Identification of a high-affinity binding site involved in the transport of endocannabinoids.

Phytocannabinoids, such as the principal bioactive component of marijuana, delta9-tetrahydrocannabinol, have been used for thousands of years for medical and recreational purposes. delta9-Tetrahydrocannabinol and endogenous cannabinoids (e.g., anandamide) initiate their agonist properties by stimulating the cannabinoid family of G protein-coupled receptors (CB1 and CB2). The biosynthesis and physiology of anandamide is well understood, but its mechanism of uptake (resulting in signal termination by fatty acid amide hydrolase) has been elusive. Mounting evidence points to the existence of a specific anandamide transport protein; however, no direct evidence for this protein has been provided. Here, we use a potent, competitive small molecule inhibitor of anandamide uptake (LY2318912, IC50 7.27 +/- 0.510 nM) to identify a high-affinity, saturable anandamide transporter binding site (LY2318912; K(d) = 7.62 +/- 1.18 nM, B(max) = 31.6 +/- 1.80 fmol/mg protein) that is distinct from fatty acid amide hydrolase. Systemic administration of the inhibitor into rodents elevates anandamide levels 5-fold in the brain and demonstrates efficacy in the formalin paw-licking model of persistent pain with no obvious adverse effects on motor function. Identification of the anandamide transporter binding site resolves a missing mechanistic link in endocannabinoid signaling, and in vivo results suggest that endocannabinoid transporter antagonists may provide a strategy for positive modulation of cannabinoid receptors.

Breast cancer in patients with residual invasive carcinoma is more accurately staged with additive tumor size assessment.

BACKGROUND: Accurate assessment of tumor size for patients with breast cancer undergoing re-excision following breast-conserving therapy is important for appropriate staging and adjuvant treatment. We investigated the accuracy of additive vs. nonadditive size assessment in determining final tumor stage. METHODS: Patients with infiltrating carcinoma in the initial excision and in at least one additional re-excision (re-excision positive; n = 89) had tumor size assessed with additive and nonadditive techniques. This group was compared with patients undergoing re-excision but without identifiable residual carcinoma (re-excision negative; n = 105) regarding rates of lymph node (LN) metastasis. RESULTS: The re-excision positive patients had a different median final tumor size depending on the size assessment technique used (nonadditive: 1.8 cm; additive: 3.0 cm; P <.0001). Both groups of patients had a median tumor size consistent with T1c staging in nonadditive size assessment. However, re-excision positive patients had a significantly higher incidence of LN metastasis (P <.05) than did re-excision negative patients. Both groups were then separated into T1 and T2 stages and the LN metastasis rates were assessed. Compared with nonadditive size assessment, additive size assessment distributed re-excision positive patients into T stages whereby the LN metastasis rates more closely approximated those of re-excision negative patients (T1, 3% vs. 6% difference; T2, 4% vs. 13% difference). CONCLUSIONS: With regard to LN metastasis, staging for patients with residual invasive carcinoma in re-excision specimens is more accurate with additive tumor size assessment.

Recurrence rates in patients with central or retroareolar breast cancers treated with mastectomy or lumpectomy.

BACKGROUND: Although breast conservation with lumpectomy and radiation treatment has become a commonly used treatment for breast cancer, there are little data to support the use of lumpectomy for central and retroareolar breast cancers. In this study, we investigate the local and distant recurrence rates of patients with central or retroareolar breast cancers treated with lumpectomy compared with mastectomy. METHODS: This study provides a retrospective analysis of 99 patients, from 1981 to 2000, with central or retroareolar breast cancers treated with mastectomy or lumpectomy to determine the frequency of local and distant recurrence. The mastectomy and lumpectomy patients were compared with respect to recurrence and other prognostic factors including: tumor location, tumor size, axillary nodal status, and final surgical margins. RESULTS: The overall frequency of local recurrence was 5 of 99 (5.0%) in the entire group, 3 of 67 (4.5%) and 2 of 32 (6.3%) of patients who underwent mastectomy and lumpectomy, respectively (P >0.99). Overall, 3 patients experienced a distant recurrence as a first event, with 2 patients (3.0%) in the mastectomy group and 1 patient (3.1%) in the lumpectomy group (P >0.99). The type of surgical management was not statistically significant related to either local or distant disease recurrence, with median time to local recurrence of 3.0 years for the mastectomy patients and 5.0 years for lumpectomy patients. Of the patients with central tumors who underwent mastectomy 2 of 42 (4.8%) developed local recurrences compared with those who had a lumpectomy, 1 of 21 (4.8%). Similarly for retroareolar tumors, the local recurrence rate was 1 of 25 (4.0%) for patients undergoing mastectomy and 1 of 11 (9.1%) for those undergoing lumpectomy (P >0.99). CONCLUSIONS: In this study there was no significant difference in local or distant failure rates of those patients with central or retroareolar tumors treated with mastectomy versus lumpectomy. We conclude lumpectomy to be a reasonable treatment option for selected patients with central or retroareolar breast cancers.

Hidden-Markov methods for the analysis of single-molecule actomyosin displacement data: the variance-Hidden-Markov method.

In single-molecule experiments on the interaction between myosin and actin, mechanical events are embedded in Brownian noise. Methods of detecting events have progressed from simple manual detection of shifts in the position record to threshold-based selection of intermittent periods of reduction in noise. However, none of these methods provides a "best fit" to the data. We have developed a Hidden-Markov algorithm that assumes a simple kinetic model for the actin-myosin interaction and provides automatic, threshold-free, maximum-likelihood detection of events. The method is developed for the case of a weakly trapped actin-bead dumbbell interacting with a stationary myosin molecule (Finer, J. T., R. M. Simmons, and J. A. Spudich. 1994. Nature. 368:113-119). The algorithm operates on the variance of bead position signals in a running window, and is tested using Monte Carlo simulations to formulate ways of determining the optimum window width. The working stroke is derived and corrected for actin-bead link compliance. With experimental data, we find that modulation of myosin binding by the helical structure of the actin filament complicates the determination of the working stroke; however, under conditions that produce a Gaussian distribution of bound levels (cf. Molloy, J. E., J. E. Burns, J. Kendrick-Jones, R. T. Tregear, and D. C. S. White. 1995. Nature. 378:209-212), four experiments gave working strokes in the range 5.4-6.3 nm for rabbit skeletal muscle myosin S1.

Methylene blue dye as an alternative to isosulfan blue dye for sentinel lymph node localization.

Isosulfan blue dye has been used with increasing frequency in localizing sentinel lymph nodes in breast cancer patients. Few alternative types of dye have been investigated. In a prospective study of 30 patients, methylene blue dye was used instead of isosulfan blue dye to localize the sentinel lymph node. The methylene blue dye localization technique was successful in 90% of patients. These results are similar to those for isosulfan blue dye. This study describes methylene blue dye localization as a successful alternative to isosulfan dye in identifying the sentinel node in breast cancer patients. The methylene blue dye technique offers a substantial cost reduction.

Models of motor-assisted transport of intracellular particles.

One-dimensional models are presented for the macroscopic intracellular transport of vesicles and organelles by molecular motors on a network of aligned intracellular filaments. A motor-coated vesicle or organelle is described as a diffusing particle binding intermittently to filaments, when it is transported at the motor velocity. Two models are treated in detail: 1) a unidirectional model, where only one kind of motor is operative and all filaments have the same polarity; and 2) a bidirectional model, in which filaments of both polarities exist (for example, a randomly polarized actin network for myosin motors) and/or particles have plus-end and minus-end motors operating on unipolar filaments (kinesin and dynein on microtubules). The unidirectional model provides net particle transport in the absence of a concentration gradient. A symmetric bidirectional model, with equal mixtures of filament polarities or plus-end and minus-end motors of the same characteristics, provides rapid transport down a concentration gradient and enhanced dispersion of particles from a point source by motor-assisted diffusion. Both models are studied in detail as a function of the diffusion constant and motor velocity of bound particles, and their rates of binding to and detachment from filaments. These models can form the basis of more realistic models for particle transport in axons, melanophores, and the dendritic arms of melanocytes, in which networks of actin filaments and microtubules coexist and motors for both types of filament are implicated.

Postgraduate training in breast surgery.

Dramatic changes have taken place in the field of breast surgery within the past ten years. Much of the change in practice is due to the advent of less invasive surgical procedures and increased technology such as the evolution of sentinel node biopsies. Many surgeons choose to undergo additional postgraduate training to gain an increased proficiency in the diagnosis and surgical treatment of benign and malignant disorders of the breast.

Skin-sparing mastectomy with immediate breast reconstruction: a critical analysis of local recurrence.

PURPOSE: The purpose of this study was to provide follow-up data regarding the incidence of local breast cancer recurrence in patients undergoing skin-sparing mastectomy versus conventional non-skin-sparing mastectomy methods. PATIENTS AND METHODS: A retrospective follow-up study and analysis were performed of patients who underwent mastectomies for invasive breast cancer at The New York Presbyterian Hospital, Cornell University Medical College and Strang-Cornell Breast Center between 1990 and 1998. RESULTS: A total of 198 patients were identified in this study, and the mean follow-up was 49 months. This group included 71 patients who underwent skin-sparing mastectomy and 127 who underwent non-skin-sparing mastectomy procedures. No statistical differences in local recurrence rates were demonstrated between patients treated with skin-sparing mastectomy and those who underwent non-skin-sparing mastectomy. Local recurrence was present in four of 71 (5.6%) patients undergoing skin-sparing mastectomy and in five of 127 (3.9%) of those undergoing non-skin-sparing mastectomy. CONCLUSIONS: The use of skin-sparing mastectomy does not lead to an increase in local recurrence rates when compared with conventional non-sparing mastectomies and provides for improved aesthetic results after immediate reconstruction.

Predictive factors associated with axillary lymph node metastases in T1a and T1b breast carcinomas: analysis in more than 900 patients.

BACKGROUND: Axillary lymph node metastasis (ALNM) represents the single most important prognostic indicator in patients diagnosed with breast cancer. The proportion of < or = 1-cm (T1a, T1b) invasive breast carcinomas is increasing. The incidence and predictive factors associated with ALNM in patients with < or = 1-cm tumors remains unclear and the role of axillary lymph node dissection in these patients has been questioned. The purpose of this study was to determine clinical and pathologic factors predictive of ALNM in patients with < or = 1-cm invasive breast carcinomas by univariate and multivariate analyses. STUDY DESIGN: Review analysis from a prospective database identified patients with < or = 1-cm invasive breast cancers treated at our institution between 1990 and 1996. All patients underwent a resection of the primary tumor and axillary lymph node dissections. Routine patient and tumor characteristics evaluated included: age, race, tumor size, histologic grade, estrogen and progesterone receptor status, and lymphatic and vascular invasion. Univariate and multivariate analyses were performed. Adjusted odds ratios (OR) and 95% confidence intervals (CI) are presented. RESULTS: A total of 919 patients were identified in this study with tumors < or = 1 cm. These included 199 patients (21.7%) with T1a tumors and 720 patients (78.3%) with T1b tumors. ALNM was detected in 165 patients with an overall incidence of 18.0%. Of the ALNM group, 32 patients (19.4%) had T1a tumors and 133 patients (80.6%) had T1b tumors. Four variables were found to be significant in univariate analysis. These included: increasing tumor size, poor histologic grade, presence of lymphatic or vascular invasion, and younger age of the patient. An increase in tumor size was associated with a significant risk of ALNM (OR = 2.66, 95% CI = 1.28 to 5.75; p = 0.01). Poor tumor grade and the presence of lymphatic or vascular invasion were also associated with an increased risk of ALNM (OR = 2.69, p = 0.003 and OR = 5.52, p = 0.0001, respectively). Patients with ALNM were more likely to have a tumor grade of 3 (25.0% ALNM versus 12.5% node-negative, p = 0.004) and lymphatic or vascular invasion (16.9% ALNM versus 3.5% node-negative, p < 0.0001). In multivariate analysis, an increased risk of ALNM was demonstrated with increasing tumor size (0.1-cm increments), poor histologic grade, and younger age. CONCLUSIONS: This study investigated clinical and pathologic factors influencing ALNM in patients with T1a and T1b breast carcinomas. We have identified three factors by multivariate analysis as significant independent predictors of ALNM in this group of patients. These include increasing tumor size, poor histologic grade, and younger age. Given the significant amount of ALNM demonstrated in this study (overall 18%) and the inability to identify a subgroup of patients that had an acceptable low risk of ALNM, the complete omission of assessing the axilla for metastatic disease in patients with small breast cancers cannot be advocated. Our recommendation for patients diagnosed with T1a and T1b tumors is to have their axilla investigated for metastatic disease either by traditional axillary lymph node dissections or by intraoperative lymphatic mapping and sentinel lymph node biopsy techniques.

Local and distant recurrence rates in skin-sparing mastectomies compared with non-skin-sparing mastectomies.

BACKGROUND: Skin-sparing mastectomies (SSMs) are being used more frequently to treat many cases of breast cancer. This type of surgery maximizes breast skin preservation and facilitates immediate reconstruction, resulting in a superior cosmetic appearance after mastectomy and a more satisfied patient. Although SSMs are becoming more common, there are few data regarding the local and distant recurrence rates. METHODS: A total of 231 patients treated with mastectomies from 1990 to 1998 were studied, including 77 SSM and 154 non-skin-sparing (NSSM) mastectomy patients. RESULTS: The local recurrence rates for SSM and NSSM were 3.90% (3 of 77 patients) and 3.25% (5 of 154 patients), respectively. The local recurrence-free survival at 5 years was 95.3% for SSM patients and 95.2% for NSSM patients (P = .28). The distant recurrence rates of SSM and NSSM were 3.9% (3 of 77 patients) and 3.9% (6 of 154 patients), respectively. The distant recurrence-free actuarial survival at 5 years was 90.2% for SSM patients and 92% for NSSM patients (P = .07). CONCLUSIONS: Mastectomies using the skin-sparing technique do not appear to result in any increase in local or distant recurrence and improve aesthetic results of the immediate reconstruction.

Characterization of neuropeptide Y-induced feeding in mice: do Y1-Y6 receptor subtypes mediate feeding?

The stimulation of food consumption after i.c.v. administration of various neuropeptide Y (NPY) receptor agonists was examined in CD-1 mice. These agonists, including endogenous peptides NPY, peptide YY (PYY), and pancreatic polypeptide, as well as several N-terminal truncated and synthetic peptides that are prototypic receptor agonists at Y1-Y6 NPY receptors ([Leu31Pro34]NPY, NPY2-36, NPY3-36, NPY13-36, PYY3-36, Pro34PYY, and D-Trp32NPY), showed varying abilities to elicit food consumption such that PYY > NPY2-36 = NPY = PYY3-36 > Pro34PYY > NPY3-36 >> [Leu31Pro34]NPY > NPY13-36 = D-Trp32NPY = pancreatic polypeptide. Published reports have suggested that NPY-induced feeding is mediated via the Y1 or the Y5 receptor subtypes. However, the relative ability of the various peptide analogs to elicit feeding differed from the relative ability of these peptides to bind to cloned Y1-Y6 receptors. The effects of prototypic Y1 receptor antagonists on NPY-induced feeding were also evaluated after i.c.v. administration. GR231118 (1229U91), a peptide Y1 antagonist, did not block NPY-induced feeding at the doses tested. BIBP3226, a nonpeptide Y1 receptor antagonist, as well as its opposite enantiomer, BIBP3435, which is inactive at Y1 receptors, blocked feeding elicited by NPY, [Leu31Pro34], or PYY at doses that did not cause overt behavioral dysfunction. The lack of effects with GR231118 and the nonstereoselective effects of BIBP3226 suggested that NPY-induced feeding in mice was not mediated via the Y1 receptor. Thus, by using currently available prototypic peptide NPY receptor agonists for Y1-Y6 receptors and peptide and nonpeptide Y1 receptor antagonists GR231118 and BIBP3226, the mediation of NPY-induced feeding cannot be unequivocally attributed to any one of the known NPY receptors. It is possible that NPY-induced feeding is mediated either by a combination of more than one NPY receptor subtype or by a unique NPY receptor subtype. Additional subtype-selective receptor antagonists, when available, will help to clarify this issue further.

Single-molecule biomechanics with optical methods.

Single-molecule observation and manipulation have come of age. With the advent of optical tweezers and other methods for probing and imaging single molecules, investigators have circumvented the model-dependent extrapolation from ensemble assays that has been the hallmark of classical biochemistry and biophysics. In recent years, there have been important advances in the understanding of how motor proteins work. The range of these technologies has also started to expand into areas such as DNA transcription and protein folding. Here, recent experiments with rotary motors, linear motors, RNA polymerase, and titin are described.

The evaluation of high risk and pre-invasive breast lesions and the decision process for follow up and surgical intervention.

Atypical epithelial hyperplasia, lobular carcinoma in situ (lobular neoplasia), radial scar, and ductal carcinoma in situ are considered high-risk lesions that predispose toward the future development of non-invasive or invasive breast cancer. Generally, those women with atypical epithelial hyperplasia, radial scar, or lobular carcinoma in situ can be managed conservatively by close surveillance. The minority of women may consider prophylactic mastectomy. Ductal carcinoma in situ can usually be managed by lumpectomy with or without radiation, with some patients requiring mastectomy due to extensive disease.

ErbB-2 protein in sera and tumors of breast cancer patients.

We compared levels of erbB-2 oncoprotein among three groups: Group I included 60 asymptomatic women; Group II had 51 women with benign breast biopsies; and Group III had 67 women with node-negative breast cancer. Serological levels of erbB-2 protein were measured in all participants; tumor levels were measured for Groups II and III. Forty-three percent of usable tumors (25/58), including three of seven lobular tumors, were erbB-2 positive. Tumor and blood oncoprotein levels were unrelated. Blood levels, however, were positively related to tumor volume, but only when the tumor had both a ductal carcinoma in situ (DCIS) component and an invasive component, suggesting a role for erbB-2 protein in progression of DCIS to invasive carcinoma. In Groups I and II serological levels of erbB-2 protein were directly related to age, and inversely related to having had a live birth. Therefore, a model that determined the threshold levels of serological erbB-2 positivity in Group III included age and nulliparity as independent variables. Only three of the 67 women (4.5%) in Group III were positive for serological erbB-2. In a multivariate model, with serological erbB-2 as the dependent variable, and in which the independent variables included Study Group, there was a statistical trend for younger women, in which Group III had the highest serological levels of erbB-2, followed by Group II, and then Group I. In women who were over the age of 50 years the trend was reversed; i.e., levels of erbB-2 tended to be lowest in Group III, followed by Group II, and finally Group I.

Kainate GluR5 receptor subtype mediates the nociceptive response to formalin in the rat.

In order to study the roles of the AMPA and kainate subtypes of non-NMDA glutamate receptors in the processing of persistent nociceptive information, compounds with varying activities at these receptors were examined for effects on the formalin-induced paw-licking behavior in rats. The selective AMPA antagonist, LY300164 and the mixed AMPA/kainate antagonist, NBQX, were compared for their effects on formalin-induced pain behavior. NBQX (3, 10, 20 mg/kg, i.p.), caused antinociception as well as ataxia whereas the selective AMPA antagonist, LY300164 (3,5,10 mg/kg, i.p.), did not cause antinociception at doses that did not produce ataxia. In view of the well documented distribution of kainate receptors on C fibres and of the kainate-preferring iGluR5 subtype on dorsal root ganglia (DRG), we tested a series of three decahydroisoquinolines with different profiles of activity between iGluR5 and AMPA receptors and all without activity on iGluR6, iGluR7 or KA2 subtypes. LY293558 (0.1, 1, 3, 5 mg/kg, i.p.), which had low micromolar affinity for both iGluR5 and 2 caused, like NBQX, both antinociceptive and ataxic effects. However, the selective iGluR5 antagonist LY382884 (5, 10, 30, 100 mg/kg, i.p.), exhibited antinociceptive actions without ataxia while the iGluR2 preferring antagonist LY302679 (5 mg/kg, i.p), caused ataxia but did not produce antinociceptive effects at that dose. These actions were stereoselective since the enantiomeric compounds, LY293559 and LY302680, were ineffective in these tests. The data strongly suggest an involvement of iGluR5 in the processing of nociceptive information.