggesting that the replication of our plasmids is, in fact, bidirectional, the two forks being active at different times.

Stability of linear DNA in recA mutant Escherichia coli cells reflects ongoing chromosomal DNA degradation.

To study the fate of linear DNA in Escherichia coli cells, we linearized plasmid DNA at a specific site in vivo and monitored its behavior in recA mutant cells deficient in recombinational repair. Earlier, we had found that in wild-type (WT) cells linearized DNA is degraded to completion by RecBCD nuclease. We had also found that in WT cells chi sites on linear DNA inhibit RecBCD degradation by turning off its nucleolytic activities. Now we report that chi sites do not work in the absence of the RecA protein, suggesting that RecA is required in vivo to turn off the degradative activities of the RecBCD enzyme. We also report that the degradation of linearized plasmid DNA, even devoid of chi sites, is never complete in recA cells. Investigation of this linear DNA stability indicates that a fraction of recA cells are recBC phenocopies due to ongoing chromosomal DNA degradation, which titrates RecBCD nuclease. A possible role for RecBCD-promoted DNA degradation in controlling chromosomal DNA replication in E. coli is discussed.

Annealing vs. invasion in phage lambda recombination.

Genetic recombination catalyzed by lambda's Red pathway was studied in rec+ and recA mutant bacteria by examining both intracellular lambda DNA and mature progeny particles. Recombination of nonreplicating phage chromosomes was induced by double-strand breaks delivered at unique sites in vivo. In rec+ cells, cutting only one chromosome gave nearly maximal stimulation of recombination; the recombinants formed contained relatively short hybrid regions, suggesting strand invasion. In contrast, in recA mutant cells, cutting the two parental chromosomes at non-allelic sites was required for maximal stimulation; the recombinants formed tended to be hybrid over the entire region between the two cuts, implying strand annealing. We conclude that, in the absence of RecA and the presence of non-allelic DNA ends, the Red pathway of lambda catalyzes recombination primarily by annealing.

Unraveling the late stages of recombinational repair: metabolism of DNA junctions in Escherichia coli.

DNA junctions are by-products of recombinational repair, during which a damaged DNA sequence, assisted by RecA filament, invades an intact homologous DNA to form a joint molecule. The junctions are three-strand or four-strand depending on how many single DNA strands participate in joint molecules. In E. coli, at least two independent pathways to remove the junctions are proposed to operate. One is via RuvAB-promoted migration of four-strand junctions with their subsequent resolution by RuvC. In vivo, RuvAB and RuvC enzymes might work in a single complex, a resolvasome, to clean DNA from used RecA filaments and to resolve four-strand junctions. An alternative pathway for junction removal could be via RecG-promoted branch migration of three-strand junctions, provided that an as yet uncharacterized endonuclease activity incises one of the strands in the joint molecules.

A mechanism for induction of the SOS response in E. coli: insights into the regulation of reversible protein polymerization in vivo.

During normal DNA replication, RecA, the principal recombinational repair enzyme of E. coli, cannot assemble its filament on SSB-bound single-stranded DNA at the replication forks. This behavior is paralleled in vitro, where at low Mg2+ concentrations RecA can not polymerize on SSB-bound single-stranded DNA. Inhibition of DNA replication in vivo renders RecA able to polymerize on SSB-bound single-stranded DNA and to activate the SOS response. Although the mechanism of SOS induction is still obscure, abundant in vitro observations indicate that RecA filament formation on SSB-bound single-stranded DNA is facilitated at elevated concentrations of ATP, Mg2+ and spermidine. It is proposed here that inhibition of DNA synthesis in vivo leads to a similar accumulation of ATP and its counter-ions, Mg2+ and spermidine, resulting ultimately in SOS induction. When DNA synthesis is restored, the concentration of ATP, Mg2+ and spermidine returns to normal levels, favoring RecA depolymerization. On the basis of the known structure of RecA, a mechanism for reversible RecA polymerization is presented. In a RecA polymer, the monomers are known to interact with each other primarily through hydrophobic, oppositely charged surfaces. In conditions suboptimal for polymerization, these hydrophobic surfaces of the monomers are possibly masked by electrostatic interactions with other, oppositely charged domains of the monomers. There are known recombinational repair proteins whose specific functions are likely to assist in RecA polymerization or depolymerization. Features of reversible polymerization of eukaryotic proteins tubulin and actin are consistent with the possibility that RecA exploits a general principle for the regulation of reversible protein polymerization.

Instability of inhibited replication forks in E. coli.

Inhibiting the progress of replication forks in E. coli makes them susceptible to breakage. Broken replication forks are evidently reassembled by the RecBCD recombinational repair pathway. These findings explain a particular pattern of DNA degradation during inhibition of chromosomal replication, the role of recombination in the viability of mutants with displaced replication origin, and hyper-recombination observed in the Terminus of the E. coli chromosome in rnh mutants. Breakage and repair of inhibited replication forks could be the reason for the recombination-dependence of inducible stable DNA replication. A mechanism by which RecABCD-dependent recombination between very short inverted repeats may help E. coli to invert an operon, transcribed in the direction opposite to that of DNA replication, is discussed.

The recombination hot spot chi activates RecBCD recombination by converting Escherichia coli to a recD mutant phenocopy.

The products of the recB and recC genes are necessary for conjugal recombination and for repair of chromosomal double-chain breaks in Escherichia coli. The recD gene product combines with the RecB and RecC proteins to comprise RecBCD enzyme but is required for neither recombination nor repair. On the contrary, RecBCD enzyme is an exonuclease that inhibits recombination by destroying linear DNA. The RecD ejection model proposes that RecBCD enzyme enters a DNA duplex at a double-chain end and travels destructively until it encounters the recombination hot spot sequence chi. Chi then alters the RecBCD enzyme by weakening the affinity of the RecD subunit for the RecBC heterodimer. With the loss of the RecD subunit, the resulting protein, RecBC(D-), becomes deficient for exonuclease activity and proficient as a recombinagenic helicase. To test the model, genetic crosses between lambda phage were conducted in cells containing chi on a nonhomologous plasmid. Upon delivering a double-chain break to the plasmid, lambda recombined as if the cells had become recD mutants. The ability of chi to alter lambda recombination in trans was reversed by overproducing the RecD subunit. These results indicate that chi can influence a recombination act without directly participating in it.

Collapse and repair of replication forks in Escherichia coli.

Single-strand interruptions in a template DNA are likely to cause collapse of replication forks. We propose a model for the repair of collapsed replication forks in Escherichia coli by the RecBCD recombinational pathway. The model gives reasons for the preferential orientation of Chi sites in the E. coli chromosome and accounts for the hyper-rec phenotype of the strains with increased numbers of single-strand interruptions in their DNA. On the basis of the model we offer schemes for various repeat-mediated recombinational events and discuss a mechanism for quasi-conservative DNA replication explaining the recombinational repair-associated mutagenesis.

Chi sites in combination with RecA protein increase the survival of linear DNA in Escherichia coli by inactivating exoV activity of RecBCD nuclease.

In Escherichia coli, unprotected linear DNA is degraded by exoV activity of the RecBCD nuclease, a protein that plays a central role in the repair of double-strand breaks. Specific short asymmetric sequences, called chi sites, are hotspots for RecBCD-promoted recombination and are shown in vitro to attenuate exoV activity. To study RecBCD-chi site interactions in vivo we used phage lambda's terminase to introduce a site-specific double-strand break at lambda's cos site inserted into a plasmid. We show that after terminase has cut cos in vivo, nucleases degrade linearized DNA only from the end that does not have a strong terminase binding site. Linearized cosmid DNA containing chi sites in the proper orientation to the unprotected end is degraded more slowly in rec+ E. coli than is chi-less DNA. Increased survival of chi-containing DNA is a result of partial inactivation of exoV activity and is dependent on RecA and SSB proteins. The linearization of chi-containing DNA molecules leads to RecA-dependent formation of branched structures which have been proposed as intermediates in the RecBCD pathway of double-strand break repair.

RuvA, RuvB and RuvC proteins: cleaning-up after recombinational repairs in E. coli.

After the completion of RecA protein-mediated recombinational repair of daughter-strand gaps in E. coli, participating chromosomes are held together by Holliday junctions. Until recently, it was not known how the cell disengages the connected chromosomes. Accumulating genetic data suggested that the product of the ruv locus participates in recombinational repair and acts after the formation of Holliday junctions. Molecular characterization of the locus revealed that there are three genes--ruvA, ruvB and ruvC; mutations in any one of the genes confer the same phenotype. Recently, the RuvC protein was found to be a Holliday junction resolvase. At first glance, the resolving activity of RuvC alone would appear to be sufficient for the separation of recombining chromosomes. However, in vitro studies show that the filament of RecA protein is unable to dissociate from the products of the recombination reaction. Thus, in vivo, even if the Holliday junctions are resolved by RuvC, RecA filament must be holding two DNA duplexes together. New findings about enzymatic activities of RuvA and RuvB proteins foster the hope that the machinery for removing the RecA filament from DNA has been found.

[Congenital angiodysplasias of the large intestine]. / Vrozhdennye angiodisplazii tolstoi kishki.

Angiodysplasia is a rare disease of the intestinal vessels. One of its types is the congenital form (so-called hemangioma or vascular hamartoma) which is mostly encountered at young age. Forty-eight patients with congenital forms of angiodysplasia of the large intestine were treated at the Scientific Research Institute of Proctology, RF Ministry of Health, beginning from 1962. According to morphological structure, there were 26 cases of the cavernous type, 11 of the capillary type, 11 of the mixed forms. In 46 of 48 patients the rectum and sigmoid were involved in the pathological process. Intestinal bleeding of various degree and intensity, occurring in 40 patients from early childhood, was the principal symptom in the clinical picture of the disease. The endoscopic picture of congenital angiodysplasia of the large intestine has characteristic macroscopic signs. Irrigoscopy and angiography are also very valuable and informative methods in the diagnosis of this form. Surgical interventions were performed on 28 patients, which accounted for 58.3% of the total number of patients with congenital angiodysplasia. Profuse bleeding leading to stable anemization of the patient is an indication for surgery. The volume of the operation was determined by the localization and spread of the pathological process. In concurrent localization of dysplasias, particularly in the pelvic cavity, and bearing in mind the seriousness of the operation itself, the degree of intraoperative blood loss, and the technical peculiarities of the intervention we prefer many-stage treatment. We encountered 18 complications, which mainly developed on the part of the perineum.