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.

Changes in the lateral filament spacing of skinned muscle fibres when cross-bridges attach.

When a skinned fibre prepared from frog skeletal muscle goes from the relaxed to the rigor state at a sarcomere length of about 2.2 micron, the 1, 0 transverse spacing of the filament lattice, measured by X-ray diffraction, decreases by about 11%. In measurements at various sarcomere lengths, the decrease in the spacing was approximately proportional to the degree of overlap between the thick and thin filaments. This suggests that the shrinkage of the lattice is caused by a lateral force produced by cross-bridges. In order to estimate the magnitude of the lateral force, the decrease of spacing between relaxed and rigor states was compared with the shrinkage caused osmotically by adding a high molecular weight polymer, polyvinylpyrrolidone, to the bathing solution. The results indicate that the lateral force produced per unit length of thick filament in the overlap zone is of the same order of magnitude as the axially directed force produced during maximum isometric contraction (10(-10) to 10(-9) N/micron). Experiments in the presence of a high concentration of polyvinylpyrrolidone (100 g/l) show that when the lattice spacing is decreased osmotically beyond a certain value, the lateral force produced when the fibre goes into rigor changes its direction, causing the lattice to swell. This result can be explained by assuming that there is an optimum interfilament spacing at which the cross-bridges produce no lateral force. At other spacings, the lateral force tends to displace the filament lattice toward that optimum value.

Changes in the X-ray reflections from contracting muscle during rapid mechanical transients and their structural implications.

During normal contractions of vertebrate striated muscle, it is believed that the cross-bridges which produce the sliding force undergo asynchronous cyclical changes in their structure. Thus, an X-ray diffraction diagram from a muscle under these conditions will give structural information averaged over the whole range of cross-bridge states. Such diagrams show characteristic and informative differences from those given by relaxed muscle, but can give little information about changes in the configuration of the cross-bridges at different stages of their working stroke. However, it is possible to effect a partial synchronization of these changes by applying very rapid changes in length, completed in less than one millisecond to an otherwise isometrically contracting muscle. If the amplitude of these length changes is comparable to the length of the cross-bridge stroke (say 100 A per half-sarcomere), then it should bring about a transient but significant redistribution of cross-bridge states, which would show up in the X-ray diagram. We have made use of synchrotron radiation as a high intensity X-ray source in order to record such patterns with the necessary time resolution (1 ms or less) and have found major changes in the intensity of the 143 A meridional reflection accompanying the rapid length changes of the muscle. These changes appear to arise from specific configurational changes in the cross-bridges during the working stroke. A model is suggested in which the 143 A meridional intensity in a contracting muscle arises mainly from attached cross-bridges and is generated by the part of the myosin head near the S1-S2 junction. During normal contraction, cross-bridges go through their structural cycle asynchronously with each other, since they start at different times, but if the S2 changes in length rather little, then the configurational changes in the myosin heads are synchronized with the actin filament movement in such a way that the S1-S2 junction remains relatively fixed in its axial position. In a quick release, it is suggested that bringing many S1 heads simultaneously to the end of their working strokes on actin disrupts the 143 A axial repeat of their distal ends near S2, and brings about the large decrease of the 143 A meridional reflection. This model therefore involves a large change in the position of part of the myosin head structure relative to actin during the working stroke of the cross-bridge.

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.

Synthesis and pharmacological characterization of 1-phenyl-, 4-phenyl-, and 1-benzyl-1,2,3,4-tetrahydroisoquinolines as dopamine receptor ligands.

A series of 1-phenyl-, 4-phenyl-, and 1-benzyl-1,2,3,4-tetrahydroisoquinolines have been prepared as ring-contracted analogues of the prototypical D1 dopamine receptor antagonist SCH23390 [(R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H- 3-benzazepine]. The affinity and selectivity of these isoquinolines for D1 receptors was determined by three biochemical endpoints in membrane homogenates prepared from rat corpus striatum: the potency to complete for [3H]SCH23390 binding sites; the potency to compete for [3H]spiperone (a D2 receptor ligand) binding sites; and effects on dopamine-stimulated adenylate cyclase. Competitive binding measurements at D1 sites showed SCH23390 to possess the highest affinity, followed by 1-phenyl greater than 1-benzyl greater than 4-phenyl for the isoquinolines. These results were highly correlated with the ability of the test compounds to antagonize dopamine-stimulated adenylate cyclase (r = 0.98). None of the compounds alone stimulated cAMP formation at concentrations of 10 nM to 100 microM. D2 competition binding showed the 1-benzyl derivative to possess the highest affinity, followed by 4-phenyl greater than SCH23390 greater than 1-phenyl. The tertiary 1-phenyl derivative was more potent than the secondary 1-phenyl analogue in all assays. Interestingly, resolution and single-crystal X-ray analysis of the tertiary N-methyl-1-phenyltetrahydroisoquinoline showed the most active enantiomer to possess the S absolute configuration, in contrast to the benzazepine (R)-SCH23390.

3-Aryl-1,2-diacetamidopropane derivatives as novel and potent NK-1 receptor antagonists.

Early structure-activity studies on racemic tryptophan ester and amide NK-1 antagonists 5-7 led to the discovery that the potency of the series could be markedly increased by moving the carbonyl function in these molecules to an off-chain position as in the 3-aryl-1,2-diacetamidopropane 9. Further medicinal chemistry incorporating this change resulted in the discovery of a novel series of highly potent aryl amino acid derived NK-1 antagonists of the R stereoisomeric series (IC50's = 100 pM to > 5 microM). Compounds in this series were shown to be competitive antagonists using an in vitro NK-1 smooth muscle assay, and this data correlated well with observed human NK-1 binding affinities. Two of these agents, (R)-25 and (R)-32, blocked intrathecal NK-1 agonist-driven [Ac-[Arg6,Sar9,Met(O2)11]- substance P 6-11 (Ac-Sar9)] nociceptive behavior in mice. Both compounds potently blocked the neurogenic dural inflammation following trigeminal ganglion stimulation in the guinea pig after intravenous administration. Further, upon oral administration in this model, (R)-32 was observed to be very potent (ID50 = 91 ng/kg) and have a long duration of action (> 8 h at 1 micrograms/kg). Compound (R)-32, designated LY303870, is currently under clinical development as an NK-1 antagonist with a long duration of action.

Binding of [3H]prazosin and [3H]p-aminoclonidine to alpha-adrenoceptors in rat spinal cord.

alpha-Adrenoceptors in spinal cord appear to play a role in a number of physiologic processes including the control of blood pressure, pain and motor function. In order to evaluate more clearly these potential roles, the characteristics of binding of [3H]prazosin ([3H]PRZ) to spinal alpha 1 adrenoceptors and [3H]p-aminoclonidine ([3H]PAC) to spinal alpha 2 adrenoceptors were determined. Binding of each ligand to their respective adrenoceptors was saturable and Scatchard analysis revealed binding of each to a single class of adrenoceptors with characteristics of [3H]PRZ binding of Bmax = 78 fmol/mg protein and Kd = 0.75 nM and [3H]PAC binding Bmax = 70 fmol/mg protein and Kd = 1.39 nM. Whereas [3H]PRZ specific binding (Bmax) was unaltered by guanine nucleotides. [3H]PAC binding was increased with addition of 10 microM guanosine triphosphate (GTP) (P less than 0.05) and decreased with either 50 microM GTP or guanyl-5'-yl-imidodiphosphate [Gpp(NH)p] (P less than 0.01). Competition for specific [3H]PRZ and [3H]PAC binding by various alpha 1 and alpha 2 adrenoceptor agonists and antagonists of known pharmacologic activity revealed that [3H]PRZ defines alpha 1 adrenoceptors (Ki = 2.1 nM for prazosin vs 4300 nM for yohimbine) and [3H]PAC defines alpha 2 adrenoceptors (Ki = 1.06 nM for yohimbine vs 15480 nM for prazosin). Regional spinal cord studies demonstrated that dorsal spinal cord in the lumbar region contains the highest density of both [3H]PRZ (Bmax = 93 +/- 14 fmol/mg protein) and [3H]PAC (Bmax = 101 +/- 6 fmol/mg protein) binding. In contrast, lowest binding was evident in thoracic cord with equal levels in both dorsal and ventral regions (Bmax = 44-48 fmol/mg protein). The regional distribution of both alpha 1 and alpha 2 adrenoceptors in spinal cord compares to the localization previously classified functionally utilizing various pharmacological agonists and antagonists at norepinephrine receptors.

5HT1A receptor antagonists enhance the functional activity of fluoxetine in a mouse model of feeding.

Fluoxetine has been reported to suppress food intake in animal models of feeding. Fluoxetine increases extracellular serotonin in the brain. 5HT1A autoreceptors regulate synaptic levels of serotonin. A combination of a 5HT1A receptor antagonist and fluoxetine has been previously reported to enhance extracellular levels of serotonin over what is obtained with fluoxetine alone. Thus, a combination of fluoxetine and a 5HT1A antagonist could enhance the ability of fluoxetine to suppress appetite. Fluoxetine was tested in a model of feeding, in which CD-1 mice were trained to drink sweetened condensed milk. Fluoxetine was found to attenuate milk drinking, in a dose-dependent manner, at doses greater than 10 mg/kg, i.p. A 10 mg/kg dose of fluoxetine, which was ineffective by itself, was then combined either with 5-hydroxytryptophan (5HTP), a serotonin precursor, or with S(-) pindolol, a 5HT1A/beta adrenergic receptor antagonist or with LY206130, a more selective 5HT1A receptor antagonist. These treatment paradigms resulted in significant attenuation of the consumption of sweetened condensed milk. Since fluoxetine has been shown to be useful in the treatment of eating disorders and to promote weight loss in obese humans, although at doses greater than those required for the treatment of depression, a combination of fluoxetine with a 5HT1A receptor antagonist could be of clinical utility in the treatment of eating disorders and obesity.

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.

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.

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.

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.

X-ray diffraction studies on muscle during rapid shortening and their implications concerning crossbridge behaviour.

In isometric contraction, a high proportion of crossbridges are always in the attached state and crossbridge cycling is slow. During shortening, crossbridges must be entering the detached state at a higher rate, as they come to the end of their working strokes. The size of the population of detached crossbridges will then depend on the re-attachment rate and it is therefore of some interest to find out whether a significant detached population can be detected. Observations on the equatorial X-ray diffraction pattern indicate that this is the case at higher speeds of shortening, for example at a speed where the detachment rate must be of the order of 200 per second. In a muscle under these conditions, the 59 A and 51 A actin layer line reflections decrease in intensity compared to their values during isometric contraction. This decrease does not appear to be associated with a change in structure of the actin-troponin-tropomyosin complex, since the second actin layer-line reflection remains virtually unchanged in intensity. Thus the change is likely to arise from either a different total number of attached crossbridges, or a different number of attached crossbridges in the tension generating state. The result provides some further evidence for specific helical labelling of the actin structure by crossbridges during contraction, as do some recent electronmicroscope studies of rapidly frozen contracting muscle.

Force on single actin filaments in a motility assay measured with an optical trap.

We have used an optical trap to measure or exert a force on single actin filaments via the attachment of polystyrene beads which were coated with NEM-modified HMM. In the simplest experiment, beads were attached to rhodamine phalloidin labelled actin filaments and observed to move on an HMM coated surface in the presence of ATP. Moving beads were steered into the vicinity of the trap using a PZT operated microscope stage. The minimum force needed to stop a moving bead was measured by lowering the trap strength until the bead resumed movement. By aligning the optical trap with the centre of a quadrant detector placed in an image plane of the microscope, it was possible to measure the force exerted on a filament by measuring the displacement of the bead position from the centre of the trap. In each of these experiments, the trap was calibrated by applying a Stokes force to a bead in free solution. The characteristics of the trap were studied, and the displacement of the bead from the centre of the trap was shown to be directly proportional to the applied force over a large part of the total range of the trap. The compliance of the trap could be substantially reduced by the use of feedback control to deflect the laser beam via an acousto-optic modulator. The advantages and limitations of this technique will be discussed.