In-vivo imaging of the microvasculature of the soft tissue margins of osteonecrotic jaw lesions.

Introduction Given the increasing incidence of medication-related jaw osteonecrosis, and recognition of the mucosal blood supply's importance, we have developed a non-invasive Real Time Optical Vascular Imaging (RTOVI) instrument. Imaging the red blood cells within the sub-mucosal capillary networks demonstrates the microcirculatory anatomy. We report a small trial, demonstrating the technique's viability, examining mucosal microcirculatory changes adjacent to osteonecrotic lesions.Aims Imaging the microvasculature of soft tissue margins of patients' exposed necrotic bone lesions in situ was intended to provide unique observational as well as quantitative data, using an image analysis routine, based on ImageJ software. Our interest was to evaluate whether this could offer valuable information for complex wound margin management.Methods Four osteoradionecrosis and four medication-related osteonecrosis patients (M:F 1:1 mean 68.25 years) were enrolled under the NRES Ethics 11/LON/0354 and KCL Research Ethics Committee (REC) BDM/14/15-14 approvals. Microvascular images from mucosal margins of exposed mandibular osteonecrosis lesions were compared with equivalent images from both uninvolved contralateral mucosa and similar mucosal sites in four healthy subjects.Results We demonstrated narrow hypo-vascularised oedematous lesion margins surrounded by a concentric inflammatory band and normal mucosa beyond. Parameters reporting individual capillary shape, via mean percentage of occupancy per capillary per field of view and capillary loop aspect ratio, differed significantly between groups (ANOVA, p = 0.0002 and p = 0.04 respectively). Values reporting capillary number and area showed expected changes but did not reach statistical significance.Conclusion This pilot study demonstrated the feasibility of mucosal microvascular imaging in assessing the microvascular changes found in the soft tissues at the margins of osteonecrotic lesions, with potential to inform therapeutic interventions and clinical decisions to continue or modify regime strategies at the earliest opportunity. Given the increasing incidence of medication-related jaw osteonecrosis, and the recognition of the importance of mucosal blood supply, we developed a non-invasive instrument demonstrating microcirculation anatomy by imaging transiting red blood cells.

Rapid Bacterial Detection during Endodontic Treatment.

Bacteria present in the root canal (RC) space following an RC treatment (RCT) can lead to persistent infections, resulting in treatment failure and the need for reintervention or extraction. Currently, there are no standardized methods in use to clinically detect bacterial presence within RC spaces. The use of paper point sampling and fluorescence staining was shown to be a rapid method, able to detect residual bacteria following treatment. The study demonstrated that Calcein acetoxymethyl (AM) proved to be a suitable dye for detecting vital bacteria within mature endodontic biofilms, with an improved sensitivity over colony-forming unit counting in a stressed biofilm model. Furthermore, in a clinical trial with primary RCTs, 53 infected teeth were sampled in vivo, and increased detection of vital cells was found when compared with colony-forming unit counting, highlighting the sensitivity of the technique in detecting low cell numbers. By combining fluorescent staining and microspectroscopy with software-based spectral analysis, successful detection of vital cells from RCs was possible after 5 min of Calcein AM incubation. Application of this technology during RCT has the potential to reduce persistent infections through vital cell detection and additional treatment. Furthermore, this technique could be applied to antimicrobial research and disinfection control in clinical settings ( ClinicalTrials.gov NCT03055975).

Detecting intratumoral heterogeneity of EGFR activity by liposome-based in vivo transfection of a fluorescent biosensor.

Despite decades of research in the epidermal growth factor receptor (EGFR) signalling field, and many targeted anti-cancer drugs that have been tested clinically, the success rate for these agents in the clinic is low, particularly in terms of the improvement of overall survival. Intratumoral heterogeneity is proposed as a major mechanism underlying treatment failure of these molecule-targeted agents. Here we highlight the application of fluorescence lifetime microscopy (FLIM)-based biosensing to demonstrate intratumoral heterogeneity of EGFR activity. For sensing EGFR activity in cells, we used a genetically encoded CrkII-based biosensor which undergoes conformational changes upon tyrosine-221 phosphorylation by EGFR. We transfected this biosensor into EGFR-positive tumour cells using targeted lipopolyplexes bearing EGFR-binding peptides at their surfaces. In a murine model of basal-like breast cancer, we demonstrated a significant degree of intratumoral heterogeneity in EGFR activity, as well as the pharmacodynamic effect of a radionuclide-labeled EGFR inhibitor in situ. Furthermore, a significant correlation between high EGFR activity in tumour cells and macrophage-tumour cell proximity was found to in part account for the intratumoral heterogeneity in EGFR activity observed. The same effect of macrophage infiltrate on EGFR activation was also seen in a colorectal cancer xenograft. In contrast, a non-small cell lung cancer xenograft expressing a constitutively active EGFR conformational mutant exhibited macrophage proximity-independent EGFR activity. Our study validates the use of this methodology to monitor therapeutic response in terms of EGFR activity. In addition, we found iNOS gene induction in macrophages that are cultured in tumour cell-conditioned media as well as an iNOS activity-dependent increase in EGFR activity in tumour cells. These findings point towards an immune microenvironment-mediated regulation that gives rise to the observed intratumoral heterogeneity of EGFR signalling activity in tumour cells in vivo.

The effect of air-abrasion on the susceptibility of sound enamel to acid challenge.

OBJECTIVE: To evaluate the effect of air-abrasion using three abrasive powders, on the susceptibility of sound enamel to an acid challenge. METHODS: 40 human enamel samples were flattened, polished and assigned to 4 experimental groups (n=10); a: alumina air-abrasion, b: sodium bicarbonate air-abrasion, c: bioactive glass (BAG) air-abrasion and d: no surface treatment (control). White light confocal profilometry was used to measure the step height enamel loss of the abraded area within each sample at three stages; after sample preparation (baseline), after air-abrasion and finally after exposing the samples to pH-cycling for 10 days. Data was analysed statistically using one-way ANOVA with Tukey's HSD post-hoc tests (p<0.05). Unique prismatic structures generated by abrasion and subsequent pH cycling were imaged using multiphoton excitation microscopy, exploiting strong autofluorescence properties of the enamel without labelling. Z-stacks of treated and equivalent control surfaces were used to generate non-destructively 3-dimensional surface profiles similar to those produced by scanning electron microscopy. RESULTS: There was no significant difference in the step height enamel loss after initial surface air-abrasion compared to the negative control group. However, a significant increase in the step height enamel loss was observed in the alumina air-abraded samples after pH-cycling compared to the negative control (p<0.05). Sodium bicarbonate as well as BAG air-abrasion exhibited similar enamel surface loss to that detected in the negative control group (p>0.05). Surface profile examination revealed a deposition effect across sodium bicarbonate and BAG-abraded groups. CONCLUSION: This study demonstrates the importance of powder selection when using air abrasion technology in clinical dentistry. Pre-treating the enamel surface with alumina air-abrasion significantly increased its susceptibility to acid challenge. Therefore, when using alumina air-abrasion clinically, clinicians must be aware that abrading sound enamel excessively renders that surface more susceptible to the effects of acid erosion. BAG and sodium bicarbonate powders were less invasive when compared to the alumina powder, supporting their use for controlled surface stain removal from enamel where indicated clinically.

Multiphoton luminescence imaging of chemically functionalized multi-walled carbon nanotubes in cells and solid tumors.

The intrinsic nonlinear photoluminescence (PL) property of chemically functionalized multi-walled nanotubes MWNTs (f-MWNTs) is reported in this study. f-MWNTs are imaged in fixed lung epithelial cancer cells (A549) and Kupffer cells in vitro, and in subcutaneously implanted solid tumors in vivo, for the first time, using multiphoton PL and fluorescence lifetime imaging (FLIM). Multiphoton imaging in the near-infrared excitation region (∼750-950 nm), employed in this study in a label-free manner, provides sensitivity and resolution optimal to track f-MWNTs within intra-cellular compartments and facilitates tumour imaging and sentinel lymph node tracking in vivo. Wider applications include employing this technique in live imaging of f-MWNTs in biological milieu to facilitate image-guided drug delivery.

Calcium silicate cement-induced remineralisation of totally demineralised dentine in comparison with glass ionomer cement: tetracycline labelling and two-photon fluorescence microscopy.

Two-photon fluorescence microscopy, in combination with tetracycline labelling, was used to observe the remineralising potentials of a calcium silicate-based restorative material (Biodentine(TM) ) and a glass ionomer cement (GIC:​Fuji​IX) on totally demineralised dentine. Forty demineralised dentine discs were stored with either cement in three different solutions: phosphate buffered saline (PBS) with tetracycline, phosphate-free tetracycline, and tetracycline-free PBS. Additional samples of demineralised dentine were stored alone in the first solution. After 8-week storage at 37 °C, dentine samples were imaged using two-photon fluorescence microscopy and Raman spectroscopy. Samples were later embedded in PMMA and polished block surfaces studied by 20 kV BSE imaging in an SEM to study variations in mineral concentration. The highest fluorescence intensity was exhibited by the dentine stored with Biodentine(TM) in the PBS/tetracycline solution. These samples also showed microscopic features of matrix remineralisation including a mineralisation front and intra- and intertubular mineralisation. In the other solutions, dentine exhibited much weaker fluorescence with none of these features detectable. Raman spectra confirmed the formation of calcium phosphate mineral with Raman peaks similar to apatite, while no mineral formation was detected in the dentine stored in cement-free or PBS-free media, or with GIC. It could therefore be concluded that Biodentine(TM) induced calcium phosphate mineral formation within the dentine matrix when stored in phosphate-rich media, which was selectively detectable using the tetracycline labelling.

Multimodal optical characterisation of collagen photodegradation by femtosecond infrared laser ablation.

Collagen is a structural component of the human body, as a connective tissue it can become altered as a result of pathophysiological conditions. Although the collagen degradation mechanism is not fully understood, it plays an important role in ageing, disease progression and applications in therapeutic laser treatments. To fully understand the mechanism of collagen alteration, in our study photo-disruptive effects were induced in collagen I matrix by point-irradiation with a femtosecond Ti-sapphire laser under controlled laser ablation settings. This was followed by multi-modal imaging of the irradiated and surrounding areas to analyse the degradation mechanism. Our multi-modal methodology was based on second harmonic generation (SHG), scanning electron microscope (SEM), autofluorescence (AF) average intensities and the average fluorescence lifetime. This allowed us to quantitatively characterise the degraded area into four distinct zones: (1) depolymerised zone in the laser focal spot as indicated by the loss of SHG signal, (2) enhanced crosslinking zone in the inner boundary of the laser induced cavity as represented by the high fluorescence ring, (3) reduced crosslinking zone formed the outer boundary of the cavity as marked by the increased SHG signal and (4) native collagen. These identified distinct zones were in good agreement with the expected photochemical changes shown using Raman spectroscopy. In addition, imaging using polarisation-resolved SHG (p-SHG) revealed both a high degree of fibre re-orientation and a SHG change in tensor ratios around the irradiation spot. Our multi-modal optical imaging approach can provide a new methodology for defining distinct zones that can be used in a clinical setting to determine suitable thresholds for applying safe laser treatments without affecting the surrounding tissues. Furthermore this technique can be extended to address challenges observed in collagen based tissue engineering and used as a minimally invasive diagnostic tool to characterise diseased and non-diseased collagen rich tissues.

De la biología al injerto de tejido adiposo: cómo mejorar el lipoinjerto / From biology to fat grafting: how to improve lipofilling

A pesar de que el uso del injerto de grasa ha ganado popularidad, no hay consenso sobre la mejor manera de manejar el tejido adiposo. Los protocolos difieren y los resultados son a menudo variables. Diversos factores influyen en la calidad de la grasa inyectada, entre los que encontramos las moléculas tóxicas provenientes de la infiltración, procedimiento previo a la liposucción. En este trabajo, hemos confirmado el efecto nocivo de los anestésicos sobre las células madre derivadas del tejido adiposo, determinando el efecto del lavado y la centrifugación en el tejido graso con el fin de proponer un protocolo simple y optimizado para mejorar la supervivencia del injerto. Evaluamos la citotoxicidad de la lidocaína sobre las células madre derivadas de tejido adiposo (ADSC) mediante ensayo de LDH. Sometimos el tejido adiposo conjunto a varios tipos de centrifugación (de 1 segundo a 10minutos y desde 0 g a 1800 g), y el volumen de líquido y el aceite liberado se midió inmediatamente después de la centrifugación. Tras la determinación de las condiciones óptimas para la manipulación de tejidos (400 g/1 minuto), inyectamos el tejido adiposo de liposucción sin o con lidocaína en ratones inmunodeficientes. Un mes después de la inyección, evaluamos la calidad de los injertos mediante histología, y en comparación con los injertos obtenidos a partir de un protocolo convencional: una simple sedimentación. La lidocaína ejerce un efecto citotóxico sobre las ADSC, y este efecto depende del tiempo de incubación y de las concentraciones. En cuanto al tejido adiposo, una centrifugación intensa (900 g, 1800 g) es perjudicial en comparación con una centrifugación suave (100 g, 400 g). Además, las secciones histológicas de los injertos de tejido adiposo no centrifugados mostraron la presencia de grandes vacuolas de aceite mientras que los injertos resultantes de lavado con protocolo de centrifugación suave (400g/1minuto) no lo hacen. En conclusión, creemos que se debe emplear un manejo adecuado del tejido adiposo, incluyendo lavado y centrifugación, con el fin de eliminar el líquido de infiltración y las moléculas tóxicas asociadas que son perjudiciales para los injertos. Sin embargo, no recomendamos una centrifugación intensa ya que conduce muy rápidamente a una mayor muerte celular. Por lo tanto, una centrifugación suave (400 g/1 minuto) precedida de lavados, parece ser el protocolo más apropiado para la reinyección del tejido adiposo

While fat grafting for soft tissue filling has gained popularity, there is no consensus on the best way how to handle adipose tissue. Protocols differ and results are often highly variable. Various factors influence the quality of injected fat, among which the toxic molecules coming from infiltration procedure prior to liposuction. In this work, we have confirmed the deleterious effect of anesthetics on adipose-derived stem cells, and determined the effect of washing and centrifugation on adipose tissue, in order to propose a simple and optimized protocol to improve graft survival. Lidocaine cytotoxicity on adipose-derived stem cells (ADSCs) was evaluated by LDH assay. Then, whole adipose tissue was subjected to various centrifugation types (from 1 sec to 10 min and from 0 g to 1800 g), and volumeof liquid and oil released were measured immediately after centrifugation. After determination of the optimal conditions for tissue handling (400 g/1 min),adipose tissue from liposuction made without or with lidocaine was injected into immunodeficient mice. One month after injection, quality of the grafts was evaluated by histology, and compared with grafts obtained from one conventional protocol: a simple settling. Lidocaine exerts a cytotoxic effect on ADSCs, and this effect is dependent on the incubation time and concentrations. Concerning adipose tissue, strong centrifugation (900 g, 1800 g) is deleterious compared to the low centrifugation(100 g, 400 g). In addition, histological sections of the non-centrifuged adipose tissue grafts shows the presence of extensive oil vacuoles, whereas the grafts resulting from washing with soft centrifugation protocol (400 g/1 min) do not. To conclude that appropriate handling of adipose tissue, including washing and centrifugation, should be done in order to remove infiltration liquid and associated toxic molecules, which are deleterious for the grafts. However, strong centrifugation is not recommended since it leads very quickly to greater cell death. Thus, soft centrifugation (400 g/1 min), preceded by washings, seems to be the most appropriate protocol for the re-injection of adipose tissue

Microbiochemical analysis of carious dentine using Raman and fluorescence spectroscopy.

The aim of this study was to evaluate and correlate objectively the microspectroscopically derived biochemical components of sound, infected and affected carious dentine with their microhardness and autofluorescence (AF) characteristics. Over 3 million high-resolution Raman spectra from 8 extracted human carious teeth were recorded using Raman spectrometer with parallel spectrum acquisition. Green AF signals across each carious lesion from all samples were acquired with a similar spatial resolution using confocal fluorescence microscopy. The Knoop microhardness (KHN) from a total of 233 co-localized areas was recorded from the same samples and allocated subjectively into the three zones. Cluster analysis of the Raman data, performed using in-house software, produced five independent spectral components representing mineral content, protein content, porphyrin fluorescence (PF), putative infected dentine signal (IDS) and affected dentine signal (ADS). The distributions of the 5 Raman components and the AF signal were matched across all samples and their average values were calculated for each corresponding KHN area. The infected dentine was defined significantly by the KHN, AF and by the relative contribution of the mineral, PF and IDS clusters. Protein cluster was not statistically related to the KHN or AF. A delineation between affected and sound dentine was observed using the KHN, AF, PF and ADS parameters. This study concludes that micro-Raman spectroscopy can provide a non-invasive and objective evaluation of different carious dentine zones. Being able to detect and assess clinically the caries-affected dentine during minimally invasive operative caries management is important to control the risk of unnecessary tissue removal.

Dentin-cement interfacial interaction: calcium silicates and polyalkenoates.

The interfacial properties of a new calcium-silicate-based coronal restorative material (Biodentine™) and a glass-ionomer cement (GIC) with dentin have been studied by confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), micro-Raman spectroscopy, and two-photon auto-fluorescence and second-harmonic-generation (SHG) imaging. Results indicate the formation of tag-like structures alongside an interfacial layer called the "mineral infiltration zone", where the alkaline caustic effect of the calcium silicate cement's hydration products degrades the collagenous component of the interfacial dentin. This degradation leads to the formation of a porous structure which facilitates the permeation of high concentrations of Ca(2+), OH(-), and CO(3) (2-) ions, leading to increased mineralization in this region. Comparison of the dentin-restorative interfaces shows that there is a dentin-mineral infiltration with the Biodentine, whereas polyacrylic and tartaric acids and their salts characterize the penetration of the GIC. A new type of interfacial interaction, "the mineral infiltration zone", is suggested for these calcium-silicate-based cements.

Plasmon resonances on metal tips: understanding tip-enhanced Raman scattering.

Calculations of the electric-field enhancements in the vicinity of an illuminated silver tip, modeled using a Drude dielectric response, have been performed using the finite difference time domain method. Tip-induced field enhancements, of application in "apertureless" Raman scanning near-field optical microscopy (SNOM), result from the resonant excitation of plasmons on the metal tip. The sharpness of the plasmon resonance spectrum and the highly localized nature of these modes impose conditions to better exploit tip plasmons in tip-enhanced apertureless SNOM. The effect of tip-to-substrate separation and polarization on the resolution and enhancement are analyzed, with emphasis on the different field components parallel and perpendicular to the substrate.

A new splicing acceptor site and poly(A)+ sequence signal within DQA1*0401 and DQA1*0501 mRNA 3'UTR contribute to increase the extraordinary diversity of mRNA isoforms.

In this paper, we have analysed the diversity of mRNA species generated by DQA1*0501 and DQA1*0401 alleles in homozygous B-lymphoblastoid cell lines. As we previously reported, six mRNA isoforms that differ in the 3'UTR have been identified in these cells. This diversity of mRNA species results both from the alternative use of two acceptor spliced sites and the differential selection of two poly(A)+ sequence signals by the processing machinery. In this report we describe a new acceptor sequence signal that allows generation of a new alternative spliced mRNA species. This acceptor sequence signal was also present in all of the seven DQA1 homozygous cell lines analysed. In addition, we have identified a previously undetected, non-conventional but functional, poly(A)+ sequence signal that lacks an identifiable AATAAA hexamer, one of the most important element of the core. This sequence signal allows the generation of two additional mRNA isoforms both in DQA1*0501 and DQA1*0401 homozygous cell lines but not in the others. We show that DQA1*0501 and DQA1*0401 primary transcripts can be processed into nine mRNA isoforms that differ in the 3'UTR. Finally, we summarized all the DQA1 mRNA species deriving from DQA1*0101, DQA1*0102, DQA1*0103, DQA1*0201, DQA1*0301, DQA1*0401 and DQA1*0501 alleles and shown in B-lymphoblastoid cell lines.

Is aggregation of beta-amyloid peptides a mis-functioning of a current interaction process?

In a previous study, Hughes et al. [Proc. Natl. Acad. Sci. USA 93 (1996) 2065-2070] demonstrated that the amyloid peptide is able to interact with itself in a two-hybrid system and that interaction is specific. They further supported that the method could be used to define the sequences that might be important in nucleation-dependent aggregation. The sequence of the amyloid peptide can be split into four clusters, two hydrophilic (1-16 and 22-28) and two hydrophobic (17-21 and 29-42). We designed by molecular modeling and tested by the two-hybrid approach, series of mutations spread all over the sequence and changing the distribution of hydrophobicity and/or the spatial hindrance. In the two-hybrid assay, interaction of native Abeta is reproduced. Screening of mutations demonstrates that the C-domain (residues 29-40 (42)), the median domain (residues 17-22) and the N-domain (1-16) are all crucial for interaction. This demonstrates that almost all fragments of the amyloid peptide but a loop (residues 23-28) and the C-term amino acid are important for the native interaction. We support that the folded three-dimensional (3D) structure is the Abeta-Abeta interacting species, that the whole sequence is involved in that 3D fold which has a low secondary structure propensity and a high susceptibility to mutations and thus should have a low stability. The native fold of Abeta could be stabilized in Abeta-Abeta complexes which could in other circumstances facilitate the nucleation event of aggregation that leads to the formation of stable senile plaques.

Interaction between the N-terminal domain of gastric H,K-ATPase and the spectrin binding domain of ankyrin III.

We screened a cDNA bank of rabbit gastric fundic mucosa by two-hybrid assays looking for binding partners of the N-terminal domain of the rabbit gastric H,K-ATPase. We extracted five clones sharing more than 90% sequence identity. The longest clone codes for a protein sharing a high identity (96 and 96.8%, respectively) with a fragment of the membrane domain, from Arg-835 to Ser-873, plus the major part of the "spectrin binding domain" going from Glu-874 to Leu-1455 of human and mouse ankyrin III. We conclude that the membrane and spectrin binding domains of the rabbit ankyrin III are candidates for the binding partner of the N-terminal domain of the rabbit gastric H,K-ATPase. To validate the ankyrin-ATPase interaction and to test its specificity, we produced both domains in yeast and bacteria, coimmunoprecipitated them with an anti-ATPase antibody, and copurified them by affinity chromatography. The sequence of rabbit ankyrin III was not known, and this is the first report demonstrating that the ankyrin III and the H,K-ATPase interact with no intermediate. The interaction involves the N-terminal domain of the ATPase on one hand and the spectrin binding domain of the ankyrin on the other.

Prediction of epitopes and production of monoclonal antibodies against gastric H,K-ATPase.

Monoclonal antibodies (mAbs) were produced against gastric H,K-ATPase using a theoretical and experimental strategy based on prediction of linear epitopes by molecular modelling followed by production of anti-peptide antibodies. By analysing the alpha subunit sequence, we predicted several epitopes corresponding to amino acids K519-L533, E543-Y553 and S786-L798 and produced monoclonal antibodies HK519, HK543 and HK786. All three react against gastric H,K-ATPase in RaLISA, immunohistochemistry and Western blots demonstrating that they recognize the native and the SDS-denatured ionic pump and that the epitopes are located at the surface of the native ATPase. Antibody Kd are in the range 6-10x10(-8) M. Monoclonal antibody HK519 is a competitive inhibitor of ATP, in agreement with ATP binding to K519. Neither mAb 543, nor mAb 786 inhibit the ATPase activity. Monoclonal antibody 95111, whose epitope is mapped between residues C529 and E561, competes with mAb HK543 but not with the other two. We suggest that the 95111 epitope is overlapping or very close to the HK543-553 sequence. Induction of E1 conformer by binding FITC to K519 increases the number of mAb 95111 and mAb HK543 epitopes but not that of mAb 786, supporting the fact that the fragment E543-Y553 changes accessibility, maybe during the E1-E2 transconformation.

1-anilino-8-naphtalene sulfonate probes a gastric HK-ATPase potassium site whose access requires ionophores.

1-anilino-8-naphtalenesulfonate (ANS) is a hydrophobic dipole previously used to demonstrate that the proton for potassium exchange by the gastric HK-ATPase is electroneutral. In this paper, we demonstrate that ANS binds to gastric membranes and probes conformational changes of the HK-ATPase independently of any active H for K exchange. Conformational changes require the presence of potassium-valinomycin and are not triggered by sodium. Potassium effect is enhanced by ATP, in the presence and in the absence of magnesium and, by ADP, in the presence of magnesium. Labeling of the pig HK-ATPase K518 by fluorescein-5-isothiocyanate inhibits the enzyme activity and knocks out the ATP effect on ANS fluorescence. Scherring 28080 and the monoclonal antibody 95-111, two competitive inhibitors of K-activated ATPase dephosphorylation, do not modify K-effect on ANS fluorescence but inhibit ATP effects. This supports that ANS does not probe K-site between the H1-H2 loop. Treatment of gastric membranes with trypsin does not inhibit the ANS response to potassium but does inhibit the response to ATP. This suggests that the ATP site inducing the ANS response is cytoplasmic and the potassium site is intramembranous. Titration reveals that one mole of ANS interacts with one mole of ATPase. We suggest that ANS probes a hydrophobic potassium site of gastric ATPase and that addition of ATP and ADP-Mg embed that site.

Topological model of membrane domain of the cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis transmembrane conductance regulator is a cAMP-regulated chloride channel. We used molecular modelling to predict 3-D models for the CFTR membrane domain. Hydropathy and residue conservation in all CFTRs as well as in other proteins suggested that the membrane domain is a 12-helix bundle. If the domain is enclosing a channel for chloride, it could be made of five helices. We propose two structural models in which both lumenal and cytoplasmic entrances to the chloride pore have a ring of positively charged residues. The inner surface of the channel is covered with neutral polar plus one or two charged residues. Helices that are not directly involved in the chloride channel could organise to form a second channel; a dimeric symmetrical structure is proposed. Analysis raised interest for helix 5: this hydrophobic fragment is conserved in all CFTRs and aligns with segments present in several different ion channels and transporters. The existence of an FFXXFFXXF motif is proposed. Helix 5 could be an important domain of CFTRs. The models agree with available data from pathological mutations but does not account for the membrane insertion of a hydrophilic fragment of NBDI.