Threshold regulation and stochasticity from the MecA/ClpCP proteolytic system in Streptococcus mutans competence.

Many bacterial species use the MecA/ClpCP proteolytic system to block entry into genetic competence. In Streptococcus mutans, MecA/ClpCP degrades ComX (also called SigX), an alternative sigma factor for the comY operon and other late competence genes. Although the mechanism of MecA/ClpCP has been studied in multiple Streptococcus species, its role within noisy competence pathways is poorly understood. S. mutans competence can be triggered by two different peptides, CSP and XIP, but it is not known whether MecA/ClpCP acts similarly for both stimuli, how it affects competence heterogeneity, and how its regulation is overcome. We have studied the effect of MecA/ClpCP on the activation of comY in individual S. mutans cells. Our data show that MecA/ClpCP is active under both XIP and CSP stimulation, that it provides threshold control of comY, and that it adds noise in comY expression. Our data agree quantitatively with a model in which MecA/ClpCP prevents adventitious entry into competence by sequestering or intercepting low levels of ComX. Competence is permitted when ComX levels exceed a threshold, but cell-to-cell heterogeneity in MecA levels creates variability in that threshold. Therefore, MecA/ClpCP provides a stochastic switch, located downstream of the already noisy comX, that enhances phenotypic diversity.

A prospective non-randomized two-centre study of patients with passive faecal incontinence after birth trauma and patients with soiling after anal surgery, treated by elastomer implants versus rectal irrigation.

AIM: This study is a prospective evaluation of patients with passive faecal incontinence and patients with soiling treated by elastomer implants and rectal irrigation. PATIENTS AND METHODS: Patients with passive faecal incontinence after birth trauma resulting from a defect of the internal sphincter and patients with soiling after previous anal surgery were included. All patients underwent endo-anal ultrasound, magnetic resonance imaging, and anal manometry. The patients with passive faecal incontinence were initially treated by anal sphincter exercises and biofeedback therapy during half a year. The patients completed incontinence scores, a quality of life questionnaire, and a 2-week diary card. RESULTS: The elastomer group consisted of 30 males and 45 females with a mean age of 53 years (25-77). The rectal irrigation group consisted of 32 males and 43 females with a mean age of 50 years (25-74). At 6 months follow-up, 30 patients with soiling of the rectal irrigation group and only nine patients of the elastomer group were completely cured (p = 0.02). Only three patients with passive faecal incontinence were cured in the rectal irrigation group and none in the elastomer group. Three distal migrations of elastomer implants required removal at follow-up. CONCLUSIONS: After patients had performed anal sphincter exercises, no clear improvement of passive faecal incontinence was obtained by elastomer implants or rectal irrigation. However, rectal irrigation is far more effective than elastomer implants in patients with soiling.

Transvaginal posterior colporrhaphy combined with laparoscopic ventral mesh rectopexy for isolated Grade III rectocele: a prospective study of 27 patients.

AIM: The aim of this study was to evaluate prospectively transvaginal posterior colporrhaphy (TPC) combined with laparoscopic ventral mesh rectopexy (LVR) in patients with a symptomatic isolated rectocele. METHOD: Patients with these complaints underwent dynamic and static MRI. All consecutive patients with a Grade III (4 cm or more) rectocele and without internal/external rectal prolapse, enterocele and external sphincter damage were operated on. The patients completed the Obstructed Defecation Syndrome (ODS) score and the Cleveland Clinic Incontinence Score (CCIS). All tests were repeated after treatment. Dynamic disorders of the pelvic floor detected by MRI were recorded. RESULTS: In 27 patients [median age 67 (46-73) years], TPC combined with LVR was feasible. Complications were limited to port site infection in two patients. Sexual discomfort (n = 8) due to prolapse diminished in six (75%) patients and in one (4%) de novo dyspareunia developed after treatment. The median follow-up was 12 (10-18) months. The median CCIS was 12 (10-16) before treatment and 8 (7-10) after (P < 0.0001). The median ODS score was 19 (17-23) before and 6 (3-10) after treatment (P < 0.0001). There was no change in urinary symptoms. CONCLUSION: TPC combined with LVR for obstructed defaecation and faecal incontinence in patients with Grade III rectocele significantly relieves the symptoms of these disorders.

Autologous platelet-derived growth factors (platelet-rich plasma) as an adjunct to mucosal advancement flap in high cryptoglandular perianal fistulae: a pilot study.

AIM: The aim of this study was to explore autologous platelet-rich plasma as an adjunct to the staged mucosal advancement flap in the treatment of perianal fistulae. METHOD: Between February 2006 and May 2007, 10 patients with fistula tracts transversing from the middle-third or upper part of the anal sphincter were treated for at least 3 months with noncutting setons prior to definitive closure by autologous platelet-rich plasma as an adjunct to a mucosal advancement flap. Five patients smoked tobacco. RESULTS: The study group consisted of six women and four men with a median age of 44 (range 30-75) years and a median follow up of 26 (range 17-32) months. One (10%) patient had a recurrent fistula. No new continence disorders developed after definitive treatment in both groups. CONCLUSION: Platelet-rich plasma as an adjunct to a staged mucosal advancement flap for the treatment of perianal cryptoglandular fistulae is a promising treatment modality and seems to establish a high healing rate.

Conservative treatment of patients with faecal soiling.

PURPOSE: A prospective evaluation of fifty patients with faecal soiling but normal sphincter function treated by a conservative treatment algorithm. PATIENTS AND METHODS: Between January 2010 and January 2011, 50 consecutive patients of two different clinical centres, with faecal soiling and normal anorectal function as assessed by endoanal ultrasound, MRI and anal manometry, were eligible for the purpose of this study. All patients started the therapy by psyllium (PS) and a fibre-rich diet daily after 2 months followed by rectal irrigation (RI) in case of incomplete response and after 4 months by 4 g colestyramine (CO), respectively. The patients completed the Vaizey incontinence score and a 2-week diary card. All tests were performed repeated after 2, 4 and 8 months, respectively. RESULTS: The study group consisted of 41 men and 9 women and a mean age of 38 years (21-70). The soiling complaints resolved completely in 37 (79%) patients. After treatment with PS, RI and CO, 12 (24%) patients, 24 (73%) patients and 1 (79%) patient, respectively, resolved completely of faecal soiling. Average weekly soiling frequency, the amount of patients wearing pads daily and the Vaizey incontinence score diminished significantly after treatment with psyllium and after treatment with rectal irrigation (P < 0.001). CONCLUSION: Conservative treatment focussed on complete evacuation or clearing the anorectal canal is effective in the treatment of patients with faecal soiling.

Characterization of monoclonal antibodies to Acanthamoeba myosin-I that cross-react with both myosin-II and low molecular mass nuclear proteins.

We characterized nine monoclonal antibodies that bind to the heavy chain of Acanthamoeba myosin-IA. Eight of these antibodies bind to myosin-IB and eight cross-react with Acanthamoeba myosin-II. All but one of the antibodies bind to a 30-kD chymotryptic peptide of myosin-IA that derives from the COOH terminus of the molecule, and to tryptic peptides as small as 17 kD, hence these epitopes are clustered closely together on the heavy chain. None of the antibodies prevent heavy chain phosphorylation by myosin-I heavy chain kinase. One antibody inhibits the K+-EDTA ATPase activity and three antibodies inhibit the actin-activated Mg++-ATPase activity of myosin-I under the set of conditions that we tested. When fluorescent antibody staining of both whole cells and isolated nuclei is done, several of these monoclonal antibodies react strongly with nuclei. These antibodies also stain the cytoplasmic matrix, especially the cortex near the plasma membrane. All nine of the monoclonal antibodies bind to polypeptides of 30-34 kD that are highly enriched in nuclei isolated from Acanthamoeba. There is no myosin-I in the isolated nuclei, so the 30-34-kD polypeptides, not myosin-I, are responsible for the nuclear staining.

Handedness: basic physics.

In the News & Comment article "Genetic testing set for takeoff" by Rachel Nowak (22 July, p. 464), Michael Liskay of Oregon Health Sciences University in Portland should have been named as the senior scientist on one of the teams that identified MLH1. The test for Charcot-Marie-Tooth disease detects the duplication of the PMP22 gene, not a deletion, as stated in the table accompanying the article (p. 466).

Protein reaction kinetics in a room-temperature glass.

Protein reaction kinetics in aqueous solution at room temperature are often simplified by the thermal averaging of conformational substates. These substates exhibit widely varying reaction rates that are usually exposed by trapping in a glass at low temperature. Here, it is shown that the solvent viscosity, rather than the low temperature, is primarily responsible for the trapping. This was demonstrated by placement of myoglobin in a glass at room temperature and subsequent observation of inhomogeneous reaction kinetics. The high solvent viscosity slowed the rate of crossing the energy barriers that separated the substates and also suppressed any change in the average protein conformation after ligand dissociation.

Expression of murine CD1 on gastrointestinal epithelium.

Cluster of differentiation 1 (CD1) in humans is a family of major histocompatibility complex (MHC) class I-like molecules expressed on the surface of immature thymocytes, Langerhans cells, and a subpopulation of B cells. The only function identified for human CD1 is as a ligand recognized by a subpopulation of T lymphocytes. In order to study the distribution and function of these molecules in the mouse, a murine CD1 complementary DNA was expressed in mouse fibroblasts and used to produce monoclonal antibodies. These antibodies revealed prominent expression of murine CD1 only on gastrointestinal tract epithelium and in the cytoplasm of hepatocytes. Low levels of expression were also detected on thymocytes and peripheral lymphocytes. The gastrointestinal distribution of murine CD1 suggests that this molecule may be important in epithelial immunity.

Epithelial basement membrane of mouse jejunum. Evidence for laminin turnover along the entire crypt-villus axis.

Little is known regarding turnover of the epithelial basement membrane in adult small intestine. Are components degraded and inserted along the length of the crypt-villus axis or selectively in the crypt region with subsequent migration of basement membrane from crypt to villus tip in concert with epithelium? We injected affinity-purified sheep anti-laminin IgG or sheep anti-laminin IgG complexed to horseradish peroxidase (HRP) into mice to label basement membrane laminin in vivo. Fluorescence microscopy revealed linear fluorescence along the length of the jejunal epithelial basement membrane 1 d after anti-laminin IgG injection. By 1 wk, small nonfluorescent domains were interposed between larger fluorescent domains. Over the next 5 wk the lengths of nonfluorescent domains increased progressively whereas those of fluorescent domains decreased. Additionally, electron microscopy revealed HRP reaction product along the length of the epithelial basement membrane after 1 d whereas unlabeled or lightly labeled domains that increased in length with time were observed interposed between heavily labeled domains by 2 and 4 wk along the entire crypt-villus axis. We conclude that laminin turnover occurs focally in the epithelial basement membrane of mouse jejunum along the crypt-villus axis over a period of weeks and that this basement membrane does not comigrate in concert with its overlying epithelium.

Two-state expansion and collapse of a polypeptide.

The initial phase of folding for many proteins is presumed to be the collapse of the polypeptide chain from expanded to compact, but still denatured, conformations. Theory and simulations suggest that this collapse may be a two-state transition, characterized by barrier-crossing kinetics, while the collapse of homopolymers and random heteropolymers is continuous and multi-phasic. A new rapid-mixing flow technique has been used to resolve the late stages of polypeptide collapse, at time scales >/=45 microseconds. We have used a laser temperature-jump with fluorescence spectroscopy to resolve the complete time-course of the collapse of denatured cytochrome c with nanosecond time resolution. We find the process to be exponential in time and thermally activated, with an apparent activation energy approximately 9 k(B)T (after correction for solvent viscosity). These results indicate that polypeptide collapse is kinetically a two-state transition. Because of the observed free energy barrier, the time scale of polypeptide collapse is dramatically slower than is predicted by Langevin models for homopolymer collapse.

Two-state expansion and collapse of a polypeptide.

The initial phase of folding for many proteins is presumed to be the collapse of the polypeptide chain from expanded to compact, but still denatured, conformations. Theory and simulations suggest that this collapse may be a two-state transition, characterized by barrier-crossing kinetics, while the collapse of homopolymers is continuous and multi-phasic. We have used a laser temperature-jump with fluorescence spectroscopy to measure the complete time-course of the collapse of denatured cytochrome c with nanosecond time resolution. We find the process to be exponential in time and thermally activated, with an apparent activation energy approximately 9 k(B)T (after correction for solvent viscosity). These results indicate that polypeptide collapse is kinetically a two-state transition. Because of the observed free energy barrier, the time scale of polypeptide collapse is dramatically slower than is predicted by Langevin models for homopolymer collapse.

Rate of intrachain contact formation in an unfolded protein: temperature and denaturant effects.

We have measured the effect of temperature and denaturant concentration on the rate of intrachain diffusion in an unfolded protein. After photodissociating a ligand from the heme iron of unfolded horse cytochrome c, we use transient optical absorption spectroscopy to measure the time scale of the diffusive motions that bring the heme, located at His18, into contact with its native ligand, Met80. Measuring the rate at which this 62 residue intrachain loop forms under both folding and unfolding conditions, we find a significant effect of denaturant on the chain dynamics. The diffusion of the chain accelerates as denaturant concentration decreases, with the contact formation rate approaching a value near approximately 6x10(5) s(-1) in the absence of denaturant. This result agrees well with an extrapolation from recent loop formation measurements in short synthetic peptides. The temperature dependence of the rate of contact formation indicates an Arrhenius activation barrier, Ea approximately 20 kJ/mol, at high denaturant concentrations, comparable to what is expected from solvent viscosity effects alone. Although Ea increases by several kBT as denaturant concentration decreases, the overall rate of diffusion nevertheless increases. These results indicate that inter-residue energetic interactions do not control conformational diffusion in unfolded states, even under folding conditions.

Immunocytochemical localization of actin in epithelial cells of rat small intestine by light and electron microscopy.

We used post-embedding immunocytochemical techniques and affinity-purified anti-actin antibody to evaluate localization of actin in epithelial cells of small intestine by fluorescence and electron microscopy. Small intestine was fixed with 2% formaldehyde-0.1% glutaraldehyde and embedded in Lowicryl K4M. One-micron or thin sections were stained with antibody followed by rhodamine- or colloidal gold-labeled goat anti-rabbit IgG, respectively. Label was present overlying microvilli, the apical terminal web, and the cytoplasm directly adjacent to occluding and intermediate junctions. Label was associated with outer mitochondrial membranes of all cells and the supranuclear Golgi region of goblet cells. Lateral cytoplasmic interdigitations between mature cells and subplasmalemmal filaments next to intrusive cells were densely labeled. The cytoplasm adjacent to unplicated domains of lateral membrane was focally labeled. Label was prominent over organized filament bundles within the subplasmalemmal web at the base of mature cells, whereas there was focal labeling of the cytoplasm adjacent to the basal membrane of undifferentiated cells. Basolateral epithelial cell processes were labeled. Label was focally present overlying the cellular ground substance. Our results demonstrate that actin is distributed in a distinctive fashion within intestinal epithelial cells. This distribution suggests that in addition to its function as a structural protein, actin may participate in regulation of epithelial tight junction permeability, in motile processes including migration of cells from the crypt to the villus tip, in accommodation of intrusive intraepithelial cells and in adhesion of cells to one another and to their substratum.

Acidic conditions ameliorate both adenosine triphosphate depletion and the development of hyperpermeability in cultured Caco-2BBe enterocytic monolayers subjected to metabolic inhibition.

BACKGROUND: We recently reported that moderate degrees of adenosine triphosphate (ATP) depletion induced by chronic glycolytic inhibition or hypoxia increase the permeability of Caco-2BBe enterocytic monolayers. Interestingly, the development of lactic acidosis induced by anaerobic glycolysis ameliorates the development of hyperpermeability caused by chronic ATP depletion. We sought to further elucidate the mechanism(s) responsible for the apparent protection against epithelial hyperpermeability afforded by mild acidosis under conditions of metabolic inhibition. METHODS: Caco-2BBe monolayers growing on permeable supports in bicameral chambers were incubated with 2-deoxyglucose (2DOG) in a glucose-free (Glu-) environment to inhibit glycolysis. Permeability was determined by measuring the transepithelial flux of fluorescein sulfonic acid. Concentrations of intracellular calcium [Ca2+]i were determined fluorometrically by using fura-2. RESULTS: When extracellular pH (pH0) was maintained at 7.4 or 5.5, incubation of monolayers for 24 hours with Glu-/2DOG increased permeability and depleted intracellular ATP levels. However, keeping pH0 at 7.0 to 6.0 ameliorated both the development of hyperpermeability and the depletion of ATP induced by Glu-/2DOG. These protective effects were observed under acidic conditions created either by addition to the medium of HCl or by incubation under an atmosphere containing 20% CO2. Incubation with Glu-/2DOG caused bulging of the apical membranes of cells (electron microscopy) and derangements in the perijunctional distribution of actin (fluorescence microscopy); however, these structural changes were ameliorated by mild acidosis. Acute chemical hypoxia at pH0 7.4 induced by Glu-/2DOG plus antimycin A decreased cellular ATP levels and elevated [Ca2+]i. Lowering pH0 to 6.8 ameliorated both the depletion of ATP and the increase in [Ca2+]i induced by Glu-/2DOG+antimycin A. CONCLUSIONS: Moderate decreases in pH ameliorate the hyperpermeability induced by metabolic inhibition, possibly by diminishing ATP depletion and blunting increases in [Ca2+]i.

Comments on the physics and chemistry of trehalose as a storage medium for hemoglobin-based blood substitutes: "from Kramers Theory to the Battlefield".

A glass of the naturally-occurring sugar trehalose may be a suitable medium for the storage of hemoglobin-based blood substitutes. Trehalose has many or possibly all of the properties required for this purpose, including solubilization of hemoglobin to a very high concentration, lack of toxicity, slowing of oxidation to the non-oxygen binding methemoglobin, stability at room temperature and above, and ease of transport. It should also be possible to prepare hemoglobin extremely rapidly for injection into the circulation in situations where blood replacement is required immediately, as in a domestic emergency room or on the battlefield. These practical considerations are briefly discussed, as well as the theoretical reasons for slowing of chemical reactions in the glassy state.

Staged Mucosal Advancement Flap versus Staged Fibrin Sealant in the Treatment of Complex Perianal Fistulas.

Background. In this prospective randomised study, the staged mucosal advancement flap is compared with staged fibrin sealant application in the treatment of perianal fistulas. Methods. All patients with high complex cryptoglandular fistulas were randomised to closure of the internal opening by a mucosal advancement flap (MF) or injection with fibrin sealant (FS) after treatment with setons. Recurrence rate and incontinence disorders were explored. Results. The MF group (5 females and 10 males) with a median age of 51 years and a median followup of 52 months. The FS group (4 females and 11 males) with a median age of 45 years and a median followup of 49 months. Three (20%) patients of the MF group had a recurrent fistula compared to 9 (60%) of the FS group (P = 0.03). No new continence disorders developed. Conclusion. Staged FS injection has a much lower success rate compared to MF.

Impairment by allyl isothiocyanate of gastric epithelial wound repair through inhibition of ion transporters.

Isothiocyanate is a transient receptor potential ankyrin 1 (TRPA1) agonist and also an inhibitor of ion transporters such as anion exchanger (AE) and Na(+)/HCO(3)(-) cotransporter (NBC). We examined the expression of TRPA1 and ion transporters in monolayers of the rat gastric epithelial cell line RGM1 and investigated the involvement of these factors in the inhibitory action of isothiocyanate on epithelial wound healing. After obtaining a confluent monolayer, a round artificial wound of constant size was induced in the center of the cell monolayer using a pencil-type mixer with a rotating silicon tip. Immediately after the wound induction, cells at the edge of the wound started to form lamellipodia, migrating towards the center of wound, and the cell-free area decreased with time. Addition of allyl isothiocyanate to standard buffer suppressed the recovery of the wound in a concentration-dependent manner without affecting the viability of the RGM1 cells. Icilin, another TRPA1 agonist, dose-dependently inhibited wound repair. Likewise, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a stilbene compound containing an isothiocyanate group, also inhibited the recovery of epithelial wounds. In addition, the repair of epithelial wounds was suppressed when the cells were incubated in Na(+), Cl(-) or HCO(3)(-) free buffer. The RGM1 cells expressed the mRNAs of AE2a and NBC1 but not TRPA1. These results suggested that isothiocyanate impairs the repair of epithelial wounds in RGM1 cells, probably through the inhibition of ion transporters such as AE2a and NBC1 and not the activation of the TRPA1 channel. It is assumed that the process of epithelial repair is associated with the regulation of cell volume and intracellular pH (pHi) by these ion transporters.

Long-term outcome following mucosal advancement flap for high perianal fistulas and fistulotomy for low perianal fistulas: recurrent perianal fistulas: failure of treatment or recurrent patient disease?

BACKGROUND: In this study, we determined the long-term outcome of perianal fistulas treated with mucosal advancement flap (MF) or fistulotomy (FT). METHODS: One hundred three patients with perianal fistulas were treated by MF for high fistulas or FT for low fistulas and were retrospectively assessed by case-note review and examined at the out-patient clinic. The localization and time of recurrence of the fistula were recorded. RESULTS: Forty-one patients [median follow-up of 72 months (range 48-99)] were treated by an MF, and 62 patients [median follow up of 75 months (range 48-99)] were treated by FT. After 12, 48, and 72 months, the fistula had recurred in 9 (22%), 26 (63%), and 26 (63%) patients of the MF group and in 4 (7%), 16 (26%), and 24 (39%) patients of the FT group, respectively. Eighteen (69%) of the recurrences in the MF group and ten (33%) of the FT group occurred within 24 months after surgery (p=0.01). Four (15%) of the recurrences in the MF group and 13 (54%) of the recurrences in the FT group were present in a different localization (p=0.007). CONCLUSION: The success rate of both FT and MF techniques decreases with time. Recurrence appears to be caused by failure of treatment and by recurrent patient disease.

Staged mucosal advancement flap for the treatment of complex anal fistulas: pretreatment with noncutting Setons and in case of recurrent multiple abscesses a diverting stoma.

OBJECTIVE: To assess the efficacy of a staged strategy for the treatment of complex perianal fistula. METHODS: Between January 1999 and April 2003 all consecutive patients with complex perianal fistulas were treated according to a staged strategy. Fistula tracks originating from the middle third or upper part of the anal sphincter were included. Patients were examined for recurrent fistulas and complaints of incontinence and soiling. Initial treatment consisted of a noncutting seton with or without a diverting stoma. Definitive surgical treatment consisted of an advancement flap or fistulotomy. RESULTS: Thirty patients were included (median age; 42 years, range 22-68 years). Seven had Crohn's disease without signs of rectal and anal involvement other than the fistula. At a median follow up of 22 months (range 8-52 months) in 29 (97%) patients, the wounds had healed completely; 7 (22%) patients subsequently developed a recurrent fistula and minor soiling occurred in 7 (23%) patients. CONCLUSION: Initial treatment with a seton with and without a diverting stoma minimizing inflammatory activity at the fistula site before definitive surgical treatment gave good results in this difficult group of patients.