Competitive mode and site of interaction of ticagrelor at the human platelet P2Y12 -receptor.

BACKGROUND: The G-protein-coupled P2Y12 -receptor plays a crucial role in platelet aggregation. Recently, ticagrelor was licensed as the first perorally active and reversible P2Y12 -receptor antagonist. OBJECTIVE: The present study investigated the site and the antagonistic mode of action of ticagrelor at wild-type or mutant human P2Y12 -receptors. METHODS: Recombinant wild-type or mutant human P2Y12 -receptors were stably expressed in Chinese hamster ovary Flp-In cells. Receptor function was assessed by quantification of ADP- and 2-methylthio-ADP-mediated inhibition of forskolin-induced cellular cAMP production either using a [(3) H]cAMP-radioaffinity assay or a cAMP response element-driven luciferase reporter gene assay. RESULTS: The natural agonist ADP inhibited forskolin-induced cAMP formation at the wild-type P2Y12 -receptor with a lower potency (EC50 209 nm) than the synthetic agonist 2-methylthio-ADP (EC50 1.0 nm). Ticagrelor shifted the concentration-response curves of both agonists in a parallel and surmountable manner to the right. Increasing concentrations of ticagrelor caused increasing shifts. Schild-plot analysis revealed pA2 values of 8.85 for ticagrelor against ADP, and 8.69 against 2-methylthio-ADP, and slopes of the regression lines not different from unity. In cells expressing a recombinant C194A(5.43) -mutant P2Y12 -receptor construct, ticagrelor lost antagonistic potency when tested against ADP or 2-methylthio-ADP. CONCLUSIONS: The experiments reveal a surmountable and competitive mode of antagonism of ticagrelor at P2Y12 -receptors activated by either the natural agonist ADP or the synthetic agonist 2-methylthio-ADP. Cys194(5.43) is likely to be involved in the interaction of ticagrelor with ADP and 2-methylthio-ADP. The data give new insights into the site and mode of action of ticagrelor at the human P2Y12 -receptor.

A prospective study of the accuracy of the surgeon's diagnosis and significance of positive margins in nonmelanoma skin cancers.

Even with a precise preoperative diagnosis, complete excision of nonmelanoma skin cancer is not always achieved. The conundrum remains the decision for appropriate secondary treatment. Many surgeons, regardless of the nature of the lesion, consider re-excision to be the only option. In a prior 4-year prospective study that ascertained the accuracy of our clinical diagnosis of skin lesions removed in an office setting, one-fifth were found to be malignant and 98 percent (n = 415) of the lesions were nonmelanoma skin cancer. Unfortunately, 65 (15.7 percent) of the malignant nonmelanoma skin cancer lesions had positive margins. The outcome of our management for these specific lesions was followed prospectively over the 7.5 years of this study to determine whether aggressive surgical intervention was justified in every case. Of 65 patients with lesions, early and complete re-excision of margin-positive nonmelanoma skin cancer was performed for 34 (52.3 percent), with residual tumor found in 11 (32.4 percent), followed by a later recurrence in one (2.9 percent). The remaining 31 patients agreed to semiannual office visits, with one (3.2 percent) recurrence in this group. Thus, the overall rate of recurrence for margin-positive nonmelanoma skin cancer was 3.1 percent, with a mean follow-up of 3.6 years (range, 0 to 7.5 years). There were no recurrences for basal cell carcinoma in either treatment group, suggesting that, at least for "simple" primary lesions without confounding risk factors, there is some validity to a "wait and see" attitude, in which treatment of a potential recurrence would be straightforward. Despite our observed infrequent local recurrences of squamous cell cancers (13.3 percent), the small risk of metastases still suggests the appropriateness of complete surgical eradication for these tumors whenever feasible.

Octyl-2-Cyanoacrylate adhesive for rapid nail plate restoration.

Following nail bed repair, returning the nail plate as a conforming stent or splint is a common technique. Especially when split, the nail plate fragments can very readily be pieced together and bonded to the nail bed using the tissue adhesive Octyl-2-Cyanoacrylate. This new formulation can expedite this maneuver, and has shown no signs of histotoxicity or adverse effect on nail plate regeneration.

Mannose receptor is expressed in normal and dystrophic retinal pigment epithelium.

In normal retinas, the phagocytosis of shed photoreceptor outer segments is mediated in part through a mannose receptor protein located in the apical retinal pigment epithelium membrane. As dystrophic rats of the Royal College of Surgeons have a defect in which the retinal pigment epithelium (RPE) is unable to phagocytize the shed outer segments, it is hypothesized that mannose receptor expression will be lost with the progression of photoreceptor degeneration. Immunohistochemical and molecular techniques have been used to study the developmental expression of the mannose receptor in normal and dystrophic retinal pigment epithelium. By immunofluorescence, the mannose receptor is localized to the retinal pigment epithelium, apical membrane region, beginning around 5 days postnatally in both normal and dystrophic retinas. In immunoblots, bands at 175 kDa are labelled by an anti-mannose receptor antibody in apical membrane samples from both normal and dystrophic RPE at all developmental times sampled. RT-PCR analysis reveals that mannose receptor message is present in normal and dystrophic RPE samples at all developmental time points examined. The present study demonstrates that the expression of the mannose receptor begins prior to outer segment differentiation and the initiation of phagocytosis in both normal and dystrophic RPE. Expression of the mannose receptor continues to be unchanged during the progression of photoreceptor degeneration in the dystrophic retina.

Prospective study of the accuracy of the surgeon's diagnosis in 2000 excised skin tumors.

Expeditious yet efficacious removal of skin tumors is a common responsibility for the plastic surgeon. The need to minimize potential risks for mortality or morbidity from undue or excessive surgical resections and to control costs by avoiding unnecessary procedures behooves us to make a precise clinical diagnosis preceding any decision even for such "minor" surgery. Just how accurate these decisions can be expected to be for a typical surgical practice was scrutinized by means of this prospective 4-year study involving the resection of 2058 skin lesions. Each lesion was initially assigned a clinical diagnosis after a brief gross examination and then compared with the pathology report, which was always considered to be the correct answer. Within these parameters, only 65 percent of all tumors were identified correctly preoperatively. Two-thirds of all lesions were benign. Three-quarters of benign lesions were as assumed, and 92 percent of all presumed benign lesions were benign even if incorrectly identified initially, whereas fortunately only 3 percent proved to be malignant. On the other hand, only three-fifths of malignant lesions were identified correctly clinically, yet only 11 percent were benign, implying that most such lesions properly deserved excision anyway. Therefore, approximately 90 percent of all lesions whether benign or malignant were removed appropriately without compromising the patient, but to expect a clinical acumen of 100 percent in this setting may not be realistic. The accuracy of the surgeon in identifying lesions as probably benign was certainly high enough that cost-containment mechanisms designed to deny authorization for their removal probably would be justifiable and difficult to appeal. Any suspicious or equivocal lesions still will require mandatory intervention despite such constraints, because often only histologic examination will allow a definitive diagnosis.

Nonoperative estimation of the soleus musculotendinous junction using magnetic resonance imaging.

Although it is recognized as the muscle flap of choice for middle-third defects of the lower limb, the capability for even more distal transposition of the soleus muscle remains controversial. Such reach depends directly on the site of insertion of the muscle and previously has not been assessed convincingly without surgical intervention. Magnetic resonance imaging (MRI) may be a noninvasive alternative for determining the distal extent of the musculotendinous junction of the soleus muscle. In our last four patients, preoperative MRI scans were obtained prior to an elective soleus muscle transfer. The distance from the ankle joint to the most distal site of the soleus insertion was measured on the MRI scan and compared with the actual intraoperative measurement, which had a significant correlation (r = 0.98, p = 0.019). A retrospective review of 42 other sagittal ankle MRI scans predicted the mean of this distance to be 1.92 +/- 1.23 cm (range -0.4 to 4.5 cm), compared with gross anatomic dissections in 30 unrelated fresh cadavers, where this was 4.06 +/- 3.11 cm (range -0.7 to 12.5 cm). These additional data are pertinent because they reinforce recognition of the great variation in soleus anatomy, which would limit clinical applications for the distal third of the leg only for those individuals with very distal insertions. The MRI scan can reliably identify the soleus muscle and provides a nonoperative method for evaluation of potential feasibility for its use as a local muscle flap for distal lower extremity defects.

Natural, high-mannose glycoproteins inhibit ROS binding and ingestion by RPE cell cultures.

Previous studies have suggested that a mannose receptor mediates the phagocytic uptake of effete rod outer segments by retinal pigment epithelial cells. In the present study, the effect of adding a soluble ligand for the mannose receptor, horseradish peroxidase, was examined. Cultured retinal pigment epithelial cells from Long Evans rats were preincubated with various concentrations of horseradish peroxidase for 20 min followed by a challenge of FITC-labeled bovine rod outer segments for 3 h. Both counts of total rod outer segments (bound and ingested) and ingested rod outer segments were determined. Rod outer segment uptake was reduced, in a concentration-dependent fashion, by an average of 60% of control values when horseradish peroxidase was added to retinal pigment epithelial cultures. Similarly, total rod outer segment values were reduced to 50% of controls in the presence of at least a 10 micrograms ml-1 horseradish peroxidase concentration. Horseradish peroxidase inhibition of retinal pigment epithelial phagocytic capacity was reversible. Other high mannose glycoproteins, such as invertase, beta-glucoronidase, and ovalbumin, were equally effective in preventing rod outer segment ingestion by retinal pigment epithelial cells. These data further support the hypothesis that a mannose receptor on the retinal pigment epithelial apical surface facilitates phagocytosis of rod outer segments.

Prospective comparison of minimal incision "open" and two-portal endoscopic carpal tunnel release.

Part of the groundswell for endoscopic plastic surgery initially gained momentum in hand surgery, with claims that endoscopic carpal tunnel release allowed less invasive surgery and a more rapid recovery due to diminished pain and scarring than was possible with traditional "open" methods. Admittedly, no ultimate difference in their efficacy as regards symptom relief had been observed. However, in our opinion, some of these conclusions may be flawed, since an "open" method employing the most minimal possible incisions was not necessarily used. Therefore, a more apropos study should compare an acceptable minimally invasive "open" technique versus endoscopic carpal tunnel decompression. A prospective, consecutive series of 96 patients with medically unresponsive, confirmed carpal tunnel syndrome with no other concomitant hand pathology was selected. Fifty-three patients (71 hands) underwent "open" release using a minimal incision, which was comparable in composition to a group of 47 patients (66 hands) who had a two-portal endoscopic release. Scar length (p = 0.999), need for hand therapy (p = 0.798), rate of complications (p = 0.359), length of time before resuming routine activities (p = 0.255), and length of time before return to work (p = 0.373) were not statistically different whether an "open" or "closed" procedure had been performed. Regardless of the technique employed, individuals receiving Worker's Compensation more often required hand therapy (p < 0.02) and had a significantly longer recovery period (p < 0.005). A subgroup of 15 patients with bilateral carpal tunnel syndrome who had decompression using opposing methods had no significant difference in preference.(ABSTRACT TRUNCATED AT 250 WORDS)

Microsurgical transfer of vascularized tissue to close problem wounds.

Interventions to achieve optimal wound healing challenge the skills of all health care professionals. Although most wounds are best managed by appropriate nursing intervention outside the OR, some complex wounds require more expeditious closure to ensure maximum preservation of vital structures and their functions. Microsurgical transfer of vascularized tissue (ie, flaps) often is the only method of achieving immediate wound coverage. Perioperative team members who treat such wounds must be aware of the distinct attributes and potential morbidity associated with flap procedures.

Characterization of rod outer segment plasma membrane proteins which bind to the mannose receptor.

Previous work by our laboratory has demonstrated that rod outer segment (ROS) phagocytosis can be mediated by mannose-receptor dependent activity. This study was designed to probe for potential ligands on the ROS surface which could interact with the mannose receptor during the phagocytic cycle. Solubilized ROS plasma membranes were passed over a mannose receptor-Sepharose column in the presence of CaCl2. Proteins specifically bound to the column were eluted using methyl-D-mannoside and EDTA and characterized by gel electrophoresis, lectin blots, and immunoblots. Silver stained gels of ROS plasma membrane proteins eluted from the mannose-receptor column demonstrated six bands: a major band at 36 kD, identified by monospecific antibodies as rhodopsin, and bands of Mr = 39 kD, 67 kD, 76 kD, 97 kD and 100 kD. Lectin blots of the eluted fractions confirmed that all six proteins in these fractions could bind concanavalin A. In summary, these results showed that rhodopsin and several other mannose-containing glycoproteins on ROS plasma membranes were bound to a mannose receptor column, and thus could serve as ligands for mannose receptor-mediated ROS phagocytosis.

Isolation of the phagocytic compartment from macrophages using a paramagnetic, particulate ligand.

Isolation of Fc receptor-dependent phagosomes from a macrophage cell line, J774 A.1, was accomplished using antibody-opsonized, paramagnetic beads. Following binding and ingestion of these beads, cells were homogenized in a standard membrane isolation buffer. Phagosomes containing the trapped paramagnetic beads were isolated by subjecting the whole cell homogenate to a magnetic field. The method is extremely simple and the preparation of an enriched phagosome fraction from a whole cell homogenate is rapid and highly selective. The method should provide useful starting material for investigators interested in cytoskeletal involvement in phagocytosis, in kinetic studies of ingestion, in phagosome-lysosome fusion, and in the ability of a particular ligand to initiate phagocytosis.

Reactivation of isolated mitotic apparatus: metaphase versus anaphase spindles.

Mitotic spindles isolated from sea urchin eggs can be reactivated to undergo mitotic processes in vitro. Spindles incubated in reactivation media containing sea urchin tubulin and nucleotides undergo pole-pole elongation similar to that observed in living cells during anaphase-B. The in vitro behavior of spindles isolated during metaphase and anaphase are compared. Both metaphase and anaphase spindles undergo pole-pole elongation with similar rates, but only in the presence of added tubulin. In contrast, metaphase but not anaphase spindles increase chromosome-pole distance in the presence of exogenous tubulin, suggesting that in vitro, tubulin can be incorporated at the kinetochores of metaphase but not anaphase chromosomes. The rate of spindle elongation, ultimate length achieved, and the increase in chromosome-pole distance for isolated metaphase spindles is related to the concentration of available tubulin. Pole-pole elongation and chromosome-pole elongation does not require added adenosine triphosphate (ATP). Guanosine triphosphate (GTP) will support all activities observed. Thus, the force generation mechanism for anaphase-B in isolated sea urchin spindles is independent of added ATP, but dependent on the availability of tubulin. These results support the hypothesis that the mechanism of force generation for anaphase-B is linked to the incorporation of tubulin into the mitotic apparatus. (If, in addition, a microtubule-dependent motor-protein(s) is acting to generate force, it does not appear to be dependent on ATP as the exclusive energy source.

Micromanipulation studies of the asymmetric positioning of the maturation spindle in Chaetopterus sp. oocytes: I. Anchorage of the spindle to the cortex and migration of a displaced spindle.

We investigated the nature of the asymmetric positioning and attachment of Chaetopterus oocyte meiotic spindles to the animal pole cortex by micromanipulation. The manipulated spindle's behavior was analyzed in clarified oocyte fragments using video-enhanced polarized light microscopy. As the spindle was drawn towards the cell interior with a microneedle, the cell surface dimpled inwards adjacent to the outer spindle pole. As the spindle was pulled further inwards, the dimple suddenly receded indicating a rupture of a mechanical link between the cell cortex and outer spindle pole. The spindle paused briefly when released from the microneedle; then it spontaneously migrated back to the original attachment site and reassociated with the cell cortex. Positive birefringent astral fibers were seen running between the outer spindle pole and the cortex during the migration. The velocity of the spindle during its migration tended to increase as it came closer to the cortex. Velocities as high as 1.25 micron/sec. were measured. If removed too far from the attachment site cortex (greater than 35 micron), the spindle remained stationary until pushed closer to the original attachment site. Spindles, inverted by micromanipulation, migrated and reattached to the cortical site by their former inner pole; thus either spindle pole can seek out and migrate to the original attachment site. However, spindle poles pushed against other cortical regions did not attach demonstrating that there is only one unique, localized attachment site for spindle attachment.

Development of nurse cell-oocyte interactions in the insect telotrophic ovary (Rhodnius prolixus).

The establishment of reorganization of intercellular bridges during larval-adult ovarian differentiation is the basis of the syncytial nature of the adult hemipteran telotrophic ovary. The formation, in the late differentiation phase, of groups of closely arranged nurse cell nuclei occupying a common cytoplasm results from membrane fusions. Oocyte-oocyte intercellular bridge systems later are modified to form the trophic cords. The trophic core, which undergoes a restructuring during the late differentiation phase, mediated nurse cell-oocyte interactions in this system. Material, transported to and accumulated by late differentiation phase pre-vitellogenic oocytes, originates from trophic core restructuring and zone III nurse cell production.

Development and cellular differentiation of an insect telotrophic ovary (Rhodnius prolixus).

Differentiation events accompanying the larval-adult ovarian transformation in Rhodnius prolixus can be divided into three phases: proliferative phase (unfed to 3 days post-feed or DPF), early differentiation phase (9-15 DPF) and late differentiation phase (16 DPF to moult at 21 DPF). Ovarioles remain morphologically larval until feeding initiates development. The unfed ovariole contains germ cells surrounding a central trophic core region with the 'germarial lumen' occupying the basal region of the tropharium immediately above the pre-follicular tissue. Mitosis of germ cells during the proliferative phase results in a progressive increase in tropharial size with no differentiation of tissues. Regional specialization within the ovariole marks the beginning of the early differentiation phase. A zone of oocytes is established at the base of the tropharium with nuclei containing synaptonemal complexes and condensing chromosomes. Nurse cell differentiation is characterized by nucleolar elaboration and nucleo-cytoplasmic transport, the cytoplasm becoming rich in ribosomes. Autoradiographic results suggest that functional nurse cell-oocyte divergence occurs concurrently with morphological divergence. Pre-follicular tissue is divided into apical and basal zones with apical zone differentiation occurring during early and late differentiation phases.

X-ray diffraction study of some normal alkyl esters of long-chain acids.

X-ray powder diffraction data are reported for 15 normal long-chain esters. The compounds represent all combinations of acid and alcohol where the acid portion is n-tetradecanoic, n-hexadecanoic, or n-octadecanoic acid, and the alcohol portion is n-tetradecanol, n-pentadecanol, n-hexadecanol, n-heptadecanol, or n-octadecanol. The individual compounds can be identified and distinguished by the diffraction data. Several of the esters have long spacings that are a linear function of the number of carbon atoms in the molecule and are consistent with a similar function for ethyl esters of long-chain acids. The remainder of the compounds crystallize in other polymorphic forms and therefore do not follow this function.

X-ray diffraction powder studies of some dithiol diesters of long chain acids.

X-ray diffraction powder data are reported for 25 mono- and dithiol diesters of straight chain aliphatic acids where the acid portion of the molecule consists of one of the following acids: octanoic, decanoic, dodecanoic, tetradecanoic, hexadecanoic or octadecanoic acids, and where the thiol portion consists of one of the following: 2-mercaptoethane, 1,2-ethanedithiol, 1,3-propanedithiol, 1,4-butanedithiol or 1,5-pentanedithiol. The individual compounds can be identified and distinguished by the long spacing data. The compounds crystallize in tilted monomolecular layers.